Comprehensive Glycoscience, Four-Volume Set: From Chemistry to Systems Biology

Preface In terms of sub-topics, the world of carbohydrates is an extremely heterogeneous world. Quite often, only the w...

Author: J. P. Kamerling

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Preface In terms of sub-topics, the world of carbohydrates is an extremely heterogeneous world. Quite often, only the word ‘carbohydrate’ forms a connection between the different continents. Although (poly) saccharides have already been in use for decades in various industrial applications, and polysaccharidebased vaccines are on the market, new developments make them challenging for deep investigations and novel (biomolecular) applications. But nowadays carbohydrate biochemistry and molecular biology focused on glycoconjugates may also rejoice in a high level of interest from the biomedical and pharmaceutical side. Carbohydrate-mediated communication between biomolecules on the molecular level, glycoprotein engineering, and oligo- and polysaccharide immunology are all aspects of this fast growing area. Structural analysis including conformational analysis and organic/enzymatic synthesis of carbohydrates are cornerstones of nearly all of the developments mentioned. It is evident that carbohydrate research/glycoscience on a high level can only be successful in a multidisciplinary approach. After genomics and proteomics, glycomics is also ready for a key position in life sciences. Comprehensive Glycoscience brings together glycochemistry, glycoanalysis, glycobiology, glycomedicine, glyco(bio)technology, food and industrial glycoscience, and glycobioinformatics – glycoproteins, glycolipids, proteoglycans, glycosylphosphatidylinositol anchors, and polysaccharides – the microbial, animal, plant, and human world – health and disease. In a collection of 118 chapters in four volumes, and in the electronic version via Science Direct, highly-recognized glycoscientists (academic and industrial scientists, polysaccharide experts and glycoconjugate experts, chemists of all disciplines, biochemists, biologists, and medical doctors) give insight into their glycoworld. Volume 1 contains 23 chapters that give an Introduction to Glycoscience and then focuses on the Organic and Enzymatic Synthesis of Carbohydrates. Volume 2, also of 23 chapters, deals with Analysis and Structural Aspects of Glycans as well as with Polysaccharide Functional Properties. Then, in Volume 3, with 36 chapters, attention is paid to the Biochemistry of Glycoconjugate Glycans and to Carbohydrate-mediated Interactions. Finally, in Volume 4, also of 36 chapters, Cell Glycobiology and Development as well as Health and Disease in Glycomedicine are the topics of interest. The glycoknowledge is discussed by the authors on a basic (tutorial) level as well as on a more advanced level, depending on the topic. Referring to older reviews and key papers discloses the glycoliterature broadly. This makes Comprehensive Glycoscience a real encyclopedia for master students, Ph.D. students, professors, and industrial scientists, interested in the state of the glyco-art. Johannis P. Kamerling Utrecht University, Utrecht, The Netherlands

vii

Editor-in-Chief

Hans Kamerling (1944) studied chemistry at Utrecht University (1962–1969), and obtained his Ph.D. degree in 1972 with Prof. J. F. Arens and Prof. J. F. G. Vliegenthart. His glycoscience research is focused on: (1) structural studies on polysaccharides and glycoprotein glycans (since 1969); (2) organic/enzymatic synthesis of (conjugated) carbohydrates (since 1984); and (3) interaction studies between carbohydrates and complementary biomolecules (since 1990). Since 1969, he is connected to the UU Department of Bio-Organic Chemistry. In 1974 he was a visiting scientist with Prof. B. Lindberg at the University of Stockholm (Sweden), and in 1981 with Prof. A. Kobata at the University of Kobe (Japan). In the period 2000–2003 he was Dean of Studies of the Faculty of Chemistry. Since 2002 he is Chairman of the Department of Bio-Organic Chemistry. In 1990 in Yokohama (Japan), he was honoured with the prestigious Roy L. Whistler Award of the International Carbohydrate Organization. He is Doctor Honoris Causa of the Lajos Kossuth University of Debrecen, Hungary (1999), and Profesor Invitado of the University of Havana, Cuba (2002). Since 2003, he is the Dutch representative in the European Carbohydrate Organization and in the International Carbohydrate Organization. He has been / is Editor and Board Member of several scientific journals, and acted as secretary of the XIIth International Carbohydrate Symposium (1984), the 9th European Carbohydrate Symposium (1997), and the XVIth International Symposium on Glycoconjugates (2001). He is (co)author of over 370 scientific papers/books/reviews, and has supervised 42 doctoral theses at Utrecht University.

ix

Editors

Geert-Jan Boons received his B.S. in chemistry in 1983 and his Ph.D. in synthetic carbohydrate chemistry in 1991 from the State University of Leiden (Netherlands). Prior to joining the faculty at the CCRC in 1998, he spent seven years in the U.K., first as a postdoctoral fellow at Imperial College, London, and the University of Cambridge, and then as a lecturer and professor at the University of Birmingham. In 2003, Dr. Boons was the recipient of the Carbohydrate Research Award for Creativity in Carbohydrate Science from the European Carbohydrate Association. Also in 2003, he was elected chairman for 2005 of the Gordon Research Conference on Carbohydrates. In 2004, he received the Horace Isbell Award from the Division of Carbohydrate Chemistry of the American Chemical Society and was appointed Franklin Professor of Chemistry in the Franklin College of Arts and Sciences at the University of Georgia. Dr. Boons serves on the editorial boards of Carbohydrate Research, the Journal of Carbohydrate Chemistry, and Advances in Carbohydrate Chemistry and Biochemistry. Research in the Boons laboratory deals with the synthesis and biological functions of carbohydrates and glycoconjugates. The diversity of topics to which Dr. Boons has significantly contributed include the development of new and better methods for synthesizing exceptionally complex molecules, the use of new methods in the synthesis and study of properties of complex carbohydrates of increasing size and complexity, the development of synthetic cancer and bacterial vaccines, the design and synthesis of glycosidase inhibitors and the use of synthetic compounds for the study of innate immunity.

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Editors

Yuan Chan Lee received his Ph.D. degree in Biochemistry from the University of Iowa in 1962. After his postdoctoral training at the University of California in Berkeley, he was appointed Assistant Professor of Biology at Johns Hopkins University, where he became Professor of Biology in 1974. Dr. Lee’s research interests encompass glycochemistry and glycobiology, especially in the area of carbohydrate recognition. He developed neoglycoproteins and other neoglycoconjugates, with which he probed the modes of carbohydrate–protein interactions. He is also well known in the areas of carbohydrate analysis. Dr. Lee has received many awards, including the Claude Hudson Award from the American Chemical Society.

Akemi Suzuki graduated from Shinshu University School of Medicine in 1971. He joined Prof. T. Yamakawa’s laboratory, Department of Biochemistry, Faculty of Medicine, University of Tokyo, and received his D.Med.Sci. in 1977. He started his career working on glycolipid biochemistry. Then, he studied immunology in the laboratory of Prof. Donald Marcus at the Albert Einstein College of Medicine from 1978 to 1980. He returned to work in the Department of Biochemistry, University of Tokyo, and started research on glycolipid genetics using mice as an experimental model. He moved to the Tokyo Metropolitan Institute of Medical Science in 1982 and then to RIKEN Frontier System in 1999 as a group director, Supra-Biomolecular System Research Group. His research interests include regulation of glycolipid expression and microdomain formation and functions supported by glycolipids.

Editors

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Naoyuki Taniguchi graduated from the Hokkaido University School of Medicine, Sapporo, Japan in 1967, and obtained his M.D. degree and then his Ph.D. at the same university in 1972. He was a visiting Associate Professor, Department of Biochemistry, Cornell University Medical College, New York and then became Associate Professor, Biochemistry Laboratory Cancer Institute, Hokkaido University in 1980. He became Professor and Chairman of the Department of Biochemistry, Osaka University Medical School from 1986 to 2006, He is now Endowed Chair, Department of Glycomics, Research Institute for Microbial Diseases, Osaka University and Professor Emeritus of Osaka University. His research interest is mainly focused on the disease-related functional glycomics using glycosyltransferase genes and non-enzymatic glycosylation.

Alphons G. J. (Fons) Voragen, born in Heerlen, The Netherlands, studied Food Science at Wageningen University, where he obtained his M.Sc. in 1968 and his Ph.D. in 1972. He then took up a lecturer position in Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University and Research Center, interrupted by stays in the laboratories of Professor Peter Albersheim, Plant Biochemistry, at UC Boulder as research associate (1974–1976) and as visiting consultant with the H. J. Heinz Company, Pittsburgh, PA (1983–1984). Since 1989 he is full Professor in Food Chemistry at Wageningen University. His scientific interests are plant cell wall polysaccharides, processing of plant raw materials to high quality foods, food enzymology and food ingredients. He is author, coauthor, editor, or coeditor of more than 400 journal articles, book chapters and books, and editorial board member of a number of scientific journals.

Permission Acknowledgments The following material is produced with kind permission of American Association for the Advancement of Science Volume 3: Figure 3 of 3.25 Carbohydrate Recognition by Cytokines and its Relevance to their Physiological Activities Figure 2, 3, 4 and 5 of 3.28 Microbial lectins Figure 48 of 3.29 Carbohydrate–Protein Interactions: Carbohydrate-Binding Modules Volume 4: Figure 4 and 5 of 4.23 Glycoconjugates and bacterial infections: Helicobacter pylori The following material is produced with kind permission of Nature Publishing Group Volume 1: Figures 13 and 20 1.11 Enzymatic approaches to O-glycoside introduction using glycosyltransferases Volume 2: Figures 6, 8a, 8b, 8c, 8d and 8e of 2.22 Biosynthesis of cellulose Volume 3: Figures 6, 7, 8, 9, 10 and 11 of 3.28 Microbial lectins Figures 19, 26, 36, 45, 46 and 53 of 3.29 Carbohydrate–Protein Interactions: Carbohydrate-Binding Modules The following material is produced with kind permission of Oxford University Press (New York) Volume 2: Figure 7 of 2.06 X-ray crystallography of lectins Volume 3: Figure 17, 27 and 54 of 3.29 Carbohydrate–Protein Interactions: Carbohydrate-Binding Modules Figure 2 of 3.33 Anticarbohydrate antibodies Volume 4: Figure 10 of 4.15 Structure and functional interaction of glycosphingolipids inducing signal transduction to affect cellular phenotype Figure 1 of 4.18 Neural functions of glycolipids

CUMULATIVE INDEX NOTES To save space in the index the following abbreviations have been used in subentries. Enzymes in subentries have also been abbreviated. CBM – carbohydrate-binding modules EPS – exopolysaccharide ERAD – endoplasmic reticulum-associated degradation GAG – glycosaminoglycan GPI – glycosylphosphatidylinositol GSL – glycosphingolipids HPAEC – high-pH anion exchange chromatography Ig – immunoglobulin LLO – lipid-linked oligosaccharides LPS – lipopolysaccharide NMR – nuclear magnetic resonance Use of a certain nomenclature in this index does not indicate the authors’ or publisher’ s support for that particular naming system. The choice of using one style has been made for sake of clarity for the reader. Cross-reference terms in italics are general cross-references, or refer to subentry terms within the main entry (the main entry is not repeated to save space). Readers are also advised to refer to the end of each article for additional cross-references - not all of these cross-references have been included in the index cross-references. The index is arranged in set-out style with a maximum of three levels of heading. Major discussion of a subject is indicated by bold page numbers. Page numbers suffixed by t and f refer to Tables and Figures respectively. vs. indicates a comparison. This index is in letter-by-letter order, whereby hyphens and spaces within index headings are ignored in the alphabetization. Prefixes and terms in parentheses are excluded from the initial alphabetization.

A A1 subgroup see ABO(H) blood groups A2 subgroup see ABO(H) blood groups A-315675 (ABT-675) 1:915–916 ABACAN 4:306 A-band, Pseudomonas LPS core polysaccharide 1:141–142 ABC-binding cassette transporter see ATP-binding cassette (ABC)-transporter Abequose, acyclic Fischer projection formulae 1:9f ABH antigens 3:344 bacterial expression systems see Bacterial expression systems, sequential multiglycosylation erythrocytes 3:344 glycoproteins 3:344 lactosaminoglycan-type oligosaccharide chains 3:346–347, 3:365f lacto-series GSLs 3:344 polyglycosyl-ceramides 3:345, 3:346f secreted vs. 3:347 type 1 chain glycoplipids 3:346f type 2 chain glycoplipids 3:344, 3:346f type 3 chain glycoplipids 3:344, 3:345, 3:346f type 4 chain glycoplipids 3:345, 3:346f GSLs 4:274–276, 4:275f immunodominance 3:344 malignant transformation 3:348 meconium 3:347 microorganisms 3:348 oligosaccharides 3:346f, 3:347 pancreas 3:347 plants 3:348

plasma 3:347 secreted 3:347, 3:348f N-acetylgalactosamine 3:347 N-acetylglucosamine 3:347 N-acetylneuraminic acid 3:347 erythrocytes vs. 3:347 fucose 3:347 galactose 3:347 peptide moiety 3:347 structure 3:347 small intestine 3:347 species-specificity 3:348 see also specific types ABO(H) blood groups 3:343 A1 subgroup 3:353 a-1,3-N-acetylgalactosaminyltransferase 3:353 antibodies to 3:353 erythrocyte density 3:353 transferase studies 3:353 A2 subgroup 3:353 alleles 3:354 erythrocyte density 3:353 incidence 3:353 transferase studies 3:353 ABH subgroups/variants 3:352 acquired B 3:355 H-deficient variants 3:355 ‘weak A’ variant 3:354 ‘weak B’ variant 3:354 see also specific subgroups A determinant 3:344, 3:345f abbreviation 1:75t Forssmann antigen cross-reactivity 3:211 source 1:75t structure 1:75t

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B determinant 3:344, 3:345f abbreviation 1:75t source 1:75t structure 1:75t biosynthesis 3:348 Cis-AB phenotype 3:354 amino acid sequences 3:354 serum transferases 3:354 structural mutation 3:354 definition 3:344 genetics 3:348 ABO gene 3:348 Hh gene 3:349 secretor gene (Se/se) 3:349, 3:350, 3:352 glycosyltransferases 3:51, 3:349 A glycosyltransferase (a3GalNAcT) 1:422, 3:39t, 3:51 amino acid sequences 3:350f, 3:351f B galactosyltransferase 1:419 see also Transferred nuclear Overhauser experiments (trNOE) B transferase 3:39t, 3:51 Cad b4-GalNAc-transferase 3:51 a3-gal-transferase 3:51 H structure as sugar acceptor 3:349 H-transferase 3:351 linear B determinant synthesis 3:51 molecular biology 3:350 O alleles 3:350, 3:351f substrate specificity 3:349 sugar nucleotide specificity 3:349–350 2-D nuclear Overhauser enhancement spectroscopy (NOESY) 3:809, 3:810f type II membrane topology 3:350 see also specific enzymes H determinant 3:344, 3:345f, 3:354 abbreviation 1:75t

1.01 Basics Concepts and Nomenclature Recommendations in Carbohydrate Chemistry J. P. Kamerling, Utrecht University, Utrecht, The Netherlands ß 2007 Elsevier Ltd. All rights reserved. 1.01.1 Introduction

1

1.01.2 General Features of Monosaccharides

2

1.01.3 Historical Background of Chirality in Carbohydrates

3

1.01.4 From Acyclic Forms to Cyclic Forms and Back

4

1.01.4.1 Fischer Projection Formulas of Acyclic Forms

4

1.01.4.1.1

Systematic names using Fischer projection formulas of acyclic forms

1.01.4.2 Cyclic Forms Using Fischer Projection Formulas 1.01.4.2.1

Systematic names Using Fischer projection formulas of cyclic forms

7

11 13

1.01.4.3 Cyclic Forms Using Haworth Representations

13

1.01.4.4 Cyclic Forms Using Mills Depictions

16

1.01.5 Structural Aspects of Monosaccharides

16

1.01.5.1 Monosaccharide Conformations

16

1.01.5.2 Mutarotation

19

1.01.5.3 Anomeric Effect

19

1.01.5.4 Rotamers

22

1.01.5.5 Glycosides

22

1.01.6 Naming Monosaccharide Derivatives, Oligosaccharides, Polysaccharides, and Derivatized Oligosaccharides

23

1.01.6.1 Naming Monosaccharide Derivatives

23

1.01.6.2 Naming Oligosaccharides

24

1.01.6.3 Naming Polysaccharides

27

1.01.6.4 Naming Derivatized Oligosaccharides

28

1.01.7 Torsion Angles for Glycosidic Linkages and Exo-Anomeric Effect

29

1.01.7.1 Torsion Angles for Glycosidic Linkages

29

1.01.7.2 Exo-Anomeric Effect

30

1.01.8 Naming Glycoproteins

31

1.01.9 Naming Glycolipids

34

1.01.10

Naming Cyclitols

35

1.01.11

List of Abbreviations and Symbol Nomenclature

37

1.01.1 Introduction Carbohydrates belong, together with nucleic acids, proteins, and lipids, to the most important classes of organicchemical natural products on earth. In terms of abundance, this class of compounds constitutes more than 50% of the dry weight of the biomass. Via a highly complex biochemical process, plants produce using CO2 and H2O, and catalyzed by light, carbohydrate C6H12O6, predominantly glucose (photosynthesis). The generic term ‘carbohydrate’ refers to former views that carbon-containing compounds, possessing hydrogen and oxygen in the molar ratio of 2:1,

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Basics Concepts and Nomenclature Recommendations in Carbohydrate Chemistry

had to be substances built up from carbon and water: Cn(H2O)n. Later structural studies indicated that these groups of compounds were not hydrates of carbon, but polyhydroxylated organic substances. Nowadays, the terms ‘carbohydrate’, ‘saccharide’, ‘sugar’, and ‘glycan’ are used interchangeably. The term ‘saccharide’ is derived from the word for ‘sugar’ in several early languages, such as sarkara in Sanskrit, sakcharon in Greek, and saccharum in Latin. They all refer to the sweet character of the carbohydrates initially discovered. In recent years, the term ‘glyco’ is often used as a prefix of another term to indicate the involvement of carbohydrate in that particular area: glycoscience, glycochemistry, glycobiology, glycomedicine, glycomics, glycoanalysis, glycosynthesis, glycoarray, and so on. Originally mainly associated with agrochemical compounds, carbohydrates have been shown to be constituents of all living organisms. They contribute to a great variety of functions, of which the most important are: (1) major source of metabolic energy, (2) structural components of cell walls and the extracellular matrix, and (3) recognition sites on cell surfaces (adhesion, signaling). They occur as such, but also in conjugated form with proteins and lipids, generally described as glycoconjugates. In these glycoconjugates, the carbohydrate part can be responsible for its solubility, stability, and life span, like in proteins, but also for guiding its trafficking. Comprehensive Glycoscience – From Chemistry to Systems Biology reviews the state of the art in the different carbohydrate fields: plant and microbial polysaccharides, glycoproteins, proteoglycans (polysaccharides belonging to the class of glycosaminoglycans), glycolipids, glycosylphosphatidylinositol anchors, lipopolysaccharides, oligosaccharides, and many conjugated forms of small oligosaccharides. Officially, the term glycoproteins includes proteoglycans, but in general the latter biopolymers are discussed separately. In the present ‘omics’ era, glycomics is a next step in differentiation after genomics and proteomics. It is defined as the full characterization of the entire repertoire of glycans in a living organism. Carbohydrates can be divided in several ways. A frequently occurring division distinguishes (1) monosaccharides, (2) oligosaccharides, (3) polysaccharides, and (4) substances derived from monosaccharides. In this classification, monosaccharides are the single carbohydrate building blocks, the monomeric constituents of glycan chains. They cannot be hydrolyzed into more simple substances with the characteristics of a carbohydrate. Oligosaccharides are defined as carbohydrates that comprise 2–10 monosaccharides, linked together in a linear or branched way. These oligomers are called di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, and decasaccharides. The term polysaccharides is used for glycan chains built up from more than 10 monosaccharides, linked together in a linear or branched way, thereby forming a huge family of biopolymers. However, it should be noted that the borderline between oligosaccharides and polysaccharides is not drawn strictly. Over the years, nomenclature committees of the IUPAC/IUBMB (IUPAC¼International Union of Pure and Applied Chemistry; IUBMB¼International Union of Biochemistry and Molecular Biology) have formulated documents with recommendations how to describe specific aspects of carbohydrates. At the moment, the following documents are available on the internet:1,2 (a) Nomenclature of Carbohydrates (recommendations 1996); (b) Symbols for Specifying the Conformation of Polysaccharide Chains (recommendations 1981); (c) Nomenclature of Glycoproteins, Glycopeptides and Peptidoglycans (recommendations 1985); (d) Nomenclature of Glycolipids (recommendations 1997); (e) Nomenclature of Cyclitols (recommendations 1973); (f) Numbering of Atoms in myo-Inositol (recommendations 1988). These documents formed the basis for writing the major part of this chapter. But traditional ways of describing carbohydrates, which deviate from these recommendations, will also receive attention, as well as recent recommendations formulated by the USA Consortium for Functional Glycomics.3

1.01.2 General Features of Monosaccharides When looking at the chemical structure of monosaccharides, the typical structural features of polyhydroxy aldehydes or polyhydroxy ketones are observed. According to the IUPAC/IUBMB recommendations, in the carbohydrate nomenclature, polyhydroxy aldehydes are called aldoses, and polyhydroxy ketones ketoses.1,2 The term ‘aldose’ is constructed from the elements ‘ald’ of aldehyde and ‘ose’, being the suffix for a sugar. In a similar way, the term ‘ketose’ is built up from the elements ‘ket’ of ketone and ‘ose’. For monosaccharides, a classification system based on the number of carbon atoms has been established. Here, in numbering the carbon atoms, the aldehyde function in aldoses is given the locant 1; in ketoses, the ketone function receives the lowest possible locant, thereby distinguishing 2-ketoses, 3-ketoses, etc. In this classification system, glyceraldehyde can be considered as the simplest aldose, an aldotriose, and 1,3-dihydroxy-acetone as the simplest ketose, a ketotriose (Figure 1). As will be seen below, only for these smallest forms the name does not end in ‘ose’. In Table 1, an overview is presented of both the aldo- and the keto-series, wherein the more generally used term ‘ulose’ instead of ‘ketose’ is included.


O

1 C

H H

C

H

OH

Name

H

D/L

O

1 C 2 C

C

2C

O

3 CH2OH

C

C C

HOH2C

O

CH2OH

1,3-Dihydroxy-acetone

D-Glyceraldehyde

H

2C

OH

3CH OH 2

CH2OH

1 HOH2C

3

OH

H

C

OH

H

OH

OH

C

Figure 1 The simplest aldose and ketose structures in Fischer projection formulas, together with different ways of presenting a tetrahedral carbon atom in glycoscience.

Table 1 Nomenclature connecting aldoses/ketoses and number of carbon atoms No. of C atoms

Aldose series

Ketose series

3 C atoms 4 C atoms 5 C atoms 6 C atoms 7 C atoms 8 C atoms 9 C atoms

Aldotriose Aldotetrose Aldopentose Aldohexose Aldoheptose Aldooctose Aldononose

Ketotriose Ketotetrose Ketopentose Ketohexose Ketoheptose Ketooctose Ketononose

Triulose Tetrulose Pentulose Hexulose Heptulose Octulose Nonulose

One of the structural features of monosaccharides from aldotriose and tetrulose on is that they are chiral molecules with one or more asymmetric carbons. Here, a basic aspect in organic chemistry is introduced, namely, stereochemistry. Chiral molecules, having nonsuperimposable mirror images, asymmetric carbons being carbons bonded to four different atoms or groups, asymmetric carbons as chirality centers.

1.01.3 Historical Background of Chirality in Carbohydrates ¨ ber die The phenomen of chirality brings us back to 1891, the year that Emil Fischer published his famous paper ‘U Configuration des Traubenzuckers und seiner Isomeren’ in the German scientific journal Berichte (‘Concerning the configuration of glucose and its isomers’), an article dealing with the stereochemistry of the monomeric building block glucose, a real milestone in carbohydrate history. After Dumas had formulated, in 1843, the empirical formula CH2O for the compound with the name glucose, and it had become evident for Fittig and Bayer in the years 1868–70 that glucose had to be an open polyhydroxy aldehyde chain of six carbon atoms, the knowledge in 1886 had so far accumulated that four different monosaccharides with the same brutoformula C6H12O6, named glucose, fructose, galactose, and sorbose, had been identified. The scientific reports about stereochemistry dating from 1874 of both Van’t Hoff and Le Bel, suggesting that the presence of asymmetric carbon atoms in certain organic chemical compounds had to be the cause of the earlier observed property of optical activity of these compounds, formed in fact the introduction to the world-famous stereochemical investigations of Fischer. In the paper mentioned above, Fischer stated clearly ‘‘Alle bisherigen Beobachtungen in der Zuckergruppe stehen mit der Theorie des asymme¨ bereinstimmung, dass man schon jetzt den Versuch wagen darf, trischen Kohlenstoffatoms in so volkommener U dieselbe als Grundlage fu¨r die Klassification dieser Substanzen zu benu¨tzen’’ (‘‘as all observations made so far for carbohydrates are in such a good agreement with the asymmetric carbon atom theory, it will be allowable to risk already now to utilize this theory as a basis for the classification of these substances’’). The fact that in theory, for glucose, eight different structural formulas could be constructed, whereby the absolute configuration was not taken

4


into account, did not form a problem for Fischer. He was able to work out structural relationships for the aldohexoses glucose, mannose, and gulose, the ketohexose fructose, and the aldopentoses arabinose and xylose, in terms of relative configurations of the hydroxy groups at the asymmetric carbon atoms. In his approaches, he made use of various chemical reactions such as osazone formation, oxidation of aldehyde functions and/or hydroxymethyl groups, and chain elongation via aldehyde functions, of course in combination with polarimetric measurements. To formulate the differences in structure, he made use of the projection formula concept introduced by himself. However, one problem he could not solve was that of the absolute configuration of the compounds. So, he made a choice with only half a probability of being correct, and many years later, in the 1950s, it turned out that his chosen structural model for dextrorotary glucose ¼ D-glucose was the right one. It were Bijvoet and co-workers who solved in 1951 the latter problem indirectly via crystallographic studies of tartrate.

1.01.4 From Acyclic Forms to Cyclic Forms and Back 1.01.4.1 Fischer Projection Formulas of Acyclic Forms The description of spatial structures on a flat page needs careful arrangements with respect to the three-dimensional conformation and the way of projection. In glycoscience, the Fischer D,L-nomenclature, as developed for monosaccharides, is still followed. In special cases, however, use is made of the Cahn–Ingold–Prelog R,S-nomenclature. As is illustrated in Figure 1, a tetrahedral carbon atom in a flat Fischer projection is represented by two crossed lines. The horizontal lines represent the bonds coming out of the plane to the viewer, whereas the vertical lines represent the bonds going out at the backside of the plane. Worked out for tetroses, pentoses, and hexoses (Figure 2), the spatial structure, drawn in a wedge-and-dash eclipsed form, is oriented in such a way that the carbon chain is seen vertically with the C atom of the carbonyl group placed on top (aldoses; carbon atom with locant 1), or as close to the top as possible (ketoses). For the projection per chiral

H

1

O

H Name D /L

H

2 3 4

C C

OH OH

O

1

H

C

H

1

C

H

C

H

OH OH

C

CH2OH

CH2OH

O

HO

2

O

1

H

C

C

C

H

HO

C

H

C

H

HO

C

H

C

OH

H

C

OH

3

Name

HO D/L

H

4 5

D-Erythrose

CH2OH

CH2OH D-Lyxose

H

1

O

C H HO Name H D/L

H

2 3 4 5 6

C C

OH H

H H HO

O

1

1

C C C

OH H

2

HO

OH

H

C

OH

H

C

OH

H

C

OH

H

D-Glucose

CH2OH 2

C

O

HO

C

H

OH

H

C

OH

OH

H

C

OH

C

O

C

H

C

4

C

CH2OH

3

CH2OH

CH2OH

5

C

6

CH2OH

CH2OH

D-Fructose

Figure 2 Fischer projection formulas together with related spatial structures, used for the projections, for a tetrose, a pentose, and two hexoses.

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C atom, the vertical C–C bonds are going into the plane and the horizontal C–H and C–OH bonds are coming out of the plane. As a next step, the structure oriented in this way is projected in a flat Fischer projection. It may be clear, that using the energetically disfavored eclipsed conformation for the projection, the Fischer projection is not intended to reflect a true representation of the energetically most favored conformation. However, over the years, it turned out to be a useful projection in presenting and discussing monosaccharide structures. When in the Fischer projection the OH group at the bottommost asymmetric C atom, in fact the chiral C atom with the highest locant, projects on the right, the compound is defined to have the D-configuration. In case of a left-side projection of the latter OH group, the compound is defined to have the L-configuration. For nomenclature purposes, the highest-numbered chiral C atom is called the configurational atom. The stereochemical orientation at the chiral centers with lower locants relative to the orientation of the OH group used for the D/L-configuration in compounds up to four chiral centers is reflected by the historically introduced names (trivial names). It should be noted that in case of the presence of a second CO group, for example, a COOH group, the aldehyde function has a higher priority in locant number than the COOH group. The same holds in case of a ketone. Finally, it is essential to realize that the D (dextro) and L (laevo) system reflects a theoretical agreement; the symbols (þ) and (–), quite often seen in connection with a saccharide, reflect a physical value, that is, the sign of the optical rotation value, measured in a polarimeter. To differentiate between stereochemically different monosaccharide structures of the same aldo- or keto-series, a number of names and connected definitions are in use. These names are: enantiomers, diastereomers, epimers, and anomers. Pairs of enantiomers are nonsuperimposable mirror-image molecules. Pairs of diastereomers are stereomers that are not enantiomers. Pairs of epimers are diastereomers that differ in configuration at only one asymmetric carbon. Pairs of anomers are pairs of epimers that differ in configuration at the (hemi-)acetal carbon (the anomeric center; see Section 1.01.4.2). In Figure 3, Fischer projection formulas of the D-enantiomers of the common aldotriose, aldotetroses, aldopentoses, and aldohexoses are presented, including their trivial names and their abbreviations when defined (presented without D). An old saying for remembering the structures of the eight aldohexoses is: ‘‘All altruists gladly make gum in gallon tanks.’’ It reflects the systematic interchange of OH groups at the three chiral centers, C2, C3, and C4, used for the trivial naming in relation to the orientation of the OH group at C5. The L-enantiomers of the various aldoses are

CHO H

C

CHO OH

CH2OH

CHO

CHO

CHO

C

H

OH

HO

C

H

HO

C

H

OH

H

C

OH

H

C

OH

HO

C

H

H

C

OH

HO

C

H

H

C

OH

H

C

OH

H

C

OH

H

C

H

C

OH

H

C

D-Erythrose D-erythro

D-Threose D-threo

CHO

CHO

H

C

OH HO

C

H

H

C

OH

H

C

H

C

OH

H

H

C

OH

H

CH2OH

OH HO

OH

CH2OH

Gro D-glycero

C

C

CH2OH

CHO

H

H

CH2OH

D-Glyceraldehyde

CHO

CHO

CH2OH

CH2OH

CH2OH

D-Ribose

D-Arabinose

D-Xylose

D-Lyxose

Rib D-ribo

Ara D-arabino

Xyl D-xylo

D-lyxo

CHO

CHO

Lyx

CHO

CHO

CHO

H

C

OH

HO

C

H

H

C

OH HO

C

H

H

C

OH HO

C

H

OH

HO

C

H

HO

C

H

H

C

OH

H

C

OH HO

C

H

HO

C

H

C

OH

H

C

OH

H

C

OH HO

C

H

HO

C

H

HO

C

H

HO

C

H

C

OH

H

C

OH

H

C

OH

C

OH

H

C

OH

H

C

OH

H

C

OH

CH2OH

H

CH2OH

CH2OH

CH2OH

D-Allose

D-Altrose

D-Glucose

D-Mannose

D-Gulose

D-Idose

D-Galactose

D-Talose

All D-allo

Alt D-altro

Glc D-gluco

Man D-manno

Gul D-gulo

Ido D-ido

Gal D-galacto

D-talo

CH2OH

CH2OH

CH2OH

Tal

Figure 3 Fischer projection formulas of the D-enantiomers of the common aldotriose, aldotetroses, aldopentoses, and aldohexoses, including their trivial names and abbreviations (presented without D). The configurational prefixes correlate with the red parts in the structures.

6


the complete mirror images of the structures presented in Figure 3, as worked out in Figure 4 for D- and L-glucose. So, following the definitions introduced above, D-erythrose and L-erythrose are enantiomers. But D-erythrose and D-threose are diastereomers or C2 epimers. And D-glucose and D-galactose are C4 epimers. In Figure 5, Fischer projection formulas of the D-enantiomers of the common tetrulose, pentuloses, and hexuloses are depicted, including their trivial names and their abbreviations when defined (presented without D).

O H

H

H

O C

C

H

C

OH

HO

C

H

H

C

H

C

H

C

OH

HO

C

H

OH

H

C

OH

H

C

CH2OH

O

O

H

C

HO

C

H

H

C

OH

H

HO

C

H

H

HO

C

H

HO

C

H

H

C

OH

OH

HO

C

OH

HO

C

CH2OH

C

CH2OH

CH2OH

D-Glucose

L-Glucose

Mirror plane

Figure 4 Fischer projection formulas for D- and L-glucose, together with their related spatial structures, used for the projections.

CH2OH

CH2OH

CH2OH

CH2OH

C

C

O

C

O

C

O

C

OH

H

C

OH

HO

C

H

H

C

OH

H

C

OH

O

CH2OH

H

CH2OH

1,3-Dihydroxy-acetone

CH2OH

D-Erythrulose

CH2OH

D-Ribulose

D-Xylulose

Rul

Xul

CH2OH

CH2OH

CH2OH

CH2OH

C

O

C

O

C

O

C

O

H

C

OH

HO

C

H

H

C

OH

HO

C

H

H

C

OH

H

C

OH

HO

C

H

HO

C

H

H

C

OH

H

C

OH

H

C

OH

H

C

OH

CH2OH

CH2OH

CH2OH

CH2OH

D-Psicose

D-Fructose

D-Sorbose

D-Tagatose

Psi

Fru

Sor

Tag

Figure 5 Fischer projection formulas of the D-enantiomers of the common tetrulose, pentuloses, and hexuloses, including their trivial names and abbreviations (presented without D).


1.01.4.1.1

7

Systematic names using Fischer projection formulas of acyclic forms

As indicated above, for the aldoses with three to six C atoms and the 2-ketoses with four to six C atoms, a system of trivial names is in use. Most of these names have a historical background. A generally applicable nomenclature system for naming monosaccharides, in which the trivial names play an important role, has been formulated by IUPAC/ IUBMB, and first recommendations were already published in 1969.1,2 In this system, systematic names are formed from a stem name and a configurational prefix or prefixes. For aldoses, the stem names are triose, tetrose, pentose, hexose, heptose, octose, nonose, decose (see Table 1). For ketoses, the stem names are tetrulose, pentulose, hexulose, heptulose, octulose, nonulose, deculose (see Table 1); to indicate the position of the keto function, ulose is preceded by the locant of the carbonyl group, for example, hex-2-ulose, non-3-ulose. The trivial names of the aldoses up to four C atoms form the basis of the configurational prefixes in both the aldoses and the ketoses. The various configurational prefixes have been included in Figure 3, being the red parts in the various Fischer projection formulas. They are printed in lowercase italics, and are preceded by either the Fischer D- or L-, as appropriate, printed in small-capital roman letters (D-glycero, D-er ythro, etc.).

1.01.4.1.1.1 Aldoses The chain of the aldose is numbered in such a way that the carbonyl group is at position 1. Following the nomenclature rules, monosaccharides with a trivial name like D-glucose, D-mannose, and D-ribose are systematically named D-glucohexose (Figure 6), D-manno-hexose, and D-ribo-pentose, respectively. When starting a new sentence, the first letter of the stem name will be typed as a capital, but not the configurational prefix: D-gluco-Hexose. Of course, for these examples, it is much more convenient to use their trivial names than their systematic names. However, the usefulness of the nomenclature system will become clear when going to aldoses with more than four chiral centers. Here, two or even more configurational prefixes are added to the stem name, as will be illustrated for two higher aldoses (Figure 6). Generally, the prefixes are assigned in order to the chiral centers in groups of four, beginning with the group proximal to C1. The prefix relating to the group of C atom(s) farthest from C1 is cited first. So, the heptose is systematically named D-glycero-D-galacto-heptose, and not D-galacto-D-glycero-heptose. In a similar systematic evaluation, the nonose is named L-ribo-D-gluco-nonose, and not D-gluco-L-ribo-nonose. Following the description in Section 1.01.4.1, it may be clear that the latter two compounds contain two configurational atoms. 1.01.4.1.1.2 Ketoses The chain of the ketose is numbered in such a way that the carbonyl function receives the lowest possible locant. In the case of 2-ketoses, the locant 2 may be omitted if no ambiguity can arise; especially in a biochemical context this is quite often seen. Following the nomenclature rules, monosaccharides with a trivial name like D-fructose and D-sorbose CHO

CHO CHO

CHO

CHO

HO

C

H

H

C

OH

H

C

OH

HO

C

H

H

C

OH

HO

C

H

HO

C

H

OH

HO

C

H

H

C

OH

H

OH

OH

H

C

OH

HO

C

H

H

C

OH

H

OH

OH

H

C

OH

HO

C

H

H

CH2OH

D-Glucose D-gluco-Hexose

OH

OH

C

HO

OH

C

H

CH2OH

H

H

HO

CH2OH

CH2OH

D-glycero- D-galacto-Heptose

CH2OH

L-ribo-D-gluco-Nonose

Figure 6 Fischer projection formulas and systematic names of a hexose, a heptose, and a nonose. Red and blue colors have been used to visualize the specific configurational prefixes.

8


CH2OH

CH2OH

C

O

C

O

HO

C

H

H

C

OH

H

C

OH

HO

C

H

H

C

OH

H

C

OH

H

C

OH

HO

C

H

CH2OH D-Fructose D-arabino -Hex-2-ulose

CH2OH H

C

OH

C

O

HO

C

H

H

C

OH

CH2OH D-xylo -Hex-3-ulose

CH2OH CH2OH H

C

OH

C

O

H

C

OH

HO

C

H

H

C

OH

H

C

OH

HO

C

H

CH2OH L-threo - D-gulo -Non-3-ulose

L-glycero - D-gluco -Oct-2-ulose

CH2OH

CH2OH HO

C

H

H

C

OH

HO

C

H

C

O

HO

C

H

H

C

OH

C

O

HO

C

H

HO

C

H

HO

C

H

HO

C

H

HO

C

H

CH2OH L-gluco -Hept-4-ulose

CH2OH L-erythro - L-gluco -Non-5-ulose

Figure 7 Fischer projection formulas and systematic names of two hexuloses, a heptulose, an octulose, and two nonuloses. Red and blue colors have been used to visualize the specific configurational prefixes.

are systematically named D-arabino-hex-2-ulose (Figure 7) and D-xylo-hex-2-ulose, respectively. Here also it holds that at the beginning of a new sentence the stem name is typed with a capital, but not the configurational prefix: Darabino-Hex-2-ulose. It should be noted that the Chemical Abstracts Ser vice (CAS) frequently uses somewhat deviating nomenclature rules. In the CAS system, D-fructose is called D-arabino-2-hexulose; here, the locant for the carbonyl group precedes the stem name. For monosaccharides with an odd number of C atoms having the carbonyl group in the middle of the chain, a choice between alternative names is made on the basis of which comes first in the alphabet. In Figure 7, a series of examples is presented that illustrate the different agreements: L-glycero-D-gluco-oct-2-ulose, D-xylo-hex-3-ulose (not L-xylo-hex-4-ulose, because the locant order of the carbonyl function is 3 > 4), L-threo-D-gulonon-3-ulose, L-gluco-hept-4-ulose (not D-gulo-hept-4-ulose, because in the alphabetical order of the configurational prefixes gl > gu), L-er ythro-L-gluco-non-5-ulose (not D-threo-D-allo-non-5-ulose, because in the alphabetical order of the configurational prefixes e > t ; at this stage of assigning a systematic name, the alphabetical order D > L does not play a role). The various examples make clear that for ketoses with the carbonyl group at C3 or a higher-numbered C atom, the carbonyl group is ignored in choosing the appropriate configurational prefix or prefixes according to the stem names in Figure 3. To illustrate again the CAS system of naming compounds, being not in accordance with the IUPAC/IUBMB system, L-threo-D-gulo-non-3-ulose is called L-glycero-D-gluco-D-glycero-3-nonulose. Here, the sets of chiral centers on either side of the carbonyl group are separated, before counting the chiral centers.

1.01.4.1.1.3 Deoxy sugars Another important group of monosaccharides are the deoxy sugars. Like many of the aldoses and 2-ketoses, several deoxy sugars also have historically determined trivial names. Typical examples comprise the 6-deoxy sugars L-fucose (¼ 6-deoxy-L-galactose; L-Fuc), D-quinovose (¼ 6-deoxy-D-glucose; D-Qui), and L-rhamnose (¼ 6-deoxy-L-mannose;


9

L-Rha). Systematic names are usually preferred when deoxygenation occurs at one of the chiral centers of the parent sugar. Here, the systematic name consists of the prefix ‘deoxy’, preceded by the locant of the position of the deoxygenated C atom and followed by the stem name with such configurational prefixes as necessary to describe the configuration(s) at the chiral centers present in the deoxy sugar. In a similar way as already discussed for the aldoses and ketoses, in deoxy sugars also the configurational prefixes are cited in order commencing at the end farthest from C1. In Figure 8, six examples are presented, wherein the rules have been worked out. Following the nomenclature system, the systematic name for L-fucose is 6-deoxy-L-galacto-hexose. The structure called 4-deoxyD-xylo-hexose is an example which illustrates that when the CH2 group divides the chiral centers into two sets, it is ignored for the purpose of assigning a configurational prefix. Therefore, this compound should not be named 4-deoxyD-glycero-L-threo-hexose. In a biochemical context, this compound is often named 4-deoxy-D-galactose or 4-deoxy-Dglucose, also illustrating that eliminating one of the chiral centers leads in fact to two trivial-derived names. It is advised that, when a (shorter) trivial-derived name is preferred in a report, the systematic name is included once as a reference name. In a similar way, the structure called 3-deoxy-D-arabino-hexose should not be named 3-deoxy-Der ythro-L-glycero-hexose. Figure 8 also includes an example of a mixed keto/deoxy sugar: 5-deoxy-D-arabino-hept-3ulose. Here, the carbonyl group receives the lowest possible locant, as already explained for the ketoses. Taking into account all rules discussed so far, it may be evident that the keto/deoxy sugar should not be named 5-deoxy-D-glyceroD-glycero-L-glycero-hept-3-ulose. Finally, to show that two enantiomers can have different trivial names by history, the structures of abequose and colitose are presented. Their systematic names are 3,6-dideoxy-D-xylo-hexose and 3,6-dideoxy-L-xylo-hexose, respectively.

1.01.4.1.1.4 Amino sugars Well-known examples of amino sugars, with generally accepted trivial names in both organic chemistry and biochemistry, are D-glucosamine (D-GlcN) (Figure 9), D-galactosamine (D-GalN), and D-mannosamine (D-ManN). Here, the

CHO

CHO

HO

C

H

H

C

H HO

H

C

OH

OH

HO

C

C

OH

H

C

H

H

CH3 L-Fucose 6-Deoxy-L-galactose 6-Deoxy-L-galacto- hexose

CH2OH

CHO HO

C

H

H

H

C

H

C

H

H

C

OH

C

OH

H

C

OH

CH2OH

CH2OH

HO

C

H

C

O

H

C

OH

H

C

H

H

C

OH

CH2OH 4-Deoxy-D-xylo- hexose

3-Deoxy-D-arabino-hexose

5-Deoxy-D-arabino-hept-3-ulose

CHO

CHO H

C

OH

HO

C

H

H

C

H

H

C

H

HO

C

H

H

C

OH

H

C

OH

HO

C

H

CH3 Abequose 3,6-Dideoxy-D-xylo- hexose

CH3 Colitose 3,6-Dideoxy-L-xylo-hexose

Figure 8 Fischer projection formulas and systematic names of various deoxy sugars. Configurational prefixes are marked red, and deoxy functions blue.

10


CHO

COOH

H

C

NH2

C

HO

C

H

CH2

H

C

OH

H

C

H

C

OH

H 2N

CH2OH D-Glucosamine 2-Amino-2-deoxy-D-glucose 2-Amino-2-deoxy-D-gluco- hexose D-GlcN

O

CHO H

C

NHAc

HO

C

H

OH

H

C

OH

C

H

H

C

OH

HO

C

H

H

C

OH

H

C

OH

CHO

CH2OH

CH2OH N-Acetyl-D-glucosamine 2-Acetamido-2-deoxy-D-glucose 2-Acetamido-2-deoxy-D-gluco-hexose D-GlcNAc

Neuraminic acid 5-Amino-3,5-dideoxy-D-glycero - D-galacto - non-2-ulosonic acid Neu

H

C

NH2

HO

C

H

H

C

NH2

H

C

OH

CH3

Bacillosamine 2,4-Diamino-2,4,6-trideoxy-D-glucose 2,4-Diamino-2,4,6-trideoxy-D-gluco-hexose

Figure 9 Fischer projection formulas and systematic names of various amino sugars. Configurational prefixes are marked red or blue, and amino functions green.

trivial names include that C2 bears the amino function instead of a hydroxy function. However, in the systematic nomenclature approach, the replacement of a hydroxy group by an amino group is seen as a substitution of the appropriate hydrogen atom of the corresponding deoxy sugar by the amino group. Furthermore, for the stereochemistry, the amino group is regarded as being equivalent to a hydroxy function. This means that although D-glucosamine is also called 2-amino-2-deoxy-D-glucose, its systematic name is 2-amino-2-deoxy-D-gluco-hexose (alphabetical order, a > d; see Section 1.01.6.1). Another biological relevant example, depicted in Figure 9, is a compound with the trivial name neuraminic acid (Neu); using the systematic nomenclature it is called 5-amino-3,5-dideoxy-D-glycero-D-galactonon-2-ulosonic acid. In fact, the compound can be considered as derived from a ketoaldose (aldoketose, aldosulose). Furthermore, it is a deoxy sugar as well as an amino sugar. The term ‘onic acid’ indicates an oxidized aldehyde function (see Section 1.01.4.1.1.5). Note that the trivial name neuraminic acid specifically refers to the enantiomer mentioned above, which means that the name D-neuraminic acid is not correct. In a similar way, the name D-muramic acid does not exist; the trivial name muramic acid (Mur) corresponds with 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. When the amino group is substituted, in case of trivial names the substitution is indicated by a prefix preceded by the locant N, for example, N-acetyl-D-glucosamine (D-GlcNAc) (Figure 9), N-acetylneuraminic acid (Neu5Ac). When the whole substituted amino group is considered as a prefix, the structure of N-acetyl-D-glucosamine is called 2-acetamido-2-deoxy-D-glucose; its systematic name is 2-acetamido-2-deoxy-D-gluco-hexose. It should be noted that such trivial names also exist that do not imply replacement of a hydroxy group by an amino function. A typical example is bacillosamine with the systematic name 2,4-diamino-2,4,6-trideoxy-D-gluco-hexose (for a discussion of the alphabetical order of the prefixes, see Section 1.01.6.1) (Figure 9).

1.01.4.1.1.5 Reduction and oxidation of monosaccharides Due to the presence of aldehyde, keto, and hydroxy functions in carbohydrates, reduction and oxidation reactions are interesting options for modifying native materials. But also, as such, reduced and oxidized forms do occur in nature. Distinct recommendations for naming these carbohydrates are found in the IUPAC/IUBMB documents, and are discussed now (Figure 10).1,2 Reduction of the aldehyde function of an aldose or the keto function of a ketose (ulose) leads to an alditol, whereby the suffix ‘ose’ is changed into ‘itol’. Typical examples are the conversion of D-glucose into D-glucitol (D-Glc-ol) (D-gluco-hexose into D-gluco-hexitol). Or the conversion of D-fructose (D-arabino-hex-2-ulose) into a mixture of D-glucitol (D-gluco-hexitol) and D-mannitol (D-manno-hexitol). The latter example demonstrates clearly how useful the trivial name nomenclature can be. However, for higher monosaccharides, the systematic nomenclature rules are followed, for example, reduction of D-er ythro-L-galacto-octose yields D-er ythro-L-galacto-octitol (the potential name D-threo-L-gulo-octitol is not correct; see Section 1.01.6.1). When the aldehyde group of an aldose is oxidized, a so-called aldonic acid is formed. Here, in both trivial and systematic names of the aldose, the suffix ‘ose’ is replaced by ‘onic acid’ (see the systematic name of neuraminic acid


CHO

CH2OH

CHO

COOH

R

R

R

R

Aldose

Alditol

Aldose

Aldonic acid

CHO

CHO

CHO

COOH

CH2OH

COOH

CH2OH

COOH

Aldose

Alduronic acid

Aldose

11

Aldaric acid

Figure 10 Naming reduced and oxidized aldoses. The changes in suffix are marked blue.

in Section 1.01.4.1.1.4). Salts are named by changing the ending ‘onic acid’ into ‘onate’, denoting the anion; the same holds for esters. Some of the examples worked out for the C1 oxidation of D-glucose are as follows: D-gluconic acid (free acid); sodium D-gluconate (salt); methyl D-gluconate (ester). In case of oxidation of the primary hydroxy group in aldoses, yielding alduronic acids, the suffix ‘ose’ of the systematic or trivial names is replaced by ‘uronic acid’. For the anion and ester the ending ‘ose’ is changed into ‘uronate’. Examples worked out for the C6 oxidation of D-glucose are as follows: D-glucuronic acid (free acid); sodium D-glucuronate (salt); methyl D-glucuronate (ester). Following rules that are discussed in Sections 1.01.4.2 and 1.01.5.5, D-glucuronic acid (D-GlcA) in its pyranose ring form with a free anomeric center is called D-glucopyranuronic acid; in its pyranose ring form as methyl b-glycoside it is called methyl b-D-glucopyranosiduronic acid. When both the aldehyde function and the primary hydroxy group of an aldose are oxidized, a so-called aldaric acid is formed. Now, the suffix ‘ose’ of the systematic or trivial names is changed into ‘aric acid’; in salts and esters, the ending ‘ose’ is changed into ‘ate’. Examples worked out for the C1/C6 oxidation of D-glucose are the following: D-glucaric acid (free acid); disodium D-glucarate (salt); 1-methyl hydrogen D-glucarate (monoester); 1,6-dimethyl D-glucarate (diester).

1.01.4.2 Cyclic Forms Using Fischer Projection Formulas Under acidic or alkaline conditions, alcohols undergo a rapid and reversible nucleophilic addition reaction with aldehydes or ketones, affording hemiacetals (hydroxy ethers). The equilibrium normally favors the aldehyde or ketone, and the yield of the hemiacetal is low. In contrast, monosaccharides with a suitable carbon-chain length, having both hydroxy and carbonyl functions, undergo intramolecular (cyclic) hemiacetal formation. Here, the equilibrium favors cyclic forms, and ‘open chain’ forms occur only in trace amounts. Stable five-(4 C atoms and 1 O atom) and six-(5 C atoms and 1 O atom) membered ring forms are the result. To illustrate the intramolecular hemiacetal formation, Figure 11 presents the cyclization of D-glucose into a six-membered ring and the cyclization of D-fructose into a five-membered ring, making use of Fischer projection formulas. A six-membered ring is called a pyranose ring (abbreviated p), a five-membered ring a furanose ring (abbreviated f ) (see Section 1.01.4.3). Carbohydrates with a ‘hidden’ aldehyde or keto group present are called reducing sugars. In the case of D-glucose, the hydroxy group at C5 reacts intramolecularly with the aldehyde group (C1). As the carbonyl carbon C1 of the open-chain form becomes an additional asymmetric carbon in the hemiacetal formation, two pyranose rings are formed in fact. To describe the stereochemistry around C1 (denoted the anomeric C atom), the terms a and b have been chosen. The symbol a is used when the OH group attached to C1 projects in the same direction as the OH group at the configurational atom (here, to the right because of the D-configuration at C5/formally cis-orientiation of the two O atoms in the projection). The symbol b is used when the OH group bonded to C1 projects in the opposite direction as the OH group at the configurational atom (here, to the left because of the D-configuration at C5/formally trans-orientation of the two O atoms in the projection). Generally, in pentoses and hexoses, the configurational atom is used as the anomeric reference atom. In naming the cyclized forms, D-glucose is preceded

12


1

HO H HO H H

C 2 3 4 5 6

H

∗

C

OH

C

H

C

OH

O

1

1

C

H 2

H O

3

HO

4

H

C

5

H

6

CH2OH

b-D-Glucopyranose b-D-Glcp

H

C

OH

C

H

C

OH

H

C

OH

H

C 2

H HO

3 4 5 6

CH2OH

∗

OH

C

OH

C

H

C

OH

a/b

O Name

D/L

C CH2OH

a-D-Glucopyranose a-D-Glcp

D-Glucose D-Glc 1

CH2OH HO HO H H

2 3 4 5 6

C

∗

1

2

CH2OH

C

H

C

OH

O

HO

3 4

H

C

H

5 6

CH2OH

b-D-Fructofuranose b-D-Fruf

1 2

C

O

HOH2C

C

H

HO

C

OH

C

H

OH

H

3 4 5 6

CH2OH

D-Fructose D-Fru

C

∗

OH

C

H

C

OH

O

C CH2OH

a-D-Fructofuranose a-D-Fruf

Figure 11 Fischer projection formulas in cyclic form (hemiacetal form), worked out for the aldohexose D-glucose (pyranose ring) and the 2-ketohexose D-fructose (furanose ring). The parts involved in the cyclization are marked red. The newly formed chiral centers are marked with an asterisk. For a detailed discussion of the naming, including the assignment of the symbols a and b, see text.

H

C*

OH

HO

C*

H

H

C

OH

HO

C

H

HO

C

H

H

C

OH

H

C

OH

HO

C

H

H

C

C

H

CH2OH

CH2OH

a -D-Glcp

O

O

Mirror plane

a-L-Glcp

Figure 12 Fischer projection formulas in cyclic form of D- and L-glucose in the a-pyranose ring form. The parts involved in the cyclization are marked red. The anomeric C1 atom is marked with an asterisk.

by a or b and the segment ‘pyrano’ is inserted in the trivial name, yielding a-D-glucopyranose (a-D-Glcp) and b-D-glucopyranose (b-D-Glcp). In view of the foregoing discussion, it should be stressed that the mirror image of a-D-glucopyranose is a-L-glucopyranose, and not b-L-glucopyranose (Figure 12). In the case of D-fructose, the hydroxy group at C5 reacts intramolecularly with the ketone group (C2). As the carbonyl carbon C2 of the open-chain form becomes now an additional asymmetric carbon in the hemiacetal


13

formation, in fact two furanose rings are formed. Here, in the cyclized form, C2 is called the anomeric C atom, and the projection rules to name the a- and b-anomers are identical to those given above for D-glucose. So, D-fructose is preceded by a or b and the segment ‘furano’ is inserted in the trivial name, yielding a-D-fructofuranose (a-D-Fruf ) and b-D-fructofuranose (b-D-Fruf).

1.01.4.2.1

Systematic names Using Fischer projection formulas of cyclic forms

When discussing the systematic naming for acyclic forms of monosaccharides in their Fischer projection formulas, the system of configurational prefixes was introduced, a system that could handle more than four chiral centers. As explained in Section 1.01.4.2, when monosaccharides in their acyclic form are converted into cyclic forms, extra chiral centers, named anomeric centers, are created. The orientations of the new hydroxy groups are described with the a/b coding system, whereby configurational atoms act as anomeric reference atoms. This connection needs for its application in defining systematic names for cyclized monosaccharides with two or more configurational prefixes, meaning two or more configurational atoms, a further precision in terms of which configurational atom is used as the anomeric reference atom. In Figure 13, the configurational atoms have been indicated with a red arrow in the pyranosidic Fischer projection formulas of D-gluco-hexose, L-glycero-D-manno-heptose, and 5-acetamido-3,5-dideoxyD-glycero-D-galacto-non-2-ulosonic acid. Green arrows have been used to indicate the anomeric reference atom in the three structures. The situation for the already-discussed D-gluco-hexose is evident: the red and the green arrows coincide, and the presented structure is a-D-gluco-hexopyranose. The choice of the anomeric reference atom in the two other examples makes clear that, when multiple configurational prefixes are used, the anomeric reference atom is the configurational atom of the group of chiral centers next to the anomeric center that is involved in the heterocyclic ring. For assigning the anomeric configuration to L-glycero-D-manno-heptopyranose, the highest-membered chiral center of the D-manno-fragment is used, leading to L-glycero-a-D-manno-heptopyranose. In a similar reasoning, the third structure in Figure 13 has to be named 5-acetamido-3,5-dideoxy-D-glycero-b-D-galacto-non-2-ulopyranosonic acid.

1.01.4.3 Cyclic Forms Using Haworth Representations Although highly important for explaining the D/L and a/b rules discussed so far under Section 1.01.4, the drawings of the cyclic forms of the Fischer projection formulas are not very realistic. They show long C–O–C bonds between the (anomeric) C atoms involved in the ring formation via the O bridge. More realistic drawings of the cyclic forms were introduced by Haworth in the 1920s, and are called Haworth representations. These representations, being planar rings, are not projections in the plane of the paper, but perspective drawings. In these drawings, the planar rings are orientated almost perpendicular to the plane of the paper, but viewed from slightly above, so that the edge closer to the viewer is drawn below the more distant edge. The atoms connected to the ring C atoms are placed in vertical positions. In Figure 14, Haworth representations of an aldohexopyranose, an aldohexofuranose, a 2-ketohexopyranose, a 2-ketohexofuranose, an aldoheptopyranose, and a 2-ketononopyranosonic acid, together with the structures of

H

∗

C

OH

H

H

C

OH

HO

C

H

H

C

H

C

∗

C

OH

HO

C

H

HO

C

H

OH

H

C

OH

O

H

C

CH2OH

HO

C

HO

C

∗

COOH

CH2 H

C

OH

AcHN

C

H

O

O

C

H

H

H

C

OH

CH2OH

H

C

OH

CH2OH a - D-Glucopyranose a - D-gluco- Hexopyranose

L-glycero- a - D-manno- Heptopyranose

5-Acetamido-3,5-dideoxy-D-glycero- b - D-galacto - non-2ulopyranosonic acid

Figure 13 Fischer projection formulas in cyclic form and systematic names of various monosaccharides. The anomeric centers (asterisks) and the parts involved in the cyclization are marked green; red arrows indicate the configurational atom(s) and green arrows the anomeric reference atoms. For a full discussion, see text.

14


6

6

5

H

H OH

4

HO

HO

CH2OH

H

C

5

H

O H

H

OH

4

OH H

Aldohexopyranose

HO

2

H

H H

5

OH

3

OH

1

H

O CH2OH

H

4

OH

3

OH

Aldohexofuranose

HOH2C 5

2

HO

1

6

1

6

H

O 1

H 2

3

CH2OH

CH2OH

O H

H

OH

OH 3

4

H

OH

2-Ketohexopyranose

2

HO

H

2-Ketohexofuranose

7

CH2OH

H

6 6

O

O

5

Tetrahydropyran

O

O

OH H

C

H

C

COOH

4

7 8

3 1

H OH

OH 5

O H

H

2

H

H H

Pyran

O OH

AcHN

C

H

H OH HO

4

1

HO

OH 2

3

H

H

OH

9 CH2OH

Furan

Tetrahydrofuran

2-Ketononopyranosonic acid

Aldoheptopyranose

Figure 14 Haworth representations of various monosaccharides in pyranose or furanose ring form. The pyran and furan structures have been included for comparison.

the compounds that functioned as models for the terms pyranose ( p) and furanose ( f ) (pyran/tetrahydropyran and furan/tetrahydrofuran), are depicted. In D-aldohexopyranose rings (example: a-D-glucopyranose), the ring O atom is placed in the back right-hand corner of the ring, with the anomeric C1 atom at the right-hand end, whereas the ring bonds closer to the viewer are thickened. When unconventional Haworth representations with the O atom in other positions are desirable, these modifications should be carried out with great care, as mistakes can be easily introduced. In D-aldohexofuranose rings (example: a-D-glucofuranose), the ring O atom is placed away from the viewer, with the anomeric C1 atom at the right-hand end, whereas the ring bonds closer to the viewer are thickened. In the D-ketohexose examples worked out for a-D-fructopyranose and a-D-fructofuranose, identical protocols are followed, now with the anomeric C2 atom placed at the right-hand end. These rules lead to a clockwise numbering of the ring atoms. As in Fischer projection formulas, the H atoms connected to the C atoms are quite often omitted in Haworth representations also. The Haworth representations of the heptose and nonose structures correlate with the Fischer projection formulas of these structures in Figure 13. For the mirror images of these Haworth representations, no general recommendations have been formulated. From Figure 15, it can be deduced that starting from a-D-glucopyranose, a-L-glucopyranose mirror image I is closest to the D representation, including the clockwise numbering of the ring atoms. In many nomenclature questions, the Fischer projection formulas are the best to generate the right names for new compounds. Therefore, knowledge of the structural relationship between Fischer projection formulas and Haworth representations is extremely important. In Figures 16–19, the bridge between the two types of drawings is visualized for a/b-D-glucopyranose, a/b-D-glucofuranose, a/b-D-galactofuranose, and a/b-D-fructofuranose, respectively. Figure 16 shows the conversion of D-glucose (Fischer projection formula) into a/b-D-glucopyranose (Haworth representation). The first step is the conversion of the projection into the related energetically disfavored eclipsed conformation. This three-dimensional structure is rotated for 90 , so that the horizontal structure has the aldehyde function C1 at the right-hand side and the primary hydroxy function C6 at the left-hand side. To create a pyranose


15

a - D -Glucopyranose CH2OH H

CH2OH

O H H OH

H O

H

HO

H OH

H

H H HO

HO

OH OH

OH

H

a - L -Glucopyranose (II) Mirror planes

H HO

O OH CH2OH H HO

H

H OH

H

a - L -Glucopyranose (I) Figure 15 Haworth representations of D- and L-glucose in the a-pyranose ring form.

O

H C H HO H H

C

OH H

C

OH

C

HO H H

OH

C

C

H

≡

O

1

H

2 3 4 5

C

OH

C

H

HOH2C

OH

C

C

6

5

OH C

C

OH

H

C

C

4

OH

H

3

H C1 2

O

OH

OH Allowed rotation of 120⬚ around C4−C5 bond

HOH2C 6

CH2OH

H

H

D-Glucose

6

CH2OH H HO

C H

O OH

CH2OH

6

5

C

OH

H

C

C

4

OH

H

3

H

5

H

C1 2

OH

Close backward to form the ring structure

b

O

4

O

OH

1

OH 3

2

OH

OH OH a

OH

OH a/b -D-Glucopyranose

OH

Figure 16 Visualization of the conversion of D-glucose (Fischer projection formula) into a/b-D-glucopyranose (Haworth representation). The important C6–C5 part in the cyclization reaction is marked red; the backward closing is indicated in blue. For a full discussion, see text.

16


H

O

H HO H H

C

OH

C C C

C

H

H

HO

≡

OH

H

OH

H

CH2OH

O

1

H

C

2 3 4 5

C

OH

C

H

H

H

C

HOH2C

C

6

OH

C

5

H

C

C

4

OH OH C

OH 3

H

H C1 2

O

OH


HOH2C 6

D-Glucose

6 6

HO HO

5

H OH

C C H

O

CH2OH

CH2OH

C 4

H

3

H

H C

HO 4

C1 2

5

b H

O

OH

1

O

OH


OH

OH O

3

OH

H a

2

OH a /b-D-Glucofuranose

OH OH

Figure 17 Visualization of the conversion of D-glucose (Fischer projection formula) into a/b-D-glucofuranose (Haworth representation). The important C6–C5–C4 part in the cyclization reaction is marked red; the backward closing is indicated in blue.

ring, a rotation of 120 around the C4–C5 bond is carried out, bringing HO5 at the left-hand side in the plane of the main carbon chain (eclipsed conformation). Then, HO5 is ring-closed at the backside with C1 (hemiacetal formation), yielding a ring structure that is easily converted into the Haworth representation. To deduce the a/b configuration in a Haworth representation is not always easily done, as the relation with the D/L coding is no longer visible like in a Fischer projection formula. Here, it is recommended to use the orientation of the functional group at C2 (when no substituent is present at C2, use C3) in a virtual Fischer projection formula. In a similar way, a Haworth representation can be created for a/b-D-glucofuranose (Figure 17) and a/b-D-galactofuranose (Figure 18). Here, rotations of 120 are carried out around the C3–C4 bond in the three-dimensional horizontal eclipsed conformation. This leads to an upward orientation of the C5–C6 part in D-glucofuranose, and a downward orientation of the C5–C6 part in D-galactofuranose. In Figure 19, the conversion of D-fructose into a/b-Dfructofuranose is worked out. Note that in this case the ring closure of HO5 is carried out with C2.

1.01.4.4 Cyclic Forms Using Mills Depictions Mills depictions are sometimes seen in reports dealing with the organic synthesis of carbohydrates, especially when additional rings are present. In these drawings, the main hemiacetal ring is placed in the plane of the paper. Dashed bonds denote substituents below the plane, and thickened bonds above the plane. Figure 20 shows a few examples of such drawings.

1.01.5 Structural Aspects of Monosaccharides 1.01.5.1 Monosaccharide Conformations In reality, pyranose ring structures can occur in two chair (C ), six boat (B), six skew (S ), and twelve half-chair (H ) conformations (Figure 21). The included map of pyranose ring interconversions was taken from Ref. 4. For energy reasons, in practice, chair conformations C strongly dominate. The B, S, and H conformations mainly occur in case of


H

O

H HO HO H

OH

C C C

HO

≡

H

HO

OH

C

C

H

H

O

1

H

C

H

2 3 4 5

C

OH

C

H

C

HOH2C

C

6

H

5

OH C

C

C 4

H

H

H

OH OH

H

C

3

C1 2

O

OH

H


HOH2C 6

CH2OH

17

D-Galactose

O

OH

O

HO

H

OH

H

C

C

C

4

5

H

OH

C 6

3

H

4

OH

OH

1

H OH

C1 2

O

OH

H

b

2

3

OH

5

O

a

6 CH2OH

CH2OH


H

OH

OH OH

a/b-D-Galactofuranose

Figure 18 Visualization of the conversion of D-galactose (Fischer projection formula) into a/b-D-galactofuranose (Haworth representation). The important C6–C5–C4 part in the cyclization reaction is marked red; the backward closing is indicated in blue. 1

CH2OH

HO

C

O

C

H

HOH2C 2

H

C

OH

H

C

OH

3

HO

≡

4

H

5

H

C

O

C

H

C

OH

C

OH

H

OH

C

C

C

1

HOH2C 6

5

3

4

OH OH

HOH2C 6

CH2OH

H

C

CH2OH

2

O

H

Allowed rotation of 120⬚ around C4−C5 bond

D-Fructose

6

HOH2C

6

CH2OH H HO

5

H

OH

3

4

OH

2

H


2

CH2OH O

OH

CH2OH

HO

1

C

C

C

C

O

5

OH O HO b

CH2OH 1

4

3

OH

a/b -D-Fructofuranose

O

CH2OH

HO

a

OH

Figure 19 Visualization of the conversion of D-fructose (Fischer projection formula) into a/b-D-fructofuranose (Haworth representation). The important C6–C5 part in the cyclization reaction is marked red; the backward closing is indicated in blue.

18


HOH2C

HOH2C

HOH2C

O

O

HO

OH

O

HO

OH

O

O

Me2C HO

OH

HO

a - D -Glucopyranose

OH

CMe2 O

b - D -Glucopyranose

O

1,2:3,4-Di-O -isopropylidene-a - D -galactopyranose

Figure 20 Mills depictions of some monosaccharide structures.

1C 4

4

1

O

O 1

4C 1

4

3H

2

1C 4

Chairs (C) OS 2

1C 4 1H 2

1

4

B2,5

O

3H

4

1S 5

OH

1,4B

1S 3 1H O

1 1C 4

5 o

1

1

B1,4 2H 1

4H 3

O

Skew (S) 5S1

OH 4C 1

1,4B

5

3S

5

4H 5

Boat (B )

1C 4

3,OB

5S

2H

3

1 5H 4

B 3,O

2S O

2,5B

1C 4

o 5H

O Half-chair (H) 5HO

O

1C 4

Figure 21 Map of pyranose ring interconversions between chair (C), boat (B), skew (S) and half-chair (H) conformations.4 Reproduced from Stoddart, J. F. Stereochemistry of Carbohydrates; Wiley-Interscience, New York, 1971.

double bonds or specific substituents, or in intermediate situations. As is illustrated in Figure 22, for b-D-glucopyranose, transformation of a Haworth representation leads theoretically to two chair conformations, called 4C1(D) and 1 C4(D), with opposing orientations of the substituents (equatorial vs axial). However, for this monosaccharide, only the 4 C1 chair conformation is of importance. It is energetically the most favored chair with the fewest nonbonded interactions, and all OH groups and the CH2OH group (driving force) in equatorial position. This conformation is also the relevant one for a-D-glucopyranose and the a/b-anomers of D-galactose and D-mannose, having one or two axial OH groups. But in other cases the preferences can be different. For example, in a-D-idopyranose, the 1C4 chair conformation dominates (the four equatorial OH groups win from the axial CH2OH group), whereas for b-Darabinopyranose both chair conformations are in equilibrium with each other (no driving force of a CH2OH group) (see also Chapter 2.11).


19

CH2OH H

O OH H OH

4

H

HO

1 H

OH

H

b -D-Glucopyranose H 4 HO

CH2OH

CH2OH H H

HO

O

OH H 4C

CH2OH HO

H OH

H

1 H

OH

3

OH HO

H

4

OH H

H

H OH 4 (D )

HO

H CH2OH C O 4

HOH2C

C

H 4 H

H 1

H H

H b -D-Glucofuranose H

OH O

1C

1 (D )

H O

4 OH

OH 1 H

OH

OH

OH 1 H

3E

O H OH

HO 3

OH

H

1 H

E3

Figure 22 Examples of conformations of b-D-glucopyranose and b-D-glucofuranose.

Furanose ring structures occur in twist (T) and envelope (E) conformations, and in Figure 23 the population of 20 relevant conformations is shown. The included map of furanose ring interconversions was taken from Ref. 4. The difference in energy between the different conformations on the circle is in general low, whereby the orientations of the substituents form the driving forces to occur in selected three-dimensional forms (see also Chapter 2.11). As an illustration, two conformations for b-D-glucofuranose are included in Figure 22. Finally, Figure 24 shows the easiest way to transform chair conformations of D-monosaccharides into those of L-monosaccharides, as illustrated for b-D-galactopyranose and b-L-galactopyranose.

1.01.5.2 Mutarotation When dissolved in water, each aldose or ketose occurs in a monosaccharide-specific equilibrium of a-pyranose, b-pyranose, a-furanose, b-furanose, and acyclic (open chain) forms. Starting from one of the pure ring forms, this equilibrium is reached by reversible ring openings of the hemiacetals (anomers) to the acyclic form, followed by reclosures (Figure 25). This process is called mutarotation, referring to changes in optical rotation to an equilibrium value when pure anomeric forms of monosaccharides are dissolved in water. Although the equilibration is slow at neutral pH, it is catalyzed by either dilute acid or base. Thermodynamic stability of the different forms is of importance for the final molar ratio in a monosaccharide. In general, the acyclic forms are only present in trace amounts, for example, in the case of D-glucose its amount is < 0.03%. Typical examples of equilibrium molar ratios for a series of aldoses are presented in Table 2.

1.01.5.3 Anomeric Effect Although equatorially oriented OH groups in monosaccharide pyranose ring forms are energetically more favored than axial ones (see Section 1.01.5.1), the anomeric OH group with a free choice for the axial or equatorial position

20


OT 1

OE

E1

OT 4

2T

1 2E

E4

3

O

4 3T 4

2T

3

1 3E

2

E3

3T 2

4T

3E

3

3 O

4E

E2

1

4 1T

4T

2 1E

1T

3T 2

O

2

EO O

Figure 23 Map of furanose ring interconversions between twist (T) and envelope (E) conformations.4 Reproduced from Wiley VCH Europe Ltd.

OH CH2OH H H

H HO

O b - D -Galactopyranose OH

OH H

4C

1 (D)

H Mirror plane

H HOH2C H

H H

OH

O

b - L -Galactopyranose OH

HO OH

1C

4 (L)

H

Figure 24 Conformational D/L mirror images of b-D-galactopyranose [4C1 (D)] and b-L-galactopyranose [1C4 (L)].

(see Section 1.01.5.2) does not follow this rule exactly. Table 2 shows for each of the examples a substantial percentage of the anomeric OH group in axial position, which means that at a first glance the preference for the equatorial position is surprisingly low. The preference of the anomeric OH group for the axial position is called the anomeric effect, a term introduced by Lemieux in the 1960s (for the exo-anomeric effect, see Section 1.01.7.2). The anomeric effect plays an important role in carbohydrate organic synthesis. It increases when the electronegativity of the substituent at the anomeric center increases, and it decreases going to solvents with a high dielectric constant. Two main explanations have been offered for the origin of the anomeric effect: (1) an explanation based on favorable/unfavorable dipole–dipole interactions between the ring O atom and the anomeric OH group (substituent), and (2) an explanation based on favorable/unfavorable orbital–orbital interactions between the ring O atom and the anomeric OH group (substituent). As nowadays the second theory is the most accepted one, this explanation is visualized in Figure 26 for D-glucopyranose (36% a-D-Glcp and 64% b-D-Glcp). Here, a parallel overlap of the axial


OH

OH O

HO HO

21

OH

O

HO HO

OH

OH OH

b -p (64%)

a -p (36%) OH OH

HO

O

HO

C

OH Aldehyde ( m (of methyl). After the substituents have been placed in alphabetical order, the lowest locant is given to the first-cited substituent: 2 (of 2-O-acetyl) > 5 (of 5-O-acetyl). In Figure 30, the alphabetization of substituents in cyclic monosaccharide derivatives is illustrated. The name of the compound in Figure 30a is methyl 3-azido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-2-fluoro-a-D-allopyranoside. The

24


CH2OH

CH2OH

H

OH

HO

H

OH

H

HO H

HO

H

HO

OH

HO

H

H

OH

OH

H

OH

HO

H

OH

HO

H

(a)

CH3

H

H

CH2OH

CH2OH

H

H

CH3

CH2OH

CH2OH 1-Deoxy-D-arabinitol

(b)

L-Glucitol

CH2OH CH2OH MeO H HO

H OH H

CH2OH H HO H

CH2OH

OH

CH2OH

MeO

H

AcO

H

MeO

H

HO

H

H

H

OH

H

OH

OMe

H

OMe

H

OMe

CH2OH

4-O-Methyl-D-xylitol (c)

CH2OH

CH2OH

2,3,5-Tri-O-methyl-D-mannitol 2-O-Acetyl-5-O-methyl-D-mannitol (d)

(e)

Figure 29 Naming different alditol(s) (derivatives). For a full explanation, see text.

alphabetical order of the groups is indicated by a > be > br > d > f, thereby ignoring the prefix ‘tri’. The IUPAC/ IUBMB recommendations1,2 say that when there are no brackets, or within any brackets, the alphabetical order ignores ‘di’, ‘tri’, etc. The name of the compound in Figure 30b is 2-acetamido-1,3,4-tri-O-acetyl-2,6dideoxy-a-L-galactopyranose, whereby the prefixes ‘di’ and ‘tri’ are ignored. The alphabetical order of the groups follows aceta > acety > d. The name of the compound in Figure 30c is methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityla-D-glucopyranoside, with a > th > tr. The name of the compound in Figure 30d is 3-O-acetyl-4-O-benzoyl-2-O-methyla-D-mannopyranuronic acid, with a > b > m. In case of the corresponding methyl glycoside, the name becomes methyl 3-O-acetyl-4-O-benzoyl-2-O-methyl-a-D-mannopyranosiduronic acid, and in case of the sodium salt it becomes sodium (methyl 3-O-acetyl-4-O-benzoyl-2-O-methyl-a-D-mannopyranosid)uronate. The name of the compound in Figure 30e is 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-a-D-glucopyranosyl trichloroacetimidate; for the alphabetical order of the groups, a > b > d > p, the prefix ‘di’ is ignored. The name of the compound in Figure 30f is 2-O-acetyl-3-O-benzyl-(R)-4,6-O-benzylidene-a-D-glucopyranosyl bromide, with a > benzyl > benzyli. Note that the residue formed by detaching the anomeric hydroxy group from a monosaccharide is named by replacing the terminal ‘e’ of the monosaccharide name by ‘yl’. Finally, Figures 30g and 30h illustrate how careful one should be when dealing with assigning anomeric configurations, thereby always going back to original Fischer projection formulas. In b-D-glucuronic acid (Figure 30g) (b-Dgluco-hexopyranuronic acid), present as a monosaccharide constituent in glycosaminoglycans, the anomeric hydroxy group is in equatorial position. However, for dehydrated b-D-glucuronic acid (Figure 30h), with a double bond between C4 and C5, also having the anomeric hydroxy group in equatorial position, the systematic name becomes 4-deoxy-a-L-threo-hex-4-enopyranuronic acid.

1.01.6.2 Naming Oligosaccharides Reaction of the hemiacetal OH group (anomeric OH group) of monosaccharide A with an OH group of monosaccharide B results in the formation of an acetal A–B and water. The formed glycoside is a disaccharide, and the acetal


25

OAc CH2Br

CH2OTr

O

BzO

CH3

AcO

O NHAc

N3

AcO

AcO

F

OAc

OAc

OMe

(a)

SMe

(b)

(c)

COOH

CH2OBn

O OMe

BnO

AcO

AcO

Ph

O

O

O O

BnO

BnO PhthN

OH (d)

OAc OC(=NH)CCl3

(e)

COOH O

O HO

OH

HO

OH (g)

Br (f)

COOH HO

O

OH OH

(h)

Figure 30 Structures of different monosaccharide derivatives, to support the discussion of the alphabetization of substituents in the text.

linkage between the two monosaccharide residues is called the glycosidic linkage. When the OH group of monosaccharide B is also a hemiacetal OH group, a nonreducing disaccharide (A$B) is obtained, called a glycosyl glycoside, or in case of pyranose ring forms, a glycopyranosyl glycopyranoside. In all other cases, a reducing disaccharide (A!B) is formed, having a nonreducing unit A (acetal) and a reducing unit B (hemiacetal); it is called a glycosylglycose or in case of pyranose ring forms, a glycopyranosylglycopyranose. The glycosidic linkage between the two monomers can exist in a or b configuration. If possible, trivial names are used for the constituting monosaccharides, otherwise systematic names. In fact, this system follows the use of trivial and systematic names already discussed for monosaccharides. It should be noted that the setup of the coupling of two monosaccharides in this way is only of theoretical importance. In carbohydrate synthesis, anomeric center activation/protection/deprotection protocols have to be followed to realize the aimed coupling of monomers. In Figure 31, three examples are presented, which will be used to illustrate the IUPAC/IUBMB nomenclature recommendations, including abbreviated forms.1,2 As is evident from the naming of oligosaccharides above, free glycose is replaced by glycosyl; in a similar way, free glycuronic acid is transformed into glycosyluronic acid and free glyculosonic acid into glyculosylonic acid. In this context, it is worthwhile to mention that the glycosyl variant of neuraminic acid/sialic acid is, according to IUPAC/IUBMB recommendations,1,2 neuraminosyl/sialosyl; however, traditionally, neuraminyl/sialyl is used (see also Section 1.01.9). The full name of the disaccharide (Figure 31a) with the trivial name maltose is a-D-glucopyranosyl-(1!4)-Dglucopyranose. Quite often maltose is called D-maltose, but this is not correct; the D/L nomenclature is only applicable to monosaccharides. Maltose is a reducing disaccharide, because the right-side D-glucose unit has a hemiacetal OH group. The abbreviation in extended form is a-D-Glcp-(1!4)-D-Glcp; in condensed form, Glc(a1-4)Glc; in short form, Glca-4Glc or Glca4Glc. So, when the ring form is a pyranosidic one, the p is not included in the condensed and short forms. When the D or L configuration of the monosaccharide is known, it is not included in the condensed and short forms. Besides the recommended abbreviations, several variants are seen in the literature; the most frequent variants are Glca1-4Glc or Glca1!4Glc. In case of the b anomer only (b-maltose), the extended form abbreviation is a-D-Glcp-(1!4)-b-D-Glcp.

26


CH2OH HO

OH

O

CH2OH

O

HO OH

HO

CH2OH

O

O (a) Maltose

OH OCH2

HO OH

CH2OH HO

O

HO

OH

HO (c) Raffinose

O

OH HOH2C

CH2OH HO

O OH

O CH2OH

HO

(b) Cellotriose

O

O

O OH

HO

O

CH2OH

HO OH

OH

OH

Figure 31 Structures of maltose, cellotriose, and raffinose, to support the discussion on naming oligosaccharides, including abbreviated forms, in the text.

The full name of the reducing trisaccharide (Figure 31b) with the trivial name cellotriose (not D-cellotriose) is b-D-glucopyranosyl-(1!4)-b-D-glucopyranosyl-(1!4)-D-glucopyranose. The abbreviation in extended form is b-D-Glcp-(1!4)-b-D-Glcp-(1!4)-D-Glcp; in condensed form, Glc(b1-4)Glc(b1-4)Glc; in short form, Glcb-4Glcb4Glc or Glcb4Glcb4Glc. Nonreducing oligosaccharides (no free hemiacetal group) are named as glycosides. In the case of a trisaccharide, this can be a glycosylglycosyl glycoside or a glycosyl glycosylglycoside, as appropriate. This means that the full name of the nonreducing trisaccharide (Figure 31c) with the trivial name raffinose is a-D-galactopyranosyl-(1!6)-a-Dglucopyranosyl b-D-fructofuranoside (sequential (end-to-end) naming) or b-D-fructofuranosyl a-D-galactopyranosyl(1!6)-a-D-glucopyranoside (alphabetical order naming: glucose preferred to fructose for citation as glycoside). The abbreviation in extended form is a-D-Galp-(1!6)-a-D-Glcp-(1$2)-b-D-Fruf; in condensed form, Gal(a1-6)Glc(a1-2b) Fruf ; in short form, Gala-6Glca-bFruf or Gala6GlcabFruf. The various presentations of a branched structure will be illustrated using a Lewis x epitope connected to mannose as part of a glycoprotein N-glycan (see Section 1.01.8). In branched oligosaccharides, terms designating branching are enclosed in square brackets; the longest chain is regarded as the parent. If two chains are of equal length, the one with the lower locant at the branch point is preferent. This means that the IUPAC/IUBMB full name of the terminal part of the N-glycan is a-L-fucopyranosyl-(1!3)-[b-D-galactopyranosyl-(1!4)]-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1!2)-a-Dmannopyranosyl-(1! (when it is an isolated tetrasaccharide, it ends with !2)-D-mannose) However, the traditionally full name, found in the literature, will be (Fuc and Gal interchanged): b-D-galactopyranosyl-(1!4)-[a-L-fucopyranosyl-(1!3)]-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1!2)-a-Dmannopyranosyl-(1! This example, whereby the branching recommendation is not followed, has been chosen to demonstrate that for historic reasons some oligosaccharides in the literature are handled otherwise. Then, this holds, of course, also for the different abbreviated forms of the Lewis x epitope, which will be presented below following the IUPAC/IUBMB recommendations, but taking into account the historical view (Fuc and Gal interchanged).1,2 The abbreviation in extended form is written as

→

b-D-Galp-(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→ 3 1 a-L-Fucp


27

or more common nowadays is

b-D-Galp-(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→ / a-L-Fucp-(1→3)

In condensed form, the abbreviations in two or one lines become

Gal(b1-4)GlcNAc(b1-2)Man(a1| Fuc(a1-3)

or

Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-2)Man(a1-

or

Gal(b1-4)GlcNAc(b1-2)Man(a1/ Fuc(a1-3) Additional condensed non-IUPAC forms, frequently seen, are

Galb1-4(Fuca1-3)GlcNAcb1-2Mana1-

or

Galb1→4(Fuca1→3)GlcNAcb1→2Mana1→

or

Galb1-4GlcNAcb1-2Mana13 / Fuca1 In short form, the abbreviations become

Galb -4(Fuca -3)GlcNAcb-2Mana -

or

Galb 4(Fuca3)GlcNAcb 2Mana

or

Galb 4GlcNAcb 2Mana | Fuca3

1.01.6.3 Naming Polysaccharides In this section, extended forms of three polysaccharides are presented, that illustrate how the IUPAC/IUBMB recommendations are used in this field.1,2 For full carbohydrate details of polysaccharides, see Chapters 1.04 and 1.05.

[3)-b-D-Glcp-(1→4)-b -D-GlcpA-(1→4)-b-D-Glcp-(1→]n [4)-a-L-Rhap-(1→6)-b -D-GalpN-(1→]n

1 a-L-Araf

→

→

[5)-a-L-Araf-(1→5)-a-L-Araf-(1→5)-a-L-Araf-(1→5)-a-L-Araf-(1→]n 3 2 1 a-L-Araf

However, polysaccharides with branching points are also written as

[5)-a-L-Araf-(1→5)-a-L-Araf-(1→5)-a-L-Araf-(1→5)-a-L-Araf-(1→]n / / a-L-Araf-(1→3) a-L-Araf-(1→2)

28


OAc

OAc

OBn

O

O

HO BnO

AcO

O PhthN

OBn

O NPhth OAc

OH O

BnO

O

TO

O

TO

BnO

O

OAll

OT O(CH2)7CH3

(a)

OAll

NPhth

(c)

OTr O

OBn

AcO

O

O OAc

BnO

O PhthN

F O

BnO BnO

(b)

O(CH2)7CH3 O

BzO BzO

O

O OBz

OAc

Me

O OAc

AcO

O OBn OBn

AcO

OBn O OAc AcO

BzO

O OAc

O

OBn O

O

BnO

AcO

O NPhth

O

O

BnO

OMe NPhth

(d) Figure 32 Structures of different oligosaccharide derivatives, to support the discussion on naming, including the alphabetization of substituents, in the text.

1.01.6.4 Naming Derivatized Oligosaccharides To illustrate the naming of derivatized oligosaccharides, specifically focusing on alphabetization, a few examples are presented in Figure 32. As already discussed regarding the naming of nonsubstituted oligosaccharides (see Section 1.01.6.2), here in branched oligosaccharides also, terms designating branches are enclosed in square brackets. Furthermore, the longest chain is regarded as the parent, and if two chains are of equal length, the one with the lower locant at the branching point is preferred.


29

The disaccharide structure in Figure 32a is called octyl (3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-(1!2)-3,4,6-tri-O-benzyl-a-D-mannopyranoside. The alphabetic order of the substituents follows from: b > d > p. So, the positions of the substituents do not play a role at this stage. As already indicated in Section 1.01.6.1 dealing with the naming of monosaccharide derivatives, the prefix ‘di’ is ignored. The trisaccharide structure in Figure 32b is called octyl (2,3-di-O-acetyl-4-O-allyl-6-O-trityl-b-D-galactopyranosyl)(1!4)-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-(1!2)-3,4-di-O-benzyl-6-deoxy-6-fluoro-a-Dmannopyranoside. The alphabetical order of the substituents in each of the three units follows from ac>al>t; b>d>p; b>d>f. The prefixes ‘di’ and the positions of the substituents are ignored. Using this example, it is interesting to also include the former, highly complicated, way of naming oligosaccharides, as this system is widely found in older literature: octyl 2-O-[4-O-(2,3-di-O-acetyl-4-O-allyl-6-O-trityl-b-D-galactopyranosyl)-3,6-di-O-benzyl-2-deoxy-2phthalimido-b-D-glucopyranosyl]-3,4-di-O-benzyl-6-deoxy-6-fluoro-a-D-mannopyranoside. In fact, it is a system based on substitutions only. The trisaccharide structure in Figure 32c is called allyl (2,3,4-tri-O-p-toluoyl-b-D-glucopyranosyl)-(1!3)-[(3,4,6-triO-acetyl-2-deoxy-2-phthalimido-b-D-galactopyranosyl)-(1!6)]-4-O-acetyl-2-deoxy-2-phthalimido-b-D-galactopyranoside. The alphabetical order of the substituents in each of the two units follows from a >d >p; a >d >p. The prefix ‘tri’ and the positions of the substituents are ignored. The full name of the heptasaccharide structure in Figure 32d is called methyl (2,3,4-tri-O-benzoyl-b-Dxylopyranosyl)-(1!2)-[(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-(1!3)][(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-(1!6)]-(4-O-benzoyl-b-D-mannopyranosyl)-(1!4)-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl)(1!4)-[(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-(1!6)]-3-O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranoside.

1.01.7 Torsion Angles for Glycosidic Linkages and Exo-Anomeric Effect 1.01.7.1 Torsion Angles for Glycosidic Linkages For the determination of three-dimensional structures of glycan chains, the orientations of the glycosidic bonds are essential. To describe these orientations, two torsion angles are in use: f and c; in case of an exocyclic C atom, the

y (i+1) y(i + 1)

w (i+1)

O O

O

O f (i )

f (i )

O

Residue i

O

Residue i

(a)

OH

t 0 (~117⬚)

O 5⬘

O HO

C 2⬘

OH

O C

1⬘ H 1⬘ 4

f 4

(b)

H

4 OH O

C

O y

HO

C 3

OH O

Figure 33 Torsion angles f and c to describe orientations of glycosidic linkages in glycan chains. For a full discussion, see text.

30


rotamer angle o is added (Figure 33a). In this nomenclature system, the anomeric C atom of residue i is coupled via an oxygen bridge with a C atom of residue (i – 1). According to the IUPAC/IUBMB recommendations, the ring O atom of residue i is the reference atom.1,2 This means that for an aldopyranose the torsion angle f is defined as O5(i)–C1(i)–Ox(i – 1)–Cx(i – 1) and for an aldofuranose as O4(i)–C1(i)–Ox(i – 1)–Cx(i – 1). The torsion angle c is defined as C1(i)–Ox(i – 1)–Cx(i – 1)–C(x – 1) (i – 1). However, in the literature, other definitions for f and c are also applied. Therefore, it is recommended to inspect very carefully that how f and c are defined, when reading a paper dealing with conformational analysis. In Figure 33b, the various definitions have been worked out for an aldohexopyranosyl-(1!4)-aldohexopyranosyl (a-D-glucopyranosyl-(1!4)-a-D-glucopyranosyl) element:

f: t (O5⬘–C1⬘–O4–C4)

y : t (C1⬘–O4–C4–C3)

IUPAC/IUBMB

f: t (H1⬘–C1⬘–O1⬘(4)–C4)

y : t (C1⬘–O1⬘(4)–C4–H4)

Non-IUPAC/IUBMB

f: t (C2⬘–C1⬘–O1⬘(4)–C4)

y : t (C1⬘–O1⬘(4)–C4–H4)

Non-IUPAC/IUBMB

Note that, as glycosylations occur at the anomeric C atom, mechanistically the glycosidic O atom belongs to the ‘reducing unit’. So, O4 is preferred over O10 . Comparable definitions hold for (1-2) and (1-3) linkages. For an aldohexopyranosyl-(1!6)-aldohexopyranose element, the various definitions are as follows:

f: t (O5⬘–C1⬘–O6–C6)

y : t (C1⬘–O6–C6–C5)

IUPAC/IUBMB

f: t (H1⬘–C1⬘–O1⬘(6)–C6)

y : t (C1⬘–O1⬘(6)–C6–C5)

Non-IUPAC/IUBMB

f: t (C2⬘–C1⬘–O1⬘(6)–C6)

y : t (C1⬘–O1⬘(6)–C6–C5)

Non-IUPAC/IUBMB

For an explanation of the rotamer angle o, see Section 1.01.5.4.

1.01.7.2 Exo-Anomeric Effect In pyranoside forms, preferred conformations have been found to exist for exocyclic, glycosidic C–O bonds. This phenomenon, formulated by Lemieux, has been called the exo-anomeric effect. The exo-anomeric effect has a strong influence on the torsion angle f, and is therefore an important factor in directing the relative positioning between two monosaccharide residues. Model studies, comprising energy calculations on the conformations of dimethoxymethane, yielded minima values for f of about 60 (Figure 34a). In Figure 34b, three Newman projections are presented for the situation around the anomeric C atom in methyl b-D-glucopyranoside, whereby C1–Oaglycone is the axis in the projection from C1 to Oaglycone. In the situations (i) and (ii), stabilizing interactions occur between one molecular orbital with a lone pair of electrons on the Oaglycone atom and the ‘back lobe’ of the molecular orbital associated with the s-bond between C1 and O5 (s* antibonding molecular orbital). Therefore, situation (iii) is energetically not favorable. Situation (i) is preferred over situation (ii), because in situation (i) lower steric interactions exist between the methyl group and the substituents at C1 (f–60 , O5–C1–Oaglycone–Caglycone, IUPAC/IUBMB nomenclature; fþ60 , H1–C1–Oaglycone–Caglycone, non-IUPAC/ IUBMB nomenclature). The parallel overlap results in a lengthening of the endocyclic C1–O5 bond and a shortening of the exocyclic C1–Oaglycone bond. Looking at the three Newman projections for the situation around the anomeric C atom in methyl a-D-glucopyranoside (Figure 34c), in the situations (ii) and (iii) stabilizing interactions occur between one molecular orbital with a lone pair of electrons on the Oaglycone atom and the ‘back lobe’ of the molecular orbital associated with the s-bond between C1 and O5 (s* antibonding molecular orbital). Therefore, situation (i) is energetically not favorable. Situation (iii) is preferred over situation (ii), because in situation (iii) lower steric interactions exist

31


f H

O H3C

C H f O

O

O H3C

CH3

CH3

C H H

(a)

CH2OH HO

CH3

O5

O5

2 1

OH

C2

O5

C2

H3C

H Methyl b-D-glucopyranoside

CH2OH

CH3 H1

H1

H1

(i)

(ii)

(iii)

CH3

O5

HO

C2

2

O5

C2

O5

C2

O5

H

HO OH

1 OCH3

(c)

O5

OCH3

HO

(b)

C2

Methyl a-D-glucopyranoside

H3C

CH3 H1

H1

H1

(i)

(ii)

(iii)

Figure 34 Exo-anomeric effect; favorable/unfavorable orbital–orbital interactions around the glycosidic linkage in the a- and b-anomeric forms of methyl D-glucopyranoside in the 4C1 conformation. For a full discussion, see text.

between the methyl group and the substituents at C1 (f þ60 , O5–C1–Oaglycone–Caglycone, IUPAC/IUBMB nomenclature; f –60 , H1–C1–Oaglycone–Caglycone, non-IUPAC/IUBMB nomenclature). The parallel overlap results in a lengthening of the endocyclic C1–O5 bond and a shortening of the exocyclic C1–Oaglycone bond.

1.01.8 Naming Glycoproteins According to the IUPAC/IUBMB recommendations (1985), the term glycation is proposed for all reactions that link a carbohydrate in a covalent way to a protein, to give a glycoprotein.1,2 This means that both glycoside formation and amination (Schiff base reaction/Amadori rearrangement) fall under this definition. In this section, attention is only paid to nomenclature recommendations for glycoproteins that include glycoside formation (Figure 35), more specifically glycoproteins that include Asn-linked carbohydrate chains. For full carbohydrate details of all classes of glycoproteins, including also the subclass of proteoglycans, and glycosylphosphatidylinositol anchors, see Chapter 1.02. Proteins that are glycosylated at L-Asn are called N-glycoproteins or N-glycosylproteins, whereby the linkage between the monosaccharide involved and the side chain of Asn is called the N-glycosyl linkage; a typical example is the linkage between b-D-GlcNAc and Asn. The carbohydrates are called N-glycans or N-linked oligosaccharides/ sugar chains. Note that the prefix N- appears in normal style, and not in italic style (IUBMB recommendation, 1992); however, this recommendation is not generally followed in the literature. In a similar way, proteins that are glycosylated at L-Ser or L-Thr are called O-glycoproteins or O-glycosylproteins, whereby the linkage between the monosaccharide involved and the side chain of Ser/Thr is called the O-glycosyl linkage; typical examples comprise the linkage between a-D-GalNAc, b-D-GlcNAc, a-D-Man or b-D-Xyl and Ser/Thr. The carbohydrates are called O-glycans or O-linked oligosaccharides/sugar chains. Proteins that are glycosylated both via N- and O-glycosyl linkages are called N-,O-glycoproteins or N,O-glycoproteins. Besides the O-glycoproteins with Ser and Thr as attachment sites, O-glycoproteins having other amino acids as attachment sites also do occur. Proteins that are glycosylated with

32


N-GLYCAN NH2

Asn-X-Ser/Thr

O-GLYCAN

Ser O Thr

C

O NH

O-GLYCAN

H2 C

H2 C

O

P

O−

O GLYCAN

O

Glycoprotein

INOSITOL O

−O

P

Glycosylphosphatidylinositol anchor

O

O

O

O

O

C

C

O

Figure 35 Schematic representation of a glycoprotein/glycosylphosphatidylinositol anchor, showing different types of glycan chains.

a-D-Man at the side chain of L-Trp are called C-glycoproteins. For more details about the different types of carbohydrate–peptide linkages in glycoproteins, see Chapters 1.02 and 2.01. The monosaccharide–amino acid coupling products are defined as glycosyl-amino-acids and the proposed abbreviations are: (GlcNAc-)Asn, (GalNAc-)Ser, (GalNAc-)Thr, and so on. The parentheses around the monosaccharide are needed to indicate that the monosaccharide is connected to the functional group in the side chain of the amino acid instead of to the a-amino group of the amino acid. Full names are, for example, 2-acetamido-N-(L-aspart-4-yl)2-deoxy-b-D-glucopyranosylamine or N 4-(N-acetyl-b-D-glucosaminyl)asparagine for (GlcNAc-)Asn; 2-acetamido-O(L-ser-3-yl)-2-deoxy-a-D-galactopyranosylamine or O3-(N-acetyl-a-D-galactosaminyl)serine for (GalNAc-)Ser. In case of oligosaccharides coupled to an amino acid, the general term glyco-amino-acid has been defined; more specific terms such as oligosaccharylasparagine have also been proposed. According to the IUPAC/IUBMB recommendations,1,2 the N-glycans can be divided into three classes, all having the same pentasaccharide core Man(a1-3)[Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc(b1-N):

Man(a 1-6) \ / Man(a1-3)

Man(b1-4)GlcNAc(b1-4)GlcNAc(b1-N)Asn

(condensed form, three lines)

Mana 6 \ Manb4GlcNAcb4GlcNAcb Asn / Mana3

(short form, three lines)

Mana1 \ 6 Manb1-4GlcNAcb1-4GlcNAc-Asn 3 / Mana 1

(frequently used non-IUPAC form)


33

The classes are (for full details, see Chapter 1.02): 1. Oligomannose type, with a variable number of D-Man residues. However, in literature, the traditional term highmannose type is mainly seen. To discriminate between the mannose-containing N-glycans with different degrees of polymerization, as generated in the biosynthetic pathways of different species, it may be better to use the term oligomannose type for up to nine Man residues (Man9GlcNAc2) and the term high-mannose type for more than nine Man residues (Man>9GlcNAc2) (this then also includes the terms polymannose or hypermannose used for yeast-glycoprotein-derived N-glycans). The term paucimannose type is used in the literature to describe the truncated N-glycans Man99% ee) with isoprenyl acetate and Candida

Synthesis of Monosaccharides and Analogs

OAc i, O2, hn HO Rose Bengal ii, Thiourea O

OR

OH Candida antarctica lipase B

i, I2, py, CCl4 ii, NaBH4, CeCl3, MeOH iii, TBSCl, imidazole iv, CO,Bu3SnH, Pd(PPh3)4 v, NaBH4⋅CeCl3, MeOH vi, TBSCl, imidazole

OR

OR

OR

HO NH

491

i, TBSCl Imidazole ii, KOH, MeOH iii, PDC, CH2Cl2

i, O3, MeOH, −78 ⬚C iii, Me2S, 20 ⬚C iii, BnNH3Cl Na(CN)BH3 RO

493

492

i, HCl, MeOH ii, H2, Pd−C

OH

AcO

561

OR OR

NBn 494

R = TBS = SiMe2(t-Bu)

HO HO (+)-495

Scheme 128 Johnson’s synthesis of 1,3-dideoxynojirimycin.

antarctica lipase B (Novo Nordisk SP 435) to give 491. After silylation of 491 and subsequent treatment with KOH and oxidation, enantiomerically pure enone 492 is obtained.267 Treatment of 492 with iodine and pyridine leads to a-iodination.268 Successive reduction under Luche conditions, alcohol silylation, carbonylation of the iodoalkene, reduction of the obtained enal, and alcohol silylation leads to 493. Ozonolysis of 493 gives the corresponding ketoaldehyde, which is then transformed into 494 via reductive amination with high diastereoselectivity (syn:anti > 20:1). Deprotection delivers (þ)-495 (Scheme 128). A similar approach has converted enantiomerically pure (2R,3R)-2,3isopropylidenedioxycyclopent-4-en-1-one derived from cyclopentadiene269a,269b into ()-1-deoxymannonojirimycin and ()-1-deoxytalonojirimycin (1,5-dideoxy-1,5-imino-D-talitol).270a Enone 492 has been converted to (2R,3S)-2,3bis[(tert-butyl)dimethylsilyloxy]cyclopent-4-en-1-one and then to (þ)-1-deoxynojirimycin.270b Mehta and co-workers have presented271a,271b a synthesis of racemic deoxynojirimycin analogs and isofagomin analogs, starting from the Diels–Alder adduct of vinyl acetate to 5,5-dimethoxy-1,2,3,4-tetrachlorocyclopentadiene.

1.13.9.2 From Benzene and Derivatives Dioxygenases present in the blocked mutants of Pseudomonas putida, a soil bacterium, degrades benzene and its derivatives into cyclohexa-3,5-diene-1,2-diols. With chlorobenzene, diol 496 is obtained with >99% ee. This compound is converted in a few chemical steps into tetrose, pentose, and hexose derivatives (Scheme 129a).272a Further applications of this methodology are shown in Scheme 129b272b and 129c.272c A recombinant strain of E. coli expressing naphthalene dioxygenase from Pseudomonas sp. NCIB 98164 has been found to oxidize specifically N-methyl-2-pyridone into the cis-5,6-dihydro-5,6-dihydro derivative.272d

1.13.9.3 From Cycloheptatriene Cycloheptatriene has been converted into L-glucose via Pseudomonas cepacia lipase-mediated desymmetrization of a meso-3-O-protected cyclohept-6-ene-1,3,5-triol using isopropenyl acetate as solvent. Cycloheptatriene is oxidized to tropone by hydride transfer to trityl cation. Reduction of tropone with NaBH4 generates cyclohepta-3,5-dienol. After protection of the alcohol moiety, singlet oxygen adds to the diene forming endoperoxide 498, the reduction of which furnishes 499. Enzyme-catalyzed monoacetylation leads to 500 in 40% yield and high enantiomeric purity. Protection of the alcohol moiety of 500, methanolysis of its acetate, and oxidation provides enone 501. After enolization, the corresponding silyl ether 502 is oxidized to give 503. Reduction of the ketone and diol protection furnishes 504. Double hydroxylation and protection of the diol generates 505. Conversion of the benzyloxymethoxy group into a mesyloxy group, followed by desilylation and Swern oxidation, provides enone 506. After reduction of ketone 506 to the allylic alcohol, ozonolysis and reduction give triol 507 which is oxidatively cleaved to 2,3:4,5-di-O-isopropylidene273a L-glucose and by acid treatment L-glucose is released (Scheme 130).

562


Cl

Cl

Cl OH

Pseudomonas putida 39D

(MeO)2CMe2

O

TsOH

O

OH 496

497

O

O O O

HO O

(i-Bu)2AlH CH2Cl2

O

i, O3 ii, Me2S

O

(i-Bu)2AlH CH2Cl2

O O

O O

HO

L-Erythrose

acetonide

Ph3PBrMe, BuLi CH2Cl2

O

OH O

HO

O

LiAlH4 Et2O

O

OH

O

O

i, O3, CH2Cl2 ii, Me2S

O O D-Erythrose

O

O O

HO HO

OsO4 Acetone

acetonide

O O

O

O HO

HO

(a)

L-Ribonic-g -lactone acetonide

Cl

O

KMnO4

497

MgSO4

HO

O

i, O3, NaHCO3

O

MeOH ii, H2/Pd–C

OH

O O

O

O

O H HO

O OH OH

O

HO Cl 497

HO

Cl

mCPBA

O

Bu4PH2F3

O

CH2Cl2 20 ⬚C

O

100 ⬚C, 24 h 75%

O

O

F OH

O

OBn

(b)

497

ii, Me2S iii, B2H6

OH

F OBn OTBS

i, KMnO4 ii, TBSCl, imidazole

i, NaH, BnBr ii, HCl, THF

OH

i, O3, CH2Cl2

OH OH

F

TBSO

i, TBSCl, imidazole TBSO ii, LiAlH4 iii, Swern TBSO H

O

iii, O3/MeOH, −78 to 0 ⬚C HO then NaBH4, 18 ⬚C

O COOMe

COOEt

TBSO TBSO

O

i, prep. HPLC ii, O3, MeOH

O

O OH

HO

COOEt +Isomer OBn 497

N3 i, mCPBA ii, N3− iii, NaH, BnBr

O

O H

OH i, O3, MeOH H2N ii, NaBH4 HO iii, H2/Pd–C

O O Cl

O

COOEt Br

i, ii, Zn, NH4Cl, THF

O

O

i, K2CO3, BnBr

O

ii, Me2CO, TfOH iii, Swern

OH

O

AcNH

O

HO NBn2

OH OH OH Neu5Ac

O O O

BnO OH

COOH

HO

O

Cl i, mCPBA 497 ii, BnOH, TfOH

O OH

O

O

(c)

OH OH OH Kdn

COOEt

O Bn2N

O

COOH

iii, CF3COOH, H2O

TBSO HO

Zn/NH4Cl, THF

OTBS O

O HO

Br

O

HO

Cl HO

O O H F

O O

i, O3, MeOH

O

ii, NaBH3CN

H HO

OH O

OH

O

H

OH

OH HO Vitamin C

Scheme 129 a and b, Hudlicky’s syntheses of tetrose, pentose, and hexose derivatives. c, Banwell’s total syntheses of Kdn, Neu5Ac, and vitamin C.


O

i, Ph3COH HBF4

OH

OTBDMS

NaBH4

i, TBDMSCl imidazole ii, 1O2

MeOH

ii, Na2CO3 MeOH, 30 8C

563

O O 498

OTBDMS Zn, AcOH HO

OTBDMS

Amano PS-30 lipase

AcO

OH 499

500

OTBDMS

O

OBOM

OH

OAc

i, BnOCH2Cl, (i-Pr)2NEt ii, KOH, MeOH iii, Pyridinium dichromate

OTBDMS TMSOTf Et3N

OTBDMS mCPBA TMSO

TMSO

OBOM

501

O

OBOM 503

502

OTBDMS i,

OTBDMS i, (i-Bu)2AlH ii, Me2C(OMe)2 TsOH

O O

OBOM

O O

O

N

ii, Me2C(OMe)2 TsOH

O

OBOM O

504

O 505

O

OH

O

i, H2, Pd–C ii, MsCl, Et3N iii, Bu4NF, THF iv, (ClCO)2, DMSO, Et3N

i, (i-Bu)2AlH ii, O3 iii, Me2S iv, NaBH4

O O

O 506

i, NaIO4, SiO2, CH2Cl2 ii, HCl

HO HO

O

O

OH

O

OH O

O

507 OH OH

HO L-Glucose

BOM: PhCH2OCH2

Scheme 130 Johnson’s total synthesis of L-glucose.

Alcohol has been converted also to 3-deoxy-D-arabino-heptulosonic acid273b and to enantio pure 2-deoxyhexoses.273c Cycloheptatrienone (tropone) has been converted into heptitol derivatives via the optical resolution of the (tropone) Fe(CO)3 complex.273d Racemic methyl b-allopyranoside and a 2C-branched analog have been derived from cyclooctatetraene.274

1.13.9.4 From Penta-1,4-Diene When applied to penta-1,4-diene, the Sharpless asymmetric dihydroxylation forms a 1:1 mixture of (2S,4S)- and (2S,4R)penta-1,2,4,5-tetrols 508 and 509, which can be converted to diepoxides 510 and 511, respectively (Scheme 131).275 A stereo- and enantioselective synthesis of 510 is possible starting from 1,5-dichloropenta-2,4-diene applying Noyori’s asymmetric hydrogenation.276 Diepoxide 510 has been converted into alditol 512 and thioalditols 513 and 514.

1.13.9.5 From Furfural Furfural (515, furfuraldehyde) is a very inexpensive starting material obtained from the left-overs of agriculture (acidic distillation of straw and brans).277 It adds MeMgCl giving racemic 1-(2-furyl)ethanol ()-516, which has been resolved easily using either enzymatic methods278a–278c or metal-catalyzed kinetic resolution.279a,279b For instance, the reaction of ()-516 with t-butyl hydroperoxide in the presence of catalytic amounts of Ti(O-i-Pr)4 and

564


AD-mix-a HO 78% (see Scheme 43) Cl

OH

O Cu

{[(R )-BINAP]RuCl2}2 Et3N, 120 ⬚C 1200 psi H2 MeOH Cl

Cl OH OH 54%

OH OH OH 509

i, TsCl, py ii, NaH, THF

Cl O

HO

+

OH OH 508

O

O

O

O

510

KOH, Et2O 89%

511 NaOH, H2O 25 ⬚C, 36 h 78%

OH

O

OH 512

S

Na2S, EtOH 0 ⬚C, 7 h

OH

S

+

(R )-BINAP: (R )-(+)-2,2⬘-bis(diphenylphosphino)-1,1⬘-binaphthyl

OH

HO

513

5:1

OH 514

Scheme 131 Conversion of penta-1,4-diene into pentitol derivatives.

O

CHO

MeMgCl THF, 0 8C 92%

O OH

515

OH

t-BuOOH (0.6 equiv.) Ti(O-i-Pr)4 (1 equiv.) L-(+)-DIPT (1.2 equiv.) isooctane, −21 8C MS 3 Å

(±)-516

O

OH (+)-516 95% ee

O

Me +

R O 517

Scheme 132 Oxidative kinetic resolution of 1-(2-furyl)ethanol. L-(þ)-diisopropyl

tartrate (L-(þ)-DIPT) gives a mixture of optically active (þ)-516 and pyranone 517. These compounds are separated readily and isolated in 32% and 53% yield, respectively (Scheme 132).280a,280b Utilizing Novozyme 435 (immobilized on acrylic resin) to catalyze the transacetylation of ()-516 with isopropenyl acetate in (i-Pr)2O, ()-516 was obtained in 36% yield and with an ee better than 95% (55% conversion rate). If the reaction was stopped at 45% conversion rate, ester 518 was obtained in >97% ee. The same enzyme catalyzed the fast hydrolysis of 518 into (þ)-516 (Scheme 133).281a,281b Oxidation of (þ)-516 with N-bromosuccinimide in THF/ H2O gave 519. Subsequent oxidation with CrO3NH4Cl in CH2Cl2 furnished 520 that was reduced into pure 521 under Luche’s conditions (NaBH4/CeCl3) at 78 C, and into a 2:1 mixture of 521 and 522 at 0 C. Compound 522 was isolated and converted into pyranoses 523–525 as outlined in Scheme 133.281a,281b C-linked disaccharides have been derived from 1,4-di(2-furyl)butane-1,4-diol in a similar way.282

1.13.10 Enantioselective Epoxidation of Allylic Alcohols This powerful method has already been presented in Schemes 68 and 69. In this section, further applications are discussed. AZT (30 -azido-30 -deoxythymidine) and other modified nucleosides have been obtained by Jung and coworkers,283 starting from crotonaldehyde (Scheme 134). The chirality is introduced via Katsuki–Sharpless epoxidation. Enolization of crotonaldehyde with TMSCl and Et3N gives a mixture of (E)- and (Z)-1-(trimethylsilyloxy) butadiene. Condensation with methyl orthoformate using ZnCl2 as catalyst gives enal acetal 526. Reduction of 526 leads to the allylic alcohol 527, the epoxidation of which affords epoxy alcohol 528 (>95% ee, 74% yield). Opening of the epoxide with TMSN3/Et2AlF in CH2Cl2 provides 529. Acidic hydrolysis of 529 gives the corresponding 2,3-dideoxypentose derivatives 530.284,285a–285c


O

CHO

Novozyme 435

MeMgCl THF, 0 8C 92%

O

515

518

(±)-516

Lipase/H2O pH = 7.0, 25 8C 90%

NBS, NaHCO3 NaOAc/THF/H2O 93%

(+)-516

+ O

O

OAc (i-Pr)2O

OH

OAc

OH (−)-516 OH

O

518

CrO3⋅NH4Cl CH2Cl2

O

O

O

NaBH4/CeCl3 75% based on 519

O

+ HO

HO 521

522 O

H2/Pd–C 96%

522

520 O

O

O

O

519

520

565

O

HO O

NaClO/pyr. 40%

O

HO O

i, (t-Bu)Me2SiCl imidazole 98% ii, NaClO/pyr. 45%

O

O

Si O O

(i-Bu)2AlH CH2Cl2 94%

HO

O

OH

523

i, NaBH4, (PhSe)2 AcOH, i-PrOH ii, (i-Bu)2AlH 65% i, NaBH4, (PhSe)2 AcOH, i-PrOH ii, (i-Bu)2AlH 65%

HO

O

OH

OH 524

HO HO

O

OH

525

Scheme 133 Synthesis of trideoxy and dideoxyhexoses starting from furfural.

OTMS OHC

TMSCl Et3N ZnCl2

t-BuOOH Ti(O-i-Pr)4 HO O D-(−)-diisopropyl H Tartrate, SiO2 − − − − Nu: : N3 , PhS , PhSe

HC(OMe)3 ZnCl2

OHC

(i-Bu)2AlH

OMe

HO

OMe

OMe 526

H

OMe OMe

OMe

HO Nu: HO Et2AlF H

528

527

H

Nu

OMe OMe

529

HCl

OH O OH Nu 530

Scheme 134 Jung’s total synthesis of 2,3-deoxy-3-substituted D-ribose derivatives.

1.13.10.1 Desymmetrization of meso-Dienols Katsuki–Sharpless desymmetrization of penta-1,4-dien-3-ol 531 gives the monoepoxide 532, which can be converted into 1,4-dideoxy-1,4-imino-D-lyxitol 533 (Scheme 135).286 Asymmetric epoxidation of allylic alcohol 535 derived from the Wittig–Horner olefination of benzyloxyacetaldehyde with 534 (Scheme 136) gave epoxide (þ)-536 in 87% yield.287 Acid-promoted rearrangement of its acetate (þ)-537 (participation of the acetoxy group to the epoxide ring opening via intermediate 538) generated orthoester (þ)-539 (41%) and a mixture diols of (þ)-540 (16%) and (þ)-541 (11%). Further hydrolysis of this mixture and methanolysis provided triol ()-542, which has debenzylated into 2-C-methyl-D-erythritol (þ)-543.288

566


OH

OH

(+)-AE

NH3

O

HO

NH2 OH

OH

i, TsCl, py ii, NBS

N Ts

OH

531 (+)-AE: see Scheme 68

OH

HO N H

Br

532

OH

533

Scheme 135 Ja¨ger’s synthesis of 1,4-dideoxy-1,4-iminopentitols.

O

BnO

Ph3P

+

COOR

H

BnO

COOR

BnO

OH

Me

535

534 (−)-AE

O

BnO

HO

CF3COOH CHCl3 20 8C

OH

BnO

+

OH OH (+)-540

BnO

O

(+)-539

OAc

BnO

O

BnO

OH

H2O+

O

538

(+)-536 (+)-537

Ac2O/pyr DMAP

O

+

O

OH R⬘O

OH OH

(+)-541

OH OH

(+)-542 (+)-543

H2/Pd–C

R⬘ = Bn R⬘ = H

Scheme 136 Synthesis of 2-D-methyl-D-erythritol.

O OH

OH (−)-AE 94%

OBn

OBn

O

OBn

PhNCO, py 90%

O

OBn

OH

O O

OBn O

i, NaOMe MeOH ii, NaH, BnBr OBn 92%

OBn OBn H

OBn O

OBn

BnOOC CrCl2, THF 95%

545 O

OBn OBn OBn

BnO OBn TBSO

O

OBn

544

BF3⋅Et2O Et2O 90%

NHPh

O

Br

OBn OBn i, O3, MeOH ii, Me2S, −78 8C OBn OBn 95%

OBn

OBn OBn

BnO OH OBn

1:1 546A/546B i, O3, MeOH ii, Me2 S 92% iii, H2, Pd(OH)2/C, MeOH iv, AcOH, H2O

i, Chromatography OBn ii, TBSCl Imidazole DMF TBS = SiMe2(t-Bu) OH

HO O

HOOC OH

Kdo Scheme 137 Schreiber’s total synthesis of Kdo.

OBn

OH OH


567

By applying a Katsuki–Sharpless asymmetric epoxidation, Schreiber and co-workers289 have obtained Kdo from the diallyl alcohol 544 (Scheme 137). The method generates 2,3,4,5-O-tetrabenzyl-D-arabinose 545 which is coupled with CrCl2 to benzyl a-(bromomethyl)-acrylate giving a 1:1 mixture of alcohols 546A and 546B. Chromatographic separation and silylation followed by ozonolysis, hydrogenolysis, and acid treatment provide Kdo.

1.13.10.2 Kinetic Resolution of Racemic Allylic Alcohols The Katsuki–Sharpless asymmetric epoxidation of racemic diol ()-547 (obtained by allylation of (E)-crotonaldehyde) gives, after chromatographic separation, the er ythro-epoxide (þ)-548 (33% yield, >95% ee). Its urethane undergoes assisted epoxide ring opening under acidic conditions providing a 10:1 mixture of arabino-carbonate (þ)-549 and ribostereomer. Carbonate hydrolysis and subsequent ozonolysis generates D-olivose (Scheme 138). Asymmetric epoxidation of the kinetically resolved dienol ()-547 (72%) leads to ()-548 (75% yield, 95% ee). Acidic hydrolysis of ()-548 gives a ribo-triol, the ozonolysis of which leads to D-digitoxose.290a–290c In a similar manner, asymmetric epoxidation of dienol ()-550 leads to (þ)-551, which can be converted into (þ)-oliose, and to ()-550, which can be converted to (þ)-cymarose.291a,291b

(−)-AE⬘

O

OH

+

OH

(±)-547

OH

(+)-548

(−)-547

i, PhNCO, py ii, BF3⋅Et2O

(+)-AE⬘

HO

Me

Me

+

i, H3O ii, O3 iii, Me2S

O OH

OH

HO (+)-D-Olivose

O O

OH

O (+)-549

OH

i, H3O+ ii, O3 HO iii, Me2S

O

OH

LiAlH4

O OH OH

(+)-D-Digitoxose

(−)-548

Me

OH

(−)-AE⬘

HO

Me

O OH

OH

O (±)-550

(+)-551 OH

OH (+)-AE⬘ O (−)-550

(+)-D-Oliose Me

HO

O

MeOH TsOH

OH

OH

HO

OMe

OMe (+)-Cymarose

(−)-551

(+)-AE⬘, (−)-AE⬘, see Scheme 68 OH

OH

OH

(+)-AE⬘

+ OBn

OBn

OBn

O

(±)-552 i, Red-Al, THF ii, O3 iii, H2, Pd–C

Me

O

OH OBn

OH

i, BnOH, HCl ii, MeI, NaH iii, H2, Pd−C

Me

O

OH OH

MeO L-Chalcose

Scheme 138 Total syntheses of 2,6-dideoxyhexoses.

568


D- and L-chalcose have been prepared in a similar way from the racemic mixtures of threo-dipropenylglycol ()-552 obtained by reductive dimerization of crotonaldehyde.292 A simple, divergent, asymmetric synthesis of the four stereomers of the 3-amino-2,3,6-trideoxy-L-hexose family has been proposed by Dai and co-workers,293 which is based on the Katsuki–Sharpless asymmetric epoxidation of allylic alcohols (Scheme 139). N-Trifluoroacetyl-L-daunosamine, N-trifluoroacetyl-L-acosamine, N-benzoyl-D-acosamine, and N-benzoyl-D-nitrosamine have been derived from methyl sorbate via the methyl 4,5-epoxy-(E)-hex-2-enoates obtained via a chemoenzymatic method.294 Application of the Katsuki–Sharpless enantioselective epoxidation to racemic mono-O-benzylated divinylglycol has allowed to prepare enantiomerically pure L-lyxo- and D-lyxo-pentoses and analogs.295,296a–296c

1.13.11 Enantioselective Sharpless Dihydroxylation and Aminohydroxylation This extremely powerful method has already been presented in Scheme 43, and selected examples of application are collected in this section. For instance (Scheme 140), tetritol and tetrose derivatives are obtained readily from asymmetric dihydroxylation of (E)-but-2-ene-1,4-diol,297 and 4-deoxy-D- and L-threose are derived from benzene1,2-dimethyl acetal of (E)-crotonaldehyde. Asymmetric dihydroxylation of the dimethyl acetal of 5-[(tert-butyldiphenylsilyl)oxy]-(E)-pent-3-enal generates a diol that is converted in three steps into 2-deoxyxylofuranosides with high ee.298 Asymmetric dihydroxylation of 2-vinylfuran gives diol (þ)-555. Its oxidation with mCPBA generates an enone that eliminates an equivalent of water with the formation of (þ)-isolevoglucosenone. This compound can be isomerized into ()-levoglucosenone, as shown in Scheme 141. The L-hexose derivatives ()-isolevoglucosenone and (þ)-levoglucosenone are obtained with the same ease.299 OH

OH

(+)-AE

O

+

CH2Cl2, −25 8C (+)-553 (35%, 90% ee) BzHN (−)-554

i, TsO− OMe OMe ii, TsOH

O

O

OBz

HO

O

NH2 OH

O Me

O

O Me NH2

OH

NH2 Acosamine (L-arabino)

OH

DEAD Ph3P, PhMe iii, MeOH, MeONa

O Me

O

NH3 MeOH

i, (−)-AE ii, p-NO2C6H4COOH

OH

NH2

Ph3P, CH2Cl2

(+)-553

O Me NH2

Ristosamine (L-ribo)

i, NaN3 NH4Cl 100 8C ii, LiAlH4 Et2O

O

i, (−)-AE ii, PhCOOH (+)-553 DEAD

HO

OH

OTs (−)-554

(−)-554 (43.5%, >90% ee)

OH

i, NH3, MeOH, 100 8C ii, BzCl, K2CO3

OH

OH

OH

i, TsO− ii, OMe OMe TsOH iii, NaN3 iv, LiAlH4, Et2O v, TsOH, H2O

HO 3-epi-Daunosamine (L-xylo) Scheme 139 Total syntheses of 3-amino-2,3,6-trideoxyhexoses.

OH

HO NH2 L-Daunosamine NH2 OH OH


OH

OTBS

OTBS HO

i, AD-mix-b ii, TBSCl, py

i, SOCl2 ii, RuCl3

OH

OBn

569

O

O

Bu4NF

S

O

O

OBn

OBn TBS = SiMe2(t-Bu)

SPh

O PhSNa

HO

OSO3−

OH

OBn

O i, mCPBA ii, AcONa, Ac2O

OBn

HO

H OH

OBn OH

AD-mix-b, 25 ⬚C, 2d t-BuOH/H2O (1:1) 96%, 82% ee

OH

OH O

O Pd(OH)2, H2 (50 psi) MeOH, pH 1.0, 65 ⬚C

H OH 4-Deoxy-D-threose

O

O O AD-mix-a, 25 ⬚C, 2 d t-BuOH/H2O (1:1) 100%, 79% ee

OH

OH

OH O

O Pd(OH)2, H2 (50 psi) MeOH, pH 1.0, 65 ⬚C

H OH 4-Deoxy-L-threose

O

Scheme 140 Total asymmetric synthesis of tetritol and tetrose derivatives.

O

AD-mix-a MeSO2NH2 aq. t-BuOH 0 ⬚C

OH O

HO

OH O OH

mCPBA CH2Cl2

TsOH PhH, 80 ⬚C

O (+)-555

H2O2 NaOH

O O

O

O

(+)-Isolevo glucosenone

O

O

O

O

H2NNH2⋅H2O AcOH (cat) MeOH, 0 ⬚C

O

OH

MnO2 CH2Cl2 20 ⬚C

O

O

O

(−)-Levoglucosenone O

AD-mix-b MeSO2NH2 aq. t-BuOH 0 ⬚C

OH HO

O

O

O

O

O

O (−)-Isolevoglucosenone

O

(+)-Levoglucosenone

Scheme 141 Total synthesis of (þ)- and ()-isolevoglucosenone and of (þ)- and ()-levoglucosenone.

Applying the same route, D- and L-mannose have been obtained in five-step synthesis (39% overall yield) from furfural. Similarly, the same methodology has been extended to the preparation of D- and L-gulose and D- and L-talose (19% yield),300a,300b and to the synthesis of 2-deoxy- and 2,3-dideoxyhexoses.301 A route to difluorosugar 561 has been developed. It includes a Stille coupling302a–302c of 556 and 557 that generates diene 558. Sharpless asymmetric dihydroxylation of 558 was chemoselective and provided diol 559 in 54% yield. The corresponding acetonide 560 was then debenzylated with H2O2/LiOH. After treatment with 12 M HCl in THF, the semiprotected 1-deoxy-1,1-difluoro-D-xylulose 562 was obtained (Scheme 142).303

570


Pd(OAc)2, CuI Ph3P, DMF OPMBz 30−40 ⬚C 41% 557 MEM = MeOCH2CH2OCH2 PMBz = 4-MeOC6H4CO

OMEM F

+

SnBu3

I

F 556 (AD-mix-a) K2OsO4⋅2H2O (DHQD)2PHAL K3Fe(CN)6, K2CO3 t-BuOH/H2O 54%

MEMO F F

OMEM F F 558

MEMO F

OH Acetone, CuSO4 TsOH, 20 ⬚C 68%

OPMBz OH

OPMBz

F

OPMBz OH

560: R = PMBz 561: R = H

559 H2O2, LiOH CF2H O

O i, HCl, THF ii, Acetone, TsOH, CuSO4

OH

O

HO 562

Scheme 142 Synthesis of 1-deoxy-1,1-difluoro-D-xylose.

O

O R

EtO

i, NMO, OsO4 t-BuOH/acetone ii, Ac2O/py

OH

AD-mix-a

R

EtO OH 565 (80%, 80% ee) 566 (89%, 89% ee)

563: R = H 564: R = OBn

O O

OAc OH R

EtO

566

OAc OH 567 55%, d.r. 6:1 568 89%, d.r. 5:1) O

O

HO OBn

569 d.r. 5:1 O EtOOCCH2P(OCH2CF3)2

t-BuOK 18-crown-6 THF

OBn O i, AD-mix-a 70% ii, t-BuMe2SiCl imidazole 80%

HO

HO

+

H

i, NMO, OsO4 t-BuOH/acetone ii, TsOH/py 53%

O OBn TBSO 571

NMO OsO4 MeOH 70%

OBn O

OEt 570

O HO

O Bu4NF THF 80%

O

HO TBSO 572 d.r. 10:1

HO

O

HO OBn

HO

OBn

573 91%, ee

Scheme 143 O’Doherty’s iterative dihydroxylation of dienoates.

Ethyl D-galactonates 567 and 568 have been prepared by two successive dihydroxylations of dienoates 563 and 564, respectively (Scheme 143). Intermediate diol 566 has been converted into L-galacto-g-lactone 569 (Scheme 143).304 When using the (2Z,4E)-dienoate 570, the same sequence of dihydroxylations provided L-talo-glactone derivative 573.305 Both cis-(()-576) and trans-2-substituted-1,2,3,6-tetrahydropyridin-3-ol ()-577 have been prepared via an aldol condensation of tosylamide 574 with acrolein and subsequent ring-closing metathesis catalyzed by Grubbs’ I catalyst (Scheme 144).306 Asymmetric dihydroxylation of trans-acetonide ()-577 with the Hsung–Vedejs AD-mix-b307 gave


Ts N

Ts H N COOEt

i, (i-Pr)2NLi THF ii, Acrolein THF, 87%

COOEt

i, LiAlH4 THF, 87% ii, Me2C(OMe)2 p - TsOH, PhH 55%

OH

574 Ts N

Ts N

Grubbs' cat. I CH2Cl2 87−90%

O O

Ts N

Hsung−Vedejs AD-mix-a 28 h

O

O

O

(±)-576

575

(±)-577

Ts N

+

O

O

(±)-577 Ts N HO

OH (+)-578 42%

O

OH (−)-579 35%

i, Red-Al, THF toluene 110 ⬚C, 8 h ii, MeOH/HCl 40% H N HO

O

+

O

HO

571

46%

H N

OH HO

OH

OH (+)-580

OH OH

OH (+)-581

Hsung−Vedejs AD-mix-a : 1:3:3:2:0.1:0.05 alkene/K3Fe(CN)6/K2CO3/MeSO2NH2/(DHQ)2PHAL/OsO4 Grubbs’ cat. I: (Cy3P)2Ru(Cl)2CHPh Scheme 144 Ring-closing metathesis and asymmetric dihydroxylation: synthesis of iminoalditols.

OEt

O

(HJQ)2PHAL(1.2 mol%) t-BuOH

582

OH 583

O CbzN H OH

(EtO)3CH TsOH

EtO

+

O

i, NaIO4 ii, NaBH4 iii, TBSCl

COOEt OH 584

583 7:1

O

OTBS O CbzN H

585 O

OH

O COOEt CbzN H

Bn OC NCl ONa OsO4 (1 mol%)

O

(i-Bu)2AlH 91%

OH

−

mCPBA CH2Cl2 0 ⬚C

HO

N Cbz OTBS

586 OH i, NaBH4,CeCl3 ii, OsO4/NMO

N Cbz OTBS

HO EtO

OH OH

N Cbz OTBS

H2/Pd−C TsOH, MeOH

HO

OH

H TSO

N H OH −

Scheme 145 Application of the Sharpless asymmetric aminohydroxylation and of the aza-Achmatowicz reaction to the synthesis of D-1-deoxymannonojirimycin.

572


COOEt

AD-mix-a CH3SO2NH2 t-BuOH/H2O 0 ⬚C 86%

OH COOEt OH

TBSCl/Et3N DMAP CH2Cl2 20 ⬚C 64%

587 i, 4-NO2C6H4COCl py, DMAP, 0 ⬚C ii, NaN3/DMF 20 ⬚C 80%

COOEt N3 589

i, CF3COOH, THF/H2O, 20 ⬚C ii, (i - Bu)2AlH, THF/acetone, −78 ⬚C iii, TsOH/py (MeO)3CH/MeOH

Me

O

TsHN (−)-591

OMe

COOEt OH 588 93% ee OTBS

i, H2/Pd−C EtOAc, 20 ⬚C ii, TsCl, Na2CO3 Toluene/H2O 20 ⬚C 80%

OTBS

OTBS

COOEt NHTs 590 TBS = SiMe2 (t-Bu) T = 4-MeC6H4SO2 DMAP = 4-Me2NC5H4N

COOEt 587

TsN=C=O Pd(Ph3P)4 THF, 86 %

NTs

O O

O i, H2/Pd−C, EtOAc, 20 ⬚C ii, NaOH/MeOH iii, CF3COOH/THF, 72%

592

ii, TsOH/py, (MeO)3CH, MeOH, 94%

NHTs 593

TsHN i, (i-Bu)2AlH, THF/toluene, −78⬚C

O

Me

O

594 OMe

Scheme 146 New route to deoxyamino sugars.

(þ)-578 and ()-579 in 42% and 35% yield, respectively. The tosyl groups and acetonides were then removed by RedAl and HCl/MeOH to give 1,5-dideoxy-1,5-imino-D-allitol (þ)-580 and 1,5-dideoxy-1,5-imino-L-mannitol (þ)-581. Similarly, reaction of trans-acetonide 577 with Hsung–Vedejs AD-mix-a afforded acetonides ()-578 and (þ)-579 in 50 and 40% yield, respectively. The latter were converted, as before, into 1,5-dideoxy-1,5-imino-L-allitol ()-580 and 1,5-dideoxy-1,4-imino-D-mannitol ()-581.307 The Sharpless asymmetric aminohydroxylation308 of the electron-deficient 2-vinylfuran 582 gives a 7:1 mixture of semiprotected amino alcohols 583 and 584 (41%). The major product 583 (>86% ee) was reduced by diisobutylaluminum hydride giving diol 585,300a,300b which can be converted into the b-hydroxyfurylamine derivative 586, an important synthetic building block for various biologically important compounds, including 1,5-dideoxy-1,5-iminoalditols (Scheme 145). A less regioselective, but shorter, way to intermediate 586 is the direct asymmetric aminohydroxylation of vinylfuran.310a–312b Sharpless asymmetric dihydroxylation of ethyl sorbate gives diol 587 regioselectively. Selective silylation of 587 provided alcohol 588 (93% ee), which was esterified as a paranitrobenzoate and displaced with NaN3 to give the allylic azide 589. Hydrogenolysis of 589 and subsequent formation of a tosylamide furnished 590, which was then converted into methyl N-tosyl-a-D-tolyposaminide ()-591. Alternatively, diol 587 was reacted with TsN¼C¼O to give 592 (Ts ¼ para-toluenesulfonyl). Hydrogenation of the alkene moiety and subsequent methanolysis and acidic treatment provided lactone 593. Reduction of lactone 593 with (i-Bu)2NH and glycosidation with methanol furnished methyl 4-epi-N-tosyl-a-D-tolyposaminide (þ)-594 (Scheme 146).313 Lindstro¨m and co-workers313 have presented an efficient asymmetric synthesis of the iminoalditol 599 (Scheme 147). The method requires only four steps in water, without the use of protecting groups. (E,E)-1,6-Dibromohexa-2,4-diene 595 undergoes Sharpless asymmetric dihydroxylation with formation of diol 596 (70% yield, 97% ee). Upon heating in water at 50 C, the allyl bromide is hydrolyzed chemoselectively giving triol 597. Epoxidation of 597 with H2O2 in the presence of dinuclear peroxotungstate catalyst K2[W2O3(O2)4(H2O)2]314 gave 598 in 99% yield and 92% de. Ammonolysis of bromide 598 in aqueous ammonia was spontaneously followed by an intramolecular ring opening of the epoxide (60% overall yield based on 595).


AD-mix-a, NaHCO3

Br

Br 595 H2O2 (1.2 equiv.) H2O, 20 ⬚C K2[W2O3(O2)4(H2O)2] (0.02 equiv.) 99%, d.r. 96:4

Br

MeSO2NH2 H2O/t-BuOH1:1 0 ⬚C,16 h 70%, 97% ee Br

OH

OH

Br

Br

H2O 50 ⬚C, 3 h 98%

OH

OH

OH

OH

596

597 HO

OH O

OH

10% NH3/H2O 4h 88%

598

573

OH OH N H H 599

OH

Scheme 147 Efficient asymmetric synthesis of an azasugar in water.

1.13.12 Conclusion For many years, carbohydrates were very difficult synthetic targets because of their complexity arising from their stereochemistry and their multifunctionality. In parallel with the recent revolution in organic synthesis, a large number of complicated and rare monosaccharides have been prepared by total, asymmetric synthesis. Methods are available that allow one to reach both enantiomers of any natural or non-natural monosaccharides, including deoxyaminosugars, thiosugars, and azasugars, and this, quite offen, in a few synthetic steps. Depending on the target, pure chemical procedures relying on asymmetric catalysis using either metallic or pure organic catalysts can be applied successfully, alone or in combination with chemoenzymatic methods.

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Biographical Sketch

Inmaculada Robina received her graduate education at the University of Seville, Spain, where she also obtained her Ph.D. (Organic Chemistry) in 1982 (Prof. J. Fdez-Bolanõs Va´zquez, Prof. J. Fuentes). In 1986, she was promoted to associate professor of organic chemistry. She carried out postdoctoral research in Edinburgh, UK (Prof. J.G. Buchanan), where she worked on the synthesis of imino sugars. In 1999, she spent three months in Cambridge, UK (Prof. S.V. Ley), where she worked on the synthesis of oligosaccharides, in particular the synthesis of glycosyl-phosphatidoinositol anchor analogs. She has been an invited professor at the University of Lausanne and at the Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Switzerland, where she worked in Prof. P. Vogel’s research group. Her research focuses on the synthesis of oligosaccharides, especially those related to the molecular signals of the symbiosis Rhizobium-legume plant, and on the design and synthesis of enzyme inhibitors, imino-C-glycosides, imino-C-disaccharides, thiosugars, and nucleoside analogs. She is also interested in the synthesis of novel oligo- and peptidomimetics. Recently, she has been also involved in the synthesis of novel glycopeptides and peptidomimetics that would interfere with HIV entry.

Pierre Vogel spent two years at Yale University with Prof. Martin Saunders after his Ph.D. studies at the University of Lausanne, Switzerland (1969, Prof. Horst Prinzbach). He then joined the research laboratory of Syntex in Mexico City and worked with Prof. Pierre Crabbe´. He returned to the University of Lausanne where he was promoted to full professor in 1977. He has been associate professor at the Ecole Normale Supe´rieure in Paris, at the University of Paris VI, and at the University of Montpellier, France. He also taught at the Universities of Rouen and Caen in Normandy and at the Ecole Polytechnique in Palaiseau near Paris. He obtained the Novartis lectureship for 2003 and was elected Boehringer-Ingelheim Distinguished Lecturer for 2005. Since 2001, Prof. Pierre Vogel chairs the Laboratory of Glycochemistry and Asymmetric Synthesis of the Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Switzerland. He has published 3 books and has co-authored more than 400 publications in the fields of physical organic chemistry, organic and organometallic synthesis, catalysis, glycochemistry, and bio-organic chemistry.

1.14 Synthesis of Glycosides of Sialic Acid C. De Meo, Southern Illinois University – Edwardsville, Edwardsville, IL, USA G.-J. Boons, University of Georgia, Athens, GA, USA A. V. Demchenko, University of Missouri – St. Louis, St. Louis, MO, USA ß 2007 Elsevier Ltd. All rights reserved. 1.14.1 Introduction

583

1.14.2 Chemical Synthesis of O-Sialosides

584

1.14.2.1 Traditional Methods

584

1.14.2.1.1 1.14.2.1.2 1.14.2.1.3

Sialyl halides Thiosialosides and other 2-thio derivatives Phosphites and other O-R derivatives

1.14.2.2 Positional Modifications 1.14.2.2.1 1.14.2.2.2 1.14.2.2.3

584 585 589

590

Modifications at C-1 Modifications at C-3 (indirect methods) Modifications at C-5

590 591 594

1.14.3 Synthesis of C- and S-Glycosides

598

1.14.3.1 C-Linked Oligosaccharides

598

1.14.3.2 S-Linked Oligosaccharides

600

1.14.1 Introduction Sialic acids are nine-carbon monosaccharides involved in a wide range of biological phenomena. Their unique structure is characterized by the presence of a carboxylic group (ionized at physiological pH), deoxygenated C-3, glycerol chain at C-6, and differently, functionalized C-5. Among the 50 derivatives so far reported, N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, Neu5Ac) is the most widespread, although N-glycolylneuraminic acid (Neu5Gc) and 2-keto-3-deoxy-nonulosonic acid (3-deoxy-Dglycero-D-galacto-non-2-ulopyranosonic acid, Kdn) are also commonly found in biological systems (Figure 1).1,2 Variations of these structures in general involve acetylation at C-9 (Neu5,9Ac2), but di-and tri-O-acetylated derivatives are also known. Lactosylation or phosphorylation may also occur at C-9, while the hydroxyl at C-8 can be methylated or sulfated. In the cell, Kdn or Neu5Ac are synthesized by the condensation of 6-phosphate mannose or N-acetylmannosamine, respectively, with activated pyruvate, followed by dephosphorylation. After activation by CTP, the nucleotide CMPSia donor is transported into the Golgi apparatus where it is then transferred to the newly synthesized glycoconjugates by the action of specific sialyltransferases. As components of glycoconjugate chains, most sialic acids are typically found at the terminus of N-glycans, O-glycans, and glycosphingolipids. The natural equatorial glycosides and their unnatural axial counterparts are classified as a-anomers and b-glycosides, respectively. Sialic acids appear essentially at the terminal position, in which they are most commonly found (a2!3)- or (a2!6)-glycosidically linked to galactosides or (a2!6)-linked to N-acetylgalactosaminides. The disialosyl structures Neu5Aca2!8Neu5Ac and Neu5Aca2!9Neu5Ac have also been found as constituents of glycoproteins and lipids. Polysialic acids of Neu5Ac and Neu5Gc, characterized by (a2!8), (a2!9), or alternating (a2!8)/(a2!9) glycosidic linkages, have been found in glycoproteins of neural cell adhesion molecule (NCAM), in fish eggs, and in the capsule of certain bacteria such as Neisseria meningitidis group B. Other sialosides, for example, (a2!4)-linked homopolymer,3 (a2!5)-linked derivatives,4,5 etc.6,7 have also been described, but these derivatives are by far less ubiquitous. Being at the terminus of natural cell-surface glycoconjugates, sialic acids are ideally positioned to mediate carbohydrate–protein interactions in cell–cell recognition phenomena. For example, sialic acids are involved in the sialyl Lewisx-selection binding that occurs in the recruitment of leukocytes during the inflammation process. In addition, sialic acids act as receptors for some toxins, bacteria, and viruses. For example, the interaction between heamaglutinin and Neu5 Ac signifies the first stage of infection by the influenza virus. Sialic acids also play important

583

584

Synthesis of Glycosides of Sialic Acid

masking roles to prevent biological recognition.8 Modifications of sialic acids can interfere with the mode of cell interaction. For example, O-acetylation or N-acetyl hydroxylation hinders the action of sialidases leading to a longer lifespan of rat erythrocytes.9 In this context, acetylation of the C-9 hydroxyl prevents this monosaccharide to act as a receptor for influenza A and B viruses.10 Striking differences have been found in the sialylation pattern of cells during development, activation, aging, and onconogenesis.11,12 For the onconogenesis, the specific expression of abnormal sialosides (either overexpression or a different glycosylation pattern) makes them potential tumor markers and anticancer agents and provides high mobility to the cell, facilitating metastatic processes. For example, the dimer Neu5Aca2!8Neu5Ac is a principal terminal constituent of a number of glycoconjugates, such as polysialic acid overexpressed in some cancer cells. The dimeric structure is important for these biological properties since removal of one of the two Neu5Ac residues often leads to a dramatic or total loss of activity.

1.14.2 Chemical Synthesis of O-Sialosides In spite of extensive efforts and notable progress, the chemical synthesis of sialosides in high yield with complete stereoselectivity remains a significant challenge. 13–16 The presence of a destabilizing electron-withdrawing carboxylic group along with a tertiary anomeric center and the lack of a participating auxiliary often drive glycosylation reactions toward competitive elimination reactions resulting in poor stereoselectivity (b-anomer) and in the formation of a 2,3dehydro derivative. To overcome these problems, different approaches have emerged. A variety of leaving groups and activation conditions have been developed. In the following subsections, the latest developments for the direct stereoselective synthesis of a-sialosides will be classified, described, and analyzed.

1.14.2.1 Traditional Methods The conventional approach to chemical O-sialylation involves the coupling of a sialosyl donor with an appropriate leaving group (LG) at the anomeric center (C-2) with a glycosyl acceptor having at least one free hydroxyl (ROH, Scheme 1). This approach has found wide application and offers a reliable and efficient way for the synthesis of the natural and unnatural sialosides.

1.14.2.1.1 Sialyl halides Throughout the 1980s, the synthesis of the sialosides from 2-chloro derivatives was the main tool for the synthesis of compounds containing N-acetylneuraminic acid.13,17,18 Currently, however, the use of these derivatives is mainly OH 1 COOH HO 9 8 O 2 OH AcHN 5 OH 3 HO

OH COOH HO O OH GcHN OH HO

OH O

HO HO OH

aNeu5Gc Gc = C(=O)CH2OH

aNeu5Ac

COOH

HO

aKdn

Figure 1 Major naturally occurring sialic acids.

OAc

COOMe

OAc

AcO AcHN

O

LG

Promoter

AcO

AcO OAc

O AcHN AcO OAc

COOMe

ROH LG = Br, Cl, F, SMe, SEt, SPh, SC(=S)OEt, OP(OR⬘⬘)2, OCOR⬘, S-benzoxazolyl, etc

Scheme 1

OAc AcO O AcHN OAc AcO

COOMe OR

OH


BnO

OAc

OH O

COOMe

AcO OBn

BnO

585

BnO

i

O AcHN O AcO OAc BnO O OBn

BnO OAc

BnO

Cl

AcO O

AcHN AcO OAc

COOMe

Ag2CO3 HO BnO

OAc

COOMe

AcO O AcHN AcO OAc

OH BnO O BnO O BnO

i: Ag2CO3, 20%; a only or Hg(CN)2/HgBr2, 84%; a/b = 3/4

OTE

O HO

BnO

O OBn

BnO O BnO O BnO

OBn O OBn

90%, traces of b Scheme 2 .

limited to the glycosylation of simple alcohols or as precursors for the synthesis of other building blocks, such as thioglycosides and 2,3-dehydroderivatives.19,20 Most commonly, activation of halo-derivatives is achieved in the presence of silver(I) or mercury(II) salts, according to Koenigs–Knoor or Helferich method, respectively.13 Typically, silver salts are less reactive than mercury salts, yet allow a marginally higher stereoselectivity. For example, an Neu5Ac2!6Gal derivative was obtained in an excellent yield of 84% as a 3/4 mixture of a/b-anomers, using Hg(CN)2/HgBr2 as promoter, whereas complete stereoselectivity but poor yield was obtained using Ag2CO3 (Scheme 2).21 Further improvement of this traditional approach has emerged with the observation by Bovin and coworkers that partially protected diol acceptors provides significantly higher conversion yields (over 80%), even in the presence of relatively unreactive Ag2CO3 (Scheme 2).22 Although a prolonged reaction time was required (3–7 days), worthy a-selectivity was achieved at room temperature in dichloromethane (DCM) or 1,2-DCE: reaction solvents that do not usually support a-sialoside formation. It should be noted that this approach is principally effective for the sialylation of primary acceptors (6-OH of Gal or GalNAc). Along with these studies, 2-bromo23,24 and 2-fluoro derivatives25,26 of Neu5Ac have also been explored as sialyl donors, yet found somewhat narrower application for synthesis than their 2-chloro counterparts. Conversely, 2-bromo derivative of Kdn found wide application for Kdn glycosidation.27–30

1.14.2.1.2

Thiosialosides and other 2-thio derivatives

Alkyl(aryl) thioglycosides have emerged as versatile building blocks for glycoside and oligosaccharide synthesis.31,32 Due to their availability and excellent chemical stability, anomeric alkyl(aryl)thio groups offer efficient protection of anomeric centers and are compatible with many reaction conditions often employed in carbohydrate chemistry. In the presence of soft electrophiles, thioglycosides can be activated and used in direct glycosylations. Another attractive feature of thioglycosides is that they can be transformed into a range of other classes of glycosyl donors, such as halides or phosphites. The 2-thiomethyl sialoside donor is commonly obtained from the corresponding 2-O-acetate by treatment with methylthiotrimethylsilane (TMSSMe) in the presence of trimethylsilyltriflate (TMSOTf) as a 1/1 mixture of a/banomers.33 Since the a- or b-linked S-methyl sialosides have very comparable glycosyl donor properties, the mixture obtained is often used in sialylation without separation of anomers. In this context, 2-thioethyl glycoside of Neu5Ac, readily available from the corresponding 2-O-acetyl derivative,34,35 or 2-thiophenyl glycoside of Neu5Ac that can be synthesized by reaction of thiophenol with 2-O-acetyl, 2-chloro, or 2-fluoro derivatives of Neu5Ac,34,36 possesses similar sialyl donor properties to corresponding S-methyl glycosides.37–40 Since it is possible to obtain the 2-a-thiophenyl donor as an individual diastereomer from the corresponding anomeric b-chloride, application of 2-SPh sialosides as glycosyl donors has been significantly enhanced in recent years. Their synthesis is commonly performed with the use of inexpensive thiophenol in the presence of a base, such as potassium hydroxide41 or diispopropylethylamine.34 Many different activation conditions have been developed for the thioglycoside activation.42,43 Among these, good yields and high a-anomeric selectivities were achieved when the synthesis of Neu5Aca2!3Gal derivatives was

586


attempted in the presence of dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST, Scheme 3).44 The best results were achieved when glycosyl acceptors were only protected at the anomeric and primary C-6 position.45–47 The complete regioselectivity of this sialylation is attributed to the greater reactivity of the 3-OH and a considerable steric bulkiness of the sialosyl donor. Typical glycosidations of sialyl donors are performed in the participating solvents such as acetonitrile and at low temperature (40 C). These reaction conditions help to better control the sialylation process and obtain the products in high or even complete a-anomeric stereoselectivity. When the same reaction conditions (DMTST) were applied to the sialylation of a lactosyl acceptor bearing a free secondary 30 ,40 -diol, the yield and anomeric stereoselectivity were significantly reduced (Scheme 3). In this case, a notable improvement came with the application of the highly reactive promoter system NIS/cat. TfOH that has proved to be especially valuable when applied for glycosylations of sterically more hindered hydroxyls.48 For example, NIS/TfOH-mediated glycosylation of the 30 ,40 -diol gave a much higher yield of the (2!3)-linked product (69%) and improved anomeric stereoselectivity (a/b-6/1, Scheme 3). A reaction mechanism for the activation of thiosialosides in MeCN has been described.48 It was proposed that these sialylations start with in situ generation of the electrophilic species (E ¼þSMe, Iþ), which then react with the lone pair of the anomeric sulfur, resulting in the formation of a sulfonium intermediate (Scheme 4). The sulfonium moiety is an

HO

OAc OBz O HO

COOMe

AcO O AcHN AcO OAc

OAc

BnO

HO

O O BnO OBn

DMTST OAc

MeSTMS TMSOTf

OTE

HO

O OBz

COOMe

AcO O AcHN AcO OAc

98%

HO

AcHN AcO OAc

OTE

HO

O

O

HO OAc

COOMe

AcO

OBn O

SMe

DMTST: 38% (a/b-4/1) NIS/TfOH: 69% (a/b-6/1)

OTE BnO

OAc

COOMe

AcO AcHN AcO OAc

OBn O

BnO

O

O

O O BnO OBn

HO

OTE BnO

Scheme 3

OR O

COOMe

b-Sialoside ROH

ROH OAc

O SMe

E

O

COOMe

MeCN N O

ROH ROH COOMe O

OR

a-Sialoside Scheme 4

N

COOMe

AcO O AcHN AcO OAc

COOMe

COOMe


587

excellent leaving group and can be either displaced by a hydroxyl of the glycosyl acceptor or, alternatively, by the nitrogen of acetonitrile to give a nitrilium ion, which preferentially adopts an axial (b) configuration. Therefore, subsequent nucleophilic substitution with an alcohol leads predominantly to the equatorially substituted a-sialoside. It has been observed that less-reactive (secondary) alcohols provide better a-selectivities than primary alcohols. Other common types of sialosides include (2!6)-linkage to Gal or GalNAc, and the dimeric units linked via (2!8) or (2!9) bonds. While the sialylation of the primary acceptors the synthesis of (2!6)Gal and (2!9)-linked dimers is fairly efficient, the synthesis of (2!8)-linked dimers has been an extreme challenge. Early attempts to directly obtain (2!8)-linked dimers gave very disappointing results. For example, glycosylation of a 8-OH sialyl acceptor with a 2-thiomethyl neuraminyl donor in the presence of DMTST in MeCN at 40 C afforded only trace amount (5%) of the desired a-linked dimer (Scheme 5).44 A possible explanation for this modest result is poor nucleophilicity of the sterically hindered secondary 8-OH of Neu5Ac. In addition, the 8-hydroxyl is capable of forming intramolecular hydrogen bonds with the nitrogen of the 5-acetamido moiety or the 1-carboxylate group (Scheme 5). Among the first efforts to access this notable challenge was the development of a non-orthodox strategy for the synthesis of oligosaccharides containing Neu5Aca2!8Neu5Ac moiety. Mild acid treatment of colominic acid, an (a2!8)-linked homopolymer [!8)-a-Neu5Ac-(2!]n, allowed controlled glycosidic bond cleavage to give the dimer along with higher oligomers and the monomer. After separation, the dimer could be converted into a glycosyl donor (most commonly 2-SPh).49 For a relevant example, see Scheme 6. In this context, higher oligomers of Neu5Ac have also been used as sialyl donors.50,51 An attempt to address the challenge of chemical 8-sialylation was performed by changing acceptor conformation. It was anticipated that the formation of an internal 1,7-lactone would facilitate a ‘break’ in the intramolecular hydrogen bonding.52 Indeed, this strategy resulted in good yields of coupling products, but, unfortunately, only modest a-anomeric selectivity was achieved. A number of other efficient protocols for direct and indirect synthesis of sialosides involving positional modification has been developed. These techniques are discussed in Section 1.14.2.2. An example of a convergent block synthesis of an oligosaccharide that contains four sialyl moieties is shown in Scheme 6.53 This example clearly illustrates three different concepts for introducing the sialic acid fragments into complex oligosaccharides. NIS/TfOH-promoted glycosylation of a 30 ,600 -diol trisaccharide with a 2-thiophenyl glycosyl donor of Neu5Ac provided excellent regioselectivity and afforded the expected (a2!6)-linked tetrasaccharide in 45% yield. Subsequent glycosylation of the resulting tetrasaccharide with a dimeric sialyl donor, obtained by controlled hydrolysis of colominic acid, was set up under similar reaction conditions allowing a hexasaccharide in 42% yield. Removal of the 3,4-O-isopropylidene acetal followed by DMTST-promoted glycosylation with a Neu5Aca2!3 GalSMe donor furnished the desired octasaccharide in excellent overall yield. The last glycosylation step cannot be formally classified as an O-sialylation, yet this convenient block strategy offers a very common approach for the synthesis of sialylated oligosaccharides. 2-Thiomethyl sialosides have also been applied in the polymer-supported synthesis of sialyl Lewisx54 and in convergent orthogonal 55,56 and active–latent 35,57 strategies.58,59 Differentiating the anomeric reactivity of glycosides offers an attractive way for synthesizing complex oligosaccharides involving a minimal number of protecting group

OAc

COOMe

AcO O AcHN OAc AcO OH BnOCH2O

OAc O AcHN AcO OAc BnOCH2O

DMTST

O OTE AcHN HO OCH2O(CH2)2TMS H

O

OX HO

HO O AcHN OH HO Scheme 5

O COOMe

O OTE AcHN HO OCH2O(CH2)2TMS 5%, a anomer

COOMe

O

COOMe

AcO

SMe

OR

O H

O

OX

O AcHN OH HO

OR

588


OBn O

BnO HO OAc

AcHN COOMe

O

AcO O AcHN AcO OAc

SPh

O OH

O

O

NIS/TfOH 45%

AcO

O

AcHN O

O AcO OAc AcHN O

COOMe O AcHN AcO OAc

O O AcHN AcO OAc

SPh

OTE BnO

BnO HO

O OAc

O O BnO OBn

O

O O BnO OBn

OBn O OTE BnO

O O

AcO AcO

MeOOC

NIS/TfOH 42% OAc AcO

O

O

COOMe OBn O BnO O AcHN O O O OTE O BnO AcO OAc AcHN BnO OBn R1O O O R1,R2 = >CHMe2 2 AcO OAc R O OAc COOMe OBz AcO AcHN O O 1 2 O O SMe R =R =H AcHN AcO O MeOOC AcO OAc AcO DMTST 85% BzO OBz O O OAc COOMe AcO OBn O BnO O AcHN O O O AcHN O OAc AcO OTE BnO O OAc AcO OAc BnO COOMe OBn OBz AcO AcHN O O O AcHN O O O AcO OAc BzO OBz HO AcO OAc AcHN O O AcO MeOOC AcO

O AcHN AcO OAc

Scheme 6

manipulations. In this context, the following thio leaving groups can be selectively activated over conventional alkyl/ aryl thioglycosides and, therefore, have great potential for subsequent investigations in convergent multistep oligosaccharide syntheses. The first class of compounds, 2-(ethoxy)dithiocarbonate or 2-xanthate of Neu5Ac, has been synthesized from the 2-chloride by reaction with potassium ethoxydithiocarbonate in EtOH.34 Activation of xanthates with DMTST 60 or NIS/TfOH37,61,62 has been successfully employed in the synthesis of sialylated oligosaccharides. Good selectivities and yields have been achieved in the presence of phenyl sulfenyl trifluoromethanesulfonate (PhSOTf) obtained from PhSCl and AgOTf, and 2,6-di(tert-butyl)pyridine (DTBP) at low temperature (70 C).63 As highlighted in Scheme 7, the trisaccharide was obtained in an excellent yield of 74% mainly as the a-anomer (a/b - 19/1). An important feature of this glycosylation protocol is that sialyl xanthates can be selectively activated in the presence of the thioglycosides. In a number of applications, sialyl xanthates outperformed their S-alkyl counterparts in direct sialylations.64 Recently, De Meo reported application of the S-benzoxazolyl (SBox) approach to a-sialylation and the convergent synthesis of a GM3 analog.65 In this synthesis, selective activation of the SBox-moiety of the sialosyl donor over the S-ethyl moiety of the galactosyl acceptor was conveniently achieved in the presence of AgOTf. The obtained disaccharide was used in subsequent coupling directly to afford the desired GM3 trisaccharide motif in good overall yield (Scheme 7).


OAc

589

COOMe

AcO O AcHN AcO OAc

S S

OAc

OEt PhSOTf

BnO

HO

O O BnO OBn

AcO OAc

OBn O

HO

OBn O

BnO O O BnO OBn

OTE BnO

74%, a/b-19/1

OTE BnO

OAc

AcO

O

O

AcHN

DTBP HO

COOMe

AcO

COOMe N O S AcHN O AcO OAc

HO

OH O SEt

BzO BzO

AgOTf

OAc

COOMe

AcO AcHN

O

AcO OAc

O HO SEt

BzO 89%, a/b-1/1

BzO

OBn O

HO BnO

O

BnO

NIS/TfOH OAc

OMe

COOMe

AcO O

AcHN AcO OAc

O HO

BzO 70%

BnO O BnO O BzO

OMe

O OBn

Scheme 7

1.14.2.1.3

Phosphites and other O-R derivatives

Sialyl phosphites were independently introduced by Wong66 and Schmidt67 and have found wide application in chemical O-sialylation.13 They are very reactive glycosyl donors for which only a catalytic amount of TMSOTf (typically 10–20 mol %) is required for activation – a substantial advantage over thioglycosides activated with stoichiometric amounts of relatively expensive and often hazardous promoters.31 Diethyl b-sialyl phosphites can be prepared in high yields (97%) by reaction of a hemiacetal derivative of Neu5Ac with CIP(OEt)2 in the presence of the hindered base i-Pr2NEt.67,68 Glycosylation of a 6-hydroxyl derivative of a glucoside in the presence of TMSOTf (0.1 equiv.) in MeCN at 40 C afforded a (2!6)-linked disaccharide as a 4/1 mixture of a/b-anomers (70%). Application of a di-O-benzyl phosphite derivative, obtained from the hemiacetal in the presence of N,N-diethylphosphoro amidite and tetrazole, to the same model reaction gave a somewhat higher yield and improved anomeric selectivity (80%, a/b ¼ 5/1,66,69 a/b-6/1,70 Scheme 8). Other sialyl phosphites such as-O(n-Bu),-OCH2CH2Cl, OCH2CCl3, O(CH2)3O-, O-CH2CH2Me2CH2O-,68 and 1,2O-cyclopentyl71 were found to be less-efficient sialyl donors than their OEt or OBn counterparts. Conversely, dimethyl phosphite proved to be rather reactive,72 especially when promoted with ZnCl2/AgClO4 in CH2Cl2 at room temperature, providing sialosides in high yields (83–85%) yet with high b-stereoselectivity (a/b-1/5–6). A number of other oxygen-based leaving groups have been investigated in sialylations, among which phenyltrifluoroacetimidates73 and C-2 hemiketals74 have recently emerged as promising novel technologies for further perusal.

590


OAc

COOMe

AcO (BnO)2PNEt2 Tetrazole, THF

OAc AcO O AcHN AcO OAc

O AcHN AcO OAc

OP(OBn)2 COOMe

BnO BnO

OH

OH O BnO OMe

TMSOTf (0.2 equiv.) MeCN, −42 8C

ClP(OEt)2 i-Pr2NEt OAc AcO

OP(OEt)2

O AcHN AcO OAc

COOMe

TMSOTf (0.1 equiv.) MeCN, −40 8C OAc

COOMe

AcO

80%, a/b-6/1

O AcHN O AcO OAc 70%, a/b-4/1 O BnO BnO BnO OMe

Scheme 8

1.14.2.2 Positional Modifications To address the challenges associated with chemical glycosylation, several new technologies have been developed in the past few years. The improvements of conventional glycosylations with hexopyranoses have primarily emerged from investigations focusing on the nature of the leaving group or promoter. Sialylations are not exceptional in this respect, and the achievements toward this direction are summarized above. Differently from the hexoses, substantial effort has been exerted in the investigation of positional modifications, including the introduction of participating auxiliaries at the C-1 and C-3 positions. Also, modifications of the C-5 acetamido group have been reported as alternative strategies to optimize glycosylation reactions. In particular, it has been shown that the nature of the protecting group at the C-5 position plays an important role in effecting the reactivity of both sialosyl donors and acceptors.

1.14.2.2.1 Modifications at C-1 An interesting technique to control the stereoselectivity of sialylation via long-range participation was proposed by Takahashi et al.75 According to this approach, the carboxyl at C-1 of 2-thiomethyl Neu5Ac was protected as a 2-thioethyl ester. It was envisaged that activation of the anomeric center of the Neu5Ac donor would give an oxoniumion intermediate, which would be stabilized by long-range participation of the thiomethyl moiety resulting in the formation of a sulfonium intermediate (Table 1). Glycosylation of the thermodynamically more stable b-sulfonium intermediate should mainly lead to the formation of a-glycosides. Reasonable yields and a-anomeric selectivities were achieved when applied to primary as well as secondary glycosyl acceptors. The proposed mechanism was supported by the finding that anomeric selectivities were not affected by reaction solvents and even when the glycosylation was performed in ethylene glycol dimethyl ether (DME), mainly a-linked disaccharides were formed in moderate yields (Table 1, entry 2). A similar concept was explored by Gin and co-workers. In this case, the N,N-dimethylglycolamido (OCH2CONMe2) ester functionality was employed as an auxiliary at C-1, allowing the synthesis of a number of sialosides with improved a-stereoselectivity (Table 1, entries 3 and 4).76 Also, this participating moiety allows the reactions to be performed in an apolar solvent. This approach was then extended to dehydrative sialylations with 2-OH sialyl donors.74 Ito and a co-worker probed a number of other participating ethers. Although the concept applied was essentially the same as the aforementioned approaches, somewhat different observations were made.77 Thus, no a-stereoselectivity improvement was noted in the sialylations when performed in DCM. These results were rationalized by the inability of the CH2CH2CN ester to directly influence the stereoselectivity of sialylations. However, notable improvement of


591

Table 1 Long-range-assisted synthesis of a-sialosides

OAc COOR1 AcO OAc O COOR1 SR AcHN AcO NIS/TfOH AcO OAc O O AcHN OAc AcO HO HO OR4 O HO R2 R3O

R3O

R2

O OR4

Entry

R

R1

R2

R3

R4

Solvent

Yield

a/b Ratio

Ref.

1 2 3 4 5 6

Me Me Et Et Me Me

Me C2H4SMe Me CH2CONMe2 Me C2H4CN

bOMe bOMe aOMe aOMe bOpMeOPh bOpMeOPh

MBn MBn Bn Bn Bn Bn

MBn MBn Bn Bn Bn Bn

MeCN DME DCM DCM MeCN MeCN

35 21 65 80 56 63

5/1 10/1 1/4 2/1 8/1 13/1

75 75 76 76 77 77

d+

N

NMe2 OAc Me S AcO O O AcHN O AcO OAc

OAc

O

AcO O AcHN AcO OAc

ROH [Takahashi]

O O

OAc Nd + AcO O O AcHN O AcO OAc

ROH [Gin]

ROH [Ito]

stereoselectivity was detected in MeCN (entries 5 and 6, Table 1). This was rationalized by the solvent-assisted indirect participation, which stabilized the kinetically generated b-nitrilium ion. Wong and co-workers determined that significant enhancement of the reactivity of sialosyl donors can be achieved by conversion of an electron-withdrawing C-1 carboxylic moiety into a protected hydroxymethyl group.78 This reduction yields glycosyl donors of comparable reactivity to reactive armed glycosyl donors of the D-hexose series. Upon glycosylation the hydroxymethyl moiety could be deprotected and oxidized. Unfortunately, this elegant indirect sialylation approach yields predominantly unnatural b-linked sialosides. The unusual stereoselectivity was rationalized by a significant anomeric effect in the C-1-reduced derivatives in comparison to regular sialosides.78

1.14.2.2.2

Modifications at C-3 (indirect methods)

Several glycosyl donors derived from Neu5Ac have been prepared that possess an auxiliary at C-3. This auxiliary should control the anomeric selectivity of a glycosylation by neighboring group participation leading to the formation of 2,3-trans-glycosides.13 Thus, a-glycosides are favored in the case of equatorial auxiliaries (Scheme 9), whereas b-glycosides are preferentially formed when the participating auxiliary is in the axial orientation. The auxiliaries should also help to prevent 2,3-elimination, which often constitutes a major side reaction in the direct O-sialylations. One of the most important requirements is that an auxiliary should be easily installed before, and removed after, the glycosylation. Usually, auxiliaries are introduced by a chemical modification of a 2,3-dehydro derivative of Neu5Ac, readily accessible from the methyl ester of acetochloroneuraminic acid.79 These transformations can be performed either through a 2,3-oxirane derivative or by a direct addition reaction to the double bond.

1.14.2.2.2.1 3-Bromo- and 3-O-auxiliaries Addition of bromine to the double bond of the glycal of Neu5Ac gives a diaxially substituted 2,3-dibromo derivative which is only suitable for the synthesis of b-sialosides.80 Differently, the addition of NBS to the glycal at low temperature (20 C) in aqueous dimethylsulfoxide (DMSO) predominantly afforded a 2,3-diequatorial substitution product.79 This compound could be converted into a C-2 diethyl phosphite derivative, which gave good anomeric

592


OAc

OAc AcO

O AcHN AcO OAc

COOMe

AcO O AcHN X LG AcO OAc Promoter

COOMe

LG = halogen, SR⬘, phosphite X = halogen, O-Acyl, SR⬘⬘, SePh

X

OAc AcO

O

COOMe

AcHN AcO OAc ROH

OAc AcO

COOMe

O AcHN AcO OAc

OR

OAc

COOMe

AcO AcHN

O

X OR

AcO OAc

Scheme 9

OAc

OP(OEt)2

AcO O

O COOMe OAc COOMe S AcO O Ph AcHN X O TMSOTf OAc AcO OH BnO O BnO COOMe O AcHN COOMe AcHN HO OAc HO OAc X = OCSPh 83%, a only Bu3SnH/AIBN X=H AcHN AcO OAc

Scheme 10

selectivities and yields when applied in glycosylation with reactive hydroxyls. Unfortunately, when the synthesis of the (2!8)-linked dimer was attempted, a predominantly b-anomer was isolated.81 Upon glycosylation, the bromo auxiliary could be removed by reduction with Bu3SnH.81 To enhance the participatory function of the C-3 moiety, several approaches for the synthesis of 3-O-derivatives of Neu5Ac have been reported. Beginning from early studies involving the unprotected 3-OH,82,83 various participating 3-O-substituents have been probed. For example, 3-O-thiocarbamate and 3-O-thiocarbonyl functionalities capable of participation through a five-membered cyclic intermediate provided excellent a-stereoselectivities and high yields.81,84 As evident from the example illustrated in Scheme 10, an excellent result was obtained even in reactions with poorly nucleophilic 8-OH of Neu5Ac. Upon sialylation, these moieties can be removed by reduction with Bu3SnH and AIBN. The disaccharide obtained offers an opportunity for iterative oligosaccharide synthesis by repeating double bond addition–glycosylation sequence.

1.14.2.2.2.2 3-thio and 3-seleno auxiliaries Neu5Ac derivatives containing a 3-thio or 3-seleno auxiliary in combination with an appropriate anomeric leaving group possess excellent glycosyl donor properties that have been extensively studied.13 It has been proposed that these glycosylations proceed through an episulfonium (or episelenium) intermediate, which induces excellent a-stereoselectivity.85 These glycosyl donors can be obtained by addition of PhSCl to the double bond of a 2,3-dehydro derivative of Neu5Ac to give predominantly the 3-equatorial diastereomer (ax/eq ¼ 1/5).86 Even higher diastereoselectivity of the addition was achieved with 2,4-dimethylbenzenesulfenyl chloride in CH2Cl2 (ax/eq ¼ 1/21, 89%).87 This example is illustrated in Scheme 11. 2-Chloro and 2-fluoro derivatives, both bearing a 3-S-phenyl auxiliary, have been employed


593

OAc AcO

O AcHN AcO OAc

COOMe OAc

Cl

AcO O

ClS

S COOMe

AcHN AcO OAc

89%

Me ax:eq = 1:21

Me

Me

Me 1. Separation of equatorial 2. MeSNa/MeCN, quant. OAc

COOMe

AcO

HO

O AcHN AcO OAc

OBn O

BnO HO

O O BnO OBn

OTE

S SMe Me

BnO

Me 1. PhSCl/AgOTf/DTBP/MeCN, −40 ⬚C 2. Ac2O/Py/DMAP 3. Ph3SnH/AIBN/Toluene, 110 ⬚C

OAc

COOMe O

O

AcHN AcO OAc

OBn O

BnO

AcO

O O BnO OBn

HO

OTE BnO

71% overall Scheme 11

OAc

OH

Cl

AcO O

AcHN AcO OAc OAc

O

S COOMe Ph

AcHN AcO OAc

O AcHN S O Ph OAc AcO AcO

O AcHN AcO OAc

F Ph

HO

COOMe

O AcHN AcO OAc OAc

S

AgOTf/CH2Cl2 49%

COOMe

AcO

AcO

COOMe

AcO

S

F HO

Ph

AcO

O OPiv

O HO

OPiv O SEt PivO

AgOTf/SnCl2/MeCN 39%

COOMe S

HO

O COOMe

Ph O

AcHN AcO OAc

S

HO

O Ph HO

O OPiv

O HO

OPiv O SEt PivO

Scheme 12

for the preparation of a tetrasaccharide using an elegant glycosylation strategy based on sequential leaving group activation (Scheme 12).88,89 It has been established that the glycosyl fluoride is stable toward the conditions required for activation of the anomeric chloride. Therefore, coupling of a 2-chloro glycosyl donor with 8-OH glycosyl fluoride acceptor in the

594


presence of AgOTf gave a (2!8)-linked dimer in 49% yield (Scheme 12). Complete a-selectivity was achieved by neighboring group participation of the 3-thiophenyl moiety. The obtained dimer was used in the subsequent glycosylation in the presence of AgOTf/SnCl2 without a need for any chemical manipulations between glycosylation steps. A glycosylation of a thio-lactosyl acceptor afforded a tetrasaccharide in 39% yield, making use of the finding that glycosyl fluorides can be selectively activated in the presence of thioglycosyl acceptors. The thioglycoside can be subsequently activated using a thiophilic reagent and, in this case, the promoter DMTST was used in a coupling with an azidosphingosine acceptor to give a glycosyl azidosphingosine. Although the anomeric chloride can be used as a glycosyl donor directly, it has been determined that better results are obtained when the chlorine is first replaced with a thiomoiety and then activated.90,91 In spite of a number of required additional synthetic steps, the results obtained compare very favorably with one-step direct sialylations. Thus, a trisaccharide was synthesized in a high overall yield and complete stereoselectivity. Nevertheless, a 2-thioethyl leaving group in combination with a 3-thiophenyl participating auxiliary afforded an (a2!8)-linked product in a disappointing yield of 28%.91,92 The usefulness of this approach was extended to a polymer-supported synthesis of sialyl glycosides.93

1.14.2.2.3 Modifications at C-5 Recently, a number of groups reported that the nature of the protecting group at the C-5 position plays an important role in controlling the yield and stereoselectivity of glycosylation reactions. This section summarizes the latest developments in the chemistry of sialic acid, which involve modifications of the natural 5-acetamido function (NHAc). It is important to note that the main scientific effort in the area of chemical sialylation has been focused on a study of amino group modification.

1.14.2.2.3.1 N-Acetylacetamido Pioneering studies by Boons and co-workers resulted in the discovery of the powerful effect of the C-5 acetamido group on the yield and stereoselectivity of glycosylation reactions.94,95 It was discovered that the introduction of an additional acetyl group at the N-5 position of a thiomethyl sialyl donor drastically improves its reactivity in glycosylation reactions. For example, an NIS/TfOH-promoted coupling of 2-(trimethylsilyl)ethyl 6-O-benzoyl-b-Dgalactopyranoside with a methylthio 5-N-acetylacetamido donor yielded the desired (a2!3)-linked disaccharide in 72% yield in less than 5 min (Scheme 13).94 For comparison, sialidation of the corresponding mono-N-acetylated derivative required significantly more reaction time (2–6h) under essentially the same reaction conditions. Also, no large excess of the N-acetylacetamido glycosyl donor is typically required, as opposed to glycosylations with monoN-acetylated donors typically requiring 2–3 equivalents with respect to the acceptor to achieve similar results. Recently, Crich and co-workers reported the use of N-acetylacetamido thiophenyl donor for the coupling to a wide range of acceptors using diphenylsulfoxide/trifluoromethanesulfonic anydride promotion system.96 HO

OBz O

OAc OTE

HO

OAc AcO

SMe

AcRN AcO OAc

OAc AcO

R = H, Ac NIS/TfOH OH BnO

COOMe O

OTE

AcRN AcO OAc R = H, Ac Scheme 13

HO

O HO

OTE O OBz

R = H: 61% (reaction time 2–6 h) R = Ac: 72% (reaction time 5 min)

COOMe O

O AcRN AcO OAc

NIS/TfOH

HO

COOMe

AcO

COOMe

O Ac2N OAc AcO BnO

O COOMe O

AcRN AcO OAc R = H: 16%, a/b-1/1 R = Ac: 50%, a/b- 2/1

OTE


595

The higher reactivity of the sialyl donor bearing an NAc2 moiety has also been proven useful for the synthesis of (a2!8) dimers. Interestingly, the positive effect of NAc2 was determined for both the glycosyl donor and the glycosyl acceptor components of this type of sialylation. Thus, when a 5-N-diacetyl neuraminyl donor was coupled with a 5-mono-N-acetyl acceptor, the desired dimer was obtained in 16% yield (Scheme 13).95 Even this modest result was a marginal improvement compared to a similar direct sialylation performed with the monoacetylated donor and a structurally similar acceptor (5%, see Scheme 5). Significantly higher yield was achieved when a 5-N-acetylacetamido neuraminyl donor was coupled with a 5-N-acetylacetamido acceptor. In this case, the desired (2!8)-linked dimer was obtained in an improved yield (50%) and stereoselectivity (a/b ¼ 2:1, Scheme 13).95 This finding suggests the crucial role of the N-5 protecting group in the control of the reactivity of 8-OH, probably due to the decrease of H-bonding between the 5-N and 8-OH. These promising results encouraged the development of different approaches where 5-N-diacetyl donors modified at C-3 have been tested. For example, sialylation of the ethylthio 3-thiophenyl-5-N-acetylacetamido donor yielded the desired (a2!8) dimer in 44% yield,97 whereas, as mentioned above, its mono-N-acetylated counterpart gave the corresponding dimer in 28% yield.92 In both cases, complete a-stereoselectivity was achieved due to the efficient participation of an S-phenyl auxiliary at C-3. Advantageously, the additional N-acetyl group does not require additional synthetic steps for its introduction or cleavage, as it can be simply introduced (and removed) upon O-acetylation (or deacetylation).

1.14.2.2.3.2 Azido The synthesis of 5-azido derivatives of N-acetylneuraminic acid has been accomplished by both enzymatic98–101 and chemical102–105 methods. For the latter, Schmidt103 and Wong104 described the introduction of the azido group using trifluoromethanesulfonyl azide (TfN3) as the diazo transfer reagent. Similarly, sialosyl acceptors bearing an azido moiety at C-5 were synthesized to reduce the unfavorable hydrogen bonds for the synthesis of (a2!8)- and (a2!9)linked dimers. 5-Azido donors and acceptors are notably more reactive than the common acetamido group containing derivatives.104,106 For example, the 2-thiocresol (STol) sialyl donor bearing an azido group at C-5 gave significantly improved a-stereoselectivity in sialylation, especially when applied to the synthesis of (2!9)-linked dimers (Scheme 14). Thus, glycosylation with an azido-bearing sialyl acceptor gave the desired a-linked dimer with complete stereoselectivity in 65% yield. Under the same reaction conditions, the corresponding N-acetyl and N-acetylacetamido donors gave the desired disaccharide as a,b-anomeric mixtures (3:1 and 4:5, respectively). When a 5-azido phosphite was selectively activated over a 5-azido thioglycoside acceptor, the (a2!9)-linked dimer was obtained in 51% yield (Scheme 14).104 Conveniently, the dimer obtained can be used in subsequent sialylation. High a-stereoselectivity OAc AcO

COOMe O

N3 AcO OAc

STol NIS/TfOH AcO

OAc

OpClBz HO

AcO OAc N3 O

OMe

OAc

COOMe N3 COOMe pClBzO OpClBz

OP(OEt)2

O N3 AcO OAc

COOMe

AcO OAc N3 O

TMSOTf AcO

OAc OpClBz

COOMe

HO O

STol N3 OpClBz pClBzO Scheme 14

OMe O

65%, a only

O

COOMe N3 pClBzO OpClBz

AcO

OpClBz O

OpClBz O

COOMe O

STol N3 COOMe OpClBz pClBzO 51%, a only

596


was observed with 5-azido derivatives in the case of highly nucleophilic primary acceptor sialylations. The observed effect was attributed to both steric and electronic effects. The linear azido group withdraws electrons and, therefore, can stabilize the acetonitrilium intermediate and allow easier access of the acceptor from the a-face of the donor. The azido group effect on yield and stereoselectivity in glycosylation reactions is less prominent with sterically hindered and/or less reactive acceptors, such for the synthesis of (a2!3) galactosyl derivatives and (a2!8) dimers. For the latter, the coupling between 5-azido perbenzylated thioglycoside donor and 5-azido sialyl acceptor did not give any dimeric product.107

1.14.2.2.3.3 Trifluoroacetamido As mentioned above, the synthesis of the (a2!8)-glycosidic bond is complicated by the low reactivity of the acceptor due to intramolecular hydrogen bonds (Table 1). Hence, a potent electron-withdrawing group such as trifluoroacetamido group should help to further decrease the unfavorable H-bonds between the acetamido function and 8-OH. The introduction of a trifluoroacetyl group can be accomplished by the reaction of the corresponding 5-amino precursor with methyltrifluoroacetate in the presence of triethylamine and methanol.24 Thus, a 5-N-trifluoracetyl sialyl donor and acceptor proved to be far more reactive than the corresponding N-acetamido and N-acetylacetamido derivatives (Scheme 15).108 While complete stereoselectivity was observed in every case, and the highest yields were achieved with 5-N-TFA acceptor, the versatility of the N-TFA donor allowed a highly efficient synthesis of the human melanoma associated antigen GD3 derivative, which has multiple (2!8)-linked Neu5Ac residues. To date, N-TFA-protected sialylations are the only examples of stereoselective synthesis of (a2!8) dimers by direct methods.108–110 High reactivity of the 5-N-TFA sialyl donor has also been reported for the synthesis of (a2!3)- and (a2!6)-linked derivatives with a wide range of galactosyl acceptors.111 Thus, complete stereoselectivity and best yields were accomplished for more sterically hindered/less-reactive acceptors. For more primary hydroxyl groups or triols, loss of stereoselectivity and/or regioselectivity was observed. Recently, phosphite sialosyl donors bearing a C-5 trifluroacetamido group were applied to the synthesis of (a2!9)linked oligomers (Scheme 16).112 Also in this case, these donors gave a higher degree of a-anomeric selectivity as compared to conventional sialyl donors. Thus, an N-TFA phosphite donor was coupled with an N-TFA thioglycoside acceptor thereby obtaining the a-linked dimer with 77% yield. The STol moiety was then replaced with phosphite. By iterating these coupling–reprotection steps, higher-order sialosides were obtained with high yield and stereoselectivity. It was noted that stereoselectivity drops as the size of the glycosyl donor increases. 1.14.2.2.3.4 N-Trichloroethoxycarbonyl (N-Troc) Conversion of the N-acetyl phenylthioglycoside derivative into the corresponding N-trichloroethoxycarbonyl (Troc) derivative was described by Wu113 and Kiso114 using succinimidyl 2,2,2-trichloroethyl carbonate and trichloroethylchloroformate, respectively. BnO

OBn O OTE

HO BnO

NIS/TfOH

OAc SMe AcO O COOMe TFAHN AcO OAc

OH BnO O RHN AcO OAc

OAc

AcO O TFAHN NIS/TfOH AcO OAc BnO COOMe OTE

R = Ac, TFA Scheme 15

OAc COOMe OBn AcO O O OTE TFAHN O AcO OAc BnO OBn 84%, a only COOMe O COOMe RHN

O

AcO OAc R = Ac: 34%, a only R = TFA: 55%, a only

OTE


OAc OP(OBn)2 AcO O COOMe TFAHN AcO OAc

AcO OAc OR2 COOMe TFAHN O O O R1 AcO TFAHN AcO MeOOC AcO OAc

TMSOTf

OH

R1 = a-STol; R2 = H (77%, a only)

COOMe

HO O TFAHN OAc AcO

597

i

STol

R1 = b-OP(OBn)2; R1 = Ac TMSOTf

OAc AcO TFAHN

COOMe AcO OAc OR2 COOMe TFAHN O O O O O STol TFAHN OAc MeOOC AcO AcO AcO OAc 70%, a/b = 4/1

Scheme 16

HO

OBn O

OAc

OMP

HO

NIS/TfOH

BnO

OAc

COOMe

AcO O RHN OAc AcO

SPh

NIS/TESOTf OH

COOMe

HO O

AcHN HO OAc

SPh

COOMe

AcO O RHN OAc AcO

BnO

O HO

OMP O OBn

R = Ac: 23%, a only R = Troc: 64%, a/b = 5.4/1

AcO OAc OH COOMe TrocHN O O O SPh AcO AcHN MeOOC AcO HO OAc 43%, a/b = 1.2/1

Scheme 17

Kiso and co-workers proved that the introduction of a Troc group at N-5 increases the reactivity of a thiophenyl sialyl donor when compared to the corresponding N-monoacetylated derivative.114 However, loss in stereoselectivity was also observed (Scheme 17). For the synthesis of the (2!9)-linked dimer, the N-Troc (armed) thiosialoside donor was coupled with an N-acetyl (disarmed) thiosialoside acceptor. The resulting disaccharide was obtained in moderate yield and stereoselectivity. Interestingly, the arming and disarming of these building blocks was achieved from a remote position. Recently, this method was applied for the synthesis of the 2,6-sialyl T antigen by one-pot glycosylation.115

1.14.2.2.3.5 Miscellaneous substituents at C-5 Recently, an interesting N-5 modification has been reported by Kiso and co-workers: specifically, a conformationally constrained C-1–N-5 lactam proved to increase the reactivity of 8-OH.116 Thus, coupling between a 5-N-Troc thiophenyl donor and 1,5 lactamized acceptor, bearing itself a thiophenyl leaving group, gave the (a2!8) dimer with 49% yield. The use of an N-TFA donor increased the glycosylation outcome up to 71% (Scheme 18). Conversion of the acetamido group in a oxazolidinone fused ring by introduction has been reported as an alternative method for the synthesis of (a2!8) and (a2!9) dimers.117 Thus, coupling between a 5-N, 4-O carbonyl-protected

598


OAc

COOMe

AcO

O

SPh

RHN AcO OAc

NIS/TfOH O

H

OAc

COOMe

AcO O

RHN AcO OAc BnO

N

OH BnO AcO

N O

AcO

SPh

O

O

H

O

SPh

OAc

R = Troc: 49%, a only R = TFA: 71%, a only

OAc

Scheme 18

BnO ClAcO ClAcO HN O

SPh O

COOMe

O NIS/TfOH

BnO ClAcO ClAcO HN O

BnO

OH COOMe

HO HN O

O

OC8H17

O

COOMe O

O BnO

O COOMe

HO HN O

O

OC8H17

O

86%, a only

Scheme 19

sialyl donor and acceptor gave the (a2!8) dimer with 86% yield (Scheme 19). Interestingly, the use of dichloromethane as a solvent did not decrease the stereoselectivity. Other known modifications of C-5 involve N-tert-butyloxycarbonyl (Boc),118 N-benzyloxycarbonyl (Cbz, Z),119 N-phthalimido (Phth),120 and N-tert-butyloxycarbonylacetamido.121 However, extensive studies of the effect of these moieties on reactivity or stereoselectivity in the syntheses of sialosides have not yet emerged. Neu5Gc is an important naturally occurring neuraminic acid derivative. Several attempts to synthesize oligosaccharides that contain this derivative have been reported and for this purpose, glycosyl donors were prepared that contain an O-acetyl-28,122–124 or O-benzyl-protected125,126 N-glycolyl moiety, C(¼O)CH2OAc or C(¼O)CH2OBn, respectively. Other functional groups at C-5 were also introduced for the purpose of biological studies.127

1.14.3 Synthesis of C- and S-Glycosides In vivo, sialic acid-containing glycoconjugates undergo desialylation by the action of the hydrolytic enzyme neuraminidase. As a result of the cleavage, the asialylated glycoconjugate chain is unmasked and therefore catabolized. The replacement of the exo-cyclic oxygen in the glycosidic bond by carbon (C-glycosides) or sulfur (thioglycosides) offers non-hydrolyzable derivatives with attractive pharmaceutical applications.

1.14.3.1 C-Linked Oligosaccharides The major problem presented by the synthesis of C-glycosides of Neu5Ac is the formation of a quaternary carbon. A general diastereoselective approach based on the samarium(II)-mediated coupling developed by Beau and co-workers128 was adopted by Linhardt and co-workers for the synthesis of C-sialosides.129 Efficient coupling was established between a neuraminic acid phenyl sulfone and ketones and aldehydes resulting in exclusively a-linked C-glycosides. Some of these compounds are potent inhibitors of the bacterial neuraminidase from Clostridium perfringens. The high stereoselectivity of this methodology was rationalized by the presence of a samarium enolate intermediate whose b-face is more sterically hindered than the a-face (Scheme 20). The methodology was applied to the synthesis of a wide variety of C-glycosides derivatives, such as analogs of gangliosides GM4, GM3, and sTn,130–132 as well as the


599

OAc COOMe AcO O SO2Ph AcHN AcO OAc SmI2 I2SmO

OAc AcO

OMe

O AcHN AcO OAc

OAc

COOMe OH AcHN AcO AcO OAc O AcO AcHN O COOBn AcO

AcO

O O

AcO AcHN

O

COOBn NHZ

O NHZ

Scheme 20

OH O

HO HO

BnO O

OBn O OBn

I

BnO

HO

BuLi O

O

O

BnO

OBn O

O O TBDMSO OTBDMS O

HO

O

OBn

Li

BnO

O

BnO

O

OH AcHN HO OH

O

OBn

O BnO TBDMSO OTBDMS

HO

BnO

O

COOH

O OBn

OH HO

O OH

OBn BnO

O OBn

SePh BnO

OH

HO

O

O HOOH

HO

O HO OO

OBn BnO

O OBn

O Scheme 21 .

synthesis of C-glycosides of Kdn.133 Similarly, Beau and co-workers employed their 2-pyridyl sulfones to C-sialylation.134 Schmidt and co-workers developed a general strategy for the synthesis of C-glycosides of Neu5Ac which was applied to the preparation of a methylene-bridged Neu5Aca2!3Gal C-disaccharide (Scheme 21).135 The key feature of this methodology is the use of an inexpensive open chain precursor of Neu5Ac and its stereoselective cyclization using phenylselenyl triflate.

600


1. S-Alkylation OAc AcO

COOMe

O AcHN AcO OAc

OAc ROTf

SAc

AcO

COOMe

O AcHN OAc AcO

SR

OAc

COOMe

2. Thioglycosylation OAc AcO O AcHN OAc AcO

Cl

HSR COOMe

AcO O AcHN OAc AcO

SR

Scheme 22

1.14.3.2 S-Linked Oligosaccharides Sialic acid thioglycosides or thiosialosides are another class of hydrolytically stable analogs of glycosides of Neu5Ac that have garnered particular interest as biological probes and potential inhibitors of sialic acid-recognizing proteins.136,137 In general, thiosialosides can be synthesized by two common pathways as follows: (1) S-alkylation of an acceptor that contains a good leaving group with a 2-thio-Neu5Ac derivative or (2) incorporation of sulfur into the glycosyl acceptor, which is then coupled with a conventional glycosyl donor (Scheme 22).138 The former has been successfully employed for a range of thiosialosides. In particular, for the synthesis of thiosialoside with (a2!3)Gal linkages, the use of 3-O-triflate-gulose furanose as the alkylation reagent provides the desired product in high yields.139 In addition, new chromatography matrix has been developed to selectively bind sialic acid-recognizing proteins. Thus, 2-thioalkyl derivatives were coupled to CNBr-activated Sepharose 4B through the terminal amino group.140 This matrix was employed to purify Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Tr ypanosoma cruzi, and sialyltransferases from rat liver in high yield. Another interesting example of the use of thiosialosides as biological probes is the synthesis of the CMP-sialic acid thio analog and its biological evaluation in comparison with the natural CMP-activated sialic acid. Interestingly, the thio-derivatives proved to be more hydrolytically stable toward neuraminidase but a poor substrate for a-2,3-sialyltransferase.141 Multivalent sialosides have proven to increase inhibitor activity by amplifying the carbohydrate–protein interactions. Thus, several examples of multivalent thiosialosides have been reported as high-affinity ligands for sialic acidrecognizing proteins.138

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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M.; Gin, D. Y. Org. Lett. 2001, 3, 1665–1668. Ishiwata, A.; Ito, Y. Synlett 2003, 1339–1343. Ye, X. S.; Huang, X. F.; Wong, C. H. Chem. Commun. 2001, 974–975. Okamoto, K.; Kondo, T.; Goto, T. Bull. Chem. Soc. Jpn. 1987, 60, 631–636. Okamoto, K.; Kondo, T.; Goto, T. Chem. Lett. 1986, 1449–1452. Castro-Palomino, J. C.; Tsvetkov, Y. E.; Schmidt, R. R. J. Am. Chem. Soc. 1998, 120, 5434–5440. Okamoto, K.; Kondo, T.; Goto, T. Tetrahedron Lett. 1986, 27, 5233–5236. Okamoto, K.; Kondo, T.; Goto, T. Tetrahedron 1988, 44, 1291–1298. Castro-Palomino, J. C.; Tsvetkov, Y. E.; Schneider, R.; Schmidt, R. R. Tetrahedron Lett. 1997, 38, 6837–6840.

602 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141.


Ito, Y.; Ogawa, T. Tetrahedron 1990, 46, 89–102. Kondo, T.; Abe, H.; Goto, T. Chem. Lett. 1988, 1657–1660. Martichonok, V.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 8187–8191. Kondo, T.; Tomoo, T.; Abe, H.; Isobe, M.; Goto, T. Chem. Lett. 1996, 337–338. Kondo, T.; Tomoo, T.; Abe, H.; Isobe, M.; Goto, T. J. Carbohydr. Chem. 1996, 15, 857–878. Ercegovic, T.; Magnusson, G. J. Chem. Soc., Chem. Comm. 1994, 831–832. Ercegovic, T.; Magnusson, G. J. Org. Chem. 1995, 60, 3378–2284. Ercegovic, T.; Magnusson, G. J. Org. Chem. 1996, 61, 179–184. Kononov, L. O.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1997, 38, 1599–1602. Demchenko, A. V.; Boons, G. J. Tetrahedron Lett. 1998, 39, 3065–3068. Demchenko, A. V.; Boons, G. J. Chem. Eur. J. 1999, 5, 1278–1283. Crich, D.; Wenju, L. Org. Lett. 2006, 8, 959–962. Hossain, N.; Magnusson, G. Tetrahedron Lett. 1999, 40, 2217–2220. Auge’, C.; Bouxan, B.; Cavaye’, B.; Gautheron, C. Tetrahedron Lett. 1988, 30, 2217–2220. Auge’, C.; David, S.; Malleron, A. Carbohyd. Res. 1989, 188, 201–205. Schrell, A.; Whitesides, G. M. Liebigs Ann. Chem. 1990, 1111–1114. Kragl, U.; Godde, A.; Andrey, C.; Kinzy, W.; Cappon, J. J.; Lugtenburg, J. Tetrahedron: Asymmetry 1993, 6, 1193–1202. Schreiner, E.; Zbiral, E. Liebigs Ann. Chem. 1990, 581–586. Schneider, R.; Freyhardt, C. C.; Schmidt, R. R. Eur. J. Org. Chem. 2001, 1655–1661. Yu, C. S.; Niikura, K.; Lin, C. C.; Wong, C. H. Angew. Chem. Int. Ed. 2001, 40, 2900–2903. Voight, E. A.; Rein, C.; Burke, S. D. J. Org. Chem. 2002, 67, 8489–8499. Mukaiyama, T.; Mandai, H.; Jona, H. Chem. Lett. 2002, 12, 1182–1183. Lu, K. C.; Tseng, S. Y.; Lin, C. C. Carbohyd. Res. 2002, 337, 755–760. De Meo, C.; Demchenko, A. V.; Boons, G. J. J. Org. Chem. 2001, 66, 5490–5497. Meijer, A.; Ellervik, U. J. Org. Chem. 2004, 69, 6249–6256. Tsvetkov, Y. E.; Nifantiev, N. E. Synlett 2005, 9, 1375–1380. De Meo, C.; Demchenko, A. V.; Boons, G. J. Aust. J. Chem. 2002, 55, 131–134. Lin, C. C.; Huang, K. T.; Lin, C. C. Org. Lett. 2005, 7, 4169–4172. Ren, C. T.; Chen, C. S.; Wu, S. H. J. Org. Chem. 2002, 67, 1376–1379. Ando, H.; Koike, Y.; Ishida, H.; Kiso, M. Tetrahedron Lett. 2003, 44, 6883–6886. Tanaka, H.; Adachi, M.; Takahashi, T. Chem. Eur. J. 2005, 11, 849–862. Ando, H.; Koike, Y.; Koizumi, S.; Ishida, H.; Kiso, M. Angew. Chem., Int. Ed. 2005, 44, 2–7. Tanaka, H.; Nishiura, Y.; Takahashi, T. J. Am. Chem. Soc. 2006, 128, 7124–7125. Fujita, S.; Numata, M.; Sugimoto, M.; Tomita, K.; Ogawa, T. Carbohyd. Res. 1992, 228, 347–370. Sparks, M. A.; Williams, K. W.; Lukacs, C.; Schrell, A.; Priebe, G.; Spaltenstein, A.; Whitesides, G. M. Tetrahedron 1993, 49, 1–12. Fujita, S.; Numata, M.; Sugimoto, M.; Tomita, K.; Ogawa, T. Carbohyd. Res. 1994, 263, 181–196. Sherman, A. A.; Yudina, O. N.; Shashkov, A. S.; Menshov, V. M.; Nifant’ev, N. E. Carbohyd. Res. 2001, 330, 445–458. Hasegawa, A.; Uchimura, A.; Ishida, H.; Kiso, M. Biosci. Biotech. Biochem. 1995, 59, 1091–1094. Simeoni, L. A.; Tuzikov, A. B.; Byramova, N. E.; Bovin, N. V. Bioorg. Khim. 1997, 23, 139–146. Sugata, T.; Kan, Y.; Nagaregawa, Y.; Miyamoto, T.; Higuchi, R. J. Carbohyd. Chem. 1997, 16, 917–925. Sugata, T.; Higuchi, R. Tetrahedron Lett. 1996, 37, 2613–2614. Higuchi, R.; Mori, T.; Sugata, T.; Yamada, K.; Miyamoto, T. Eur. J. Org. Chem. 1999, 145–147. Kelm, S.; Brossmer, R.; Isecke, R.; Gross, H. J.; Strenge, K.; Schauer, R. Eur. J. Biochem. 1998, 255, 663–672. Mazeas, D.; Skrydstrup, T.; Doumeix, O.; Beau, J. M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1383–1386. Du, Y.; Linhardt, R. J. Carbohyd. Res. 1998, 308, 161–164. Bazin, H. G.; Du, Y. G.; Polat, T.; Linhardt, R. J. J. Org. Chem. 1999, 64, 7254–7259. Kuberan, B.; Sikkander, S. A.; Tomiyama, H.; Linhardt, R. J. Angew.Chem. Int.Ed. 2003, 42, 2073–2075. Ress, D. K.; Sultan, N. B.; Wang, Q.; Munoz, E. V.; Tokuzoki, K.; Tomiyama, H.; Linhardt, R. J. J. Org. Chem. 2005, 70, 8197–8200. Du, Y.; Polat, T.; Linhardt, R. J. Tetrahedron Lett. 1998, 39, 5007–5010. Abdallah, Z.; Doisneau, G.; Beau, J. M. Angew. Chem. Int. Ed. 2003, 42, 5209–5212. Notz, W.; Hartel, C.; Waldscheck, B.; Schmidt, R. R. J. Org. Chem. 2001, 66, 4250–4260. Suzuki, Y.; Sato, K.; Kiso, M.; Hasegawa, A. Glycoconj. J. 1990, 7, 349–356. Wilson, J. C.; Kiefel, M. J.; Angus, D. I.; von Itzstein, M. Org. Lett. 1999, 1, 443–446. Kiefel, M.; von Itzstein, M. Chem. Rev. 2002, 102, 471–490. Turnbull, W. B.; Field, R. A. J. Chem. Soc., Perkin Trans. 2000, 1, 1859–1866. Abo, S.; Ciccotosto, S.; Alafaci, A.; von Itzstein, M. Carbohydr. Res. 1999, 322, 201–208. Cohen, S. B.; Halcomb, R. L. J. Org. Chem. 2000, 65, 6145–6152.


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Biographical Sketch

Cristina De Meo was born in Catania, Italy where she graduated with a M.Sc. in Organic Chemistry in 1996. One year later she joined Geert-Jan Boons’ group at the University of Birmingham (United Kingdom) as a Ph.D. student. In 1998 she moved with Professor Boons to the Complex Carbohydrate Research Center, University of Georgia, where she was awarded her Ph.D. in 2001. Her Ph.D. work focused on the synthesis of N-derivatives of N-acetylneuraminic acid, which resulted in the finding of N-trifluoroacetamido moiety as an activating and stereocontrolling group in O-sialylation. In 2002–2003 she was BLANCEFLOR Post-doctoral Research Associate at the University of Missouri – St. Louis under the direction of Professor Alexei Demchenko prior to joining the faculty at the Southern Illinois University Edwardsville as assistant professor of chemistry in 2003. Professor De Meo is a recipient of the Great Teacher Award from the SIUE Alumni Association (2004) and Cottrell College Science Award from Research Corporation (2005). Her current research program is mainly focused on the chemistry of sialic acid and its conformational and structural modifications to improve the stereoselectivity of chemical sialylations.

Dr. Boons received his B.S. in chemistry in 1983 and his Ph.D. in synthetic carbohydrate chemistry in 1991 from the State University of Leiden (Netherlands). Prior to joining the faculty at the CCRC in 1998, he spent seven years in the United Kingdom, first as a postdoctoral fellow at Imperial College, London, and the University of Cambridge, and then as a lecturer and professor at the University of Birmingham. In 2003, Dr. Boons was the recipient of the Carbohydrate Research Award for Creativity in Carbohydrate Science from the European Carbohydrate Association. Also in 2003, he was elected chairman for 2005 of the Gordon Research Conference on Carbohydrates. In 2004, he received the Horace Isbell Award from the Division of Carbohydrate Chemistry of the American Chemical Society and was appointed Franklin Professor of Chemistry in the Franklin College of Arts and Sciences at the University of Georgia, USA. Dr. Boons serves on the editorial boards of Carbohydrate Research, the Journal of Carbohydrate Chemistry, and Advances in Carbohydrate Chemistry and Biochemistry. Research in the Boons lab deals with the synthesis and biological functions of carbohydrates and glycoconjugates. The diversity of topics to which Dr. Boons has significantly contributed include the development of new and better methods for synthesizing exceptionally complex molecules, the use of new methods in the synthesis and study of properties of complex carbohydrates of increasing size and complexity, the development of synthetic cancer and bacterial vaccines, the design and synthesis of glycosidase inhibitors and the use of synthetic compounds for the study of innate immunity.

604


Alexei Demchenko was born (1965), raised, and educated in Moscow, Russia. He graduated from Mendeleev University of Chemical Technology of Russia with a M.Sc. in Chemical Engineering (1988) before joining the laboratory of Nikolay Kochetkov at Zelinski Institute of Organic Chemistry, Russian Academy of Sciences, Moscow. Under direction of Professor Kochetkov he made the first steps in the area of carbohydrate synthesis. In 1993 he was awarded a Ph.D. for his work on the development of thiocyanate methodology for stereospecific glycosylation. After two post-doctoral years under Kochetkov, he joined Geert-Jan Boons’ group at the University of Birmingham (United Kingdom) as a BBSRC post-doctoral research fellow. In 1998 he moved with Professor Boons to the Complex Carbohydrate Research Center, University of Georgia as a research associate, where his research was focusing on the synthesis of complex oligosaccharides for immunological studies and exploration of chemical O-sialylation. In 2001 he joined the faculty at the University of Missouri St. Louis as an assistant professor of chemistry and biochemistry. In 2005 he received CAREER award from the National Science Foundation. Dr. Demchenko is serving on the editorial board of Carbohydrate Research. Professor Demchenko co-authored over 50 articles, reviews, and book chapters; his current research interests are in the area of synthetic carbohydrate chemistry and include: novel glycosylation methods; stereocontrol and other aspects of the glycosidic bond formation; chemoselectivity and orthogonality of modern glycosyl donors; strategies for convergent assembly of complex oligosaccharides and glycoconjugates; transition metal complexes in synthetic carbohydrate chemistry; regioselective protection of carbohydrate molecules; fully synthetic vaccines based on glycostructures with potential biological activity; application of solid phase and surface chemistry to stereoselective glycosylation and rapid assembly of complex oligosaccharides and glycoconjugates. His research program is currently funded by NSF, NIH, AHA, and PRF.

1.15 Synthesis of Glycopeptides and Glycoproteins D. P. Gamblin, E. M. Scanlan, and B. G. Davis, University of Oxford, Oxford, UK ß 2007 Elsevier Ltd. All rights reserved. 1.15.1 Introduction

605

1.15.1.1 Aims

606

1.15.1.2 Why Does Nature Employ Carbohydrates?

607

1.15.1.3 The Biological Importance of Glycopeptides/Glycoproteins

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1.15.1.4 Potential Applications

608

1.15.1.5 The Need for Homogeneity/Single Glycoforms

610

1.15.2 Glycopeptide Assembly

610

1.15.2.1 Strategies for Forming the Glycosidic Linkage

610

1.15.2.1.1 1.15.2.1.2 1.15.2.1.3

Linkage variety Natural linkage types Unnatural linkages

610 611 612

1.15.2.2 Assembly Strategies 1.15.2.2.1 1.15.2.2.2 1.15.2.2.3 1.15.2.2.4

614

Linear assembly Convergent assembly Elaborative and mixed assembly strategies Native ligation assembly

614 616 618 618

1.15.3 Chemical Glycoprotein Synthesis

622

1.15.3.1 Indiscriminate Glycosylation

623

1.15.3.2 Chemoselective and Site-Specific Glycosylation

625

1.15.3.3 Site-Selective Glycosylation

626

1.15.4 Enzymatic Glycoprotein and Glycopeptide Synthesis

630

1.15.4.1 Elaboration of Glycans

630

1.15.4.2 Trimming of Glycans

631

1.15.4.3 Alteration of Glycans (Glycoprotein Remodeling)

631

1.15.4.4 Enzymatic Formation of the Glycan–Protein/Peptide Link

633

1.15.4.5 Glycopeptide Ligation

634

1.15.5 Molecular and Cell Biological Techniques

635

1.15.5.1 Biosynthesis Augmentation

635

1.15.5.2 Biosynthesis Inhibition

636

1.15.5.3 Non-Coded Methods

636

1.15.5.4 Expressed Protein Ligation

637

1.15.1 Introduction It is becoming ever clearer that the presence of carbohydrate motifs, and their mimetics, in naturally occurring structures has a dramatic effect on their chemical, physical, and biological properties. The myriad of glycoproteins in nature reflects their broad functions as markers in cell–cell communication events that determine microbial virulence,1 inflammatory,2,3 and host immune responses.4–6 In addition, the correct glycosylation of proteins is critical to their

605

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Synthesis of Glycopeptides and Glycoproteins

expression and folding,7–9 increases their thermal and proteolytic stability,10 and modulates in vivo half-life;11–13 therefore, access to well-defined scaffolds to probe the nature of these processes is paramount. Their precise manipulation is a dominant primary goal in glycoscience14,15 and has driven and continues to drive synthetic methodology toward the controlled synthesis of glycoconjugates and in particular glycoproteins.

1.15.1.1 Aims The aim of this chapter is to highlight various methodologies and synthetic strategies that have been applied to the preparation of glycoproteins and, where relevant, glycopeptides as model systems. These procedures encompass chemical, enzymatic, and molecular biological approaches used independently or in combination with one another. The net result is a formidable range of techniques that enables the crossover between the ever-diminishing boundaries between chemistry and biology. Glycopeptides/glycoproteins may be considered quite simply to be polyaminoacid–poly/oligosaccharide conjugates. As such, synthetic strategies may be summarized as depicted in Scheme 1.16 Many of the terms that have been used to describe the construction of linkages A, B, C, and D are arbitrary ones that have been coined over time by various workers in the field, but nonetheless are useful and will be used where appropriate in this review. It is not the intention of this chapter to provide a complete coverage of all the methods for the construction of carbohydrate–amino acid conjugates but to instead focus on the formation of glycosylated-polypeptide structures. Consequently, detailed discussion into glycopeptide synthesis and assembly will only be covered in passing and where it is of potential or direct relevance to glycoproteins. Many excellent reviews have appeared on the topic of synthesizing shorter-length glycopeptides and the reader is referred to these.17–30 Similarly, although they are crucial prerequisite building blocks for glycoprotein synthesis, the synthesis of pure proteins31–35 or oligosaccharides 36–43 will not be covered unless of direct relevance. This chapter builds on our first comprehensive review in 2002 and adopts a similar structure,16 and the reader is also referred to other excellent reviews that have also covered aspects of glycoprotein synthesis, either independently or as part of larger reviews on glycoconjugates.14,44–52 The biological significance of both natural and synthesized53 glycopeptides/glycoproteins will not be discussed in detail, but of course is the primary driver for much of this work.54 Furthermore, the somewhat artificial distinction between ‘chemical’ and ‘biological’ techniques is unhelpful and has been avoided where possible. All sources of glycoproteins have potential function; therefore, there will be no distinction drawn between synthetic analogs, sometimes called neoglycoproteins (neo simply means new, and adds nothing to the nomenclature of this field), and those occurring naturally. Indeed, with the advent of an array of techniques adapted from nature, the distinction between neo and natural has already blurred. A

O

O

X

O

HO

Y

Disconnection A Amino acid or peptide glycosylation O

O

X

O

HO

Y

B

Disconnection B Native chemical ligation Enzymatic ligation D O HO

O O

A or C B X Y

O

O O

HO

B

Y

X C

Disconnection C Protein glycosylation Glycan Protein O

O HO

HO D Disconnection D Glycoprotein remodelling

Scheme 1 Retrosynthetic analysis of glycoprotein synthesis.

X

Y


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1.15.1.2 Why Does Nature Employ Carbohydrates? Carbohydrates are unparalleled in the number of structures they can adopt; as a consequence, nature exploits this structural diversity to convey information at a molecular level. In terms of oligomerization, proteins and nucleic acids are effectively linear in structure (amide bonds in proteins, and 30 –50 linkage of phosphodiesters within DNA), where limited building block sets (4 for DNA, 20 for amino acids) give rise to limited variations. Carbohydrates on the other hand are totally unrivalled in the structures that they can adopt due to variation in configuration (e.g., Glc O-2eq. vs Man O-2ax.) and connectivity (e.g. 1-2, 1-3, 1-4, 1-6 between two pyranoses). This point is illustrated well by the comparison between a hexamer of the respective biopolymers. Peptides and DNA have 6.4107 and 4096 possible permutations, respectively, whereas carbohydrates have access to some 1.051012 combinations.55 Furthermore, additional variety caused by ring size, branching, anomeric configuration, and increased functionality (e.g., acylation, sulfation, and phosphorylation) gives carbohydrates almost unlimited potential for diversity. This inherent structural variation of carbohydrates gives them a role in a wide number of functions,4,5,9,56,57 ranging from sources of energy and metabolic intermediates, to the structural components of plants (cellulose), animals (chitin), and nucleic acids (DNA, RNA), the latter being a frequently unrecognized example. Above all, the almost unlimited variation of oligosaccharides is truly exploited by nature through the combination of carbohydrates with proteins (glycopeptide/glycoprotein and in other glycoconjugates), the products of which have caused an explosion of interest within the scientific community. This structural diversity has been christened glycocode16,58 – a term that well represents the potential level of complex information that carbohydrate structures are able to convey. It should also be noted that the vast number of potential permutations represents a high technological barrier and means that no longer can the oligosaccharide portions of glycoproteins be made on an iterative basis since there are far too many possible synthetic targets. It is therefore crucial that the design of new glycoproteins is guided by the identification of the associated functions and activities of existing structures.

1.15.1.3 The Biological Importance of Glycopeptides/Glycoproteins This section briefly highlights the biological significance of glycopeptides/proteins. The functions and versatility of glycosylated biomolecules are, as a consequence of their ability to transmit sophisticated information, incredibly broad. For example, glycoproteins have been implicated in physiological processes ranging from receptor-mediated endocytosis, and protein quality control, to the interaction and subsequent invasion of pathogens, and the triggering of effects that lead to the release of biomodulators. A thorough understanding of these processes is essential to their successful exploitation in pharmaceutical therapies that either block lectin-mediated process through inhibition or those that exploit this binding to target designed glycoconjugates to lectin-expressing cells. Indeed, the recent failure of a number of carbohydrate-based drugs may be attributed to a poor understanding of their supposed mechanism of action rather than due to any inherent flaws associated with carbohydrate therapeutics.59 When good understanding has been achieved, the results have been impressive.60,61 In addition to their critical role in communication events, an interesting aspect of N-linked protein glycosylation is highlighted by its post-translational role in the ‘quality control’ of protein synthesis.9 Without correct glycosylation, many proteins fail to fold correctly. Nascent glycoproteins emerge from the ribosome and interact with the molecular chaperones calnexin and calreticulin. This process promotes correct protein folding, resulting in the eventual release of native proteins into the secretory pathway. Misfolded glycoproteins re-enter the calnexin/calreticulin cycle through repeated glucosylation–deglucosylation following the actions of glucosidase II and UDP-Glc:glycoprotein glucosyltransferase.62 Persistently misfolded proteins are eventually trimmed of their terminal mannose by the action of a-mannosidase I, causing the incompetent protein to enter the endoplasmic reticulum-associated protein degradation (ERAD) pathway via the critical recognition by mannose-binding lectins EDEM and EDEM2.63 It has been suggested that, if nascent protein fails to fold properly, these glycans are incorrectly displayed and cannot be processed in these trimming steps, leading to rejection and ERAD. Therefore, these apparently superfluous trimming steps may not simply be a means to glycan structure but steps along a ‘quality controlled’ protein production line and suggest a novel role for added glycans as indicators of correct protein structure.7–9,64 It has also been suggested that glycans aid the folding and transport of proteins by protecting them from proteolysis10 and chemical glycosylation has also been linked to increased stability.65 Proteolytic stability, as a result of glycosylation, has been observed through comparison of RNase A, an unglycosylated pancreatic ribonuclease, with RNase B, which bears a single high-mannose oligosaccharide at Asn34.66–68 Carbohydrates have also been shown to alter the physiochemical properties of proteins found in arctic fish.69 In such fish, O-glycan-rich proteins act as an in vivo ‘antifreeze’, preventing the nucleation of ice allowing

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deep sea fish to survive temperatures of 2 C. Glycopeptide analogs of these proteins have also demonstrated significant antifreeze properties.70 In addition to their role in protein folding, glycans have been shown to stabilize the eventual tertiary structure of proteins,71 such as the human glycoprotein CD2.72 This conformational effect has also been observed on a variety of glycopeptide models. For example, in aqueous solution a turn is induced in a SYSPTSPSYS segment of the C-terminal domain of RNA polymerase II when it contains a threonine O-linked to an a-D-GlcNAc monosaccharide, whereas the corresponding non-glycosylated peptide adopts a randomly coiled structure.73 This result tallies well with suggestions that reversible glycosylation of this site might act as a regulatory mechanism for the control of transcription akin to phosphorylation.74 Similar observations with glycopeptides have also been reported from NMR studies, which showed conformational bias and stabilizing effects upon glycosylation.75,76 However, it should be noted that the method of glycosylation may also play a significant role; conformational changes observed for the glycosylation of lysine side chains through amide bond formation appeared to have been largely due to charge neutralization rather than glycosylation per se.77 Another important function of glycosylation is in the recognition of sperm by egg during fertilization.78 The coating of the mammalian ovum contains a large number of both O- and N-glycans. In mice, it is believed that the sperm binds to O-linked oligosaccharides of the ovum glycoprotein ZP3.79 Removal of these oligosaccharides results in spermbinding ablation and ZP3 knockout female mice are infertile. A similar mechanism has also been observed in sea urchins.57 Carbohydrate recognition also plays a vital role in immunity.6 The inflammatory response caused by either infection or injury results in the adhesion of leukocytes via the specific interactions of O-linked glycans containing sialyl Lewisx and newly expressed selectins (E, I, and P) upon the surface of specialized endothelial cells and on leukocytes themselves.80,81 Over recruitment of leukocytes to the traumatized site can lead to chronic inflammatory diseases, for example, rheumatoid arthritis. Consequently, compounds displaying sialyl Lewisx-type structures are viewed as potential anti-inflammatory drugs.82 In theory, these compounds competitively bind to the expressed selectins, decreasing the interaction of leukocytes with the endothelial cells, therefore reducing the inflammatory response.

1.15.1.4 Potential Applications The future of carbohydrate science will be honed by the application of its products; the applications therefore of glycoproteins are an important backdrop to this review and are the context in which their synthesis should rightly be judged.14,47,54,83,84 As early as 1929 it was identified that immunological activity toward carbohydrates may be greatly enhanced through conjugation to protein carriers,85,86 and in 1936 their use as a strategy to combat pneumococci was first described.87 However, until the 1970s this work was limited by the often-minute amounts of oligosaccharides isolated from natural sources. In a series of four papers in 1975,88 Lemieux and co-workers completed the total synthesis of the Lewis-a (Lea) trisaccharide using newly developed glycosidation techniques and conjugated it to BSA using the acyl azide method (see Section 1.15.3) which they pioneered for this purpose. Remarkably, it took another 12 years for the first carbohydrate–protein vaccine to be licensed and since then the development of bacterial capsular polysaccharide– protein conjugate vaccines has blossomed.89 The specificity of the hepatic asialoglycoprotein Gal/GalNAc-specific receptor90 has been widely exploited for liver targeting and in many cases has been the model for targeted drug delivery91 and gene delivery 92–95 therapies. A recent example termed lectin-directed enzyme-activated prodrug therapy (LEAPT), has been designed to exploit endogenous carbohydrate lectin binding through the combination of biocatalysis with novel glycosylated enzymes and prodrugs.65 In the first step of this bipartite strategy, a glycosylated enzyme is targeted to specific cell types within the body that are predetermined by the selected carbohydrate ligand; next, a prodrug capped with a non-mammalian sugar is administered. The use of linkages in the prodrug that can only be cleaved by the activity of the glycosylated enzyme ensured that it was only released at the target site (Figure 1). This first example system employed a rhamnosidase enzyme which was first deglycosylated following exposure to the enzyme endo H and then chemically reglycosylated with chosen glycans.96 When co-administered with model rhamnose-capped prodrugs, in vivo analysis showed a high level of drug in the target organ, the liver. Moreover, use of a prodrug of anticancer compound doxorubicin allowed promising treatment of an animal tumour model. The methodology shows possible adaptation to other disease targets by varying the sugar, or to target other suitable receptors of medical relevance. Although such synthetic glycoproteins are non-natural, their immunogenicities may be low if prepared with a high degree of homogeneity.94 This approach used for the targeting of drugs may also be used to target physiologically


Glycoenzyme O X

1

O X

RME 3

Drug

Drug

609

Rhamnosidase enzyme O X

Sugartargeting ligand

Drug

Drug

Drug O

RME

O

O

HO HO

OH

O

O

HO

Prodrug

Rha-capped prodrug

HO

OH

O L-rhamnose

HO

2

HO

OH

(Rha)

Glycoenzyme

Glycoprotein/enzyme localization

Drug release

Figure 1 Schematic showing the principles of LEAPT and the resulting drug targetting to hepatocytes. Reproduced with permission of Chemistry a European Journal 2006, 12, 656–665 by Wiley.

beneficial enzymes in so-called enzyme replacement therapy (ERT). For example, the mannosylation of enzymes has allowed the targeting of enzymes to particular diseased cells. Thus, the beneficial antioxidant effects of superoxide dismutase (SOD) have been directed to macrophages.97 As an approach to potential anticancer vaccines, Danishefsky and co-workers98 have used reductive amination to construct a hexasaccharide–keyhole limpet haemocyanin (KLH) protein conjugate.99 The hexasaccharide moiety, termed globo H, was originally identified from a glycoceramide associated with breast cancer and was synthesized using glycal methodology. The synthetic globo H–KLH conjugate was successfully administrated to induce high antiglobo H antibody titers and induced cell lysis, in the presence of human complement, at levels approaching those of monoclonal antibodies raised against cancerous cells. Other potential anticancer vaccines have been reported based on other tumour-associated epitopes,100 such as the sialyl-Tn motif; which as a KLH conjugate has shown promisingly higher survival rates in clinical trials.101 Recently, Verez-Bencomo has developed a glycoconjugate vaccine composed of a fully synthetic capsular polysaccharide antigen of Haemophilus influenzae type b (Hib),102 highlighting that access to powerful synthetic complex carbohydrate-based vaccines is now feasible. Initial results from clinical trials demonstrated long-term protective antibody titers as compared to licensed products. This work has highlighted the need for further development of similar approaches to other human pathogens. The importance of carbohydrates in anticancer strategies has been reviewed elsewhere.103 Helicobacter pylori, the bacteria responsible for gastric ulcers, attach themselves to gut cells by binding to extracellular sialylated glycoproteins. This adhesion has been effectively inhibited, as part of an antiulceritic strategy, by albumin glycosylated with 30 -sialyllactosyl residues and illustrates how glycoproteins might serve a useful role as antiadhesives.104 It is interesting to think that the array of sialylated conjugates in human milk might serve the same antiadhesive function as these synthetic glycoconjugates and once again highlights the importance of studying nature’s tactics as models for our own.

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A much less widely explored aspect is the use of glycosylated enzymes as custom-made biocatalysts.105 Examples include carbohydrate–protease conjugates, which show greater stabilities at high temperatures106 and in organic solvents,107,108 and those that catalyze high-yielding peptide synthesis109 with sometimes greatly enhanced synthetic utility, altered stereospecificity,110,111 and increased activities.112 For example, RNase A that had been chemically mono- and di-glycosylated showed slightly lower (80% of unglycosylated) catalytic activity but greatly enhanced thermostability.113 Novel analytical techniques have also utilized glycoproteins. For example, layer-by-layer deposition of a mannose-specific lectin and mannosylated enzymes (glucose oxidase and lactate oxidase) on a platinum surface allowed the preparation of a sensitive active-enzyme electrode.114

1.15.1.5 The Need for Homogeneity/Single Glycoforms As previously discussed, glycoproteins are fundamental to a variety of biological processes. However, the study of these events is made difficult by the fact that naturally expressed glycoproteins appear as heterogeneous mixtures.115 The biosynthesis of the amino acid sequence of proteins is under direct genetic control. However, the biosynthesis of the glycan portion is not template mediated; the glycan structure is influenced by competition between various glycosyltransferases, glycosidases, and their substrates, the substrate specificity of these processing enzymes, and the substrate/donor availability. This variable post-translational processing of glycans results in the synthesis of a variety of possible glycan structures called glycoforms, which are glycoproteins that possess the same protein backbone, but differ in the oligosaccharide component and the site of glycosylation. Therefore, the expression of glycoproteins in mammalian cell culture often leads to complex mixtures of glycoforms. The different properties demonstrated by each glycoform within the heterogeneous mixture present not only regulatory difficulties for therapeutic glycoproteins,48 but also problems in determining exact structure–activity relationships (SARs). Rudd and co-authors have suggested that these heterogeneous mixtures of glycoforms provide a broad range of activities that can be biased in one direction or another by fine tuning of the glycan structure.68 Therefore, there is an urgent need to develop novel methodologies to provide alternative sources of homogeneous glycoproteins. The development of highly successful small-molecule carbohydrate-containing ligands has often involved careful SAR refinements.116 If we are to achieve the same successes with glycoproteins, then homogeneity must be one of the first priorities. This goal is one that the field of glycoprotein synthesis is now beginning to address.

1.15.2 Glycopeptide Assembly The need for homogeneous samples (single glycoforms), outlined above, has resulted in great effort in the field of de novo synthesis of glycoproteins. The preparation of glycopeptides requires a combination of synthetic methods from both carbohydrate and peptide chemistry. Central to any glycopeptide synthetic strategy is the formation of the glycan–amino acid bond (Scheme 1, disconnection A). Thus, the required carbohydrate structure is attached to an amino acid residue (typically serine and threonine for O-linked glycopeptides and asparagine for N-linked glycopeptides). An excellent review of methods for the formation of the glycosidic link between peptides and glycans (Scheme 1, glycopeptide synthesis) has been published.23 Arsequell’s reviews also contain good summaries of approaches to N-25 and O-linked24 glycopeptide systems. Suitably protected, the glycoaminoacid/peptide is then used as a building block in strategies that often rely heavily on standard solid-phase peptide synthesis (SPPS) techniques. As a proof of concept, Ramage and co-workers have reported the first total synthesis of deglycosylated human erythropoietin via SPPS methodology.117 Two factors limit this approach: first, the requirement not only for extensive carbohydrate protection but also amino-acid protection regimes; and second, the acid and base lability of glycosylated amino acid residues.17 This feature of glycoproteins has long been exploited to strip glycans from protein surfaces. The necessary protection and deprotection regimes, the use of particular supports, including the introduction of specific linkers, and coupling methods have all been tailored to be compatible with the presence of carbohydrates. Several excellent reviews17–28,118 cover these aspects in detail but some recent examples to highlight the available strategies are included.

1.15.2.1 Strategies for Forming the Glycosidic Linkage 1.15.2.1.1 Linkage variety Most glycan–protein linkages observed in nature are O-(Ser/Thr) and N-(Asn) glycosides. The majority of motifs for N-linked are GlcNAc(b1-N)Asn, and for O-linked are GlcNAc(b1-O)Ser/Thr or GalNAc(a1-O)Ser/Thr, although


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other unusual linkage forms are also observed,119 for example, GlcNAc(a1-N)Asn,120 Glc-Asn,121 GalNAc-Asn,122 Rha-Asn,123 or even C-linked Man(a1-C)Trp.124 The Complex Carbohydrate Structure Database (CCSD or CarbBank) (http://bssv01.lancs.ac.uk/gig/pages/gag/carbbank.htm) acts as the oligosaccharide equivalent to GenBank by cataloging many of these structures. It provides access to information about structure, author, etc. and the compilation of records that may be submitted to the CCSD. A guide to its use has recently been published.125,126 O-GLYCBASE127,127a is a database of over 170 glycoproteins with experimentally verified O-linked glycosylation sites, compiled from protein sequence databases and literature. It contains information about the glycan, the sequence, literature references, and is http-linked to other databases. The information in this database has also provided the basis for a predictive neural network that may be applied to predictions of mucin-type GalNAc O-glycosylation sites in mammalian proteins and is available at [http://www.cbs.dtu.dk/services/NetOGlyc/]. Given the apparent lack of consensus sequences for O-glycosylation, which contrasts with the fairly good predictability in N-glycosylation, such tools can provide very useful information.

1.15.2.1.2

Natural linkage types

The earliest examples of formation of N-linked glyco-Asn motifs through carbodiimide-mediated coupling of glycosylamines (Scheme 2a) with Asp side-chain carboxylate were reported in 1961128 and have been widely exploited. Although by far the most popular method, it should be noted that amide-bond formation of an anomeric glycosylamine

[P]O

O

[P]O

DCC

H N

O

NH2 COOH [P⬘]-Asp-O[P⬘⬘]

(a)

[P]O

[P⬘]-Asn-O[P⬘⬘]

DME, 45 ⬚C O

[P]O

NCS

H N

O

COOH [P⬘]-Asp-O[P⬘⬘]

(b)

O

O

[P⬘]-Asn-O[P⬘⬘] [P]O H2O

[P]O

O

NBS, CH3CN [P]O

O

NHAc

N C CH3

COOH Z-Asp-OBn [P]O Ac O N Piperidine

O

[P]O

O

(c)

H N

O

O Z-Asn-OBn

Z-Asn-OBn [P]O

O

(d)

[P]O

(e)

O

O

Ph Tf2O, DCM [P]O O S(O) CONHSiMe3 [P⬘]-Asn-O[P⬘⬘] PPh3, Various [P]O solvents O N3 COOH [P⬘]-Asn-O[P⬘⬘]

H N

O


H N

O


Scheme 2 Principle methods for the formation of naturally linked N-linked glycopeptides. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.

612


with an aspartate carboxylate side chain is often plagued by the ready anomerization of the glycosylamine and the resulting formation of anomeric mixtures of glycopeptides.129,130 Some reports have suggested that such anomerization is reduced by the use of propanedithiol as a reductant.131 The glycosylamine approach may also be complicated by intramolecular aspartimide formation if the Asp to be coupled is one residue toward the N-terminus of a glycinyl or alaninyl residue. A number of other valuable methods exist. As a landmark achievement, a protected Man(b1-4)GlcNAc(b1-4) GlcNAc(b1-N)Asn motif was first synthesized by the reaction of the isothiocyanate of the protected trisaccharide with the carboxylate side chain of a partially protected Asp (Scheme 2b).132 In the key amidation step, N,N-bisglycosylthiourea formation also competes. Use of the Ritter reaction has allowed preparation of the key GlcNAc(b1-N)Asn linkage (Scheme 2c).133 Thus, reaction of some nitriles followed by hydrolysis of the nitrilium intermediates results in direct amide formation. More complex amides, such as side-chain N-linked asparagine derivatives, were formed indirectly from attack of the appropriate carboxylate side chain on a b-acetonitrilium followed by deacetylation. Neighboring group participation by a C-2 phthalimide is an important factor in preventing preferential a-amide formation.134 Subsequent glycosylation of the GlcNAc introduced in this way allowed GlcNAc(b1-4)GlcNAc(b1-N)Asn motif synthesis. Other aromatic carboxylic acids and the effect of variations in glycosyl substituents/protection on the glycosyl nitrilium have also been investigated.135 A silylated amide asparagine side chain has been directly N-glycosylated with glycosyl sulfoxide donors (Scheme 2d).136 Reaction of glycosyl azides with Asp side chain carboxylates in the presence of PPh3137 in DCM or MeOH provides good yields of b-Asn N-glycosides (Scheme 2e).138 Recently, this approach has usefully been expanded to incorporate deprotected sugars.139 While the majority of the above examples have been applied to glyco-Asn synthesis prior to assembly, the methodology has also been successfully applied to convergent N-glycosylation of peptides (Section 1.15.2.2.2). The majority of O-linked glycoaminoacids have been prepared via traditional glycosylation methodology with glycosyl donors.24 For example, O-glycosylation of Ser or Thr with, for example, a trisaccharide trichloroacetimidate glycosyl donor allows O-linked trisaccharide-amino acid building blocks to be synthesized.140 Recently, the glycosylation of hexafluoroacetone-protected hydroxyl-bearing Ser, Hyp, and Tyr allowed preparation of glycosylated, dipeptides in just three steps from readily available unprotected amino acids, for example, [GlcNAc4(b1-O)]TY in 70% overall yield from T and Y.141 Other examples include the glycosylation of resin-bound peptides with a variety of glycosyl trichloroacetimidate donors.142,143

1.15.2.1.3 Unnatural linkages Some valuable techniques, developed for the preparation of unnatural glycan–peptide linkages, may be considered in the greater context of glycoprotein synthesis. A number of the techniques that follow have either only been applied to, or indeed are limited strategically to peptide systems. However, many others may be usefully adapted to larger protein systems. The use of a serine-derived sulfamidate (Scheme 3a) based on previous methods,144 allows reaction with thiohexoses to create S-linked glycoaminoacids in water and this is a method that could potentially be adapted to convert N-terminal serines to glycosylcysteines in peptides, although some amino acid a-epimerization was noted and this may limit its general applicability.145 Oxidative elimination of phenylselenocysteine provides dehydroalanine in small peptides which can then act as a,b-unsaturated conjugative addition acceptors for various protected and unprotected thiosugars, although often with poor stereoselectivity (Scheme 3a).146 Solid-supported unprotected thioglucose can be used as a nucleophile to substitute a number of side-chain amino acid iodides to give S-linked glycoaminoacids.147 Similarly, iodide displacement of an iodoserine or Mitsunobu coupling using 1-deoxy-1thio-GlcNAc gave Fmoc-(GlcNAc-)Cys, although in this study racemization was observed during iodide displacement due to in situ elimination and subsequent conjugate addition.148 Peptides containing b-bromoalanine and g-bromohomoalanine were prepared by the direct bromination of peptides or via the incorporation of a bromo amino acid into the peptide synthesis. These were then coupled with 1-thio sugars to furnish S-linked glycopeptides in good yield (Scheme 3a).149 One major advantage of this methodology is that due to the mild reaction conditions, epimerization at the a-carbon of the amino acid was not observed. The methodology could also be applied to deprotected 1-thio sugars resulting in the formation of partially protected glycopeptides. Aziridine-containing peptides (or Azy-peptides) have also been employed as reagents for the preparation of glycopeptides (Scheme 3a).150 Azy-containing peptides can be prepared by standard SPPS technology using FmocAzyOH as a novel amino acid monomer. Unfortunately, attempts to extend Azy-containing peptides by sequential coupling of single Fmoc-amino acids were unsuccessful, due to intramolecular aziridine N-deacetylation by the liberated amine upon Fmoc removal at the [Azyþ2] position. This problem was overcome via incorporation of a dipeptide building block for simultaneous


RHN

HO

O

COONHR⬘

O O S O N H

SH

HO COOH

X (CH2)1-3 RHN (a)

O

S

(CH2)1-3

N H

X = Br or I

C O

COONHR⬘

N O

O

OAc O

(i) O3, DCM, MeOH, py then Me2S NHBoc, THF, −78⬚C (ii) (MeO)2P(O)

AcO

O

NHAc

OAc

OAc AcO

COOCH2CH2TMS NHBoc 1

(b) [P]O

O

[P]O

(i) H2, Pd-C N3

O

[P]O

PPh3, CBr4,

H N

O

H

(ii) AcOCHO

NC

Et3N, DCM O

[P]O

Py-N-oxide

H2N O

Peptide

[P]O

NCO

O

H N

HN

Y N H HO

O

X

NH2 HO

O

X = O or N

O H N N H

O

e.g., Y = (CH2)4NHC(O)CH2

OH

N O

(d)

O

N3

n

+ HN [P]

CuI

[P]O

O

CO2R

O Y

OM catalyst

O + N H

CH2Cl2 O

N N

N n

CO2R HN

[P]O

(f)

NH

NH

(e) [P]O

Y

or

OH HO

[P]O

Peptide

O

(c)

[P] O H

N H

OAc

O

O O

AcO

O

O O

AcO

COOCH2CH2TMS

tetramethylguanidine (iii) H2, (S,S)-Et-DuPHOS-Rh*, THF O

OAc O

OAc

OAc OAc

O

Scheme 3 Principle methods for the formation of nonnaturally linked glycopeptides.

OBz

=

613

614


incorporation of the [Azyþ2] and [Azyþ3] residues, avoiding aziridine deacetylation and allowing for further sequential peptide elongation. The resulting Azy-peptides were treated with 1-thio-sugars to furnish glycopeptides in 43% yield over 10 steps in a convergent manner. More recently, Wong and co-workers have described a one-pot general strategy for S-linked glycopeptide synthesis. Peracetylated thiosugars have been converted to S-linked amino acid building blocks with retention of anomeric integrity.151 Danishefsky and co-workers have created unnaturally O-glycosylated glyco-amino acids decorated with various tumor-associated antigens.152 This method relies on a pentenyl glycoside-derived aldehyde and a glycine phosphonate based on a Horner–Emmons method first devised by Toone and co-workers.153 The resulting –(CH2)4O-glycoside side-chain amino acids, for example, 1, (Scheme 3b) were then coupled to create a ‘multiantigenic peptide’. Glycosylisocyanates prepared by oxidation of isocyanides, in turn prepared from anomeric formamides, react with amines to give urea linkages and this has allowed one example of a glycosyl–amino acid conjugate to be synthesized (Scheme 3c).154 Chemoselective ligation155 approaches have been applied to unnaturally linked glycopeptide synthesis. Both a- and e-lysine amino groups were functionalized with aminooxyacetyl groups before reaction of the free amine introduced with reducing sugars to generate oxime linkages (Scheme 3d). The use of orthogonal N-protection also allowed regioselective glycosylation.156 In a similar manner, reaction of a 12-aa peptide containing an N-terminal aminooxyacetyl function has also been described.157 The use of a basic peptide allows this to be used as a method for enhancing the MALDI-MS sensitivity of oligosaccharides for carbohydrate sequencing work. Good aminooxy-over lysine-N-selectivity, attributable to a-effect enhanced nucleophilicity, was observed. Aminooxyacetyl Lys capping and oxime formation with the reducing end of lactose has also been applied to a sulfopeptide.158 This type of oxime formation can be hindered by E versus Z oxime formation and equilibria between cyclic hemiaminal pyrano- or furano-forms and open-chain oxime. In contrast, N-methyl aminooxyacetyl-capped lysines react with the reducing ends of various glucosyl saccharides to give cyclic b-N-linked glycopyranosides with moderate yield and good selectivity;159 indeed, as observed previously,157 Lys side chains in the peptide do not react. Imperiali has evolved these ideas by developing non-natural tripeptides containing b-hydroxylamine and alanine-b-hydrazide (Scheme 3d) and reacting them with N-acetylglucosamine, also via its reducing terminus.160 Double chemoselective thioether and hydrazone formation in SPPS-derived lysine clusters has allowed glycosylation of lysine side-chain termini using thioethyl glycosides while extending the keto-functionalized C-terminus with an antigenic hydrazide peptide sequence.161,162 Glycosylmaleimides can be used to modify cysteinyl-containing peptides163 (see also Section 1.15.5.2, Scheme 11e) and in a reverse sense maleimido-peptides react with unprotected 2-thioethyl N-glycosides; the latter were easily prepared from unprotected parent carbohydrate.164 The Cu(I) catalyzed [3þ2]-cycloaddition between organic azides and acetylenes (so called ‘click reactions’), as reported by the groups of Meldal165 and Sharpless,166 generally result in the formation of the corresponding 1,4-disubstituted 1,2,3-triazoles in high yields. Application of this reaction to azidoglycosides and acetylenic amino acids has allowed the preparation of triazolelinked glycopeptides (Scheme 3e).167 This methodology, in combination with a Tyc TE catalyzed macrocyclization has also been exploited in the preparation of libraries of cyclic glycopeptide variants of the antibiotic tyrocidine.168 Josephson and co-workers have recently applied ‘click’ chemistry to chemical modification of a peptide containing an unnatural alkyne amino acid. The resulting thymidine and glucopyranosyl ligated products were detected by MALDI-TOF MS.169 Olefin metathesis (OM) has been employed in synthetic strategies towards C-linked glycopeptides (Scheme 3f).170–172 Tripeptides incorporating an allyl functionality were prepared and successfully ligated with C-allyl glycosides to furnish the C-linked glycopeptides in yields of up to 68%.170 OM, using the Grubbs secondgeneration catalyst, has been successfully employed between a- and b-C-allyl glycosides and protected vinyl glycines to furnish olefin products in 57–94% yields, subsequent palladium-catalyzed hydrogenation gave C-glycosyl amino acids in good yield.173 A similar approach has also been employed to produce glycomimetics of globo H.174 Interestingly, Nolen and co-workers report increased efficiency with O-allyl glycosides than the corresponding C-allyl derivatives.175

1.15.2.2 Assembly Strategies 1.15.2.2.1 Linear assembly These techniques rely heavily on SPPS methodology. Impressive examples include the synthesis of a cyclic 47-aa HIV-1 V3 domain containing two GlcNAc residues176 and the synthesis of the RNA polymerase II C-terminal domain heptapeptide repeat containing a single GlcNAc(b1-O)Ser and variously N-acetylglucosaminidated-Asn 11-aa sequence from the C-terminal domain of mammalian neurofilaments.177 Preparative scale hydrazinolysis allows N-glycans to be stripped from N-linked glycoproteins, such as fetuin, on scales of up to 500mg for the construction


615

HO O HO HO

O

HO HO

HO

NH

OH O

H2N

O AcHN

HO HO OH O O O HO HO HO AcHN OH O O O O HO HO HO AcHN HO HO

HO HO

O HO

O HN

OH O

O

O

NH

O

O

O OH

O HO O

OH OH O

OH O HO

O

O HO NHAc

HN

OH O

O

H N

NHAc

NH O O HN O

2

NH O HN O NH2

Figure 2 Glycononapeptide prepared from CDI coupling between peptide and complex type oligosaccharides. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.

of glyco-Asn building blocks to be used in linear assembly. In this way, glycononapeptide 2 (Figure 2) was synthesized from carbodiimide-mediated coupling of oligosaccharyl-glycosylamines, derived from the corresponding glycosylhydrazide stripping product after purification, with a protected Asp.178 This building block was used in linear assembly; where product yields were lower for the synthesis of the bulkier undecasaccharide-bearing glycosyl amino acid than for a monosaccharide-bearing glycoaminoacid building block (35% cf. 78%, respectively). The first example of the synthesis of a glycophosphopeptide 3 is shown in Scheme 4. The enzyme labile group PhAcOZ was used to protect the N-terminus of a serine residue which was glycosylated with GlcNAc. This formed the critical building block 4 in the synthesis of 3. Penicillin acylase allowed the removal of PhAcOZ and the free amine was coupled using carbodiimide to a glycosylated dipeptide, itself prepared from key block 4. Again, enzymatic cleavage allowed the N-terminus to be released before further couplings with peptides and finally a serine residue bearing a protected phosphorylated side chain, where global deprotection gave 3.179 Protease-mediated peptide ligation has typically not been capable of general coupling of glycopeptide blocks due to the often stringent specificities of these enzymes (see Section 1.15.4.5).180,181 This has limited the utility of such ligations to those that do not include glycosylated amino acids directly next to the ligation site. However, the recent application of precise chemical modification to proteases has generated catalysts capable of this type of direct ligation.111 A hybrid chemical synthesis strategy in which a glycosylated tripeptide was oligomerized up to 12 repeating units has been reported. The tripeptide Z-AAT-Bn was glycosylated with Gal(b1-3)GalNAc, deprotected, and then treated with diphenylphosphoryl azide as an activator to form an oligopeptoid which demonstrated significant antifreeze properties.70 Block coupling strategies may also be applied to the construction of non-proteolyzable (and therefore with potentially higher oral bioavailability) peptide mimics (glycopeptoids),182 such as the L(GlcNAc-) NFKA mimic 5,183,184 (Figure 3) which may also show interesting conformational restriction as a result of rotamer formation. Similarly, a linear glycopeptoid mimic of the Tn-antigen (GalNAc(a1-O)Ser/Thr) was prepared using a reiterative TBTU-mediated coupling of a single orthogonally protected aminoester building block.185 This method

616


AcO AcO

OAc PhAcOZ Ser OH O PhAcOZ Ser OH OAc O AcO O N AcO O NHAc 4

H3C (i) H-Pro-OAll, EDC, HOBt (ii) (Ph3P)4Pd PhAcOZ Ser Pro OH OAc O AcO O AcO NHAc

AcO AcO

(i) t-Bu isourea (ii) Penicillin acylase

H Ser OBut OAc O O NHAc

NHAc

EDC, HOBt O O OAc PhAcOZ Ser Pro Ser OBut OAc O AcO O AcO NHAc (i) Penicillin acylase (ii) PhAcOZ-Pro-Thr-OH, EDC, HOBt (iii) Penicillin acylase (iv) Aloc-Ser(OPO(OAll)2)-OH, EDC, HOBt NHAc (v) (Ph3P)4Pd then TFA then HO O HO NH2NH2, MeOH O OH H Ser Pro Thr Ser Pro Ser OH OH O O HO O 3 P OH HO NHAc O OH AcO AcO

Scheme 4 The synthesis of the first example of a glycophosphopeptide. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.

has been elegantly extended to the concept of glycopeptoids in which both the inter-residue and side chain distance may be varied. Through the incorporation of different aromatic, amine, and peptide spacer units the spatial presentation of C-glycosides in these structures may be optimized.186

1.15.2.2.2 Convergent assembly As has been described above, a linear strategy in glycopeptide synthesis is more usual since direct peptide glycosylation is often unsuccessful, given the variety of functional groups that would be required to protect a given oligopeptide of any significant length. However, Lansbury and co-workers have adapted the use of glycosylamine coupling with carboxylates in a convergent approach to glycopeptide synthesis. For example, HBTU-mediated coupling of GlcNAc glucosamine with the side-chain aspartate carboxylate in pentapeptide 6 allowed the formation of an Asn-linked N-acetylglucosaminyl-containing glycopeptide (Scheme 5).187 This method was successfully extended to peptides containing more complex glycans such as the high-mannose core of N-linked glycoproteins, Man5GlcNAc2.188 Furthermore, this method has been expanded to encompass solid-phase bound glycosylamines, which are then coupled to side-chain carboxylates in pentapeptides before the peptide chain is further extended.189,190 As part of their investigation into the preparation of the prostate-specific antigen (PSA) glycopeptides, Danishefsky and co-workers have developed a ‘universal’ strategy for the preparation of complex N-linked glucopeptides from a common glycal precursor.191 It was found that a sequence consisting of Kochetkov amination192 of an oligosaccharide bearing a free reducing terminus, followed by application of Lansbury aspartylation188 and thence by native chemical ligation (NCL), provided a route to complex N-linked polypeptides.


R

O

H N

N H

O

O

H N

N H O

617

R

N H

O

O

HN +

O

NHAc NH3

HO

L(GlcNAc-)NFKA OH OH O R

N

O N

N O

N

N O

O

R O

HN +

NHAc NH3

O HO

OH OH

5

Figure 3 A non-proteolyzable glycopeptoid. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.

OH

O N H

O

H N

N H OH

O OH

O

O

O

HO HO

H N

O N H OH

OH NH2 O

6

NH2 HBTU, HOBT

NHAc OH

O N H

H N O

O

H N

N H NH OO

O

O N H OH

OH NH2 O

NHAc

HO OH OH Scheme 5 Convergent assembly of glycopeptides using HBTU-mediated coupling. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.

A glycosylamine convergent strategy has also been applied to the synthesis of a high-mannose core glycopeptide; again, anomerization of the pentasaccharide glycosylamine used led to the formation of a mix of a- and b-Asn-GlcNAc linkages.130 It is worth noting that use of microwave irradiation in the acceleration of the Kotchekov animation.193 In a strong demonstration of convergent glycosylamine methodology, a 15-mer glycosylamine corresponding to a

618


high-mannose H-type 2 blood group determinant was coupled to the carboxylate of a LADVT pentapeptide. Although the low yield for this step (20%) indicates the apparent difficulties of this type of convergent coupling, this is a testament to the type of structures that can now be constructed.194 The mutarotation that often accompanies the use of glycoylamines in convergent glycopeptide synthesis may be avoided through the use of configurationally stable glycosyl azides. Indeed, the Staudinger reaction has allowed efficient convergent use of such azides in conjunction with aspartyl peptides without the need for carbohydrate protection.139 Despite these successful examples of convergent N-linked glycopeptides, it should be noted that the convergent O-glycosylation of peptides to create O-linked (e.g., Ser/Thr) glycopeptides has not yet been achieved in a general manner in solution, although rare convergent solid-phase examples exist.142,143

1.15.2.2.3 Elaborative and mixed assembly strategies A mixed strategy of initial linear assembly coupled with convergent elaboration may also be successful. For example, in an elegant combination of both chemical and glycosyltransferase-mediated glycosylation, the synthesis of an undecasaccharide-linked asparagine residue 7 (Figure 4) demonstrated that highly glycosylated glycopeptide building blocks were accessible.195 This was an important step towards the first synthesis of a glycopeptide fragment bearing this same full N-linked oligosaccharide, which was achieved through linear assembly of a heptasaccharide bearing Asn into a pentapeptide followed by galactosyltransferase and sialyltransferase-catalyzed elaboration.196 A sulfated N-terminal octapeptide from P-selectin glycoprotein ligand (PSGL-1) bearing a pentasaccharide sLex(b1-6)GalNAc(a1-O)Thr has been synthesized using a combination of initial SPPS with a disaccharide-amino acid building block and then chemical tyrosine sulfation followed by glycosyltransferase-mediated elaboration.197 The presence of sulfotyrosine makes this a particularly difficult glycopeptide to elaborate and in this study alternative glycosyltransferases, more tolerant than those used previously, had to be found. In an excellent display of the power of glycosyltransferases in glycopeptide elaboration (Scheme 6), the sulfated N-terminal domain of PSGL-1 has been synthesized on a small scale and characterized by HPLC and MS in two glycoforms that differ significantly in their binding to P-selectin as a result of only a subtle alteration of internal glycan structure.198 Isolation of the required six glycosyltransferases and one sulfotransferase allowed 9 and 10 to be prepared from the linearly SPPS-assembled 23-aa monosaccharide glycopeptide 8.

1.15.2.2.4 Native ligation assembly The concept of native chemical ligation (NCL) which involves the ligation of C-terminal thioesters with N-terminal cysteinyl peptides was introduced by Dawson and Kent199 and is based on observations made by Wieland in the 1950s.200 Two fully unprotected synthetic peptides react under aqueous conditions at near neutral pH to form an amide bond, creating a linkage as in the ‘native’ peptide backbone. This reaction essentially proceeds by a transthioesterification by a cysteinyl peptide such as 13 and a subsequent S!N shift allowing intramolecular amidation (Scheme 7b). Peptide thioesters required for NCL may be synthesized through SPPS using Boc-based strategies that are compatible with a direct thioester linkage to the support; Fmoc methods cannot be used in the same way as this typically leads to

OH HO AcHN

HOOC OH HO O O HO

OH HO AcHN

HO

HOOC OH HO O O HO

HO

O

O HO HO

OH O AcHN

HO HO OH O O O HO HO AcHN HO HO

O OH O 7 O

O OH O HO O

OH O

OH O HO

O

O HO NHAc

OH O

H N

NHAc

COOH O

NH2

Figure 4 An example of a possible glycoamino acid structure obtained from a combination of chemical and enzymatic synthesis. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.


OH HO HO

O

OH O

HO

8

AcHN

AcHN GQATEYEYLDYDFLPETEPPEML UDP-Gal b1,3-GalT OH

HO OH O O HO O HO AcHN HO

i, UDP-Gal, b1,4-GalT HO ii, CMP-NeuAc, a2,3-SiaT iii, GDP-Fuc, a1,3-FucT iv, PAPS, sulfotransferase

UDP-GalNAC b1,3-GlcNAcT HO O HO

GQATEYEYLDYDFLPETEPPEML UDP-GicNAC b1,6-GlcNAcT OH HO O O HO OH NHAc HO O O HO O HO AcHN HO

OH

HO

HO

O HO

HO HO

O O OH NHAc

HO

O

HO HOOC OH O O HO O

GQATEYEYLDYDFLPETEPPEML

O O NHAc GQATEYEYLDYDFLPETEPPEML −

HO HOOC OH O O

i, GDP-Fuc, a1,3-FucT ii, PAPS, sulfotransferase

AcHN

OH

O O HO O AcHN HO

O

OH

HO

O

OH

OH

− − O3S SO3 SO3

O O NHAc GQATEYEYLDYDFLPETEPPEML

AcHN

CMP-NeuAc a2,3-SiaT HO

HO

9

HO

O O HO O AcHN HO

O O

HO

O OH

OH

OH O HO

OH

O

O O HO O AcHN HO

UDP-Gal b1,4-GalT

HO

OH

OH

OH


OH

HO HOOC OH O O

619

OH

OH O

OH

O O HO O NHAc OH O OHOH O O HO OH O O HO HO AcHN HO GQATEYEYLDYDFLPETEPPEML O

−

10

OH

− − O3S SO3 SO3

O

O O O HO HO OH NHAc HO O O HO O HO AcHN HO

O


Scheme 6 The enzymatic synthesis of a sulfated N-terminal of PSGL-1. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.

thioester degradation. However, due to the acidity of the trifluoroacetic acid (TFA) treatment required, Boc-based methods are incompatible with glycosidic linkages,201 although in some cases acetylation provides TFA stability.202 Applications of NCL in glycopeptide synthesis include linear assembly of a 46-aa C-terminal segment containing eight GalNAc(a1-O)Ser/Thr residues followed by native chemical ligation with a 47-aa N-terminal peptide fragment; this allowed the first synthesis of a mucin-type glycoprotein segment, the chemokine lymphotactin.203 Use of sulfonamide safety-catch linkers also allows the synthesis of the required glycopeptide thioesters via Fmoc strategy.204–206 To allow the use of NCL in syntheses, additional modifications are also often needed. For example, alteration of residue 25 in 13 from Gly!Cys was necessary to allow native chemical ligation by incorporating the required Cys at the N-terminus of the C-terminal portion. In addition, to prevent intramolecular aspartimide formation during SPPS, two Asp-Gly and Asp-Asn motifs were altered Asp!Glu. Finally, after ligation, hydrazine treatment allowed deprotection of acetylated glycans. Interestingly, expressed ligation, that is, recombinant preparation (see below and Section 1.15.5.4) of the N-terminal thioester failed in this system and lower yields (38%) were also obtained from the native ligation due to the presence of a valinyl residue at the C-terminus of the N-terminal fragment. In this case, glycosylation appeared to have little or no effect on the structure or activity of lymphotactin. Expressed protein ligation (Section 1.15.5.4) has been used to incorporate variously modified cysteines at the Cterminus of bacterially expressed mannan-binding protein (MBP) including Cys-[GlcNAc(b1-N)]Asn 12.207 Thus, MBP was expressed in Escherichia coli as a fusion 11 (Scheme 7a) to the N-terminus of a widely used intein from Saccharomyces cerevisiae; this intein also bears a chitin-binding domain at its C-terminus. Inteins are protein sequences that are self-spliced from protein sequences. The first step of splicing is an N!S acyl shift at the Cys N-terminal amino acid of the intein with a suitable C-terminus of the MBP (Scheme 7a). The resultant thioester can be purified on chitin beads before being transthioesterified to give a soluble thioester of MBP for use in NCL. MBP can then be natively ligated199 (Scheme 7b) with a cysteinyl N-terminus peptide such as 12. Using the NCL methodology, Bertozzi and co-workers have completed the total synthesis of a chemically defined version of diptericin208 which is a 82-residue antimicrobial glycoprotein isolated from insects. Since diptericin lacks cysteine, Gly25 was mutated to the cysteine residue necessary for the native chemical ligation. The 58-mer

620


Expressed protein HS O Peptide A

H2N

Intein

N H

CO2H 11

Intramolecular intein thioesterification O Peptide A

H2N

S

Intein thioester Intein

H2N (a)

− SO3

HS

CO2H

Transesterification

O −

Peptide A

H2N

SO3 OH O HO

S HS

Transesterification Step 1 of native ligation

OH O

O Peptide A

S

HO

where HS

OH O HO

OH O

HS

Peptide A

H2N

HO

X

X

Peptide B

or HS (b)

H2N

CO2H

Y CO2H

HS H N

=

Y

H2N H2N

Y

Peptide B

N H

X

CO2H

Peptide B

H2N Intramolecular amidation Step 2 of native ligation O

Y

Peptide B

H2N

H 2N

X

CO2H

OAc OAc O AcO AcHN O Cys25 Diptericin 25-82

HO HO

OH O

O HN

NHAc

CO2H

O 12

13 CO2H

Scheme 7 Native chemical ligation (NCL) approaches. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society.

C-terminal glycopeptide was prepared by standard SPPS protocol. The ligation of the 24-mer thioester and the 58-mer cysteinyl glycopeptide followed by O-deacetylation furnished the fully deprotected 82-mer glycopeptide. Recently, a glycosylated fragment of bovine RNase B was successfully prepared by Unverzagt and co-workers using SPPS methodology, thus demonstrating the first example of the synthesis of a complex-type N-linked glycopeptide.209 An Fmocprotected asparagine, glycosylated with a complex unprotected biantennary heptasaccharide, was introduced using PyBOP in the presence of DIPEA onto a pentapeptide linked to a Rink-amide safety catch linker bound201 to a solid


Standard SPPS methodology

AcO AcO

OAc O Boc

AcHN AcO O AcO AcO

AcO AcO AcO AcHN

O O O O

AcO AcO AcO

O AcO

O HO HO HO HO HO AcHN

Peptide

O

O S N O R

= GlcNAc2Man3GlcNAc2

O

N H

Spacer

Ot Bu

OAc AcHN AcO O AcO O O O O AcO AcHN OAc

H N

(i) ethyl 3-sulfanylpropionate, sodium thiophenolate, DMF (ii) TFA, H2O, TES, 46% O

H–Met-Leu-Ser-Arg-Asn-Val-Thr-Lys-Asp-Arg–SPh OH O

AcHN HO O HO HO HO HO HO

Boc

621

HS (i) O O

O O

O HO O

H2N

RNase 41-68

O (i) thiophenol, phosphate buffer pH 7.6 (ii) NH2NH2.H2O, DTT.

OH AcHN HO O HO O H O N O O HO O AcHN OH H–Met-Leu-Ser-Arg-Asn-Val-Thr-Lys-Asp-Arg-Cys

RNase 41-68

Scheme 8 Unverzagt’s approach towards a glycosylated fragment found in RNase B.

support. This was subsequently further extended by SPPS and released from the safety catch linker by sodium thiophenolate (Scheme 8). The resulting thioester was coupled to RNase41–68 through NCL. Danishefsky and co-workers have introduced an interesting alternative to the difficult preparation of glycopeptide thioesters.210 It was found that a peptide phenolic ester equipped with an ortho disulfide moiety can be ligated to a peptide containing an N-terminal cysteine residue after the disulfide end is reductively cleaved (Scheme 9). The suggested mechanism for this attractive ligation reaction involves a ‘dynamic’ O ! S acyl transfer in the reduced phenolic ester intermediate, this thioester can then transesterify to take part in NCL. The relative stability of a glycopeptide ester over a glycopeptide thioester toward Fmoc SPPS conditions renders this an extremely useful native ligation method. Using this strategy, Danishefsky and co-workers have successfully prepared several model glycopeptides containing two glycosylation sites.210 A limitation of NCL methodology is its intrinsic reliance on having a cysteine residue at the ligation juncture. Cysteine is relatively uncommon and comprises only 1.7% of all residues in proteins.16 In the absence of a cysteine residue near to or at the glycosylation site, other strategies have been employed. The majority of these emerging strategies involve removable auxilaries that act as cysteine surrogates to mediate the chemical ligation of peptide fragments. The challenge of these approaches is to obtain fast ligation while maintaining a cleavable handle. Native chemical ligation combined with desulfurization to furnish linear and cyclic polypeptides has been described by Dawson and co-workers.211 Up to now, very few of these removable auxiliary methods have been applied to the preparation of glycopeptides. Recently, Macmillan and co-workers have reported a rapid synthesis of acyl transfer auxilaries for cysteine-free native glycopeptide ligation.212 It was demonstrated that auxiliary introduction and cleavage were compatible with the presence of glycosidic linkages and to function in cysteine-free ligation across Gly–Gly junctions. Unfortunately, the auxiliaries failed to deliver ligation products at more general linkages such as Leu–Gly and Ser–Gly due to steric factors. NCL followed by S-glycosylation has proved to be a versatile method for S-linked glycopeptide synthesis. Homocysteine-containing peptides prepared by NCL were glycosylated in the presence of Na2CO3 with glycosyl halides to furnish S-linked glycopeptides in good yield. In this fashion, the synthesis of an S-linked glycopeptide analog carrying two sugar residues derived from Tamm–Horsfall glycoprotein, the most abundant glycoprotein present in human urine, was achieved (Figure 5).213

622


HO HO

O

HO AcHN O

EtSS

SPPS Fmoc chemistry

Ac-Arg-Ala-Arg-Ser-Gly-Phe O Excess R-SH HO HO

O

HO AcHN O

HO HO

HS

HO

Ac-Arg-Ala-Arg-Ser-Gly-Phe O

OH O

O

O AcHN HO

NH

AcHN

O

+ H2N-Cys-Ala-Asn-Val-Ser-NH2

HO HO

O

HS

HO AcHN O

HO

Ac-Arg-Ala-Arg-Ser-Gly-Phe S

Excess MES-Na 0.2 M PBS pH 7.4 HO HO

HO HO

O

HO

HO

OH O

O

O AcHN HO

NH

AcHN

O

AcHN O Ac-Arg-Ala-Arg-Ser-Gly-Phe

Cys-Ala-Asn-Val-Ser-NH2

Scheme 9 Modified native chemical ligation with phenolic peptide ester.

AcO AcO

OAc O

OAc

O AcO AcO

O AcO +

H3N

AcO AcO

S O N H

N O O

N H

OAc O

AcHN OH O H N O

N H OH

S H N

O O−

O

Figure 5 An S-linked analog of a fragment of Tamm–Horsfall glycoprotein.

1.15.3 Chemical Glycoprotein Synthesis Typically syntheses of glycoproteins adopt one of two strategies. The first is the formation of the putative glycanprotein link early to form glycopeptide building blocks that may then be assembled (Scheme 1, disconnection B). The other is the construction of the link late on in the synthesis once the protein scaffold for its presentation is in place


623

(Scheme 1, disconnection C). Given the requirements for protection that need to be considered in the use of glycosylated building blocks, and the instability that may be associated with the link,17 it is clear why the latter approach has often seemed the most attractive option. However, although the convergent glycosylation of oligopeptides may be successful, it is limited by a lack of chemo-and/or regio-selectivity when applied to proteins.17 For this reason, alternative glycoprotein synthesis techniques have been more widely applied.14,44,48,84,105,214,215 Protein glycosylation methods have been divided here according to classes of chemoselective: site-specific and site-selective. This distinction is arbitrarily based on the overall glycosylation strategy and does not necessarily reflect the inherent specificity or selectivity of a given reaction. For example, the use of the same chemoselective (e.g., thiol-reactive) reaction will give rise to: (1) indiscriminate glycosylation with a variety of the available cysteine thiols; (2) sitespecificity when used to glycosylate a single, naturally occuring, cysteine thiol; (3) site-selectivity, when used to modify a single, cysteine thiol that is artificially introduced to a preselected position.

1.15.3.1 Indiscriminate Glycosylation The use of 2-iminomethoxymethyl thioglycosides (IME)96 and reductive amination methods216 are still, after 20 years, the most frequently used strategies for glycoprotein preparation. The former may be readily prepared by the action of methoxide on cyanomethyl thioglycosides, themselves readily derived from 1-thiosaccharides (Scheme 10a). Recent applications to this method have been extended to in vivo bipartite drug delivery65 and the incorporation of 20 000 glycans to the surface of adenovirus.217 Gray originally used reductive animation to modify albumin with lactose through NaBH3CN-mediated reduction (Scheme 10b),216 although borane has also been used. Reductive animation is amenable to other sources of aldehyde functionality such as the hydrolysis of acetal-containing spacer arms,218 or those generated by ozonolysis of unsaturated spacer arms,219 or through periodate cleavage of diols. However, conjugations through reductive amination are often accompanied by low protein loading levels, presumably due to steric hindrance induced by short spacer arms. In a pragmatic approach to overcome this problem, a second hydrazide spacer arm can be used to extend an existing aldehyde-terminated linker.220 Reaction of the maleimido terminus of the resulting longer spacer arm with thiols introduced to the surface of the KLH allowed a fivefold greater loading of the sialyl-GalNAc disaccharide. The use of glycosidic aromatic diazonium salts, derived from p-aminoaryl glycosides, as electrophiles to functionalize a wide range of electron-rich side chains within protein structures was first demonstrated as early as 1929 (Scheme 10c).85,221 p-Aminoaryl glycosides may also be elaborated to phenylisothiocyanates, which react selectively on amino residues (Scheme 10d).222 A one-pot, two-step preparation of anomeric para-nitroanilide (pNA) pyroglutamates from free carbohydrates also provides an alternative route to aromatic isothiocyanates.223 Following glycosylamine formation with the a-amino group of pNA glutamic acid, the side-chain g-carboxylic acid readily reacts with the resulting secondary amine to give a pyroglutamate which can be further elaborated (Scheme 10e). Glycosyl isothiocyanates have also been used in this fashion as protein-labeling reagents to elucidate carbohydrate transport mechanisms.224 In 1975, Lemieux and co-workers developed highly activated acyl azides as reagents for the formation of amides from proteinaceous amines and carboxylate ester-terminus spacer-arm carbohydrates.88 These esters were transformed to the corresponding acyl hydrazides, prior to oxidation with nitrous acid, to give acyl azides (Scheme 10f ). Mixed anhydride methods are well established for the activation of carboxylic acids to form carboxyl derivatives and allow aldonic acids to be coupled to protein-based amines in this way (Scheme 10g).225,226 Similarly, carbodiimide chemistry227 and the use of N-carboxyanhydrides228 has allowed the coupling of aldonates and glycosylated amino acids as sources of glycans bearing carboxylic acids. In a reverse sense, carbodiimide chemistry has also been employed in an attempt to activate and therefore glycosylate protein carboxylates (Scheme 10h).229 RNase A was mono- and diglycosylated (mixtures of mono 45%, di 34%, and unglycosylated 21% were obtained) by EDC-mediated coupling of D-glucosamine via N-2. Glycosylation sites were predicted to be Asp53 and Glu49 side-chain carboxylates by analogy with previous side-chain carboxylate reactions in RNase, but are probably a distribution among several of the 12 available carboxylates.113 Treatment of the protein with hydroxylamine was assumed to have reversed concomitant tyrosine modification. A similar modification of a-chymotrypsin led to broad heterogeneity and the formation of mixtures bearing glucosaminyl residues on 1–7 of the available 17 carboxylates; however, long reaction times (18–21h) led, in some cases, to up to 75% degradation through autoproteolysis.230 Tietze, Gabius, and co-workers have described the use of diethyl squarate for the coupling of carbohydrates bearing N-terminus spacer arms to amines in BSA (Scheme 10i).231 2-Chloroethyl-1-thioglycosides have also been used to indiscriminately alkylate protein amino and hydroxyl groups;232 an essentially analogous technique using 2-bromoethyl

624


AcO

(i) (H2N)2CS AcO O

O Br

(a)

HO

O

S

(ii) ClCH2CN

H2N

Lys (CH2)4 Protein

OH

(b)

HO

HO

MeO−

CN

OH NH

NaBH3CN

H2N

NH

O

S

Lys (CH2)4 Protein HO

Lys

NH

O

S

OMe

NH

(CH2)4

Protein

Lys (CH2)4 Protein

Tyr Protein

HO

O

O

HO

HNO2

NH2

Tyr

HO O

HO

+ N2

O

Protein

O

O

N N HO

(c) Lys

HO

O

(d)

HO

O

O

NH2

L-Glu-pNA

HO

OH

O

O

HO

pNA

O

O

O

O

N O

NC6H4NCS

O

NH NH

(CH2)4

Protein

O HO

N

O

O

Lys (CH2)4 Protein

H2N

Lys

S

HO

H2N

O

HO

R = OMe HNO2 R = NHNH2

(CH2)8COR

O

(i) BOP, imid (ii) Pd, H2 (iii) as for (d)

N2H4

(CH2)4 Protein

NCS

O

H N

O

HO

H2N O

O

HO

(e)

HO

(Im)2CS

Lys

S NC6H4NH

NH


O

(CH2)8CON3

O

(CH2)8 NH

(CH2)4

Protein

Lys (CH2)4

Protein

O

(f) Lys

O HO

O

(i) Hal oxidation (ii) NaOH (aq.) HO OH

(g)

O

OH O−Na+

Cl

H2N

(CH2)3

Lys

Protein

HO

OH

NH

(CH2)4

Protein

Et3N

O

O Asp, Glu (CH2)1,2

HO HO

H2N

(h) O

(i)

HO

O

NH2(CH2)2NH2 EtO R = OMe O R = NH(CH2)2NH2 (CH2)8COR O

O (j)

O

H2N

Lys (CH2)4

N Protein

O

O N O O

O

OEt

O

−

O

O

Asp, Glu OH (CH2)1,2 Protein

EDC

OH

O


O

O

O

(CH2)8 NH(CH ) NH 2 2

O Lys (CH2)4 NH

Protein

O

H N

N

HO

H2N

EtOH

SO3 O

O

H2N

Protein

Protein

(CH2)4 Lys

HO

O

H N

O O

N HO

O O

Scheme 10 Current methodologies developed for indiscriminate protein glycosylation.

O

O

Protein

(CH2)4 Lys


625

glycosides with short peptide sequences containing cysteine or homocysteine and no basic residues allowed some selectivity for S-alkylation.233 A novel high-temperature ‘baking’ method has been described for the modification of proteins that are lyophilized with reducing oligosaccharides.234 Remarkably, despite being heated with the reducing sugar in air at 95–120 C for up to 40 min both trypsin and an IgG antibody survived with minimal loss of biological activity. The results of tryptic digests and conjugate hydrolyses suggest that the mechanism of conjugation involves an Amadori rearrangement with protein lysines which destroys the integrity of the reducing end residue. Pozsgay and co-workers have recently described the use of Diels–Alder chemistry to glycosylate human serum albumin with a variety of sugar-based dienes (Scheme 10j).235 It should be noted that the installation of electron-deficient double bonds as dienophiles required initial chemical modification on the protein with 3-sulfosuccinimidyl 4-maleimidobutyrate linkers, and so sacrificed potential chemoselectivity. Although high levels of functionalization are thus easily accessible using the above methods, a lack of residue selectivity is often a drawback in the synthesis of well-defined conjugates. In addition, these techniques may alter the isoelectric point of the protein and/or destroy the cyclic nature of glycans introduced.

1.15.3.2 Chemoselective and Site-Specific Glycosylation In an attempt to increase the selectivity and predictability of protein glycosylation, various novel approaches have been described, all of which exploit the chemoselectivities of various enzymatic and traditional methods. Bertozzi and co-workers have described the use of galactose oxidase to selectively introduce an aldehyde tag to the C-6 of a GalNAc residue in the antimicrobial 19-residue peptide drosocin (Scheme 11a).236,237 This aldehyde tag was then selectivity conjugated with aminooxy glycosides to introduce further oligosaccharides via the formation of an oxime, in a manner previously demonstrated for the conjugation of spacer-arm hydrazides with cell surface aldehydes.238 The resulting non-native glycopeptide showed comparable biological activity to the native form illustrating that certain unnatural linkages can in certain circumstances be tolerated. A similar chemoselective ligation approach has been applied to various glycopeptide syntheses (see Section 1.15.2.3).156–158,160 These approaches still require the linear construction of an initial glycopeptide and therefore suffer from the same disadvantages of protection and lability outlined in Section 1.15.2. However, they do hold the advantage that its application may be coupled with other methods for the introduction of a ketone tag (see below and Section 1.15.3.3). In addition, the ready synthesis of hydrazides, thiosemicarbazides, and aminooxyglycosides of lactose, Gal, GalNAc, GlcNAc, and sLex allowed their attachment to a ketone-containing 19-aa peptide.239 Flitsch’s iodoacetamide methodology (see below)240 has been cleverly adapted by Bertozzi and co-workers to convergently ligate Glc- and Galiodoacetamides to a C-3 sugar thiol in a GalNAc(a1-O)Thr peptide.241 The required thiol-protected glycosylated amino acid was synthesized via SPPS to give a glycosylated 17-aa glycopeptide that corresponds to the N-terminal region of P-selectin glycoprotein (PSGL-1). Deprotection of the thiol and treatment with Glc- and Gal-iodoacetamide allowed the synthesis of a Glc/Gal(b1-3)GalNAc(a1-O)Thr mimetic. It should be noted that no selectivity was observed if a cysteine was present in the peptide backbone. Furthermore, the alkylation of C-2 modified terminal mannoses of the N-linked core pentasaccharide with bromoacetamido trisaccharides allowed the preparation of a biantennary N-linked glycopeptide of CD52.242 In a strategic sense, although chemoselective, these approaches have avoided the key issue in glycoprotein synthesis – the formation of the carbohydrate–protein link – by relying on the presence of an existing glycan in the peptide/protein structure or an artificially introduced reactive group as a tag for reaction. Similarly, the elegant enzymatic methods of Wong and co-workers, Takegawa and co-workers, and Wang and co-workers described in Section 1.15.4.3 still require that a protein–N-glycan link be present from the start.176,190,243–248 As these latter methods alter one glycan structure for another, they are therefore better described as glycoprotein remodeling (GPR) and they offer the glycoscientist no general choice over the site of glycosylation. Several methods have been proposed that tackle this central issue by exploiting specificity for functionality already found in proteins. Among the first approaches was that of Flitsch and co-workers, who reacted the a-iodoacetamide of N-acetyl-D-glucosamine with bovine serum albumin (BSA) to modify the single free cysteine present (Scheme 11b).240 Later this method was applied by Wong and co-workers to introduce chitotriose and a heptasaccharide stripped from the surface of horseradish peroxidase to BSA.249 Boons and co-workers have used either dithiopyridyl methodology to make disulfide-linked BSA-N-acetyl-D-glucosamine constructs (Scheme 11c) or aerial oxidation and disulfide exchange to form a variety of glycopeptides/proteins (Scheme 11d).250–252 More recently,

626


Protein

O HO

HO

R

O

X

NH2 X = O or N

O

Protein

X N

O e.g. HO

R O

HO

AcHN O

(a)

Cys

HO HO

O

N3

PtO2, H2

HO

O

I

HO

O

NH2

Protein

HS

H N

O

EEDQ

(b) S HO

O

O HO

2

Cys

H N

O

Protein

S O

Cys

NO2 N

SH

HO

I

Protein

O

HS S S

HO

NO2

Cys O

N

(c)

Protein

S S

Cys HO (d)

O

O (i) [P]O

O

N3

Pd, H2

[P]O

O

HO

SH

Cys O

HO

O

Protein

S S

Cys

O

Protein

HS

N

(ii) deprotect

(e)

O

O

then HMDS, ZnCl2 NH2

Protein

HS

O HO

O

Cys Protein

S

N

O

O Cys

[P]O

(f)

O

Y

NaSSO2R [P]O R = Me or Ph

Y = Br or O(CH2)2Br [P] = Ac or H HO

O

SH

Protein

O HS X S R O X = S or O(CH2)2S

HO

O

Cys O

Protein

X S

Cys PhSeBr

HO

Protein

O

HS S Se Ph

(g)

HO

Cys O

Protein

S S

Scheme 11 Methods developed for site specific protein glycosylation.

glycosylmaleimides have also been developed as cysteine-reactive glycosylation reagents for the direct functionalization of a cysteinyl-containing 11-aa peptide and BSA (Scheme 11e),163 a method that is similar in concept to earlier spacer-arm maleimide-terminated reagents.220

1.15.3.3 Site-Selective Glycosylation For full control of glycosylation, both choice of site (site-selectivity) and glycan are needed. A combined site-directed mutagenesis and chemical modification approach has solved this fundamental problem.16,105,112,253 This two-step approach provides a general method that allows both regio- and glycan-specific glycosylation of proteins (Scheme 12). The strategy involves the introduction of cysteine, via mutagenesis, as a chemoselective tag to preselected positions within a given protein. The free thiol is subsequently modified with thiol-specific carbohydrate reagents (Scheme 11b–f), such as glycosyl methanethiosulfonates, GlycoMTS,254,255 or, more recently, glycosyl phenylthiosulfonates, GlycoPTS (Scheme 11f ).256 Four sites on the representative serine protease subtilisin Bacillus lentus (SBL), which does not naturally contain cysteine, at different locations and of different characteristics were selected for mutation to cysteine in order to provide a broad test of the glycosylation methodology. Wide applicability with respect to the sugar moiety was evaluated by using a representative library of tethered or untethered, protected or deprotected, mono- and disaccharide methanethiosulfonates. The homogeneous glycoproteins formed allowed the first systematic determinations of the properties of novel glycoforms, thus providing detailed glycan structure–hydrolytic activity relationships for a library of 48 glycosylated forms of SBL to be determined.253 These glycosylated enzymes displayed enhanced utility as


HO

O X S R or O HO O H N

O

Cys Site-directed mutagenesis Protein

Protein

HS

HO

627

I

O Cys O

Protein

X S

X = NHC(O)CH2 or S or O(CH2)2S R = Me or Ph

Scheme 12 The pioneering strategies in site-selective protein glycosylation.

catalysts in peptide synthesis and allowed syntheses of dipeptides that were not possible using the unglycosylated catalyst.110,111,257 Subsequently, MTS reagents, including glyco-MTS, have been employed to tether a variety of biological ligands onto SBL that successfully target it to receptor proteins, increasing selectivity to over 350-fold.258 Initially, the appropriate ligand acts as a homing device to bind the target protein which is then catalytically degraded by the attached serine protease (SBL), thereby producing enzymes that selectively destroy protein function. More recently, the glycoMTS method has also allowed the synthesis of the first examples of a homogeneous protein bearing symmetrically branched multivalent glycans in which both the site of glycosylation and the structure of the glycan introduced has been predetermined.259 This new class of glycoconjugate, the glycodendriprotein, allows for increased protease localization and was demonstrated by a high level of co-aggregation inhibition between the pathogen Actinomyces naeslundii and co-pathogen Streptococcus oralis (Scheme 13).260 A recent addition to cysteine site-selective protein glycosylation strategy includes the use of selenenylsulfidemediated glycosylation (GlycoSeS) (Scheme 11g). In this approach, site-selective glycoconjugation is accomplished between cysteine-containing proteins and glycosyl thiols via phenyl selenenylsulfide intermediates. Pre-activation of either the cysteine mutant protein or thiosugar is possible following exposure to PhSeBr (Scheme 14). This procedure was demonstrated on simple cysteine-containing peptides and was shown to be successful on a variety of different proteins highlighting the compatibility of the strategy with a variety of protein thiol environments. This high-yielding procedure also demonstrated the first example of multi-site-selective glycosylation, the coupling of a heptasaccharide (the largest to date), and the first enzymatic extension of a disulfide-linked glycoprotein. Furthermore, a novel thionation reaction of unprotected reducing sugars allows the direct synthesis of glycosyl thiols,262 which when combined with Glyco-SeS allows for a direct one-pot protein glycosylation method. An impressive extension by Flitsch and co-workers of this mutagenesis-modification approach has allowed the glycosylation of erythropoietin (EPO) using glycosyl iodoacetamides (Scheme 11b).263 Several cysteine mutations were introduced at natural N-glycan glycosylation sites N24C, N38C, and N83C, where it is known that carbohydrate motifs are critical to the function of the mammalian hormone. Although requiring forcing conditions, 500 equiv. of sugar reagent, to obtain partial glycosylation (>60% on N38C, 30% on N24C and N83C as determined by proteolytic digest), the glycosylated EPO was purified using lectin affinity column chromatography to give glycoprotein. However, under these forcing conditions additional nonspecific glycosylation of histidine residues, probably in the His10-tag, was also observed thereby indicating that such glycosyliodoacetamides may not be absolutely selective; this lack of selectivity was circumvented by carrying out the modification in the presence of excess imidazole. It was possible to confirm glycosylation of N83C unambiguously by proteolytic digest and it should be noted that, as in the case of BSA, the three disulfide bonds in these EPO mutants were untouched by glycosylation of the single free cysteine. The use of iodoacetamides has been further extended by Alleman and co-workers to include the siteselective modification of dihydofolate reductase (DHFR), where mono-glycosylated forms of DHFR showed increased stability to thermolytic denaturation.264 The Cys120 and Cys87 mutant of DHFR, when exposed to glycosyliodoacetamides at pH 9, resulted in the uncontrolled glycosylation of lysine residues in addition to cysteines. Fortunately, this problem could be circumvented at pH 7, giving a mixture of mono-glycosylated and unmodified protein. Purification of this mixture was accomplished using biotinylated thiosulfonates,265 to scavenge the unmodified DHFR cysteine mutant, followed by avidin-based affinity chromatography to yield pure glycoproteins, using a procedure depicted previously.258 More recently, Withers and co-workers have employed glycosyl-iodoacetamides in conjunction with glycosynthases to chemically glycosylate and then enzymatically extend glycosylation on proteins.266

628

H N

Cl N

NH2 a, b

H2N

O

N

NHBoc

Gal-S C

NH

Cl

CH3O2SS(CH2)4C(O)HN

Gal = HO N

NHR

HO

OH N H

R = Boc d,e d,f R = C(O)(CH2)3SH 1 H g,h R = C(O)(CH2)4SSO2CH3 N Gal-S H O N N H O N NHBoc S NH N Gal-S NHR O N O H N

Gal-S O

N

H N O

NH

Gal-S O

H N

Gal-S O

N

SS

H N O

NH

Gal-S

NH Gal-S O R = Boc d,f R = C(O)(CH2)4SSO2CH3 H SS N NHR O N NH O R = Boc R = C(O)(CH2)4SSO2CH3

d,f

N H S

Protease

Glycodendrimer

OH O

NH O

CH3O2SS(CH2)4C(O)HN h

O

Gal-S

O

a, k

H N


H2N

+ −SO

2Me

N

Synthetic glycoprotein protease

N H O

(CH2)n

Boc = ButOC(O)-

Pathogen A. naeslundii

NHR-butyl-MTS = O O SSO2CH3 N O (CH2)4 O

Degradation

Control

Inhibition of aggregation

Reagents and conditions: a, Boc2O, DCM, −78 ⬚C, b, (CICH2CO)2O, DCM; c, 2 equiv. Gal−S−Na+, DMF; d, CF3COOH DCM; e, thiobutyrolactone, dithiothreitol, NaHCO , water, EtOH; NHS−butyl−MTS, DMF; 3 g, 1 equiv. Gal−S−Na+, DMF; h, 1, DMF

O

Scheme 13 The synthesis of glycodendriproteins and the resulting inhibition between oral pathogens Actinomyces naeslundii and Streptococcus oralis. Reproduced with permission of Chemistry a European Journal 2006, 12, 656–665 by Wiley.


2.

PhSe–S

HO HO

1. PhSeBr

HS

SH NHAc

OH O

HO HO

HO HO

OH O

S−S NHAc

HO OH O HO HO OUDP HO OH O O HO Gal-T HO HO

629

OH O

S−S NHAc

OH O

SH NHAc

2.

1. PhSeBr

HS

OH O

HO HO

S−SePh NHAc

Scheme 14 Selenenyl sulfide mediated glycosylation and subsequent enzymatic extension. Reproduced with permission of Chemical Reviews 2002, 102(2), 579–601 by American Chemical Society. N297C

S

HO HO HO

Fc S

Fc N297C

OH O

+ OH O O HO

O HO O O OH

HO HO HO

0.25 M NH4OAc, pH 8.5 OH OH O O O SH HO NHAc NHAc

OH O OH O O HO

O HO O O OH

OH O O HO NHAc

OH O

N297C

Fc

S S NHAc

OH OH OH

OH OH OH

Scheme 15 Disulfide exchange leading to glycosylated fragments of IgG1.

Boons and co-authors have produced homogeneous disulfide-linked glycoproteins through a cysteine mutation into the Fc region of immunoglobin G (IgG1). Well-defined glycoforms of IgG1-Fc were prepared by disulfide exchange between thiol containing sugars and oxidized protein in yields of up to 60% using 50 equiv. of carbohydrate (Scheme 15).251 Unfortunately, previous dithiopyridyl methodology (Scheme 11c) was not compatible with disaccharides and larger carbohydrates.250 Importantly, proteolytic digest showed that the procedure had not disrupted the internal cystine disulfide bridges and that the carbohydrate motif was located at N297C. The glycosylated antibodies were subsequently screened for their ability to inhibit the JY cell superoxide burst achieved when FcgRI receptors, found on U937 leukocytes, interact with Fc receptors on JY cells. As expected, the naturally glycosylated Fc fragment completely inhibits this process, while the aglycosylated showed minimal activity, reiterating the importance of the carbohydrate motif. Interestingly, the artificial glycoconjugates displayed partial superoxide inhibition, indicating that protein activity can be restored, or more importantly generated, by the in vitro attachment of oligosaccharides. In addition to controlled cysteine modification, selective lysine modification has also been achieved. For example, the use of para-nitrophenylester-linked thiogalactoside allowed an interesting site-selective glycosylation in 60% yield of one lysine among four potential lysines in a designed helix–loop–helix 42-aa polypeptide. The selectivity of the amide formation was confirmed by peptide digest mapping, and was achieved by fine-tuning reaction conditions. Initially, a histidinyl-11-ester intermediate is formed followed by intramolecular acyl transfer to the iþ4 lysine-15 to compete successfully with direct background amidations with all lysines (the rates are such that under these optimal conditions 94% of amidation should be intramolecular).267 Some conformational effects of this glycosylation have also been noted.77 While these strategies have focused on site-selective glycosylation of naturally occurring amino acids, Schultz and co-workers have employed triplet amber codon suppression methodology to introduce unnatural ketone ‘handles’ (p-acetyl-L-phenylalanine) into the Z domain of staphylococcal protein A.268,269 Successful in vivo incorporation of

630


O

OH

O

O

OH HO AcHN HO

OH

OH HO O O O HO AcHN OH OH

N O

(i) GalT, UDP-Gal (ii) SiaT, CMP-Sia

O O E.coli evolved MjTyrRS

HO HO HO

O AcHN

O NH2

HO HO HO

In vivo expression

O O

N

AcHN

CO2H H2N Unnatural amino acid

Scheme 16 A rare example of electrophilic site-selective protein glycosylation.

unnatural amino acids has been accomplished by the development of a tRNA synthetase–tRNA pair from Methanococcus jannaschii which is able to introduce unnatural amino acids at amber-suppressed codon sites. The incorporated nonnatural electrophilic amino acid was then targeted by an N-acetyl glucosamine aminooxy saccharide,270 developed by Bertozzi and co-workers (Scheme 16, see Section 1.15.3.2),236 to form glycoproteins in excellent yield following HPLC purification. Furthermore, the corresponding glycoconjugate was subsequently extended with the wellestablished transformation sequence of galactosyltransferase and sialyltransferase to form sialylLacNAc, showing that the nonnatural oxime linkage tolerates a variety of enzymatic extension conditions.

1.15.4 Enzymatic Glycoprotein and Glycopeptide Synthesis The two distinct sets of strategies outlined in the two previous sections are equally applicable to enzyme-catalyzed techniques.

1.15.4.1 Elaboration of Glycans Enzyme-catalyzed techniques for elaboration of existing glycans on glyco-amino acids/peptides/proteins have proved particularly successful. One of the earliest examples of the use of glycosyltransferases in glycoprotein synthesis was reported by Paulson and co-workers who employed a sialyltransferase and CMP-N-acetylneuraminic acid to restore 95% of the sialic acids to a fully desialylated protein.271 Bertozzi and co-workers have impressively employed enzymatic approaches to directly modify cell surface proteins.272 The use of a milk fucosyltransferase, that displays a broad substrate specificity, allowed modification of glycans with fucosyl residues bearing a range of substituents at C-6. More recently, Ito and co-workers have employed a glucosyltransferase and UDP-Glc in the monoglucosylation of a chemically synthesized Man9GlcNAc2-containing glycopeptide.273 Glycosyltransferases are also often used to elaborate glycopeptide structures either prior to or after linear assembly (see Section 1.15.2.2.3). Indeed, highly glycosylated glycopeptide building blocks, such as 7, for use in linear glycopeptide assembly were first made accessible through the use of glycosyltransferases.195,196 Much of this work has strong resonance with the use of glycosyltransferases in oligosaccharide synthesis.43,274–279,280–282 Glycosyltransferase-mediated elaboration as a chemoselective technique is particularly useful for challenging structures rich in functionality such as sulfopeptides,197 although strategic considerations are important with regard to the often stringent substrate specificity of glycosyltransferases. The presence of sulfation in a PSGL-1 octapeptide made this a particularly challenging glycopeptide to elaborate, and alternative glycosyltransferases, more tolerant than those used previously, had to be identified. It should be noted that enzyme availability is sometimes a stumbling block


631

in such techniques; in particular, the number of readily available branching GlcNAc-transferases is particularly limited. In an excellent display of the power of glycosyltransferases in glycopeptide elaboration (Scheme 6), the sulfated N-terminal domain of PSGL-1 has been synthesized on a small scale and characterized by HPLC and MS in two glycoforms that crucially differ in their binding to P-selectin as a result of only a subtle alteration of internal glycan structure.198 This was achieved by the isolation of the required six glycosyltransferases and one sulfotransferase and allowed 9 and 10 to be synthesized from a linearly SPPS-assembled 23-aa monosaccharide-bearing glycopeptide. In an early use of enzymatic glycosylation on the solid phase, the sLex-Asn-Phe dipeptide was synthesized using aminopropyl silica as a support and subsequently cleaved from a glycine linker by the peptidase chymotrypsin.283 In this context, it is interesting to note that the ability of glycosyltransferases to modify glycans on glycopeptides may be greatly influenced by the peptide backbone and sites that are already glycosylated.284 For example, prolines, negatively charged residues, and the presence of a disaccharide all inhibited the glycosylation of an adjacent GlcNAc-Thr residue. Enzymatic extensions have also been performed on a water soluble polymer producing a sialyl-Lewisx-modified 12- and 11-mer.285,286 Initially, the glycopeptide is constructed through standard SPPS to include a keto-containing ‘transporter group’ via a modified C-terminal proline residue. The glycopeptide is then released from the resin and trapped onto a water-soluble polyacrylamide derivative through chemoselective ligation. Subsequent enzymatic extension, followed by photolytic cleavage of the transporter moiety, furnished the glycopeptide in 12% overall yield requiring minimal purification.

1.15.4.2 Trimming of Glycans It is also possible to purify mixtures of glycoforms through selective enzymatic degradation of unwanted glycoforms.287 Endoglycosidase-mediated trimming of glycan strutures plays a critical role in the initial step of ‘glycoprotein remodeling’ (see Section 1.15.4.3). The glycosylation of antibodies affects activity and function288 and their manipulation often provides some good case studies in glycoprotein synthesis methods. For example, terminal deglycosylation of human IgGs with b-galactosidase exposed GlcNAc residues and thereby introduced interactions with MBP.289 An in vitro enzyme cycle that ‘trims’, using a specific a-glucosidase, and ‘adds’, using a glucosyltransferase, a Glc(a1-3) unit to the tip of the glucosylated branch in the initial N-linked glycoprotein glycan core has been created. 290 This in vitro system elegantly mimics an in vivo cycle that plays a critical role in binding to lectin chaperones, calnexin and calreticulin, during folding in the endoplasmic reticulum (ER).8

1.15.4.3 Alteration of Glycans (Glycoprotein Remodeling) The combined trimming (typically enzymatic) of existing glycan structures followed by elaboration to alternative ones has become known as ‘glycoprotein remodeling’ (GPR). Central to this technique, as a result of the difficulty in making natural glycan-protein links, at least one glycan must remain to serve as a tag for specific elaboration. For example, a synthesis of a single unnatural glycoform of ribonuclease B (RNase B) 16 was achived by Endo-H degradation down to a single N-linked GlcNAc motif followed by elaboration with the well-established sequential system of galactosyltransferase, fucosyltransferase, sialyltransferase to construct a sLex glycoform (Scheme 17).291 Takegawa and co-workers have applied endoglycosidase-mediated transglycosylation (the synthetic utility of which was originally demonstrated using Endo-F and Endo-H as early as 1986)292 to the same partially deglycosylated RNase B 15 in the synthesis of the Man6GlcNAc2 glycoprotein 17.293 EndoA-catalyzed transglycosylation has also allowed the addition of decasaccharide Man9GlcNAc from naturally derived glyco aminoacid Man9GlcNAc2-Asn onto a chemically synthesized native N-linked GlcNAc-Asn containing pentapeptide,294 and a non-native C-linked analog244 in yields of 25–26%.243 This transglycosylation activity of endoA is usefully improved by the use of partially organic solvent systems, such as 35% aqueous acetone.295,296 Similar yields are also reported by Wang and co-workers in the Endo-A catalyzed transglycosylation of a variety of high-mannose motifs to a similar peptide backbone.176,245,247 Interestingly, all studied donors showed comparable reactivity with Endo-A highlighting the broad specificity of this endoglycosidase towards high-mannose motifs. This work has later been extended to include a 34-mer, representing a section of the HIV-1 glycoprotein envelope gp41. Such EndoA transglycosylation has also been cleverly combined with the chemical glycoprotein synthesis techniques.297 For example, transglycosylation using Man9GlcNAc2-Asn as a donor onto p-isothiocyanatophenyl-b-D-Glc as an acceptor gave a high-mannose isothiocyanate Man9GlcNAcGlc reagent that was used to indiscriminately glycosylate lysines of RNase A, lysozyme, and a-lactalbumin. Endo-M endoglycosidase shows a moderate, broad substrate specificity toward a variety of oligosaccharides in transfers to GlcNAc-bearing peptides (up to pentapeptides), but only in yields of up to 20%.298,299 Complex-type

632


Asn34 O

OH HO HO

O

NH NHAc

Subtilisin 8397

OH HO HO

(i) GalT, UDP-Gal (ii) FucT-V, GDP-Fuc (iii) SiaIT, CMP-Sial

O

O

NH NHAc 15

Endo A, Man6GlcNAc2

O

O sLex

Asn

Man6GlcNAc2

NH 16

NH 17

Scheme 17 Glycopeptide ligation combined with enzymatic remodelling offers avenues to glycoprotein synthesis.

glycans were transferred more efficiently than high-mannose type. It should be noted that synthetic yields are inversely related to ability to hydrolyze indicating that post-transglycosylation hydrolysis of a product may be an important factor in determining overall yield. Endo-M also catalyzes the transfer of (NeuAcGalGlcNAcMan)2 ManGlcNAc to an octapeptide bearing a single GlcNAc in HPLC-determined yields of approximately 10%300 and allows the synthesis of two different glycoforms of substance P, an undecapeptide,301 all formed using the same combined SPPS and transglycosylation strategy. The use of a dimethylphosphinothioic anhydride coupling Mpt-MA (which avoided the need for protection of the glycan) and Ag-catalyzed condensation of an N-terminal fragment thioester with a C-terminal portion (which required only side chain Lys and Cys protection) was coupled with Endo-M catalyzed transglycosylation to readily yield a C-terminal sequence of eel calcitonin in a non-natural glycoform that bears a disialo biantennary-type undecasaccharide: CS[(NeuAc-Gal-GlcNAc-Man)2Man-GlcNAc-GlcNAc-] NLSTCVLGKSQELHKLQTYPRTDVGAGTP-NH2.302 A recent example by Takegawa and co-workers demonstrates the combined use of Endo-M and Endo-A in the successful transglycosylation of a 32-mer peptide. N-glycosylation was demonstrated at two sites with both high-mannose and complex glycans, representing the first example of a differential multiple glycosylation to a glycopeptide using endoglycosidases.303 This procedure relies on the efficiency (yields up to 38%) of Endo-M to transglycosylate complex glycans and the specificity of Endo-A toward high-mannose moieties which prevents hydrolysis of the initial complex. More recently, Wang and co-workers have produced a 47-mer glycopeptide through an Endo-A catalyzed glycosylation in yields of up to an impressive 86%.176,304 This strategy simultaneously added two N-glycan motifs to HIV-1 V3 domain glycopeptides via novel oligosaccharide oxazoline donor substrates (Scheme 18). The key to this work has been the ingenious use of oxazolines which act as good substrates for glycosylation, but the fully incorporated motif is a poor substrate for hydrolysis, thus minimizing the degradation observed with transglycosylation. A similar result has been reported by Fairbanks and co-workers.305


HO HO HO

O

H N

633

O

AcHN Ac-W-M-E-W-D-R-E-I-N-N-Y-T-S-L-I-H-S-L-I-E-E-S-Q-N-Q-Q-E-K-N-E-Q-E-L-L-NH2

Endo-A 75%

HO HO HO

HO HO HO

O OH

OH O

HO O

AcHN O OH

HO O HO

O

OH O O HO O

HO HO HO

OH O O HO O

HO HO HO

H N

OH O

OH O HO

O N O

O OH

O

AcHN

Ac-W-M-E-W-D-R-E-I-N-N-Y-T-S-L-I-H-S-L-I-E-E-S-Q-N-Q-Q-E-K-N-E-Q-E-L-L-NH2

Scheme 18 The use of oxazolines and Endo-A in the formation of glycopeptides.

Nature employs a similar ‘remodeling’strategy in the case of Trypanosoma cruzi, the protozoan responsible for Chagas’ disease. The parasite does not synthesize sialic acid, but instead expresses a transsialidase that catalyzes the transfer of sialic acid from glycoconjugates found in the host to its own surface proteins. The resulting sialylated glycoproteins are then bound by host sialic acid-binding receptors, thereby allowing cellular invasion.306 This observation has been adapted by Contreras and co-workers through the engineering of the yeast cells of Saccharomyces cerevisiae, to express cell surface-exposed Trypanosoma cruzi trans-sialidase (TS) via fusion to Aga2.307 The ‘biocatalyst’ demonstrated the conversion of non-sialylated synthetic oligosaccharides to mono- and disialylated variants through the exposure of acceptors with suitable donors; furthermore, the reaction could be driven to completion using a large excess of the 30 -sialyllactose donor. Although only demonstrated on oligosaccharides, this procedure offers a potentially powerful tool when combined with other biological augmentation pathways for the synthesis of homogeneous glycoproteins.

1.15.4.4 Enzymatic Formation of the Glycan–Protein/Peptide Link An attractive approach to enzymatic glycoprotein synthesis is to exploit the biomachinery responsible for the formation of the sugar–protein link in N-linked glycoprotein biosynthesis.308 The enzyme responsible, oligosaccharyltransferase (OST), co-translationally transfers a high-mannose core oligosaccharide from a fatty acid pyrophosphate carrier to the side chain amide of an asparagine (Asn) residue in the consensus sequence Asn-X-Thr/Ser of the nascent glycoprotein; although rare, other sequences have been reported (e.g., Asn-Ala-Cys).309 The use of this enzyme in isolated form in in vitro glycoprotein synthesis has, however, been met with limited success. While transfer of carbohydrates to a 17-residue peptide containing an unusual Asn-Asn-Thr-Ser sequence was possible, direct glycan transfer to RNase A failed.310 In addition, transfer to sequences in which X¼Pro are not possible and those in which X¼Trp, Asp, Glu, Leu are inefficient.311 In a more recent example, Imperiali and co-workers have obtained glycopeptides using PglB, part of the oligosaccharyl transferase complex from Campylobacter jejuni.312 This enzyme was able to transfer unusual bacterial disaccharides from prenylated donors onto the heptapeptide KDFNVSK and an octapeptide, being a segment from known glycoproteins of C. jejuni. Investigations into the peptide specificity of PglB highlighted the requirement of amino acid determinants beyond the sequon of N-X-S/T. Other enzymes have also been explored, for example, an endo-galactosaminidase has been used to transfer Gal (b1-3)GalNAc(a1- to the side-chain hydroxyl of a serinyl residue in a hexapeptide.313 Furthermore, the use of a microbial transglutaminase (TGase) to transamidate the side-chain g-carboxamide group in the dipeptide Z-Gln-Gly with -O(CH2)3S(CH2)2NH2 glycosides (shorter spacers were unsuccessful) to form an unnaturally N-linked glycopeptide has been described.314,315 Such transglutamination has been further developed by Nishimura and co-workers in combination with enzymatic elongation in the production of mono-, di-, and tri-antennary glycopeptides.316

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Impressively, this work has been combined with site-directed mutagenesis in the first fully enzymatic method for the site-selective synthesis of homogeneous glycoproteins. Mutagenesis was employed to introduce glutamine into the Nterminus of the B chain of insulin which subsequently underwent a TGase-catalyzed transamidation with aminecontaining oligosaccharides (Scheme 19).281 Disappointingly, after the TGase reaction proteolytic digestion revealed a lack of selectivity: both Q1 and Q4 residues were modified. Fortunately, both isomers could be separated by HPLC and extended with a2,3-SiaT producing two different sialyllactose-modified insulins. Furthermore, this procedure was also developed to include dendrimeric displays of sialic acid, thus creating glycodendriproteins.282 All modified glycoproteins demonstrated a lower binding affinity to the insulin receptor; however, overall in vivo activity was increased due to the incorporation of sialic acid, with the glycodendriproteins demonstrating significantly enhanced half-life caused by the higher degree of sialic acid incorporation.

1.15.4.5 Glycopeptide Ligation Subtilisin peptidases have been elegantly used to catalyze the synthesis of glycopeptides,180,181 in spite of the fact that the natural specificity of these enzymes has limited these peptide ligations to those in which the glycosylated residues 0 0 are typically at least one residue distant (P2,P3. . . or P2, P3 . . .) from the amide bond formed. Thus, while ligation of ZGly-OBz with H-Gly-[GlcNAcAc3(b1-O)]Ser-NH2 was successful, no yield of product was obtained with H[GlcNAcAc3(b1-O)]Ser-NH2. The use of this ligation method coupled with other enzyme-mediated strategies culminated in a truly elegant synthesis of a single unnatural glycoform of ribonuclease B (RNase B) 16 using a protease-catalyzed ligation of fragments of the protein backbone, including a fragment bearing a single GlcNAc(b1-N) Asn, followed by glycosyltransferase-catalyzed elaboration reactions of that glycan (Scheme 17).291 The limitations of the natural specificity of the subtilisins in this regard have also been overcome by the logical redesign through siteselective chemical modification.111 H2N

Mutation site that differs from WT

B-1

O A-1 Q

Q Q

Q

OH OH HO AcHN

a. Transglutaminase

COOH O

HO

HO

HO O

HO

OH O OH

HO

O O OH HO

OH O OH

O HO

OH O

O

NH2

OH

b. a-2,6-Sialyltransferase O

HO HO AcHN

HN

B-1 Q Q Q

OH

HO

O NH

−

O O P O O O COOH HO

O

N

O

OH

O A-1

Q

Scheme 19 TGase and a2,6-SiaT allow for the preparation of insulin demonstrating increased in vivo activity.


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1.15.5 Molecular and Cell Biological Techniques In vivo methods, that alter the natural machinery of glycosylation, offer promising opportunities.317–319 Although, prokaryotic, for example, bacterial systems, do not typically glycosylate proteins, the use of eukaryotic systems can circumvent this problem. Unfortunately, as yet, this leads to heterogeneous products, although excitingly homogeneity is almost within reach.320–323 This daunting task is made difficult by the large array of biosynthetic glycosylation products and thus the corresponding array of pathways that need to be controlled or adapted.324 These pathways, the levels of enzymes that drive them and their activities, differ subtly according to species, cell-type, and protein. By expressing a particular glycoprotein in one organism, different glycosylation patterns may arise to those found by expression of the same enzyme in another. In this way, glycosylation patterns may be guided in a particular direction. Thus, expression in, for example, plants325 or mice326 may allow the production of patterns similar but subtly altered compared to those in mammalian systems. The use of yeast to study the N-linked biosynthetic pathway and associated congenital diseases has been reviewed.53,321

1.15.5.1 Biosynthesis Augmentation Broad changes in glycosylation patterns can be achieved through ‘glycosylation engineering’ that regulates levels of the glycosyltransferases involved in post-translational elaboration of protein-linked glycans. For example, cell lines or cultures in which extra glycosyltransferase-expressing genes have been introduced may be used to enhance the presence of particular sugars in glycan structures. The addition of a sialyltransferase to a Chinese hamster ovary (CHO) cell line resulted in the increased ‘misglycosylation’ of N-linked glycoproteins to give glycan motifs bearing (a2-6) Gal-in replacement of the normal (a2-3)Gal-linked sialic acid terminated residues.327 Similarly, tetracycline-regulated expression of the GlcNAc-transferase involved in ‘bisecting’ (i.e., glycosylation of OH-4 of the b-Man residue in N-linked glycans) the core of N-linked glycans in CHO cells resulted in an increase in bisected glycoforms from 25% to 50%, thereby increasing cytotoxicity of the IgG.328 Transfection of CHO cells with GlcNAc-transferase and fucosyltransferase activity either using two separate plasmids vectors329 or one polycistronic vector330 allows the construction of sLex on PSGL-1. The use of one plasmid prevented disparate expression levels. A rare example involving the re-engineering of the glycosylation pathway in the yeast Pichia pastoris,321 to mirror the processing of human N-glycans, has resulted in the secretion of glycoproteins with complex N-glycosylation.322,323,331 The procedure required elimination of the ‘natural’ glycosylation pathway and the introduction and correct localization of mannosidase I and II, N-acetylglucosaminyltransferases I and II, and uridine 50 -diphosphate-N-acetylglucosamine transporter. Successful incorporation of these eukaryotic enzymes produced essentially homogeneous glycoproteins bearing the complex human N-glycan GlcNAc2Man3GlcNAc2. More recently, Wildt and co-workers have engineered strains of P. pastoris in a similar manner to synthesize complex glycans bearing GlcNAc2Man3 GlcNAc2. Furthermore, a fusion protein containing the catalytic domain of GalT, UDP-galactose-4-epimerase and a yeast leader sequence was anchored into the Golgi compartment. This synthetic fusion protein was able to mediate the epimerization of UDP-glucose to UDP-galactose, and catalyze the transfer of the resulting donor to terminal GlcNAc residues producing a bi-antennary galactosyl-tipped glycan. A similar strategy has also been employed in P. pastoris to provide a general platform for the production of recombinant monoclonal antibodies with human N-glycosylation patterns with high homogeneity.332 Further engineered glycosylation pathways have been comprehensively reviewed elsewhere.321 Baculovirus vectors allow introduction of mammalian glycosyltransferases into insect cell systems that normally produce N-linked glycoproteins with limited glycan diversity.333 For example, the introduction of human GlcNActransferase I allowed a high degree of further processing that is normally lacking in insect cells;334 furthermore, early expression of bovine b-1,4-galactosyltransferase gave galactosylated N-glycans.335 Plants have also proved suitable hosts: again additional b-1,4-galactosyltransferase activity is the key to altering plant N-glycosylation patterns toward those more like mammalian systems.336 Thus, expression of human b-1,4-galactosyltransferase in tobacco plants resulted in N-linked glycoproteins of which 15% bore terminal Gal(b1-4) residues. Interestingly, crossing of this transgenic plant with one expressing mouse antibody (‘plantibody’) allowed in planta glycosylation engineering to produce galactosylated antibodies. The prospects are also good for the glycosylation of larger biomolecular complexes by taking advantage of the oftenrelaxed specificities of biosynthetic pathways. Indeed, the use of an unnatural N-levulinoylmannosamine (Man–Lev) as a precursor in preference to natural precursor ManNAc can be achieved simply by feeding it to cells and this has allowed the introduction of a ketone tag into sialic acid residues found at cell surfaces.337 This allowed the selective introduction of further glycans through reaction with aminooxy and hydrazide-functionalized carbohydrates to form imines and hydrazones according to the manner described in Sections 1.15.2 and 1.15.3 (Scheme 11a).338 This

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strategy is similar to one previously applied to aldehydes introduced chemically to cell surfaces.238 In a similar manner, neural cell surfaces have been also engineered by introducing an unnatural N-propanoylneuraminic acid precursor and an N-thioacetyl.339,340 There is also good evidence that in certain cases the level of glycosylation is also influenced by levels of the intracellular nucleotide-mono/diphosphate donor substrates for glycosyltransferases that may in turn be increased simply by increasing the concentration of their biosynthetic precursors in the incubation media. Thus, increasing glucosamine concentration increased GlcNAc-UDP levels in CHO cells and hence levels of bisecting GlcNAc structures.341 The powerful Man–Lev technique described above has also been used to display biotin on cell surfaces.342 This ‘cell surface engineering’ utilized a biotinylated hydrazide to give cells that were far more readily transfected by adenovirus when pre-treated with an avidin-(anti-adenovirus antibody) conjugate. In addition, an aminooxyfunctionalized Eu3þ-complex MRI contrast reagent was constructed and localized through ligation.343 The use of peracetylated N-azidoacetylmannosamine as a precursor allowed the cell surface display of azido-sialic acids through uptake and intracellular hydrolysis of the acetate groups and the biosynthetic processing.344 Staudinger-type reaction with a biotinylated phosphine-ester results in an intramolecular amide formation and hence biotinylation of the cell surface. The advantage of this method is that azides and phosphines are extraneous to nature and are therefore unlikely to react in the absence of one another; therefore, this method can also allow intracellular ligation.345 A traceless variant which has extrudes the phosphine oxide formed has also been published to allow ‘clean’ amide formation.346 A further example follows a salvage pathway in CHO cells that allows the incorporation of a 2-keto analog of GalNAc into cell surfaces glycoprotein but not a 2-keto analog of GlcNAc.347

1.15.5.2 Biosynthesis Inhibition The inhibition of enzymes involved in the biosynthesis308 of glycoproteins offers an alternative way of controlling their structure. For example, the glycosyltransferase inhibitor tunicamycin inhibits the synthesis of the lipid-linked pyrophosphate oligosaccharide precursor that is used as a glycosyl donor in the formation of N-linked glycoproteins.348 The resulting lack of donor prevents formation of Asn-linked glycans and results in only O-glycosylated proteins. Less drastic inhibition of later trimming steps, which are mediated by glycosidases in the ER and Golgi, can be used to create smaller than natural ranges of Asn-linked glycoforms rather than none at all. For example, the use of the glucosidase inhibitor N-butyl deoxynojirimycin (NBDNJ) resulted in a reduction in the number of glycoforms of the HIV surface protein gp120 that were produced from more than 100 to 3.5 NBDNJ shuts down the early glucosidase triming in HIV glycoprotein biosynthesis and results in poorly processed N-glycans of gp120.349 Inhibition of trimming glycosidases also alters the function of IgGs by altering glycosylation pattern.350 Swainsonine, a branch trimming a-mannosidase II inhibitor, reduces the formation of the GlcNAc(b1-6) branch in N-linked glycans.351 Mutation of the gene that encodes for a-mannosidase II in mice leads to lupus-like automimmune disease thereby showing the importance of N-glycosylation.352

1.15.5.3 Non-Coded Methods In the late 1990s Hecht353,354 and Schmidt355 have both proposed the adoption of the in vitro use of misacylated tRNAs in non-sense codon suppression read-through techniques356 to exploit the natural mechanism of protein biosynthesis (translation). Unlike natural protein glycosylation, which occurs co- and post-translationally, this method requires the synthesis of AUCtRNA acylated with glycosylated-amino acids (Scheme 20). In this way Glc(b1-O)Ser was incorporated in place of Ser at position 286 of firefly luciferase.353,354 Schmidt and Wieland have also described the preparation of a hARF-protein altered to contain GlcNAcAc3(a1-O)Ser instead of a Lys in this way.355 To this end, Hecht and co-workers demonstrated the synthesis of tRNAs acylated with Gal,Glc,Man,GlcNAc-Ser, albeit in very low overall yields due to difficulties in achieving efficient acylation and deprotection steps.357 More recently these problems have been overcome by Schmidt and co-workers in a novel synthesis of aminoacylated dinucleotides which have been used in an efficient synthesis of dinucleotides loaded with protected glycosyl amino acids.358 Although they have low suppression efficiency, these glycosylated amino acids, including the Tn-antigen, have been incorporated into human granulocyte-colony stimulating factor through noncoded read-through techniques. Impressively, Schultz has taken this approach one step further through in vivo use of the M. jannaschii tRNA synthetase-tRNA system to allow the incorporation of the key glycosylamino acids, b-GlcNAc serine and a-GalNAc threonine, into a selection of proteins.359,360 Although these procedures are still in their infancy, they offer the scientific community a powerful tool for the synthesis of homogenous glycoproteins. Once a routine method, it will be interesting to see if glycoproteins made this way through ‘pre-translational glycosylation’ will differ from natural glycoproteins, which are co-/post-translationally


UAG AUC tRNA

NH2 OH O N O P O− O O N O C

HO O HO

OH O O P O− O O O O

H2 N

Stop codon

mRNA

NH2 O N − O P O O O N O C

637

HO

NH2 N

O HO

C C A 3⬘-O O

O

N

N

H2N

N A

OH

O

H N R

O

O

R⬘ N H

O

3⬘-O ACC

O

Nascent protein

Scheme 20 The use of misacylated tRNA in non-sense codon suppression in glycoprotein synthesis.

glycosylated, especially given the apparent role of glycosylation in correct protein folding. It is also interesting to see the further possibilities for combining read-through techniques with those of chemical glycoprotein synthesis. To this end, read-through techniques have also allowed the introduction of un-natural amino acids as tags for subsequent site-selective glycosylation (see Section 1.15.3.3).270

1.15.5.4 Expressed Protein Ligation Expressed protein ligation (Scheme 7a) has been used to create bacterially expressed peptides for use in NCL.361 For example, mannan-binding protein (MBP) was expressed in E. coli as a fusion to the N-terminus of a widely used intein from Saccharomyces cerevisiae; this intein also bears a chitin-binding domain at its C-terminus for purification. Once expressed, this portion self-spliced the binding domain, and the resulting peptidothioester was used in native chemical ligation (see Section 1.15.2.2.4) with glycosylated peptides such as Cys(b-GlcNAc-)Asn.207 Imperali and co-workers have developed a semisynthetic route toward the immunity protein Im7.362 In this procedure, a glycopeptide bearing an N-terminal Cys, obtained from standard SPPS, was coupled using NCL to a thioester-containing fragment, obtained from recombinant methods using the commercially available IMPACT system, a technique based on a pH-dependent intein cleavage. Bertozzi and co-workers have also used expressed protein ligation to construct two model non-natural glycoproteins:363 a 42-aa GlyCAM-1 central fragment, a stretch which is not glycosylated in full GlyCAM-1, was expressed as an intein–chitin binding domain fusion protein in bacterial culture and purified on chitin beads. This was released as a C-terminal thioester and then coupled with a 15-residue glycopeptide through native chemical ligation. Interestingly, a second unglycosylated 77-aa domain was also ligated to a polyglycosylated 56-mer, constructed from SPPS and NCL, in this manner. The final result was the preparation of three well-defined glycoforms of GlyC-1 with as many as 13 N-acetylgalactosamine residues present.364 More recently, Wong and co-workers have employed tobacco etch virus protease (TEV protease) cleavable fusion proteins to produce N-terminal cysteine-containing peptides.365 These peptides were then coupled to a synthetic thioester glycopeptide via NCL. N-terminal cysteines have also been produced using the more common Xa proteasecatalyzed cleavage through the introduction of a Ile-Glu-Gly-Arg (IEGR) cleavage site into a section of GlyC-1.364 N-terminal cysteine-containing peptides have also been cleanly produced chemically through cyanogen bromidemediated cleavage at methionine sites.366

Glossary armed and disarmed donors Terms first used by Prof. B. Fraser-Reid in the description of the relative reactivities of differently protected glycosyl donors; armed are more reactive typically by virtue of etheric protection (e.g., Bn). calnexin Also referred to as IP90, p88, and p90, is a 88kDa chaperone protein found in the endoplastmic reticulum having a similar role to calreticulin. It binds transiently and selectively with nascent partially folded monoglucosylated N-linked glycoproteins and retains them in the endoplasmic reticulum until they are completely folded.

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calreticulin Calreticulin is an important multifunctional calcium binding protein found in the membranes of smooth muscle and non-muscle endoplasmic reticulum (ER). The principle role of calreticulin’s is its function in the folding and peptide-loading nascent N-linked glycoproteins. C-glycoside Sugar derivative where the exocylic anomeric oxygen has been replaced with a methylene group. EDEM and EDEM II Mannose binding lectins responsible for entry into ERAD. Endo A, Endo H and Endo M A family of Endoglycosidase’s that cleave within the chitobiose core of high mannose (Endo H and A) and complex type (Endo M) N-linked glycoproteins, leaving only a single GlcNAc residue bound to the glycoprotein. EPO Generally used short form for erythropoietic glycoprotein hormone secreted from the renal gland in response to oxygen depletion in tissues, involved in the regulation of the production of red blood cells. ERAD (Endoplasmic reticulum-associated protein degradation) An intracellular trafficking process involving the translocation of misfolded N-linked glycoproteins to the cytoplasm, which undergo subsequent enzymatic degradation. Gal Abv. galactose. GalNAc Abv. N-acetylgalactosamine. Glc Abv. glucose. GlcNAc Abv. N-acetylglucosamine. glycan Strictly defined as polysaccharide, but loosely used in literature to refer to any carbohydrate structure for example the carbohydrate component of a glycoprotein or glycolipid. glycocode The term used to describe the information conveyed by the structural diversity of carbohydrates. glycoconjugate Molecules that contain carbohydrate structures covalently bound to another molecular structure, e.g., glycolipids, glycopeptides, and glycoproteins. glycoform Variants of a glycoprotein in which both the composition of the carbohydrate and/or the site of glycosylation exhibit heterogeneity while the peptide remains uniform. inteins A section that is removed in a protein that reacts through self-splicing and rejoining. Inteins are extensively used in biotechnology particularly within native chemical ligation. lectin Carbohydrate-binding proteins that are not enzymes or antibodies. Lewis X (see Lewis sugars). sialyl Lewis X (see Lewis sugars). Lewis sugars Lewis system sugars are a family of fucosylated blood group sugars, to include Lewis a (Lea), Lewis b (Leb), Lewis c (Lec), Lewis d (Led), Lewis x (Lex) and sialyl Lewis (sLex (sLex)). Man Abv. mannose. NeuAc Abv. N-acetylneuraminic acid. N-linked glycoprotein A glycoprotein where a carbohydrate is bound to the protein via the nitrogen of the amide side chain of asparagine. The Asn residue is typically found in the sequence Asn-X-Ser/Thr (X¼any amino acid except proline). O-linked glycoprotein A glycoprotein where a carbohydrate is bound to the protein via an the alcohol side chain of typically serine or threonine. PTM (Post-translational modification) The chemical modification of a protein after its translation. Modifications include lipids, carbohydrates, sulfation, phosphorylation. receptor-mediated endocytosis The process whereby cells absorb material (molecules such as proteins) outside the cell by engulfing them within their cell membrane through the specific interactions of receptors with ligands on the substrate. selectins A family of lectins that are expressed as part of the inflammatory response promoting leukocyte homing to sites of trauma. SPPS Solid Phase Peptide Synthesis. TCA Trichloroacetimidate, the anomeric leaving group found in one of (if not the) leading glycosyl donor type. thiosugars Carbohydrate in which an oxygen (here largely used to indicate the anomeric O-1) is replaced by sulfur. Tn antigen (motif ) GalNAc(a1-O)Ser/Thr.

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Biographical Sketch

David Gamblin was born in Taunton, England in 1980. He was awarded his M.Chem. in 2002 and later obtained his D.Phil. in 2005 from the University of Oxford. His doctoral thesis focused on the development and design of novel chemoselective reagents for site-selective protein modification, for which he was awarded the Europa medal in 2004. In 2005 he took up a postdoctoral research fellowship working in collaboration with Professors Ben Davis and John Simons FRS. David’s research interests include precise protein modification, glycosylation chemistry and glycan analysis using mass spectrometry. David is currently a Lecturer in Organic Chemistry at St Edmund Hall, Oxford.

Eoin Scanlan was born in Galway in 1978 and from 1996–2000 studied chemistry at The National University of Ireland Galway. He received his Ph.D. in 2003 from the University of St. Andrews, working under the supervision of Prof. John Walton. His doctoral research focused on the use of oxime oxalate amides as tin free precursors for the synthesis of biologically active lactams. In January 2004 he took up a postdoctoral research fellowship at the University of Bern, Switzerland where he worked with Prof. Philippe Renaud on the use of organoboranes as free radical precursors. In July 2005 he moved to the University of Oxford where he is currently working with Prof. Ben Davis on a study of carbohydrate binding modules and novel regioselective glycosylation strategies.

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Professor Ben Davis is a Professor of Chemistry, and Fellow and Tutor in Organic Chemistry, Pembroke College. After his B.A. (1993) and D.Phil. (1996) at Oxford he spent 2 years at Toronto, exploring protein design and biocatalysis. In 1998 he returned to the U.K. to take up a lectureship at the University of Durham. In 2001 he returned to Oxford and was made a Professor in 2006. His group’s research centres on chemical biology with an emphasis on carbohydrates and proteins. In particular, the group’s interests encompass synthesis and methodology, inhibitor design, protein engineering, drug delivery, molecular modeling, molecular biology, and glycoscience with the goal of medicinal application. He has published >80 papers, >20 patents and has given >110 invited lectures. This work has been funded by participation in >£9m of research income and has received the 1999 Royal Society of Chemistry Meldola medal, the 2001 Royal Society of Chemistry Carbohydrate Award, an AstraZeneca Strategic Research Award, a DTI Smart Award, a Mitzutani Foundation for Glycoscience Award, the 2002 Philip Leverhulme Prize in Molecular Biology & Biochemistry and in 2005 the Royal Society’s Mullard Prize and Medal. Ben Davis is co-founder of Glycoform, a small biotechnology company aimed at exploiting the therapeutic potential of glycoproteins. In 2003 he was named as among the 100 top young innovators in the world by Technology Review. In 2006, he was awarded one of the Royal Society of Chemistry’s Corday-Morgan Medals for 2005 and the 2006 International Association for Protein Structure Analysis and Proteomics Young Investigator Award.

1.16 Synthesis of C- and S-Glycosides F. Nicotra, C. Airoldi, and F. Cardona, University of Milano-Bicocca, Milan, Italy ß 2007 Elsevier Ltd. All rights reserved. 1.16.1 Introduction

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1.16.1.1 C-Glycosides of Biological Relevance

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1.16.1.2 S-Glycosides of Biological Relevance

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1.16.2 Procedures of C-Glycosidation

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1.16.2.1 Reaction of Glycosyl Halides with Organometallic Reagents and Carbanions

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1.16.2.2 C-Glycosylation via Glyconolactones

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1.16.2.3 C-Glycosylation Involving Lewis Acid-Catalyzed Formation of Oxonium Ions

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1.16.2.4 C-Glycosylation via Olefination–Cyclization

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1.16.2.4.1 1.16.2.4.2

C-glycosylation using stabilized ylides C-glycosylation using not stabilized ylides

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1.16.2.5 C-Glycosides by Cyclization of Diols and Derivatives

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1.16.2.6 C-Glycosylation of Glycals

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1.16.2.6.1 1.16.2.6.2 1.16.2.6.3

Acid-catalyzed addition of carbon nucleophiles to glycals Transition metal-catalyzed addition of carbon nucleophiles to glycals Addition of carbon nucleophiles to glycals via 1,2-episulfonium intermediates

661 662 663

1.16.2.7 C-Glycosylation via 1,2-Anhydrosugars

664

1.16.2.8 C-Glycosylation via Glycosyl Radicals

664

1.16.2.9 C-Glycosylation via Glycosyl Anions

665

1.16.2.10 C-Glycosylation via Ramberg–Backlund Reaction

666

1.16.3 Procedures of S-Glycosylation

667

1.16.3.1 S-Glycosylation by Acid-Promoted Displacement at the Anomeric Center of Glycosyl Donors

668

1.16.3.2 S-Glycosylation via SN2 Reaction of 1-Thiosugar Donors with Activated Acceptors

669

1.16.3.3 S-Glycosylation via SN2 Reaction of Thiolate Anions on Glycosyl Halides

670

1.16.3.4 S-Glycosylation via Michael Addition of 1-Thiosugars to Unsaturated Acceptors

673

1.16.3.5 Enzymatic S-Glycosylation: The Use of Thioglycoligases

673

1.16.3.6 Synthetic Applications of S-Glycosides

675

1.16.3.6.1 1.16.3.6.2

Thioglycosides as glycosyl donors Toward the synthesis of glycoproteins: Synthesis of S-linked glycoconjugates

1.16.4 Conclusions

675 676

677

1.16.1 Introduction Carbohydrates present an interesting feature: the electrophilic character of the anomeric center allows the sugar to link different molecules presenting a functional group that acts as a nucleophile. Usually an alcohol, an amino, or an amido group perform this linkage in Nature; however, occasionally, a sulfur or a carbon nucleophile are also involved, generating respectively S-glycosides and C-glycosides. Figure 1 shows example of natural S- and C-glycosides. Glucosinolates 1 are examples of S-glycosides widely present in the Brassica family (cauliflower, sprouts, cabbage, and other), and are hydrolyzed by enzymes to volatile flavors when cellular structure is disrupted. C-Glycosides are formed in Nature when a compound with a particularly nucleophilic carbon atom reacts with an NDP-sugar; an example is the glucosyltransferase reaction of resorcinol with UDP-glucose that affords compound

647

648

Synthesis of C- and S-Glycosides

OH

OH

O

HO HO

O

HO HO

R

S OH

HO

OH 2 HO a natural C-glycoside

NOSO3 − Glucosinolates, natural S-glycosides 1

Figure 1 Examples of natural S- and C-glycosides.

N N O

OH

O

Glucuronyl transferase

O

O

HO HO

O

OH

HO HO

O O

N N O

OH OH

OUDP

4

3 Scheme 1 Generation of a drug metabolite C-glucuronide.

OH SH R

SH N OH O

R

SH R

NO

NOH

H Thiohydroxamic acid

UDP-Glc

O

HO HO

OH S 5

R

N Desulfoglucosinolate OH

Scheme 2 S-glycosylation reaction in glucosinolate biosynthesis.

2. Also exogenous compounds, such as drugs containing a carbon atom with a strong nucleophilic character, can undergo an enzymatic reaction which generates C-glycosylated drug metabolites. Scheme 1 describes the enzymatic conversion of Butazolidin® into the corresponding C-glucuronate.1 S-Glycosides are synthesized in Nature starting from an NDP-sugar and a compound containing a nucleophilic sulfur atom. An example is the S-glycosylation of thiohydroamic acids with UDP-glucose that is catalyzed by a UDPGthiohydroximate glucosyltransferase and leads to desulfoglucosinolates, precursors of glucosinolates (Scheme 2).2 A variety of methods have been developed to synthesize in laboratory S- and C-glycosides, most of which exploit, like in Nature, a nucleophilic sulfur or carbon atom that displaces a leaving group at the anomeric center of the sugar. Like any glycosylation reaction, two anomers can be obtained, and the possibility to control the stereochemical outcome of the reaction, generating stereoselectively each of the two stereoisomers, is an important synthetic goal. The interest in S- and C-glycosides lies on the metabolic stability of the glycosidic linkage, which cannot be hydrolyzed by glycosidases. The metabolic stability of these compounds finds application in the inhibition of carbohydrate-processing enzymes, the S- and C-glycosides being able to fit into the active site of glycosidases and glycosyltransferases without being modified by the enzyme. This behavior allows also mapping the active site in order to study the recognition and binding processes, and to investigate the mechanism of action of these enzymes.3a,3b The metabolic stability of S- and C-glycosides is also exploited in the generation of glycomimetic drugs resistant to in vivo hydrolysis, such as integrine ligands or glycidic immunostimulators.

1.16.1.1 C-Glycosides of Biological Relevance Since 1959, a number of C-nucleosides, such as pirazomycin 6 or showdomycin 7 (Figure 2), in which the carbon atoms substitute the nitrogen of the base, have been isolated from natural sources.


649

Those compounds showed a variety of interesting biological properties such as antitumor, antiviral, or antibacterial activities.4a,4b The combination of metabolic stability and relevant biological activities generated a great interest in the synthesis of a variety of C-glycosides designed in order to inhibit specific enzymes, act as agonists or antagonists in receptorial phenomena, and induce immunogenic response. Aryl C-glycosides, in particular antraquinone-carbohydrate hybrids such as undamycinone B 8 (Figure 3), have shown DNA-binding properties and cytotoxicity, therefore being promising antitumor agents.5a,5b C-Glycosides presenting reactive a-bromoketone functionality (9 in Figure 4)6 have been described as irreversible inhibitors of glycosidases. More sophisticated C-glycosidic structures have been synthesized to mimic NDP-sugars and therefore as specific inhibitors of glycosyltransferases. An example, reported in Figure 5, shows the nucleoside-diphospho-exo-glycal 10 displacing time-dependent inactivation of UDP-galactopyranose mutase.7

HO

O

CONH2

HN N

NH HO

OH

O

OH OH 6 Pirazomycin (antiviral)

O

O

OH OH 7 Showdomycin (antibacterial, antitumor)

Figure 2 Examples of biologically active C-nucleosides.

Me O Me

OH

O

HO HO 8

OH O

Urdamycinone B (a angucyclin) Figure 3 Urdamycinone B, an aryl C-glycoside with promising antitumor activity.

OH

OH O

HO HO

OH

O

O HO

OH

Br O

9 Figure 4 A C-glycoside that irreversibly inhibits glycosidases.

O O O P O P O OH OH

O OH HO

OH

OH

10

NH O

N

O

OH OH

Figure 5 An NDP-sugar mimic displacing time-dependent inactivation of UDP-galactopyranose mutase.

650


C-Glycosides related to tumor-associated antigens are another relevant synthetic target. Mimics of GalNAca1-O-Ser (Tn antigen) linked to immunogenic peptides in order to elicit both B- and T-immunoresponse have been synthesized (11 and 12 in Figure 6). In order to achieve increased stability toward in vivo degradation, the two moieties are connected by a C-glycosidic bond, in place of the natural O-glycosidic linkage. In addition, the concept of chemoselective ligation has been applied to the site-specific attachment of the sugar moiety to peptides. Chemoselectivity was achieved coupling an aminoxy group properly introduced in the peptide backbone with a keto function present on the C-glycosyl analog of the Tn antigen, affording the corresponding oxime.8a,8b In compound 12, the presence of two sugar moieties acts as an adjuvant on dendritic cells by upregulating the expression of B7.2 molecule and increasing the presentation of the peptide to T-cells. This suggests that the N-acetylgalactosamine analog not only can be used as a B-cell epitope, but in multiple copies, it can target the vaccine to dendritic cells by binding and cross-linking its surface receptor. C-Glycosides related to the Neu5Aca2-6GalNAca1-O-Ser, the sTn antigen, have been also synthesized; Figure 7 describes the sTn mimic 13 in which the neuraminic acid is linked to the N-acetylgalactosamine unit through a C-glycosidic linkage.9a,9b C-Glycoside mimics of important glycidic ligands, such as sialyl Lewis X, which are implicated in selectin interaction as an initial event in inflammation response and in tumor metastasis generation, have been synthesized. In the example reported in Figure 8,10 the C-disaccharide 14 contains the structural elements responsible for selectin-sialyl Lewis X recognition (the L-fucose and D-galactose units and the carboxylic group of sialic acid). An interesting example of biological application of C-glycosides has been reported by Tietze et al., which generated novel carboranyl C-glycosides for the treatment of cancer by boron neutron capture therapy.11 Boron neutron capture therapy is a binary system used for the treatment of cancer, which involves administration of a boron compound and subsequent irradiation with slow neutrons. It relies on the specific ability of the isotope 10B to react with thermic neutrons to give an a-particle. If boron is present in a tumor cell, irradiation with a beam of slow neutrons will cause destruction of the malign tissue. OH

HO

O

HO

B-epitope

AcHN OH

HO

N O

O

HO

T-epitope

O

HN VHAAHAEINEAGRG

AcHN

Spacer HO HO

N O

OH O

O 11

NH

AcHN N O

O

H N

HN

AVHAAHAEINEAG

OH

O 12 Figure 6 Two C-glycopeptides with interesting immunogenic properties.

HO OH HO AcHN

HO

CO2H

O OH

OH O

13

HO

Figure 7 A C-disaccharide mimicking the sTn antigen.

AcHN

CO2R O

NHR⬘

OH


HO HO

HO HO OH OH O OH

HO

HO

O HO AcHN

HO

OH OH

OH O

O

O

HO

OH

OH O

O

O

O

651

OH

O OH 14

Sialyl Lewis X

C -disaccharide mimic

Figure 8 Sialyl Lewis X and a C-disaccharide mimic.

OH HO HO

O S OH Sinigrin

NOSO3 −

Figure 9 Sinigrin, a naturally occurring glucosinolate found in seeds of black mustard.

A C-glycoside analog of the immunostimulant a-galactosylceramide has been synthesised by Frank and co-workers, and resulted 1000 times more protective than the O-glycoside in mouse against malaria (Plasmodium yoelii) infection, and 100 times more effective against lung melanoma.12 From the reported examples, it is clear that the presence of a C-glycosidic linkage has been exploited, taking advantage of the metabolic stability, either for inhibiting an enzymatic process or to generate potential glycidic drugs avoiding the intrinsic metabolic lability of the O-glycosidic linkage.

1.16.1.2 S-Glycosides of Biological Relevance Several thioglycosides presenting interesting biological activities have been isolated from many different organisms. Glucosinolates, previously introduced, are one of the most important classes. Their unique properties were first observed in the seventeenth century and, nowadays, about 120 different glucosinolates have been identified in 16 families of dicotyledonous angiosperms.13 Glucosinolates are b-thioglucoside N-hydroxysulfates differing for the side-chain R (Figure 1) and resulting to be stable water-soluble precursors of isothiocyanates. In particular, upon tissue disruption, a specific enzyme called myrosinase (EC 3.2.3.1) is released and it can reach its glucosinolate substrate. Myrosinase hydrolyzes the b-glucosyl moiety, affording the thiohydroxyamate-O-sulfonate that rearranges giving isothiocyanates or other breakdown products, such as thiocyanates and nitriles, responsible for the highly distinctive flavor of the Brassicaceae family. Glucosinolates and their breakdown products have been extensively studied and characterized also for their bacteriocidal,14a–14i fungicidal,15a–15g nematocidal,16a,16b and alleopathic17 properties. In addition, cruciferous vegetables, such as cabbage and mustard, have been used as wound poultices and antitumor agents for centuries (Figure 9). As a consequence of the fact that an important correlation between increased consumption of vegetables (in particular cruciferous ones) and fruits and reduced incidence of many types of cancer has been demonstrated,18 glucosinolate/ isothiocyanate cancer chemoprotection activities are now attracting great interest for the development of new anticancer drugs. Lincomycines are other examples of remarkable thioglycosides occurring in Nature. Lincomycin A and B (Figure 10) are thioglycoside-based antibiotics produced by Streptomycies lincolnensis.19 They consist of an aminooctose moiety, the a-methylthiolincosaminide, linked by an amidic bond to an N-propilhygric acid (lincomycin A) or an ethylhygric acid (lincomycin B) unit. Lincomycins present a bacteriostatic activity based on the inhibition of the bacterial proteic synthesis. Lincomycin A in particular have been largely employed in clinical therapy of infections caused by micoplasmas, Gram-positive bacteria, and cocci, but it presents a lot of side effects; for this reason, it has been substituted by 7-chloro-7deoxylincomycin or clindamycin (Figure 10), a semisynthetic more lipophilic compound having larger spectrum of action and major antibacterial activity.

652


CH3 R

CH3

N

R9 H N OH

O

O HO

15

OH Lincomycin A: R = Pr; R9 = OH SCH3 Lincomycin B: R = Et; R9 = OH Clindamycin: R = Pr; R9 = Cl Figure 10 Lincomycin A and B and clindamycin, thioglycosides with antibiotic activity.

O

PO

OH

n 16 OH

PO n

OH

PO n

20

19

18

OH PO OH n R

O

PO R⬘

n

21

:_

n

22

O

PO

O

PO

O

n

X

n

17

O

PO

O

PO CHO

n 24

23

O

PO n O

RO n

O

PO R

27

R

R OH

25

n 26

C-glycoside Scheme 3 General scheme for C-glycosylation procedures.

1.16.2 Procedures of C-Glycosidation The synthesis of C-glycosides can be effected in a great variety of procedures, and has been reviewed by many authors.20a–20h Here we will highlight the most important general methods, and describe specific interesting examples in which biologically relevant or particular complex C-glycosides have been generated, or examples in which particular original methods have been adopted. A general overview of the most common strategies, intermediates, and synthons in C-glycosylation starting from an aldose or a ketose is showed in Scheme 3. The carbonyl function 17 of the sugar, which is masked in the hemiacetalic form 16, can undergo either nucleophilic addition by way of a carbon nucleophile, or olefination reaction. In the first case, the obtained diol 20 must be cyclized with elimination of a molecule of water affording the C-glycoside 27. In the second case, the enitol 21 can be cyclized by activation of the double bond and attack of the hydroxyl group. If the olefination is performed with a stabilized Horner–Emmons reagent, the double bond is already activated by the electron-withdrawing group, and a Michael cyclization occurs.


653

Alternatively the anomeric center of the sugar can be activated by conversion of the hydroxyl group to a better leaving group X (18 in Scheme 3), usually an halide, an imidate, a thioderivative. Depending on the nature of X, anomeric cations (oxonium ions) 22, anomeric carbanions 23, and anomeric radicals 24 can be generated. Those intermediates respectively react with proper carbon nucleophiles, carbon electrophiles, and carbon radical scavengers, generating C-glycosides 27. Oxidation of the anomeric hydroxyl group affords the sugars lactone 19, which reacts with organometallic reagents affording the lactol 25, which in turn can easily generate the anomeric carbocation 26, the reduction of which with triethylsilane affords C-glycoside 27. Glycals 28, that can be easily generated from activated sugars 18 by elimination of HX, are useful intermediates in the synthesis of C-glycosides. The double bond can be activated either by epoxidation (29 in Scheme 4) or with transition metal catalysts (30 in Scheme 4), in order to undergo the attack of proper carbon nucleophiles affording C-glycosides 31 and 32. All these C-glycosylation procedures present specific stereochemical outcomes that depend on many factors, the reaction mechanism, the preferred conformation of the sugar substrate and/or the reaction intermediate, the presence of ‘participating’ groups adjacent to the anomeric center, and even the nature of the solvent.

1.16.2.1 Reaction of Glycosyl Halides with Organometallic Reagents and Carbanions The first example of synthesis of a C-glycoside goes back to 1945, when Hurd reacted 2,3,4,6-tetra-O-acetyl-Dglucopyranosyl chloride 33 with different arylmagnesium halides, obtaining, after acetylation, the corresponding polyacetylated C-glucopyranosyl derivatives 34 and 35 (Scheme 5).21

O

PO n

P⬘O

O

PO

28 O

RO

n

n

O

M

29

P⬘O

30 O

PO

P⬘O

O

PO

R

n

R

n

31 OH

32

Scheme 4 Strategies of C-glycosylation starting from glycals.

AcO AcO

OAc O

i, PhMgBr ii, Ac2O, Py

OAc Cl 33 AcO

OAc

AcO AcO 36

34 58% i, PhMgBr ii, Ac2O, Py

O Cl

AcO AcO

OAc O

AcO

AcO Ph + AcO

OAc

OAc 35 27% AcO

OAc O

AcO AcO

+ 37 41%

OAc O

AcO AcO

Ph

Scheme 5 Examples of C-glycosylation using Grignard reagents and glycosyl halides.

Ph

OAc O Ph 38 24%

654


The same author experimented the Grignard reaction on different sugar substrates, such as 2,3,4,6-tetra-O-acetyl-Dmannopyranosyl chloride 36 (Scheme 6).22a,22b In these examples, the presence of an acyl ‘participating’ group at C2 in the polyacetylated glycosyl halide directs the attack of the nucleophile from the opposite side, as in O-glycosylations (Scheme 6). In the case of glucopyranosyl chloride 33 the prevalent product is therefore the b-C-glucoside 34, whereas in the case of mannopyranosyl chloride the main product is the a-C-mannoside 37. In the absence of a participating group, the C-glycosylation occurs mainly with inversion of the anomeric configuration, as the reaction intermediate is probably a ‘ion pair’ 40, as shown in Scheme 7. So, the stereochemistry of the 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl chloride 39 with 2,4-dimethoxyphenyl magnesium bromide affords the corresponding b-C-glucopyranosyl derivative 41 (60% d.e.; 4:1 b: a).23 Other organometallic reagents such as organolithium,24 organocadmium,25 organozinc,26 lithium dialkyl cuprates,27 organomercurium,28 and organoaluminum29 have also been experimented. Table 1 reports examples of the reaction of glycosyl halides with the different organometallic reagents, providing yields and references. Stabilized carbanions, such as malonates, react with glycosyl halides affording C-glycosides.30a–30f The reaction usually occurs with inversion of the configuration. It has been reported that 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl bromide 42 reacts with sodium dibenzyl malonate affording the corresponding b-C-glucoside 44, whereas the same reaction performed in the presence of tetrabutyl ammonium bromide affords the a-C-glucoside 45 (Scheme 8). The bromide ion in fact displaces the glycosyl bromide, giving rise to an equilibrium in which the a-anomer 42 is more abundant (anomeric effect), but the less stable b-anomer 43 is more reactive. It reacts faster with the malonyl anion, affording the product with the a-anomeric configuration.

AcO AcO

OAc O

36

AcO AcO

OAc Cl

OAc O O +

OAc O

AcO AcO

Nu OAc

O

H3C Scheme 6 Effect of an acyl-participating group in the stereochemistry of the nucleophilic displacement of a glycosyl halide.

OBn O

BnO BnO

OBn

ArMgBr BnO BnO

OBn O OBn

Cl

39

40

ArMgBr BnO BnO

+

OBn O OBn

Cl

−

OMe

OMe

41

Scheme 7 Generation of an aryl C-glycoside by reaction of a glycosyl halide with an ArMgBr; an ion-pair intermediate is generally involved.

Table 1 Reaction of glycosyl halides with different organometallic reagents Organometallic reagents

Leaving group

Yields (%)

References

RMgBr, RMgCl PhLi Ar2Cd Ph2Zn R2CuLi furanylHgCl M3Al

Cl, Br Cl Cl Cl Br Br F

50–85 27 20–83 72 40–60 58 95

22a,22b,23 24 25 26 27 28 29


OBn O

BnO BnO

−

−

OBn O

Br BnO BnO

OBn 42 Br

Br 43 OBn −

CH(CO2Bn)2

OBn O

BnO BnO

655

CH(CO2Bn)2 OBn

OBn O

BnO BnO

45

44

CH(CO2Bn)2

OBn CH(CO2Bn)2

Scheme 8 Reaction of an a-glucosyl bromide 42 with a malonate anion generates the b-C-glycoside 44; in presence of bromide ions, the more reactive b-bromide 43 is formed, which generates the a-C-glucoside 45.

BnO BnO

OBn O

46

BnO

−

OBn

CH(CO2Et)2

OBn 47 CH(CO2Et)2 60% d.e. −

CH(CO2Et)2

BnO OBn 48

BnO BnO

I

OBn O

OBn O

OBn O

BnO BnO

CH(CO2Et)2 49 OBn 80% d.e.

I

Scheme 9 Stereochemical outcome of the reaction of different glycosyl iodides with malonate anions.

BnO BnO

OBn O

47

OBn CH(CO2Et)2

OBn OH

BnO BnO

C(CO2Et)2 50

OBn

BnO BnO

OBn O CH(CO2Et)2 51

OBn

Scheme 10 Epimerization of C-glycosyl malonates.

Many factors however can influence the stereochemical outcome of the reaction, among which are the reactivity of the glycosyl halide and the kinetic or thermodynamic control of the reaction. The reaction of the less reactive 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl iodide 46 with 1.3 equiv. of diethyl malonate previously treated with sodium hexamethyldisilazane in the presence of 15-crown-5 resulted in the preferential formation of the a-C-glucoside 47 (5:1 a:b). This result suggests that the glycosyl iodide is not particularly susceptible to the attack of the malonate anion, whereas the b-iodide that is formed by in situ anomerization, being more reactive, undergoes the nucleophilic attack affording the product with the a-anomeric configuration.30f In the case of 2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl iodide 48, which is more reactive, the same reaction affords the preferentially b-C-galactoside 49 (1:10 a:b) (Scheme 9). On the other hand, the possibility of a malonyl C-glycoside such as compound 47 to perform a retro-Michael reaction, affording an unsaturated open-chain derivative 50, followed by a Michael cyclization, affords the thermodynamic product, which is the b-C-glycoside 51 (Scheme 10).

656


BnO BnO

OBn O

RLi O

52

BnO BnO

OBn O

Et3SiH BF3 OEt2 •

R

BnO BnO

OBn OH 53

OBn

OBn O R 54

OBn

Scheme 11 Generation of C-glycosides by reaction of a glyconolactone with an organolithium reagent followed by Lewis acid-catalyzed reduction with triethylsilane.

RO

BrCH2CO2R

O n

O

RO O

CH2COOR

n

OH

L4Co 55

56 Et3SiH, BF3 OEt2 •

RO O CH2COOR

n 57

Scheme 12 Co(0)-mediated addition of a-bromoacetates to sugar lactones for the generation of C-glycosides.

1.16.2.2 C-Glycosylation via Glyconolactones Organometallic reagents can generate C-glycosides also reacting with glyconolactones 52. In this process, usually performed with organolithium compounds due to the hardness of the lactone, a lactol 53 is formed, the Lewis acidcatalyzed reduction of which with triethylsilane affords the C-glycoside 54 (Scheme 11).31 The stereocontrol of the reaction depends on the stereochemical requirements of the reductive step.32 In the case of glucopyranosides and more in general in pyranosides in 4C1 conformation, the reducing agent approaches the anomeric carbon from the a-face, therefore affording a b-C-glucoside. In furanosides, the stereochemical outcome cannot be predicted due to the conformational flexibility of the intermediate oxonium ion. This procedure has been exploited with different vinyl lithium,33 aryl lithium,34 alkinyl lithium,35 alkyl tin,36 and Grignard reagents.37 Recently, a mild and easy Co(0)-and/or Mg-mediated addition of a-bromoacetates to sugar lactones 55 to give the corresponding lactols 56, and the reduction to a C-glycoside 57, has been reported (Scheme 12).38 The Co(0)-mediated addition of a-haloesteres and a-haloketones to carbonyl compounds represents a mild and convenient procedure for the synthesis of b-hydroxy esters and b-hydroxyketones.39 A similar approach, applied to the reaction of a-halophosphonates and carbonyl compounds, yields b-hydroxyphosphonates, stable biomimetics of the corresponding phosphates.40 A different approach that allows to generate C-glycosides from glyconolactones consists in the olefination of the lactone. Glyconolactones (58 in Scheme 13) undergoes Tebbe methylenation,41a,41b difluoromethylenation,42 and dichloromethylenation,43 and also the Wittig olefination44 to afford different glycoexoenitols 59–63, which can be manipulated to generate different C-glycosides. In particular, it is worthy of note that a stabilized Grignard reagent, such as Ph3P¼CHCN, can be efficiently used in olefination of glyconolactones with the use of microwave activation.45 Treatment of the lactone with ethyl isocyanoacetate46 affords a useful synthon 63 to obtain C-glycosyl aminoacids, a class of compounds that has attracted great interest.47

1.16.2.3 C-Glycosylation Involving Lewis Acid-Catalyzed Formation of Oxonium Ions One of the most efficient and widely used methods of C-glycosylation consists in the treatment of a glycoside 64 with a Lewis acid, resulting in the displacement of the leaving group (OR, OCOR, Cl, F, SR, trichloroacetimidate)48 with formation of an oxonium ion 65, which can react with a variety of proper carbon nucleophiles, such as allylsilanes,49 propargylsilanes,50 enolethers51a,51b and enamines,52 trimethylsilyl cyanide,53 aromatic compounds,54a,54b and even alkenes,55 affording the C-glycosides 66–71 (Scheme 14).


657

RO O RO

O CH2

n

CCl2

NC C CO2Et

Ph3PCHCN

P(NMe)3 CF2Br2 Zn

O n

O n

58

P(NMe)3 CCl4

59 RO

O CNCH2CO2Et KOH RO

n

Tebbe

63 RO O

RO

CHCN

n

O CF2

n

62

60 61 Scheme 13 General scheme of glyconolactones’ olefination.

BnO BnO

OBn O 66 OBn

SiMe3

BnO BnO

OBn O

BnO BnO

OBn O OBn

Lewis acid BnO BnO

OBn X

64 X = Cl, Br, F, OMe, OCOR, SR, trichloroacetimidate

BnO BnO

SiMe3

OBn

67 OSiMe3

65

BnO BnO

OBn Me3SiCN

HO R

OBn O

71

OBn + O

HO

R

BnO BnO

OBn HO O

BnO BnO

OBn O OBn

OBn O

69

OBn

C

O

68

CN

OBn 70

HO

Scheme 14 General strategies of Lewis acid-catalyzed C-glycosylation procedures.

The stereochemistry of the process is determined by the anomeric effect which favors the transition state in which the lowest unoccupied molecular orbital (LUMO) of the orbital that will link the nucleophile overlaps with the ion pair of the ring oxygen. In the case of glycopyranosides with a 1C4 conformation, such as glucopyranosides, mannopyranosides, and galactopyranosides, the attack of the nucleophile occurs from the a-face generating the a-C-glycoside with high stereoselectivity. In the case of furanosides, the prediction or the stereochemical outcome of the reaction is much more difficult, and it depends on the nature of the solvent and the reaction temperature. It is interesting to note that this procedure is very efficient also in the formation of C-glycosides of ketoses, such as fructose, in which the reaction occurs on a tetrasubstituted carbon atom, which makes more difficult to apply other procedures. The reaction of methyl 1,3,4,6-tetra-O-benzyl-a-D-fructofuranoside 72 with allyltrimethylsilane and the Lewis acid trimethylsilyltriflate stereoselectively afforded the a-allyl C-glycoside 73 in about 60% d.e., independently of the nature of the solvent and the temperature.56 Manipulation of the a-allylic appendage allows to obtain different a-C-furanosides and even b-C-fructosides. To do that, the double bond of the allylic appendage of 73 was exploited to deprotect the primary hydroxyl group at C1, so that it can be further manipulated as C-glycosidic appendage, whereas the allylic substituent was converted into an hydroxymethylene group (Scheme 15).

658


BnO

OBn

O BnO OBn

BnO

OBn

O

i

BnO

O

ii

BnO

OMe OBn

72

O

BnO

I

74

OBn

73

iii BnO

OSiMe2tBu

O BnO OBn

OH

BnO

BnO

OSiMe2tBu

O

BnO

BnO

v, vi

OH

O

iv OBn

77

OBn

76

75

Scheme 15 i, Me3SiCH2CH¼CH2, Me3SiOTfMeCN, 0 C; ii, I2, THF; iii, Zn, AcOH; iv, t-BuMe2SiCl-TEA-DMAP, CH2Cl2; v, PdCl2(MeCN)2, PhH, reflux; vi, O3, CH2Cl2, –78 C, then Zn(BH4)2.

BnO

O

BnO

OH OBn

78

Ph3P=CHCO2Et

BnO

OH

CO2Et

BnO EtONa

O

CH2CO2Et

EtOH DME

BnO

OBn 79

OBn

BnO 80

Scheme 16 C-glycosylation by reaction of an aldose with a stabilized ylide.

The strategy, in other words, consists in having two hydroxymethylene substituents at the ‘anomeric’ center of the sugar, differently protected so that they can be selectively manipulated. In details, treatment of C-glycoside 73 with iodine results in the attachment of the benzylated oxygen at C1 to the oxonium ion intermediate with debenzylation and formation of the cyclic iodoether 74. Treatment of the cyclic iodoether 74 with zinc and acetic acid resulted in a reductive elimination, with formation of 75, which is molecule 73 deprotected at C1. Once deprotected, this hydroxyl group of 75 can be functionalized in different ways, so acting as a b-oriented C-glycosidic substituent. The a-oriented allylic anomeric appendage of 75 can be in turn converted into an hydroxymethyl group, taking the place of the original C1 of the fructose skeleton. The conversion of the allylic substituent into an hydroxymethyl group was effected by isomerization of the double bond ozonolysis and reduction of the ozonide to a hydroxyl group (77 in Scheme 15). The procedure required a delicate choice of protecting groups and experimental conditions, as the proximity of the two appendages can cause reciprocal interference. The Lewis acid-catalyzed C-glycosylation procedure has been applied to the synthesis of a variety of aryl C-glycosides, some of which showed interesting antibiotic activities.57 This is a sort of Friedel–Craft electrophilic aromatic substitution in which the glycosyl oxonium ion acts as nucleophile. The reaction has been extended to a variety of heteroaromatic compounds such as pyrrole.58 Recently, the environmentally friendly C-glycosylation of phloroacetophenone with unprotected glucose using scandium(III) trifluoromethanesulfonate in aqueous media has been reported.59 This procedure can be applied to the synthesis of C-glycosides of amino sugars, such as glucosamine, which is all but trivial, provided that the amino group is fully protected as phthalimido,60 or is substituted by an azido group,61 from which the amine can be generated by reduction at the end of the synthesis.

1.16.2.4 C-Glycosylation via Olefination–Cyclization Another possibility to perform a C-glycosylation exploiting the electrophilic character of the anomeric center consists in the olefination of the free aldehyde, in equilibrium with the hemiacetal, followed by cyclization of the obtained enitol.

1.16.2.4.1 C-glycosylation using stabilized ylides Aldoses, such as 2,3,5-tri-O-benzyl-D-ribofuranose 78 in Scheme 16, react with stabilized ylides, affording an openchain elongated sugar 79 that can undergo, spontaneously or by treatment with bases, Michael-type cyclization with generation of the C-glycoside 80.62a–62h


659

This reversible reaction favors the thermodynamic product, in the case of glucopyranosides or galactopyranosides in the b-anomer due to the less-hindered equatorial orientation of the anomeric substituent. In the case of mannopyranosides, the disfavored cis-interaction of the b-anomeric substituent with the subsituent at C2 makes more complicated the prediction of the stereochemical outcome of the reaction. An interesting result has been obtained with N-acetylglucosamine protected as 4,6-benzylidene, the reaction of which with carboethoxymethylenetriphenylphosphorane afforded the one-pot cyclized product stereoselectively with the a-configuration.63 This is an interesting approach for the stereoselective synthesis of a-C-glycosides of glucosamine that has been shown effective also with a 4,6-ethylidene protection.64 Suppression of the electron-withdrawing group, for example, by reduction of the ester function to an hydroxyl group, prevents the Michael cyclization; therefore, a kinetic control of the cyclization reaction can be obtained by activation of the double bond with a proper electrophile. This activation can be performed by Sharpless epoxidation (Scheme 17),65 or by iodination.66 In the Sharpless procedure, the a,b-unsaturated ester 82 has been reduced with diisobutyl aluminum hydride, after protection of the free hydroxyl group, to generate the a,b-unsaturated alcohol 83. The allylic alcohol 83 was submitted to stereoselective Sharpless epoxidation in presence of (þ)diethyl tartrate to afford the epoxide 84, and (–)diethyl tartrate to afford the epoxide 85. Deprotection of the hydroxyl group that must perform the cyclization, and treatment with sodium hydride in order to generate an alcoholate that efficiently attacks the epoxide, results in the formation of the b-C-glycoside 86 from epoxide 84, and the a-glycoside 87 from epoxide 85.

1.16.2.4.2

C-glycosylation using not stabilized ylides

In Section 1.16.2.4.1, it had been shown that enitols such as compound 83 can generate C-glycosides by activation of the double bond. More in general, Wittig reaction followed by cyclization via electrophilic activation of the double bond is an interesting C-glycosidation procedure. Different electrophiles, such as I2, Br2, NBS, NIS, PhSeCl, or metachlorperoxybenzoic acid, have been used in the cyclization of glycoenitols,67 but the best stereochemical results have been obtained with mercuric salts and iodine. Properly protected aldoses, such as tetrabenzyl glucose 88 in Scheme 18, react with methylene triphenylphosphorane to afford an enitol 89. In order to cyclize this enitol, activation of the double bond with mercury salts or with iodine is required.68a–68e If the activation of the double bond is performed with iodine (or with N-iodosuccinimide), one must take care of the fact that also the oxigens of the benzyl ethers can act as nucleophiles, undergoing debenzylation. In this case, the preferred 5-exo-cyclization, according to the Baldwin rules,69 occurs with formation of a C-furanoside 91.

Ph

O O BnO

O

Ph3CHCO2Et

OBn OH

O O BnO

i, Et3SiOTf ii, DIBALH

81 Ph

Ph

O O BnO

82

OBn

CO2Et

OSiEt3 OH OBn

83

(+) DET

OH

(−) DET

Sharpless epox. Ph

O O BnO

Ph

OSiEt3 OH 84

OBn

O

O O BnO

OSiEt3 OH 85

−

OBn

O

i, F ; ii, NaH Ph

Ph

O O BnO

O

OH OH

O O BnO

O OBn

OBn 86

87

HO

OH

Scheme 17 Stereoselective C-glycosylation exploiting the Sharpless epoxidation of allylic alcohols.

660


BnO BnO

OBn O

Ph3PCH2

BnO BnO

OBn OH 88

i, Hg(OAc)2 ii, KCl

OBn OH

BnO BnO

OBn

89 BnO

OBn O

90

OBn HgCl

I2

HO

O

BnO OBn 91

I

Scheme 18 C-Glycosidation via methylenation–cyclization of tetrabenzyl glucose. Iodine generates the C-furanoside 91 by debenzylation.

E+

H

R OR

Figure 11 Preferential attack of the electrophilic reagent on allylic ethers.

BnO

BnO

O BnO

Zn(CH=CH2)2 OH

BnO BnO

OBn OH

i, Hg(OAc)2 ii, KCl

BnO BnO

OBn O OH

OH 92

93

94

HgCl

Scheme 19 C-Glycosylation by stereoselective generation of the enitol 93 using divinylzinc and subsequent electrophilic cyclization.

This process is highly stereoselective, the double bond being attached by the electrophile from the less-hindered face of the most stable conformation, the one in which the allylic hydroxyl group lies on the same plane of the double bond (Figure 11). A limit of this procedure is that the yield of the Wittig reaction is often very low, as the b-elimination is a competitive reaction. To overcome this problem, the open-chain enitols have been generated by vinylation (Scheme 19).70 With divinylzinc, the vinylation occurs in a highly stereoselective manner affording the threo-products, according with a Cram-kelated model. The process has been applied also to the synthesis of C-glycosides of D-glucosamine,71 exploiting the stereoselective reaction of vinyl magnesium bromide with the benzylglycosylamine 95 obtained by reaction of tribenzyl arabinose 92 with benzylamine in presence of molecular sieves as dehydrating agents (Scheme 20). Mercurio cyclization of the aminoenitol 96 afforded stereoselectively the a-C-glucosaminoside 97.

1.16.2.5 C-Glycosides by Cyclization of Diols and Derivatives Furanosides and pyranosides can be respectively obtained from a g- or d-diol by intramolecular dehydration. The cyclization can be performed by acid catalysis or by conversion of one of the two hydroxyl groups into a leaving group.


BnO

O BnO

BnO

CH2=CHMgBr BnO BnO

NHBn

i, Hg(OCOCF3)2 ii, KCl

OBn OH

BnO BnO

OBn O BnHN

NHBn 95

661

96

97

HgCl

Scheme 20 Generation of glucosamine C-glycosides by reaction of arabinosyl amine 95 with divinylzinc and subsequent electrophilic cyclization.

OP

OP

OP

OH

O OH 98

PO

PO

PO OH

OH

O

Route a

CHO

CHO

100

99 Route b RM

OP

OP

O

OH PO

PO

R OH 101

OH

OH

R

102

Scheme 21 General scheme of C-glycosylation via dehydration of a diol.

BnO BnO

OP

OBn O

103

OTf

DMS-Py OH

160 ⬚C

O

BnO BnO

CHO 104

Scheme 22 An example of C-glycosylation by displacement of a triflate.

This process has been applied to the synthesis of C-glycosides, with two main synthetic strategies: (1) exploiting in the cyclization two hydroxyl groups already present in the open-chain starting sugar 99, with contraction of the sugar ring (route a in Scheme 21); (2) generating a new alcoholic function by addition of an organometallic reagent to the anomeric carbonyl group of 99, therefore maintaining the original ring size of the sugar (route b in Scheme 21). The C-glycoside formation by ring contraction has been proposed in very mild condition, heating a 2-O-trifluoromethanesulfonyl glycoside in pyridine-DMS (DMS ¼ dimethyl sulfide; Scheme 22).72 Instead of the triflates, epoxides,73 organothallium,74 organoselenium,75 and diazonium salts76 have been used. The reaction of the aldose with an organometallic reagent affords a diol, the cyclization of which has been effected by acidcatalyzed dehydration77 or by treatment with p-toluenesulphonyl chloride.78 In these cases, however, a mixture of stereoisomers is obtained, as each of the two hydroxyl groups can act as leaving group.

1.16.2.6 C-Glycosylation of Glycals The easy accessibility and versatile functionality of glycals also provides advantages in C-glycoside synthesis.79 Glycals represent a very important class of compounds where the double bond can be easily modified, for example, by hydroxylation, hydrogenation, epoxidation, and amino hydroxylation. C-Glycosylation of glycals usually occurs by allylic substitution with carbon nucleophiles, furnishing 2,3-unsaturated C-glycosides or C-pseudoglycals 106 (Scheme 23).

1.16.2.6.1

Acid-catalyzed addition of carbon nucleophiles to glycals

Allylic substitution of glycals with carbon nucleophiles can be catalyzed by a strong Lewis acid such as BF3OEt280,81 or TiCl4.82 Other reagents such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ),83 TMSOTf,84 montmorillonite K10,85 and InCl386 and InBr87 are also known to promote C-glycosylation of glycals (TMS ¼ trimethylsilyl).

662


OAc

OAc O

AcO AcO

O

AcO

Nu 105

106

Scheme 23 3-O-Acetyl glycals undergoes nucleophilic allylic displacement.

OAc

OAc O

AcO

10 mol% Yb(OTf)3

AcO

Nu

106 OAc

Br

OSiMe3

mainlya

C-Nucleophile

105

SiMe3

O

AcO

SiMe3 OSiMe3

SiMe3

SiMe3 OSiMe3

Ph

OSiMe3 O OBn

N

Boc

O O

107

Scheme 24 Description of the different carbon nucleophiles used in the allylic substitution on 3-O-acetyl glycals.

Schmidt and co-workers described the synthesis of C-pseudoglycals starting from tri-O-acetyl glucal and different allylsilanes or silylenol ethers as nucleophiles in the presence of catalytic amounts of Yb(OTf )3 (Scheme 24). They demonstrate that Yb(OTf)3 is an efficient catalyst for high stereoselective C-glycosylation procedures to produce a variety of functionalized C-pseudoglycals, which are useful intermediates for various applications.88 More recently, Das and collaborators reported a novel synthesis of 2,3-unsaturated glycosides using microwaveinduced, InCl3-catalyzed Ferrier rearrangement of acetylglycals. This method provides an efficient alternative to the existing methodologies to synthesized 2,3-unsaturated C-glycosides.89 Misra and co-workers explore the potential of HClO4–SiO2, an inexpensive catalyst for the preparation of 2,3unsaturated C-glycosides through Ferrier rearrangement of glycals. They studied the use of HClO4–SiO2-catalyzed allylic rearrangement of acylated and alkylated glycals with silylated C-nucleophiles and active methylene compounds. The reaction proceeds smoothly at room temperature producing excellent yield of C-pseudoglycals, without the formation of any by-products.90 Triflate salts release trifilic acid, which catalyze the allylic rearrangement of acylated or alkylated glycals leading to the Ferrier rearrangement product.

1.16.2.6.2 Transition metal-catalyzed addition of carbon nucleophiles to glycals An interesting alternative for C-glycosylation of glycals consists in the use of palladium or palladium salts as catalysts. Following this approach, proper carbon nucleophiles are b-dicarbonyl compound91 and aromatic systems.54a In palladium-catalyzed reactions, different products (109–111) can be obtained, as the intermediate organopalladium adduct 108 decomposes in different ways92 (Scheme 25). Maddaford and co-workers described the synthesis of C-arylglycosides, using arylboronic acids as nucleophiles. These reagents are attractive for their air and moisture stability, availability, and low toxicity, and generate a wide variety of s-aryl-Pd complexes by transmetalation when treated with a palladium(II) salt under very mild conditions (Scheme 26). The efficiency of the transmetalation from boron to palladium was demonstrated in the cross-coupling reaction of organoboron compounds with organic electrophiles.93 The mechanism of the reaction is believed to involve a transmetalation of the phenylboronic acid to Pd(II)(AcO)2 to give PhPdOAc, which then undergoes syn-addition to the a-face of the glycal double bond followed by antielimination of palladium(II) acetate.


663

OAc O

AcO

Ar OAc

OAc

O

AcO AcO

109

105

OAc

O

AcO AcO

O

AcO AcO 108

Ar

Ar 110

Pd

OAc O

AcO

Ar 111

OAc

Scheme 25 C-Glycosylation of glycals using palladium catalysts.

PhB(OH)2

Pd(OAc)2

PhPdOAc + (OAc)B(OH)2

MeCN AcO

O

PhPdOAc AcO MeCN

AcO OAc

Ph

O

AcO

O

Ph + Pd(OAc)2

PdOAc

AcO

AcO

OAc

110

105 Scheme 26 An example of generation of a C-glycoside via a s-aryl-Pd complex.

O But Si O But

O

RCOOH DCC

OH 112

O

O But Si O But

Tebbe

But Si O But

O

O

113

O

R

O But Si O But

O

O 114

R

R

O O 115

Scheme 27 C-Glycosylation of a glycal via Claisen rearrangement.

A two-step process involving Tebbe methylation and thermal Claisen rearrangement to produce C-b-pseudoglycals has been reported.94 In this process, the hydroxyl group at C3 of 4,6-diprotected D-glucal 112 was esterified with different carboxylic acids to afford compounds 113, which were then submitted to Tebbe methylenation in order to generate the enol ethers 114 that underwent a Claisen rearrangement with formation of the 2,3-unsaturated C-glycosides (Scheme 27). The reaction mechanism implies that the orientation of the substituent at C3 is maintained at C1 after the rearrangement; therefore, the process is totally stereoselective.

1.16.2.6.3

Addition of carbon nucleophiles to glycals via 1,2-episulfonium intermediates

Treatment of glycals with arylsulfonyl chloride results in the formation of a 2-deoxy-2-arylthio-glycosyl chloride which in presence of Lewis acids generates a 1,2-episulfonium intermediate that can be attached by different nucleophiles, including carbon nucleophiles such as enol ethers, silyl enol ethers, or Grignard reagent (Scheme 28).95

664


RO

O

ArSCl

RO

O

RO

Cl

O SAr

RO

RO

SAr

RO

RO

C-Nu

O

Nu SAr

RO

OR

OR

OR

OR

116

117

118

119

Scheme 28 C-Glycosylation of a glycal via a 1,2-episulfonium intermediate.

BnO

O

O O

BnO

O

O C-Nu

BnO

BnO OBn 120

BnO

O

OH

BnO OBn 121

Nu

OBn 122

Scheme 29 C-Glycosylation of a glycal via the corresponding epoxide.

1.16.2.7 C-Glycosylation via 1,2-Anhydrosugars Among the many glycosyl donors that have been utilized in C-glycoside synthesis, 1,2-anhydrosugars (sugar epoxides) have received a significant amount of attention (Scheme 29). This is largely due to their utility in the synthesis of C-glycosides having a trans-relationship between the C2 hydroxyl group and the anomeric substituent. Furthermore, the experimental improvement in the synthesis of sugar oxiranes, which can be obtained by epoxidation of glycal with dimethyldioxirane under very mild experimental conditions, strongly enhanced the potentiality of this procedure.96a,96b This procedure allows to use the crude epoxidation product in the subsequent reaction, bypassing the isolation of relatively unstable epoxide prior to the addition of carbon nucleophiles. 1,2-Anhydrosugars (epoxides) have been reacted with organocuprates97a–97c to afford C-glycosides since 1982. There are few examples of reaction of glycal epoxides with other carbon nucleophiles such as Grignard reagents,98 organolithium reagents,98 sodium malonates and alkinylzinc reagents,99 allylstannanes,98 organoaluminum and organoboron reagents.100 The topic has been reviewed.

1.16.2.8 C-Glycosylation via Glycosyl Radicals Treatment of glycosyl halides with allyl-n-butylstannane results in the formation of a glycosyl radical that can be exploited in the synthesis of C-glycosides. The first example goes back to 1982 when Keck reported the C–C bond formation via the reaction of trialkylstannanes with different organic halides among which 2,3:4,6-diisopropylidene-a101 D-mannopyranosyl chloride. One year later, Baldwin102 reported the reaction of glycosyl radical, obtained from a 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl phenylselenide or bromide with methyl acrylate, and Giese103a,103b the reaction with acrylonitrile. Scheme 30 summarizes the most different approaches that have been reviewed in 1998.104 The glycosyl radical can be generated by irradiation or with radical promoters such as 2,20 -azobisisobutyronitrile (AIBN), starting from glycosyl halides, phenylselenides, methylthiocarbonates, or p-anisyltellurides. Allystannanes, allylthioethers, and variety of carbon–carbon double bonds activated with an electron-withdrawing group have been used as radical scavengers, including the glycoderivative 128 in order to afford the C-disaccharide 129.105a,105b One serious limitation of the procedure lies in the fact that an excess of scavenger is required in order that the reaction proceeds in acceptable yields. Taking into account that the intramolecular radical reactions are much more efficient, the scavenger has been attached to the hydroxyl group at C2 of the sugar.106a,106b This approach has been used by in the synthesis of C-disaccharides using temporay ketal107 or silylketal108a,108b connections between the two sugars. The stereochemistry of the reaction of glycosyl radicals is strongly influenced by the anomeric effect. Glucopyranosides and mannopyranosides afford stereoselectively the a-C-glycoside, whereas in furanosidic structures the stereochemistry is not always predictable. Little and Parrish used a titanocene-mediated ring opening of carbohydrate-derived epoxides to generate glycosyl radicals which in turn have been reacted with different radical-trapping agents among which is the N-Boc-protected 2-amino-2-propenoate, which affords a C-glycosyl amino acid 131 (Scheme 31).109


665

OP O PO Bu3Sn

125 OP O

OP O

123

CO2R

OP Bu3SnH

PO

O

OP O

124 HO

X = Cl, Br, I, SePh,

CO2R 126

CN

PO

Irradiation X or AIBN

PO

PO OH

Te-p-anisyl, O O

S

O 128

CN

127

OP

SMe

O

HO

PO

OH

129

O O

Scheme 30 General scheme of C-glycosylation via a glycosyl radical.

OBn

O O

BnO BnO

OBn

O

OBn O

BnO BnO

O 120

Cp2TiCl2, Mn

O BnO BnO Cp2ClTiO

121 NHBoc CO2Me OBn O

BnO BnO

OH 130

CO2Me NHBoc

Scheme 31 Generation of a glycosyl radical from the epoxide 121, and formation of a C-glycosyl aminoacid by reaction with N-Boc-protected 2-amino-2-propenoate.

The C-glycosylation via glycosyl radicals can be applied also to the synthesis of 2-amino-2-deoxy sugars. Taking into account that an easy way to generate 2-amino-2-deoxy sugars consists in the azidoseleniation of glycals, and that phenylseleno glycosides efficiently generate glycosyl radicals, Gallagher and co-workers110 proposed the procedure reported in Scheme 32.

1.16.2.9 C-Glycosylation via Glycosyl Anions The anomeric center of the sugars has an electrophilic character; however, many attempts have been made to reverse making it nucleophilic. The first problem that has been encountered in these attempts is the presence of a leaving group in b-position (usually an alkoxy or acyloxy group) that undergoes b-elimination if a carbanion is generated at the anomeric center. The elimination is prevented if the anomeric anion is strongly stabilized, for example, by a nitro group (Scheme 33),111a–111c or in presence of a bad leaving group, such as the anions –O–112a,112b or –N-Ac–,113 generated from a free hydroxyl group or an acetamido group at C2 (Scheme 34), and obviously in 2-deoxy sugars.

666


(PhSe)2, NaN3, PhI(OAc)2

AcO OAc O AcO

AcO OAc O AcO N3 SePh

105

131

AcO OAc

CO2R

O

Reduction N-protection

AcO OAc O

AcO AcO

AcHN CO2R

133

AcHN 132

SePh

Scheme 32 Generation of N-acetylgalactosamine C-glycoside from triacetyl galactal.

OBn

i, HCHO, K2CO3

O

BnO BnO

OBn

OBn

O

BnO BnO

NO2 ii, Ac2O

NO2 OBn

134

OAc

135

Scheme 33 The presence of a nitro group at the anomeric position allows the formation of a glycosyl carbanion preventing the b-elimination.

i, SOCl2 ii, Bu3SnH iii, BuLi

OBn O

BnO BnO

OH NHAc

OBn O

BnO BnO

Li NLiAc

136 CO2

OHC

OBn COOH NHAc 137

NHCbz

OBn O

BnO BnO

O

BnO BnO

CO2Me

OH CO2Me

NHAc 138

NHCbz

Scheme 34 Example of anomeric metalation, avoiding b-elimination, in the generation of C-glycosides.

Anomeric metalations have been effected in different ways, starting generally from glycosyl halide or from glycals, that were converted into glycosyl-SnBu3,114a,114b glycosyl-SO2Ph,115 glycosyl-SO2Py, glycosyl-Mn(CO)5,116 that reacted with carbon electrophiles such as aldehydes or a,b-unsaturated carbonyl compounds, usually by in situ conversion into the more reactive organolithium reagent. Glycosyl-SO2Py allows the in situ reaction with a carbon electrophile catalyzed by samarium iodide.117a–117d This is probably the most efficient method to generate C-glycosides, including 2-amino-2-deoxy-C-glycosides and C-disaccharides (Scheme 35), exploiting a C-glycosyl nucleophilic intermediate. The synthesis of C-disaccharides using this and other methods has been reviewed by Postema et al.20f and the carbanionic reactivity of the anomeric center of carbohydrates by Somsa`k.118

1.16.2.10 C-Glycosylation via Ramberg–Backlund Reaction Reaction of a protected aldose 144 with sulfolylphosponate reagent 145 in presence of NaH results in the formation of the C-glycosyl sulfone 146, whereas with reagent 145 affords the glycoside 148 that can further react with an aldose to


OAc

OAc

OAc O

AcO AcO AcHN

PySH

MCPBA

O

AcO AcO

SPy

O

AcO AcO

SO2Py

NHAc

Cl

141

140

139

667

NHAc

SmI2

OAc

OHC O

O

AcO AcO AcHN

OH O

BnO BnO

OH O

BnO BnO

142 OMe

OMe

143

Scheme 35 Synthesis of a C-disaccharide exploiting the SmI2-catalyzed reaction of a glycosyl pyridylsulfone.

OP

O2 S

(EtO)2OP

O

145

PO

OP

Ph

OH

O

O2 S

PO

Ph

NaH

144 (EtO)2OP

O2 S

147 PO(OEt)2

KOH

146

OP O PO

O2 S

OP O PO

PO(OEt)2

Ph

148

149 144

OP O PO

OP

OP O2 S

KOH O

OP

150

OP O

PO

O

OP

151

Scheme 36 C-Glycosylation via Ramberg–Backlund reaction.

afford the C-disaccharide 150. Ramberg–Backlund rearrangement of sulfones 147 and 150 generates the C-glycoside 149 and the C-disaccharide 151. Following this approach, different C-glycosides and C-disaccharides have been synthesized (Scheme 36).119a–119c

1.16.3 Procedures of S-Glycosylation The chemical synthesis of S-glycosides has been reviewed by many authors. In this chapter, we focus on the most important general methods, and examples reported are focused on the synthesis of non-natural thioglycosides of biological interest. Several methods have been published for the synthesis of thioglycosides. These include the glycosylation of thio-acceptors with activated glycosyl donors, such as trichloroacetimidates, an SN2-type reaction involving the displacement of a good leaving group by a 1-thio-glycopyranose or the nucleophilic attack of a thiolate anion on a glycosyl halide, the Michael-like addition of a 1-thiosugar to an a,b-unsaturated system. Recently, a new approach based on the enzymatic synthesis of thioglycosides, exploiting engineered glycosidases, has been introduced.

668


1.16.3.1 S-Glycosylation by Acid-Promoted Displacement at the Anomeric Center of Glycosyl Donors A classical method for glycosyl donor activation is the preparation of the corresponding trichloroacetimidates; it can also be applied to the synthesis of thioglycosides. In Scheme 37, two glycosylation reactions for the preparation of thio-analogs of kojibioside, employing glucopyranose trichloroacetimidates as donors, activated with a Lewis acid (TESOTf ), and a 2-thio-glucopyranoside as acceptor, are reported.120 The use of both the benzilated a- and b-glucopyranose trichloroacetimidate resulted in a highly stereoselective glycosylation reaction that afforded exclusively the a-anomer, while the acetylated a-trichloroacetimidate donor gave a mixture of a- and b-anomers. Also 1,6-anhydro derivatives of mono- and disaccharides can be used as glycosyl donors. With this approach, Wang et al.121 achieved, under acidic conditions, the disaccharide 159, a precursor for the synthesis of 4-thiomaltose (Scheme 38). A mixture of the a-linked and the b-linked isomers was also obtained in this case. The same procedure, applied to the disaccharide 160 as acceptor, gave rise only to the a-linked thio-trisaccharide (Scheme 39).

O

BnO BnO

OBn

OBn

OBn OAll SH 152

TESOTf

O

+ BnO BnO

CCl3 OBn O 153

NH

O

BnO BnO

CH2Cl2 −40 ⬚C to −10 ⬚C

OBn S AllO

OBn OBn

O OBn

154

O

BnO BnO

OAc

OAc

OBn OAll

+

SH

AcO AcO

152

TESOTf

O OAc O 155

CH2Cl2 −40 ⬚C to −10 ⬚C

CCl3

OBn OBn

O

AcO AcO

OAc

OBn

S O

156 OAll

NH

Scheme 37 Glycosylation reactions for the synthesis of a thio analog of kojibioside using the trichloroacetimidate method.

OH OBn O OBn

O

OBn i, ZnI2 CH2Cl2

O

TMSS + BnO

OBn OMe 158

OBn 157

BnO BnO

ii, K2CO3 MeOH

O OBn

OBn O

S BnO

OBn OMe

159

Scheme 38 Glycosylation reaction for the synthesis of a thio analog of maltose using a 1,6-anhydro derivate of glucose as donor.

OH O

OBn O OBn

OBn +

TMSS BnO

OBn O

OBn 157

BnO i, ZnI2 BnO CH2Cl2

O OBn O

160

ii, K2CO3 MeOH O

O OBn S BnO 161

OBn O OBn O

OBn O

BnO

O BnO

Scheme 39 Glycosylation reaction for the synthesis of a trisaccharide containing a thio linkage using a 1,6-anhydro derivate of glucose as donor.


OAc

OAc O

AcO AcO

669

i, PhSH, BF3 OEt2 •

OAc OAc 162

O

AcO AcO

ii, PhSH, InCl3 (20 mol.%), TiCl4 (0.2 equiv.) in AcCN

SPh

OAc 163

Scheme 40 Synthesis of a thiophenyl glucoside starting from an acetate glycosyl donor.

(AcO)n

(HO)n Ac2O, Cu(OTf)2

O

164

OH

(AcO)n Thiol

O

0 8C to RT 165

OAc

O

BF3 • OEt2, RT 166

SR

Scheme 41 One-pot synthesis of per-O-acetylated thioglycosides from unprotected reducing sugars.

It has been demonstrated that this reaction does not work when 1,6-anhydro-di-and-trisaccharides are employed as glycosyl donors. Another possibility is represented by the use of protected sugars bearing anomeric acetates, under Lewis acid catalysis. One of the first examples was reported by Ferrier and Furneaux122 and concerns on the synthesis of a thiophenylglucoside (i, Scheme 40). Recently, Das et al. described the use of indum(III) halides, in particular InCl3, with TiCl4 as co-activator, as new alternative promoter for the highly yielding synthesis of thioglycosides starting from acetate donors (ii, Scheme 40).123 In 2003, Tai and co-workers proposed the one-pot synthesis of per-O-acetylated thioglycosides from unprotected reducing sugars (Scheme 41).124 In this method, the use of a stoichiometric quantity of acetic anhydride, with the addition of catalytic amount of Cu(OTf )2, avoiding the neutralization of reagent in excess followed by work-up and purification before the second step, makes possible the sequential per-O-acetylation-anomeric thiolysis in order to obtain a per-O-acetylated thioglycoside in good yields by a one-pot procedure. According to the experimental data reported, in most of the case, only the 1,2-trans-thioglycoside was recovered, as a consequence of the neighboring group participation of the acetyl group present on C2. A similar result was achieved by Mukhopadhyay et al. that substituted Cu(OTf )2 with iodine as catalyst for the per-O-acetylation step.125 Agnihotri and co-workers presented a variation of this procedure that employed BF3OEt2 as catalyst for both acetylation and thiolysis reaction.126

1.16.3.2 S-Glycosylation via SN2 Reaction of 1-Thiosugar Donors with Activated Acceptors This method takes advantage of the fact that sulfur atom is less basic but more nucleophilic than oxygen. As example, the synthesis of methyl 4-deoxy-4-thio-maltoside 170 and methyl 4-deoxy-4-thio-cellobioside 173 (Scheme 42) performed by Driguez and co-workers is illustrated.127a,127b 1-Thiolates 168 and 172 reacted with the proper acceptor 169 affording, through a SN2 mechanism, the thiodisaccharides 170 and 173 with a thio-a and thio-b linkage, respectively, depending on the stereochemistry of the donor. The anomeric S-alkylation strategy was also used by Rye and Withers that synthesized a thiodisaccharide based on the structure of the glycosaminoglycan chondroitin (Scheme 43).128 This compound was designed as potential inhibitor of chondroitin AC lyase from Flavobacterium heparinum, useful for the structural analysis of the enzyme active site. A method for the exclusive preparation of 1,2-trans-thioglycosides uses S-glycosyl isothiouronium salts 180 for the introduction of the thio function in the anomeric position. S-glycosyl isothiouronium salts are synthesized from glycosyl halides by treatment with a thiourea (Scheme 44).129a–129d These compounds present the additional advantage that, in contrast with glycosyl halides from which they are generated, they are stable solids. Falconer and co-workers demonstrated that thioglycosides can be obtained reacting a 1-thiosugar and an alcohol via a Mitsunobu condensation, obviously with retention of configuration at the anomeric center (Scheme 45).130

670


OAc O

AcO AcO

OAc SAc MeONa MeOH

167 OH

168

O BzO

O

HO HO

OH

S−

OH

OTf OBz

OBz 169 OMe

O

HO HO

OH OH

O

S HO

i, HMPA ii, Ac2O in pyridine iii, MeONa (cat.) in MeOH

OH OMe

170

OAc O

AcO AcO

SAc 171

OAc MeONa MeOH

OTf OBz O

BzO

OH O

HO HO

S− OH 172

OBz OMe 169

i, HMPA ii, Ac2O in pyridine iii, MeONa (cat.) in MeOH

OH O

HO HO

OMe OH

HO S

O

OH OH 173

Scheme 42 Synthesis of methyl 4-thio-maltoside and methyl 4-thio-cellobioside via an SN2 reaction employing a 1-thiodonor.

OAc OAc

OAc OAc O AcO 174

NHAc

O

NaH SH THF, 0 ⬚C to RT

S−

AcO 175

NHAc

i, NaH in THF, 0⬚C to RT ii, DMF OTf AcO i, DMF ii, 10% TFA in CH2Cl2 iii, CrO3, H2SO4, acetone Water, sonication, 358C iv, MeONa in MeOH

OH OH O HO

S NHAc HO 178

COOH O OBn OH

176

OAc OAc O AcO

OPMB O OBn OAc

S NHAc AcO 177

OPMB O OBn OAc

Scheme 43 Synthesis of a thio-linked analog of chondroitin using a 1-thiolate glycoside as donor.

1.16.3.3 S-Glycosylation via SN2 Reaction of Thiolate Anions on Glycosyl Halides This method is based on the displacement of an electrophilic group at the anomeric position of the donor with a thiolate present on the acceptor. For comparison, the synthesis of methyl 4-deoxy-4-thio-maltoside and methyl 4-deoxy-4-thiocellobioside, previously described for the case of thiodonors, is presented.131 The thiodisaccharides 170 and 173 show a thio-a and a thio-b linkage, respectively, which are opposite to the stereochemistry of the donor (Scheme 46). This kind of S-glycosylation offered an alternative approach to the synthesis of the Neu5Aca2-3-S-Gal 189 (Scheme 47), as described by Schmidt et al.132a,132b

671


S OAc

OAc H2N

O

AcO AcO

OAc Br 179

NH2

Acetone

O

AcO AcO

+

NH2 Br −

S

OAc

NH2

180 K2CO3 aq.

OAc

OAc MeI, DIPEA, Et3N

O

AcO AcO

SMe

OAc 182

O

AcO AcO

OAc

SH

181

Scheme 44 Synthesis of thioglycoside employing an S-glycosyl isothiouronium salt.

R1 OAc

R1 OAc ADDP, Me3P

O

R2 AcO

R3

+ R OH

SH

THF

183a: R1 = H; R2 = R3 = OAc 183b: R2 = H; R1 = R3 = OAc 183c: R1 = H; R2 = OAc; R3 = NHDde

O

R2 AcO

SR

R3

184a: R1 = H; R2 = R3 = OAc 184b: R2 = H; R1 = R3 = OAc 184c: R1 = H; R2 = OAc; R3 = NHDde

Scheme 45 Use of Mitsunobu condensation for the synthesis of thioglycosides.

OH OAc

OAc O

AcO AcO

Cl 185

O

+ HS AcO

OAc 186

OAc AcO AcO

i, MeONa in MeOH ii, HMPA iii, Ac2O in pyridine iv, MeONa in MeOH

OAc Br 179

+ HS AcO

OH OH

i, MeONa in MeOH OH ii, HMPA iii, Ac2O in pyridine iv, MeONa in MeOH HO HO

O OAc OMe 186

O

S HO

OAc OMe

OAc O

O

HO HO

OH OMe

170

O

OMe OH

HO S

O

OH OH

173

Scheme 46 Synthesis of methyl 4-deoxy-4-thio-maltoside and methyl 4-deoxy-4-thio-cellobioside via an SN2 reaction employing a thio acceptor.

Ph

AcO

OAc OAc

Cl O

AcHN AcO 187

O

O O

CO2Me + HS

OBz

i, NaH, Kriptofix 21 THF, 75% OAc OAc ii, TFA, H2O:Ac2O AcO Pyr, 89% AcHN OTDS AcO

188

CO2Me O

189

S

OAc OAc

BzO

O OTDS

Scheme 47 Synthesis of Neu5Aca2-3-S-Gal via an SN2 reaction employing a thio acceptor.

672


Neu5Aca2-3Galb1-4(Fuca1-3)GlcNAc, the Lewis X epitope, is involved in inflammatory processes. The insertion of a thioglycosydic linkage in the Neu5Aca2-3Gal moiety increases the metabolic stability maintaining the biological activity. Another example is the synthesis of 4-deoxy-4-thio-lactoside (Scheme 48) described by Goodman et al., which used the approach already described.133 An interesting variation of this method uses of disulfide sugars as glycosyl acceptors. Schmidt and co-workers applied this strategy to the synthesis of b1-4-linked thiodisaccharides (Scheme 49).134 The reaction of disulfide 192 with acetobromogalactose 190 in presence of a mixture of Hg(CN)2 and HgBr2 achieved the thiodisaccharide 193 in 50% yield, with additional formation of the trisaccharide 194. According to the proposed model, cyanide opens the disulfide bond, with the generation of a thiolate able to react with the anomeric position of the glycosyl donor substituting the bromide by an SN2 mechanism; as a consequence, in this case also, the thioglycosidic bond formation resulted as diastereospecific, affording the b-linkage. Very recently, Kumar et al. developed a new one-pot strategy for the synthesis of thioglycosides in excellent yield through in situ generation of per-O-acetylated glycosyl bromides.135 As pictured in Scheme 50, the use of stoichiometric quantity of acetic anhydride in the presence of HBr/AcOH (30%) allows the preparation of a per-O-acetylated glycosyl bromide directly from a free sugar, that is then submitted to a phase transfer-catalyzed anomeric thiolysis by addition of the proper thiol in order to achieve the target thioglycoside.

OAc OAc

OAc O

AcO

i, MeONa in MeOH ii, HMPA iii, Ac2O in pyridine iv, MeONa in MeOH v, BzCl in pyridine

OAc Br 190

O

+ HS AcO 186

OMe OH

OH OH O HO

OAc OMe

HO S

O

OH 191

OH

Scheme 48 Synthesis of methyl 4-deoxy-4-thio-lactoside via an SN2 reaction employing a thioacceptor.

S

S

OAc OAc O +

HO AcHN 192

O AcO

O

190

OAc Br

Hg(CN)2, HgBr2 Toluene, nitromethane OAc OAc

OAc OAc

O O

AcO

AcO

S SCN OAc

O

+

HO 193

AcHN

AcO

S SCN OAc OAc O

O O

OAc

O

AcO

AcHN

O

194

Scheme 49 Synthesis of a b1-4-linked thiodisaccharide using disulfide sugars.

(HO)n O

164

Ac2O

(AcO)n

(AcO)n Thiol, TBAHS

O

O SR

33% HBr/AcOH, 0 8C to RT OH

CH2Cl2/sat. NaHCO3 Br

196

195 Scheme 50 One-pot synthesis of per-O-acetylated thioglycosides from unprotected reducing sugars under phase transfer reaction conditions.


673

The products were obtained with complete inversion of stereochemistry. This procedure can be applied also to the synthesis of glycosyl azides.

1.16.3.4 S-Glycosylation via Michael Addition of 1-Thiosugars to Unsaturated Acceptors This approach, introduced in 1995 by Witczak et al.,136 consists in the stereoselective Michael addition of a 1-thiosugar to the a,b-conjugated system of a convenient levoglucosenone, thus achieving the formation of an 1,4-thio linkage between two sugars. Here, we report the synthesis of L-fucopyranosyl-4-thiodisaccharides that present inhibitory activity on a-L-fucosidase (Scheme 51). Uhrirg and co-workers employed this methodology for the synthesis of 3-deoxy-4-S-(1!4)-thiodisaccharides 208–211 that showed b-glycosidase inhibitory activity.137 2,3,4,6-Tetra-O-acetyl-1-thio-b-galactose 205 or 2,3,4,6tetra-O-acetyl-1-thio-b-glucose 181 were coupled to (2S,6S)-6-acetoxymethyl-2-(2-propyloxy)-2H-pyran-3(6H)-one 204, using a catalytic amount of triethylamine. Subsequent reductions with sodium borohydride of the crude ulosides led to two pairs of isomers 208–211 (Scheme 52). Compounds 208 and 210 were tested as inhibitors of a b-galactosidase from Escherichia coli. Compound 208 resulted in a competitive inhibitor with Ki ¼0.16mM and Km ¼1.17mM, while compound 210 is a mixed-type inhibitor with Ki ¼0.12mM. In contrast, thiodisaccharides 209 and 211 did not show any b-glucosidase inhibitory activity.

1.16.3.5 Enzymatic S-Glycosylation: The Use of Thioglycoligases All routes for chemical thioglycosides synthesis previously described require different protection and deprotection steps and a good control of anomeric stereochemistry. A suitable and very interesting option is the use of enzymatic activities able to catalyze the formation of an S-glycosidic bond. A new approach for the synthesis of (1-4)-b-Sdisaccharides138a,138b explores the use of thioglycoligases, the alanine acid/base mutants of two retaining b-glycosidases, the b-glucosidase from Agrobacterium sp. Abg E171A (Scheme 53a) and the b-mannosidase from Cellulomonas fini Man2 E429A (Scheme 53b). This strategy employs unprotected activated donor glycosides, in particular dinitrophenyl glycosides, and deoxythiosugars as acceptors. O O

O

SH O AcO OAc 197

198

OAc

O

O

Et3N, benzene

O

S O

AcO OAc i, L-selectride 199 ii, BF3OEt/Ac2O

OAc i, NH2OH:HCl/EtOH/pyridine ii, Ac2O, pyridine O

OH

O AcO OAc

O

O

S OAc 200

OAc

OAc O

MeOH/H2O/Et3N

NOAc

S

AcO OAc

202 i, 9-BBN/H2O2/NaOH ii, BF3OEt/Ac2O iii, MeOH/H2O/Et3N

OAc

OH

O HO OH

OH O

S

OH

S

OH O

OH 201

HO OH

O OH NHAc

OH 203

Scheme 51 Synthesis of S-disaccharides inhibitors of a-L-fucosidases via Michael addition.

674


R1

OAc O

O

O

R2 AcO

+

R1 OAc

OAc Et3N

SH

O

R2 AcO

OAc

S OAc

OAc

O

OCHMe2 205: R1 = OAc; R2 = H 181: R1 = H; R2 = OAc

204

206: R1 = OAc; R2 = H 207: R1 = H; R2 = OAc

O

OCHMe2

NaBH4, MeOH, 0 ⬚C R1 OH R2 HO

O S OH

OH R3

O

R4 R1

R2

R3

R4

208: = OH; = H; = H; = OH 209: R1 = H; R2 = OH; R3 = H; R4 = OH 210: R1 = OH; R2 = H; R3 = OH; R4 = H 211: R1 = H; R2 = OH; R3 = OH; R4 = H

OCHMe2

Scheme 52 Synthesis of b-glycosidase inhibitors via addition of 2,3,4,6-tetra-O-acetyl-1-thio-b-galactose or glucose to sugar-derived (2S,6S)-6-acetoxymethyl-2-(2-propyloxy)-2H-pyran-3(6H)-one used as Michael acceptor.

OH

OH O

HO HO

+

ODNP

O

HS HO

OH 212

OH

OpNP

213 i, Abg E171A, phosphate buffer, pH 6.8 ii, Ac2O, pyr OAc

OAc AcO AcO

O

O

S AcO

OpNP

OAc

OAc

214

(a) OH HO HO

OH

OH O ODNP

+

O

HS HO

215

OH

OpNP

213 i, Man2A E429A, phosphate buffer, pH 6.8 ii, Ac2O, pyr OAc

AcO AcO (b)

OAc OAc O

S AcO

O OpNP OAc

216

Scheme 53 Enzymatic synthesis of (1-4)-thiosaccharides catalyzed by engineered thioligases Abg E71A and Man2A E429A.


OH

OH O

HO HO

675

OH

O

HS HO

+

OpNP

OH

F

217

213 Abg E171A, E358G, pH 7.0 OH

OH O

HO HO

OH

O

S HO

OpNP OH

218 Scheme 54 Enzymatic synthesis of (1-4)-thiosaccharides catalyzed by the double mutant Abg E71A E358G.

OAc

OAc

OAc O

AcO AcS

213

OAc

SPh

219

AcO AcO

i, NaOMe, MeOH ii, E171A, 80 mM phosphate buffer pH 7.4 iii, Ac2O, pyr OAc

SAc

213 O

AcO AcO

O

SPh

O SPh OAc

O

AcO AcO

OAc 221

AcO S OAc 220

222

S OAc AcO AcO

O SPh OAc

Scheme 55 Enzymatic synthesis of (1-3)- and (1-6)-thiosaccharides catalyzed by thioligase Abg E71A.

Also, the double mutant (acid/base and nucleophile) Abg E71A E358G was developed.139 It efficiently catalyzes thioglycoside formation from a glycosyl fluoridine donor with inverted anomeric stereochemistry (Scheme 54). This represents a significant improvement, because glycosyl fluoride donors are more easily synthesized than dinitrophenyl ones. In addition, whereas acid/base mutant slowly hydrolyzes the donor, the acid/base and nucleophilic mutant is completely hydrolytically inactive. More recently, Stick and co-workers140 extended the use of this methodology to the synthesis of (1-3)- and (1-6)-bS-linked disaccharides (Scheme 55). In principle, this enzymatic methodology could allow the synthesis of thioglycosidic linkages in oligosaccharides under mild, aqueous conditions.

1.16.3.6 Synthetic Applications of S-Glycosides 1.16.3.6.1

Thioglycosides as glycosyl donors

Thioglycosides are largely employed as glycosyl donors for the synthesis of oligosaccharides. This is the consequence of the fact that the thioacetal function can act at the same time as an anomeric protecting group and as an efficient leaving group. In fact, the sulfur atom is a soft nucleophile and can react with soft electrophiles such as halogens, alkylating and acylating reagents, and heavy metal cations. In contrast, the oxygen atom of the hydroxyl groups is a hard nucleophile and shows a good reactivity mainly with hard electrophiles. Thioglycosides offer sufficient temporary protection of the anomeric position, showing a good stability toward a wide range of reaction conditions, thus allowing the introduction of many different functional and protecting groups on the alcoholic functions of sugars. Moreover, there are several alternative methods, based on the use of electrophilic reagents, for thio function activation in order to obtain a glycosylating species.

676


Taking advantage of these features, thioglycosides play a crucial role in different sequential glycosylation routes.141a,141b The versatility of thioglycosides allowed the development of chemoselective, orthogonal, and iterative glycosylation strategies recently reviewed by van der Marel et al.142

1.16.3.6.2 Toward the synthesis of glycoproteins: Synthesis of S-linked glycoconjugates Glycoproteins are involved in a number of biological processes, such as cell adhesion, cell differentiation, signal transduction, host–pathogen interactions, and immune response.143a–143f Chemical synthesis of glycosylated proteins is thought to be a useful tool to study and understand glycan function and to obtain new potential therapeutic agents. In the light of these considerations, the development of new methodologies for the synthesis of natural and unnatural glycoproteins (neoglycoproteins) arouses great interest. Glycopeptides occurring in Nature present N-glycosidic or O-glycosidic linkages between the side chain of an appropriate amino acid and the carbohydrate moiety. The substitution of the anomeric nitrogen or oxygen atom by sulfur achieves the corresponding thioglycopeptides. This kind of carbohydrate–protein linkage, tolerated by most biological systems, is more stable to chemical and enzymatic degradation and allows the synthesis of thio-oligosaccharides employed as enzyme inhibitors,144a,144b which are considered better immunogens than the natural ones.145a–145c The most common synthetic procedure for thio-linked glycosyl amino acids is based on the reaction between an anomeric thiolate and an aniline residue functionalized with a good leaving group (Scheme 56). In this approach, a b-elimination side reaction, followed by a Michael addition, can occur, affording a diasteroisomeric mixture at the a-carbon of the amino acid obtained. Schmidt and co-workers proposed an alternative method in order to overcome this drawback.146 They performed the reaction in an ethyl acetate/water two-phase system, in the presence of tetrabutylammonium hydrogen sulfate (TBAHS) and NaHCO3, or in a one-phase system of dimethylformamide (DMF)/water, in the presence of NaHCO3, using b-bromoaniline-containing peptides. The thiolate anion is generated in situ by NaHCO3 action and the a-thioglycoside is formed by nucleophilc displacement of the b-bromo leaving group. The use of a solution of NaHCO3 at pH 8.5 reduced drastically the risk of the bromoaniline derivatives undergoing b-elimination. At the same time, Davis et al. developed a very original two-step strategy, in order to perform a one-pot site-selective protein glycoconjugation (Glyco-SeS).147 According to this procedure, a cysteine residue is incorporated into the protein backbone in a specific position. The protein is then activated to thioglycosylation with the transformation of this cysteine thiol function into a phenylselenyl sulfide by treatment with phenylselenenyl bromide. The electrophilic X R2HN

RS

RS-

CO2R1

R2HN

223

CO2R1 224

Scheme 56 The most common synthetic approach for the synthesis of S-linked glycosyl amino acid.

+ S SH

PhSe

225

226

S 227 Scheme 57 Protein glycosylation via Glyco-SeS strategy.


(OH)n

(OH)n O

LR

(OH)n SBL-Cys156-SePh

O

677

SBL-Cys156-SePh O S S

OH 228

SH 229

230

Scheme 58 Two-step procedure for direct SBLCys156-SePh conjugation to a deprotected reducing sugar through LR and Glyco-SeS.

character of the sulfur atom in the resulting S–Se bond makes possible its nucleophilic substitution by 1-thio mono- or oligosaccharides (Scheme 57). With the aim to apply this strategy on oligosaccharides directly isolated from natural sources, they tried to develop a chemo- and regioselective procedure for the thionation of the anomeric center in the presence of other functional groups. In particular, they investigated the use of Lawessons’s reagent (LR),148 achieving a two-step strategy for the direct protein glycoconjugation, starting from deprotected reducing sugars (Scheme 58).149 This procedure was tested on different sugars, among which are the oligosaccharides isolated from N- and O-glycoproteins occurring in Nature. They were treated with LR and then submitted to reaction with SBLCys156SePh, a selenenylsulfide-activated mutant subtilisin from Bacillus lentus, giving rise to the corresponding glycoproteins with percentages of conversion superior to 95%.

1.16.4 Conclusions The chapter shows how relevant glycomimetics such as C-glycosides and S-glycosides are in the development of metabolically stable inhibitors, agonist, and antagonist, and how many efficient and various synthetic methods have been developed for their generation. Some problems of control of the stereochemistry still remain, but undoubtedly, taking advantage of the many synthetic methods reported, it is now possible to obtain almost all the desired C-glycosides and S-glycosides. It is expected therefore that these glycomimetics will not only help the scientists in spreading light on the complex and multifarious physiological roles of carbohydrates, but also in finding carbohydrate-based new drugs.

Glossary aglycon A not glycidic compound linked to a sugar. agonist A molecule that performs the same physiological effect as a reference drug or a natural regulator. anomeric center The carbon atom involved in the glycosidic linkage. antagonist A molecule that prevents the physiological effect of a reference drug or a natural regulator. C-glycosylation A process in which the anomeric center of the sugar links a carbon atom instead of on oxygen atom. chemoselective ligation A process that allows to link selectively two molecules involving two functional groups that react without requiring the protection of the other functional group present in the molecule. dendritic cell Cells responsible for the immunoresponse. epitope Part of a molecule that generate an immunoresponse. glucosinolate Natural glycoconjugates present in Brassicaceae that presents S-glycosidic linkage. glycomimetic A compound that mimes the structure and/or the functional group distribution of a natural saccharidic structure. glycosidases Enzymes involved in the hydrolysis of the glycosidic linkage. glycosyltransferases Enzymes involved in the formation of the glycosidic linkage. myrosinase An enzyme that hydrolyzes glucosinolates. selectin Receptorial protein that recognizes and links carbohydrate ligands. S-glycosylation A process in which the anomeric center of the sugar links a sulfur atom instead of on oxygen atom.

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140. Sick, R. V.; Stubbs, K. A. Tethraedron Asymmetry 2005, 321–335. 141a. Pachmathu, K.; Schmidt, R. Chem. Rev. 2006, 106, 160–187. 141b. Codeé, J. D. C.; van den Bos, L. J.; Litjens, R. E. J. N.; Overkleeft, H. S.; Van Boom, J. H.; van der Marel, G. A. Org. Lett. 2003, 5, 11, 1948–1950. 142. Codeé, J. D. C.; Litjens, R. E. J. N.; van de Bos, L. J.; Overkleeft, H. S.; van der Marel, G. A. Chem. Soc. Rev. 2005, 34, 769–782. 143a. Varki, A. Glycobiology 1993, 3, 97–130. 143b. Dwek, R. A. Chem. Rev. 1996, 96, 683–720. 143c. Rudd, P. M.; Elliot, Y.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370–2376. 143d. Davis, B. G. Chem. Rev. 2002, 102, 579–601. 143e. Lowe, J. B.; Marth, J. D. Annu. Rev. Biochem. 2003, 72, 643–691. 143f. Doores, K. J.; Gamblin, D. P.; Davis, B. G. Chem. Eur. J. 2006, 12, 656–665. 144a. Driguez, H. Top. Curr. Chem. 1997, 187, 85–116. 144b. Witczak, Z. J. Curr. Med. Chem. 1999, 6, 165–178. 145a. Bousquet, E.; Spadaro, A.; Pappalardo, M. S.; Bernardini, R.; Romeo, R.; Panza, L.; Ronsisvalle, G. J. Carbohydr. Chem. 2000, 19, 527. 145b. J. R. Rich, J. R.; Bundle, D. R. Org. Lett. 2004, 6, 897–900. 145c. Kuberan, B.; Sikkander, S. A.; Tomiyama, H.; Linhardt, R. J. Angew. Chem. 2003, 115, 2119–2121. 146. Zhu, X.; Schmidt, R. R. Chem. Eur. J. 2004, 10, 875–887. 147. Gamblin, D. P.; Garnier, P.; van Kasteren, S.; Oldham, N. J.; Fairbanks, A. J.; Davis, B. G. Angew. Chem., Int. Ed. 2004, 43, 828–833. 148. Pedersen, B. S.; Scheibye, S.; Nilson, W. H.; Lawesson, S. O. Bull. Soc. Chim. Belg. 1978, 87, 223–228. 149. Bernardes, G. J. L.; Gamblin, D. P.; Davis, B. G. Angew. Chem., Int. Ed. 2006, 45, 4007–4011.

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Biographical Sketch

Francesco Nicotra was born in 1950 in Catania, Italy, where he received his bachelor’s degree in chemistry in 1973. He is actually full professor of organic chemistry at the University of Milano-Bicocca and director of the Department of Biotechnology and Biosciences. He is the Italian representative in the International Carbohydrate Organisation and the European Carbohydrate Organisation, member of the Committee of IUPAC Division III, Organic and Biomolecular Chemistry, and chairman of the subcommittee of biotechnology of the same division. Prof. Nicotra has been involved in the organization of numerous scientific events as organiser and member of the international scientific committee. He has presented numerous plenary and keynote lectures in international conferences and has published over 120 papers in international journals, 9 chapters of books, 3 books, and 4 patents. Prof. Nicotra is editor for the UNESCO encyclopedia EOLSS (www.eolss.net) of the theme ‘Organic and Bio-Molecular Chemistry’. The research interests range across the synthesis of various biologically active compounds, in particular carbohydrates and structural analogs, the development of new biomaterials, synthetic methods, and the use of biocatalysis.

Cristina Airoldi was born in 1979 in Bergamo, Italy. She received her bachelor’s degree in biotechnology in 2003 at the University of Milano-Bicocca, where she is finishing her Ph.D. in chemistry under the supervision of Prof. Francesco Nicotra. Her research interests concern the synthesis of carbohydrate-based compounds presenting biological activity and NMR studies of their interaction with targeted receptors.


683

Francisco Cardona was born in 1979 in Covilha˜, Portugal, where he received his bachelor’s degree in chemistry in 2004. He is actually doing a Marie Curie Fellowship under the supervision of Prof. Francesco Nicotra at the University of Milano-Bicocca, Department of Biotechnology and Biosciences. His research interests are based on the synthesis of carbohydrate-based compounds such as glycodendrimers, glycopeptides, and glycoproteins.

1.17 Synthesis of Lipopolysaccharide, Peptidoglycan, and Lipoteichoic Acid Fragments S. Kusumoto, Suntory Institute for Bioorganic Research, Osaka, Japan K. Fukase and Y. Fujimoto, Osaka University, Osaka, Japan ß 2007 Elsevier Ltd. All rights reserved. 1.17.1 Introduction

685

1.17.2 Synthesis of Peptidoglycan

685

1.17.3 Synthesis of Lipid A and Lipopolysaccharide

691

1.17.3.1 Synthesis of Lipid A, the Lipophilic Component of Lipopolysaccharide

692

1.17.3.1.1 1.17.3.1.2 1.17.3.1.3 1.17.3.1.4

The first synthesis of natural-type lipid A Synthesis of lipid A analogs via improved routes Synthesis of phosphonooxyethyl analog of lipid A Synthesis of lipid A containing an unsaturated fatty acyl group

692 693 696 697

1.17.3.2 Synthesis of Re-Type Lipopolysaccharide

699

1.17.4 Synthesis of Lipoteichoic Acid

702

1.17.4.1 Synthesis of Kojibiose-Containing LTA

702

1.17.4.2 Synthesis of Gentiobiose-Containing LTA

705

1.17.5 Closing Remarks

706

1.17.1 Introduction The surface of a bacterial cell is covered by a stable layer called cell envelope. The major constituents of this envelope are macromolecular glycoconjugates which are characteristic of bacteria in terms of both their building molecules and structural architecture. Typical of such cell surface glycoconjugates are peptidoglycan (PGN), lipopolysaccharide (LPS), and lipoteichoic acid (LTA), which constitute the theme of this chapter. These complex molecules are essential for the survival of bacteria as they form a strong structure which maintains the cell shape and protects the life process carried out inside: the cytoplasmic cell membrane alone is too fragile to protect the cells from osmotic and other mechanical stresses.1,2 Among the three mentioned above, PGN is common to both Gram-positive and-negative microbes with some minor structural variations. By contrast, LPS and LTA are ubiquitous but typical components of Gram-negative and-positive cells, respectively. Intensive chemical synthesis of PGN and LPS, and later LTA as well, was undertaken mainly in relation to the strong potency of these glycoconjugates to activate the immunological systems of their hosts.3,4 These bacterial cell components which never occur in higher organisms are recognized by their hosts as definite signals of invading microorganisms and trigger the defense systems of the hosts: this is a widespread phenomenon now known as innate immunity.5 Chemical synthesis provides homogeneous compounds with known definite structures free from the influence of any bacterial contaminants. Synthetic preparations corresponding to various partial structures of PGN, LPS, and LTA, therefore played decisive roles in unequivocal determination of the structures required for triggering the innate immunity systems. Identification of the specific receptors on host cells which recognize individual bacterial components became possible on the basis of the synthetic works described in this chapter. Chemical synthesis has now become a powerful tool in the research field of biologically active glycoconjugates because of its high potency to construct complex molecules built up from various types of components including carbohydrates, amino acids, fatty acids, and phosphates.

1.17.2 Synthesis of Peptidoglycan PGN is a macromolecule with characteristic three-dimensional network structure consisting of glycan chains and peptides.1,2,6 The glycan chain is a (b1-4)-linked linear polymer of alternating N-acetylglucosamine (GlcNAc) and

685

686

Synthesis of Lipopolysaccharide, Peptidoglycan, and Lipoteichoic Acid Fragments

N-acetylmuramic acid (MurNAc). Short peptide chains of alternating L- and D-amino acids of a constant sequence interlink between the carboxyl groups of MurNAc residues of different glycan chains forming the network structure. This rigid structure is important to protect bacterial cells against various environmental stresses.1,6 Any compounds such as typical antibiotics able to inhibit the biosynthesis of PGN are of value to protect mammals from invading microoraganisms.7 The same structure of the glycan chain is shared by most bacteria, but some variations are found in the peptide part. The major variation is observed at the third amino acid of the peptide interbridge. Most Gram-negative bacteria and Gram-positive bacilli have meso-diaminopimelic acid (meso-Dap), whereas other Gram-positive bacteria use L-lysine (L-Lys) at this position. Some other minor variations are also observed but basic architecture is well conserved in practically all bacterial species (Figure 1). PGN is therefore utilized by innate immunity systems of higher organisms ranging from invertebrate to mammals to discriminate microorganisms and self.4,8 The early synthetic works on PGN were focused to confirm the proposed structure of PGN in relation to its biosynthesis and the action mechanisms of antibiotics. Bricas et al. synthesized many Dap-containing peptides corresponding to partial structures of PGN and established the configurations of the Dap residue involved in particular linkage of Dap-type PGN. They discriminated the L- and D-centers of meso-Dap by the aid of selective enzymatic cleavage of N2-t-butoxycarbonyl hydrazide at the L-configurated carboxyl group. The structure of a tetrapeptide 1 synthesized is shown below.9,10 NH2 CH3 H2N

H N

O

H N

D

N H

L

O

CO2H

D

CO2H

D

CO2H

L

O

CH3

1

Early observation by Freund11 that heat-killed Mycobacterium cells strongly enhance the immunological responses of higher animals initiated intensive investigations toward elucidation of the active principle for this unique function of bacterial cells. This function then proved to be not unique to Mycobacterium cells but common to various bacterial species. Controlled enzymatic degradation of PGN and careful separation of the hydrolysis products were effective to conclude that a small and soluble partial structure of PGN is responsible for this activity. The final evidence was obtained by the aid of chemical synthesis. Synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide, MDP, 2) free from any contaminants of bacterial origin showed definite activity comparable to that of PGN (Scheme 1). MDP, a common partial structure of all PGN, was thus concluded to be the minimal structure required for the immunostimulatory function of PGN.12–15 Elongation of the peptide chain from MDP to muramyl tri- and tetrapeptide has no obvious effect on the biological activity. OH O

HO Me O C

OH NHAc

D

O

C NH L

O

Me

O HN

2

D

NH2

CO2H

Muramyl peptides containing meso-Dap and several of their unnatural diastereomers and analogs were also synthesized for the precise study of their biological activities.16,17 In these synthesis, meso-Dap was constructed by the coupling of two different components for easier differentiation of the two stereogenic centers (L and D, respectively) of meso-Dap. Scheme 2 illustrates one of the syntheses of meso-Dap derivatives where an olefin methathesis

GlcNAc

MurNAc

MurNAc

OH

OH

OH O

O

NHAc

O O NHAc DC

O HO

O O

NHAc

Me

O C

GlcNAc

O

O

OH NHAc

DC

Me

g a

NHAc

L-Ala

O C DC

CO

D-Ala

L

CO2H

HN C H

O OH

GlcNAc

O

D-Ala

(Gly)5

e

O

NH

g

L-Ala

O OH MurNAc

DC

O

DC

Me

NHAc

O O

OH GlcNAc

Lys-type PGN of Stapylocuccus aureus

O OH

NHAc HO O

O OH

GlcNAc

O

MurNAc

OH

OH

D-Ala

D-Glu-OH

a

g

O

HO O

OH MurNAc

GlcNAc

D-Ala

HN C H meso-Dap D CO2H

MurNAc

MurNAc Dap-type PGN of Escherichia coli

Figure 1 Schematic structures of cell wall peptidoglycan. Arrows in the structures represent the direction of peptide bonds.

NHAc DC

O C

a

D-Glu-OH

DC Me NHAc NHAc O O O O OH OH

NHAc

O

OO

NHAc

O C

Me

O

O

O HO

CO L HN C H (CH2)3

O C

NHAc

HO O

L-Ala

D

HN C H meso-Dap (CH2)3 HN C H L CO

O C

Me

NHAc Me

O C

(Gly)5

HN C H (CH2)4 L-Lys g e D-Glu-NH2 a NH

NHAc

NHAc HO O

CO

O DC

(CH2)4 L-Lys

a

O

O

D-Glu-NH2

L-Ala

GlcNAc

OH O

O HO

O C

a

MurNAc

Me


OH O HO

GlcNAc

687

688


Cl

Me C

OH

O O HO

Ph O

HO HO

NH2 OH

Ph

O C

DCC−HOSu

AcNH OBn

H2−Pd

C NH Me

87%

AcNH OBn

CO2H

O

Me

O

C

93%

O

O

O L-Ala-D-Glu(OBn)NH2

Me

NaH

AcNH OBn

O O O

Ph

CO2Me

O

2

85% C

O O

HN

C

NH2

CO2Bn Scheme 1 An early synthesis of N-acetylmuramyl-L-Ala-D-isoGln (MDP).

BocHN

D

CO2But

MesN NMes Cl FmocHN Ru Cl Ph PCy3 Metathesis

+ FmocHN

L

CO2Bn

64%

L(S)

FmocHN

CO2Bn

L(S)

CO2Bn

H2 CO2But D(R)

NHBoc

Fmoc: fluorenyloxymethyl

Pt/C 97%

CO2But D(R)

NHBoc 3

Scheme 2 Synthesis of a stereoselectively protected meso-Dap derivative.

reaction was employed to give a stereoselectively protected meso-Dap 3.17 The other utilized Wittig–Hornor coupling of C5 and C2 units.16 The stereoselectively protected meso-Dap derivatives (3 and 4) thus obtained were used for the synthesis of PGN fragments. Stepwise coupling of appropriately protected amino acids with 2-azido-2-deoxy-4,6-Oisopropylidene derivative of muramic acid b-thexyldimethylsilyl glycoside 5 and conversion of the azide into an acetamido group followed by final deprotection gave various N-acetylmuramyl di-, tri-, and tetrapeptides (6) (Scheme 3).16 A small molecule, MDP, represents the active center responsible for the immunostimulating activity of macromolecular PGN, but nothing was known on the effect of the molecular size, in particular the length of the glycan chain, on the biological activity and manner of recognition by receptors of host cells. To access this issue, PGN fragments with longer glycan chains were synthesized based on a simple and efficient strategy (Scheme 4). Direct coupling of muramic acid and glucosamine components was achieved by employing a 3-O-benzyl-4,6-Obenzylidene-N-2,2,2-trichloroethoxycarbonyl (N-Troc) glucosamine trichloroacetimidate 7 as the donor and the a-allyl glycoside of 6-O-benzyl-N-Troc-muramic acid ethyl ester 8 as the acceptor: the reaction proceeded smoothly to form the desired (b1-4)-disaccharide 9 directly.18 N-Troc derivatives of 2-amino sugar were known to be good glycosyl donors for the formation of b-glycosidic linkages of 2-amino-2-deoxy sugars as demonstrated in the earlier synthesis of lipid A derivatives described later in this chapter.19,20 The other protecting groups were so designed that the disaccharide obtained can be converted to both disaccharide donor and acceptor by simple procedures. Thus, the disaccharide donor was obtained by selective cleavage of the allyl glycoside followed by conversion to the glycosyl trichloroacetimidate 10, whereas selective reductive opening of the benzylidene ring of 9 directly afforded the disaccharide acceptor 11. Both were coupled under similar conditions to give the tetrasacharide 12 of alternating glucosamine and muramic acid. The same deprotection and coupling procedures were again applied to the

i, LiOH ii, L-AlaOMe EEDQ, Et3N

OTf

Me C OH

CO2Me

O O HO

O

HO HO

NH2 OH

O

OTDS

N3

O O O

Me

NaH

O

C

70−95%

OTDS

N3

CO2Me

O O O

Me C

85%

5

O

N3 NH CO2Me

NHBoc CO2TMSE

O

O

CO2Me

O

C

NH PyBOP NMM O

CO2H

90%

CO2Me

Me O H2N

C

N H

C

NH

O

CO2Me

CO2Me

Me C

O O C

O

N H

C

NH

O

Me CO2Me NHBoc

OH O OH NHAc CO2H

Me C

O O

HN

C

O

C

NH

N H

TMSE: trimethylsilylethyl

CO2H NH2

CO2H 6

CO2Me

689

Scheme 3 Synthesis of a meso-Dap-containing PGN fragment.

CO2Me

NH

CO2Me

Z: benzyloxycarbonyl

CO2Me

NH

C

NHBoc

TDS: thexyldimethylsilyl

C

HO O Me C

76%

NHAc NH

Me

O

NHBoc

Me

i, LiOH ii, TFA

OTDS

C

HN O

C

H N

86%

O O O

NHAc Me

H2−Pd/C

i, H2−Pd/C

OTDS

C Me

CO2Me ZHN ii, Z-D-GluOMe PyBOP, NMM

CO2Me

NH

Me

92%

4

89%

C

ii, D-AlaOMe O PyBOP, NMM

CO2Me

i, H2S, pyr−H2O O ii, Ac2O, pyr O O Me iii, LiOH C

NHBoc


ZHN

ZHN

OTDS

C Me

i, TBAF

O

O O TrocNH

CCl3 NH

7 i, Ir complex then, I2, H2O 93% ii, CCl3CN, Cs2CO3 Ph

O O HO

Me3N.BH3, BF3.Et2O CH3CN 73%

Ph 81%

74%

O O BnO

O O TrocNH CH CH TrocNH 3 CO2Et 10 O

O

O O BnO

Ph TMSOTf

NH

79%

BnO

O

O

O BnO

O TrocNH CH CH TrocNH 3 CO2Et

BnO HO BnO

CCl3

O

BnO O O O O TrocNH CH3CH TrocNH OAllyl CO2Et 11

BnO

O

O O BnO

BnO

O

O O TrocNH CH CH TrocNH OAllyl 3 CO2Et 9 O

BnO

O

BnO HO BnO

Ph

BnO O

O O TrocNH CH CH TrocNH 3 CO2Et

O

O

O TrocNH TrocNH CH3CH CO2Et

O BnO

O O O TrocNH CH CH TrocNH OAllyl 3 CO2Et

NH

O O BnO

Ph

TrocNH

BnO

70%

O

H2–Pd(OH)2 48%

HO HO HO

O

O

O

O

AcNH CH3CH CO Peptide

O HO

Troc: 2,2,2-trichloroethoxycarbonyl

Scheme 4 Synthesis of a lysine-type peptidoglycan fragment containing a repeating octasaccharide.

O

O BnO

TrocNH

O O

O AcNH

O O

O

TrocNH OAllyl CH3CH CO2Et 3

HO

HO O

BnO

BnO

O

CH3CH TrocNH CO2Et

HO

O AcNH

BnO

O

13: Peptide : -L-Ala-D-isoGln

Ir complex: [Ir(cod)(Ph2MeP)2]PF5

BnO

O

O CCl3

O O TrocNH CH3CH TrocNH OAllyl CO2Et

ii, LiOH quant. iii, protected peptide WSCI-HCl, HOBt 31%

BnO O

BnO

O

i, Zn–Cu, AcOH then Ac2O, pyr 82%

BnO O

12

O O TrocNH CH CH TrocNH 3 CO2Et

BnO O BnO

O

O

AcNH OPr CH3CH CO Peptide 3


BnO TMSOTf O HO O 88% TrocNH OAllyl CH3CH CO2Et 8

+

690

O O BnO

Ph


691

tetrasaccharide to give the octasaccharide. Removal of all the Troc groups followed by N-acetylation and coupling with L-alanyl-D-isoglutamine benzyl ester and final hydrogenolysis afforded the oligosaccharide peptide fragment 13 of PGN.18,21 In the meantime, Inohara et al. and others characterized two cytoplasmic proteins named Nod1 and Nod2 which belong to the family of NOD-LRR proteins.22,23 Nod1 was concluded to be a receptor which specifically recognizes g-D-glutamyl-meso-Dap (14) and induces cellular responses of innate immunity.24 Compound 14 is a common partial structure of Dap-type PGN and this represents the second minimal immunostimulating active entity of this type of PGN. Nod2, by contrast, turned out to be a specific receptor of MDP 2,25 which has been looked for almost 30 years after the identification of MDP as the first minimal active entity of PGN. There was some confusion due to heterogeneity of test components isolated from bacterial cells until the true ligands of Nod1 and Nod2 were unequivocally identified by the use of synthetic MDP and Dap peptides.22–25 It should also be mentioned here that MDP activates Nod2 more efficiently than any of its di-, tetra-, and octasaccharide dipeptide analogs as clearly confirmed with the synthetic MDP and its oligosaccharide analogs.21,25 Identification of Nod1 also gave substantial base for the immunositimulating activity of FK156 (15) isolated as a microbial metabolite and chemically synthesized.26,27 FK156 has a lactyl tetrapeptide structure corresponding to another partial structure of Dap-type PGN. Discovery of Nod1 revealed that MDP is not a sole active entity of peptidoglycan recognized by the innate immune system of higher animals, which monitors invading microorganisms by multiple channels. NH2 D

O H2N

CO2H

D

N H

CO2H

CO2H

L

14 NH2 O Me

CH3

D

OH

N H

H N

O D

N H

L

O

H N

CO2H

D

CO2H

D

CO2H

L

O

Me

15

1.17.3 Synthesis of Lipid A and Lipopolysaccharide On the outermost surface, that is, outside the peptidoglycan (PGN) layer, of Gram-negative bacterial cells, another lipid bilayer called the outer membrane which differs from the cytoplasmic membrane is present.28–30 Lipopolysaccharide (LPS) constitutes, as a component characteristic of this group of bacteria, the outer leaflet of the outer membrane. LPS has been synonymously called endotoxin after a German microbiologist, Pffeifer, who first recognized LPS as a toxic material firmly bound to the cell of Vibrio cholerae and gave the name ‘endotoxin’ to the material.31 Endotoxin, or LPS, induces high fever, tissue damage, septic shock and, in serious cases, even death of experimental animals and thus causes related serious problems also in humans. Besides these harmful so-called endotoxic activities, LPS exhibits beneficial functions such as antitumor activity and high potency to stimulate immunological responses of host animals. All of these functions of LPS are now known to be attributed to its ability to activate immunocompetent cells such as monocyte and macrophage to induce cytokine production, which results in activation of the entire immunological system. This reflects the important self-defense ability of higher animals against invasion of Gram-negative bacteria occurring ubiquitously around us. The overexpression of this defense function to lead to uncontrolled activation is observed as the toxic action of LPS. Chemically, LPS consists of a polysaccharide and a glycolipid which are covalently linked together. The latter glycolipid is localized in the lipid bilayer of the outer membrane, anchoring the LPS molecule to bacterial cells, whereas the polysaccharide is extended outward from the cell surface. The polysaccharide can be divided again in two distinct parts. The distal one is called O-antigenic polysaccharide consisting of repeating oligosaccharide units, which

692


n

O-antigenic polysaccharide

Outer core Inner core Core oligosaccharide

Lipid A

Figure 2 Schematic architecture of an enterobacterial lipopolysaccharide (LPS).

is variable depending on species and strains and represents the antigenic determinant of the bacteria. The rest is an oligosaccharide, having more conserved structures, called core oligosaccharide, which connects the O-polysaccharide and the glycolipid designated lipid A (Figure 2).29,32

1.17.3.1 Synthesis of Lipid A, the Lipophilic Component of Lipopolysaccharide 1.17.3.1.1 The first synthesis of natural-type lipid A The lipophilic component, that is, the glycolipid part, of LPS was named ‘lipid A’ by Westphal and Lu¨deritz, who found that the linkage between the core oligosaccharide and glycolipid can be selectively cleaved by mild acidic hydrolysis and observed that the latter is the endotoxic principle of LPS.33,34 The chemical structure of enterobacterial lipid A was then intensively studied.35,36 Lipid A itself is not present naturally in a free form in bacterial cells but obtained as a hydrolysis product only after mild acid treatment of LPS. The basic structure of enterobacterial lipid A is an N,O-polyacylated (b1-6)-glucosamine disaccharide bisphosphorylated at the 1- and 40 -positions. Lipid A has a strong tendency to aggregate owing to the amphiphilic nature of the molecule. Efficient purification and spectroscopic and structural analysis of lipid A were thus very difficult. The number, composition, and exact positions of acyl groups in the disaccharide backbone had not been strictly determined until the complete chemical structure of Escherichia coli lipid A 16 was finally proposed.37 The structure contains four (R)-3-hydroxytetradecanoic acids and each one dodecanoic and tetradecanoic acids in an unsymmetrical manner as shown in the structural formula. The first chemical synthesis of lipid A was achieved with the structure 17.37,38 This compound corresponding to one of early biosynthetic intermediates to LPS which contains only 4 mol of 3-hydroxytetradecanoic acids in a symmetrical distribution on the disaccharide bisphosphate, so that the synthesis was simpler than that required for 16. OH

O (HO)2P O

O

O O

O

HO

HN

O

O

O

O

(HO)2P O

O

O

O

OH

O O

O HO

O

HN O P(OH) 2

O

O

O HO

O HO

O

O HN O P(OH) 2

HO

HO

16

O

HO

HN

O HO

O

O

17

The mature lipid A of E. coli 16 was then synthesized as summarized in Scheme 5.39,40 The basic strategy of the synthesis was designed based on the following principles: (1) Only those persistent protecting groups were employed that can be removed by hydrogenolysis at the final step. This was expected to facilitate the purification of the final amphiphilic product. (2) All the acyl groups and the protected 40 -phosphate were introduced already at the stage of monosaccharide precursors in order to reduce the number of protecting groups required. (3) The N-acyl group of the distal glucosamine residue was, however, introduced after the formation of the disaccharide. N-3-acyloxyacyl groups on 2-amino-sugar moieties readily undergo b-elimination to form a,b-unsaturated acyl derivatives when the glycosidic position of the sugar was activated for glycosylation. The respective 2-amino group was protected during glycosylation with a


TrocO

HO O OCOR1

O

+ Br

(PhO)2P O 18

HO

NHTroc

19

O 70% OAllyl (PhO)2P O NHCOR2

i, Zn−AcOH ii, R3CO2H DCC

O

TrocO Hg(CN)2

O OCOR2

O OCOR2

O OCOR1 HO NHTroc

693

OAllyl NHCOR2

71%

i, BnOCH2Cl O (Pri)2NEt BnOCH2O O O O O ii, Ir complex OCOR2 OCOR1 O OCOR2 OCOR1 OH O HO OAllyl iii, I2, H2O (PhO)2P O HO O (PhO) P 2 NHCOR2 41% NHCOR3 NHCOR2 NHCOR3 O

HO

i, n-BuLi

ii, (BnO)2POCl

O OCOR2

O OCOR1

O

i, H2–Pd ii, H2–Pt

O

BnOCH2O

HO NHCOR3

(PhO)2P O

CH3(CH2)12

HO NHCOR3

O CO

OH CO

R2CO: CH3(CH2)10

O

O P (OH)2 NHCOR4 16 O

OBn

CH3(CH2)10

R4CO: CH3(CH2)10

O OCOR4

O OCOR1

O P (OBn)2 (HO)2P O NHCOR2 27% (for 4 steps)

O R1CO:

O

O

O

HO

CO


R3CO:

CH3(CH2)10 CH3(CH2)10

O CO

Ir complex: [Ir(cod)(PCH3Ph2)]PF6

Scheme 5 The first synthesis of E. coli lipid A.

2,2,2-trichloroethoxycarbonyl (Troc) group which assures the formation of desired b-glycosides and can be selectively removed after the formation of the disaccharide. (4) The glycosyl phosphate moiety was introduced at the final synthetic stage because of the chemical lability of this particular phosphate. The optically pure (R)-3-hydroxy fatty acid, one of the important components of lipid A, was prepared by enantioselective reduction of the corresponding ketoester.41 The actual synthesis started with mercury salt-assisted coupling of a 2-N-Troc-glycosyl bromide 18 with an acceptor 19 having two free hydroxy groups at the 4- and 6-positions. Selective glycosylation at the 6-hydroxy group proceeded to give the desired b(1-6) backbone disaccharide. Cleavage of the N- and O-Troc groups was followed by N-acylation to give the fully acylated disaccharide, whose 60 -hydroxy group was again protected by benzyloxymethylation for the subsequent phosphorylation. Phosphorylation of the glycosidic hydroxy group was then achieved by selective lithiation and reaction with dibenzyl phosphorochloridate. Hydrogenolytic removal of all the benzyl-type protecting groups with a palladium catalyst was followed by hydrogenolysis with a platinum catalyst to remove the phenyl esters of the 40 -phosphate to give the desired synthetic E. coli lipid A. Biological test proved that the synthetic 16 exhibits all the endotoxic activities, including both beneficial and detrimental ones, described for bacterial LPS and lipid A obtained from it.42 This was the first and final direct proof that lipid A is the real active entity of bacterial endotoxin.

1.17.3.1.2

Synthesis of lipid A analogs via improved routes

The successful syntheses of lipid A 16 and 17 opened a new possibility to prepare various definite structural analogs of lipid A for precise study on the relationship between the chemical structures and biological activities of this family of glycoconjugates. Thus, many analogous structures including both natural and unnatural ones were prepared where the synthetic routes were modified at various points to improve yields and reduce reaction steps.43 The basic strategy in the above early synthesis was also followed in most of the new syntheses, too. The major points followed were: (1) use of benzyl groups for persistent protection and final hydrogenolytic deprotection; (2) use of N-Troc glycosyl donors; and (3) introduction of the glycosyl phosphate at the latest stage.

694


BnO O O P O O

i, Zn−Cu AcOH ii, R3CO2H DCC 82%

O OCOR1

CCl3

O

C NH NHTroc

TMSOTf

O OCOR2

+

O OCOR2

O OCOR1 HO NHCOR3

O O P O O

85%

OAllyl NHCOR2

i, n-BuLi

O O BnO ii, (BnO)2POP(OBn)2 56%

O O P O O

HO NHTroc

O OCOR2

BnO NHCOR3

H2−Pd O

93%

O P(OBn)2 NHCOR2

O OCOR2

HO NHCOR3

OH

NHCOR2

O O OCOR1

O

OAllyl NHCOR2

O O OCOR1

HO

O O OCOR1

O OCOR2

O OCOR1

BnO

i, Ir complex ii, I2, H2O

O

O O P O O

O O P O O

82% OAllyl NHCOR2

HO

BnO

O

BnO

HO

(HO)2P O

O OCOR4

HO NHCOR3

O

O P(OH)2 NHCOR4 20 O

O R1CO:

OBn

CH3(CH2)12

O

CH3(CH2)10

CO

CH3(CH2)10

CH3(CH2)10

CO

R3CO:

CH3(CH2)10

O CO

Ir complex: [Ir(cod)(PCH3Ph2)]PF6

OH R4CO:

R2CO:

CH3(CH2)10

CO


Scheme 6 Synthesis of E. coli-type lipid A containing (S)-3-hydroxy acids.

One of such improved synthesis is shown in Scheme 6 as a typical example, where E. coli-type lipid A 20 has unnatural (S)-3-hydroxytetradecanoic acids in place of the natural (R)-acids.44 The major points improved include (1) the regioselective reductive opening of a 4,6-O-benzylidene ring to directly lead to a 6-O-benzylated glucosamine derivative in one step; (2) the use of a cyclic benzyl-type diester45 for the protection of the 40 -phosphate; and (3) the use of an N-Troc-glucosamine imidate as a glycosyl donor. The cyclic benzyl-type xylidene ester of a phosphate is more stable than a simple dibenzyl ester which is partly cleaved during the many reaction steps of the total synthesis. More importantly, the use of the xylidene protection for the 40 -phosphate enabled the final hydrogenolytic deprotection in one step with palladium catalyst alone. Several new routes to optically pure b-hydroxy fatty acids also became available.44,46,47 The method was also improved for the purification of the final free lipid A after hydrogenolysis. Liquid–liquid partition chromatography was applied for this purpose. For example, E. coli-type lipid A with (S)-hydroxytetradecanoic acid 20 was purified by the use of centrifugal partition chromatography (CPC) with a two-phase solvent system of butanol/THF/water/triethylamine¼45:35:100:22.44 More convenient and more effective purification can then be achieved by the same partition principle with a column of Sephadex LH-20 with similar solvent systems. Newly synthesized 16 was purified successfully with a solvent system of chloroform/methanol/2-propanol/water/triethylamine¼40:40:5:45:0.01.48 Several other lipid A analogs were also synthesized to obtain information on the relationship between their chemical structures and biological activities. Described below are typical of those works, where efficient synthetic routes were designed for the construction of the respective target structures. LPS of Salmonella R595 contains a heptaacyl lipid A 21. Controlled acidic and alkaline hydrolysis of the former results in the preferential formation of an artificial hexaacyl derivative 22, which has reduced toxicity but retains sufficient immunositimulating activity.49,50 This bacterial preparation, named Monophosphoryl lipid A, was thus recommended as an adjuvant for vaccination in veterinary fields. For the evaluation of the intrinsic immunositimulating activity, however, this bacterial preparation never meets the present criteria owing to its twofold heterogeneity: one is the


695

inherent heterogeneity of the lipid A part produced by bacteria and the other is the heterogeneity caused by incomplete selectivity of the hydrolysis reactions. Recently, several structural analogs of this monophosphoryl lipid A 22 containing normal fatty acyl groups with various chain lengths were synthesized and their immunositimulating activity compared.51 OH

O (HO)2P O

HN

O

HO O

O

O

HO

O

O HN O

O

HN O P(OH) 2

O

O

O

O

O

O

O O

(HO)2P O

O

O

O

OH

O O

O

O

HO HO

HN

O O

O

OH

O O O

O

21

22

Another example of unique lipid A analog synthesized is one from Rhizobiumm sin-1. This lipid A 23 contains a (b1-6)disaccharide of glucosamine and 2-aminogluconolactone and lacks both phosphates. The acylation pattern is unique as well. This lipid A is reported to lack endotoxic activity and, instead, it inhibits the cellular responses against E. coli lipid A (like lipid A of Rhodobacter sphaeroides and Rh. capsulatus do as described later in this chapter). A divergent synthetic route was recently described, and the antagonistic activity to suppress the effect of E. coli lipid A was confirmed for a synthetic product.52 There are some other interesting contributions based on the chemical syntheses of lipid A (such as the synthesis of Helicobacter pylori lipid A), which attracted much interests in relation to the pathogenicity of this particular bacteria which causes gastric diseases. The structure of this lipid A was elucidated to be 24; it has been synthesized and its function investigated.53–55 OH O (HO)2P O

HO

HN

O

O

O HN

O

O

O O

HO

HN

O

O

( )n HO

O O

HO HO

HN

O

O

O ( )n

O

HO HO

OH

O

O

O O P O OH

NH2

HO

O

O ( )n HO

( )n

( )21 HO

n = 10, 12, 14 23 24

The above three natural types of lipid A lack either both or one of the phosphates on the disaccharide as compared to the typical enterobacterial lipid A which shares the same bisphosphorylated disaccharide backbone structures. The effects of the phosphate groups on the biological activity of lipid A had been discussed since the time of the first synthesis of E. coli lipid A.42 The fatty acyl groups in lipid A also vary depending on the bacterial species and sometimes also growing conditions.35,36,56 The number, chain length, and location of the acyl groups have striking influences on the activity of lipid A, as seen in the case of biosynthetic precursor and those from Rhodobacter sphaeroides

696


described below. In the case of Yelsinia pestis, the number of acyl groups changes depending on the growing temperatures, which is described to be responsible for the strong virulence of this bacteria to cause plaque.57,58 For every synthetic lipid A analog, its biological activity is examined so that many data have been accumulated until now. But simple discussion of the relationship between the chemical structure and biological activity is not possible because the activity is markedly influenced by physicochemical properties of respective compounds. There are discussions on the influence of acylation patterns through the overall molecular shape,59 which is expected to reflect in the manner of supramolecule formation and recognition. Various physicochemical measurements, such as small-angle X ray diffraction, IR spectra, and NMR spectra,59–61 are used to elucidate the molecular conformation and manner of aggregation of mainly natural and synthetic homogeneous compounds. Biological activities of synthetic structural analogs are summarized in a review article.62 The receptor protein which is present on the host animal cells and recognizes LPS and lipid A was recently characterized.3 It belongs to the family of so-called Toll-like receptors (TLRs) and among them TLR4 is responsible for the recognition of lipid A and later another small protein MD2 proved to be required for recognition.3,63 The stoichiometry of the TLR4 and lipid A as well as the occurrence of ternary complex of TLR4, MD2, and lipid A was proved by the use of synthetic radio-labeled lipid A derivatives synthesized as described below.64 Participation of some other proteins forming a multiple receptor system in the recognition is discussed.65,66 The signal transduction within the cells is well understood.3

1.17.3.1.3 Synthesis of phosphonooxyethyl analog of lipid A The chemical lability of the glycosyl phosphate functionality makes the synthesis and purification of lipid A derivatives difficult tasks, especially when dealing with a small amount of this amphiphilic substance which tends to aggregate. During intensive investigation toward novel analogs of lipid A to be used clinically as antitumor agents, an artificial phosphonooxyethyl (PE) analog 25 was created.67 Contrary to the original expectation to have a less endotoxic compound which yet retains antitumor activity, 25 exhibited potent endotoxic activity indistinguishable from that of natural counterpart 16. But this fact brought about unexpected merits. Because the phosphonooxyethyl group is stable enough as compared with the glycosyl phosphate, the synthetic routes to 25 can be quite flexible and the purification of both synthetic intermediates and the final product becomes much easier. These facts were utilized in the first chemical synthesis of a radio-labeled endotoxic lipid A analog 25a.68 OH

O (HO)2P O

O

O

O

O

O

O O

HN

O

O O

O

HO

HN

O

HO

O

O

O O P(OH)2

HO

25

Radio-labeled lipid A has been desired for a long time for the elucidation of the action mechanism of lipid A. In view of its strong endotoxicity, very high specific radioactivity is required to detect a minute amount of lipid A which suffices to induce reasonable response of host cells without killing them. Such a high radioactivity can never be attained by a biosynthetic procedure. In addition, labeling of the fatty acyl groups and phosphate should be avoided in order to exclude false information because these functionalities can be cleaved off in living systems when used as a tracer. A phosphonooxyethyl analog 25a tritium-labeled at the ethylene glycol unit that meets all the above requirements was prepared as illustrated in Scheme 7.68 A fully acylated disaccharide 40 -phosphate 26 corresponding to a synthetic intermediate to E. coli lipid A was used as the starting material to the tritium-labeled PE-type lipid A. Contrary to the ordinary synthesis of lipid A, where the allyl group is cleaved and the resulting 1-hydroxy group phosphorylated to give the lipid A structure, the allyl group


697

O i, OsO4, O

O

BnO

OCOR2 BnO O R2CONH

O

BnO

Me

O ii, Pb(OAc)4

O OCOR1

OO P O R3CONH O

N

84%

OO P O R3CONH O

BnO O R2CONH

O

i,

Quant.

O

OCOR2 BnO O R2CONH 3H

O

1H-tetrazole

OO P O R3CONH O

ii, mCPBA 85%

OO

OCOR2 BnO O R2CONH 3H

OP O

O O

O OCOR1

(HO)2P O R3CONH

O

OCOR4 HO O R4CONH 3H

3H-labeled

O P(OH)2 O

25a

R1CO:

OBn CO

R3CO:

CH3(CH2)10 CH3(CH2)10

CH3(CH2)12

O CO

CH3(CH2)10

O CH3(CH2)10

O

O OCOR1

OH

HO 88%

O

BnO

P NEt2

O

H2–Pd

R2CO:

CHO

27

O O OCOR1

NaB3H4

OCOR2

OO P O R3CONH O

26 BnO

O

O OCOR1

OH O CO

R4CO:

CH3(CH2)10

CO

Scheme 7 Synthesis of tritium-labeled phosphonooxyethyl (PE) analog of E. coli-type lipid A.

was oxidized to an aldehyde 27 in this synthesis. Reduction of the aldehyde with a tritium-labeled borohydride reagent gave the a-glycosidically linked radio-labeled hydroxyethyl function, which was then phosphorylated. Hydrogenolysis after intensive purification of the protected precursor gave the product 25a with high specific radioactivity in pure form. The corresponding biosynthetic precursor-type labeled PE derivative was also similarly obtained.68

1.17.3.1.4

Synthesis of lipid A containing an unsaturated fatty acyl group

In all the syntheses described above, the final deprotection steps were carried out by catalytic hydrogenolysis. This general strategy has been satisfactory from the following two reasons. First, most lipid A isolated from natural bacterial sources contain only saturated fatty acids as their components. The second reason is that hydrogenolysis of benzyl, xylidene, and, in earlier cases, phenyl groups forms only volatile byproducts which are readily removable leaving the final free lipid A in pure states without damaging other functionalities including the unstable glycosidic phosphate. The synthetic strategy based on the benzyl-type protection is not applicable to lipid A analogs which contain unsaturated acyl groups as is the case of Rhodobacter sphaeroides lipid A 28. This lipid A is reported to share the same hydrophilic backbone consisting of the 1,40 -bisphosphorylated (b1-6) glucosamine disaccharide as in those from other bacteria but contains unusual fatty acids: a 3-keto acid on the 2-amino group and an unsaturated acid in the 3-acyloxyacyl group linked to the 20 -amino group of the backbone.69,70 Because R. sphaeroides lipid A was reported to have a potent antagonistic activity to suppress the endotoxic function of LPS, clinical application of this particular lipid A or its derivatives was planned for therapy against sepsis and shock syndrome caused by Gram-negative infections. Christ et al.71 elaborated a new synthetic route based on allyl-type protections. These groups can be removed by transition metal-catalyzed reactions leaving the isolated double bond in the acyl group intact. The authors employed the allyloxycarbonyl (Alloc) group for persistent protection of hydroxy groups and the diallyl ester for the 40 -phosphate. Thus, the glycosyl trichloroacetimidate 29 of a 2-azide sugar was used as a donor for the disaccharide formation. The glycosidic position of the acceptor 30 was temporarily protected as the t-butyldimethylsily (TBS) glycoside. After formation of the disaccharide 31, its azide function was converted to the acylamido group. The TBS group was then cleaved and the glycosyl phosphate introduced as its diallyl ester by means of the phosphoramidite method. The all allyl-type protecting groups were successfully removed in one step with a palladium(0) catalyst to give the first synthetic lipid A analogs 28a and 28b having a double bold (Scheme 8).

698


OH

O (HO)2P O

O O HN

O O

HO

O O

HO O O

O

O

HO

O

O HN O P(OH) 2

O

28

AllocO

HO O OCOR1

O

O

CCl3 C NH

(AllylO)2P O N3

+ AllocO

29

AgOTf O (AllylO)2P O

OTBS NHCOR2

AllocO N3

O (AllylO)2P O

56%

O OCOR1

O OCOR1 AllocO

O

AllocO

NHCOR3

(Ph3P)4P Ph3P

O O P(OAllyl)2

AllocO

(AllylO)2P O

O

HO O OCOR1

O OCOR1

NHCOR2

AcOH 90%

31

80%

OTBS NHCOR2

NHCOR3

OTBS NHCOR2

i, HF ii, (AllylO)2PN(Pri)2 iii, mCPBA

O

AllocO

O OCOR1

O OCOR1

30

i, Sn(II) reagent ii, R3CO2H, DCC

O

O

AllocO O OCOR1

O

O OCOR4

O OCOR4 NHCOR3

O O P(OH)2

HO

(HO)2P O

NHCOR2 28a, b

O

AllocO R1CO:

CH3(CH2)6

CO

R2CO:

CH3(CH2)10

OH CO R4CO:

CH3(CH2)6

CO

O R3CO:

CH3(CH2)5CH=CH(CH2)5 CH3(CH2)10

O

Alloc: allyloxycarbonyl

TBS: t-butyldimethylsilyl

CO

a: cis, b: trans Scheme 8 Synthesis of a proposed structure of R. sphaeroides lipid A containing an unsaturated fatty acid.

None of the synthetic products 28a and 28b prepared separately, corresponding to the cis- and trans-isomers of the proposed structure, were, however, identical with the natural lipid A obtained from R. sphaeroides cells. Nevertheless, the synthetic compounds proved to have potent activities to suppress the toxic effect of LPS in a human monocyte system. This indicates a potential of derivatives of 28 to be used for clinical treatment of sepsis patients. In fact, the same authors then designed a novel artificial derivative 32 which is more resistant than 28 against biological degradation and expected to be of therapeutic value in future.72


OMe

O (HO)2P O

O O HN

O O

699

O O

O O

O

HO

HO

O

O HN O P(OH) 2

O

32

1.17.3.2 Synthesis of Re-Type Lipopolysaccharide Lipid A was unequivocally proved to be the chemical entity responsible for the endotoxic activity of bacterial LPS as described in the preceding sections. There still remained, however, some discussions on the possible role of the saccharide portion of LPS on the biological function of LPS. LPS present on the surface of wild-type bacteria contains a long O-specific antigenic polysaccharide chain. Mutant strains of Gram-negative bacteria are also present which lack the enzyme system producing the O-polysaccharide. These are called rough form, or in short R-form, mutants because of the appearance of their cells as compared to the smooth surface of wild-type bacteria. LPS of R-mutant bacteria lacks the O-polysaccharide chain and thus consists of the core oligosaccharide and lipid A. Among R-mutants, many types such as Ra to Re have been described depending again on the partial defects of biosynthetic enzyme systems leading to various chain lengths of the core oligosaccharide part. The simplest naturally occurring LPS is the one observed on the cells of Re-mutant bacteria.29 Re LPS 33 of E. coli contains only two additional sugar units linked to the 60 -hydroxy group of the lipid A molecule.73 The sugar linked to lipid A is a unique eight-carbon acidic sugar, 3-deoxy-D-manno-oct-2-ulosonic acid abbreviated Kdo based on its old name, 2-keto-3-deoxyoctonic acid. The ketosidic linkage of Kdo is quite labile to acidic treatment, which is the reason that lipid A can be selectively cleaved from the rest of LPS by mild acid hydrolysis.

700


Total chemical synthesis of Re LPS 33 was undertaken to unequivocally investigate the effect of the oligosaccharide part on the biological activity of lipid A under conditions where possible influences from other bacterial components are avoided. This work was a synthetic challenge because of the complex molecular structure of the target which contains both acid-labile ketosidic linkages and glycosidic phosphate and alkaline-labile O-acyl moieties.

O Ph

O

HO

O OCOR1

NHTroc

O

CCl3 +

O

O

NH

OH 35

NHCOR2

O O OCOR1

Ph

BnO BnO TBSO

O

i, Zn–Cu, AcOH O Ph ii, R3COOH OCOR2 DCC OAlly l BnO 85% NHTroc NHCOR2

O

BnO BnO TBSO

O CO2Bn OTBS F

41

ii, TESCl Imidazole 77%

BnO BnO HO

O

i,

NHCOR3

NHCOR2

BnO BnO O

O CO2Bn OTES O

O

42

BnO BnO O

O CO2Bn

O CO2Bn BnO BnO HO

O

F 39

O

BnO BnO HO OH i, Ir complex ii, I2, H2O 82% O CO2Bn O

iii,

O

37

BnO BnO TBSO O CO2Bn O OTBS PNEt2 O O O 1H-tetrazole O O O OCOR1 OCOR2 ii, mCPBA O OAllyl P O BnO 96% O NHCOR3 NHCOR2

O O BF3·Et2O O OCOR1 OCOR2 75% O OAllyl P O BnO O NHCOR3 NHCOR2

O CO2Bn OH BnO BnO HO

O O O OCOR2 OCOR1 OAllyl HO BnO NHCOR3 NHCOR2

O ii, TESCl OCOR2 OAllyl Imidazole BnO NHCOR3 NHCOR2 77%

O OCOR2 OAllyl BnO

HO

89%

i, HF

TESO i, TFA, H2O

O OCOR1

O CO2Bn OTBS O

BF3·Et2O

O O O R2COOH, DCC OCOR1 OH DMAP OAllyl O BnO 85% NHTroc NHCOR2 36

O

O O OCOR1

40

BnO BnO HO

73%

OAllyl

BnO

34 O

Nafion®–TMS Ph

TFA O CO2Bn O 43

O CO2Bn BnO BnO HO

O O OBn BnO POP OBn BnO LiN(TMS)2

22% O

O O OCOR1

O P O

O 44

O OCOR2 O O P(OBn)2 BnO

NHCOR3

NHCOR2

O

O CH3(CH2)12

Re LPS 33

H2–Pd

O CO2Bn O

O

R1CO:

82%

O

O

CH3(CH2)10 TES: triethylsilyl

Scheme 9 Synthesis of Re LPS.

CO

R2CO:

p-CF3C6H4CH2O CH3(CH2)10

TBS: t-butyldimethylsilyl

CO

R3CO:

CH3(CH2)10

O

CH3(CH2)10

Ir complex: [Ir(H)(cod)(MePh2P2)]PF6

CO


701

Stereo- and regio-selective formation of the two a-ketosidic linkages of pyranosidic Kdo was also an additional point to be elaborated. The synthetic route was so designed to introduce the two Kdo units stepwise to a glucosamine disaccharide which already has all acyl groups at the required positions. The glycosyl phosphate was introduced at the latest stage just before the final hydrogenolytic deprotection, as in the case of lipid A synthesis described above. The other phosphate on the 40 -position was also introduced after coupling of the first Kdo with the glucosamine disaccharide, because preliminary experiments proved that the presence of a protected 40 -phosphate inhibits the glycosylation at the 60 -hydroxy group of the disaccharide. 40 -Phosphorylation was smoothly achieved after introduction of the first Kdo moiety by the aid of the phosporamidite method. Coupling of 4,6-O-benzylidene-N-Troc-glucosamine trichloroacetimidate 34 with another glucosamine unit 35 having free 3- and 6-hydroxy groups gave a (b1-6) glucosamine disaccharide 36 selectively. After stepwise introduction of the acyl groups at the 3-O- and 20 -N-positions, the benzylidene group was removed and the primary 6-hydroxy group was selectively activated by converting into the triethylsilyl ether 37 for the subsequent reaction with a Kdo donor (Scheme 9).74 Protected pyranosidic Kdo fluorides were used as glycosyl donors as described in previous works of the same group,75,76 where construction of the eight-carbon skeleton of Kdo by Umpolung coupling of a D-mannitol derivative with a glyoxylic acid unit was first described. The Kdo fluorides used in the present work was prepared as exemplified in Scheme 10 utilizing the modulation described by van Boom et al. thereafter.77 Thus, after appropriate selective protection of D-mannose and reduction to the corresponding mannitol derivative, coupling of a latent C2 ketoacid unit gave the carbon skeleton 38 of Kdo. Oxidative removal of the dithioacetal group followed by reaction with (diethylamino)sulfurfrifluoride (DAST) gave 4,5-O-isopropylidene fluroride 39 to be used as a Kdo donor. The corresponding 4,5-di-O-TBS fluoride 40 was prepared from 39.74 Preliminary experiments showed that Lewis acidcatalyzed reaction of Kdo fluorides proceeds smoothly with hydroxy groups activated as silyl ethers in satisfactory yields. High a-selectivity was obtained when the donor has sterically demanding protecting groups such as isopropylydene or TBS groups on the 4- and 5-positions, which inhibit the access of the glycosyl acceptor from the b-face of the Kdo donor. Coupling of 37 and 40 gave the desired trisaccharide 41 containing a-linked Kdo in excellent stereoslectivity and high yield. The free 40 -hydroxy group of 41 was then phosphorylated by the phosphoramidite method using the cyclic xylidene protection. The TBS groups of the resultant 42 were removed and the 40 -hydroxy group of the product were selectively converted to its triethylsilyl ether for the introduction of the second Kdo residue, which was achieved by using the isopropylidene-type Kdo fluoride 39. The second Kdo residue was successfully introduced again with perfect a-selectivity to give the tetrasaccharide backbone of Re LPS. In this reaction the TBS protected donor 40 gave a lower yield probably because of its too high steric hindrance. After removal of the isopropylidene groups of the tetrasaccharide derivative 43 by careful acidic treatment followed by cleavage of the allyl glycoside, the free glycosidic position was selectively phosphorylated to give the desired protected Re LPS 44. The final hydrogenolytic deprotection of 44 proceeded smoothly to give the first synthetic Re LPS 33. The synthetic route can be modified in many ways in a manner suitable for the preparation of various partial structures and analogs of 33. For example, from the synthetic intermediate 42, a trisaccharide partial structure 45 was also prepared which can never be obtained from natural sources.

HO O OHHO HO

O O

i, acetone conc. H2SO4

OH

O

ii, Ac2O, pyr

i, 70% AcOH BnO ii, BnBr, Ag2O BnO OAc

O O

OH O O

O

OAc

LiAlH4

O O OH OBn OBn

90% (from D-mannose)

D-mannose

i, O i, SOCl2, TEA ii, RuCl3·nH2O NaIO4 56% (for 3 steps)

O O

O S

O O OBn OBn

S

CO2Bn S BuLi, HMPA ii, H2SO4 51%

S O O

S NBS CO2Bn 74%

OH OBn OBn

BnO BnO O

DAST O CO2Bn O

66%

BnO BnO O

O CO2Bn O F

OH 38

Scheme 10 Preparation of a Kdo donor from D-mannose.

39

702


Biological test of the synthetic preparations provided with clear evidence that addition of the Kdo residue(s) enhances the ability of lipid A to activate the innate immunity system. Such a strict comparison was never possible with the use of natural specimens even after intensive purification.74

1.17.4 Synthesis of Lipoteichoic Acid Lipoteichoic acid (LTA) is another typical component of the cell envelope of Gram-positive bacteria. The chemical structure of LTA has been extensively studied by Fischer et al. and others.78,79 According to them, LTA consists of a glycolipid and a hydrophilic polymer of glycerophosphate covalently bound to the former. The glycolipid usually consists of oligosaccharide of D-glucose (D-Glc) with glycosidically linked 1,2-diacyl-sn-glycerol. Structural variations are observed mainly in the glycolipid part depending on bacterial genera and species. For example, LTAs from some Enterococcus and other species contain kojibiose (an (a1-2)-disaccharide of D-Glc) and a-glycosidically bound 1,2-diacyl-sn-glycerol. LTAs of Streptococcus aureus and Bacillus subtilus, by contrast, are composed of gentiobiose (a (b1-6)-disaccharide of D-Glc) and b-linked diacylglycerol. The hydrophilic polymer of repeating glycerophosphates is linked to a hydroxy group of the glycolipid via a phosphodiester: in many cases, the linkage position is the distal glucose residue of the gentiobiose or kojibiose moiety of the glycolipid. LTA on the cell anchors itself with the glycolipid being located in the lipid bilayer of the cell membrane. The hydrophilic glycerophosphate part is thereby thought to be directed outside from the cells.78 At the 2-hydroxy groups of the glycerols, nonstoichiometric D-alanyl or, less frequently, glycosyl substituents are observed. LTA has also been assumed to be a stimulant which triggers the innate immunity system of higher animals, and several chemical syntheses were undertaken in this regard.

1.17.4.1 Synthesis of Kojibiose-Containing LTA LTAs obtained from Streptococcus pyogenes and Enterococcus hirae cells have similar structures sharing the same glycolipid consisting of a-kojibiosyl diacylglycerol (46 and 47). The latter one of E. hirae has an additional phosphatidyl moiety on the 6-position of the disaccharide. These two LTAs were synthesized to confirm the immunostimulating activity described for natural LTA fractions from these particular bacterial cells.80,81 These syntheses share some basic strategic points with a previous work of the group of van Boom, who described a synthesis of a partial structure proposed for Staphylococcus aureus LTA as mentioned in the next section,82 though a combination of new protecting groups described below was essential to construct the more complex architecture of 46a and 47a.

703


HO O HO

O O

HO O

P O

O O

X

P

HO O O

O O

X

n

X

P

O HO HO

O

O

HO O O HO HO

O O

O

R

R O

OH 46

X: H or D-Ala

46a

X: H

n=2

HO O HO

O O

RCO: fatty acyl RCO: CH3(CH2)14CO

HO O

P O

O O

X

P

HO O O

O O

X

n

X

P

O HO HO

O

O

HO O HO HO

X: H or D-Ala

47a

X: H

n=2

RCO: fatty acyl RCO: CH3(CH2)14CO

O O

O O OH O

47

O

R

R O

O

P O

O O

R

R O

Of the two LTAs, synthesis of more complex E. hirae LTA 47a is summarized (Scheme 11) because both LTAs were constructed basically via similar routes.83,84 To make the synthesis accessible, substituents in the glycerophosphate chain were omitted and only hexadecanoic acid was employed for O-acylation at the four hydroxy groups of the two glycerols. The glycolipid was constructed by stepwise condensation of a chiral glycerol derivative and two glucose residues to ensure selective formation of both a-glycoside linkages. Thereby appropriate glucosyl fluorides (48 and 49) with nonparticipating benzyl-type protecting groups at the respective 2-hydroxy groups were employed as glycosyl donors. The 6-hydroxy group of the disaccharide obtained was then selectively liberated for the introduction of the phosphatidyl moiety. This synthetic strategy became possible by the use of two novel benzyl-type protecting groups, that is, p-pivaloylaminobenzyl (PAB) and p-nitrobenzyl (4-nitrophenylmethyl, NPM) groups which are independently and selectively removable under mild oxidative conditions but are more stable than the so far known p-methoxybenzyl (4-methoxyphenylmethyl, MPM) group.85,86 The MPM group is particularly versatile because it can be removed oxidatively and used in numerous syntheses for selective protection of hydroxy groups. MPM ethers are, however, sometimes too labile to be retained through Lewis acid-catalyzed conditions of glycosylation reactions. For the formation of the a-glycosyl diacylglycerol part, an indirect route via a glycoside of 1,2-di-O-allyl-sn-glycerol 50 was employed to exclude possible migration of the acyl groups on the optically active glycerol moiety during acidcatalyzed glycosylation reaction. After coupling and manipulation of the protecting groups, the two allyl groups were removed and the two hexadecanoyl groups introduced. The NPM group on the 2-position was selectively removed without damaging the PAB ether function in the molecule. This was effected by anodic oxidation after reduction to the corresponding aminobenzyl derivative 51. The second protected glucose unit was introduced to the 2-hydroxy group of the first glucose moiety forming the second a-glycoside linkage of the glycolipid part. The PAB group on the 6-position of the disaccharide 52 was then removed by DDQ oxidation, and the phosphatidyl moiety was introduced by the phosphoramidite method at the resultant 6-hydroxy group to form the fully protected glycolipid 53. The protected glycerophosphate 54 prepared separately by iterative phosphoramidite reactions83 was then coupled at the free 60 -hydroxy group liberated by selective cleavage of the Troc function of 53. Now that all the components had been coupled together at the desired positions in correct configurations to give fully protected LTA structure 55 of E. hirae, all the benzyl protecting groups were removed by catalytic hydrogenolysis to give a molecule which corresponds to the proposed fundamental structure of E. hirae LTA 47a.

704


O

O

OAllyl

OBn

OH

OH

BnO

HO

TMSO

i, Ir complex ii, I2, H2O iii, FMPT, Et3N

OTroc

OH

OAllyl

O

OAllyl F

OBn 82%

O BnO

NH TfOH 79%

NPMO

ii, 90% AcOH, 70 ⬚C iii, RCOCl pyr, DMAP OAllyl 60% OAllyl

O

HO

O-COR O-COR

O OBn

OBn BnO

BnO

O

OH O

O

O-COR O-COR

O P O O O OBn

O OBn BnO

BnO

71%

O-COR O-COR

BnO

98% O-COR O-COR

53 i,

O

BnO MPMO

O P BnO

O

Zn−AcOH

O O

O BnO O

1H-tetrazole

O

56% (for 2 steps)

O O

OTroc

OBn BnO

DDQ

OBn

O P OBn

O-COR O-COR

O

OPAB O OBn

52

O-COR O-COR

BnO

O-COR O-COR

51

BnO

OBn

BnO

O CH2 O

H2N

BnO

BnO

O P O

OBn

BnO

OTroc O OBn

ii, MCPBA

O

BnO

O-COR O-COR

O-COR O-COR 1H-tetrazole

OH

O

Quant.

ZrCl2(Cp)2 AgClO4

O

i, Pri2N

OTroc

O OBn

O

BnO 49

OBn BnO

OBn

BnO

O

72%

OAllyl

OPAB

O

NPMO

OAllyl

50

Zn−Cu Acetylacetone

BnO

O

OTroc O OBn F

OPAB Anodic oxidation HClO4

NPMO

OPAB

OBn

CCl3

BnO

48

OPAB

ii, PABO

O

81%

NPMO

D-glucose

i, MeONa

OTroc OBn

TMOTf

BnO

NPMO

HO

OTroc

NPri2 P OBn 3

54

ii, MCPBA 22% (recovery 66%)

O-COR O-COR OBn O

O BnO MPMO

O P BnO BnO O

O

O P O O O OBn

P O OBn 3

O OBn BnO

BnO

O

NPM: 4-nitrophenylmethyl (p-nitrobenzyl) =

X: H

n=4

RCO: CH3(CH2)14CO

CH2 O2N

47a O-COR O-COR

55 CH3 CH3 C CONH CH3

H2−Pd 93%

O

BnO

PAB: 4-pivaloylaminobenzyl =

O-COR O-COR

CH2

Scheme 11 Synthesis of the fundamental structure of E. hirae LTA.

Ir complex: [Ir(H)(cod)(MePh2P2)]PF6


705

Contrary to the initial expectation, the synthetic 46a and 47a showed no immunostimulating activity. Extensive analysis of LTA fractions from E. hirae cells has led to the conclusion that observed cytokine-inducing potency of natural LTA fraction be attributable not to LTA itself having the structure 47 but to another cell wall component such as lipopeptides,87 which is still to be characterized.

1.17.4.2 Synthesis of Gentiobiose-Containing LTA Synthesis of a fragment of Staphylococcus aureus LTA 56a, in which a glycolipid of b-gentiobiosyl diacylglycerol is linked to a glycerophosphate chain at the 60 -hydroxy group of the disaccharide, was reported by the group of van Boon.60 In this early synthesis, bifunctional phosphorylating reagents with 2,2,2-tribromoethyl and 2-chlorophenyl phosphates frequently used at that time in nucleic acid synthesis were employed for the preparation of the glycerophosphate trimer and its coupling with the glycolipid component. The alanyl substituents were omitted as in the synthesis of 46a and 47a above. The benzyl group was used for persistent protection and the selectively cleavable pivaloyl group for temporary protection of hydroxy functions, respectively. HO O P O O

HO O

HO O P O O O

X1

X2

n

HO O P O O HO O HO X3

56

X1, X2, X3: H, D-Ala, or a-D-GlcN

56a

X1,

56b

X1: H, X2: D-Ala; X3: a-D-GlcN

X2,

X3:

H

O O

O

O

OH HO HO

O

O

O H

R O

R O

RCO: fatty acyl n=1

RCO: CH3(CH2)12CO

n=4

RCO: CH3(CH2)12CO

Recently, another synthesis of the same LTA was also reported by Schmidt et al. (Scheme 12).88,89 The authors planned to prove the role of D-alanine residues present in the glycerophosphate chain. The same group recently reported that the D-alanyl ester which is readily cleaved during the isolation procedure of LTA from cells is essential for the cytokine-inducing activity of the isolated LTA.90 Synthesis of the glycolipid started from gentiobiose, which was converted to a b-glycoside 57 of 1,2-O-isopropylidene-sn-glycerol. After conversion of the protecting groups on the disaccharide, the isopropylidene group was removed and the resulting hydroxy groups were acylated. The glycolipid 58 was then converted to phosphoramidite 59 at the 60 -position and subjected to coupling with the glycerophosphate chain with six glycerol units 60 prepared separately. The latter hexamer 60 was constructed by stepwise phosphoramidite reactions, where four glycerol units were temporarily protected with MPM groups at the central 2-hydroxy groups for the later introduction of alanine esters. The 2-hydroxy group of the proximal glycerol in 60 bears a-glycosidically bound N-acetylglucosamine. The substitution pattern of the hexamer was so designed as to reflect the 4:1:1 ratio of alanine, glucosamine, and hydrogen in the glycerophosphate part of natural LTA. After the coupling of 59 and 60, selective oxidative cleavage of the MPM ethers in the product 61 followed by coupling with N-benzyloxycarobonyl-D-alanine under conventional conditions gave the fully protected target structure, which in turn was subjected to hydrogenolytic deprotection to give the final product 56b. The synthetic compound corresponding to the proposed fundamental structure of LTA from S. aureus was reported to have immunostimulation activity to induce cytokine production in human leukocytes as the natural LTA dose.91 An analog of 56b which has L-alanyl substituents in place of the D-alanine exhibits 10- to 100-fold less than its naturaltype counterpart 56b but definite activity. The synthetic glycolipid devoid of the glycerophosphate part was also reported to have similar weak activity. Thus, the definite structural entity responsible for the immunostimulation of LTA seems to be not yet very specifically characterizable. Under this situation, it should also be noted that another research group has the opinion that instead of the lipoteichoic acid, a lipoprotein is the dominant immunobiologically active component in Staphylococcus aureus cells.92,93

706


OH

O

OH

OH

HO

HO

O

OBz BzO

i, 75% AcOH 70 ⬚C 78% ii, RCOCl, Et3N 81%

OH

O

OTBDPS

O

O

OBn

iii, TBAF 76% BzO

BzO

OBn

OBn

O

O

O

P MPMO BnO O 1H-tetrazole

ButO

2H

75%

BzO OBn

O O-COR O-COR

OBn BzO OBn

59

OH

O BnO BnO

O O

O

O

P BnO 4

P MPMO BnO O

R1-O O

O P O OBn

O

O

O

O

OBn

O-COR O-COR

OBn

BzO

61

OBn

BzO OBn

O O

O P HO

O D-Ala-O

O

O P HO 4

R2-O O

56b

RCO: CH3(CH2)12CO Bz: benzoyl

O

OBn

P R1-O BnO O 4 60

i, CAN 67% ii, Z-D-Ala, PyBOP 70% iii, H2–Pd(OH)2 47% HO HO

O

BzO

OBn

58

O O

OBn

Pri2N O P NPri2 O P OBn OBn OBn 79%

O

O

OBn

O-COR O-COR

OBn

80%

O

O BzO

Pri2N

O

BF3·Et2O

NH OBz

OBz

57

ii,

BzO

60%

BzO

OBz

CCl3

O

OBz

i, NaOMe ii, TBDPSCl, pyr iii, BnBr, NaH

O O

OBz

O

OBz OBz

O

O O

O

BzO

O

O

HO

O

O

OH

OBz

BnO BnO

OBz

OH 74%

OH

i,

i, BzCl, pyr ii, N2H4·AcOH iii, CCl3CN, DBU

O

O

O P O OH

O

O O

OH

OH

HO

R1: tri-O-benzyl-a-D-GlcNAc

MPM: p-methoxyphenylmethyl = p-methoxybenzyl

O

OH

O-COR O-COR

HO OH

R2: a-D-GlcNAc TBDPS: t-butyldiphenylsilyl

Scheme 12 Synthesis of the fundamental structure of S. aureus LTA.

1.17.5 Closing Remarks In this chapter chemical syntheses are described of various complex glycoconjugates of bacterial origin. Rationally designed condensations of protected components led to strictly defined target compounds, whose structures are confirmed spectroscopically at every key step, so that even after multistep reactions the structures of the products are well guaranteed. The molecular weights of some final products are over 2000, or sometimes close to 3000. Even in those cases, their molecular formulas can be precisely confirmed by matrix-assisted laser desorption ionizaion (MALDI) or electrospray ionization (ESI) mass spectrometry (MS) techniques. One may agree that at present well-trained chemists are able to construct such complex, large molecules at will by the aid of various protecting groups which can be selectively and independently introduced/removed to or from each other at desired steps to distinguish individual hydroxy groups. In some cases novel methods for protection had to be


707

elaborated as exemplified in some of the syntheses described above. The great methodological developments in glycosylation, phosphorylation, as well as peptide bond formation during the recent decades also very much contribute to increase the power of synthetic chemistry in this field. As mentioned frequently, synthetic compounds played decisive roles in the identification of the structures responsible for the biological activities of the typical bacterial glycoconjugates dealt with in this chapter. In the biological studies carried out after that, various synthetic structural analogs of both natural and unnatural structures contributed to the understanding of the structure–activity relationships. The latter works not described in detail in this chapter are summarized in several review articles 61.61,62,94 One of few but obvious weak points of chemical synthesis at present is the limitation of the amounts of materials to be supplied, in particular, when the preparations require too many steps of reactions to reach the targets. A limited number of research laboratories cannot thus afford to offer sufficient amounts of materials for biological studies worldwide. Commercial supply of key compounds is very important. MDP 2 whose preparation is not too complicated, for example, has been commercially available from several sources. In the case of lipid A, by contrast, synthetic 16 and 17 are available only from a single source. The group of Raetz recently described a procedure for extensive purification of LPS from an E. coli Re mutant on the basis of his long basic research on the biosynthesis of LPS.95,96 According to their MS analysis, the purified natural LPS consists exclusively of compound 33 with a minor component having a shorter fatty acyl chain. They propose to use the purified natural Re LPS as a characterized standard for biological investigations under the situation where sufficient amounts of synthetic preparations are not available. As far as LPS and lipid A are concerned, the receptor system on host cells has been unequivocally identified by the use of synthetic lipid A and the major signaling pathway after the receptor also elucidated. In view of such a situation, the purified natural LPS may be accepted for further biological investigations. Nevertheless, it should yet be noted here that when the biological activity of the synthetic Re LPS 33 was compared with that of a purified natural counterpart in which only a trace amount of unidentified impurities was detected by MS, the natural preparation showed definitely higher activity very probably due to the impurities of bacterial origin.74 One has to pay attention to this point as far as bacterial products are used. The important missions of chemical synthesis, therefore, never end by unequivocal identification of the structure responsible for the biological activity of such complex glycoconjugates. Rather, supply of pure standard preparations is strongly required for subsequent researches on the mechanism of biological functions. To answer this, the efficiency of chemical synthesis has to be continuously improved. This can be achieved by reduction of the numbers of protecting groups employed, which necessarily requires elaboration of efficient and more highly selective couplings of components like enzyme reactions. Entirely new strategies may also be important for efficient improvements. A wide space still remains open where new chemistry is to be developed. Collaboration and frequent exchange of information between synthetic chemists and scientists working in the biological fields are therefore quite important.

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Oikawa, M.; Wada, A.; Yoshizaki, H.; Fukase, K.; Kusumoto, S. Bull. Chem. Soc. Jpn. 1997, 70, 1435–1440. 49. Qureshi, N.; Mascagni, P.; Ribi, E.; Takayama, K. J. Biol. Chem. 1985, 260, 5271–5278. 50. Meyers, K. R.; Truchor, A. T.; Wald, J.; Hudson, Y.; Ulrich, J. T. A Critical Determinant Lipid A Endotoxic Activity. In Cellular and Molecular Aspects of Endotoxin Reactions; Nowotony, A.; Spitzer, J. J.; Ziegler, E. J., Eds.; Endotoxin Research Series; Elsevier: Amsterdam, 1990; vol. 1, pp 145–156. 51. Johnson, D. A.; Keegan, D. S.; Sowell, C. G.; Livesay, M. T.; Johnson, C. L.; Taubner, L. M.; Harris, A.; Meyers, K. R.; Thompson, J. D.; Gustafson, G. L.; Rhodes, M. J.; Ulrich, J. T.; Ward, J. R.; Yorgensen, Y. M.; Cantrel, J. L.; Brookshire, V. G. J. Med. Chem. 1999, 42, 4640–4649. 52. Demchenko, A. V.; Wolfelt, M. A.; Santhanam, B.; Moore, J. N.; Boons, G.-J. J. Am. Chem. Soc. 2003, 125, 6103–6112. 53. Suda, Y.; Ogawa, T.; Kashihara, W.; Oikawa, M.; Shimoyama, T.; Hayashi, T.; Tamura, T.; Kusumoto, S. J. Biochem. 1997, 121, 1129–1133. 54. Sakai, Y.; Oikawa, M.; Yoshizaki, H.; Ogawa, T.; Suda, Y.; Fukase, K.; Kusumoto, S. Tetrahedron Lett. 2000, 41, 6843–6847. 55. Ogawa, T.; Asai, Y.; Sakai, Y.; Oikawa, M.; Fukase, K.; Suda, Y.; Kusumoto, S.; Tamura, T. FEMS Immunol. Microbiol. 2003, 36, 1–7. 56. Knirel, Y. A.; Dentovskaya, S. V.; Sentchenkova, S. N.; Shaikhutdinova, R. Z.; Kocharova, N. A.; Anisimov, A. P. J. Endotoxin Res. 2006, 12, 3–9. 57. Kawahara, K.; Tsukano, H.; Watanabe, H.; Lindner, B.; Matsuura, M. Infect. Immun. 2002, 70, 4092–4098. 58. Knirel, Y. A.; Lindner, B.; Vinogradov, E. V.; Kocharova, N. A.; Sentchenkova, S. N.; Shaikhutdinova, R. Z.; Dentovskaya, S. V.; Fursova, N. K.; Bakhteeva, I. V.; Titareva, G. M.; Balakhnov, S. V.; Holst, O.; Gremyakova, T. A.; Pier, G. B.; Anisimov, A. P. Biochemistry 2005, 44, 1731–1743. 59. Seydel, U.; Wiese, A.; Schrom, A. B.; Brandenburg, K. A. Biophysical View on the Function and Activity of Endotoxin. In Endotoxin in Health and Disease; Brade, H.; Opal, S. M.; Vogel, S. N.; Morrison, D. C., Eds.; Marcel Dekker: New York, 1999; pp 195–219. 60. Wang, Y.; Hollingworth, R. I. Biochemistry 1996, 35, 5647–5654. 61. Oikawa, M.; Shintaku, T.; Fukuda, N.; Seklijic, H.; Fukase, Y.; Yoshizaki, H.; Fukase, K.; Kusumoto, S. Org. Biomol. Chem. 2004, 2, 3557–3565. 62. Takada, H.; Kotani, S. CRC Critical Rev. Microbiol. 1989, 16, 477–523. 63. Akashi, S.; Nagai, Y.; Ogata, H.; Oikawa, M.; Fukase, K.; Kusumoto, S.; Kawasaki, K.; Nishijima, M.; Hayashi, S.; Kimoto, M.; Miyake, K. Int. Immun., 2001, 13, 1595–1599. 64. Kobayashi, M.; Saitoh, S.; Tanimura, N.; Takahashi, K.; Kawasaki, K.; Nishijima, M.; Fujimoto, Y.; Fukase, K.; Akashi-Takamura, S.; Miyake, K. J. Immunol. 2006, 176, 6211–6218. 65. Triantafilou, M.; Brandenburg, K.; Kusumoto, S.; Fukase, K.; Mackie, A.; Seydel, U.; Triantafilou, K. Biochem. J. 2004, 381, 527–536. 66. Triantafilou, M.; Triantafilou, K. J. Endotoxin Res. 2005, 11, 6–11. 67. Kusama, T.; Soga, T.; Shioya, E.; Nakayama, K.; Nakajima, H; Osada, Y.; Ono, Y.; Kusumoto, S.; Shiba, T. Chem. Pharm. Bull. 1990, 38, 3366– 3372. 68. Fukase, K.; Kirikae, T.; Kirikae, F.; Liu, W- C.; Oikawa, M.; Suda, Y.; Kurosawa, M.; Fukase, Y.; Yoshizaki, H.; Kusumoto, S. Bull. Chem. Soc. Jpn. 2001, 74, 2189–2197. 69. Salimath, P. V.; Weckesser, J.; Strittmatter, W.; Mayer, H. Eur. J. Biochem. 1983, 136, 195–200. 70. Qureshi, N.; Honovich, J. P.; Hara, H.; Cotter, R. J.; Takayama, K. J. Biol. Chem. 1988, 263, 5502–5504.


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71. Christ, W. J.; McGuiness, P. D.; Asano, O.; Wang, Y.; Mullarkey, M. A.; Perez, M.; Hawkins, L. D.; Blythe, T. A.; Dubuc, G. R.; Robidoux, A. L. J. Am. Chem. Soc. 1994, 116, 3637–3638. 72. Christ, W. J.; Asano, O.; Robidoux, A. L.; Perez, M.; Wang, Y.; Dubuc, G. R.; Gabin, W. E.; Hawkins, L. D.; McGuiness, P. D.; Mullarkey, M. A.; Lewis, M. D.; Kishi, Y.; Kawata, T.; Bristol, J. R.; Rose, J. R.; Rossignor, D. P.; Kobayashi, S.; Hashinuma, I.; Kimura, A.; Asakawa, N.; Katayama, K.; Yamatsu, I. Science 1995, 268, 80–83. 73. Za¨hringer, U.; Lindner, B.; Seydel, U.; Rietschel, E. Th.; Naoki, H.; Unger, F. M.; Imoto, M.; Kusumoto, S.; Shiba, T. Tetrahedron Lett. 1985, 26, 6321–6324. 74. Yoshizaki, H.; Fukuda, N.; Sato, K.; Oikawa, M.; Fukase, K.; Suda, Y.; Kusumoto, S. Angew. Chem. Int. Ed. 2001, 40, 1475–1480. 75. Imoto, M.; Kusumoto, S.; Shiba, T. Tetrahedron Lett. 1987, 28, 6235–6238. 76. Imoto, M.; Kusunose, N.; Matsuura, Y.; Kusumoto, S.; Shiba, T. Tetrahedron Lett. 1987, 28, 6277–6280. 77. van der Klein, J. H.; Boons, G. J. P. H.; Veeneman, G. H.; van Boom, J. H. Tetrahedron Lett. 1989, 30, 5477–5480. 78. Fischer, W. Bacterial Phospholipids and Lipoteichoic Acid. In Glycolipids, Phosphoglycolipids, and Sulfoglycolipids; Handbook of Lipid Research; Kates, M., Ed.; Plenum: New York, 1990; pp 123–234. 79. Fischer, W. Med. Microbiol. Immunol. 1994, 183, 61–76. 80. Usami, H.; Yamamoto, A.; Sugawara, Y.; Hamada, S.; Yamamoto, T.; Kato, K.; Kokeguchi, S.; Takeda, H.; Kotani, S. Br. J. Cancer 1987, 56, 797–799. 81. Tsutsui, O.; Kokeguchi, S.; Matsumura, T.; Kato, K. FEMS Microbiol. Immun. 1991, 76, 211–218. 82. Oltvoort, J. J.; Kloosterman, M.; Constant, A. A.; van Boeckel, A. A.; van Boom, J. H. Carbohydr. Res. 1984, 130, 147–163. 83. Fukase, K.; Matsumoto, T.; Ito, N.; Yoshimura, T.; Kotani, S.; Kusumoto, S. Bull. Chem. Soc. Jpn., 1992, 65, 2643–2654. 84. Fukase, K.; Yoshimura, T.; Kotani, S.; Kusumoto, S. Bull. Chem. Soc. Jpn. 1994, 67, 473–482. 85. Fukase, K.; Tanaka, H.; Torii, S.; Kusumoto, S. Tetrahedron Lett. 1990, 31, 389–392. 86. Fukase, K.; Yoshimura, T.; Hashida, M.; Kusumoto, S. Tetrahedron Lett. 1991, 32, 4019–4022. 87. Suda, Y.; Tochio, H.; Kawano, K.; Takada, H.; Yoshida, T.; Kotani, S.; Kusumoto, S. FEMS Imunol. Med. Microbiol., 1995, 12, 97–112. 88. Stadejmeier, A.; Morath, S.; Hartung, T.; Schmidt, R. R. Angew. Chem. Int. Ed. 2003, 42, 916–920. 89. Figueroa-Perez, I.; Stadejmeier, A.; Morath, S.; Hartung, T.; Schmidt, R. R. Tetrahed. Asymm. 2005, 16, 493–506. 90. Morath, S.; Stadelmaier, A.; Geyer, A.; Schmidt, R. R.; Hartung, T. J. Exp. Med. 2002, 195, 1635–1640. 91. Deoninger, S.; Stadejmeier, A.; von Aulock, S.; Morath, S.; Schmidt, R. R.; Hartung, T. J. Immunol. 2003, 170, 4134–4138. 92. Hashimoto, M.; Tawaratsumida, K.; Kariya, H.; Aoyama, K.; Tamura, T.; Suda, T. Internat. Immunol. 2005, 18, 355–362. 93. Hashimoto, M.; Tawaratsumida, K.; Kariya, H.; Kiyohara, A.; Suda, Y.; Kirikae, F.; Kirikae, T.; Goetz, F. J. Immunol. 2006, 177, 3162–3169. 94. Takada, H.; Kotani, S. Immunopharmacological Activities of Synthetic Muramyl-Peptides. In Immunology of the Bacterial Cell Envelope; StewartTull, D. E. S., Davies, M., Eds.; John Wiley and Sons: Chichester, 1985; pp 119–152. 95. Raetz, C. R. H.; Whitfield, C. Annu. Rev. Biochem. 2002, 71, 635–700. 96. Raetz, C. R. H.; Garrett, T. A.; Reynolds, C. M.; Shaw, W. A.; Moore, J. D.; Smith, D. C., Jr.; Ribeiro, A. A.; Murphy, R. C.; Ulevitch, R. J. C.; Fearns, D.; Reichart, C. K.; Glass, C.; Benner, S.; Subramaniam, R.; Harkewicz, R.; Bowers-Gentry, C.; Buczynski, M. W.; Cooper, J. A.; Deems, R. A.; Dennis, E. A. Lipid Res. 2006, 47, 1097–1111.

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Biographical Sketch

Shoichi Kusumoto was born in 1940 and received his B.S. from Osaka University on 1963. He then joined the Faculty of Osaka City University as a research associate. After receiving his Ph.D., he stayed two years at the German Cancer Research Center (Prof. Erich Hecker), Heidelberg, being supported by the Alexander von Humboldt Foundation. In 1972, he moved to Osaka University and joined the group of Prof. Tetsuo Shiba, where he started chemical and functional research on bacterial glycoconjugates. He was promoted to professor of organic chemistry in the same university in 1988. Since April 2004, he is the director of Suntory Institute for Bioorganic Research, Osaka. His research has been mainly focused on the structure analysis, synthesis, and biological function of bacterial glycoconjugates such as peptidoglycan, lipopolysaccharides, and lipoteichoic acids. For these works, he received the International Society of Immunopharmacology Award in 1983, Chemical Society of Japan Award in 1999, and The F. B. Bang Award of International Endotoxin Society in 2004.

Koichi Fukase was born in Okayama in 1960, and received his B.A. in 1982 and Ph.D. in 1987 from Osaka University under the direction of Professor Tetsuo Shiba. After receiving a one-year fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists, he was appointed as research associate of Dr. Kusumoto’s group at Osaka University in 1988. He was promoted to assistant professor (1996), associate professor (1998), and full professor (2004). He was a recipient of The Chemical Society of Japan Award for Young Chemists in 1994. His research interest centers around chemical and biological study of oligosaccharides and glycoconjugates and combinatorial chemistry.


711

Yukari Fujimoto received her B.S. in 1989 from Osaka University under the direction of Prof. Shoichi Kusumoto. After industrial experience at Sumitomo Chemical and postgraduate studies at Columbia University (Prof. Koji Nakanishi), she received her Ph.D. from Osaka University in 2002. After postdoctoral studies at Nagoya University, she joined the faculty of Osaka University as a research associate in 2003, where she is currently an assistant professor of Prof. Koichi Fukase’s group. Her current major interest is in the area of synthesis and function of bacterial glycoconjugates.

1.18 Synthesis of Glycosaminoglycans and Their Oligosaccharides R. J. Linhardt and M. Weı¨wer, Rensselaer Polytechnic Institute, Troy, NY, USA ß 2007 Elsevier Ltd. All rights reserved. 1.18.1 Introduction: Glycosaminoglycans Structure and Biology

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1.18.1.1 Structure: HA, CS/DS, HP/HS, KS

713

1.18.1.2 Biosynthesis

715

1.18.1.3 Biology

715

1.18.2 Overview of Synthetic Approaches

715

1.18.2.1 Glycosaminoglycans without Uronic Acid: KS

717

1.18.2.1.1 1.18.2.1.2

Chemical synthesis Enzymatic synthesis

717 719

1.18.2.2 Glycosaminoglycans with Glucuronic Acid: HA and CS 1.18.2.2.1 1.18.2.2.2 1.18.2.2.3

Chemical synthesis Chemoenzymatic synthesis Enzymatic synthesis

719 725 727

1.18.2.3 Glycosaminoglycans with Iduronic Acid: DS, HS, and HP 1.18.2.3.1 1.18.2.3.2 1.18.2.3.3

719

Chemical synthesis Chemoenzymatic synthesis Enzymatic synthesis

728 728 736 737

1.18.3 Conclusions and Future Prospects

741

1.18.1 Introduction: Glycosaminoglycans Structure and Biology 1.18.1.1 Structure: HA, CS/DS, HP/HS, KS Glycosaminoglycans (GAGs) represent an important family of polyanionic, linear, highly functionalized bioactive oligosaccharides that are ubiquitous components of animal connective tissues. Although hyaluronic acid and heparin are also found in their free form, GAGs often exist as long chains covalently linked to a protein core, called a proteoglycan (PG).1 GAGs are major structural components of PGs and are believed to be responsible for the many biochemical events mediated by PGs. The GAG family are comprised of keratan sulfate (KS), hyaluronic acid or hyaluronan (HA), chondroitin sulfate (CS), dermatan sulfate (DS), heparin (HP), and heparan sulfate (HS). These polysaccharides all consist of repeating disaccharide units composed of a 2-amino-2-deoxy sugar (hexosamine) and, with the exception of KS (containing D-galactose), a uronic acid residue (Table 1). KS can occur in two different main forms. KSI is linked to its core protein by an N-glycosidic linkage between N-acetyl-Dglucosamine and L-asparagine and KSII is linked to the core protein by an O-glycosidic linkage between N-acetyl2,3 D-galactosamine and L-serine or L-threonine. A third type of KS has been reported with an O-linkage through 4 mannose to a serine residue. KS is a polymer of D-galactose and N-acetyl-D-glucosamine disaccharide repeating units [3)-b-D-Gal-(1!4)-b-D-GlcNAc-(1!]n (530

20

18

0 40 45 50 55 60 65 70 75 80 Vo

Effluent volume (ml)

Figure 6 Restricted polymerization by recombinant EXT1 (a) and EXT1/2 complex (b).

oligomers resembled that of EXT1 alone. This indicated that the catalytic activity of EXT1/2 was due to complex formation of the two proteins. Sugahara et al. studied the elongation of the recombinant glypican-1 core protein and a synthetic linkage region analog (GlcA-Gal-O-C2H4NH-benzyloxycarbonyl).129 Recombinant soluble enzymes expressed by co-transfection of EXT1 and EXT2 were able to polymerize the heparan backbone with average molecular weights >1.7105. Truncated forms (devoid of the transmembrane and cytoplasmic region) of EXT1 and EXT2 were expressed because a previous work suggested that truncated forms of EXT1 and EXT2 are sufficient for complex formation.145 First, N-acetylheparosan was used as acceptor substrate for the co-expressed truncated soluble forms of EXT1 and EXT2 but only a few numbers of sugar units were incorporated at the nonreducing end. Then, since it was reported that concurrent N-deacetylase/N-sulfotransferase reactions enhanced the polymerization of heparin chains,146 partially Nsulfo derivatives of the K5 oligosaccharides were evaluated as acceptors but did not show better efficiency. The authors suggested that polymerization reactions require acceptor core proteins and tested a synthetic linkage region analog (GlcAb1!3Galb1-O-C2H4NHCbz) and glypican 1. In both cases, chain polymerization occurred, leading to the formation of polymers having molecular weight of 98 600 and 28 600, respectively. a-Thrombomodulin and the linkage region tetrasaccharide–hexapeptide GlcA-Gal-Gal-Xyl-Ser-Gly-Trp-Pro-Asp-Gly also showed good activity as acceptors and the results altogether are consistent with the notion that a certain hydrophobic sequence peptide sequence is required for the chain initiation and polymerization. No polymerization was observed on any acceptor tested by using only one of the soluble forms of EXT1 or EXT2 or by using a mixture of separately expressed soluble forms of EXT1 and EXT2. In 2002, DeAngelis et al. identified and cloned from P. multocida type D a heparosan synthase, a dual-action glycosyltransferase responsible for polymerizing the heparosan backbone component of the type D capsular polysaccharide.130 The recombinant E. coli-derived P. multocida heparosan synthase ( pmHS1) catalyzed the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcA and no other structurally related sugar nucleotides were used as donor by the enzyme. Although the presence of a starting polymer acceptor was not necessary to observe the polymerization reaction, it was found to stimulate about 7–25-fold the synthase activity allowing the production of heparosan-type polymers composed of 500–3000 sugar residues. More recently, DeAngelis and White identified a new heparosan synthase ( pmHS2) from P. multocida types A, D, and F, encoded by the gene hssB.131 As P. multocida types A and F were previously found to possess hyaluronan ( pmHAS) and chondroitin ( pmCS) synthases, respectively,67,147 the authors suggested that type A and F organisms utilize either the capsule locus synthase gene (encoding pmHAS and pmCS, respectively) or the hssB gene to produce their camouflage, depending on environmental conditions or stage of infection. From a synthetic point of view, the pmHS2 enzyme was not stimulated greatly by the addition of an exogenously supplied polymer acceptor and yielded smaller molecularweight-product size distributions. One of the more elegant application of enzymatic synthesis of HS oligosaccharides was reported in 2003 by Rosenberg et al. and describe the preparation of ATIII-binding pentasaccharide (Figure 7).148 N-acetylheparosan 150 was harvested from E. coli K5 bacterial cells and further modified by sequential enzyme-catalyzed transformations. N-Deacetylation followed by N-sulfonation was accomplished using N-deacetylase N-sulfotransferase 2 (NDST2) in the presence of PAPS. Next, polysaccharide 151 was treated with heparitinase I, resulting in a mixture of oligosaccharides of different sizes that were purified to homogeneity by preparative HPLC. Hexasaccharide 152 thus obtained was then subjected to treatment with C5 epimerase and 2-O-sulfotransferase 1 (2-OST1) to prepare

740

Synthesis of Glycosaminoglycans and Their Oligosaccharides

OH

OH

−OOC

O O

O HO

OHO

O

−OOC

O NHAc HO

OH

O HO

OH

−OOC

O

O

O NHAc HO

OH

O

O HO

150

OH

NHAc O

N-Deacetylase N-Sulfotransferase 2 OH

OH

−OOC

O O

O HO

O HO

O HO OH

−O

OH

−OOC

O

O NHAc HO

OH

O

−OOC

3SHN

O

O HO

OH

OH O O HO

O

−OOC

O HO− OH

O O

HO

OHO OH

O O HO

O3SHN

−OOC

OH

OH

OHO − O3SHN

OH O

O −O

152

NHSO3−

OH

O

O NHAc HO

O

O HO

OH

C5-Epimerase 2-O-Sulfotransferase 1 OH −OOC O

OH

−OOC

O

OH

−OOC

O

O NHAc HO

OH

3SHN

OH O

HO

151 −O

Heparitinase I −OOC

O

O HO

O

O HO

OH

153

NHSO3−

3SO

6-O-Sulfotransferase 1 and 6-O-Sulfotransferase 2a OSO3−

−OOC

O O

HO

O HO OH

OSO3− −OOC

O

−OOC

OH OH

O

O NHAc HO

O HO − OH

O

O

O3SHN

−O

O

O HO −O

3SO

OH

154

3SHN

∆ -Glycuronidase 4,5

OSO3− O HOHO

OSO3− −OOC

O

OH

−OOC

OH

O O NHAc HO

OHO

−O SHN 3

OH

O

O −O

O HO

O OH 155 NHSO3−

3SO

3-O-Sulfotransferase 1 PAP34S OSO3− O HO

OSO3− O

−OOC

OH

−OOC

OH

O HO

O NHAc HO

O OH

−O 34SO 3

O NHSO3−

O −O

3SO

O HO

O OH

156

NHSO3−

Figure 7 Enzymatic synthesis of ATIII-binding pentasaccharide.

hexasaccharide 153. Fortunately, the C5 epimerase can only act on a GlcA unit flanked by N-sulfoglucosamine residues.133 2-OST1 preferentially promote the sulfonation of IdoA located at the reducing side of N-sulfoglucosamine residue.149 Thus, after epimerization, preferential sulfonation occurs on the newly generated IdoA and lead to the formation of hexasaccharide 153. Then, selective 6-O-sulfonation of two glucosamine units, located at

Synthesis of Glycosaminoglycans and Their Oligosaccharides

741

the nonreducing end and in the middle of the hexasaccharide, was accomplished in the presence of 6-OST1 and 6-OST2a.150 Removal of the unsaturated uronic acid residue at the non-reducing end by treatment with D4,5glycuronidase afforded pentasaccharide 155 in quantitative yield. The last step, involving the 3-O-sulfonation of the central glucosamine unit was accomplished by treatment with 3-OST1 and allowed the generation of ATIIIbinding pentasaccharide. This enzymatic approach generated the ATIII-binding pentasaccharide in six steps with an overall yield of 1.1%, at least twofold better than for the chemical approach. The enzymatic preparation of dermatan sulfate oligosaccharides and polysaccharides has been less studied. Habuchi et al. have reported the enzymatic synthesis of oversulfated DS by sulfonation of dermatan sulfate using GalNAc4S-6-OST in the presence of PAPS.73,151 Recently, Lauder et al. characterized oligosaccharides obtained by enzymatic degradation of chondroitin and dermatan sulfate using NMR spectroscopy.152

1.18.3 Conclusions and Future Prospects The ability of GAGs to regulate a wide number of biological processes is only beginning to be understood at a molecular level. In this context, the synthesis of well-defined GAG oligosaccharides is essential. The arsenal of methodologies developed in synthetic organic chemistry over the past century has allowed the preparation of important targets. However, the number of steps involving selective protection/deprotection sequences and the stereoselectivity of the critical glycosylation reactions still remains problematic. Enzymatic and chemoenzymatic syntheses are an area of growing interest. Mutant synthases form P. multocida (pmHAS, pmCS, pmHS1, and pmHS2) allowing the in vitro synthesis of HA, CS, and HS oligosaccharide backbones has been studied by DeAngelis and are able to polymerize appropriate UDP-sugar nucleotides (UDP-GlcA and UDP-GlcNAc or UDP-GalNAc). A wide number of enzymes involved in the modification (N-sulfonation, O-sulfonation, C5 epimerization) of theses oligosaccharide backbones have also been identified and used for the synthesis of well-defined sulfated oligosaccharides and polysaccharides. As a representative example, the enzymatic synthesis of ATIII-binding pentasaccharide has been described in six steps (60 for the chemical approach) with an overall yield of 1.1% (1000 µM) 49: R = NHC(NH)NH2 (130 µM)

O

CO2H

HO AcHN

AcHN

AcHN

H

897

R

47: R = NH2 (270 µM) 50: R = NHC(NH)NH2 (9 µM)

48: R = NH2 (13 µM) 51: R = NHC(NH)NH2 (0.55 µM)

Figure 9 Analogs of 4-amino-4-deoxy-Neu5Ac2en 8 and 4-deoxy-4-guanidino-Neu5Ac2en (9) modified by removal of C7–C9. IC50 values for 46–51 against isolated influenza A virus sialidase are given in parentheses.

1.22.3.1.5.1 C7 modifications The hydroxyl group at C7 of Neu5Ac2en is the only polar group on this scaffold that does not appear to make contact with the active site, and is in fact directed toward bulk solvent. Modifications at this position have primarily aimed to enhance the pharmacokinetic properties of Neu5Ac2en-based inhibitors by introducing lipophilic character or attaching the ligand to a multivalent scaffold. A series of 7-O-carbamoyl derivatives of 4-amino-4-deoxy and 4-deoxy-4-guanidino-Neu5Ac2en were prepared and screened for their ability to inhibit sialidase activity and viral replication (influenza virus A and B).104 Potent inhibitors 52 and 53 were identified that rivaled the activity of Zanamivir 9 in enzyme assays, but proved slightly less effective in plaque reduction assays (plaque IC50 influenza virus B: 52 0.023mgml–1, 53 0.009mgml–1, 54 0.52mgml–1, 9 0.002mgml–1). In general, monosubstitution of the carbamate nitrogen proved superior to disubstitution, and bulky and/or lipophilic N-substituents were inferior to those bearing polar functional groups. These results were rationalized in terms of interactions between the substituents and polar amino acid residues on the surface of the protein or with solvent. In keeping with trends observed for other Neu5Ac2en derivatives, 7-O-carbamoyl C4 guanidino derivatives were significantly more potent inhibitors than the corresponding 7-O-carbamoyl C4 amine derivatives. The ability to derivatize the C7 hydroxyl group of Neu5Ac2en and related compounds without affecting binding to the NA active site has allowed for the development of a carbamate-based Zanamivir–biotin conjugate for potential application as an influenza diagnostic.105 R1 O

N

R2 O

HO

H

O

CO2H

HO AcHN

52: R1 = H, R2 = (CH2)6NH2 53: R1 = H, R2 = (CH2)6CH3 54: R1 = R2 = (CH2)2CH3

HN NH H2N

Modification of the substituent at C7 in 4-deoxy-4-guanidino-Neu5Ac2en by O-alkylation has also been reported.106,107 Ethers containing a linear alkyl chain ranging from 1 to 14 carbons in length were evaluated against influenza A virus in both sialidase inhibition and plaque reduction assays. While none proved superior to Zanamivir against NA in isolated enzyme inhibition assays, alkyl ethers up to 12 carbon atoms in length performed better in the viral replication assay. Alkylation of the C7 hydroxyl with two-carbon substituents bearing terminal hydroxyl, amino, azido, and acetamido groups yielded inhibitors 55–58 with antiviral properties that exceeded those of the parent triol 9 in the plaque reduction assay, although their potency did not differ substantially from ethyl or propyl ethers 59 or 60. The superior effectiveness of 7-O-alkyl ethers bearing hydrophobic substituents (2–12 carbon atoms) stands in contrast to results reported for hydrophobic groups among the 7-O-carbamoyl series. These findings have not been rationalized to date. Derivatives of 4-deoxy-4-guanidino-Neu5Ac2en such as 61, O-alkylated at C7 and deoxygenated at both C8 and C9 were prepared and evaluated against influenza A and B virus sialidases and used in a viral replication assay.107 While these molecules displayed similar efficacy to the parent triol against influenza A virus sialidase, they were relatively ineffective against influenza B virus (Figure 10). NA inhibitors formed by replacement of the hydroxyl at C7 of 4-deoxy-4-guanidino-Neu5Ac2en with fluorine, azide, or amine for the most part maintained both their ability to inhibit the enzyme and to prevent influenza

898

Design and Synthesis of Sialidase Inhibitors for Influenza Virus Infections

X

n O

HO

H

O O

CO2H

HO AcHN

H

CO2H

O

AcHN HN

HN NH

NH

H2N 55: 56: 57: 58: 59: 60:

H2N

n = 2, X = OH (0.21) n = 2, X = NH2 (0.63) n = 2, X = N3 (0.13) n = 2, X = NHAc (0.20) n = 2, X = H (0.14) n = 3, X = H (0.35)

61 (1.9 )

Figure 10 Zanamivir derivatives modified through alkylation of the C7 hydroxyl. Values in parentheses reflect IC50 values against influenza A virus in a plaque reduction assay, and are relative to a reference value of 1.0 for Zanamavir, 9.

R HO

H

F F O

CO2H

HO AcHN

H

HO

O

HO AcHN HN

HN NH

H2N 62: 63: 64: 65: 66:

CO2H

R = F (0.4) R = N3 (0.5) R = NH2 (4.0) R = NHC(O)(CH2)6CH3 (5.1) R = NHC(O)(CH2)14CH3 (52)

NH H2N 67 (10.0)

Figure 11 Zanamivir derivatives modified by replacement of the C7 hydroxyl. Values in parentheses reflect the relative ability of the compounds to inhibit influenza A virus sialidase. The reference value for Zanamivir 9 is 1.0.

replication in vitro.106 In particular, the fluoro and azido derivatives 62 and 63 demonstrated superior characteristics to Zanamivir. In keeping with trends observed for 7-O-alkyl derivatives of Zanamivir, amides prepared by acylation of amine 64 were effective inhibitors when shorter alkyl groups (2000-fold) in the ability of this derivative 144 of 121 to interfere with catalysis. The less sterically demanding fluoro-substituted cyclohexene 145 retained its activity for the most part. A methyl group was appended to C3 in an effort to capitalize on potential hydrophobic contacts in this region.139 Instead, inhibition by 146 was diminished by over three orders of magnitude relative to 121. Cyclohexane-based substrate mimetics such as 147 have shown greatly reduced ability to inhibit NA relative to the cyclohexene transition state analogs (Figure 24).147

1.22.3.4.6

Development of Oseltamivir, a cyclohexene-based therapeutic

Despite the intuitively apparent enhancement of lipophilicity accompanying replacement of the glycerol side chain in the cyclohexene-ether series, both the guanidino derivative 140 and amine 121 displayed poor oral bioavailability,

X O

115: NHAc (130 nM)

H

CO2H

141: NHC(O)CF3 (100 nM) 142: NHC(O)CH2CH3 (1500 nM)

X NH2

143: NHSO2CH3 (2500 nM)

Figure 23 A comparison of influenza virus A sialidase inhibition by derivatives of 115 with various C5 nitrogen substituents. IC50 values are given in parentheses.

O

H

X CO2H

AcHN

O

H

CO2H Me

AcHN NH2

121: X = H (1 nM) 144: X = Me (2 µM) 145: X = F (3 nM)

O

O CO2H

AcHN

NH2 146 (1.5 µM)

H

NH2 147 (75 µM)

Figure 24 Inhibitory activity of GS4071 121 and related compounds against influenza A virus sialidase. IC50 values are given in parentheses.

910


likely as a consequence of their zwitterionic character. A prodrug approach in which the carboxylate was converted to an ethyl ester was investigated for 140 and 121, and proved successful only in the latter case.148,149 The ethyl ester of 121 was found to display considerably superior pharmacokinetic properties to the parent carboxylic acid. Oseltamivir 148, the orally administered ethyl ester form of GS4071 121, is now produced under the trade name TamifluTM by Roche.150 TamifluTM and RelenzaTM are currently the only NA inhibitors approved for clinical use. The development of new synthetic routes for the production of Oseltamivir remains the subject of intensive research in academic and industrial settings since demand currently outstrips production capacity.87,151–154 O

CO2Et

AcHN NH2 Oseltamivir 148

1.22.3.5 Influenza Virus NA Inhibitors Based on an Aromatic Scaffold One of the earliest strategies employed in the search for noncarbohydrate-based sialidase inhibitors involved replacement of the dihydropyran core of Neu5Ac2en with an aromatic ring. These efforts were based in part on the observation that crystal structures of Neu5Ac2en and related inhibitors in complex with influenza virus NA did not suggest significant contacts between the cyclic scaffolds and the protein.155 In its bound conformation Zanamivir 9 adopts a half-chair orientation, with both the carboxylate and the normally equatorial substituents at C4, C5, and C6 essentially coplanar with the ring.52 Therefore, it appeared reasonable to replace the sugar ring with a modified benzoic acid-bearing substituent oriented for optimal interaction with the protein in key binding pocket subsites. Efforts to design benzoic acid-based NA inhibitors were pursued by a number of groups that advanced other purported benefits such as enhanced stability, lipophilicity and bioavailability, reduced molecular complexity, and ease of synthesis.156–163 Early investigations afforded the aromatic counterpart 149 of Zanamivir 9, which displayed no appreciable interaction with sialidase.164 Removal of the glycerol side chain gave the lead compound 150 (BANA113, BCX-140), a modestly active inhibitor of influenza A virus sialidase (IC50 ¼ 2.5 mM)156–158 that reduced enzymatic activity at a level comparable to Neu5Ac2en. X-ray structure determination of 150 bound to influenza virus sialidase revealed a bound orientation distinct from that of Neu5Ac2en-based inhibitors. Whereas the guanidino group of Zanamivir is enclosed by subsite S2, the same functional group in 150 was found to bind at S5, engaged in charge–charge interactions with Glu276.157–159 Efforts to occupy the S2 subsite by addition of a guanidino group at C5 of the benzoic acid template 151 resulted in even worse inhibitory activity (IC50 ¼ 70 mM),159 attributed to inefficient interaction of the newly introduced guanidino group with Glu119 and Glu227. Addition of a hydrophobic residue at C5 [152, R ¼ OCH(C2H5)2] led to activity (IC50 ¼ 3 mM) that did not exceed that of 150.161,163 OH OH CO2H

HO AcHN

CO2H AcHN

HN

149

CO2H

AcHN HN

HN NH

NH H2N

R

H2N 150

NH H2N 151: R = NHC(=NH)NH2 152: R = OCH(C2H5)2

Computational methods were used in the design of a second generation of benzoic acid-based NA inhibitors, including di-, tri-, and tetrasubstituted derivatives.158,161–163 Although a large number of candidates were synthesized, none of the resulting compounds demonstrated significantly increased activity against influenza virus sialidase. Moreover, the interactions of individual substituents on the benzene ring with the active site were


911

not found to be additive. The overall interaction of the molecules with the active site of the enzyme was dependent upon the electronic and steric interaction of each unique substituent, which made the design of inhibitors difficult.158 Replacement of the guanidino group of 150 with various 2-amino-imidazole residues gave structures of type 153.160 The activity of the reported compounds (R ¼ aromatic) was only moderate, though comparable to 150, suggesting that neither the imidazole ring nor its substituents achieved additional binding contacts with NA. However, X-ray analysis of 153 (R ¼ Ph) with influenza B virus sialidase revealed that in addition to the expected contacts made between the 2-amino-imidazole nitrogen atoms and those residues that bind the guanidino group in 150, the phenyl group occupied a hydrophobic cleft formed by Ile220 and Ala244.160 CO2H R AcHN

153

N HN

NH2 N

CO2H

HO

N N

R

HO

CO2H

R

O H2N

HN NH

154

O

155

Replacement of the C4 acetamido moiety of 150 with, for example,-NHSO2CH3,-CONHCH3,-SO2NH2, orCH2SOCH3 did not enhance activity, although each of these groups were found to bind to the same pocket as the acetamido group, albeit in a different manner.158 Another group of benzoic acid-based sialidase inhibitors in which the acetamide of 150 was replaced with substituted 2-pyrrolidinones was disclosed.162,163 Compound 154 (R ¼ H) was 10-15-fold less active than its acetamido counterpart 150, consistent with the loss of a hydrogen-bonding interaction from the acetamide NH to an ordered water molecule. However, molecular modeling studies revealed that incorporation of a hydroxymethyl substituent onto C50 of the pyrrolidinone could provide an adequate means of replacement of the ordered water. Compound 154 (R ¼ hydroxymethyl) showed low micromolar activity against both NA(A) and NA(B) (IC50 ¼5 and 8mM, respectively).165 X-ray crystallography was employed to confirm the modeling-based assumptions that the methylene groups of the 2-pyrrolidinone moiety interacted with the hydrophobic portion of the S3 pocket, that the hydroxymethyl group displaced a buried water molecule buried in S2, and that the guanidino group remained bound in subsite S5. In light of these findings it was anticipated that exchange of the polar guanidino substituent with a more lipophilic function might result in effective inhibitors, owing to the potential for nonpolar contacts in S4 and S5. Replacement of the guanidino group in 154 (R ¼ hydroxymethyl) by a hydrophobic 3-pentylamino group gave 155, a 100-fold more potent inhibitor of NA(A) (IC50 ¼ 0.048 mM), though an ineffective inhibitor of NA(B) (IC50 ¼ 104 mM). Analysis of the X-ray structure of the NA(A)–155 complex showed, as expected, significant differences in the S5 binding pocket when compared to the structure of NA(A) bound to the guanidino derivative 154. Interaction with the 3-pentylamino group resulted in conformational reorganization of Glu276 to form an intramolecular salt bridge with Arg224, as previously observed in the case of the C6 carboxamide series of Neu5Ac2en analogs, GS4071, and other inhibitors. As a result of this arrangement a small hydrophobic pocket is formed into which one branch of the pentyl chain binds, while the other half interacts with the existing, extended lipophilic cleft of subsite S4 created by Ala246 and Ile222. It is believed that an entropy gain as a result of these hydrophobic interactions is mainly responsible for the enhanced NA(A) inhibitory activity.163 For influenza B NA, these rearrangements result in distortion of the protein backbone near Glu276 and in the second amino acid shell, which is energetically unfavorable and consequently lowers activity.114 More recently, investigations of new tetrasubstituted benzene and pyridine derivatives have been reported.166 The first inhibitor in this series was 156 (3-guanidino-4-N-acetyl-5-hydroxyethyl benzoic acid). Although it displayed poor in vitro activity (IC50 ¼0.4mM), analysis of the 156–NA cocrystal structure revealed that the hydroxyethyl group (R1) occupied subsite S5, the preferred site of the glycerol side chain of Neu5Ac2en. Replacement of the hydroxymethyl substituent with an oxime afforded 157, and resulted in a 10-fold increase in activity. The pyridine-based analog of 150 was also prepared, and inhibition of NA(A) by 158 was found to be in the low micromolar range, but did not exceed the potency of the original carbocycle (IC50 ¼ 6 mM for 158, compared to IC50 ¼ 2.5 mM for 150). Both 150 and 158 were found to bind to NA in a similar orientation. The tetrasubstituted pyridine derivative 159 (IC50 ¼4mM) also showed no significant increase in activity compared to the benzene series. Overall, the interactions between the pyridine nitrogen and the active site were not relevant to activity.166

912


R1

X

CO2H

R2 R3

156: 157: 158: 159:

X = CH, R1 = CH2CH2OH, R2 = NHCOCH3, R3 = NHC(=NH)NH2 X = CH, R1 = CH=NOH, R2 = NHSO2CH3, R3 = NHC(=NH)NH2 X = N, R1 = NHC(=NH)NH2, R2 = NHCOCH3, R3 = H 1 2 3 X = N, R = NHC(=NH)NH2, R = NHCOCH3, R = NH2

To date no inhibitor based on an aromatic scaffold has approached the potency of Zanamivir or Oseltamivir against influenza virus sialidase. No compound of this family has proceeded to clinical trials.

1.22.3.6 Inhibitors Based on a Five-Membered Ring Scaffold Whereas cyclohexene or benzene ring systems are intuitively apparent mimics of Neu5Ac2en and related inhibitors, it is less obvious that five-membered ring systems can play the same role. However, it has been demonstrated that compounds based on different five-membered cyclic scaffolds can engage each of the identified subsites (S1–S5) of influenza virus NA, resulting in potent inhibitory activity.

1.22.3.6.1 Cyclopentane derivatives as influenza virus sialidase inhibitors The nine-carbon furanose 160, one of the first inhibitors of influenza virus NA described that contained a five-membered ring, displayed activity that approached that of Neu5Ac2en.167 Comparison of the crystal structures of 160 and Neu5Ac2en bound to N9 influenza virus sialidase revealed that the key functional groups (carboxylate, glycerol side chain, N-acetyl group, and C4 hydroxyl group) in both complexes shared similar orientations within the active site, despite the fact that the space occupied by the furanose ring differed from that filled by the dihydropyran backbone.168 This observation reinforced the notion that the inhibition of influenza virus sialidases is highly dependent on the relative position of the pendant functional groups within the active site rather than the absolute position of the central ring. OH OH

HO

O AcHN

COOH OH

COOH AcHN HN

HO H2N 160

COOH AcHN HN

NH 161

H2N

NH 162

The encouraging NA inhibitory properties of nonulosonic acid 160 served as a starting point for the structure-based design of a new class of sialidase inhibitors at BioCryst Pharmaceuticals.168 Evaluation of racemic 161 in complex with sialidase identified a single active isomer. As expected, the interactions of the guanidino group with the enzyme in S2 were found to parallel those observed for Zanamivir. This trisubstituted structure showed 10-fold greater inhibition of influenza A sialidase than did Neu5Ac2en. Further development efforts led to 162, containing an n-butyl side chain designed to generate additional contacts with the small hydrophobic surface in the active site formed by Ala246, Ile222, and the side chain of Arg224 (S4). To determine the identity of the active form of 162, a stereoisomeric mixture was soaked into crystals of N9 sialidase. Surprisingly, the relative stereochemistry of the active forms of 161 and 162 differed, and the absolute configuration at C4 was found to be R in compound 162 and S in compound 161. As a consequence, the guanidino groups are arranged differently in the S2 binding subsite and the hydrophobic n-butyl side chain of 162 adopts different positions in the active site of influenza virus sialidase A compared to B, as was reported for other Neu5Ac-type inhibitors bearing hydrophobic groups in place of the glycerol side chain.115,136 Encouraged by the successful inhibition of influenza virus NA by 162 [IC50 ¼ 50 and 900 nM against NA(A) and NA(B), respectively],168,169 compound 163 (BCX-1812) was synthesized. The n-butyl side chain of 162 was replaced by a 20 -ethylbutyl group, intended to take advantage of both the hydrophobic pocket created by the rearrangement of Glu276 and the hydrophobic surface of S4. Indeed, the crystal structure of the enzyme bound to active isomer 163 revealed both of the desired interactions. Once the isomer of highest binding affinity had been identified, the stereoselective synthesis of this compound was undertaken.169 This new cyclopentane-based compound BCX-1812 163 was the first highly potent inhibitor of its class, inhibiting both influenza A and B virus sialidases at a nanomolar level (IC50 ¼ 1.1 and 0.2 nM against NA(A) and NA(B), respectively). The functionalized cyclopentane 164 (BCX1827) displayed similar activity to 163, exhibiting potent anti-influenza virus effects both in vitro in MDCK cells and in vivo in a mouse model.170–172


913

OH CO2H

AcHN

HN

HN H2N

CO2H

AcHN

NH

H2N

NH

Peramivir, BCX-1812 163

164

BCX-1812, also known as Peramivir and RWJ-270201, successfully completed animal studies and Phase I and Phase II clinical trials in which the compound was administrated orally showing neither major side effects nor toxicity.65 However, in a Phase III trial, Peramivir did not show statistical efficacy, presumably because of a lack of bioavailability. Recently, the efficacy of intramuscular injection of Peramivir in a mouse influenza model was demonstrated (Figures 25 and 26).173 It is known that C6-carboxamide derivatives of Zanamivir are very effective against NA(A) but only modestly active against NA(B) (see Section 1.22.3.2.1.1).112,113 An analogous modification was applied to cyclopentane-based inhibitors.174 A series of multisubstituted cyclopentane amides such as 165–168 were prepared, including 14 alkyl or aralkyl monosubstituted amides, 13 alkyl or aralkyl disubstituted amides, and 12 examples of cycloaliphatic or substituted cycloaliphatic amides. The complexity of this library was increased by the initial synthesis and screening of stereoisomeric mixtures, arising from chiral centres at C1 in the ring and C10 on the side chain. Those diastereomeric mixtures that demonstrated significant inhibition of NA were synthesized separately in a stereoselective manner and reassayed in order to identify the most active component. R1 N R2

O

CO2H AcHN

1 2 166: R = CH2CH3, R = CH2CH3

167: R1 = (CH2)2CH3, R2 = (CH2)2CH3

HN H2N

165: R1 = CH(CH2CH3)2, R2 = H

168: R = −(CH2)2O(CH2)2− NH

Arg371

Arg292

Glu276

Tyr406

Arg224 Arg118

Asp151 Trp178

Figure 25 Superimposition of inhibitors and key active site residues from crystal structures of BCX-1812 163 (magenta carbons, PDB – 1L7F) and Neu5Ac2en 2 (green carbons, PDB – 1F8B) in complex with influenza virus A sialidase.45

914


Glu276

Arg292 NH

O − H2N HN

H2N

O

NH2

+

+ S5

NH2

H N

H2N

Arg224

Arg371

+ O

HO Ile222

NH2

S1

− O

S4

+

NH

H2N

S3

Trp178

H2N

NH

O

NH2 NH Arg118

+ NH2

NH

O

−

O

S2 H2N

NH2

−

−

+ O

O

O

O

Asp151

NH

Arg152

Glu227

Glu119

Figure 26 A schematic representation of some key interactions of BCX-1812 163 with conserved influenza virus A sialidase active site residues.

In vitro test data for the best candidates 165–168 showed low micromolar activity for the inhibition of NA(A) but diminished activity towards NA(B), although it remained in the micromolar range. Specifically, secondary amide 165 was active at a level of 0.08mM against influenza virus sialidase A and 3 mM against sialidase B, disubstituted amide 166 yielded IC50 values of 0.015 and 3 mM against type A and B NA, 167 displayed IC50 values of 0.06 and 9.2 mM, and 168 inhibited NA(A) at a level of 0.04 mM and NA(B) at 7.8 mM, respectively.174 These results were comparable to the results reported for the amides of Zanamivir type. The active stereoisomer of 166 was identified through X-ray diffraction after soaking the racemic cyclopentane amide into crystals of the enzyme. The carboxylic acid and the guanidino group were found to be oriented trans to each other, while the carboxylic acid and the substituted amide group were cis. Thus, the absolute configuration at the carbon bearing the guanidino group (C4) is S in 166, whereas it is R in BCX-1827. As a result of these differences in stereochemistry, both the guanidino group and the acetamido groups of 166 interact differently with the active site residues compared to their counterparts in BCX-1827. However, the interactions of the guanidino group of 166 with active site amino acids are similar to those observed in the dihydropyran series.112,113 Substrate analogs of 165 and 167 were also prepared, but showed significantly reduced inhibition.174

1.22.3.6.2 Pyrrolidine derivatives as influenza virus sialidase inhibitors Another remarkable approach to the design of influenza virus sialidase inhibitors, in this case based on a pyrrolidine core, was reported from Abbott Laboratories.60,175–177 Directed screening of selected chemical libraries using a sialidase biochemical assay, identified a variety of compounds with sialidase inhibitory activities less than 100mM. The selected lead compound in this series, cis-N-t-butoxycarbonyl-3-amino-pyrrolidine-4-carboxylic acid 169, had an activity of 58 mM against influenza virus A sialidase. The strategy for the optimization of this lead structure involved a combination of traditional medicinal chemistry including X-ray crystallographic analysis, computational modelling and chemical synthesis, and combinatorial chemistry (high-throughput parallel synthesis) allowing rapid and comprehensive exploration of a given scaffold. Docking studies of 169 with NA structures suggested addition of a substituent on the ring nitrogen, with the intention of creating additional contacts in both subsites S3 and S5. Thus, large numbers of trisubstituted pyrrolidine analogs such as amides (250), carbamates (25), sulfonamides (25), and ureas (250)


915

were synthesized by automated solid phase parallel synthesis. From within this pool of compounds, urea analogs with various alkyl groups demonstrated low micromolar level inhibition. In particular compound 170 was approximately 50fold (1.1 mM against NA(A)) more active than the lead 169. Through analysis of the structures of 169 and 170 bound to influenza virus A sialidase, it was observed that neither of the important subsites S3 (acetamide pocket of Zanamivir) and S2 (guanidino pocket of Zanamivir) were occupied properly. Efforts to improve binding via variation of the exocyclic amine were frustrated by reorientation of the inhibitor within the binding site. Furthermore, molecular modeling of this class of trisubstituted pyrrolidines ceased to be predictive. As a consequence, a series of tetrasubstituted pyrrolidines was synthesized, and a method for high-throughput protein crystallographic X-ray analysis was established in order to verify design strategies. The rapid availability of structural information had a profound impact on this program, allowing for adjustment in response to unexpected binding modes of new inhibitors and for facile identification of active isomers from among diastereomeric mixtures. Of the tetrasubstituted pyrrolidine series of type 171–173 only the acetamide 171 [7.5 mM against NA(A)] and the trifluoroacetamide 172 [0.28 mM against NA(A)] were active in the low micromolar range, though selectivity for inhibition of NA(A) over NA(B) was pronounced. Evaluation of the NA-172 complex confirmed the expected interaction of the carboxylate with the triarginyl cluster in S1, identified binding of the hydrophobic portion of the urea in S5 and the concomitant conformational reorganization of Glu276 observed previously,115,148,168 and revealed that the exocyclic amine did not bind at S2 due to an unexpected rotation of the pyrrolidine ring. Carbocyclic analog 173 exhibited similar binding but weaker activity (24mM) against NA(A) and no activity against NA(B). NH2 R N

NH2

R O

CO2H

X

CO2H

N O 169: R =

O 171: X = N, R = NHC(O)CH3 172: X = N, R = NHC(O)CF3 173: X = CH2, R = NHC(O)CH3

O 170: R =

N

The breakthrough in the development of high-affinity inhibitors for influenza virus sialidase in this series was the surprising 180 reorientation (relative to 171–173) of cyclopentane derivative 174 within the active site of the sialidase [IC50 ¼ 0.7 mM against NA(A)]. Crystallographic analysis of 174 bound to NA showed an unpredicted hydrophobic interaction of the methyl ester with subsite S2, normally the site of charge–charge interactions. Compound 174 was considered as a three-site binder, since the exocyclic amine made no significant interaction with the protein. Four-site binders were obtained through introduction of aliphatic alkyl groups on the side chain methylene carbon of a pyrrolidine template, as illustrated by 175, affording significant enhancement in potency against both NA (A) and NA(B) [IC50 ¼41nM against NA(A) and IC50 ¼56nM against NA(B)].60,178 X-ray crystallographic studies indicated that 175 retained the binding mode of 174, with the methyl ester interacting at S2, and in addition to the binding of the carboxylic acid at S1 and the acetamido group at S3, the iso-butyl group occupied S5. This new binding of the hydrophobic methyl ester arises mainly through strong van der Waals interaction with the side chain methylene groups of Asp151 and Leu135 in subsite S2, as well as a potential p–p stacking with the C¼O of Glu119. Additionally, the ester displaces two water molecules from S2, affording 175 an entropic advantage.179 O

O

O

O AcHN

AcHN

AcHN COOH NH2 174

N H 175

COOH O

N H

COOR

176: R = H 177: R = CH2CH3

Extensive efforts were devoted to replacement of the metabolically labile methyl ester in 175. Potentially suitable functionalities were identified including 2-pyrazolyl or short alkene residues such as cis-propenyl.179 The latter alkene was incorporated into an inhibitor that ultimately led to development of the clinical candidate 176 (ABT-675, A-315675) after further optimization of the hydrophobic side chain.175,180,181 The crystal structures of ABT-675 bound

916


Figure 27 Superimposition of inhibitors and key active site residues from crystal structures of pyrrolidine derivative 175 (magenta carbons, PDB – 1XOE) and Neu5Ac2en 2 (green carbons, PDB – 1F8B) in complex with influenza virus A sialidase.45

to NA(A) and (B) confirmed the expected hydrophobic interactions of the cis-propenyl group with S2. The n-propyl group of the side chain was observed to interact with S4, while the methyl and methoxy groups were associated with S5. In contrast to results obtained with other inhibitors that occupy both S4 and S5, Glu276 was observed to maintain its native conformation in the 176-NA(B) complex and underwent conformational reorganization during binding by NA(A). This pyrrolidine-based inhibitor effectively inhibits both influenza virus A and B sialidase [IC50 ¼0.2nM against NA(A) and IC50 ¼ 0.1 nM against NA(B)], and represents a 107-fold improvement in binding affinity relative to the original pyrrolidine screening hit 169. Due to its zwitterionic character ABT-675 has limited oral bioavailability.175 Attempts to remedy this drawback via replacement of the pyrrolidine core with a furan or cyclopentane ring resulted in a significant loss of activity.178 A second approach involving use of the ethyl ester prodrug ABT-667 (177) has proven more successful, with this compound currently in clinical studies (Figure 27).

1.22.4 Outlook Influenza virus sialidase is a challenging target in drug design that required over three decades of intense research before yielding the two inhibitors that are now used clinically. A plethora of structural information will assist in the development of new, more potent and longer-lasting inhibitors, and ongoing efforts will undoubtedly reveal additional drugs. As the enormous efforts to date have shown, there are many facets to the challenge of NA inhibitor drug design. It is particularly apparent that incorporation of hydrophobic character in an effort to reduce overall polarity and complement nonpolar regions of the active site, brings with it dangers in terms of selective inhibition of sialidase from influenza virus A over influenza virus B. Avoiding inhibitor-induced active site reorganization will likely feature prominently in the design of new drugs owing to the increased vulnerability that such treatments may have towards mutation-induced redundancy. Given the prominent role of computational chemistry in NA inhibitor design to date, it is not surprising that some of the more recent efforts have been theoretical in nature.109,182–184

Glossary Ic50 Inhibitor concentration at which 50% inhibition is observed. Ki Inhibition constant.


917

Acknowledgments Prof. von Itzstein gratefully acknowledges the financial support of the Australian Research Council (ARC) and the National Health and Medical Research Council and the ARC for the award of a Federation Fellowship. Dr. Dietmar Gehle gratefully acknowledges the award of a Deutsche Forschungsgemeinschaft (DFG) Fellowship. Dr. Jamie Rich gratefully acknowledges the Natural Sciences and Engineering Research Council of Canada for a postdoctoral fellowship.

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Agents Chemother. 1998, 42, 647–653. Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.; Li, W.; Sidwell, R. W.; Huffman, J. H.; Sweet, C.; Jakeman, K. J.; Merson, J.; Lacy, S. A.; Lew, W.; Williams, M. A.; Zhang, L.; Chen, M. S.; Bischofberger, N.; Kim, C. U. Antimicrob. Agents Chemother. 1998, 42, 640–646. Doucette, K. E.; Aoki, F. Y. Exp. Opin. Pharmacother. 2001, 2, 1671–1683. Yeung, Y. Y.; Hong, S.; Corey, E. J. J. Am. Chem. Soc. 2006, 128, 6310–6311. Fukuta, Y.; Mita, T.; Fukuda, N.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 2006, 128, 6312–6313. Harrington, P. J.; Brown, J. D.; Foderaro, T.; Hughes, R. C. Org. Process Res. Dev. 2004, 8, 86–91. Abrecht, S.; Harrington, P.; Iding, H.; Karpf, M.; Trussardi, R.; Wirz, B.; Zutter, U. Chimia 2004, 58, 621–929. Luo, M.; Air, G. M.; Brouillette, W. J. J. Infect. Dis. 1997, 176, S62–S65. Jedrzejas, M. J.; Singh, S.; Brouillette, W. J.; Laver, W. G.; Air, G. M.; Luo, M. Biochemistry 1995, 34, 3144–3151. Singh, S.; Jedrzejas, M. J.; Air, G. M.; Luo, M.; Laver, W. G.; Brouillette, W. J. J. Med. Chem. 1995, 38, 3217–3225. Chand, P.; Babu, Y. S.; Bantia, S.; Chu, N. M.; Cole, L. B.; Kotian, P. L.; Laver, W. G.; Montgomery, J. A.; Pathak, V. P.; Petty, S. L.; Shrout, D. P.; Walsh, D. A.; Walsh, G. W. J. Med. Chem. 1997, 40, 4030–4052.

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159. Sudbeck, E. A.; Jedrzejas, M. J.; Singh, S.; Brouillette, W. J.; Air, G. M.; Laver, W. G.; Babu, Y. S.; Bantia, S.; Chand, P.; Chu, N.; Montgomery, J. A.; Walsh, D. A.; Luo, M. J. Mol. Biol. 1997, 267, 584–594. 160. Howes, P. D.; Cleasby, A.; Evans, D. N.; Feilden, H.; Smith, P. W.; Sollis, S. L.; Taylor, N.; Wonacott, A. J. Eur. J. Med. Chem. 1999, 34, 225–234. 161. Brouillette, W. J.; Atigadda, V. R.; Duarte, F.; Luo, D.; Air, G. M.; Babu, Y. S.; Bantia, S. Bioorg. Med. Chem. Lett. 1999, 9, 3259–3259. 162. Finley, J. B.; Atigadda, V. R.; Duarte, F.; Zhao, J. J.; Brouillette, W. J.; Air, G. M.; Luo, M. J. Mol. Biol. 1999, 293, 1107–1119. 163. Atigadda, V. R.; Brouillette, W. J.; Duarte, F.; Ali, S. M.; Babu, Y. S.; Bantia, S.; Chand, P.; Chu, N.; Montgomery, J. A.; Walsh, D. A.; Sudbeck, E. A.; Finley, J.; Luo, M.; Air, G. M.; Laver, G. W. J. Med. Chem. 1999, 42, 2332–2343. 164. Williams, M.; Bischofberger, N.; Swaminathan, S.; Kim, C. U. Bioorg. Med. Chem. Lett. 1995, 5, 2251–2254. 165. Brouillette, W. J.; Atigadda, V. R.; Luo, M.; Air, G. M.; Babu, Y. S.; Bantia, S. Bioorg. Med. Chem. Lett. 1999, 9, 1901–1906. 166. Chand, P.; Kotian, P. L.; Morris, P. E.; Bantia, S.; Walsh, D. A.; Babu, Y. S. Bioorg. Med. Chem. 2005, 13, 2665–2678. 167. Yamamoto, T.; Kumazawa, H.; Inami, K.; Teshima, T.; Shiba, T. Tetrahedron Lett. 1992, 33, 5791–5794. 168. Babu, Y. S.; Chand, P.; Bantia, S.; Kotian, P.; Dehghani, A.; El Kattan, Y.; Lin, T. H.; Hutchison, T. L.; Elliott, A. J.; Parker, C. D.; Ananth, S. L.; Horn, L. L.; Laver, G. W.; Montgomery, J. A. J. Med. Chem. 2000, 43, 3482–3486. 169. Chand, P.; Kotian, P. L.; Dehghani, A.; El-Kattan, Y.; Lin, T. H.; Hutchison, T. L.; Babu, Y. S.; Bantia, S.; Elliott, A. J.; Montgomery, J. A. J. Med. Chem. 2001, 44, 4379–4392. 170. Bantia, S.; Parker, C. D.; Ananth, S. L.; Horn, L. L.; Andries, K.; Chand, P.; Kotian, P. L.; Dehghani, A.; El Kattan, Y.; Lin, T.; Hutchison, T. L.; Montgomery, J. A.; Kellog, D. L.; Babu, Y. S. Antimicrob. Agents Chemother. 2001, 45, 1162–1167. 171. Sidwell, R. W.; Smee, D. F.; Huffman, J. H.; Barnard, D. L.; Bailey, K. W.; Morrey, J. D.; Babu, Y. S. Antimicrob. Agents Chemother. 2001, 45, 749–757. 172. Smee, D. F.; Huffman, J. H.; Morrison, A. C.; Barnard, D. L.; Sidwell, R. W. Antimicrob. Agents Chemother. 2001, 45, 743–748. 173. Bantia, S.; Arnold, C. S.; Parker, C. D.; Upshaw, R.; Chand, P. Antiviral Res. 2006, 69, 39–45. 174. Chand, P.; Babu, Y. S.; Bantia, S.; Rowland, S.; Dehghani, A.; Kotian, P. L.; Hutchison, T. L.; Ali, S.; Brouillette, W.; El-Kattan, Y.; Lin, T.-H. J. Med. Chem. 2004, 47, 1919–1929. 175. Kati, W. M.; Montgomery, D.; Carrick, R.; Gubareva, L.; Maring, C.; McDaniel, K.; Steffy, K.; Molla, A.; Hayden, F.; Kempf, D.; Kohlbrenner, W. Antimicrob. Agents Chemother. 2002, 46, 1014–1021. 176. Wang, G. T.; Chen, Y.; Wang, S.; Gentles, R.; Sowin, T.; Kati, W.; Muchmore, S.; Giranda, V.; Stewart, K.; Sham, H.; Kempf, D.; Laver, W. G. J. Med. Chem. 2001, 44, 1192–1201. 177. Kati, W. M.; Montgomery, D.; Maring, C.; Stoll, V. S.; Giranda, V.; Chen, X. Q.; Laver, W. G.; Kohlbrenner, W.; Norbeck, D. W. Antimicrob. Agents Chemother. 2001, 45, 2563–2570. 178. Wang, G. T.; Wang, S.; Gentles, R.; Sowin, T.; Maring, C. J.; Kempf, D. J.; Kati, W. M.; Stoll, V.; Stewart, K. D.; Laver, G. Bioorg. Med. Chem. Lett. 2005, 15, 125–128. 179. Maring, C. J.; Stoll, V. S.; Zhao, C.; Sun, M. H.; Krueger, A. C.; Stewart, K. D.; Madigan, D. L.; Kati, W. M.; Xu, Y. B.; Carrick, R. J.; Montgomery, D. A.; Kempf-Grote, A.; Marsh, K. C.; Molla, A.; Steffy, K. R.; Sham, H. L.; Laver, W. G.; Gu, Y. G.; Kempf, D. J.; Kohlbrenner, W. E. J. Med. Chem. 2005, 48, 3980–3990. 180. DeGoey, D. A.; Chen, H. J.; Flosi, W. J.; Grampovnik, D. J.; Yeung, C. M.; Klein, L. L.; Kempf, D. J. J. Org. Chem. 2002, 67, 5445–5453. 181. Hanessian, S.; Bayrakdarian, M.; Luo, X. J. Am. Chem. Soc. 2002, 124, 4716–4721. 182. Verma, R. P.; Hansch, C. Bioorg. Med. Chem. 2006, 14, 982–996. 183. Steindl, T.; Langer, T. J. Chem. Inf. Comp. Sci. 2004, 44, 1849–1856. 184. Zhang, J.; Yu, K.; Zhu, W.; Jiang, H. Bioorg. Med. Chem. Lett. 2006, 16, 3009–3014.


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Biographical Sketch

Dr. Jamie Rich hails from Vancouver, Canada. He completed his Bachelor’s degree in Chemistry and Biochemistry at the University of Northern British Columbia in 2000, where he worked with Dr. Kerry Reimer on the synthesis of oligosaccharides of plant cell wall origin. In the same year he began doctoral studies with Dr. David Bundle at the University of Alberta, investigating the synthesis of sulfur-containing ganglioside analogs and their potential use in conjugate vaccine applications. Following completion of his Ph.D. in 2005, Jamie moved to Australia where he is a Natural Sciences and Engineering Research Council of Canada postdoctoral fellow in the laboratory of Dr. Mark von Itzstein at the Institute for Glycomics, Griffith University. He is currently involved in the design and synthesis of new inhibitors of influenza virus sialidase.

Dr. Dietmar Gehle completed his undergraduate studies, majoring in chemistry, at the University of Paderborn, Germany in 2001. His Ph.D., in the field of carbohydrate chemistry and synthesis, was completed at the same University under the supervision of Prof. Dr. K. Krohn in 2005. Upon completion of his Ph.D. he took up a postdoctoral position in the group of Prof. Dr. M. von Itzstein at the Institute for Glycomics, Griffith University, Australia. In 2006, Dr. Gehle was awarded a Deutsche Forschungsgemeinschaft (DFG) Fellowship to continue his research interests in antiviral drug discovery in the Institute for Glycomics at Griffith University.

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Professor Mark von Itzstein completed his undergraduate and Ph.D. degree at Griffith University, Australia in 1984. His Ph.D., in the field of organophosphorus chemistry, reaction mechanisms and aspects of carbohydrate chemistry, was under the supervision of Professor Ian Jenkins and Professor Gus Guthrie. Upon completion of his Ph.D., Professor von Itzstein was awarded an Alexander von Humboldt Fellowship to work in the group of Professor Dr. Manfred Reetz. In 1986 Professor von Itzstein returned to Australia to establish his research group in the Department of Medicinal Chemistry, Monash University. During this time Professor von Itzstein and his group discovered the first designer anti-influenza drug RelenzaTM. In 2000 Professor von Itzstein established the Institute for Glycomics at Griffith University (Gold Coast Campus), Australia and is the Institute’s inaugural Director. Professor von Itzstein is the recipient of a number of awards including the Australia Prize, Centenary Medal, Adrien Albert Award, UQ Gatton Gold Medal, Alexander von Humboldt Forschungspreis, ARC Federation Fellowship, and was elected a Fellow of the Australian Academy of Sciences in 2003. Professor Dr. von Itzstein’s research interests cover a broad range of activities that focus on the chemistry and biology of carbohydrates as related to disease and drug discovery.

1.23 Production of Oligosaccharides in Microbes E. Samain, Centre de Recherche sur les Macromolećules Ve´ge´tales (CERMAV-CNRS), Grenoble, France ß 2007 Elsevier Ltd. All rights reserved. 1.23.1 Introduction

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1.23.1.1 Advantages of Oligosaccharide Production in Whole Cells

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1.23.1.2 Basis of the Different Available Technologies

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1.23.2 Technologies Using Nongrowing, Permeabilized Whole Cells

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1.23.2.1 Orotic Acid as Nucleoside Phosphate Precursor

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1.23.2.2 Use of a Single Organism

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1.23.3 Technologies Using Growing Living Cells

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1.23.3.1 Endogenously Biosynthesized Acceptors

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1.23.3.2 Internalization of Exogenous Acceptors

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1.23.4 Glycosyltransferase Genes

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1.23.4.1 Eukaryotic Genes

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1.23.4.2 Bacterial Genes

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1.23.5 Metabolic Engineering of Sugar Nucleotide Biosynthesis

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1.23.5.1 Metabolic Engineering of UDP-Gal Biosynthesis

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1.23.5.2 Metabolic Engineering of UDP-GlcNAc Biosynthesis

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1.23.5.3 Metabolic Engineering of UDP-GalNAc Biosynthesis

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1.23.5.4 Metabolic Engineering of CMP-Neu5Ac Biosynthesis

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1.23.5.5 Metabolic Engineering of GDP-Man and GDP-Fuc Biosynthesis

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1.23.5.6 Metabolic Engineering of Other Sugar Nucleotides

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1.23.6 Production of Complex Oligosaccharides by Sequential Multiglycosylation

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1.23.6.1 ABH and Lewis Antigen Oligosaccharides

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1.23.6.2 Ganglioside Oligosaccharides

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1.23.6.3 Globoside Oligosaccharides

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1.23.6.4 Nod Factor Oligosaccharides

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1.23.7 Production and Purification of Oligosaccharides in Living E. coli

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1.23.7.1 High Cell Density Culture

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1.23.7.2 Product Recovery

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1.23.8 Drawbacks and Undesirable Side Reactions

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1.23.8.1 Leakage of Intermediates during Sequential Multiglycosylation

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1.23.8.2 Repeated Glycosylation Cycles

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1.23.8.3 Unspecific Glycosylations

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1.23.8.4 Indigenous Enzymatic Modifications

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1.23.1 Introduction 1.23.1.1 Advantages of Oligosaccharide Production in Whole Cells Most of the complex carbohydrate structures of biological interest were elucidated more than 30 years ago but the difficulty in obtaining reasonable quantities of pure material from natural sources by purification has since contributed to delays in the development of glycobiology and its numerous potential applications. The synthesis of

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Production of Oligosaccharides in Microbes

complex oligosaccharides has thus become a challenge for the chemistry community. Following the pioneering work of Lemieux on the synthesis of human blood group determinants in the 1970s,1 the chemical synthesis of the major glycosyl structures was successfully achieved during the 1980s. However, these syntheses required multiple protection/deprotection steps and the resulting compounds were generally obtained only in low quantities on the milligram scale. In addition, each synthesis required several months of labor-intensive work and chemists were reluctant to repeat them when stocks were exhausted. Therefore, many chemists turned to enzymatic methods and investigated the use of different classes of enzymes, including glycosidase, glycosyl phosphorylase, transglycosidase, and glycosyltransferase.2,3 In nature, the formation of glycosyl bonds is catalyzed in a very specific manner by the glycosyltransferases of the Leloir pathway, which uses activated sugar nucleotides as substrate donors. The majority of genes encoding individual glycosyltransferases were cloned and characterized during the 1990s, thus enabling their production as recombinant proteins for enzymatic synthesis purposes. The major drawbacks of this approach were the cost of the sugar nucleotides and the inhibition by nucleoside diphosphate generated in the reaction. These problems were partially solved by recycling the sugar nucleotides using multiple enzymatic systems that mimicked the natural sugar-nucleotide biosynthesis pathway. These strategies were successfully used to produce various complex oligosaccharides on the multigram scale.4 However, the cost of synthesis still remained high for several reasons. First, the different enzymes had to be bought or produced and purified separately. For example, the galactosylation system with its integrated cofactor regeneration required seven different enzymes when glucose or galactose was used as a precursor.5 Secondly and most importantly, the formation of each glycosidic bond required the consumption of at least two energy-rich bonds that were generally provided by the addition of relatively expensive energy-rich phosphate compounds, such as phosphoenolpyruvate. Recently, new strategies have emerged to reduce these costs by carrying out the glycosylation in whole bacterial cells, which can provide the biological energy to power oligosaccharide synthesis and can be genetically engineered to produce the necessary enzymes.6 The high production yield of these systems and the low cost of the substrate they use offer the possibility of producing complex oligosaccharides on the multikilogram or even multiton scale at a reasonable price.

1.23.1.2 Basis of the Different Available Technologies In bacteria, all glycosylation reactions, which are catalyzed by sugar nucleotide-dependent glycosyltransferases take place in the cytoplasm, where all the enzymatic machinery for sugar nucleotide regeneration is also located. Synthesis of a defined oligosaccharide structure is theoretically possible under three conditions: (1) bacteria must functionally express the appropriate recombinant glycosyltransferase genes in the cytoplasm, (2) bacteria must be able to maintain the pool level of the sugar nucleotide that is used as substrate by the glycosyltransferases, and (3) the glycosyltransferase acceptor must be present in the cytoplasm. This last condition is the most difficult to satisfy and to circumvent this barrier, two conceptually different types of technology have been developed. The first type is based on the use of chemically permeabilized cells into which the acceptor molecule can freely enter by diffusion. This treatment kills the cells, and the different systems of oligosaccharide synthesis by permeabilized cells are all two-step processes. In the first step, bacteria are grown and harvested after induction of the genes required for glycosylation. In the second step, the cells are permeabilized and incubated with the acceptor that is glycosylated by the enzymes produced in the first step. Since permeabilization breaks the proton motive force across the cytoplasmic membrane, permeabilized cells cannot produce ATP by oxidative phosphorylation. However, they remain metabolically active to a certain degree and can produce energy-rich phosphate compounds by substrate level phosphorylation in glycolysis and other catabolic pathways. The second type of technology consists of using nonpermeabilized living bacterial cells. In this case, the acceptor must be either endogenously biosynthesized or internalized by the bacterial transport system. The advantage of this approach is that the cells are fully metabolically active and can efficiently produce ATP by oxidative phosphorylation. In addition, the production of recombinant glycosyltransferases and oligosaccharide synthesis occur simultaneously in a one-step process, which is associated with bacterial growth.

1.23.2 Technologies Using Nongrowing, Permeabilized Whole Cells Permeabilization of cells removes the barrier for the free diffusion of substrate/product across the cell membrane, but it also empties the cell of most small molecular weight cofactors. Larger molecules such as proteins stay inside the cells and permeabilized cells have been extensively exploited as a very economical source of enzyme for simple bioconversions such as hydrolysis, isomerization, and oxidation reactions that do not require a cofactor-regeneration system.


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On the other hand, glycosylation reactions require sugar nucleotide regeneration and are therefore critically affected by the dilution of cofactors resulting from their diffusion into the extracellular reaction medium. This dilution can be minimized by carrying out the reaction in very concentrated cell suspensions, but efficient glycosylation can only be achieved by supplementing the reaction medium with nucleoside phosphates or by providing precursors for their biosynthesis.

1.23.2.1 Orotic Acid as Nucleoside Phosphate Precursor Orotic acid is an intermediate in the biosynthesis of pyrimidine nucleotides. Mutant strains of Corynebacterium glutamicum accumulate orotic acid and therefore industrial processes have been developed for its low-cost production. Orotic acid is normally converted into UMP in two enzymatic steps: the transfer of ribose-50 -P from phosphoribosylpyrophosphate (PRPP) to orotic acid and the subsequent decarboxylation of the resulting orotidine 50 -monophosphate intermediate to yield UMP (Figure 1). Permeabilized cells of C. ammoniagenes have been reported to efficiently convert orotic acid into a mixture of UMP, UDP, and UTP in the presence of glucose as the energy source.7 Taking advantage of this unique property, researchers at the Kyowa Company have designed a process for the synthesis of sugar nucleotides by combining C. ammoniagenes cells with the permeabilized cells of recombinant Escherichia coli, used as a source of kinase, pyrophosphorylase, and other enzymes necessary for the activation of simple sugars into sugar nucleotides.8 In this process, the different strains are first individually grown in separate fermentors. After induction of the recombinant genes, the different cultures are harvested by centrifugation and the cells are permeabilized by the addition of xylene and polyoxyethylene octadecylamine (Nymen S-215). The different cells are then incubated together in the presence of orotic acid as UTP precursor, glucose or fructose as energy source, and the appropriate sugar precursor for the synthesis of the sugar nucleotide. This system has been successfully used for the high-yield production of UDP-Gal (44gl1)8 and UDP-GlcNAc (7.4gl1).9 Orotic acid can also be used as a precursor for the synthesis of CTP by coupling C. ammoniagenes cells with E. coli cells expressing the pyrG gene for CTP synthase, which converts UTP into CTP in the presence of glutamine and ATP. The formation of CMP-Neu5Ac is then possible by adding E. coli cells containing CMP-Neu5Ac synthase.10 Orotic acid is not an intermediate in the biosynthesis of purine nucleotides and cannot be used for the production of GTP and GDP-Fuc. However, permeabilized cells of C. ammoniagenes have been shown to phosphorylate GMP into GTP and a system for the production of GDP-Fuc from GMP through bacterial coupling has been described.11 All these systems for sugar nucleotide synthesis can be coupled with glycosylation by adding E. coli cells containing the appropriate glycosyltransferase to the reaction medium (Figure 2) and the resulting high-yield synthesis of the following structures has been reported: globotriose,8 N-acetyllactosamine,12 30 -sialyllactose,10 the sialyl-Tn epitope Neu5Aca-6GalNAc,13 and the Lewis x trisaccharide.11

1.23.2.2 Use of a Single Organism Oligosaccharide production has also been carried out in permeabilized E. coli cells without using the C. ammoniagenes system for UTP production. In this case, all the genes necessary for sugar nucleotide regeneration and oligosaccharide synthesis are overexpressed in a single strain. This system has only been described for the galactosylation reaction and first used for the synthesis of the a-gal epitope14 (Gala-3Galb-4Glc) and globotriose.15 The addition of catalytic amounts of UDP-Glc seems to be an absolute requirement to make the system work and the addition of energy-rich phosphate compounds such as ATP and phosphoenolpyruvate significantly improves the yield. These compounds are presumably recycled during the catabolism of glucose, which is provided as an energy source. Alternatively, UDP-Glc can be generated from sucrose and UDP by the reverse action of sucrose synthase.16 This latter strategy has previously been used in different enzymatic galactosylation systems with purified enzymes and was recently adapted for metabolically engineered Pichia pastoris.17

1.23.3 Technologies Using Growing Living Cells 1.23.3.1 Endogenously Biosynthesized Acceptors In bacteria most complex carbohydrate structures such as lipopolysaccharides (LPSs) and exopolysaccharides are assembled on lipid carriers and there are normally no free cytoplasmic oligosaccharide intermediates that could be used as acceptors for heterologous glycosyltransferases. The only exception is disaccharide trehalose (Glca-aGlc),

926


O NH

O

COOH

N H Orotic acid

Phosphoribosylpyrophosphate

Pyrophosphate O NH

O

N

COOH

Ribose-5⬘-P Orotidine 5⬘-phosphate (OMP)

CO2 O NH

O

N

Ribose-5⬘-P Uridine 5⬘-phosphate (UMP) Figure 1 Enzymatic conversion of orotic acid into UMP.

which is produced under osmotic or desiccation stress, and chitooligosaccharides that are produced in rhizobia as precursors for the synthesis of Nod Factors. These signal molecules are secreted by rhizobia to specifically induce the formation of plant nodules. The bacteria then invade the nodules, where they fix atmospheric nitrogen in a syntrophic association with the plant. The genes involved in the biosynthesis of Nod Factors have been identified and the nodC gene has been shown to encode a chitooligosaccharide synthase that uses UDP-GlcNAc as substrate donor.18 The mechanism of synthesis is still not fully understood but it has been clearly established that the undecaprenyl pyrophosphate-linked intermediate is not involved in the reaction. Some experimental data suggest that free GlcNAc could be used as a primer.19 In E. coli, GlcNAc is generated during recycling of the cell wall muropeptide by the b-N-acetylglucosaminidase encoded by nagZ.20 However, GlcNAc is re-used by the cell through phosphorylation to GlcNAc6P by the cytoplasmic kinase encoded by nagK.21 Consequently, GlcNAc concentrations are always maintained at an extremely low level and the role of GlcNAc as a primer remains questionable. Chitobiose and chitotriose


927

Figure 2 Oligosaccharide synthesis in permeabilized E. coli cells. Acceptors and sugar nucleotides freely diffuse across the membranes to be intracellularly used as substrates by recombinant glycosyltransferase. Efficient glycosylation was obtained by coupling glycosylating cells with sugar nucleotide production systems that use orotic acid as an inexpensive precursor.

are not used as acceptors and chitooligosaccharide synthesis is believed to occur with no dissociation of the enzyme substrate complex during elongation. Expression of nodC in living E. coli has been shown to result in the intracellular synthesis of chitooligosaccharide22 and this property was exploited to develop a process for the large-scale production of chitopentaose, by the high cell density culture (HCDC) of E. coli overexpressing nodC.23 Once produced in the cytoplasm, chitooligosaccharides can serve as acceptors for glycosyltransferases that recognize a terminal nonreducing GlcNAc residue. This strategy was first used for the synthesis of the hexasaccharide Galb-4[GlcNAcb-4]4GlcNAc by an E. coli strain that co-expressed the Azorhizobium caulinodans nodC gene and the Neisseria meningitidis lgtB gene for b-1,4-galactosyltransferase.24 The terminal N-acetyllactosamine motif of this hexasaccharide is a precursor for the synthesis of many complex carbohydrate structures and it has recently been shown that the a-gal epitope, which is responsible for hyper-acute rejection in xenotransplantation, could be produced with this system by expressing the additional gene for a-1,3-galactosyltransferase.25 A disadvantage of this strategy is the large size of the chitopentaose primer, which considerably increases the molecular weight of the target structures. A possible strategy to reduce this size is to enzymatically hydrolyze the chitopentaose in the living bacteria as soon as it is produced by NodC.26 The chitinase A1 encoded by the chiA gene from Bacillus circulans has been shown to hydrolyze one molecule of chitopentaose into two molecules of chitobiose and one molecule of GlcNAc.27 As the chitinase is normally secreted in the intracellular medium, the chiA gene was expressed under a truncated form that did not encode the peptide signal sequence. The co-expression of the lgtB gene for b-1,4-galactosyltransferase with nodC and the truncated form of chiA led to the formation of the trisaccharide Galb-4GlcNAcb-4GlcNAc, which could be used as a type 2 acceptor for the synthesis of blood grouprelated antigens. Surprisingly, no chitooligosaccharide formation was observed when chiA and nodC were co-expressed in the absence of lgtB. This was explained by the fact that chitobiose and GlcNAc, which were generated by chitopentaose hydrolysis, were catabolized by the living cells. It is known that E. coli is able to grow on chitobiose and the genes responsible for its assimilation have been identified to the cryptic cel operon, which has subsequently been renamed the chb operon.28 Chitobiose is transported and phosphorylated by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) encoded by the chbABC genes.29 The phosphoryl group is attached to the C-6 position of the nonreducing GlcNAc residue and the phosphorylated chitobiose was then hydrolyzed by the ChbF phospho-chitobiase to enter the GlcNAc6P and GlcNAc metabolic pathway. Deletion of the chb operon prevents the cells from degrading chitobiose, and a chb mutant co-expressing nodC and the chitinase gene chiA have been shown to accumulate high concentrations of chitobiose (4g l1) in the extracellular medium.26 Chitobiose is usually obtained by

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acid or enzymatic hydrolysis of chitin. However, yields are often low and irreproducible due to the poor solubility and reactivity of chitin. Therefore, the production of chitobiose in E. coli represents an interesting alternative for the preparation of this compound, which is an important building block for the synthesis of many complex carbohydrates.

1.23.3.2 Internalization of Exogenous Acceptors The other way to provide cells with an acceptor for glycosyltransferase is to add this acceptor to the extracellular medium and then find a way to internalize it into the bacterial cytoplasm. Monosaccharides substituted with small hydrophobic groups can diffuse through the membrane and the synthesis of an allyl derivative of N-acetyllactosamine by an E. coli strain expressing the lgtB gene for b-1,4-galactosyltransferase was achieved by growing this strain in the presence of GlcNAcb-allyl.30 However, the yield was low and the acceptor had to be chemically synthesized. An alternative is to internalize the acceptor by an active transporter (Figure 3). Most of the acceptors for glycosyltransferases are simple carbohydrate structures and some of these compounds are used as carbon and energy sources by bacteria that have developed efficient systems to transport them. There are three main systems for the active uptake of sugars in bacteria. In E. coli all the hexosamines, most of the hexoses (glucose, fructose, mannose), and some disaccharides, such as sucrose and cellobiose, are transported by PTSs.31 In these systems, carbohydrate translocation across the membrane is coupled with phosphorylation of the sugar and the energy for this process is provided by the conversion of phosphoenolpyruvate into pyruvate. Consequently, the sugar enters the cell in a phosphorylated form that cannot be used as an acceptor by glycosyltransferases. On the other hand, sugars that are internalized by ion-coupled transporters and by periplasmic-binding proteindependent ABC transporters are not phosphorylated during their uptake and can be used as intracellular acceptors for glycosyltransferases if their subsequent catabolism steps are abolished. In ion-coupled transport systems, the passage of sugar through the membrane is coupled to ion entry.32 The energy that drives sugar uptake comes from the electron chemical gradient across the membrane and most of the sugar carriers that use this mechanism are proton symporters. Examples of sugar proton symporters include the lactose permease LacY, the sucrose permease CscB, the raffinose permease RafB, and the low-affinity carriers for galactose (GalP), arabinose (AraE), and xylose (XylE). The melibiose symporter (MelB) has broader cation specificity and accepts Naþ or Liþ as coupling ions. Periplasmic-binding proteindependent ABC transporters are systems in which specific periplasmic-binding proteins act as high-affinity receptors to trap the sugars that are then translocated into the cytoplasm in an active process coupled to ATP hydrolysis.33 E. coli has an ABC transport system for maltose but also for most of the sugars that are also internalized by symporters, such as galactose, arabinose, and xylose.

Figure 3 Oligosaccharide synthesis from exogenous acceptors in living E. coli cells. Acceptors are transported across the intact membrane by sugar carriers to be glycosylated by recombinant glycosyltransferase using the endogenous sugar nucleotide pool of the living cells as substrate donor. ATP required for glycosylation is efficiently produced by oxidative phosphorylation.


929

The sugars that are not internalized by PTSs in E. coli and can therefore be used as potential acceptors for recombinant glycosyltransferases, are listed in Table 1. Among these compounds, lactose is of particular interest because a large number of complex sugars, such as human milk oligosaccharides, contain the lactose structure at their reducing end. First attempts to glycosylate exogenous acceptors in living E. coli were thus carried out with lactose. Once in the cytoplasm, lactose is normally hydrolyzed into galactose and glucose by the b-galactosidase encoded by lacZ. The strategy is thus to use a strain that lacks b-galactosidase, but functionally expresses the lactose permease LacY. This strain could be cultured on an alternative substrate, such as glucose or glycerol, which could serve as both carbon and energy sources for bacterial growth, the active uptake of lactose, and the glycosylation reaction. It has been reported that the rapid and excessive uptake of lactose can kill the cells by dissipating the proton motive force and disrupting membrane function.34 However, this phenomenon of lactose killing was not observed when lactose (5gl1) was added to a HCDC of a lacZ null strain growing on glycerol.35 On the contrary, the cells continued to grow normally as compared to a control culture without the addition of lactose. Extracellular lactose was rapidly consumed by the lacZ mutant and a concomitant accumulation of lactose was observed inside the cells. High intracellular lactose concentrations could be maintained for more than 24h without significantly disturbing the cells, which remained fully metabolically active throughout the culture. This result demonstrates that lactose can be made available intracellularly as an acceptor for glycosyltransferases over a long period. This strategy was then applied to the synthesis of several lactose-derived oligosaccharides. Although the strain was a lacZ mutant, a very slow degradation of lactose was still observed. This degradation could be due to the presence in E. coli of a second b-galactosidase encoded by the ebg gene, which has a very low activity on lactose.36 As lactose is an inexpensive substrate, which could be supplied in excess, no attempt was made to inactivate Ebg b-galactosidase in order to entirely prevent lactose degradation. Galactose is another interesting acceptor that enters the cells without being modified. Cytoplasmic galactose is normally phosphorylated to Gal1P by the kinase encoded by the galK gene. Disruption of galK allows exogenous galactose to be accumulated intracellularly in cells grown on glycerol or glucose and this strategy was recently used for the synthesis of Lewis x tetrasaccharide (Galb-4(Fuca-3)GlcNAcb-3Gal).37 The ability of certain E. coli strains to grow on raffinose (Gala-6Glca-bFruf) is conferred by special plasmids containing the rafB gene that encodes a specific raffinose carrier. Raffinose has also been shown to be a fortuitous substrate for the E. coli lactose permease LacY,38 indicating that LacY has a broad specificity which could be used to internalize various trisaccharides having an a- or b-linked galactose as terminal nonreducing residue. This broad specificity has been exploited for the synthesis of the tetrasaccharide globotetraose (GalNAcb-3Gala-3Galb-4Glc) from exogenously added globotriose in living E. coli cells.39 On the other hand, all attempts to use N-acetyllactosamine as an exogenous acceptor have been unsuccessful.40 An E. coli strain expressing the lacZ gene for b-galactosidase was shown to grow slowly on N-acetyllactosamine in the presence of IPTG as inducer of the lac operon. However,

Table 1 Sugars that can be internalized in an unmodified form to be used as acceptor for glycosyltransferases in living E. coli cells Sugar

Transport system

Genes

Hþ symporter ABC transporter Hþ symporter ABC transporter Hþ symporter ABC transporter Cation symporter

galP mglABC araE araFGH xylE

Disaccharides Maltose Lactose Melibiose Sucrose

ABC transporter Hþ symporter Hþ, Naþ, Liþ symporter Hþ symporter

malEFK lacY melB cscB

Trisaccharides Raffinose Globotriose

Hþ symporter Hþ symporter

lacY rfaB lacY

Monosaccharides Galactose Arabinose Xylose Sialic acid

nanT

930


no intracellular accumulation of free N-acetyllactosamine was detected in lacZ-deficient strains suggesting that N-acetyllactosamine was transported by the PTS. The broad specificity of the lactose permease can also be used to internalize lactose derivatives carrying latent chemical functions to produce conjugatable oligosaccharides. This strategy has been successfully applied to the synthesis of the oligosaccharide portions of GM2 and GM3 gangliosides with an allyl or a propargyl aglycon.41 The alkyne function makes possible an azido addition under aqueous conditions and the alkene function can either be converted into an aldehyde to be linked to proteins by reductive amination, or be transformed into a versatile amino group by the addition of cysteamine.

1.23.4 Glycosyltransferase Genes 1.23.4.1 Eukaryotic Genes Eukaryotic glycosyltransferase genes are difficult to express in E. coli and, although several hundreds of genes have been identified from various organisms, only a few of them have been reported to be functionally expressed in E. coli. Mammalian glycosyltransferases contain an N-terminal cytoplasmic tail, a transmembrane domain, and a stem region connected to the luminal C-terminal catalytic domain. The stem region can be cleaved by proteases in the lumen to release active soluble enzymes and a straightforward strategy to produce the active soluble domain in the cytoplasm of E. coli is to express the truncated form of the gene encoding only the C-catalytic domain. This strategy has been successfully used for the expression of the bovine gene for a-1,3-galactosyltransferase and applied to the enzymatic synthesis of oligosaccharides, containing the a-gal epitope, by whole E. coli cells.25,42 The a-1,3-galactosyltransferase belongs to glycosyltransferase family 6, which includes the blood group A and B glycosyltransferases that have also been successfully expressed in E. coli.43 It has been reported that the active forms of these proteins do not contain any disulfide bridges44,45 and this could be related to the fact that they correctly fold in E. coli. The bacterial cytoplasm is a reducing environment that prevents the formation of disulfide bridges that are necessary for the correct folding of most eukaryotic glycosyltransferases. For this reason, recombinant eukaryotic protein production is often directed to the periplasm, which is an oxidizing environment where disulfide bond formation is actively catalyzed by the Dsb system.46 However, this strategy cannot be applied to the synthesis of oligosaccharides by living cells in which the glycosylation reaction must take place in the cytoplasm. An alternative is to use mutant strains that lack both thioredoxin reductase (trxB) and glutathione reductase (gor) and have a more oxidizing cytoplasm that allows the formation of disulfide bonds.47 However, to our knowledge, no successful expression of a glycosyltransferase by this system has been reported so far. Another problem frequently encountered during the production of eukaryotic glycosyltransferase is the formation of inclusion bodies. Culture at temperatures as low as 13 C has been shown to improve the solubility of recombinant human sialyltransferase,48 but the use of such temperatures dramatically reduces the metabolic activity of living E. coli cells and consequently the productivity of oligosaccharide synthesis. A number of procedures have been described to recover active proteins from purified inclusion bodies but, of course, none of these procedures can be used in whole cells.

1.23.4.2 Bacterial Genes In contrast to their eukaryotic counterparts, bacterial glycosyltransferase genes are generally well expressed in E. coli. They are naturally designed to be intracellularly expressed and their active site faces the cytoplasm when they are membrane bound. One notable exception is the periplasmic O-antigen glucosyltransferase GtrX of Shigella flexneri, which uses a lipid-linked glucose as substrate donor to attach a glucose onto the O-antigen repeating unit.49 There is a very wide diversity of carbohydrate structures expressed by bacteria as major components of their cell envelopes. Structures found in pathogens have been particularly well studied and many of them have been shown to mimic mammalian antigens to evade the host immune response. Terminal sialylated lacto-N-tetraose and globotriose are found in the LOSs of Neissseria meningitis and N. gonorrheae and these structures are the major determinants of their different immunotypes.50 Globotetraose has been detected in the LPS of the Haemophilus influenzae strain RM11851 and several ganglioside oligosaccharide mimics have been identified in LOS of various Campylobacter jejuni strains.52 Lewis antigen structures are found in the O-antigen of Helicobacter pylori and are believed to promote bacterial adhesion and colonization.53 Mimics of mammalian carbohydrate structures are also found in the capsular polysaccharides of gram-positive bacteria and sialylated lacto-N-neotetraose and lacto-N-tetraose have been identified as the repeat units of capsular polysaccharides of different serotypes of Steptococcus agalactiae.54 The genetic loci responsible


931

Table 2 Glycosyltransferases genes used in processes of oligosaccharide synthesis by whole E. coli cells Activity

Acceptor

Origin

Gene

Reference

b-1,4-galactosyltransferase

GlcNAcb-R

lgtB

b-1,3-galactosyltransferase a-1,4-galactosyltransferase

GalNAcb-R Galb-R

a-1,3-galactosyltransferase b-1,3-galactosyltransferase

Galb-R Galb-R

b-1,4-galNAc transferase b-1,3-galNAc transferase

Neu5Aca-3Galb-R Gala-R

55 56 57 55 58 59 55 58 57 60 55

a-1,3-fucosyltransferase

Galb-4GalNAcb-R Galb-3GalNAcb-R Galb-R Galb-R

Neisseria gonorrhoeae N. meningitidis Campylobacter jejuni gonorrhoeae N. meningitidis Bos taurus N. gonorrhoeae N. meningitidis C. jejuni Haemophilus influenzae N. gonorrhoeae N. meningitidis Helicobacter pylori

a-1,2-fucosyltransferase a-2,3-sialyltransferase Bifunctional a-2,3a-2,8-sialyltransferase a-2,6-sialyltransferase

Galb-R Galb-R GalNAcb-R

H. pylori N. gonorrhoeae N. meningitides C. jejuni Photobacterium damsela

cgtB lgtC a3GalT lgtA cgtA lgtD

futA futB futC nst

61 62 63 64

cstII

52 65

for the synthesis of these structures have been characterized and most of the glycosyltransferase genes have been identified. They represent an extremely valuable source of genes for enzymatic synthesis of oligosaccharides in whole cells; the most interesting recombinant bacterial glycosyltransferases that have been used in practice are listed in Table 2 with their acceptor specificity.

1.23.5 Metabolic Engineering of Sugar Nucleotide Biosynthesis 1.23.5.1 Metabolic Engineering of UDP-Gal Biosynthesis In E. coli, UDP-Gal is used as sugar donor for the synthesis of LPS and of the extracellular polysaccharide colanic acid. As shown in Figure 4, the production of UDP-Gal from central metabolism proceeds through the activation of Glc1P into UDP-Glc by UDP-Glc pyrophosphorylase and the subsequent epimerization of UDP-Glc into UDP-Gal. UDP-Gal is also an intermediate in the catabolism of galactose, which has to be converted into Glc1P to join the glycolysis pathway. To carry out this conversion, cytoplasmic galactose is phosphorylated by the kinase GalK and the resulting Gal1P is converted into UDP-Gal by the transferase GalT, with the concomitant formation of Glc1P from UDP-Glc coming from the epimerization of UDP-Gal. The same enzyme, UDP-Glc C4 epimerase, encoded by galE, is used in the forward reaction for UDP-Gal biosynthesis from UDP-Glc and in the reverse reaction for galactose degradation. The galE gene is part of the galactose operon which also includes the galT and galK genes. The galactose operon is fully induced by galactose and repressed by glucose. However, bacteria have evolved a sophisticated regulation system with two different promotors. A first promotor PG1 requires AMPc to operate and the second promoter PG2 works in cells devoid of AMPc, under conditions of catabolic repression. This system ensures a minimal expression of the gal operon for biosynthetic purposes under different physiological conditions.66 In addition to being a precursor for UDP-Gal biosynthesis, UDP-Glc has a number of other important metabolic functions. The galU gene encoding UDP-Glc pyrophosphorylase is therefore independently regulated from the Gal operon and located elsewhere in the E. coli chromosome.67 UDP-Glc is involved in the synthesis of LPS, trehalose, and membrane-derived oligosaccharides (MDO). It is also the precursor of UDP-GlcA, which serves as the glycosyl donor for the production of colanic acid. The galF gene, which is located in the colanic acid gene cluster, encodes a protein having a strong amino-acid sequence identity to GalU. The GalF protein has no UDP-Glc pyrophosphorylase activity but has been shown to interact with GalU to increase UDP-Glc formation.68 Since galF is co-regulated with

932


Figure 4 Metabolism of UDP-Gal in living E. coli cells. UDP-Gal is normally produced by isomerization of UDP-Glc to serve as a precursor for the synthesis of cell envelope structures. It is also an intermediate in Gal catabolism. The Galactose operon is regulated by a dual promotor system that enables UDP-Gal biosynthesis in different culture conditions.

the gene for colanic acid biosynthesis, it is tempting to speculate that the stimulatory effect of GalE on UDP-Glc synthesis allow cells to compensate for extra UDP-Glc demands when colanic acid production is induced. Living E. coli are thus normally able to maintain a minimal biosynthetic flux of UDP-Gal for the synthesis of LPS and colanic acid. E. coli mutants lacking galE have altered forms of LPS and cannot produce colanic acid. However these mutants are perfectly viable, indicating that a shortage of UDP-Gal is not detrimental to the cells and that the biosynthetic flux of UDP-Gal can be entirely diverted from its natural use towards the synthesis of unnatural oligosaccharides, by overexpressing recombinant galactosyltransferase in living cells. Another question is to know whether the galactosylation reaction can be limited by the UDP-Gal biosynthetic capacity of the cells. Several galactose-containing oligosaccharides have been produced by living recombinant E. coli cells and the maximal rate of galactosylation has been observed during the synthesis of globotriose from lactose in the HCDC of cells expressing the lgtC gene for a-1,4-galactosyltransferase. Globotriose production rates were 1.2gh1 l1 of culture medium, corresponding to a rate of galactose transfer of 2.7mmolh1 l1.39 In the synthesis of complex oligosaccharides such as lacto-N-neotetraose or the oligosaccharide portion of GM2 ganglioside, lower galactosylation rates were observed. In the case of lacto-N-neotetraose (Galb-4GlcNAcb-3Galb-4Glc), the rate of galactose transfer was estimated to be around 0.5mmolh1 l1 35 and the limiting step was clearly the synthesis of the trisaccharide intermediate LNT2 (GlcNAcb-3Galb-4Glc). In the case of the GM2 sugar, the limitation came from a low expression of the b-1,3-galactosyltransferase encoded by cgtB.69 These examples show that, in most cases, the natural capacity of UDP-Gal production is high enough for an efficient galactosylation and that genetic engineering of UDP-Gal biosynthesis is not necessary. In addition, while one particular galactosylation reaction proved to be limited by the availability of UDP-Gal, a simple way to increase UDP-Gal production rates would be to feed the bacteria with galactose. In contrast, systems using permeabilized whole cells require the overexpression of all the genes involved in UDPGal biosynthesis. The most commonly used strategy is to produce UDP-Gal from galactose by overexpressing the galK, galT, and galU genes. In the Kyowa technology, these three genes were overexpressed in one E. coli strain and UDP-Glc was recycled from Glc1P, using UTP produced by C. ammoniagenes cells from orotic acid.8 In systems using only one single E. coli strain, the additional gene pykF encoding pyruvate kinase was overexpressed to allow UTP and ATP to be regenerated using phosphoenolpyruvate, which was presumably generated during glycolysis, but which could also be added to the reaction medium to improve the efficiency of the synthesis.14 An alternative is to produce UDP-Gal from UDP-Glc by expressing the galE gene and to regenerate UDP-Glc from sucrose by co-expressing the Anabaena sp. gene susA for sucrose synthase, which catalyzes the following reversible reaction: sucrose þ UDP $ UDP-Glc þ fructose.16


933

1.23.5.2 Metabolic Engineering of UDP-GlcNAc Biosynthesis UDP-GlcNAc is a vital precursor for the formation of the bacterial cell envelope. In enteric bacteria, UDP-GlcNAc resides at a complex branch point that initiates the formation of the enterobacterial common antigen (ECA), peptidoglycan, O-antigen, and the lipid A moieties of LPS. Both peptidoglycan and lipid A are absolutely essential for bacterial survival, but mutants lacking O-antigen and ECA can grow normally under laboratory conditions. Peptidoglycan makes up to 2.5% of the cell dry weight and is continuously recycled during bacterial growth with a turnover rate of about 50% per generation. Although murein peptides coming from peptidoglycan degradation are efficiently recycled, this turnover puts an additional charge on the demand of UDP-GlcNAc, which is used directly (and indirectly as an UDP-MurNAc precursor) for peptidoglycan formation. As shown in Figure 5, UDP-GlcNAc is produced from GlcN6P which can have three different origins: GlcN6P is mainly produced from Fru6P by the GlcN6P synthase encoded by the glmS gene; a significant part of the GlcN6P pool probably also originates from GlcNAc, resulting from the hydrolysis of recycled muropeptides by the b-N-acetylglucosaminidase encoded by nagZ.20 GlcNAc is then converted into GlcN6P by the action of the kinase NagK and the N-deacetylase NagA. GlcNAc6P and GlcN6P can also be formed during the active transport of exogenous GlcNAc and GlcN by specific components of the phosphoenolpyruvate-dependent phosphotransferase system. To produce UDP-GlcNAc, GlcN6P is converted into GlcN1P by the phosphoglucosamine mutase encoded by the glmM gene.70 The GlmM enzyme must be phosphorylated to be active and acts according to a ping-pong mechanism involving GlcN1,6P2 as an intermediate.71 The level of phosphorylated enzyme in cells could be one of the factors adjusting the flow of UDP-GlcNAc to the specific requirements for peptidoglycan and LPS. The formation of UDP-GlcNAc from Glc1P is catalyzed by GlmU, which is a bifunctional enzyme having both GlcN1P acetyltransferase and GlcNAc1P uridyltransferase activities.72 The acetyltransferase activity of the GlmU protein was reported to be strongly inhibited by UDP-MurNAc and to a lesser extent by GlcNAc1P.72 This feedback inhibition allowed the cells to regulate the intracellular UDP-GlcNAc pool which was shown to remain fairly constant, whatever the growth rate and bacterial substrate.73 The biosynthesis of UDP-GlcNAc is also controlled at the transcriptional level and the expression of the glmU and glmS genes, which are part of the same operon, are partially repressed when the bacteria are grown on GlcNAc or GlcN.74 The synthesis of GlcNAc-containing oligosaccharides in E. coli overexpressing heterologous genes for N-acetylglucosamine transferases could be potentially harmful to growing cells by diverting a large part of the UDP-GlcNAc pool from its normal use and causing a shortage of UDP-GlcNAc that could limit the vital formation of peptidoglycan and lipidA. However, reports of large-scale production of chitooligosaccharide and LNT2 trisaccharide (GlcNAcb3Galb-4Glc) by recombinant E. coli strains demonstrates that living cells can cope with an extra UDP-GlcNAc demand.23,35 The maximal rate of transferred GlcNAc was observed during the production of LNT2 from lactose by the HCDC of a strain overexpressing the lgtA gene for b-1,3-GlcNAc transferase. This rate was estimated to be around 1.25mmolh1 l1.35 These results show that the genetic engineering of UDP-GlcNAc biosynthesis is not necessary for high production levels of GlcNAc-containing oligosaccharides in living cells. It is however important to note that GlcNAc-containing oligosaccharides were produced in fed-batch cultures under conditions of low bacterial Anhydro muropeptide nagZ

LPS

Murein

anhMurNAc peptide UDP-MurNAc

GlcNAc nagK

nagA glmM glmU glmU GlcN1P GlcN1P GlcNAc6P GlcN6P

GlcNAc

nagE

GlcN

manXYZ

nagB

glmS

UTP

Inside

PP

UDP

Fru6P Outside

UDP-GlcNAc

GlcNAc~Acceptor

Acceptor Recombinant GlcNAc transferase

Figure 5 Metabolism of UDP-GlcNAc in living E. coli cells. UDP-GlcNAc is a vital precursor for cell envelope biosynthesis. Its pool level remains fairly constant whatever the growth conditions and is likely to be constantly replenished in case of extra consumption by recombinant GlcNAc transferase.

934


growth rate. E. coli is able to grow at a very high growth rate and has therefore the enzymatic machinery to generate a strong biosynthetic flux of UDP-GlcNAc. In slow-growing cells, these enzymatic activities are in excess due to a lower UDP-GlcNAc demand for the synthesis of cell envelope components and this excess capacity can be directed to the production of GlcNAc-containing oligosaccharides. Production of UDP-GlcNAc by permeabilized whole cells has also been reported.9 In this system, C. ammoniagenes UTP-producing cells were combined with cells of several E. coli strains overexpressing the different genes required for the synthesis of UDP-GlcNAc from glucosamine. Fructose served as the energy source and orotic acid was added as a precursor for UTP production. The first E. coli strain expressed the glk gene for the E. coli glucokinase, which is able to phosphorylate GlcN into Glc6P. Two different strains were used for the expression of the glmM and glmU genes for the conversion of Glc6P into UDP-GlcNAc. Additional strains were also needed to express the genes for phosphoglucomutase and phosphofructokinase, in order to provide Glc1,6P2, which was required to activate the phosphoglucosamine mutase GlmM. GlcN1P acetyltransferase activity requires acetylCoA as substrate but the overexpression of the genes for acetate kinase and phosphate transacetylase was not necessary.

1.23.5.3 Metabolic Engineering of UDP-GalNAc Biosynthesis UDP-GalNAc is enzymatically produced by the epimerization of the hydroxyl group at C4 of UDP-GlcNAc. This UDP-GlcNAc C4 epimerase ativity was first identified in mammalian cells and was shown to reside in the same enzyme that catalyzes UDP-Glc epimerization into UDP-Gal.75 In contrast, the GalE enzyme from E. coli K12 is active on UDP-Glc, but not on UDP-GlcNAc.76 In fact, most bacteria do not have the enzymatic machinery to synthesize UDP-GalNAc for the simple reason that GalNAc is not a common constituent of the bacterial cell envelope. GalNAc is, however, found in the cell surface structure of a few pathogenic bacteria and genes encoding UDP-GlcNAc epimerase have been identified in Pseudomonas aeruginosa,77 E. coli O55:H7,78 and C. jejuni.79 The WbpP enzyme from P. aeruginosa has been shown to be a genuine UDP-GlcNAc epimerase with extremely low UDP-Glc epimerase activity. On the other hand, the Campylobacter enzyme was reported to be bifunctional and to catalyze both UDP-Glc and UDP-GlcNAc C4 epimerization. Production of GalNAc-containing oligosaccharides in E. coli K12 requires complementation with genes encoding UDP-GlcNAc C4 epimerase. The synthesis of the GM2 oligosaccharide (GalNAcb-4(Neu5Aca-3)Galb-4Glc) has been achieved by overexpressing the wbpP gene from P. aeruginosa.69

1.23.5.4 Metabolic Engineering of CMP-Neu5Ac Biosynthesis Only a few bacterial species are able to synthesize sialic acid for its subsequent incorporation in cell surface structures such as the capsular polysialic acid of E. coli K1 and the LOSs of N. meningitidis. All other bacteria, including E. coli K12, do not have the enzymatic machinery for the biosynthesis of CMP-Neu5Ac, which is the donor substrate used by sialyltransferases. Many bacteria, however, can use sialic acid as a source of carbon and nitrogen. The catabolic system for sialic acid has been identified in E. coli and has been shown to be induced by Neu5Ac. A specific permease encoded by nanT transports Neu5Ac into the cytoplasm, where it is cleaved into ManNAc and pyruvate by the aldolase encoded by nanA.80 Sialic acid enters cells in an unmodified form and the NanT permease has been suggested to be a cation symporter.81 ManNAc is phosphorylated by the NanK kinase into ManNAc6P, which is subsequently converted into GlcNAc6P by the NanE protein.82 GlcNAc6P is then deacetylated by NagA into GlcN6P to join the glycolysis pathway or to be used as a precursor for UDP-GlcNAc biosynthesis. The nanT, nanA, nanK, and nanE genes are part of the same operon, which is regulated by the DNA-binding protein NanR.83 For safety reasons, pathogenic E. coli strains that naturally synthesize CMP-Neu5Ac cannot be used for the synthesis of sialylated oligosaccharides and laboratory strains of E. coli K12 have been genetically engineered to produce CMP-Neu5Ac. A straightforward strategy consists of expressing the gene neuA for CMP-Neu5Ac synthase, which has been identified in E.coli K184 and N. meningitides,85 in a mutant strain lacking the gene nanA for sialic acid aldolase (Figure 6). Culture of this strain on glycerol as carbon and energy source in the presence of sialic acid, resulted in the synthesis of CMP-Neu5Ac, which could serve as a substrate donor for recombinant sialyltransferase. This strategy was first used for the production of 30 -sialyllactose35 and then extended to the formation of different ganglioside oligosaccharides.69,86 In permeabilized cells, the strategy for CMP-Neu5Ac biosynthesis is different and based on the association of UTPproducing C. ammoniagenes cells, with cells of two different E. coli strains: one expressing the pyrG gene for CTP synthase to convert UTP into CTP and the second expressing the CMP-Neu5Ac synthase gene for producing CMP-Neu5Ac from CTP and Neu5Ac.


Neu5Aca-Acceptor

Recombinant sialyltransferase CMP

GlcN6P nagA

ADP ATP

GlcNAc6P

ManNAc6P nanE

Acceptor

CMP-Neu5Ac PP

acetate

NeuA

CTP nanT

Neu5Ac

ManNAc nanK

nanA

nanT

Neu5Ac

nanA Pyruvate Inside

nanR

935

nanE

Outside

nanK

Figure 6 The Neu5Ac catabolic pathway can be engineered in an anabolic pathway for CMP-Neu5Ac biosynthesis by expressing the neuA gene for CMP-Neu5Ac synthase and by disrupting the nanA gene for Neu5Ac aldolase.

1.23.5.5 Metabolic Engineering of GDP-Man and GDP-Fuc Biosynthesis In E. coli and in some enteric bacteria, L-fucose is a major constituent of the extracellular polysaccharide colanic acid, which is assembled from hexasaccharide repeat units synthesized on undecaprenyl pyrophosphate. Each unit contains two fucose residues that are incorporated in a reaction that uses GDP-fucose as substrate donor. The four genes involved in GDP-fucose biosynthesis from Man6P have been identified in the colanic acid gene cluster, which comprises a total of 20 genes.87 Man6P is converted into GDP-Man by the phosphomannomutase and Man1P guanosyltransferase encoded by the manB and manC genes. The next step is the dehydratation of GDP-Man into GDP-4-keto-6-deoxy-D-mannose by the product of the gmd gene. The production of GDP-Fuc is then carried out by the fucose synthetase encoded by wcaG in a reaction requiring NADPH to reduce the GDP-4-keto-6-L-deoxygalactose intermediate, which is produced by the epimerase activity of WcaG.88 The GDP-Fuc biosynthetic flow is controlled by the GMD enzyme, which is strongly inhibited by the end product GDP-Fuc.89 Colanic acid is synthesized in abundance in response to stress conditions such as desiccation but its production is normally low for wild-type cells under normal growth conditions. It has however been observed that cells carrying mutations in the ATP-dependent protease lon gene become mucoid due to capsule overproduction. The synthesis of colanic acid was later shown to be under the control of two positive protein regulators encoded by the rcsA and rcsB genes.90 RcsA cooperates with RcsB to stimulate the transcription of capsular polysaccharide genes, but RcsA protein has been shown to be unstable and rapidly degraded by the Lon protease. Therefore, colanic acid is usually expressed at low levels under laboratory conditions except in the lon mutant or in strains overexpressing the rcsA gene in a multicopy plasmid.91 Since the genes for GDP-Fuc biosynthesis are part of the colanic acid gene cluster, they are subjected to the same regulation mechanism and their basal expression is expected to be fairly low under standard laboratory conditions. However, their expression can easily be increased by overexpressing the rcsA gene (Figure 7). This strategy was successfully used to produce fucosylated oligosaccharide in living E.coli cells expressing heterologous fucosyltransferase genes.92 Overexpression of rcsA was indeed confirmed to be absolutely necessary to achieve a high fucosylation yield but was also shown to result in a considerable accumulation of colanic acid in the culture medium when the cells were cultured at a high cell density. As a consequence of this accumulation, the viscosity of the medium increased dramatically causing a decrease in the rate of oxygen transfer, which reduced the cell yield of the culture considerably. In addition, this polysaccharide production created an important metabolic and energetic burden for the cells and the glycosyltransferase responsible for colanic acid biosynthesis competed with recombinant glycosyltransferase for the available GDP-Fuc. To prevent the overproduction of colanic acid, a mutant strain lacking the wcaJ gene was used. The wcaJ protein is the glucosyltransferase which adds Glc1P onto undecaprenyl-P and its inactivation completely abolishes colanic acid synthesis. An alternative is to overexpress gmd, wcaG, manC, and manB in a multicopy plasmid under the control of the Plac promoter.37 This system has been shown to give fucosylation yields similar to those

936


Outside Und-PP Inside wcaJ GalPyrb-4GlcAb-3Galb-4Fucb-4FucAca-3Glc Colanic acid Fuca-acceptor GDP repeating unit Recombinant fucosyltransferase Fru6P GDP-fucose

manA

Acceptor

NADP

Man6P wcaG

manB manC

Man1P

GTP gmd

GDP-Man

gmd −

NADPH

GDP-4-keto-6-deoxyMan

PP wcaG

wcaH

wcaI

manC manB wcaJ

+ Colanic acid genes cluster RcsA positive control protein Figure 7 Genes for GDP-Fuc biosynthesis can be induced by expressing the rcsA gene for a control protein that activates the whole colanic acid genes cluster. Overproduction of the polysaccharide colanic acid is prevented by the disruption of the wcaJ gene.

obtained with the wcaJ mutant overexpressing rcsA. On one hand, this alternative system has the advantage of not requiring the wcaJ mutation and can therefore be used in any E. coli strain. On the other hand, it requires the expression of four genes instead of only one and this could be a problem for the synthesis of complex structures that necessitates the expression of several heterologous genes. A system with permeabilized cells has also been described for the synthesis of GDP-Fuc and fucosylated oligosaccharides.8 C. ammoniagenes cells, which were used to regenerate GTP from added GMP, were coupled with cells of three different E. coli strains. Mannose was converted into GDP-Man by a first strain expressing the glk gene for glucokinase and the manB and manC genes. This strain also expressed the pgm and pfkB genes to produce the GL-1,6-diP required to activate ManB mutase. A second strain, expressing the gmd gene, was responsible for the conversion of GDP-Man into GDP-4-keto-6-deoxy-D-mannose and the last strain expressed the wcaG fucose synthetase gene. To prevent inhibition of the GMD enzyme by GDP-Fuc, the production of GDP-Fuc was carried out in two steps. In the first step, cells containing fucose synthetase were omitted and the GDP-4-keto-6-deoxy-D-mannose intermediate was produced. In the second step, this intermediate was reduced to GDP-Fuc by adding the wcaG expressing cells to the reaction mixture. When the system was coupled with a strain expressing a fucosyltransferase gene, the two-step procedure was unnecessary because GDP-Fuc was used constantly and did not accumulate.

1.23.5.6 Metabolic Engineering of Other Sugar Nucleotides Other sugar nucleotides that are produced naturally by E. coli K12 for the synthesis of its cell envelope include UDPGlcA for colanic biosynthesis, TDP-rhamnose and UDP-galactofuranose for O-antigen formation, and TDP-Fuc4NAc and UDP-ManNAcA for the production of the ECA. UDP-GlcA is a precursor for the synthesis of many carbohydrate structures of great biological interest, such as the HNK-1 epitope(GlcA3Sb-3Galb-R) and heparin-like oligosaccharides whose synthesis could be imagined in metabolically engineered bacteria. In E. coli K12, UDP-GlcA production is catalyzed by the UDP-Glc dehydrogenase encoded by the ugd gene which is not located in the colanic acid gene cluster. It is interesting to note that E. coli K5, which produces a capsular polysaccharide with a basal repeat structure similar to that of heparin, has an additional UDP-Glc dehydrogenase encoded by the kfiD gene.93 The large diversity of carbohydrate structures found at the bacterial cell surface is reflected by the existence of a large number of different sugar nucleotides. Many of the genes involved in their biosynthesis have been identified and could be used in the future to metabolically engineer E. coli strains for the production of antigenic carbohydrate structures that could be useful for therapeutic applications such as vaccine preparation.


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1.23.6 Production of Complex Oligosaccharides by Sequential Multiglycosylation To be able to carry out sequential multiglycosylation reactions with different glycosyltransferases, using different sugar nucleotide precursors in the same system, offers the possibility of increasing considerably the size, complexity, and diversity of the oligosaccharide structures that can be made by bacteria. Systems using permeabilized cells are based on the overexpression of the whole sugar-nucleotide biosynthesis pathway and require the expression of a relatively large number of genes for the production of each sugar nucleotide. Consequently, it is technically difficult to overexpress in the same system all the genes necessary for the biosynthesis of two or three different sugar nucleotides and until now permeabilized cells have only been used for monoglycosylation reactions using one type of sugar nucleotide as glycosyl donor. Multiglycosylation could possibly be carried out by the sequential use of several independent monoglycosylation systems, for example, N-acetyllactosamine, produced by a first galactosylation system,12 can be fucosylated by a second system, leading to the formation of the Lewis x trisaccharide.11 However, the diffusion rate of oligosaccharides through the cell wall of permeabilized cells decreases rapidly as the size of the oligosaccharide increases and the use of acceptors longer than trisaccharides have been shown to pose problems.14 In contrast, sugar nucleotide biosynthesis in living cells necessitates little or no genetic engineering and multiglycosylation can be carried out in a single living cell overexpressing a reasonable number of genes. Several multiglycosylation systems have been described for the synthesis of a wide range of complex oligosaccharides (Table 3) and these systems are reviewed below.

1.23.6.1 ABH and Lewis Antigen Oligosaccharides Two systems have been developed for the production of oligosaccharides containing the N-acetyllactosamine motif, which can be used as type 2 acceptors for the synthesis of ABH and Lewis antigens. As described in Section 1.23.3.1, the first system is based on the synthesis of the hexasaccharide Galb-4[GlcNAcb-4]4GlcNAc by strains co-expressing the lgtB and nodC genes. Additional expression of the chitinase gene chiA results in the formation of the trisaccharide Galb-4GlcNAcb-4GlcNAc (Figure 8). This system has been applied to the synthesis of the Lewis x (Lex) tetrasaccharide Galb-4(Fuca-3)GlcNAcb-4GlcNAc in a strain that was metabolically engineered to overexpress the gene for GDP-Fuc biosynthesis and the a-1,3-fucosyltransferase futA gene from H. pylori.37 The same system was also employed for the production of the sialylated tetrasaccharide (Neu5Aca-3Galb-4GlcNAcb-4GlcNAc) by expressing the neuA and nst genes for CMP-Neu5Ac and a-2,3-sialyltransferase (unpublished result). Attempts are underway to combine both fucosylation and sialylation in this system to produce the sialyl-Lewis x epitope. An alternative system to provide living cells with type 2 precursors is the synthesis of lacto-N-neotetraose (LNnT, Galb-4GlcNAcb-3Galb-4Glc) from exogenous lactose by strains overexpressing lgtA and lgtB genes, as illustrated in Figure 9.35 In addition to LNnT, longer oligosaccharides were also produced and identified as LNnT derivatives containing between one and three additional N-acetyllactosamine units. The formation of these polylactosamine structures was due to the fact that LNnT can serve as an acceptor for the GlcNAc transferase LgtA to form an intermediate pentasaccharide that is converted into lacto-N-neohexaose (LNnH) by the galactosyltransferase LgtB. Additional cycles of N-acetyllactosamine incorporation led to the formation of lacto-N-neooctaose and lacto-Nneodecaose. There was no accumulation of tri-, penta-, and hepta-saccharide intermediates, indicating that GlcNAc transfer was the limiting reaction. The relative distribution of LNnT and of its longer derivatives depended on the amount of lactose supplied as initial acceptor. LNnT was the major product when the initial lactose concentration was 5gl1, whereas LNnO was preponderantly formed when the initial lactose concentration was lowered to 1gl1. Strains co-expressing lgtAB with the futA gene for a-1,3-fucosyltransferase have been shown to produce fucosylated derivatives of LNnT (Figure 9). There are two potential fucosylation sites for FutA in LNnT61 and low futA expression led to the preferential fucosylation of the terminal reducing glucose, resulting in the recovery of lacto-Nneofucopentaose V (LNnFP-V, Galb-4GlcNAcb-3Galb-4(Fuca-3)Glc) as the major oligosaccharide.92 Small amounts of lacto-N-difucohexaose II (LNnDH-II, Galb-4(Fuca-3)GlcNAcb-3Galb-4(Fuca-3)Glc) were also detected and LNnDH-II was later produced as the main compound by increasing futAt expression levels.95 H. pylori possesses a second fucosyltransferase gene (futB), which could be involved in the fucosylation of the distal GlcNAc residue of the polylactosamilyl chain of H. pylori LPS, whereas FutA would preferentially fucosylate internal GlcNAc residues.97 The production of significant amounts of LNnFP-II (Galb-4(Fuca-3)GlcNAcb-3Galb-4Glc) by the E. coli strain expressing futB in place of futA confirmed this hypothesis.95 However, LNnFP-II was produced in a mixture with LNnFP-V and at low yield due to the toxicity of the futB gene, which inhibited the growth of the E. coli cells. Attempts were made to replace the lactose acceptor by lactulose (Galb-4Fru), Galb-allyl, and Gal to prevent the fucosylation of the terminal reducing residue and obtain monofucosylated oligosaccharides containing the Lewis x (Lex)

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Table 3 Complex oligosaccharides produced in living E. coli cells Estimated productivity*

Ref.

A. caulinodans nodC A. caulinodans nodC,nodB nodC, lgtB nodC, lgtB, a3GalT S. meliloti nodC S. meliloti nodC

2.5

23

2.0

23

1.0 1.0

24 25

0.85 0.70

94 94

S. meliloti nodC, nodL, nodH A. caulinodans nodC, chiA nodC, chiA, lgtB nodC, chiA, futA, nanBC, gmd, wcaG nodC, chiA, lgtB, nst, neuA

0.35

94

4.0

26

2.2 1.5

26 37

1.4

Unpubl. result

5 ND ND ND 3

35 35 35 35 92

ND

95

ND

95

ND 6

37 37

Structure

Name

Genes

Chitooligosaccharides and derivatives GlcNAcb-4[GlcNAcb-4]3GlcNAc

Chitopentaose

GlcNb-4[GlcNAcb-4]3GlcNAc Galb-4[GlcNAcb-4]4GlcNAc Gala-3Galb-4[GlcNAcb-4]4GlcNAc GlcNAcb-4[GlcNAcb-4]2GlcNAc GlcNb-4[GlcNAcb-4]2GlcNAc

Tetra-N-acetylchitopentaose Type 2 acceptor a-gal epitope

GlcNAcb-4GlcNAc

Chitotetraose Tri-N-acetylchitotetraose S. meliloti Nod Factor precursor Chitobiose

Galb-4GlcNAcb-4GlcNAc Galb-4(Fuca-3)GlcNAcb-4GlcNAc

Type 2 acceptor Lex

Neu5Aca-3Galb-4GlcNAcb-4GlcNAc

Sialylated type 2 structure

GlcNOAcb-4[GlcNAcb-4]2GlcNAcS

Lactose as exogenous acceptor Galb-4GlcNAcb-3Galb-4Glc [Galb-4GlcNAcb-3]2Galb-4Glc [Galb-4GlcNAcb-3]3Galb-4Glc [Galb-4GlcNAcb-3]4Galb-4Glc Galb-4GlcNAcb-3Galb-4(Fuca-3)Glc Galb-4(Fuca-3)GlcNAcb-3Galb-4(Fuca-3)Glc Galb-4(Fuca-3)GlcNAcb-3Galb-4Glc Galb-4GlcNAcb-3Gal Galb-4(Fuca-3)GlcNAcb-3Gal

Fuca-2Galb-4GlcNAcb-3Galb-4Glc Fuca-2Galb-4Glc

Lacto-N-neotetraose Lacto-N-neohexaose Lacto-N-neooctaose Lacto-N-neodecaose Lacto-Nneofucopentaose V Lacto-Nneofucohexaose II Lacto-Nneofucopentaose II Lex tetrasaccharide

Lacto-NneofucopentaoseI 20 fucosyllactose

GalNAcb-4(Neu5Aca-3)Galb-4Glc

30 sialyllactose GM3 sugar GM2 sugar

Galb-3GalNAcb-4(Neu5Aca-3)Galb-4Glc

GM1 sugar

Neu5Aca-8Neu5Aca-3Galb-4Glc [Neu5Aca-8]2Neu5Aca-3Galb-4Glc Gala-4Galb-4Glc

GD3 sugar GT3 sugar Globotriose

Neu5Aca-2Galb-4Glc

lgtA, lgtB lgtA, lgtB lgtA, lgtB lgtA, lgtB lgtA, lgtB, futA rcsA lgtA, lgtB, futA rcsA lgtA, lgtB, futB rcsA lgtA, lgtB lgtA, lgtB, futA nanBC, gmd, wcaG lgtA, lgtB, futA rcsA futA rcsA nst, neuA nst, neuA, wbpP cgtA nst, neuA, wbpP cgtA, cgtB cstII, neuA cstII, neuA lgtC

96 14

96

2.6

35

1.2

69

0.9

69

0.8 0.9 7

86 86 39

Galactose as exogenous acceptor Galb-4GlcNAcb-3Gal Galb-4(Fuca-3)GlcNAcb-3Gal

Type 2 acceptor Lex tetrasaccharide

lgtA, lgtB lgtA, lgtB, futA nanBC,gmd, wcaG

ND 6

37 37

Globotriose as exogenous acceptor GalNAcb-3Gala-4Galb-4Glc

Globotetraose

lgtD, wbpP

0.9

39

*In gl

1

in high cell density culture.


939

GlcNAc ? 4 UDP-GlcNAc

nodC

4 UDP GlcNAcb-4[GlcNAcb-4]3GlcNAc chiA GlcNAcb-4GlcNAc + GlcNAc UDP-Gal

lgtB

UDP Galb-4GlcNAcb-4GlcNAc GDP-Fuc

nst

futA

GDP Galb-4GlcNAcb-4GlcNAc

CMP-Neu5Ac CMP

Neu5Aca-3Galb-4GlcNAcb-4GlcNAc

Fuca-3 Figure 8 Production of Lewis x or sialylated type 2 structures in living E. coli cells using endogenously biosynthesized chitobiose as a precursor.

Galb-4Glc Outside LacY

lacZ

Inside Galb-4Glc

Gal + Glc

UDP-GlcNAc

lgtA

UDP GlcNAcb-3Galb-4Glc UDP-Gal UDP

lgtB

Galb-4GlcNAcb-3Galb-4Glc GDP-Fuc GDP

futA

Galb-4GlcNAcb-3Galb-4Glc GDP-Fuc GDP

Fuca-3 futA

Galb-4GlcNAcb-3Galb-4Glc Fuca-3

Fuca-3

[Galb-4GlcNAcb-3]nGalb-4Glc lgtA

UDP-GlcNAc

UDP GlcNAcb-3[Galb-4GlcNAcb-3]n Galb-4Glc lgtB

UDP-Gal UDP

[Galb-4GlcNAcb-3]n+1Galb-4Glc GDP-Fuc futA GDP Fucosylated poly-N-acetyllactosaminyl glycan

Figure 9 Production of lacto-N-tetraose and its polylactosamine derivatives in living E. coli cells using lactose as exogenous acceptor. These structures can be used as endogenous type 2 acceptor for a-1,3-fucosyltransferase to form Lewis x structures.

motif. The use of lactulose led to the preferential formation of a lacto-N-neotetraose analog with a fucosylated terminal fructose residue.92 Galb-allyl was efficiently converted into the trisaccharide Galb-4GlcNAcb-3Galb-allyl by LgtA and LgtB, providing that the host strain was a lacA mutant (see Section 1.23.8.4). However, the target molecule Galb-4(Fuca-3)GlcNAcb-3Galb-allyl was not efficiently produced due to the rapid leakage of the intermediate Galb-4GlcNAcb-3Galb-allyl into the extracellular medium (see Section 1.23.8.1).37 The best results were

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obtained with Gal as exogenous acceptor and the Lex tetrasaccharide (Galb-4(Fuca-3)GlcNAcb-3Gal)was produced with an excellent yield. These different systems for the production of a-3-fucosyl-linked oligosaccharides can be adapted to the synthesis of H-2 antigen oligosaccharides by expressing an a-1,2-fucosyltransferase gene instead of futA. Unfortunately neither of the two mammalian genes fut1 and fut2 has been reported to be functionally expressed in E. coli. Two bacterial a-1,2-fucosyltransferase genes have been cloned from H. pylori (futC)63 and from E. coli O86 (wbnK).98 However, in vitro assays indicated that WbnK only fucosylated the Galb-3GalNAc structure99 and that FutC had the highest activity on type 1 precursors but was almost inactive on type 2 structures unless they contained an a-3-linked fucose (Lewis x antigen). The production of lacto-N-neofucopentaose1 (LNnFP-I, Fuca-2Galb-4GlcNAcb-3Galb-4Glc) was nevertheless attempted in E. coli strains co-expressing lgtAB anf futC and an efficient conversion of lactose into both LNnFP-I and 20 -fucosyllactose was observed.96 This result showed that the activity of FutC on the type 2 precursor, which was barely detectable in the in vitro experiments, was high enough in living cells to enable the synthesis of large amounts of the H antigen oligosaccharide. This discrepancy in fucosyltransferase activity could be explained by the previously reported high instability of FutC after cell lysis,63 but could also be due to the fact that the cytoplasm of living E. coli represented a more appropriate environment for the activity of bacterial glycosyltransferase than synthetic reaction media. The formation of large amounts of 20 -fucosyllactose indicated that FutC efficiently used lactose as an acceptor. On one hand, this resulted in a significant decrease in the expected yield of LNnFP-I, which contains the H2-antigen motif and could be used as a precursor for the synthesis of other antigens of the ABH histoblood group system. It should however be possible to improve LNnFP-I yields by delaying FutC expression until the LNnT is already synthesized, in order to minimize 20 -fucosyllactose production as a side-product. On the other hand, 20 -fucosyllactose is a valuable oligosaccharide that inhibits the Campylobacter colonization of human intestinal mucosa and is present at high concentrations in human milk. By expressing only futC, 20 -fucosyllactose was produced at high yield (14gl1) as the sole oligosaccharide and was recovered mainly in the extracellular medium.

1.23.6.2 Ganglioside Oligosaccharides Gangliosides are a group of sialic acid-containing glycosphingolipids that are abundant notably in brain tissues. 30 -sialyllactose (30 -SL, Neu5Aca-3Galb-4Glc) is the oligosaccharide moiety of GM3 gangioside and a precursor for the synthesis of most ganglioside sugars. Sialyllactose has been produced by culturing a metabolically engineered strain (see Section 1.23.5.4) overexpressing the N. meningitidis gene for a-2,3-sialyltransferase in the presence of sialic acid and lactose as precursors (Figure 10). At the end of the culture, the majority of 30 -SL was recovered in the cells but a significant part (40%) was also found in the extracellular medium. Additional expression of the C. jejuni cgtA gene for b-1,4-GalNAc transferase resulted in the formation of the GM2 oligosaccharide (GalNAcb-4(Neu5Aca-3)Galb-4Glc),

Galb-4Glc LacY Galb-4Glc

Outside Inside

CMP-Neu5Ac nst or cstII CMP-Neu5Ac CMP CMP Neu5Aca-8Neu5Aca-3Galb-4Glc GM3 sugar Neu5Aca-3Galb-4Glc cstII GD3 sugar UDP-GalNAc cgtA CMP-Neu5Ac UDP cstII CMP GalNAcb-4(Neu5Aca-3)Galb-4Glc GM2 sugar

UDP-Gal

cgtB

UDP

(Neu5Aca-8)2Neu5Aca-3Galb-4Glc GT3 sugar

Galb-3GalNAcb-4(Neu5Aca-3)Galb-4Glc GM1 sugar Figure 10 Production of the oligosaccharide portion of various gangliosides in living E. coli cells using lactose as exogenous acceptor.


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as long as the cells were complemented with the P. aeruginosa wbpP gene to enable the formation of UDP-GalNAc.69 Interestingly, small amounts of a GM2 oligosaccharide analog containing a terminal galactose instead of GalNAc (Galb-4(Neu5Aca-3)Galb-4Glc) was also produced, indicating that CgtA transferase was able to use UDP-Gal as substrate donor. This system was extended to the production of the GM1 oligosaccharide (Galb-3GalNAcb-4 (Neu5Aca-3)Galb-4Glc) by expressing the supplementary cgtB gene encoding b-1,3-galactosyltransferase. Synthesis of the disialyltated GD3 oligosaccharide (Neu5Aca-8Neu5Aca-3Galb-4Glc) was carried out using the same system as for GM3 sugar production, except that the N. meningitidis sialyltransferase gene was replaced by the Campylobacter cstII gene, which encodes a bifunctional enzyme having both a-2,3- and a-2,8-sialyltransferase activities.52 After a transient formation of 30 -SL, the GD3 sugar accumulated to a maximal concentration of 0.83gl1.86 However, prolonged incubation resulted in a decrease in GD3 oligosaccharide concentrations due to the formation of a larger oligosaccharide which was identified as the GT3 oligosaccharide (Neu5Aca-8Neu5Aca8Neu5Aca-3Galb-4Glc). The production of this compound was unexpected as no such trisialylated structure had been detected in C. jejuni LPS and CstII has not been reported to use GD3 sugar as acceptor.

1.23.6.3 Globoside Oligosaccharides Another interesting class of glycosphingolipids is the globo series. Globotriosylceramide constitutes the rare Pk blood group antigen, which is the receptor for Shiga toxins and its glycan portion (globotriose, Gala-4Galb-4Glc) can be efficiently produced from lactose by strains overexpressing the Neisseria lgtC gene for a-1,4-galactosyltransferase and lacking the melA gene for a-galactosidase.39 After lactose exhaustion, LgtC galactosyltransferase was shown to repetitively add Gal residues to the terminal nonreducing Gal, leading to the synthesis of a series of polygalactosylated oligosaccharides. Formation of these side products could be easily prevented by stopping the culture before the complete consumption of lactose. In contrast, polygalactosylation could not be avoided in attempts to produce globotetraose (GalNAcb-3Gala-4Galb-4Glc) by co-expressing lgtC with the H. influenzae lgtD gene for b-1,3-GalNAc transferase in the same strain. As LgtD was able to use polygalactosylated glycans as acceptors, a complex mixture of oligosaccharides was obtained in addition to the globotetraose target structure. Since it was observed that globotriose could be taken up by the E. coli transport system, this problem was solved by carrying out the globotetraose production in two steps. Globotriose was first produced under conditions that limit polygalactosylation by culturing a strain expressing only lgtC. Then, in a second step, after being partially purified from globotriose-producing cells, globotriose was supplied as exogenous acceptor to a second culture of a strain expressing only lgtD. Globotetraose is the oligosaccharide portion of globotetraosylceramide, which is known as globoside or P antigen. Globoside is an intermediate in the synthesis of more complex structures of the globo series of glycosphingolipids such as the Forsmann antigen, the stage-specific embryonic antigen 4 and globo-H. These glycolipids are involved in important developmental and pathological processes and synthesis of their carbohydrate portion by living E. coli is currently being investigated.

1.23.6.4 Nod Factor Oligosaccharides Nod factors are lipochitooligosaccharides that are secreted by rhizobia to trigger the developmental process that leads to the formation of nitrogen-fixing root nodules in leguminous plants.22 Host specificity is determined by the structure of the fatty acid chain, the length of the chitooligosaccharide and by various substituents that can be attached to the two terminal residues of the chitin backbone. As mentioned in Section 1.23.3.1, chitooligosaccharide synthesis is catalyzed by the NodC protein and the HCDC of E. coli strain overexpressing the nodC gene has been exploited as an efficient way to produce chitooligosaccharide.23 Use of the nodC gene from A. caulinodans resulted in the formation of chitopentaose as the only oligosaccharide, whereas the expression of the gene from Sinorizobium meliloti led to the synthesis of a mixture of chitopentaose (30%) and chitotetraose (70%). In rhizobia, the terminal nonreducing GlcNAc residue was specifically N-deacetylated by the NodB protein to generate a free amine group to which the fatty acid chain was attached by acyltranferase encoded by nodA. The co-expression of nodB and nodC in E. coli resulted in the production of chitooligosaccharides, which were preponderantly N-deacetylated. These different chitooligosaccharides could be further modified by expressing additional nod genes. NodL is an acetyltransferase that attaches an acetyl group to the 6 position of the terminal nonreducing GlcNAc residue using acetyl-CoA as a substrate.100 Acetyl-CoA is naturally present in the living cell and more than 60% of the chitooligosaccharides produced by an E. coli strain co-expressing nodL and nodC are shown to be O-acetylated.94 In S. meliloti, the nodH and nodPQ are required for the sulfation of Nod factors.101 NodPQ encode ATP sulfurylase and adenosine 50 -phosphosulphate (APS) kinase, respectively. These two enzymes catalyze the formation of 50 -phosphoadenosine 50 -phosphosulphate (PAPS), which is used

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as substrate donor for the sulfation of Nod Factors by the sulfotransferase encoded by nodH.102 In bacteria, PAPS is also the sulfate donor for the biosynthesis of sulfur-containing amino acids and is generated from sulfate by the housekeeping proteins CysC, CysD, and CysN. The cysCDN genes are part of the cysteine operon, which is repressed by cysteine and other reduced sulfur compounds. This means that the expression of the nodPQ genes is not necessary for the sulfation of chito-oligosaccharides in living E. coli cell expressing nodH if these cells are cultured in a mineral medium with sulfate as the sole sulfur source. Sulfated chitooligosaccharides have been shown to be produced by cells co-expressing nodH with nodC, where they represent around 50% of the total amount of chitooligosaccharides found in the bacteria. By using strains expressing different combinations of the nod genes mentioned above a large diversity of O-acetylated and/or sulfated chitooligosaccharide structures were synthesized.94 Among these structures, the deacetylated compounds could be regarded as advance intermediates for the preparation of synthetic natural Nod factors by chemical acylation.103 In addition, chemical acylation offers the possibility of synthesizing Nod Factor analogs with an unnatural lipid chain.104

1.23.7 Production and Purification of Oligosaccharides in Living E. coli 1.23.7.1 High Cell Density Culture The yield of oligosaccharides that can be produced in E. coli cells represents generally between 1 and 10% of the dry cell weight (DCW) of the culture. This means that oligosaccharides can be produced at the 10100mgl1 scale in shaker Erlenmeyer flask cultures in which cell concentrations of around 1gl1 (DCW) can normally be reached. Shaker flasks are convenient for screening a large number of strains or to test valuable precursors that are only available in the milligram scale. However if one wishes to produce oligosaccharides at the gram scale, the volumetric productivity of the culture can be increased by up to a 100-fold by growing the bacteria in fermentors at high cell densities. The main problem encountered in HCDC is the accumulation of growth-inhibiting acidic metabolites such as acetic acid, which are formed in response to oxygen limitation or/and excess of carbon source. As the oxygen delivery capacity of fermentors is limited, the easiest way to prevent an oxygen shortage is to limit bacterial growth by controlled feeding of the carbon source. Different feeding strategies have been proposed,105,106 but many of them require sophisticated feedback control of substrate concentration to determine the feeding rate. On the other hand, reasonably high cell densities of more than 100gl1 (DCW) can be reached with simple fed-batch techniques using pre-determined feeding rates to maintain carbon-limited growth.107 These simple techniques require minimal fermentation machinery: a lab-scale fermentor with temperature, pH and oxygen controls, and a peristaltic feeding pump. The fed-batch strategy, which has been routinely used for the production of oligosaccharides in HCDCs of E. coli, comprises three phases. In the first phase, cells grow exponentially until the entire initial carbon source added to the starting media has been consumed (glycerol is often used as carbon and energy source but can be replaced by any substrate which is catabolized by E. coli). Growth is then carbon-limited by the continuous feeding of a glycerol solution to prevent oxygen limitation. Glycerol is first supplied at a high feeding rate for 5h to quickly reach a high biomass concentration (phase 2). In the last phase, the glycerol-feeding rate is decreased to slow growth and maintain a minimal metabolic activity to sustain oligosaccharide production over a long period (up to 48h).

1.23.7.2 Product Recovery It has often been observed that a proportion of the oligosaccharides produced by living cells is recovered in the extracellular medium. The mechanism of oligosaccharide exit has not been identified but is believed to proceed by passive diffusion across the bacterial membranes. The diffusion rate depends on the size, hydrophobicity, and intracellular concentration of a given oligosaccharide. Neutral trisaccharides are almost entirely recovered in the extracellular medium, whereas longer, ionized oligosaccharides accumulate at high intracellular concentrations. It is often interesting to purify separately the intracellular and extracellular fractions which may have different relative compositions of oligosaccharides. The cells are separated from the extracellular fraction by centrifugation, resuspended in water, and permeabilized by heat treatment at 100 C for 30min to disrupt the bacterial membranes. After this treatment, the oligosaccharides can rapidly diffuse outside the cell and the intracellular oligosaccharide fraction is recovered after a second centrifugation step that removes the emptied cells. Both intracellular and extracellular fractions are purified by adsorption on charcoal-celite and a selective elution with aqueous ethanol. The charcoal purified fraction is generally composed of a mixture of oligosaccharides which have to be further purified by classical chromatographic procedures.


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1.23.8 Drawbacks and Undesirable Side Reactions 1.23.8.1 Leakage of Intermediates during Sequential Multiglycosylation The synthesis of complex oligosaccharides involves several glycosylation steps and the transient accumulation of intermediates can often be observed when the formation rate of an intermediate is higher than its conversion rate in the next glycosylation step. As mentioned above, oligosaccharides can diffuse outside the bacteria and small intermediates are likely to exit the cells rapidly. In some cases, this leakage can be extremely important and considerably reduce the yield of target molecules. For example, the Lex tetrasaccharide could not be produced from Galb-allyl because of the rapid leakage of the trisaccharide Galb-4GlcNAcb-3Galb-allyl intermediate.37 In this case, the presence of the allyl group increased the hydrophobicity of the trisaccharide intermediate and explained its rapid diffusion across bacterial membranes.

1.23.8.2 Repeated Glycosylation Cycles The repeated addition of the same disaccharide motif occurs when two recombinant glycosyltransferases are produced in the same strain and when each glycosyltransferase can use the product of the other glycosyltransferase as an acceptor. This phenomenon was first observed in a strain that was shown to produce a mixture of polylactosaminyl oligosaccharides by co-expressing the lgtA and lgtB genes (see Section 1.23.6.1). On one hand, this could be an advantage if the polymeric structures are interesting products, as in the case of polylactosamine. On the other hand, the formation of this longer structure reduces the yield of the target molecule and makes its purification more difficult. Other repeated glycosylation cycles are likely to occur as new systems of complex oligosaccharide synthesis are developed and could possibly result in the repeated incorporation of units longer than disaccharides. The formation of repeating units can be limited by lowering the expression level of a particular glycosyltransferase gene, increasing the concentration of the initial acceptor, and reducing the culture period. It should also be possible to search for more specific glycosyltransferases.

1.23.8.3 Unspecific Glycosylations Although the glycosyltransferases of the Leloir pathway are regarded as very specific enzymes, several unwanted side reactions have been observed in different systems of oligosaccharide synthesis by living E. coli cells. As mentioned in Section 1.23.6.3, the LgtC a-1,4-galactosyltransferase, which naturally uses b-4-linked galactose as acceptor, is able to galactosylate oligosaccharides with a terminal a-3-linked galactose and this lack of specificity has led to the formation of a series of unwanted polygalactosylated compounds. During the production of the GM2 oligosaccharide (see Section 1.23.6.2) a small amount of a GM2 analog was produced by a side galactosyltransferase activity of the cgtA GalNAc transferase. The same behavior had previously been observed in the human blood group A GalNAc transferase, which was reported to have a low activity with UDP-Gal as substrate donor.108 Overexpression of glycosyltransferases can also lead to unexpected side activities. In an attempt to optimize LNnT production, strains that strongly overexpressed lgA and lgtB were tested and were shown to produce a series of oligosaccharides with an uneven number of residues (unpublished results). Characterization of these compounds indicated that they all contained a galactose linked to the ‘reducing’ terminal glucose residue by a 1–1 linkage. The involvement of LgtB in this reaction was confirmed by the formation of the nonreducing trisaccharide (Galb-4Glc11Gal) from lactose by a strain overexpressing lgtB. Similar trehalose-type oligosaccharides have been identified in the mammary gland of lactating cows,109 indicating that other galactosyltransferases can attach galactose to the C1 of reducing sugars.

1.23.8.4 Indigenous Enzymatic Modifications Oligosaccharide synthesis in E. coli cells clearly requires the inactivation of indigenous enzymatic activity that could degrade or modify the target structures. Mutant strains lacking b- and a-galactosidase have therefore been used for the production of b- and a-galactosides. Athough E. coli has a b-N-acetylglucosaminidase (NagZ), which is involved in the murein recyling process, significant degradation of GlcNAc-containing oligosaccharides such as LNT2 (GlcNAcb3Galb-4Glc) and chitooligosaccharide has never been observed when these compounds have accumulated intracellularly. This is due to the strict specificity of NagZ which was shown to cleave GlcNAc from muropeptide but not chintobiose.20 E. coli K12 is able to grow on sialic acid and fucose but is not known to express fucosidase and

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neuraminidase. Both fucosylated and sialylated oligosaccharide can thus be produced at high yield in E. coli without requiring the inactivation of glycosidase genes. The acetyltransferase LacA, which was first described as thiogalactoside transacetylase, catalyzes the addition of an acetyl group to the 6 position of certain b-galactosides. This activity is believed to serve as a detoxification mechanism for nonmetabolizable b-galactosides which can diffuse into the extracellular medium when they become acetylated.110 Galactosides such as LNnT have never been found to be acetylated, even when such compounds accumulated at high intracellular concentrations. On the other hand, it has been observed that Galb-allyl was entirely converted into Gal6Acb-allyl in the first attempt to use it as an exogenous acceptor.37 Disruption of the lacA gene completely abolished the acetylation of Galb-allyl, which could therefore be converted into Galb-4GlcNAcb-3Galb-allyl by the concerted action of the LgtA and LgtB glycosyltransferases.

Glossary glycosphingolipids Glycolipids made of a carbohydrate portion attached on a hydrophobic tail called ceramide. Depending on the structure of the oligosaccharide portion different groups of glycosphingolipids (such as the gangliosides and the globosides) can be distinguished. high cell density culture (HCDC) Culture in which densities of 50gl1 (dry cell weight) and above are reached by using various strategies of substrate feeding. human milk oligosaccharides Lactose-derived complex oligosaccharides that resemble the ABH and Lewis antigen structure. They are abundantly present in human milk and are believed to act as anti-infective agents. Lewis x antigen Also known as SSEA-1 (stage-specific embryonic antigen 1), Lewis x is highly expressed on the embryo cell surface during early mammalian development. It is involved in cellular recognition during embryogenesis, neural development, fertilization, and tumorigenesis. metabolic engineering Modification of cellular metabolism and properties through the introduction, deletion, and modification of metabolic pathways by using recombinant DNA and other molecular biological tools. nod factor Lipooligosaccharides that are secreted by rhizobia to trigger nodulation in leguminous plants. permeabilized cells Cells whose membrane has been chemically or physically disrupted to let small molecules freely diffuse across the cell envelope. Large molecules stay in the cytoplasm and permeabilized cell can be regarded as an enzymes bag to carry out economical bioconversion reactions.

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Biographical Sketch

Eric Samain is a research engineer at CERMAV (Centre de Recherche sur les Macromolećules Ve´ge´tales), which is a publicly funded laboratory run by the CNRS (Centre National de la Recherche Scientifique) and located in Grenoble (France). He obtained his PhD in biochemistry at the University of Lille in 1983 on the microbiology of methanogenic fermentation. He then obtained a position at the INRA (Institut National de la Recherche Agronomique) to work on the bacterial production of glycosylhydrolases for agricultural uses. He joined CERMAV in 1990 to develop new processes for oligosaccharide synthesis by bacterial fermentation.

2.01 Strategies for the Structural Analysis of Carbohydrates J. P. Kamerling and G. J. Gerwig, Utrecht University, Utrecht, The Netherlands ß 2007 Elsevier Ltd. All rights reserved. 2.01.1 Introduction

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2.01.2 Primary Structural Analysis of Carbohydrates

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2.01.2.1 Polysaccharides

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2.01.2.2 Glycoprotein Glycans

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2.01.3 Monosaccharide Analysis

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2.01.3.1 Monosaccharide Analysis after Hydrolysis

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2.01.3.2 Monosaccharide Analysis after Methanolysis

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2.01.3.3

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D/L

Configuration Determination

2.01.4 Linkage/Methylation Analysis

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2.01.5 Modification/Degradation Procedures for Polysaccharides

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2.01.5.1 Uronic Acid Reduction

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2.01.5.2 Partial Acid Hydrolysis

24

2.01.5.3 Partial Alkaline Hydrolysis

26

2.01.5.4 Acetolysis

26

2.01.5.5 Uronic Acid Degradation

26

2.01.5.6 Periodate Degradation/Smith Degradation

28

2.01.5.7 De-N-acetylation – Deamination

31

2.01.5.8

32

R/S Configuration Determination of Pyruvic Acid Acetals

2.01.5.9 Enzymatic Degradation Procedures

34

2.01.6 Generation of Glycopeptides and Glycoprotein Glycans

36

2.01.6.1 Proteolytic Digestion of Glycoproteins

36

2.01.6.2 Chemical Release of Glycoprotein N- and O-Glycans

37

2.01.6.3 Enzymatic Release of Glycoprotein N-Glycans

40

2.01.6.3.1 Peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidases 2.01.6.3.2 Endo-b-N-acetylglucosaminidases

40 41

2.01.6.4 Enzymatic Release of Glycoprotein O-Glycans

42

2.01.6.5 Enzymatic Release of Oligosaccharide Fragments by Endo-b-galactosidases

43

2.01.7 Glycoprotein Glycan Analysis Tools

44

2.01.7.1 Separation Procedures

44

2.01.7.1.1 2.01.7.1.2

High-pH anion-exchange chromatography Lectin affinity chromatography

44 45

2.01.7.2 Labeling Procedures

48

2.01.7.3 Exoglycosidases

50

2.01.7.4 Profiling of Glycoprotein Glycans

53

2.01.7.5 NMR Spectroscopy of Glycoprotein Glycans

56

2.01.7.6 Mass Spectrometry of Glycoprotein Glycans

57

2.01.7.7 Glycan Detection and Differentiation Assays/Lectin Microarrays

57

2.01.7.8 Analysis of Sialic Acids

58

2.01.8 Final Remarks

59

1

2

Strategies for the Structural Analysis of Carbohydrates

2.01.1 Introduction Structural analysis of carbohydrates has always been a cornerstone of progress in glycoscience. Already at the end of the nineteenth century, the structural elucidation of the monomeric chiral building blocks, the monosaccharides, got much attention, with key contributions especially from Fischer and Van ‘t Hoff. Many present nomenclature recommendations are in fact based on the classical open chain Fischer projection formulas for monosaccharides (Chapter 1.01). Over the years, the structural knowledge about these monomers was growing, with, among others, contributions from Haworth and co-workers (1920; pyranose and furanose forms; Haworth representation of monosaccharide structures) and Bijvoet and co-workers (1950; absolute configuration). For a long time the structural analysis of larger carbohydrate chains remained an underdeveloped area, mainly due to the limitations in suitable and sensitive analytical approaches. Fortunately, during the 1960s three new developments came together: (1) the introduction of a new rapid and low-scale permethylation protocol;1 (2) developments in the application of gas–liquid chromatography (GLC) for mono- and oligosaccharides;2,3 and (3) developments in the application of electron impact mass spectrometry for derivatized mono- and oligosaccharides.4,5 At first instance, great progress was seen in the structural analysis of bacterial polysaccharides,6 soon followed by contributions from the plant polysaccharide7 and the glycoconjugate (especially glycoprotein8–10 and glycolipid11 glycans) fields. The incorporation of one-dimensional (1-D) 1H and 13C nuclear magnetic resonance (NMR) spectroscopy in the 1970s gave rise to a new explosion of possibilities,12–18 and the results obtained in those years greatly influenced the whole carbohydrate research field, including the areas of carbohydrate biosynthesis, organic synthesis, enzymatic synthesis, molecular interactions, theoretical chemistry, and molecular biology. Especially in the field of glycoprotein glycan analysis, the introduction of 1-D 1H NMR spectroscopy caused a real revolution,19 that has been of major importance in further developing this field in terms of biochemistry and biology. The 2-D/3-D NMR techniques, introduced in the 1980s, made the possibilities for structural analysis and interaction studies even larger.20 The introduction of soft-ionization techniques in mass spectrometry (MS) at the end of the twentieth century created completely new possibilities for the analysis of high-molecular-mass glycans. Starting with fast-atom-bombardment mass spectrometry,21,22 nowadays matrix-assisted laser-desorption-ionization time-of-flight and electrospray ionization are firm technologies in glycoanalysis. Especially in the field of glycoconjugates, with a focus on glycoproteomics, glycomics, and high-throughput analysis, these techniques have been broadly incorporated.23 In parallel with the improvements in NMR spectroscopy and mass spectrometry (MS), also the improvements in high-pressure liquid chromatography (HPLC), capillary electrophoresis (CE), the development of suitable microchemical degradation and derivatization protocols, the incorporation of exo- and endo-glycosidase digestions, and the development of microarray technologies, greatly contributed to the whole array of structural analysis approaches for glycan chains, as available nowadays. This chapter gives an overview of the various strategies used in carbohydrate analysis. The selected references reflect clearly the different time periods in which these methodologies were developed. Although instrumental techniques are included in this chapter, they will be broadly reviewed in several other chapters in the Section Analysis of Glycans of this book series. General structural background information about the different classes of carbohydrates that get attention in this chapter is reviewed in the Section Introduction to Glycoscience of this book series.

2.01.2 Primary Structural Analysis of Carbohydrates The primary structure of carbohydrate chains is defined by several parameters (Figure 1): 1. 2. 3. 4. 5. 6.

nature and number of the constituent monosaccharides; sequence and ring size of the monosaccharides; type and anomeric configuration of the glycosidic linkages; nature of the noncarbohydrate substituents; type of the carbohydrate–peptide linkages (glycoproteins); and nature and position in the polypeptide backbone of the amino acids involved (glycoproteins).

It may be clear that in general several analytical strategies have to be followed to reach final conclusions about a carbohydrate structure. Of course, the selected strategies depend greatly on the glycan under study. Table 1 presents an overview of methodologies that are currently in use in polysaccharide and/or glycoconjugate glycan analysis. In the following sections, these technologies will be worked out in more detail. As in the discussion of the various strategies several monosaccharide structures will pass, to support the reader, in Figure 2 a survey of current structures is compiled.


3

L-Arabinose

(furanose) L-Araf

O OH COOH a O

HO CH2OH

OH

O

(1→6) CH2

(1→4)

O

O HO

b a O

O

OH

O D-Gulose

(pyranose) D-Gulp

HO

a O

D-Mannuronic

acid (pyranose) D-ManpA

OH

(1→4) (1→3)

OH

NHCOCH3 N-Acetyl-D-glucosamine (pyranose) D-GlcpNAc

Figure 1 Schematic presentation of the parameters, which define the primary structure of a carbohydrate chain.

2.01.2.1 Polysaccharides Bacterial polysaccharides are mainly defined as capsular polysaccharides (CPSs), exopolysaccharides (EPSs), lipopolysaccharides (LPSs), teichoic acids (TAs), and peptidoglycans.24–32 Over 100 different monosaccharide constituents have been identified: pentoses, hexoses, deoxyhexoses, aminodeoxyhexoses, diaminodideoxyhexoses, diaminotrideoxyhexoses, hexuronic acids, aminodeoxyhexuronic acids, heptoses, octulosonic acids, nonulosonic acids, branched monosaccharides, monosaccharides with noncarbohydrate substituents (e.g., O-methyl, O-(1-carboxyethyl), O-acetyl, O-lactyl, O-(2-hydroxypropionyl), O-(2,4-dihydroxybutyryl), phosphate, sulfate, N-acetyl, N-glycolyl, N-(2-aminopropionyl), pyruvate), etc. See for instance the BCSDB database.32a The majority of the CPSs is built up from repeating oligosaccharide units; in general, the CPSs elucidated so far turned out to be heteropolysaccharides. EPSs occur both as hetero- and homopolysaccharides; the heteropolysaccharides are built up from repeating oligosaccharide units, whereas the majority of the homopolysaccharides is quite often heterogeneous/random in structure. In general, LPSs consist of an O-antigenic polysaccharide, built up from oligosaccharide repeating units, a core oligosaccharide, and an acylated disaccharide, called lipid A. Different types of TAs exist: common fragments are polyolphosphates, glycosylpolyolphosphates, polyolphosphate-glycosylphosphates, and polyolphosphate-glycosylpolyolphosphates; free OH functions can bear mono- or oligosaccharides, or ester-bound amino acids. Peptidoglycans are built up from ‘linear repeating disaccharide’ polysaccharides, crossed-linked via oligopeptide fragments. The repeating disaccharide consists of !)-b-D-GlcpNAc-(1!4)-b-D-MurpNAc-(b1! elements, wherein MurNAc is 2-acetamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. An upgraded overview of bacterial polysaccharide structures is included in Chapter 1.04. Plant polysaccharides are much more heterogeneous, and homo- as well as heteropolysaccharides do occur. Complete structures are difficult to define, and in general structural studies are focused on creating statistical models. Thanks to the availability of highly specific enzymes, over the years these models have been highly upgraded. An upgraded overview of plant polysaccharide structures is included in Chapter 1.05. Several approaches are available for the structural analysis of polysaccharides (Table 1). Use is made of native as well as specifically degraded polysaccharides. Important chemical degradation procedures comprise partial solvolysis, acetolysis, uronic acid degradation, periodate oxidation, and de-N-acetylation – deamination. Important enzymatic degradation procedures make use of endo-glycosidases/endo-glycanases and lyases. Although MS plays an important role in identifying oligosaccharides at many stages of the analysis, NMR spectroscopy is of the utmost importance in arriving at full structures of polysaccharides.

2.01.2.2 Glycoprotein Glycans Glycoproteins are found in a wide variety of natural sources. They are defined as proteins that bear a carbohydrate in a covalent way, a coupling that is called glycation. The general term glycation is used for both glycoside formation and

4


Table 1 Overview of methodologies that are currently in use in polysaccharide and/or glycoconjugate glycan analysis Monosaccharide analysis Gas–liquid chromatography – (electron impact mass spectrometry) (GLC-EI/MS) High-performance liquid chromatography – fluorescence detection (HPLC-FD) High-pH anion-exchange chromatography – pulsed amperometric detection (HPAEC-PAD) Capillary electrophoresis (CE) Linkage/methylation analysis Gas–liquid chromatography – (electron impact mass spectrometry) (GLC-EI/MS) Chemical degradation procedures Partial solvolysis Acetolysis Uronic acid degradation Periodate oxidation De-N-acetylation – deamination Enzymatic degradation procedures Exo-glycosidases Endo-glycosidases/endo-glycanases Lyases Release of glycans from glycoproteins Proteolytic digestion: N- and O-linked (! glycopeptides) Hydrazinolysis: N- and O-linked (! oligosaccharides) Peptide-N 4-(N-acetyl-b-glucosaminyl)asparagine amidases: N-linked (! oligosaccharides) Endo-b-N-acetylglucosaminidases: N-linked (! oligosaccharides) Alkaline borohydride treatment: O-linked (! oligosaccharide-alditols) Release of glycans from glycolipids Endoglycoceramidase (! oligosaccharides) Ozonolysis/alkaline fragmentation (! oligosaccharides) Fractionation and purification Gel-permeation chromatography (Bio-Gel, Superdex) HPLC Normal phase (Lichrosorb-NH2, TSKgel Amide-80, GlycoSep N) Reversed-phase (Hypersil ODS C18, GlycoSep R) Anion-exchange (Resource Q, Vydac 301VHP575, GlycoSep C, CarboPac) Lectin affinity chromatography Mass spectrometry (MS) using different ionization techniques Electron impact (EI) Chemical ionization (CI) Fast atom bombardment (FAB) Matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) Electrospray ionization (ES) 1-D/2-D Nuclear magnetic resonance (NMR) spectroscopy using 1H, 13C, and/or 31P probes Correlation spectroscopy (COSY) Total correlation spectroscopy (TOCSY) Heteronuclear multiple quantum coherence (HMQC) Heteronuclear single quantum coherence (HSQC) Heteronuclear multiple bond correlation (HMBC) Rotating frame Overhauser enhancement spectroscopy (ROESY) Nuclear Overhauser enhancement spectroscopy (NOESY) Profiling protocols for glycoprotein glycans HPLC profiling (anion-exchange; normal phase; reversed-phase) HPAEC profiling CE profiling Fluophore-assisted carbohydrate electrophoresis (FACE) profiling LC-MS profiling Glycan detection and differentiation assays/lectin microarrays

amination (Schiff base reaction). Besides these types of couplings, other covalent linkages do occur between carbohydrate and amino acids, such as ester linkages. Table 2 gives an overview of reported linkages between monosaccharides and amino acids; Figure 3 summarizes a series of glycoamino acids. The majority of the methodologies developed for the analysis of glycoprotein glycans is dealing with GlcNAc(b1-N)Asn and GalNAc(a1-O)Ser/Thr linkages, and


5

OH

OH

COOH

O

O HO HO

HO HO

OH

OH

NH

O

O

HO HO

OH

OH

OH

HO HO OH

OH

Ac

N-Acetyl-D-glucosamine D-GlcpNAc

D-Glucose D-Glcp

OH

D-Glucuronic

OH OH

acid

D-Xylose

D-GlcpA

D-Xylp

OH

O

OH

OH

O

COOH

O

HO

HO

OH

OH

HO

OH

NH

OH

OH

OH

Ac D-Galp

OH

OH D-Galacturonic

N-Acetyl-D-galactosamine D-GalpNAc

D-Galactose

OH

O

acid

D-Galactose

D-GalpA

D-Galf

OH OH O

OH

H3 C

HO HO

OH

H3 C

O

O

HO OH

OH HO

OH OH

D-Mannose

OH

L-Rhamnose

L-Fucose

L-Rhap

L-Fucp

D-Manp

HO O

O OH

OH

OH

HO OH

OH

OH

D-Ribose

L-Arabinose

D-Ribf

L-Araf

HO

HO HO HO

HO

OH O

OH O

HO

COOH

HO O

OH HO

OH

COOH L-glycero-D-manno-Heptose LD-Hepp

OH

AcHN

HO

3-Deoxy-D-manno-octulosonic acid Kdo

HO

HO

N-Acetylneuraminic acid Neu5Ac

Figure 2 Survey of monosaccharide structures.

these approaches will be discussed in more detail. However, many of the protocols are directly applicable to other types of linkages. In general, four groups of monosaccharides can occur as constituents of glycoprotein glycans: (1) neutral monosaccharides – D-mannose (Man), D-galactose (Gal), D-glucose (Glc), L-fucose (Fuc), L-arabinose (Ara), D-xylose (Xyl); (2) amino sugars – N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc); (3) uronic

6


Table 2 Naturally occurring covalent linkages between monosaccharides and amino acidsa; all monosaccharides, except Rha, Fuc, and Ara, have D-configuration Type/linkage

Type/linkage

N-Glycosylation GlcNAc(b1-N)Asn GalNAc(b1-N)Asn Glc(a/b1-N)Asn Rha(1-N)Asn Bac(b1-N)Asn Man(1-N)Trp O-Glycosylation GalNAc(a1-O)Ser/Thr GlcNAc(b1-O)Ser/Thr Man(a1-O)Ser/Thr Xyl(b1-O)Ser/Thr Fuc(a1-O)Ser/Thr Gal(a1-O)Ser/Thr Glc(b1-O)Ser/Thr Ara(b1-O)Ser Gal(b1-O)Hyl Araf(b1-O)Hyp Glc(a/b1-O)Tyr DATDH(1-O)Ser Gal(b1-O)Hyp S-Glycosylation Gal(1-S)Cys Glc(1-S)Cys Glypiation (GPI anchors) Xxx-CONH-(CH2)2-P-6)Man

C-Glycosylation Man(a1-C)Trp ADP ribosylation ADP-Rib(3-N)Arg ADP-Rib(3-N)Asn ADP-Rib(3-N)His ADP-Rib(3-O)Glu ADP-Rib(3-S)Cys Phosphoglycosylation GlcNAc(a1-P-O)Ser Man(a1-P-O)Ser Xyl(a1-P-O)Ser/Thr Fuc(b1-P-O)Ser Amide bond (in polysaccharides) GlcA/GalA(6-Na)Lys GalA(6-Na)Ser GalA(6-Na)Thr GalA(6-Na)Ala GlcA(6-Na)Glu MurNAc(3-Na)Ala Glycation (Schiff base) Glc-Lys Rib-Lys

a References concerning these linkages: General, Chapter 1.02 and reviews;33–36 Bac(b1-N)Asn; Bac¼bacillosamine, 2,4diacetamido-2,4,6-trideoxyglucose;37,38 Man(1-N)Trp/N-mannosyl tryptophan;39 Man(a1-C)Trp/C-mannosyl tryptophan;40 ADP-ribosylation;41–43 DATDH(1-O)Ser; DATDH¼2,4-diacetamido-2,4,6-trideoxyhexose;44 Amide bond;45–47 S-Glycosylation;48,49 Glycation.50 A further polysaccharide example, different from those in the Table: 2-(L-alanylamido)-2-deoxy51 512 D-glucose. Added in proof: FucNAc(b1-O)Ser.

acids – D-glucuronic acid (GlcA), D-galacturonic acid (GalA), L-iduronic acid (IdoA); and (4) sialic acid(s) (analogs) – Nacetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and 2-keto-3-deoxynonulosonic acid (Kdn). In addition, noncarbohydrate substituents, such as O-methyl, O-acetyl, O-lactyl, pyruvate, sulfate, or phosphate groups, can be present. Furthermore, particularly, in microorganisms and lower organisms, rare monosaccharide derivatives have been found to occur. An overview of such derivatives is presented in Table 3. It should be noted that Chapter 3.15 includes a comprehensive overview of rare monosaccharide derivatives in glycosphingolipids as found in lower animals. The glycosylation patterns of GlcNAc(b1-N)Asn-containing glycoproteins, so-called N-glycoproteins, are divided in three major classes: (1) oligomannose or high-mannose type; (2) complex type, including N-acetyllactosamine (LacNAc) type, N,N0 -diacetyllactosamine (LacdiNAc) type, and xylose type; and (3) hybrid type. The LacNAc and LacdiNAc type comprise up to five antennary systems. LacNAc as well as LacdiNAc can also occur in oligomeric form, interconnected via (b1-3) linkages. A full description of these glycosylation patterns is discussed in Chapter 1.02 (see also Chapter 1.01). The glycosylation patterns of GalNAc(a1-O)Ser/Thr-containing glycoproteins, so-called O-glycoproteins, are divided into eight core types: (1) core 1, Gal(b1-3)GalNAc; (2) core 2, Gal(b1-3)[GlcNAc(b1-6)]GalNAc; (3) core 3, GlcNAc(b1-3)GalNAc; (4) core 4, GlcNAc(b1-3)[GlcNAc(b1-6)]GalNAc; (5) core 5, GalNAc(a1-3)GalNAc; (6) core 6, GlcNAc(b1-6)GalNAc; (7) core 7, GalNAc(a1-6)GalNAc; (8) core 8, Gal(a1-3)GalNAc. A full description of these glycosylation patterns is reviewed in Chapter 1.02. For the structural analysis of glycoprotein N- and O-glycans, several approaches are available (Table 1). The level of structural detail depends on the scientific questions researchers have. Glycoproteins are studied as such, but


COOH

OH OH

OH O

HO

7

O

H2N NH

HO

CH2

COOH

HO

NH

NH Ac

O

Ac

GlcNAc(b1-N)Asn

O

COOH

H2N CH2

HN

GalNAc(a1-O)Ser

O

O OH COOH OH

OH HO O

HO

H2N O

HO

COOH

HO O

CH2

OH L-Ara(a1-O)Hyp

HO O

HO NH

H2N

Ac

CH2

Man(a1-O)Ser

COOH

GlcNAc(b1-O)Ser

OH H2N HO O CH2

HO COOH

COOH

H2N O HO HO

OH

H2N

OH CH2 O

O

(CH2)2 O

HO

OH

HO

HN

CH

Man(a1-C)Trp

OH CH2NH2

Xyl(b1-O)Ser

Gal(b1-O)Hyl

Figure 3 Survey of a series of glycoamino acid elements as occurring in glycoproteins.

also in glycopeptide or released oligosaccharide form. Free oligosaccharides can be generated via chemical or enzymatic release procedures. Enzymatic degradation procedures make use of exo- and endo-glycosidases. 1H NMR spectroscopy and nowadays especially MS play major roles in the unraveling of the highly complex glycosylation patterns. In addition, in many analytical projects, profiling methodologies are frequently used, especially in glycoproteomics research. To become familiar with many aspects of glycoprotein glycan research, the reader is referred to two excellent books on a graduate level.95,96 For a series of basic reviews, covering the glycoprotein literature up to 1995, see Refs. 97–99.

2.01.3 Monosaccharide Analysis Nowadays, the approaches to determine the monosaccharide composition of carbohydrate chains (oligosaccharides, polysaccharides, glycoprotein glycans, glycolipid glycans, proteoglycans, etc.) are mainly based on GLC and liquid chromatography (LC) procedures. To this end two solvolysis protocols are currently applied, namely, hydrolysis and methanolysis. Figure 4 summarizes both protocols for a model galactan.

2.01.3.1 Monosaccharide Analysis after Hydrolysis The hydrolysis procedure involves cleavage of the glycosidic linkages by mineral acid. As is visualized in Figure 4 for galactan ! D-galactose, each released monosaccharide constituent of an oligo/polysaccharide will give rise to a monosaccharide-specific anomeric mixture of a,b-pyranose and a,b-furanose ring forms. The reaction mechanism behind the hydrolysis is quite complicated. In principle, both the glycosidic O atom and the ring O atom can be protonated. Protonation of the glycosidic O atom leads to cleavage of the glycosidic linkage and formation of

8


Table 3 Rare monosaccharide constituents of glycoproteins Monosaccharide

Source

Refs.

2-Acetamido-4-amino-2,4,6-trideoxyglucose 6-Deoxyaltrose 3-Deoxy-D-glycero-D-galacto-nonulosonic acida

Clostridium symbiosum Salmonid fish eggs Salmonid fish eggs Amphibian eggs Bacillus stearothermophilus Nematodes Chlamydomonas eugametos Clostridium thermocellum Bacteroides cellulosolvens Trypanosoma Yeasts Snails Chlamydomonas eugametos Nematode Snail Alga Halobacteria Methanothermus fervidus Chlamydomonas eugametos Thyroglobulin Mucins Tamm-Horsfall glycoprotein Recombinant tissue plasminogen activator Mucin Pituitary glycohormones Tamm-Horsfall glycoprotein Clostridum thermocellum Thyroglobulin Zona pellucida glycoprotein Mucin HNK-1 epitope Alga Snail Chlamydomonas eugametos Ovalbumin Ovalbumin Dictyostelium discoideum Snail Dictyostelium discoideum Clostridum symbiosum Different sources Trichinella spiralis Locusta migratoria Locusta migratoria Campylobacter jejuni Bovine lung glycoprotein Nematodes Nematodes Vespula germanica

52 53 54 55 52 56 57 58 59 60,61 62 63–65 57 56 65 66 67 68 57 69 70 71,72 73 74 75 71 58 69 76 74 77,78 57,79 63,80 57 81 81 82 83 84 52 85–87 88,89 90 90 37 91 92 93 94

2,3-Diacetamido-2,3-dideoxymannuronic acid 2-O-Methyl-fucose 4-O-Methyl-xylose Galactofuranose

3-O-Methyl-galactose 4-O-Methyl-galactose 6-O-Methyl-galactose 3-O-Methyl-galacturonic acid 3-O-Methyl-glucose 3-O-Methyl-L-glucose 3-O-Sulfo-galactose

6-O-Sulfo-galactose N-Acetyl-4-O-sulfo-galactosamine N-Acetyl-3-O-methyl-glucosamine N-Acetyl-6-O-sulfo-glucosamine

3-O-Sulfo-glucuronic acid Gulose 3-O-Methyl-mannose 6-O-Methyl-mannose 4-O-Sulfo-mannose 6-O-Sulfo-mannose

Man6(PMe2) N-Acetylmannosamine Sialic acid derivatives 3,6-Dideoxy-D-arabino-hexose (D-tyvelose) 6-O-AEP-N-acetylglucosamineb 6-O-AEP-mannoseb Bacillosaminec Glutamyl-(N-glycan) 6-O-PC-N-acetylglucosamined PC-mannose 6-O-(Etn-P)-N-acetylgalactosaminee a

Kdn. AEP¼2-aminoethylphosphonate. c 2,4-Diacetamido-2,4,6-trideoxyglucose. d PC¼phosphorylcholine. e Etn-P¼2-aminoethylphosphate. Upgraded from Lis, H.; Sharon, N. Eur. J. Biochem. 1993, 218, 1–27. b


CH2OH

CH2OH

O

HO

9

CH2OH

O

OH

O

O

OH

O

OH OH

OH

OH

n OH

Solvolysis CH2OH Hydrolysis (R = H) Reduction Acetylation

HO

Methanolysis (R = Me) Trimethylsilylation

O

O OH

OR

+

OR

OH

OH OH

OH CH2OH CH2OAc

CH2OTMS

OAc Me = CH3 Ac = COCH3 TMS = Si(CH3)3

AcO AcO

TMSO

O OTMS

O OMe

+

OTMS

OMe

OAc CH2OAc

OTMS

OTMS OTMS CH2OTMS

Figure 4 Monosaccharide analysis by GLC-(EI/MS) of a model (a1-4)-galactan: Schematic presentation of the hydrolysis and methanolysis procedure, yielding alditol acetates and perTMS methyl glycosides, respectively.

an anomeric carbocation, which can react with water to give the mixture of a,b-anomers of the original ring form (Figure 5). However, pyranose/furanose ring conversions via protonation of the ring O atom in an early stage and/or mutarotation of the released original ring form will finally yield the mixture of a,b-pyranose and a,b-furanose ring forms. In view of the different stabilities of the monosaccharides and the occurring glycosidic linkages, in principle, the conditions for complete hydrolysis with minimal destruction of the constituents have to be determined experimentally. The following examples are meant to illustrate these differences in stability against mineral acid. The hydrolysis of the glycosidic bonds of 2-acetamido sugars [HexNAc(1-x)] and uronic acids [HexA(1-x)] can give rise to severe problems, due to incomplete cleavage. In the case of the 2-acetamido sugars, this is due to a competition between the hydrolysis of the N-acetyl linkage, yielding an inhibiting protonated amino group at C2, and the hydrolysis of the glycosidic linkage. This also means that the glycosidic bonds of 2-amino sugars are difficult to hydrolyze. The glycosidic bonds of pyranose forms [Hexp(1-x)] are more stable than those of furanose forms [Hexf(1-x)], and aldose forms are more stable than ketose forms. For instance, for the quantification of the 2-keto sugars sialic acid and fructose, very mild conditions have to be applied. Depending on their structure, acidic solutions of specific monosaccharides can give rise to anhydro or lactone formation. Typical examples comprise the formation of 1,6-anhydrob-D-idopyranose from b-D-idopyranose, the formation of 1,6-anhydro-a-D-talofuranose from a-D-talofuranose, the formation of D-glucofuranurono-6,3-lactone from D-glucuronic acid, and the formation of D-galactopyranurono6,3-lactone from D-galacturonic acid. Among the many proposed conditions for the hydrolysis of polysaccharides and glycoproteins,100–104 the use of 4M trifluoroacetic acid (4h, 100 C) has shown to give reasonable results. It should be noted that 2-acetamido sugarcontaining polysaccharides can be hydrolyzed in very good yields with anhydrous HF (2h, room temperature).102 For the quantification of sialic acids, a hydrolysis with 2M propionic acid (4h, 80 C) is proposed.87

10


CH2OH

CH2OH

O OR

HO

+H+

O O+

HO

OH

H O +

OH

OH

OH

CH2OH

O

HO

OH

OH

CH2OH +H2O

+ O

HO

+

OH

OH

CH2OH

O

HO

−ROH R

OH

OH b-D-Galp-(1→R)

CH2OH

H

−H+

a,b-D-Galp

O

HO

OH

OH

H

+ a,b-D-Galf

OH a,b-D-Galp

Figure 5 Simplified reaction mechanism of the hydrolysis of a model (b1-4)-galactan.

Among the various high-performance liquid chromatography (HPLC) protocols, used for the analysis of underivatized monosaccharide mixtures, high-pH anion-exchange chromatography (HPAEC) combined with pulsedamperometric detection (PAD) on CarboPac PA-1 columns is currently applied (see Section 2.01.7.1.1). HPLC combined with fluorescent detection (FD) is used in the analysis of fluorescently labeled (at the anomeric center) monosaccharide mixtures (see Section 2.01.7.2). Before analysis by GLC (flame-ionization detection), neutral and amino sugars are most commonly converted into their corresponding alditol acetates.5,100,105 Other reported derivatives are trimethylsilylated (TMS) alditols, trifluoroacetylated alditols, aldononitrile acetates, and oxime derivatives (for a review, see Ref. 104). For verification/analysis of the GLC peaks, MS can be applied using combined GLC-EI/ MS or GLC-CI/MS instruments (EI/MS ¼ electron impact mass spectrometry; CI/MS ¼ chemical ionization mass spectrometry). The analysis of sialic acids is discussed in Section 2.01.7.8. For the alditol acetate analysis, the mixture of released monosaccharides is reduced with NaBH4 (2 h, room temperature) and the formed alditols are acetylated with acetic anhydride in pyridine (1:1 mixture; 1 h, 100 C) (Figure 4). Figure 6 shows the reaction mechanisms of the reduction and acetylation reaction. A typical GLC example of a monosaccharide analysis using alditol acetates is presented in Figure 7. Extensive reviews with respect to the EI/MS of alditol acetates are available in the literature.4,5 Some basic information about the EI fragmentation patterns is presented in Figure 8.

2.01.3.2 Monosaccharide Analysis after Methanolysis The methanolysis procedure involves cleavage of the glycosidic linkages by methanolic HCl. As is visualized in Figure 4 for galactan ! D-galactose, each released monosaccharide constituent of an oligo/polysaccharide will give rise to a monosaccharide-specific mixture of methyl a,b-pyranoside and a,b-furanoside ring forms. As already discussed for the hydrolysis of glycosidic bonds (Section 2.01.3.1), also the reaction mechanism behind the methanolysis is quite complicated. Also here the first steps are the possible protonations of the glycosidic O atom and the ring O atom. Protonation of the glycosidic O atom, followed by cleavage of the glycosidic linkage, and reaction with methanol gives the mixture of 1-O-methylated a,b-anomers of the original ring form (Figure 9a). However, during the glycosidation process, also pyranose/furanose ring conversions and new glycosidations can occur via protonation of the ring O atom or of the already formed anomeric methyl groups, respectively, thereby yielding finally the monosaccharide-specific mixture of methyl a,b-pyranoside and a,b-furanoside ring forms. When compared with the hydrolysis procedures (Section 2.01.3.1), the methanolysis procedure causes less destruction of the monosaccharides, but it is very effective in cleaving glycosidic linkages. Therefore, it is applied


H H

O H

C

B

H −

H

R

H

H

C

R

Na+

O

B

H

H

−

H H

R

O C

3

Na+

R

H 4

H

C

OH

+

4H2O

NaB(OH)4

B R

C

−

O Na+

≡

H

11

H 4

H3BO3 + NaOH → NaH2BO3 + H2O (a)

O H3C

C

O O

C

O CH3

H3 C

C

O + N

+

R

C

CH3

H

O C

O

O

N

H3C

−

O O

R

+

+ HN

C

H3C +

(b) R

+ N

O H

Figure 6 Reduction mechanism (NaBH4) for the conversion of aldoses into alditols (a), and acetylation mechanism (acetic anhydride in pyridine) for the conversion of alcohols into acetylated alcohols (b).

Figure 7 Gas chromatogram of alditol acetates on an AT-1 column (30 m0.25mm, Alltech). Oven temperature program: 140–240 C at 4 Cmin1; 3min at 240 C. The peaks are numbered in their order of elution and are assigned as follows: (1) Rib-ol; (2) Fuc-ol; (3) Xyl-ol; (4) Man-ol; (5) Glc-ol; (6) Gal-ol; (7) GlcNAc-ol; (8) ManNAc-ol; and (9) GalNAc-ol.

12


CH3 H H3C

+ O

C

H3C

C

O +

C

O m/z 43 (base peak)

+ M

minus

CH3

H3C

O m/z 103

+ M

OCOCH3

H

C

O

minus

R1

COCH3

H

COCH3

H

H

C

O

minus

C

COCH3

C

O

COCH3

+

O

C

O

COCH3

H

C

O +

COCH3

COCH3

H

C

H

C

O

C

CH

COCH3

O

H

C

OH

R2

C

+ O

CH

O

O O

−CH2CO

CH3CO

−CH3COOH

H

H2C

COCH3

or

COCH3

C

+

R1

−CH3COOH

CH3CO

H

R2

R2

+ O

HOCOCH3

R1

+ O

R1

R2

CH3CO

O

or

+ O

CH3

R2

R1 C

+ M

NH2COCH3

R2

H

C

m/z 145

+ O

O +

Other possibilities

R1 C

O

O

First detectable ion in the high mass range

H

C

C

O CH3

H2C

m/z 217

C

C

CH3CO −CH2CO

+ O

CH

O

C

CH2

CH3

H

m/z 157

m/z 115

CH3 C + H2C CH3CO

O

C H

COCH3

O

−O

O CH

O

COCH3

C H

O

COCH3

+

COCH3

H2C CH3CO

m/z 289 Figure 8 A selection of EI fragmentation patterns of alditol acetates.4,5

O

C H

O CH C H

m/z 187

O

COCH3


CH2OH O OR

HO

HO

+ H+

OH

CH3

OH

O

+

OH

OH

−H+

R

H N

3

+

OH

Cl

Methyl a-D-Galp Methyl b-D-Galp Methyl a-D-Galf Methyl b-D-Galf

OH Methyl a-D-Galp

CH3 R

(CH3)3Si

OH OCH3

Si(CH3)3

OH

O

HO

Methyl b-D-Galp

(CH3)3Si

OH

CH2OH

O OCH OH

H + O CH3 OH

+HOCH3

OH

CH2OH HO

(a)

(b)

+

OH

CH2OH HO

O+

HO

OH

OH

CH2OH H O O+

CH2OH

O

HO

−ROH

R

OH

OH b-D-Galp-(1→R)

HO

CH2OH

CH2OH H OO

13

O

Si

CH3 + NH4Cl

CH3

Figure 9 Simplified reaction mechanism of the methanolysis of a model (b1-4)-galactan (a), and trimethylsilylation of a hydroxyl group (b).

to the simultaneous analysis of neutral monosaccharides, acetamido sugars, uronic acids, octulosonic acids, and sialic acids.106 Over the years, the use of 1M methanolic HCl (24h, 85 C) has shown to give excellent results.104 Applying this methodology, a few features should be taken into account: (1) carboxyl groups of acidic monosaccharides are blocked by methyl ester formation; (2) N-acyl groups in amino sugars are cleaved nearly completely, and therefore a re-N-acetylation step has to be incorporated in the procedure; (3) in glycoprotein N-glycan analysis, the linkage between GlcNAc and Asn is split only to a very limited extent and mainly the free monosaccharide is liberated instead of its methyl glycoside; (4) in oligosaccharide-alditol analysis (e.g., glycoprotein O-glycan analysis after alkaline borohydride treatment), alditols such as GalNAc-ol (35%), Xyl-ol (23%), Gal-ol (14%), Fuc-ol (10%), Ara-ol (5%), and Glc-ol (20%) give rise to anhydro derivatives;107 (5) in glycolipid glycan analysis, the ceramide residues are degraded to sphingosine and fatty acid methyl esters; (6) 2-ketoses like Fru are completely degraded, several uronic acids give rise to 3,6-lactone formation, Gul yields 30% of 1,6-anhydride,108 Neu5Ac gives 3% of 2,7-anhydride, whereas anhydro-formation has also been observed for heptoses;109 (7) Kdo gives rise to six peaks (6:8:27:15:32:12), probably belonging to two pyranose, two furanose, and two anhydro and/or lactone forms. The mixture of methyl glycosides of the different types of monosaccharides can be quantified by GLC after re-N-acetylation followed by trimethylsilylation104,106,107 or trifluoroacetylation.110 For the TMS (methyl ester) methyl glycoside analysis, the neutralized (solid silver carbonate) mixture of released glycosides in methanol is N-acetylated with acetic anhydride (24h, room temperature, dark). The trimethylsilylation is carried out with a 5:1:1 mixture of pyridine–hexamethyldisilazane–chlorotrimethylsilane (30min, room temperature) (Figure 9b). A typical example of a monosaccharide analysis using TMS (methyl ester) methyl glycosides is presented in Figure 10. It should be noted that the characteristic GLC peak pattern of methyl glycosides for each monosaccharide highly facilitates the identification of a monosaccharide. The availability of a peak pattern in a constant ratio is an important control to check if a GLC peak reflects more than one compound. Such a controlling mechanism is absent in the GLC analysis of alditol acetates. For quantification purposes, molar adjustment factors of monosaccharides, except Neu5Ac, are determined by application of the methanolysis procedure on standard mixtures of free monosaccharides and internal standard (Man-ol). For Neu5Ac, the molar adjustment factor is determined by subjecting a known sialo-oligosaccharide to methanolysis. When necessary, this is also done for uronic acid-containing material. For accurate quantification of uronic acids, the carboxyl function should be in the acid or alkyl-esterified form before carrying out methanolysis.

14


Figure 10 Gas chromatogram of TMS (methyl ester) methyl glycosides on an AT-1 column (30m0.25mm, Alltech). Oven temperature program: 140–240 C at 4 Cmin1; 3min at 240 C. The peaks are numbered in their order of elution and are assigned as follows: (1) Ara (a-p); (2) Ara (b-p); (3) Xyl (a,b-f ); (4) Fuc (b-f ); (5) Ara (f ); (6) Fuc (a-p); (7) Fuc (b-p); (8) Fuc (a-f ); (9) Xyl (a-p); (10) Xyl (b-p); (11) Man (a-p); (12) Gal (b-f ); (13) Man (b-p); (14) Gal (a-p); (15) Gal (a-f ); (16) Gal (b-p); (17) Glc (a-p); (18) Glc (b-p); (19) Man-ol (internal standard); (20) GlcNAc (a-f ); (21) GalNAc (a,b-f ); (22) Man1/6Ac1-ol (due to the re-N-acetylation step); (23) GlcNAc (b-p); (24) GalNAc (a,b-p); (25) GlcNAc (a-p); (26) GlcNAc (a,b-p; no methyl glycoside); (27) 2,7-anhydroNeu5Ac; (28) Neu5Ac (a-p); (29) Neu5Ac (b-p); and (30) Neu5,9Ac2 (b-p) (due to the re-Nacetylation step). Insert: (10 ) GlcA (lactone-f ); (20 ) GlcA (lactone-f ); (30 ) GalA (b-f ); (40 ) GalA (a-f ); (50 ) GalA (a-p); (60 ) GalA (b-p); (70 ) GlcA (b-p); and (80 ) GlcA (a-p). f, furanoside; p, pyranoside.

The EI mass spectra of TMS methyl glycosides have been studied in great detail.5,106,111–113 Some basic information about the EI fragmentation pattern is presented in Figure 11. In the high mass region, the spectra are usually characterized by [M-CH3]þ fragment ions. Just as for the alditol acetates, differences in stereochemistry within one class of monosaccharides are not reflected in the mass spectra. In general, it is possible to discriminate between pyranose and furanose ring forms in one class of monosaccharides. For TMS methyl aldohexosides, it has been found that the ratio of the intensities of the peaks at m/z 204 and m/z 217 is useful.106 In pyranose ring forms, the ion m/z 204 corresponds with the fragments TMSO2CH-3CHOTMS (major) and TMSO3CH-4CHOTMS (minor); m/z 217 corresponds mainly with TMSO2CH-3CH-4CHOTMS. In furanose ring forms m/z 204 can be explained as TMSO2CH-3CHOTMS and m/z 217 as TMSO2CH-3CHOTMS-4CH. The studied pyranose rings give rise to a ratio of I204/I217 >1, whereas the furanose rings show a ratio 1. In addition, the presence of a relatively intense peak at m/z 205 [TMSO6CH2-5CHOTMS] is characteristic of a furanose ring. The concept of discrimination between pyranose and furanose ring forms on the basis of the intensities of m/z 204 and m/z 217 is also applicable to TMS methyl glycosides of uronic acid methyl esters and to most aldopentoses. Furthermore, the discrimination system is usuable for methyl aldohexosides in which one of the trimethylsilyl groups is replaced by another substituent, for example, HexNAc (m/z 173, m/z 186, m/z 205) or natural occurring O-methylated methyl aldohexosides. The introduction of deuterated trimethylsilyl, N-acetyl and/or methyl groups in the monosaccharide can be very helpful for the interpretation of mass spectra of unknown monosaccharide residues.

2.01.3.3

D/L

Configuration Determination

Another aspect of the monosaccharide analysis is the determination of the absolute (D or L) configuration of the constituting monosaccharides. In principle, the separation of enantiomers by GLC can be achieved (1) directly on a chiral stationary phase, and (2) by conversion of the enantiomers into diastereomers using a chiral reagent, and separation on a nonchiral stationary phase. For the determination of the D and L configuration of monosaccharides, the latter approach is mainly used. To this end one extra chiral center is added to a monosaccharide via a glycosidation reaction with a chiral alcohol, for example, (–)-2-butanol (Figure 12) or (þ)-2-octanol. In this way protocols have been developed for the GLC analysis of TMS (–)-2-butyl glycosides114,115 and acetylated (þ)-2-octyl glycosides.116 Due to


CH2OTMS O+ OTMS

m/z 467 +

OMe

CH3 (from a TMS group) m/z 435 + MeOH

m/z 482

TMSO

m/z 377 + TMSOH

OTMS CH2OTMS

CH2OMe

+ O OTMS

+ O

−OMe

OMe

OTMS

m/z 482

TMSO

m/z 361 + TMSOH m/z 451

TMSO

OTMS

OTMS

CH2OTMS + O OTMS

+ O

−CH2OTMS

OMe

OTMS

m/z 482

TMSO

m/z 379

TMSO

OTMS

m/z 289 + TMSOH

OMe

OTMS

CH3 + O OTMS

−CH3CHO

OMe

TMSO

+ OMe

OTMS

m/z 394

TMSO

m/z 335 + CH3

m/z 350

OTMS

OTMS

COOMe

COOMe

+

O

OTMS

−MeOCHO

OMe

OTMS

m/z 438

TMSO

m/z 363 + CH3

TMSO

OTMS

m/z 378 OTMS

+

CH2OSi(CH3)2 HC

HC

OTMS

TMSO

Si(CH3)2

CH3

O

m/z 117

m/z 117

m/z 147

CH2

OTMS

TMSO

Not specific for a primary hydroxyl function m/z 103

H C

OTMS

C H

m/z 204 Major C2-C3, minor C3-C4 in pyranose ring forms

OTMS TMSO

C H

C H

C H

OTMS

m/z 217 Major C2-C3-C4 in pyranose ring forms

TMSO CH2 C H

OTMS

m/z 205 C5-C6 in furanose ring forms

TMSO

C H

C

C H

OTMS

m/z 305 Mainly C2-C3-C4 in pyranose ring forms

TMSO

CH2 OTMS

OTMS

CH3CONH

H C

C H

OTMS

CH3CONH

m/z 173

C H

C H

m/z 186

OTMS TMSO

C H

C H

C H

C H

OTMS

m/z 319 Furanose ring forms

m/z 319 Major contribution in pyranose ring forms

Figure 11 A selection of EI fragmentation patterns of TMS methyl glycosides.5,106,111–113

C H

OTMS

15

16


OH

HO O

O

HO

OH OH

HO OH

HO

CH3

O

* CH2

O

*

H

CH2

CH3

(a)

CH3

(b)

H

CH3

Figure 12 Conversion of enantiomers into diastereomers: a, (–)-2-butyl a-D-glucopyranoside; b, (–)-2-butyl a-L-glucopyranoside.

2 L

100

D 3 LD

1 LD

D L

%

DL

0

DL

*

*

*

*

rt

6

8

10

12

14

16

18

20

22

24 min

Figure 13 Gas chromatogram of TMS (–)-2-butyl glycosides of (1) L,D-Fuc, (2) L,D-Man, and (3) L,D-Glc on an AT-1 column (30m0.25mm, Alltech). Oven temperature program: 140–240 C at 4 Cmin1; 3min at 240 C. *, noncarbohydrate contaminants.

solubility problems of biopolymers, when directly subjected to butanolysis or octanolysis, the most convenient approach is to convert part of the mixture of methyl glycosides, generated during methanolysis for monosaccharide analysis, into a mixture of (–)-2-butyl or (þ)-2-octyl glycosides. A typical example of a D/L configuration analysis using TMS (–)-2-butyl glycosides is presented in Figure 13. It may be clear that the method allows the analysis of mixtures of monosaccharides, even in the presence of contaminants.

2.01.4 Linkage/Methylation Analysis Methylation analysis including GLC-EI/MS has proved to be of great importance for the analysis of the substitution pattern (positions of glycosidic linkages, terminal units, branching points) and the ring size of the monomeric units of a glycan chain. A general scheme of the complete protocol, used for neutral and acetamido sugars, is presented in Figure 14. The first step of the linkage analysis involves the methylation of the free hydroxyl groups. In this step, the acetamido group of acetamido sugars is also methylated, and the carboxyl functions of uronic acids and sialic acids are esterified. Detailed information about the various developed methylation methods have been reported in the literature. The reaction is usually carried out with methyl iodide (CH3I) in alkaline medium using sodium methylsulfinylmethanide in dimethyl sulfoxide,1,117–119 potassium tert-butoxide/potassium methylsulfinylmethanide in dimethyl sulfoxide,120 potassium methylsulfinylmethanide in dimethyl sulfoxide,121 lithium methylsulfinylmethanide in dimethyl sulfoxide,122 butyllithium in dimethyl sulfoxide,123 or solid NaOH in dimethyl sulfoxide.124 Nowadays,

17


CH2

CH2

O

O OMe

OH O

HO

CH2OH

OH

O

O OMe

CH2OMe

Me2SO/solid NaOH MeI

O

O

OH

CH2

OMe

O

OH

HO

O

MeO

CH2

MeO OMe

OH

O

O

O

OMe

OMe

OH

OH

OMe

+

+

OH

OMe

OMe

OMe

OMe

Ac2O/pyridine

OH

OMe

O

HO

MeO

NaBD4

MeO

O

O Hydrolysis

CH2OMe

CH2OH

CH2OH

CHDOAc

CHDOAc

CHDOAc

OMe

OMe

OMe

MeO

MeO

MeO

+

+

OMe

OAc

OAc

OAc

OAc

CH2OAc

CH2OAc

CH2OMe

MeO

Figure 14 Schematic presentation of the methylation analysis of an artificial polysaccharide.

the latter conditions are seen in most research papers. Although a complete methylation in a single operation is essential for reliable results, it is sometimes impossible to overcome a certain degree of undermethylation, even after several successive methylation steps. It should be noted that when uronic-acid-containing glycan chains are investigated, repeated treatment with base leads to b-elimination reactions (see Section 2.01.5.5). Such alkaline degradations can also be expected if reducing oligosaccharides are methylated in the presence of strong bases as mentioned above. In the latter case, sometimes protocols are followed using milder bases.3 In the second step, the permethylated glycan is hydrolyzed, yielding a mixture of partially methylated monosaccharides. To this end several hydrolysis conditions have been applied, for example, 90% formic acid/0.13M H2SO4117 and 2-4M trifluoroacetic acid.119 The last chemical step comprises a reduction with NaBH4 or NaBD4, followed by acetylation of the generated hydroxyl groups with acetic anhydride in pyridine. From a mass spectrometric point of view, it is advantageous to use NaBD4, because it enables the differentiation between primary hydroxyl groups (Figure 14). The volatile partially methylated alditol acetates of neutral and amino sugars are usually analyzed by GLC in combination with EI/MS. Uronic acids, sialic acids, and Kdo cannot be analyzed in this way, because of the hydrolysis of their methyl ester groups. However, specific approaches, including the conversion of alduronic acids into aldoses, or the analysis of TMS or acetylated methanolysates are available.104 For the analysis of acetylated, methylated methyl glycosides, see Ref. 125. For the analysis of (partially) methylated methyl ester b-methyl glycosides of sialic acids, after treatment with reagents for acetylation or N-acetylation/trimethylsilylation, see Refs. 126 and 127. EI/MS of partially methylated alditol acetates gives rise to very characteristic mass spectra, however, mostly without molecular ion peaks. The peak patterns obtained from the various derivatives yield information on the positions of the O-methyl and O-acetyl groups in the alditol chains (Figure 14). The O-methyl groups reflect the free hydroxyl groups in the corresponding monosaccharides of the native material. The mass spectra of the stereoisomeric, partially

18


methylated alditol acetates show only minor differences making an assignment of the sugar configuration (gluco, galacto, etc.) impossible. However, the nature of the parent monosaccharides can be derived from the retention times of the alditol derivatives on GLC. The highly characteristic EI mass spectra of partially methylated alditol acetates have been studied in great detail.5,104,117–119,128–138 The relevant possibilities for the fragmentation of these derivatives are presented in Figure 15. Primary fragmentations are depicted in Figures 15a–15g. Primary fragments are formed by a-cleavage, resulting in fission between the C atoms in the alditol chain. In principle, either of the two fragments formed can carry the positive charge. In the case of alditols derived from neutral monosaccharides, the charge is preferentially located on an ether O atom instead of on an ester O atom. Taking this feature into account, the following rules can be formulated:5 (1) formation of ions of lower mass is preferred; (2) formation of ions from cleavage between two methoxylated C atoms is predominant, with no marked preference for one of the two possible cations (Figure 15a); (3) in the formation of ions from cleavage between a methoxylated and an acetoxylated C atom, there is a high preference for the methoxyl-bearing cations (Figure 15b); and (4) ions formed by cleavage between two acetoxylated C atoms are generally of low abundance (Figure 15c). For alditols derived from N,N-methyl,acetyl amino sugars, the same rules hold as mentioned above. However, the most preferable a-cleavage in partially methylated alditol acetates derived from 2-acetamido sugars stems from the scission between C2, bearing the N,N-methyl,acetyl amino group, and C3, bearing the methoxyl or acetoxyl function, with predominant localization of the charge at the amino fragment (Figures 15d and 15e). a-Cleavages adjacent to deoxygenated C atoms are only significant when the neighboring C atom bears a methoxyl group. The primary fragments give rise to secondary fragments, generally by single or successive eliminations of formaldehyde, methanol, ketene, acetic acid, methyl acetate, methoxymethyl acetate, or acetoxymethyl acetate (Figures 15h–15k). For an overview of the fragment ions of several partially methylated alditol acetates of neutral and amino sugars, see Table 4. Mass fragmentography has been shown to be of high value for the analysis of complex mixtures. It should be noted that the availability of reference spectra of authentic compounds recorded with the same GLC-EI/MS combination is useful. Typical examples of EI mass spectra, related to the protocol shown in Figure 14, are depicted in Figure 16. For further EI mass spectra, see the CCRC database.138a To determine the substitution patterns and ring sizes of uronic acid residues, it is necessary to reduce these units to the corresponding aldoses. The application of deuterated reducing agents leads to the incorporation of two D atoms at the carboxyl-group-derived primary hydroxyl functions. In this way, alditols derived from uronic acids can be distinguished by EI/MS from the corresponding native aldoses. For details of the reduction, see Section 2.01.5.1. When the glycan chain contains native methoxyl groups, the use of trideuteromethyl iodide (CD3I) is important to discriminate between originally present and chemically introduced methyl groups. In specific degradation procedures in which methylation analysis plays a role, both trideuteromethyl iodide and ethyl iodide can be applied. The location of a methoxyl function at C4 or C5 in aldoses defines the ring size of the monosaccharide residue. When acetoxyl groups are present at C4 and C5, 4-linked aldopyranosyl cannot be distinguished from 5-linked aldofuranosyl. To solve this problem, in the past specific degradation methods in combination with alkylation analysis have been reported;139 nowadays, the discrimination between both possibilities is mainly based on 1H NMR investigations. Since the use of methylsulfinylmethanide causes only marginal desulfation in the methylation procedure, the location of ester sulfate groups can be established by application of the methylation technique before and after desulfation. The same holds for acetals. For phosphate substituents it has to be taken into account that they may migrate under alkaline conditions via intramolecular cyclic esters. Because native O-acyl groups are cleaved during base treatment, other procedures should be followed to determine their positions, for example, applying a methylvinyl ether protocol;140 nowadays, in glycan analysis 1H NMR spectroscopy plays a major role in assigning native O-acyl groups. Finally, a series of notes in relation to the methylation analysis is presented:104 (1) an excess of methylsulfinylmethanide can be checked with triphenylmethane, whereby a red color is produced; (2) when de-N-acetylation of a methylated 2-acetamido-2-deoxyhexose unit occurs as the first step in the hydrolysis of permethylated material, the adjacent glycosidic linkage at C1 becomes resistant to acid hydrolysis; (3) hydrolysis of permethylated carbohydrate, containing a 2-acetamido-2-deoxyhexitol unit, can give rise to some demethylation of the alditol residue;137 (4) the quantitative aspects of methylation analysis procedures are poorly understood. Several factors influence the recovery of partially methylated alditol acetates, for example, undermethylation, incomplete hydrolysis, degradation, and demethylation during hydrolysis, incomplete reduction, incomplete acetylation, and contaminants. In general, molar ratios of the different monosaccharide derivatives are calculated on the basis of flame ionization detection


+

R1 H

OMe

H

OMe

(a)

R1

H

OMe

or

H

OAc

H

OAc

H

OAc

H

(c)

OAc

>>

R1 OAc

H

OAc

or

+

H

OMe

H

OMe

OAc

>

+

R1

H

NAcMe

H

H

OAc

H

OAc

NAcMe

H

OMe

>>

CH2OMe

H

NAcMe

H

OAc + R2

CH2OAc

45

OMe 117

249 HC

89

HC

HC

OMe 161

205 HC

OMe 133

261 HC

OMe

HC

OMe

161 HC

OMe

HC

OAc

HC

OAc

HC

OAc

HC

OAc

HC

CH2OAc

CH2OMe 290 HC

45

NAcMe130

205 HC

OMe

HC

OAc

HC

OMe

CH2OMe

Figure 15 (continued)

174

290

OMe

CH2OAc

CH2OAc

CH2OAc 219 HC

CH2 HC

OMe 131

HC

OAc

161 HC

HC

OAc

HC

CH2OAc

OMe 117

CH2OMe

CH2OAc

m/z 45

R1

R2

CH2OAc

+ OMe

R2

+ NAcMe

R2

m/z 43

Not characteristic for a primary OMe group, but can also be formed by migration of a H atom to any charged one-carbon fragment (f) bearing an OMe group

H

R2

R1

45

H

+ O

R1 NAcMe

89

OAc

C

Generally, the most intense ion, derived from the acetyl function by a-cleavage H 2C

H

R1

R2

H3 C

R2

H

189

OAc

R1

NAcMe

233

H R2

H

(e)

OMe

R2

R1

(d)

H

R2

H

R2

OMe

R1 OMe

+

H

R1 H

R1

OMe

R2

OMe

R2

H

R2

H

(b)

(g)

OMe

+

R1

R1

H

R2

19

161

OMe 117

CH2

45

OMe 175 OAc

CH2OMe

HC

NAcMe158

233 HC

OMe

HC

OAc

HC

OAc

45

202

CH2OMe

CH2OAc HC

OMe

117

175 HC

OMe

161

131 HC

OMe

HC

OAc

CH3

20


O CH2

CH2OMe

(h)

H

O +

m/z 89

O +

CH3

H2C AcO

−CH2O

H

OMe +

HC

CH3

OMe

CH2 −MeOH b

H

C

AcO

O

OMe +

HC

m/z 161

CH3 −CH2CO

C

OMe +

HC

m/z 59

m/z 129

H

CHOMe

O

C

C H

CHOMe −HOAc a

C

OMe +

HC

HC C

H OMe +

HC

m/z 161 O H2C MeO

(i)

C

C

H O

HC

OMe +

CH3

CH2 MeO

+ OMe

HC

OMe

HC

OAc

Elimination of MeOH is only observed when the methoxyl group is situated at the b-position OMe to the C atom having the formal charge. Elimination of HOAc is possible from b- and H a-positions, in the latter case provided that the OMe resulting ion is stabilized by resonance. When an acetoxyl group is linked to an unsaturated C atom, ketene is eliminated with the formation of a carbonyl group. CH2

−CH2O

C HC

m/z 161

HC

HC m/z 101

−HOAc b

OMe +

m/z 87

O H3C

C HC

C

OMe +

O + m/z 71

m/z 101

AcO

OMe

CH2OAc

+ OMe

HC

OAc

O +

Ac m/z 159 + OMe

HC

MeO

−CH3COOCH2OCH3

O HC

OMe

CH2OMe

MeO

O

+ OMe

HC

OAc

m/z 101 + OMe

HC

MeO

−CH3COOCH2OCOCH3

OMe

CH2OAc

(j)

(k)

AcO

O

HC

NAcMe +

+ OMe

CH HC

OMe

CH3

m/z 233

CH2OR

OMe

CH3

O HC

+ OMe

CH HC

m/z 205

HC

OMe

−CH3COOCH3

m/z 233

HC

Me

AcO

OMe

+ O

H

HC

m/z 101

−CH2CO

CH2OR HC

NHMe +

R = Ac : m/z 158 → m/z 116 R = Me : m/z 130 → m/z 88 Intense and highly characteristic peaks for assignments of amino sugar derivatives

Figure 15 (a–k) A selection of EI fragmentation patterns of partially methylated alditol acetates.5,104,117–119,128–138


21

Table 4 Primary fragment ions (EI/MS) characteristic for the substitution pattern of partially methylated alditol acetates (nonlabeled with D at C1)5,104,117–119,128–138 Position of OMe groups Pentitol 2(4) 3 5 2,3 (3,4) 2,4 2,5 3,5 2,3,4 2,3,5 Hexitol 2 (5) 3 (4) 6 2,3 2,4 (3,5) 2,5 2,6 3,4 3,6 4,6 5,6 2,3,4 2,3,5 2,3,6 2,4,6 2,5,6 3,4,6 3,5,6 1,3,4,6 2,3,4,6 2,3,5,6 1,2,3,4,5 1,2,3,5,6 1,2,4,5,6 1,3,4,5,6 2-Deoxy-2-(N-methyl) acetamidohexitol 3 4 6 3,4 3,6 4,6 1,3,5 1,4,5 3,4,6 1,3,5,6 1,3,4,5 1,4,5,6

m/z values 261, 117 189 45 189, 161, 117 233, 117 233, 117, 45 189, 161, 45 161, 117 161, 117, 45 333, 117 261, 189 45 261, 161, 117 305, 233, 189, 117 305, 117 305, 117, 45 233, 189 233, 189, 45 261, 161, 45 333, 89, 45 233, 189, 161, 117 233, 161, 117 277, 233, 161, 117, 45 277, 233, 161, 117, 45 117, 89, 45 233, 205, 189, 161, 45 305, 205, 189, 89, 45 205, 161, 45 205, 161, 117, 45 277, 205, 161, 117, 89, 45 177, 161, 133, 117, 89, 45 249, 205, 133, 89, 45 249, 205, 133, 89, 45 249, 205, 161, 133, 89, 45

261, 202, 158 274, 189, 158 158, 45 246, 233, 202, 189, 158 233, 202, 158, 45 274, 161, 158, 45 318, 290, 233, 174, 130, 117, 45 318, 290, 246, 161, 130, 117, 45 246, 205, 202, 161, 158, 45 290, 205, 174, 130, 89, 45 290, 218, 174, 161, 130, 117, 45 290, 246, 133, 130, 89, 45

Position of OMe groups 6-Deoxyhexitol 2 3 4 2,3 2,4 3,4 2,3,4 2,3,5 Heptitol 3 2,6 2,7 3,6 4,6 6,7 2,3,6 2,3,7 2,4,6 2,4,7 2,6,7 4,6,7 2,3,4,6 2,3,4,7 2,3,6,7 2,4,6,7 3,4,6,7 2,3,4,6,7

m/z values 275,117 203, 189 261, 131 203, 161, 117 247, 233, 131, 117 189, 131 175, 161, 131, 117 175, 161, 117, 59 333, 189 117 117, 45 189, 117 261, 233, 117 89, 45 305, 161, 117 305, 161, 117, 45 233, 117 233, 117, 45 377, 349, 117, 89, 45 261, 205, 89, 45 321, 277, 233, 161, 117 277, 233, 205, 161, 117, 45 277, 161, 117, 89, 45 349, 321, 233, 205, 117, 89, 45 205, 189, 89, 45 249, 205, 161, 117, 89, 45

22


102

100

118 %

129

101

59

145 161 162

87

71 75

130

113

146

205 206

174

0

(a) 50

100

150

200

250

300

350

250

300

350

300

350

102

100

118 %

87

129

99

162 130

59 71 75

189 159

113

173

233

0

(b) 50

100

150

200

129

100

161 101 %

128

86 87 100 59

74 71

262

160

112

143

188

202 218

0

(c) 50

100

150

200

250

118

100

% 99 85 87

102 127 142

111

59

159 162

261

187 201

0

(d) 50

100

150

200

250

305 300

350

300

m/z 350

129

100

87 %

128

86 71 74

100 99 101

189

130

190 159 160

202

150

200

262

0

(e) 50

100

250

Figure 16 EI mass spectra of: a, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-hexitol-1-d; b, 1,5,6-tri-O-acetyl-2,3,4-tri-Omethyl-hexitol-1-d; c, 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl-hexitol-1-d; d, 1,4,5,6-tetra-O-acetyl-2,3-di-O-methylhexitol-1-d; and e, 1,2,3,5,6-penta-O-acetyl-4-mono-O-methyl-D-glucitol-1-d.


23

(FID) responses, taking into account similar molar adjustment factors for the various partially methylated alditol acetates. A better approach is the use of molar response factors based on the effective-carbon-response theory.141 For amino sugar derivatives quite often lower FID responses have been observed, as compared to the neutral sugar derivatives.

2.01.5 Modification/Degradation Procedures for Polysaccharides When analyzing polysaccharide chains, the study of modified polysaccharides or fragments of the native/modified polysaccharides form important parts of the total strategies to construct the ultimate polysaccharide structures. Over the years several chemical/enzymatic modification and degradation procedures have been developed. Figure 17 gives an impression of a number of these methodologies, which will be discussed in more detail in this section. Modification/ degradation procedures are generally chosen on the basis of initial monosaccharide linkage analyses. Comprehensive reviews are available from the older literature.142–144

2.01.5.1 Uronic Acid Reduction In planning strategies for the analysis of uronic-acid-containing polysaccharides, it can be useful to reduce the carboxyl function of the uronic acid constituents. As indicated already in Section 2.01.4, uronic acid derivatives are not detected in the GLC-based methylation analysis, when using partially methylated alditol acetate protocols. This is due to the low volatility of these derivatives, having a nonesterified carboxyl function at the end of the chemical derivatization procedure. In order to circumvent these problems, over the years mainly two reduction protocols have become in use, which will be illustrated below. In the first approach (Figure 18a), the native uronic-acid-containing polysaccharide is converted into the corresponding neutral polysaccharide via activation of the carboxyl function(s) with a carbodiimide, for example, N1-cyclohexyl-N3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) or N1-ethyl-N3-(dimethylaminopropyl)carbodiimide (EDC) in water, and subsequent reduction with NaBH4 or NaBD4.145,146 Frequently, several consecutive treatments are needed to obtain complete reduction. The application of NaBD4 as reducing agent leads to the incorporation of two D atoms at the carboxyl-group-derived primary hydroxyl function(s). In this way, in the

Uronic acid degradation Lyase

OH

Partial acid hydrolysis O

O HO

O

OH OH

O NH

O

O HO OH

O

O HO

Acetolysis OH O

CH3

O

De-N-acetylation − deamination

HO HO

OH OH

Periodate oxidation Reducing end Figure 17 Impression of a number of chemical/enzymatic degradation approaches.

24


COOH

O

O HO

CD2OH

Carbodiimide NaBD4

O

CD2OMe O

O

O HO

O

OH

O MeO

Permethylation

O

OH

OMe O R

C

D

R1 N d−

O R

C d+

C OH

(a)

Acid

COOH

R

C

NH R1 O

R

Nd −

Activated ester

R2

O MeO

Permethylation

OH

CD2OH

R2 Alcohol

COOMe O O

NaBD4

C N

O

O HO (b)

O

CD2OH

LiAlD4 O

OMe

Ether or THF

O

O MeO

O OMe

Figure 18 Carbodiimide-activated reduction of a uronic-acid-containing polysaccharide (a), and reduction of a permethylated uronic acid-containing polysaccharide (b).

methylation analysis, alditols derived from alduronic acids can be distinguished by EI/MS from alditols derived from the corresponding native aldoses. The pseudonative polysaccharide can be analyzed in parallel with the native polysaccharide. In the second approach (Figure 18b), directly related with the methylation analysis protocols, the uronic-acidcontaining polysaccharide is permethylated, then the methyl ester groups are reduced with LiAlH4 or LiAlD4 in diethyl ether or tetrahydrofuran,147 with NaBH4 or NaBD4 in 95% ethanol-oxolane (27:73, v/v),119 or with Li(Et3)BH or Li(Et3)BD in tetrahydrofuran.148,149 Again the deuterium-labeled reducing agent is used to mark the monosaccharide, derived from the corresponding uronic acid. In case small oligosaccharides have to be reduced, NaBH4 or NaBD4 in p-dioxane-ethanol is usable. An application of both approaches can be found in the study on the structural analysis of the Klebsiella type 57 CPS.150 This polysaccharide will also be used in some of the following sections to illustrate different degradation procedures. [→ 3)-a -D-GalpA-(1→ 2)-a -D-Manp-(1→ 3)-b -D-Galp-(1→ ]n

a -D-Manp-(1→ 4)

2.01.5.2 Partial Acid Hydrolysis In principle, partial acid hydrolysis of polysaccharide chains generates mixtures of oligosaccharides that can be fractionated by size-exclusion chromatography and different forms of HPLC. The identification of the oligosaccharides follows from monosaccharide analysis, linkage analysis, different forms of mass spectrometry (see Chapter 2.02), and different forms of NMR spectroscopy (see Chapter 2.03). Nowadays, partial acid hydrolysis is mainly used in the analysis of bacterial and plant polysaccharides. In the case of bacterial polysaccharides with repeating units, overlapping fragments can be created. Often the rate of hydrolysis of the various glycosidic linkages does not differ significantly, so that complex mixtures are obtained. In several cases, however, attractive differences between the stability/lability of glycosidic linkages do occur. A typical example comprises the partial acid hydrolysis of the EPS of Lactococcus lactis subspecies cremoris H414, having the structure [→ 4)-b -D-Galp-(1→ 3)-b -D-Galp-(1→ 4)-a -D-Galp-(1→ ]n .

b -D-Galp-(1→ 3)-b -D-Galp-(1→ 3)


25

The methodologies to establish the complete structure of this EPS included monosaccharide analysis with D/L determination, methylation analysis, Smith degradation, partial acid hydrolysis, MS, and 1H NMR spectroscopy.151 In the partial acid hydrolysis, a series of eight different linear and branched fragments were isolated, varying from tri- to heptasaccharides (hydrolysis conditions: 0.5 M trifluoroacetic acid, 24 h, 60 C), illustrating the absence of a strong specificity. Partial acid hydrolysis of polysaccharides, containing uronic acids, yields, due to the relative stability of HexA (a/b1-x) linkages, oligosaccharide fragments with HexA as the nonreducing unit. The increased stability of the aldobiuronic acids to acid hydrolysis is still not well understood. When applied to the CPS of Klebsiella type 57, of which the structure [→ 3)-a -D-GalpA-(1→ 2)-a -D-Manp-(1→ 3)-b -D-Galp-(1→ ]n

a -D-Manp-(1→ 4)

was elucidated by using monosaccharide analysis including D/L determination, linkage analysis, uronic acid degradation, partial acid hydrolysis, modified Smith degradation of carboxyl-reduced CPS, MS, and 1H NMR spectroscopy,150 two oligosaccharides were isolated, namely, a-D-GalpA-(1!2)-D-Manp and a-D-GalpA-(1!2)-a-D-Manp-(1!3)-DGalp (hydrolysis conditions: 0.25 M sulfuric acid, 8 h, 100 C). When applied to the CPS of Streptococcus pneumoniae serotype 3, having the structure [!3)-b-D-GlcpA-(1!4)-b-D-Glcp-(1!]n a series of fragments containing one to seven [!3)-b-D-GlcpA-(1!4)-b-D-Glcp-(1!] repeating units could be isolated (hydrolysis conditions: 0.6 M trifluoroacetic acid, 3 h, 100 C).152 Partial acid hydrolysis of polysaccharides, containing 2-amino sugars, yields, due to the relative stability of HexN (a/b1-x) linkages, oligosaccharide fragments with HexN as the nonreducing unit. The stability of the glycosidic linkage is due to the inductive effect of the positive-charged nitrogen function formed in acid solution. In most polysaccharides the amino function is N-acylated; to take advantage of the stability of HexN(a/b1-x) linkages, the polysaccharide should be de-N-acetylated (see Section 2.01.5.7). Typical examples wherein use can be made of relatively labile glycosidic linkages in the partial acid hydrolysis are polysaccharides containing 6-deoxyhexoses (e.g., Rha), 2-deoxyaldoses, 3,6-dideoxyhexoses (e.g., Abe), 2-keto sugars (e.g., Fru, Sia, and Kdo), and aldofuranoses (e.g., Araf, Ribf, and Galf ). In this context, it is interesting to note that the polysaccharide component of an LPS can be separated from the lipid component by selective hydrolysis of the acid labile glycosidic linkages of Kdo residues (0.1M acetic acid, 90min, 100 C).153 For the release of sialic acids from glycoconjugates, relatively mild acidic conditions can also be applied, for example, 2M propionic acid (4h, 80 C).86,87 An example associated with Rha is the EPS of Lactobacillus sake 0–1, of which the structure b -D-Glcp-(1→ 6)

Ac0.85→ 2)

[→ 4)-b -D-Glcp-(1→4 )-a -D-Glcp-(1→ 3)-b -L-Rhap-(1→ ]n sn-glycerol-(3→ P → 4)-a -L-Rhap-(1→ 3)

has been elucidated by using monosaccharide analysis including D/L determination, methylation analysis, partial acid hydrolysis, de-O-acetylation, deglycerophosphorylation, and 1-D/2-D NMR (1H, 13C, and 31P) studies.154 In the context of these studies, partial acid hydrolysis (0.3M trifluoroacetic acid, 2h, 100 C) yielded besides glycerol, 1-phosphoglycerol, Rha, and Glc, the tetrasaccharide b-D-Glcp-(1!4)[b-D-Glcp-(1!6)]-a-D-Glcp-(1!3)-L-Rhap. Several cases of solvolysis of polysaccharides with 48% HF have been reported in the literature. A typical example is the degradation of the CPS of Neisseria meningitidis serogroup H, having the structure [!4)-a-D-Galp-(1!2)-Glycerol-(3!P!]n. Incubation of the polysaccharide with 48% HF (48h, 4 C) yielded a-D-Galp-(1!2)-glycerol.155 Using 48% HF (4 days, –16 C), the CPS of S. pneumoniae serotype 6B, having the structure [!2)-a-D-Galp-(1!3)-a-D-Glcp-(1!3)-a-L-Rhap-(1!4)-D-Rib-ol-(5!P!]n yielded an octasaccharide built up from two repeating units: a-D-Galp-(1!3)-a-D-Glcp-(1!3)-a-L-Rhap-(1!4)-DRib-ol-(5!P!2)-a-D-Galp-(1!3)-a-D-Glcp-(1!3)-a-L-Rhap-(1!4)-D-Rib-ol.156 When carrying out the reaction for 48h at 4 C, besides the octasaccharide, the nonphosphorylated repeating unit, a-D-Galp-(1!3)-a-D-Glcp-(1!3)-a-LRhap-(1!4)-D-Rib-ol, was obtained.

26


2.01.5.3 Partial Alkaline Hydrolysis Alkaline conditions are very useful in case of the presence of phosphate groups. Incubation of the CPS of S. pneumoniae serotype 6B (for structure, see Section 2.01.5.2) with 10 mM NaOH (72 h, 85 C) yielded the phosphorylated repeating unit: a-D-Galp2P-(1!3)-a-D-Glcp-(1!3)-a-L-Rhap-(1!4)-D-Rib-ol.156

2.01.5.4 Acetolysis Acetolysis is generally carried out in a mixture of acetic anhydride, acetic acid, and sulfuric acid.157 Under such conditions (1-6)-linkages between pyranosidic ring forms are preferably cleaved. Typical examples have been reported for dextrans and for yeast mannans,142 as is shown for the acetolysis of a highly branched mannan → 6)-a -D-Manp-(1→ 6)-a -D-Manp-(1→ 2 2 1 a -D-Manp

1 a -D-Manp 2 1 a -D-Manp

yielding peracetylated Man(a1-2)Man and Man(a1-2)Man(a1-2)Man, and after de-O-acetylation the free oligosaccharides. A mechanism has been proposed, whereby the cleavage of the glycosidic bond in the peracetylated carbohydrate chain is initiated by the reaction of an acetyl cation with the ring O atom, leading to ring opening.

2.01.5.5 Uronic Acid Degradation Over the years, in the structural analysis of carbohydrate chains use has been made of chemical degradations based upon b-elimination reactions.142,143,158 On treatment with base several kinds of groups in b-position to an electronwithdrawing group, such as a carbonyl, a carboxylic ester, an amide, or a sulfone group, are eliminated. In these reactions the presence of an H atom in a-position to these groups is essential. The alkaline degradation of reducing sugars, which also includes epimerization reactions at C2, and the alkaline lability of Hex(1-3)Hex bonds at the reducing end form classical examples of this type of reactions. In the context of this overview, only two degradations based upon b-elimination will be worked out, as they are very important in carbohydrate analysis strategies: (1) the uronic acid degradation of permethylated uronic-acid-containing polysaccharides, and (2) the alkaline borohydride treatment of O-glycoproteins. The latter methodology will be discussed in Section 2.01.6.2. During the permethylation of uronic-acid-containing polysaccharides, the carboxyl function of the uronic acids is esterified, and an electron-withdrawing group is created. As is illustrated in Figure 19, treatment of such a polymer with base (sodium methoxide in methanol; sodium or potassium methylsulfinylmethanide in dimethyl sulfoxide) leads to the elimination of the substituent at C4. It should be noted that elimination does not occur during the permethylation step, presumably because the strong base is rapidly decomposed when excess methyl iodide is added. The needed anhydrous conditions are created by the addition of 2,2-dimethoxypropane and a catalytic amount of p-toluenesulfonic acid. After the base treatment, a mild acid hydrolysis step is introduced to cleave the glycosidic bond at C1, as well as to eliminate the substituents at C2 and C3.159 The necessity of the mild acid treatment as a follow-up of the alkaline treatment to realize the additional cleavages has been discussed several times.160,161 As shown in Figure 20, alkaline treatment of the permethylated CPS of Klebsiella type 57 (see the following structure)150 yielded partially methylated a-D-Manp-(1!3)-D-Galp. In fact, the free OH group at C2 of the Manp residue reflects the (1-2)-linkage between GalpA and Manp, whereas the reducing Gal unit reflects the connection with GalpA. [→ 3)-a -D-GalpA-(1→ 2)-a -D-Manp-(1→ 3)-b -D-Galp-(1→ ]n

a -D-Manp-(1→ 4)

A second example is the uronic acid degradation applied to the EPS of Lactobacillus acidophilus LMG9433, having the structure


COOMe

COOMe

COOMe O OR1

−

Base

H OR3

O OR1

O OR1 + R4OH

OR3

OR3

R4O

27

R4O OR2

OR2

OR2 COOMe O OR1 Mild acid

R1OH + R2OH + R3OH + Degradation products

OR3

OR2 Figure 19 Simplified reaction mechanism of the uronic-acid degradation of permethylated uronic-acid-containing glycans.

CH2OMe

CH2OMe CH2OMe

O OMe

COOMe O OMe MeO

O

O

CH2OMe O

O

OH

MeO

MeO

O

MeO

OMe

COOMe

O

MeO

O O

MeO

O Base

CH2OMe

CH2OMe

OMe

MeO

O O

MeO

O O

O

OMe

O

OMe

OMe

CH2OMe MeO

O

CH2OMe Base/acid

OH

O OMe OH

O

OMe

MeO

Figure 20 Schematic presentation of the uronic-acid degradation of the permethylated CPS of Klebsiella type 57.

b -D-GlcpNAc-(1→ 3) [→ 4)-b -D-GlcpA-(1→ 6)-a -D-Glcp-(1→ 4)-b -D-Galp-(1→ 4)-b -D-Glcp-(1→ ]n .

The methodologies to establish the complete structure of the EPS comprised monosaccharide analysis including D/L determination, methylation analysis, uronic acid degradation, de-N-acetylation – deamination, partial acid hydrolysis,

and 1-D/2-D 1H and 13C NMR spectroscopy.162 In the context of these studies, following the uronic acid degradation protocol, permethylation (CH3I) of the EPS, followed by base treatment and subsequent remethylation with CD3I yielded permethylated b-D-GlcpNAc-(1!3)[a-D-Glcp6OCD3-(1!4)]-b-D-Galp-(1!4)-D-Glcp-(1!OCD3.

28


2.01.5.6 Periodate Degradation/Smith Degradation One of the most classical chemical degradation procedures in glycoscience is the periodate oxidation.142,143 In the early days mainly the periodate consumption and the quantities of the small molecules generated by the oxidation, such as formic acid and formaldehyde, were determined, and the data extrapolated to propose native carbohydrate structures. Nowadays, having available the MS and NMR methodologies, the products that remain after the periodate oxidation are analyzed, and used in establishing native glycan structures. Periodate cleaves selectively C–C linkages in 1,2-diols, a-hydroxy-aldehydes, a-hydroxy-ketones, diketones, and a-hydroxy-amines. When used in polysaccharide analysis, it is very important that the oxidation of the glycol groupings is complete, and that no overoxidation takes place. The latter can be excluded when the reactions are carried out at 4 C in the dark, using sodium metaperiodate at pH 4. The reaction mechanism comprises the formation of cyclic intermediates, yielding finally dialdehydes. This means that in pyranose ring forms only complexes can be formed for vicinal OH groups in axial–equatorial and equatorial–equatorial orientations, although in the latter case the formation occurs more slowly. In rigid axial–axial orientations the complex formation cannot take place. Similar rules hold also for furanose ring forms. Generally, the oxidation is directly followed by a reduction with NaBH4, also in order to inhibit the formation of inter-residue hemiacetals. The most applied methodology is the Smith degradation, involving periodate oxidation followed by reduction of the oxidized material with NaBH4, and subsequent mild acid hydrolysis.163 In this reaction sequence, before reduction the excess of periodate is destroyed by ethylene glycol; in the mild acid hydrolysis step, generated acyclic acetal groupings are much faster hydrolyzed than native glycosidic linkages. The final carbohydrate products contain small fragments, such as C2–C4 polyols, glycolaldehyde, and glyceraldehyde, derived from the oxidized residues. These aglycones provide structural information concerning the monosaccharide residues from which they are derived, for example, a 2-substituted tetritol stems from a 4-substituted hexopyranose, a 2-substituted glycerol from a 4-substituted pentopyranose, a 1-substituted glycerol from a 6-substituted hexopyranose or a 5-substituted pentofuranose, and a 2-substituted glyceraldehyde from a 2-substituted hexo- or pentopyranose. In the following a few examples of periodate oxidation protocols will be presented, including information on the formation of byproducts, due to acetal migration. Figure 21 depicts the Smith degradation of the carboxyl-reduced CPS of Klebsiella type 57. It includes periodate oxidation and subsequent reduction with NaBH4, followed by mild acid hydrolysis, and reduction with NaBH4, yielding a product that can be analyzed by linkage analysis, MS, and NMR spectroscopy. However, in order to exclude possible acetal migration during the mild hydrolysis step, in the original paper,150 a modified Smith procedure was applied (Figure 22). Here, the oxidized (0.2M metaperiodate/0.1M sodium acetate buffer, pH 3.9; 120h, 40 C) and reduced (NaBH4) biopolymer was permethylated with CH3I, followed by mild acid hydrolysis, reduction with NaBD4 (in p-dioxane-ethanol), and remethylation with CD3I, yielding a product that has been identified by linkage analysis, MS, and NMR spectroscopy. In both cases, the partial structure b-D-Galp-(1!3)-a-D-Galp-(1!2)-glycerol is generated, contributing to the elucidation of the final structure of the native polysaccharide. [→ 3)-a -D-GalpA-(1→ 2)-a -D-Manp-(1→ 3)-b -D-Galp-(1→ ]n

a -D-Manp-(1→ 4)

Note that the methylated product in Figure 22 reflects original substitutions at O3 of Galp and O4 of GalpA-derived Galp (presence of OCD3 groups); the deuterated glycerol unit reflects the original 2-substituted Manp. A second example deals with the Smith degradation of the EPS of L. lactis subspecies cremoris H414 (for the structure, see below).151 Periodate oxidation followed by reduction with NaBH4, mild acid hydrolysis, and finally NaBD4 reduction will give b-D-Galp-(1!3)[b-D-Galp-(1!4)]-a-D-Galp-(1!2)-threitol, that could be identified by monosaccharide analysis, methylation analysis, MS, and NMR spectroscopy. However, besides this product, during the mild acid hydrolysis step three side-products were formed: two cyclic O-(2-hydroxyethylidene) derivatives of the tetrasaccharide-alditol (acetal migration) and the trisaccharide-alditol b-D-Galp-(1!3)[b-D-Galp-(1!4)]-D-Gal-ol-1-d (glycosidic linkage hydrolysis) (Figure 23). [→ 4)-b -D-Galp-(1→ 3)-b -D-Galp-(1→ 4)-a -D-Galp-(1→ ]n

b -D-Galp-(1→ 3)-b -D-Galp-(1→ 3)

A third example focuses on the Smith degradation of the EPS of Streptococcus thermophilus S3, having the structure b -D-Galf 2Ac0.4-(1→ 6) [→ 3)-b -D-Galp-(1→ 3)-a -D-Galp-(1→ 3)-a -L-Rhap-(1→ 2)-a -L-Rhap-(1→ 2)-a -D-Galp-(1→ ]n .

The methodologies to establish the structure comprised monosaccharide analysis including D/L determination, linkage analysis, 1-D/2-D 1H and 13C NMR studies on the native and de-O-acetylated material, together


CH2OH

CH2OH CH O CH2OH CH O

O CH2OH

OH

CH2OH

O

O

OH HO

O

CH O

i, NaIO4 ii, NaBH4

HO

CH2OH

CH OH

CH2OH

OO

OH

OO O

O

O

CH O

CH2OH

O

HO

CH2OH

O

HOCH2

O

CH2OH

CH2OH

HO

HO

29

Mild acid

OH

OH CH2OH HO

O

HO

CH O

CH2OH

CH O

CH2OH

OH

CH2OH

O

CH2OH

NaBH4

OO

HO

CH2OH

CH2OH

OH

C H

O

OH

OO

HO OH

OH

OH

Figure 21 Schematic presentation of the Smith degradation of the carboxyl-reduced CPS of Klebsiella type 57.

with nanoES-CID tandem MS studies on oligosaccharides generated by a modified Smith degradation protocol.164 Figure 24 depicts the structure of the tetrasaccharide-alditol obtained after periodate oxidation of the EPS, and subsequent reduction with NaBH4, methylation with CH3I, mild acid hydrolysis, reduction with NaBD4, and finally perdeuteromethylation with CD3I. Note that the native Galf residue is converted into an Araf residue, demonstrating the resistance of the trans-oriented OH groups in the furanose ring form of Gal during the oxidation; only the side chain had been oxidized. The deuterated methyl group at O3 of the b-Gal residue reflects the original hexosyl substitution. It should be noted that in theory a pentasaccharide-alditol should be expected (included in the figure), but as Rha(1-x) linkages are relatively labile to acid treatment, the residual glyceraldehyde is released during the mild acid hydrolysis, yielding Rha as free reducing unit. Besides the main product several other products were detected, that is, oligosaccharides missing Araf (corresponding with Galf ). A fourth example shows the use of the Smith degradation in the analysis of complex glycoprotein N-glycans of the aD-hemocyanin of Helix pomatia.65 These N-glycans contain, among others, partially methylated antennas having the structure Gal3Me(b 1- 6) Gal3Me(b 1-6)Gal3Me(b 1-3)GalNAc(b 1-4)GlcNAc(b 1-2)Man(a 1- .

The total N-glycosylation pattern analysis included studies on released oligosaccharides (see Section 2.01.6.3) using monosaccharide analysis, linkage analysis, Smith degradation, FAB-MS, and 1H NMR spectroscopy. Subjection of a pronase digest of the glycoprotein to periodate oxidation, followed by reduction with NaBH4, mild acid hydrolysis, and reduction with NaBH4, yielded two major oligosaccharides that turned out to be Gal3Me(b 1-6) Gal3Me(b 1-6)Gal3Me(b 1-3)GalNAc(b 1-4)GlcNAc(b 1-2)Gro

and the product containing glyceraldehyde 30 -hydroxypropylene diacetal instead of glycerol (Gro).

30

CH2OMe CH

O CH2OH

O

CH

O CH

O

i, NaIO4 ii, NaBH4 iii, CH3I/base

OH

CH2OMe CH

CH2OMe

O

O

CH2OMe

O

O

HO

HO

O

MeOCH2

O

CH2OH

CH

HO

O OH HO

CH2OMe

OH

CH2OH

O

CH2OMe

O

OMe

MeO

O O

O

OMe

CH2OMe

O O Mild acid

OH CH2OMe CD3O

O

MeO

O O OCD3

HO

CH O

CH2OMe

CH2OMe

CH2OMe

OMe

CH2OMe

O CH

CHDOCD3

O CH2OMe i, NaBD4 ii, CD3I

O O

MeO OH

OMe Figure 22 Schematic presentation of a modified Smith degradation of the carboxyl-reduced CPS of Klebsiella type 57.

OMe

OMe

C H

O


CH2OH


CH2OH

CH2OH

O

R1O

31

O

R1O CH2OH

OR2

O

ii, NaBH4

OH

OH

CH

OR2

i, NaIO4

O O

O

CH2OH

OH

O CH O

CH HOH2C

CH2OH

iii, Mild acid hydrolysis OH

CH2OH

CH2OH R1O

CH2OH

O

R1O

CH

OR2

OH

OR2

OH

CH

O OH

O

OH

HOH2C

CH2 O

CH2OH O

R1O

CH

OR2

CH2OH R1O

CH

O

CH2OH

O

CH

OR2 O OH

CH H2C

OH

O CH

CH2OH

CH2OH

O

CH HOH2C

3,4-O-(2-hydroxyethylidene) derivative

O

1,3-O-(2-hydroxyethylidene) derivative

Figure 23 Formation of side-products in the Smith degradation of the EPS of L. lactis subspecies cremoris H414.

O

O OMe

O

O OMe CH3

CH2

MeOCH2 OMe

MeO

O

CH2OMe

OMe

MeO

O

MeO

CH2

MeOCH2 OCD3

O

O CHDOCD3

CH3

OMe CH2OMe MeO

O OCD3

CH2OMe

O

O MeO

MeO

MeO CHDOCD3

OMe

O

O

O

OMe

MeO

OCD3

OMe

Figure 24 Structures of the theoretically expected pentasaccharide-alditol and the isolated tetrasaccharide-alditol, after periodate oxidation of the EPS of S. thermophilus S3.

2.01.5.7 De-N-acetylation – Deamination One of the classical organic chemical reactions is the conversion of primary amines into primary diazonium salts by using aqueous nitrous acid (diazotization). The subsequent release of N2 yields final products that are quite varied, being dependent upon the nature of the organic substituent attached to the amino group. In carbohydrate chemistry, the reaction of nitrous acid with 2-amino-2-deoxyhexoses has been broadly reviewed.142,143,165,166 Deamination of 2-amino-2-deoxyhexopyranosides with the amino group in equatorial position in the most stable chair conformation

32


yields 2,5-anhydrohexose residues. In this way, 2-amino-2-deoxy-D-glucopyranosides and 2-amino-2-deoxy-D-galactopyranosides are converted into 2,5-anhydro-D-mannose and 2,5-anhydro-D-talose, respectively. The rearrangement involves the attack of O5 upon the intermediate diazonium ion, release of N2 and cleavage of the C1-O5 bond, yielding a resonance-stabilized C1 carbocation. By reaction with water, the latter intermediate is converted into an aldehyde function, thereby releasing the aglycone (Figure 25a). Besides this major pathway, in the case of 2-amino2-deoxyglucopyranoside a minor pathway has been observed, involving an attack of C4 upon the intermediate diazonium ion, yielding a 2-deoxy-2-C-formyl-D-ribo-(and D-arabino-)furanosyl unit (Figure 25b). An eventual substituent at C3 will be released during this rearrangement. Note that both O5 and C4 occur in a trans- and antiparallel orientation to the Nþ 2 function. The deamination of 2-amino-2-deoxyhexopyranosides with the amino group in axial position in the most stable chair conformation is more complex. Starting from a 2-amino-2-deoxyD-mannopyranosyl unit in an oligosaccharide, besides complex rearrangement products, a D-glucopyranosyl unit has been detected. As 2-amino-2-deoxyaldoses mainly occur as N-acetylated derivatives in polysaccharides, to incorporate the deamination reaction in structural analysis strategies, de-N-acetylation has to precede deamination. N-Sulfated amino sugar residues can be deaminated without desulfation. Several de-N-acetylation protocols are available, for example, treatment with anhydrous hydrazine-hydrazine sulfate, or NaOH in aqueous dimethyl sulfoxide with sodium thiophenolate as catalyst/oxygen scavenger.167 In view of the formed aldehydes after deamination, usually a reduction with NaBH4 or NaBD4 is included. In a first example, the use of the de-N-acetylation – deamination protocol will be illustrated for the debranching of a branched EPS from Streptococcus macedonicus Sc136.168 The structure of the EPS was determined by monosaccharide analysis including D/L determination, methylation analysis, mild acid hydrolysis, de-N-acetylation – deamination, and 1-D/2-D 1H and 13C NMR spectroscopy, showing the repeating unit to be b -D-Galf-(1→ 6)-b -D-Glcp-(1→ 6)-b -D-GlcpNAc-(1→ 3) [→ 4)-a -D-Glcp-(1→ 4)-b -D-Galp-(1→ 4)-b -D-Glcp-(1→ ]n .

In the context of these studies, de-N-acetylation, carried out with anhydrous hydrazine containing hydrazine sulfate, and subsequent deamination, carried out with NaNO2 in aqueous acetic acid, yielded a debranched polysaccharide with the structure [!4)-a-D-Glcp-(1!4)-b-D-Galp-(1!4)-b-D-Glcp-(1!]n and (after reduction) the modified side-chain trisaccharide b-D-Galf-(1!6)-b-D-Glcp-(1!6)-2,5-anhydro-D-Man-ol. The additional finding of b-D-Glcp-(1!6)-2,5-anhydro-D-Man-ol was due to the acid lability of the Galf-Glc linkage. The second example shows the use of the de-N-acetylation – deamination protocol in generating a repeating unit from the EPS of S. thermophilus 8S,169 having the structure [!4)-a-D-Galp-(1!2)–b-D-Ribf-(1!4)-b-D-Galp-(1!4)-b-D-Glcp-(1!7’)-Sug-(1!4)-b-D-GalpNAc-(1!]n in which Sug is 6-O-(30 ,90 -dideoxy-D-threo-D-altro-nononic acid-20 -yl)-a-D-Glcp. The structural analysis involved monosaccharide analysis including D/L determination, methylation analysis, de-Nacetylation – deamination, MS, 1-D/2-D 1H and 13C NMR spectroscopy, and molecular modeling. In the context of this study, the de-N-acetylation – deamination (anhydrous hydrazine containing hydrazine sulfate) – borodeuteride reduction protocol yielded the modified repeating unit a-D-Galp-(1!2)-b-D-Ribf-(1!4)-b-D-Galp-(1!4)-b-D-Glcp-(1!7’)-Sug-(1!4)-2,5-anhydro-D-Tal-ol-1-d. Figure 26 shows a schematic picture of the degradation reactions, whereas Figure 27 presents the positive-ion mode nanoES-CID tandem mass spectrum of the pseudomolecular ion of the oligosaccharide-alditol.

2.01.5.8 R/S Configuration Determination of Pyruvic Acid Acetals Pyruvic acid, linked as a cyclic acetal to a monosaccharide constituent, occurs frequently in polysaccharides, but it has also been detected in glycoconjugate glycan chains. Typical examples are 4,6-pyruvated D-Glc, D-Man, and D-Gal, 3,4-pyruvated D-Gal and L-Rha, and 2,3-pyruvated D-Gal and D-GlcA. The pyruvic acid acetal carbon C2 is chiral,

OH OH HO

OH O

HO

HNO2 OR1

R3O

O

+

OH

N2

1 HC OR1 +

HO

HO

+ OR1

O R3O

NaBH4

_ H+

C

O

HO

CH2OH

HO H

H H (a)

OH OH HO R3O

OR1 N + 2

O

10−20%

O

OH OR1 1

_N

HO

4

(b) Figure 25 (a, b) Reaction mechanisms of deamination reactions.

3 HC +

OH OR1

O

O

NaBH4

OR1

_H+

2

2

+ H2O _R OH 3

OR3

HO

C H

O

HO

CH2OH


H

O+

3

OH O R3O

_R OH 1 CH

OR1

4

+H2O

2

OH O R3O

CH

2

HO

OH O R3O

HO

_N

OR1

R3O

NH2

O R3O

80−90%

33

34


OR1

OR1 O

HO HO

O

HO HO

OH O

OH O

Anhydrous hydrazine hydrazine sulfate

OH O O

HO

NaNO2 / HOAc

O O

HO

OH

NHAc

OH

NH2

O

HO

OH O

HO OH

OH OR2

OR2

OR1 OR1 −N2

HO HO

O OH O

OH O HO

HO HO

HC ⊕

OH O

H2 O O

OH O HO

O

C

O

H

OH O

OH

HO

OH O

OH OR2

HO OH OR2

Figure 26 Schematic presentation of the de-N-acetylation – deamination of the EPS of S. thermophilus 8S.

and both the R- and S-configuration have been found in pyruvated monosaccharides, for example, 4,6-O-[(R)1-carboxyethylidene]-a-D-galactopyranose and 4,6-O-[(S)-1-carboxyethylidene]-b-D-mannopyranose. As is evident from Figure 28, in both examples the methyl group occupies the equatorial position. The R/S-configuration of pyruvates can be determined by 1H and 13C NMR spectroscopy.170 In the case of 4,6-Opyruvated D-Glc residues, the R-configuration corresponds with a 1H CH3 signal at d 1.65–1.68, and the S-configuration with a 1H CH3 signal at d 1.48–1.50. For 4,6-O-pyruvated D-Gal residues the values for the R- and S-configuration are d 1.46–1.52 and d 1.66, respectively. Note that in fact the range d 1.46–1.52 corresponds with an equatorial orientation of the methyl group, and the range d 1.65–1.68 with an axial orientation. The 13C values of the methyl groups are even more pronounced: d18 for axial methyl groups (R in D-Glc and S in D-Gal), and d 26–27 for equatorial methyl groups (S in D-Glc and D-Man, and R in D-Gal). In the case of 3,4-O-pyruvated D-Gal residues the situation is more complicated. In their reduced form (3,4-O-hydroxyisopropylidene group), significant differences have been observed for the R- (dCH3 1.30; dCH3 21.8) and S- (dCH3 1.42–1.46; dCH3 23.5–24.3) configuration; for naturally occurring 3,4-O-pyruvated D-Gal (methyl group in endo-orientation) only data corresponding with the S-form are known: dCH3 1.41–1.60; dCH3 24.6–25.5. NMR data of synthetic model compounds have been reported;171,172 included references on several polysaccharide studies give insight into the natural stereochemical orientations. A typical study of an extracellular polysaccharide, containing two pyruvated hexoses, is a further example of such analyses.173

2.01.5.9 Enzymatic Degradation Procedures When available, polysaccharides can be converted very efficiently into oligosaccharide mixtures by endo-glycanases (hydrolases) or lyases. In subsequent steps, the generated mixtures are fractionated and investigated by monosaccharide analysis, methylation analysis, MS, and NMR spectrosocopy. Especially in the field of plant polysaccharides, a plethora of endo-glycanases are known: families of xylanases, amylases, cellulases, pectinases, polygalacturonases, arabinanases, galactanases, pullulanases, etc. In many structural studies of plant polysaccharides, these hydrolytic enzymes have shown to be of great value; the identified fragments, derived from these incubations, are essential in the creation of structural models. But also lyases play firm roles in


Y5 = 1042

Y3 = 748

Y2 = 586

CH2OH O

CH2OH O HO OH

Y4 = 910

O

O OH

CH2OH O O

O

OH

O

OH

CH3 HO

OH

OH HO HO HO

OH

CH2OH

B 3 = 479

B 4 = 641

CH2

O

CH2

CO2H

586

100

35

HO

O

HOH2C O HO O

OH

CHDOH

OH

641 B 5 = 877

B 6 = 1039

% +

[M+Na] 1204 877

479

748

421

910 1039

1042

0 200

300

400

500

600

700

800

900

1000

1100

1200

1300 m /z

Figure 27 Positive-ion mode nanoES-CID tandem mass spectrum of the pseudomolecular ion (m/z 1204) of the oligosaccharide-alditol, obtained by de-N-acetylation – deamination of the EPS of S. thermophilus 8S. Faber, E. J.; van Haaster, D. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Eur. J. Biochem. 2002, 269, 5590–5598.

CH3 HOOC

O

COOH

O

O

H3C

O

O

HO

HO

H3C

O

OH O

OH O

O

HOOC

OH

O OH O

O 4,6-O -[(S)-1-carboxyethylidene]-b-D-mannopyranosyl 4,6-O-[(R)-1-carboxyethylidene]-a-D-galactopyranosyl

3,4-O -[(S)-1- carboxyethylidene]-a-D-galactopyranosyl

Figure 28 Examples of pyruvated monosaccharide residues.

creating oligosaccharide mixtures. Typical examples are pectate lyase that cleaves R1-4)GalpA(b1-4)GalpA(b1-R2 linkages in pectins, thereby generating R1-4)GalpA and D4,5HexpA(a1-R2 fragments, and chondroitinase or heparinase that cleave R1-x)HexpNAc(b1-4)GlcpA(b1-R2 linkages in chondroitin sulfate or heparin, respectively, thereby generating R1-x)HexpNAc and D4,5HexpA(a1-R2 fragments (D4,5HexpA¼4-deoxy-L-threo-hex-4-enopyranuronic acid). Furthermore, bacteriophage-associated endoglycanases and lyases have been used in the structural analysis of bacterial polysaccharides from different strains.174 The hydrolytic or lyatic (at HexA; see above) cleavage leads to a depolymerization of the polysaccharides into repeating units. Unfortunately, so far the availability of this type of enzymes is very limited, but that is mainly due to the lack of investigations.

36


2.01.6 Generation of Glycopeptides and Glycoprotein Glycans The generation of information about glycoprotein glycosylation can be carried out at different levels of increasing detail. The choice of the level depends largely on the research request. Quite often researchers are only interested in the question: Is my protein glycosylated? Here, procedures such as enzymatic release of N-glycans (see Section 2.01.6.3) in combination with SDS-PAGE or MALDI-TOF MS, N,O-glycan detection assays based on lectins (see Section 2.01.7.7), or amino acid analysis before and after alkaline borohydride treatment for O-glycans (see Section 2.01.6.2) are sufficient. Another level can be the interest in the type of N-glycosylation: oligomannose type versus complex type. Here, the results of incubation with N-glycan releasing enzymes, oligomannose-type specific versus enzymes with a broad specificity, can be evaluated by SDS-PAGE, and specific lectin microarrays (see Section 2.01.7.7) can also be applied. On a higher level, researchers can be interested in detailed structures, and here fractionation procedures, profiling technologies, MS, and NMR spectroscopy come into the picture (see Section 2.01.7). For quality control of therapeutic glycoprotein glycosylation or changes in glycosylation in health and disease, HPLC, CE, or MS profiling technologies are perfect approaches, however, at a stage that in previous research the basic patterns have been elucidated. Figure 29 presents a schematic setup of a fractionation/purification strategy, that can be followed up by a great variety of analysis protocols, as discussed in Sections 2.01.5 and 2.01.7 (see also Table 1).

2.01.6.1 Proteolytic Digestion of Glycoproteins In the structural analysis of glycoprotein N- and O-glycans, the proteolytic digestion yielding glycopeptides plays an important role. Although quite often a first interest is focused on the knowledge of the total ensemble of glycan chains, for site-specificity studies, that is, the glycosylation pattern per glycosylated Asn or Ser/Thr residue, glycopeptides are a prerequisite. In order to prepare glycopeptides, enzymatic as well as chemical procedures can be followed, with a preference for enzymes. Proteolytic hydrolysis may be performed either on native or on denatured glycoproteins.175 For the denaturation, convential protocols are applied from the protein field, such as reduction of S–S bridges followed by carboxymethylation of the generated thiol functions. Table 5 presents an overview of proteolytic enzymes that are used to digest (glyco)proteins. In the case of glycan-site-specificity studies, in general enzymes with a restricted amino

Glycosylation sites analysis

Glycoprotein analysis

Glycan analysis

Glycoprotein Monosaccharide analysis Proteolytic digestion

Release of N-glycans

De-N-glycosylated protein Amino acid analysis

Glycopeptides

Release of glycans

Release of O-glycans

Peptides

Glycans

Amino acid sequence analysis Deglycosylated peptides

Glycans

Figure 29 Strategies for the preparation of glycoprobes for structural analysis.

Fractionation Purification Structural analysis


37

Table 5 Some proteolytic enzymes to generate glycopeptides from glycoproteins36 Enzyme

Source

pH optimum

Specificity

Carboxypeptidase A

Bovine pancreas

7.0–8.0

Carboxypeptidase B

Porcine/hog pancreas

7.0–9.0

Chymotrypsin (a)

Porcine/bovine pancreas

7.5–8.5

Endoproteinase Arg-C

Mouse submaxillaris glands Clostridium histolyticum Pseudomonas fragi (mutant) Clostridium histolyticum Staphylococcus aureus V8

8.0–8.5

Successive cleavage from C-terminus Slow for Gly, Asp, Glu, Cys Does not act at Arg, Lys, Pro, Hyp Successive cleavage from C-terminus of basic amino acids (Lys, Arg) C-terminal bonds of Tyr, Phe, Trp Slow for Met, Leu, Ala, Asp, Glu Carboxylic side of Arg

4.0 and 7.8

Lysobacter enzymogenes Carica papaya

8.5–8.8 6.0–7.0

Pepsin

Pig gastric mucosa

2.0–4.0

Pronase Subtilisin

Streptomyces griseus Bacillus subtilis Bacillus licheniformis

7.5–8.0 7.0–8.0

Thermolysin

Bacillus thermoproteolyticus

7.0–9.0

Trypsin

Bovine/hog pancreas

7.5–8.5

Endoproteinase Asp-N Endoproteinase Glu-C (V-8 protease) Endoproteinase Lys-C Papain

7.0–8.0

Amino side of Asp (Glu) and cysteic acid Carboxylic side of Glu (and Asp) Carboxylic side of Lys Arg, Lys, Glu, His, Gly, Tyr Total hydrolysis on prolonged incubation Does not act at acidic residues Preferentially carboxylic side of Phe, Met, Leu, Trp Tyr-X and X-Val/Ala/Gly are relatively resistant Total hydrolysis Total hydrolysis Preferentially Asp, Glu, Ala, Gly, Val Low cleavage specificity Ile, Leu, Met, Phe, Trp, Val, Ala Carboxylic side of Arg, Lys

Reproduced with permission from Gerwig, G. J.; Vliegenthart, J. F. G. Proteomics in Functional Genomics. Jolle`s, P.; Jornvall, H., Eds. Birkhauser Verlag: Basel, Switzerland, 2000; pp 159–186.

acid specificity are chosen, so that larger peptides are created. Especially trypsin, chymotrypsin, and endoproteinase Glu-C are frequently seen. But it should be noted that often incomplete and aspecific proteolytic cleavages do occur that give rise to unexpected complex mixtures of (glyco)peptides. Furthermore, when O-glycans are linked to neighboring Ser/Thr residues, enzymatic cleavage does not lead to glycopeptides containing individual glycosylation sites. The nonselective enzyme pronase, which is a mixture of endo- and exo-peptidases, is most often used to obtain glycoamino acids or glycopeptides with a very short polypeptide chain. The generated mixtures of (glyco)peptides can be used in different ways for further studies. Quite often isolation protocols are followed by using anion-exchange or (reversed-phase) HPLC (see Section 2.01.7.1), and analysis by LC-MS methodologies is also used (see Section 2.01.7.6). Furthermore, affinity chromatography using lectins (see Section 2.01.7.1.2) and CE (see Section 2.01.7.1) are applied. Classical examples using the glycopeptide approach comprise the structural analysis of glycopeptides by monosaccharide analysis, methylation analysis, and 1H NMR spectroscopy of, for instance, a1-acid glycoprotein176 and soybean agglutinin.177 Specific glycopeptides can be subjected to chemical (see Section 2.01.6.2) and/or enzymatic (see Sections 2.01.6.3 and 2.01.6.4) procedures that release the ensembles of carbohydrate chains, in order to elucidate the already-mentioned glycan-site specificity.

2.01.6.2 Chemical Release of Glycoprotein N- and O-Glycans Hydrazinolysis or alkaline hydrolysis can be applied to release N-glycans from Asn and O-glycans from Ser or Thr in the peptide backbone. O-Linkages, wherein hydroxylysine or hydroxyproline are involved, are stable in alkaline solution.

38


H2N NH2

OH

OH

O H N

RO HO

C C

NH

Anhydrous NH2−NH2

CH

NH2

Ac2O sat. NaHCO3

OH O NH2

NH2

+NH2−NH2 −NH3

+ amino acid hydrazides + CH3CONHNH2

NH2

RO HO

O

RO HO

NH

O

Ac

H2N

CH2

O

NH

NH2

OH

OH RO HO

sat. NaHCO3

O NH Ac

NH

NHAc

O RO HO NH

OH

Ac

Figure 30 Schematic presentation of the chemical release of glycoprotein N-glycans, using the hydrazinolysis/reN-acetylation protocol.

In the hydrazinolysis procedure applied to N-glycoproteins, the protein is completely destroyed. The peptide linkages are cleaved, yielding initially amino acid hydrazides, and the N-glycans are released as depicted in Figure 30. The N- and O-acetyl groups present are lost, whereas a partial loss of sulfate, phosphate, and N-glycolyl groups has been observed. The method was introduced in 1974.178 Subsequently, a modified and optimized procedure for releasing Asn-linked N-glycans, including a workup protocol to obtain oligosaccharide-alditols, was developed.179,180 The procedure comprised incubation of glycoprotein or glycopeptide material with anhydrous hydrazine for 8–12 h at 100 C, followed by re-N-acetylation in mild alkaline medium. For structural analysis, besides the conversion of the released oligosaccharides into their corresponding oligosaccharide-alditols (reduction with NaBH4, NaBD4, or NaBT4), the free oligosaccharides and their corresponding fluorescent derivatives are usual probes (see Section 2.01.7.2). Classical examples using the hydrazinolysis/re-N-acetylation/NaBH4 approach comprise the structure elucidation of the N-glycans of, for instance, human chorionic gonadotropins,181 g-glutamylpeptidases,182 and immunoglobulins183 by monosaccharide analysis, methylation analysis, and size-exclusion chromatography combined with sequential exoglycosidase digestions. Although being a successful methodology, over the years side reactions have been reported, especially those occurring at the reducing end (e.g., modification or absence of the proximal GlcNAc residue).184–188 Hydrazinolysis under oxygen-free argon seems to reduce this side-product formation.189 The presence of mucin-type O-glycans in the glycoprotein sample may cause problems, since part of the O-linked chains is released and partly degraded by the hydrazinolysis conditions as used for the release of N-glycans.190 When focusing on O-glycoproteins, a milder variant of the hydrazinolysis procedure as mentioned above for the generation of N-glycans has been developed. The release of Ser/Thr-linked O-glycans has been realized by incubation with anhydrous hydrazine for 6h at 60 C, followed by re-N-acetylation in mild alkaline medium.191–193 Although the exact reaction mechanism for the release of O-glycans is not known, an initial b-elimination reaction, followed by the formation of hydrazone derivatives, is generally accepted; in principle, it is possible that traces of water catalyze the release. It should be noted that peeling reactions occur to some extent. Typical examples using this approach comprise the structural analysis of the O-glycans of, for instance, human glycophorin A and serum IgA1,192 and human neutrophil gelatinase B194 by HPLC/exoglycosidase analysis of fluorescent 2-aminobenzamide-labeled derivatives. The most frequently used methodology to generate O-glycans (cleavage of, e.g., GalNAc-Ser/Thr; Xyl-Ser; ManSer linkages) is the alkaline hydrolysis in the presence of NaBH4 (alkaline borohydride treatment). As the alkaline hydrolysis is in fact a b-elimination reaction, additional peeling reactions at C3 of the originally Ser/Thr-linked monosaccharide can occur. Therefore, the reduction with NaBH4 is highly essential (Figure 31). In the case of GalNAc-Ser/Thr linkages, it stabilizes the reducing end of the released oligosaccharides via conversion of GalNAc into GalNAc-ol.195,196 Hereby, the initially formed 2-aminoacrylic acid in the case of Ser and 2-aminocrotonic acid in the case of Thr are transformed into DL-Ala and DL-2-aminobutyric acid. Generally, the incubation is carried out with 0.1M NaOH/2M NaBH4 for 16h at 45 C,197 but many deviations in the exact reaction conditions are known in the literature. Figure 32 shows the results of a peeling reaction, leading to the elimination of Gal in case of Gal(b1-3) GalNAc(a1-O)Ser/Thr. Under the cleavage conditions used, O-acyl groups are lost, whereas the remaining protein is

39


OH

OH

OR1

OR1 O

O R 2O

R

NH Ac

O

C

CH C

R 2O

O H

OH

Ac

NH

O H

OH

OR1 O

R

O

C

NaBH4

+

R 2O

O

CH C

O

R = H: L-Ser R = CH3: L-Thr OH

C

H

NH

GalNAc

R

NH

NaOH

NH

OH

HC

Ac

C

OR1 OH

R

C

O

+

R 2O

NH

CH2OH

H2C

CH

Ac

NH

NH

GalNAc-ol R = H: 2-aminoacrylic acid R = CH3: 2-aminocrotonic acid

R = H: DL-Ala R = CH3: DL-2-aminobutyric acid

Figure 31 Schematic presentation of the chemical release of glycoprotein O-glycans, using the alkaline borohydride treatment protocol.

OH

HO−

OR1 O

H

O

H

NHAc H

Gal O

Gal O

NH

OH

HO

Ac

H

H

Gal(b1-3)GalNAc

H

O−

H

O NHAc

NHAc O

Gal

HO

OH

HO

H

H

CH2OR1

H

H

H

H

OH

−OH

OH CH2OR1

CH2OR1

+ Gal

OH

H H

R1OH2C

O−

O NHAc H

H

NHAc

OH

OR1 O

HO H

OH CH2OR1

NH

OH

Ac

Figure 32 Carbohydrate peeling reactions, that can occur during the chemical release of glycoprotein O-glycans, using alkaline hydrolysis.

destructed to a high extent. The b-elimination reaction will not proceed satisfactorily if the linkage amino acid has a free a-amino or a-carboxyl group.175 Classical examples using the alkaline borohydride approach comprise the structural analysis of the O-glycans of, for instance, bronchial-mucus glycoproteins,198–200 proteoglycans,201 and zona pellucida glycoproteins76 by monosaccharide analysis, methylation analysis, and 1H NMR spectroscopy. Although the problems of degradation have been generally recognized, a few studies have been reported dealing with nonreductive alkaline hydrolysis. In one approach, the glycoprotein is attached to a solid support using hydrophobic interaction with

40


alkali-resistant reversed-phase beads, and a flow of alkali was used to release the O-glycans; only a limited amount of peeling was observed.202 In another approach, aqueous ammonium hydroxide (for b-elimination)-ammonium carbonate (for converting released oligosaccharides into glycosylamines) was used to release O-glycans as stable glycosylamines; conversion into oligosaccharides was realized through the addition of boric acid. The absence of significant peeling was suggested.203,204 It should be noted that N-glycans may also be liberated from N-glycoproteins by alkaline borohydride treatment. Here, the alkaline hydrolysis results in the cleavage of the amide bonds in the side chain of Asn and in the protein backbone. As a result, oligosaccharide-alditols as well as glycopeptides are obtained.205,206

2.01.6.3 Enzymatic Release of Glycoprotein N-Glycans 2.01.6.3.1 Peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidases For the enzymatic release of intact glycoprotein N-glycans, two peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidases (PNGases) are commercially available, namely, PNGase-F from Flavobacterium meningosepticum207–210 and PNGase-A from almond emulsin.211,212 Nowadays, PNGase-F is available in recombinant form expressed in Escherichia coli. Besides these two preparations, PNGases have been shown to occur widely in plant and animal cells.213,214 PNGases catalyze the hydrolysis of the b-aspartylglycosylamine linkage (GlcNAc-Asn), forming an intermediate 1-aminooligosaccharide, which is hydrolyzed nonenzymatically to ammonia and the free oligosaccharide (Figure 33). The occurrence of the intermediate was indicated by 1H NMR analysis after N-acetylation.215 Therefore, the enzyme is an amidase, and not an N-glycosidase. Note that in this cleavage Asn is converted into Asp, a change that will influence the overall charge of the remaining protein. PNGase-F is generally applicable for the release of all types of N-glycans, whether they are neutral, sialylated, sulfated, or phosphorylated, except those bearing a Fuc(a1-3) residue at the Asn-bound GlcNAc. Of importance is that (1) the glycosylation site is accessible to the enzyme, (2) both the carboxyl- and the amino-terminal group of the Asn residue, bearing the glycan chain, are in peptide linkage, and (3) the length of the carbohydrate chain attached to a glycoprotein is at least two residues. In the case of a glycopeptide, even a single GlcNAc residue linked to Asn can be liberated, provided that both the a-amino and a-carboxyl function of the Asn residue are in peptide linkage.216 Also for PNGase-A holds that the enzyme releases all types of N-glycans, now including those with a Fuc(a1-3) residue at the Asn-bound GlcNAc.217 However, for optimal release, it is advisable to use as substrate (carboxymethylated) glycoprotein proteolytic digests or purified glycopeptides instead of intact glycoproteins. It is generally recognized that denaturation of a glycoprotein prior to PNGase-F incubation greatly enhances the efficiency of the oligosaccharide release,216,218,219 and significantly decreases the amount of enzyme needed to obtain quantitative deglycosylation. Buffer solutions contain typically ethylenediamine tetraacetic acid (EDTA), sodium dodecylsulfate (SDS), 2-mercaptoethanol, and detergents such as Nonidet NP-40 or octyl b-D-glucopyranoside. For OH

OH C

O RO HO

H N NHAc

O PNGase

C O

CH2

CH

C

O RO HO NHAc

NH

NH2 + HO

C O

CH2

O

CH NH

OH +H2O −NH3

---- Man(a 1-6)

O RO HO NHAc

OH

Fuc(a 1-6) \ \ Man(b1-4)GlcNAc(b1-4)GlcNAc(b1-N)Asn /

-----Man(a1-3)

Figure 33 Schematic presentation of the enzymatic release of glycoprotein N-glycans, using peptide-N4-(N-acetyl-bglucosaminyl)asparagine amidases.


41

each glycoprotein/glycopeptide the PNGase incubation conditions should be tested and, if necessary, optimized. Therefore, it is advised to check the cleavage (optimal release) by SDS-PAGE, CE, and/or monosaccharide analysis. Quite often, before incubation, glycoproteins are carboxymethylated. Although denaturation of glycoprotein material is generally needed, several examples exist demonstrating that N-glycans can also be released under nondenaturing conditions.220 Typical examples of incubation conditions can be found in studies on the N-glycosylation patterns of, for instance, recombinant human follitropin,221 human Tamm–Horsfall glycoprotein,71,72 recombinant human erythropoietin,222 human chimeric plasminogen activator,223 vegetable foodstuffs,224 and recombinant human C1 inhibitor.225 For the structural analysis, several approaches are followed, including HPLC profiling studies combined with exoglycosidases after conversion into fluorescently labeled derivatives (see Section 2.01.4), 1H NMR spectroscopy (see Section 2.01.7.5), and mass spectrometry (including MS profiling) (see Section 2.01.7.6).

2.01.6.3.2

Endo-b-N-acetylglucosaminidases

Besides the PNGases that cleave the GlcNAc-Asn linkages in N-glycoproteins, another series of enzymes is frequently used to release N-glycans: the endo-b-N-acetylglucosaminidases. These endo-glycosidases cleave the glycosidic linkage between the two GlcNAc residues of the N,N0 -diacetylchitobiose core moiety (Figure 34). This means that after incubation with such enzymes, the remaining glycoprotein still bears one GlcNAc residue at each Asn-glycosylation site. Endo-b-N-acetylglucosaminidases have been isolated from animal organs, plants, as well as microorganisms, and a limited set is used for structural analytical purposes. For a comprehensive review, see Ref. 226. As is evident from Table 6, different endo-b-N-acetylglucosaminidases may have different specificities.180,227 The endo-b-N-acetylglucosaminidases CI (Endo-CI; from Clostridium perfringens) and D (Endo-D; from Diplococcus pneumoniae) require the Man(a1-3)Man(b1-4)GlcNAc element as their specific glycon.228–231 Their activity is optimal for the release of oligomannose-type N-glycans. For Endo-D, the terminal Man(a1-3) residue may bear a (b1-4)-linked

OH OH

O RO HO

C

O O HO

NHAc

H N

C

CH2 CH

O

NH

O

NHAc

Endo-b-N-acetylglucosaminidase

OH

OH O

O +

RO HO NHAc

OH

---- Man(a1-6)

HO HO

C H N

C

CH2 CH

O

NH

O

NHAc

[Fuc(a1-6)] Man(b1-4)GlcNAc(b1-4)GlcNAc(b1-N)Asn

-----Man(α1-3)

Figure 34 Schematic presentation of the enzymatic release of glycoprotein N-glycans, using endo-b-N-acetylglucosaminidases.

42


Table 6 Specificities of some endo-b-N-acetylglucosaminidases Endo-CI

R-6)

R-6) Man(b 1-4)GlcNAc(b 1-4)GlcNAc(b 1-N)Asn

Man(a1-3) Endo-D

R-6)

R-6)

Man(b 1-4)GlcNAc(b 1-4)GlcNAc(b 1-N)Asn R-4)Man(a1-3) Endo-H

R-6) Man(a1-6) R-2)Man(a1-3)

R-6)

R-4)Man(b 1-4)GlcNAc(b 1-4)GlcNAc(b 1-N)Asn R-3)

Endo-CII

Man(a1-6) R-2)Man(a1-3)

R-4)Man(b 1-4)GlcNAc(b 1-4)GlcNAc(b 1-N)Asn

R-2)Man(a1-3) Endo-F1

R-2,3,6)Man(a1-6) Man(b 1-4)GlcNAc(b 1-4)GlcNAc(b 1-N)Asn R-2)Man(a1-3)

R is the position where (a) mono/oligosaccharide residue(s) may be attached.

GlcNAc, but the presence of a GlcNAc(b1-2) residue will inhibit its action. In principle, the Asn-bound GlcNAc residue can be substituted by a Fuc(a1-6) unit. The endo-b-N-acetylglucosaminidase H (Endo-H; from Streptomyces plicatus or Streptomyces griseus) can be used to release all oligomannose-type and most of the hybrid-type N-glycans. It requires the Man(a1-3)Man(a1-6)Man(b1-4) GlcNAc element as its specific glycon.232–235 The terminal Man(a1-3) residue may be substituted at O2. The hybridtype N-glycans may possess a bisecting GlcNAc(b1-4) substituent at the core b-Man unit. In principle, the Asn-bound GlcNAc unit can be substituted by a Fuc(a1-6) residue. The endo-b-N-acetylglucosaminidase CII (Endo-CII; from C. perfringens) requires the branched Man(a1-3)Man (a1-6)[Man(a1-3)]Man(b1-4)GlcNAc element as its specific glycon.233,236 The two terminal Man(a1-3) units may be substituted at O2, but substitution at O4 of the Man(a1-3) residue linked to b-Man inhibits the Endo-CII action. The enzyme can be used to release oligomannose-type and hybrid-type structures, which may possess a bisecting GlcNAc (b1-4) substituent at the core b-Man unit. Furthermore, a set of endo-b-N-acetylglucosaminidases F (Endo-F; from F. meningosepticum) exists that can be applied for the release of different types of N-glycan structures. Endo-F1 releases oligomannose- and hybrid-type N-glycans, provided that no bisecting GlcNAc is present. (a1-6)-Fucosylation of the Asn-bound GlcNAc reduces the rate of hydrolysis over 50-fold relative to oligomannose-type structures. Endo-F2 releases oligomannose- and [sialylated; (a1-6)-fucosylated] diantennary complex-type N-glycans, although oligomannose-type chains are poor substrates. Endo-F3 releases [sialylated; (a1-6)-fucosylated] di- and tri- [extra (1-6) branch at the Man(a1-6) arm] antennary complex-type N-glycans; the presence of (a1-6)-linked Fuc increases the rate of hydrolysis.210,237

2.01.6.4 Enzymatic Release of Glycoprotein O-Glycans So far, only a few endo-a-N-acetylgalactosaminidases have been described, and their use for structural analytic purposes is very limited.226 The commercially available endo-a-N-acetylgalactosaminidases D (from D. pneumoniae)238 and A (from Alcaligenes spp.),239 that hydrolyze the GalNAc-Ser/Thr O-glycosidic linkage have a highly restricted specificity, only releasing Gal(b1-3)GalNAc units. No extensions of this disaccharide element appear to be tolerated.197,240 The A enzyme seems to have a preference for Ser-linked Gal(b1-3)GalNAc. Endo-a-N-acetylgalactosaminidase S (from Streptomyces sp. OH-11242) was found to cleave not only the Gal(b1-3)GalNAc(a1-O)Ser/Thr linkage, but also the Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc(a1-O)Ser/Thr linkage.241,242


43

2.01.6.5 Enzymatic Release of Oligosaccharide Fragments by Endo-b-galactosidases The presence of ensembles of repeating N-acetyllactosamine units (PNLs) in glycoprotein and glycolipid glycans often complicates the purification, and therefore the structural analysis, of these glycans. PNLs may vary not only in length, but also in charge when Neu5Ac or sulfate groups are present. Furthermore, the position of substituents in the PNLs may be variable. Endo-b-galactosidases, isolated from various biological sources, are useful tools to generate oligosaccharides that contain one or more N-acetyllactosamine units. The enzymes cleave b-galactosidic linkages in Gal(b1-3/4)GlcNAc elements of PNLs when the Gal residue is not terminal, affording terminal (peripheral) fragments, internal (backbone) fragments, and core fragments. Analysis of these structural elements provides information about the intact glycan structures. Table 7 gives an overview of a series of endo-b-galactosidases, including their substrate specificities. Endo-b-galactosidase from Escherichia freundii (Endo-Gal EF) is capable of hydrolyzing a large variety of PNLcontaining oligosaccharides and glycolipids.243–245 The rate of cleavage for Gal(b1-4)GlcNAc linkages is higher than for Gal(b1-3)GlcNAc linkages.246 Endo-Gal EF is inactive when the Gal residue is sulfated at O6, but sulfation is allowed at O6 of GlcNAc on either side of the residue to be cleaved247 (compare structures 4 and 5 in Table 7). The presence of Fuc in internal Gal(b1-4)[Fuc(a1-3)]GlcNAc elements inhibits the hydrolysis of the b-galactosidic linkage248 (compare structures 6 and 7 in Table 7). It has been suggested that Endo-Gal EF has a higher affinity for longer PNLs than for shorter ones, when both are present, although here steric hindrance could be the cause.249 Linear (b1-6)-linked N-acetyllactosamine sequences have been shown to be poor substrates for Endo-Gal EF.250 When present in a GlcNAc(b1-3)[GlcNAc(b1-6)]Gal(b1-4)GlcNAc element, Endo-Gal EF seems to be able to hydrolyze the Gal(b1-4)GlcNAc linkage to some extent, whereby the enzyme concentration used is important244,248 (structure 3 in Table 7). It turned out that branching points present in the terminal regions of long PNLs were susceptible, whereas branching points located close to the core portion were minimally hydrolyzed.251

Table 7 Substrate specificities of endo-b-galactosidases from various biological sources242a Susceptible to endo-b-galactosidase from b,c Structural element a 1. R-GlcNAc(b1-3)Gal(b1-3/4)GlcNAc(b12. R-GlcNAc(b1-6)Gal(b1-4)GlcNAc(b13. R-GlcNAc(b 1-6)

EF

BF

FK

DPDI

DPDII

CP

PS

þ þ

þ þ/d

þ þ þ

þ n.d.

þ þ

þ þ

þ þ

þ

þ

þ

þ

þ þ

þ

þ

þ

þ

þ

Gal(b 1-4)GlcNAc(b 1R⬘-GlcNAc(b 1-3) 4. R-GlcNAc6S(b1-3)Gal(b1-4)GlcNAc6S(b15. R-GlcNAc6S(b1-3)Gal6S(b1-4)GlcNAc6S(b16. Gal(b 1-4)GlcNAc(b 1-3)Gal(b 1-4)GlcNAc(b 1Fuc(a1-3) 7. Gal(b 1-4)GlcNAc(b 1-3)Gal(b 1-4)GlcNAc(b 1-

Fuc(a1-3)

Fuc(a1-3)

8. R-GlcNAc(b1-3)Gal(b1-4)Glc(b1-Cer 9. Gal(a1-3)Gal(b1-4)GlcNAc(b1-

Fuc(a1-2) 10. GalNAc(a 1-3)Gal(b 1-4)GlcNAc(b 1-

Fuc(a 1-2) 11. Gal(a1-3)Gal(b1-4)GlcNAc(b1a

A larger part of the oligosaccharide structure may have to be considered to reveal more subtle differences in specificity (see text). b The susceptibility might be concentration dependent (see text). c The abbreviations used are: EF, Escherichia freundii; BF, Bacteroides fragilis; FK, Flavobacterium keratolyticus; DPDI and DPDII, Diplococcus pneumoniae; CP, Clostridium perfringens; PS, Pseudomonas species. d Only partial cleavage has been observed. n.d., not determined.

44


The specificity of endo-b-galactosidase from Bacteroides fragilis (Endo-Gal BF) is quite similar to that of E. freundii.248,252 However, Endo-Gal BF is definitely inactive on branched PNL sequences253 (structure 3 in Table 7). In the case of complex-type N-glycans, a difference in specificity between Endo-Gal BF and EndoGal-EF has been observed for the antennary systems. Whereas di- and tetraantennary N-glycans having PNLs attached to the branches arising from the Man(a1-6)-arm are good substrates for Endo-Gal BF, triantennary N-glycans having PNLs attached to the branches arising from the Man(a1-6)-arm hardly react.222,254 However, incubation of the latter N-glycans with Endo-Gal EF resulted in a major release of the elongations;222 the developed analysis protocol included digestion with endo-b-galactosidase, followed by treatment with N-acetyl-b-glucosaminidase and 1H NMR analysis. Other examples illustrating the successful use of Endo-Gal BF in combination with 1H NMR spectrosocopy are the structural analysis of the porcine zona pellucida glycoprotein O-glycans76 and the Tamm–Horsfall glycoprotein N-glycans.255 Finally, it has been shown that at a high enzyme concentration, Endo-Gal BF is able to hydrolyze to some extent linear -6)Gal(b1-4)GlcNAc(b1- sequences,256 but at a lower concentration complete resistance was observed257 (structure 2 in Table 7). Neu5Ac(a 2-3)Gal(b 1-4)GlcNAc(b 1-3)Gal(b 1-4)GlcNAc(b 1-6) Neu5Ac(a 2-3)Gal(b 1-4)GlcNAc(b 1-3)Gal(b 1-4)GlcNAc(b 1-2)Man(a 1-6) Man(b 1-4)....... Neu5Ac(a 2-3)Gal(b 1-4)GlcNAc(b 1-2)Man(a 1-3) Neu5Ac(a 2-3)Gal(b 1-4)GlcNAc(b 1-4)

The endo-b-galactosidase from Flavobacterium keratolyticus (Endo-Gal FK) seems to have the broadest specificity of the endo-b-galactosidases. Its specificity resembles that of Endo-Gal EF,258,259 but in addition, it can hydrolyze b-galactosidic linkages in repeating -6)Gal(b1-4)GlcNAc(b1- sequences. It is also active on blood group A and B elements containing Gal(b1-4)GlcNAc260 (structures 9 and 10 in Table 7). Endo-b-galactosidase DI from D. pneumoniae (Endo-Gal DPDI) is specifically active on the blood group A and B elements containing Gal(b1-4)GlcNAc261 (structures 9 and 10 in Table 7). In contrast to Endo-Gal DPDI, endo-bgalactosidase DII from D. pneumoniae (Endo-Gal DPDII) does hydrolyze the Gal(b1-4)GlcNAc linkage in a PNL sequence.262 Endo-b-galactosidase from C. perfringens (Endo-Gal CP) releases specifically Gal(a1-3)Gal from the terminal sequence Gal(a1-3)Gal(b1-4)GlcNAc in glycoproteins and glycolipids.263,264 Endo-b-galactosidase purified from the extracts of Pseudomonas species (Endo-Gal PS) has been shown to degrade different types of keratan sulfate. Apparently, it requires sulfated GlcNAc for its activity, since lacto-N-tetraose is not hydrolyzed.265

2.01.7 Glycoprotein Glycan Analysis Tools 2.01.7.1 Separation Procedures For the separation/purification of glycopeptides, free oligosaccharides, oligosaccharide-alditols, and fluorescently labeled oligosaccharides, many protocols have appeared in the literature; they are cited in the present review at different places. Quite often research groups have developed their own strategies, being very successful in their hands. The approaches are, in general, based on size-exclusion chromatography, low-pressure liquid chromatography (LPLC), HPLC, HPAEC, CE, and lectin affinity chromatography. Detection systems vary from refractive index, intrinsic UV absorbance, pulsed amperometric detection, to radioactivity and fluorescence detection. A series of interesting reports are focused on reversed-phase HPLC separations of glycoprotein proteolytic digests;266 general analysis methodologies for glycopeptides;267 HPLC of carbohydrates on graphitized carbon columns,268,269 including their use in desalting;270 HPLC of glycosaminoglycan-derived oligosaccharides;271 serial affinity chromatography using immobilized lectin columns;272–276 HPLC of glycopeptides and glycoprotein glycans;277 O-glycan fractionation;197 general glycoprotein glycan fractionation;278,279 separation technologies in glycomics;280 N-glycan sample clean-up procedures;281,282 CE of glycopeptides and glycoprotein-derived oligosaccharides;283 electrophoretic approaches to the analysis of proteoglycans.284

2.01.7.1.1 High-pH anion-exchange chromatography Ion-exchange chromatography at alkaline pH on pellicular quaternary amine-bonded resins combined with highly sensitive pulsed amperometric detection (HPAEC-PAD) of the eluted glycans forms a very important part of the


45

arsenal of fractionation and identification methodologies.71,277,285–295 Due to the high pH, the separation is based on negative charges stemming from not only, for instance, sialic acids, but also from oxyanion formation. Care should be taken with alkaline-sensitive oligosaccharide (derivatives), as they degrade when they are subjected to this fractionation methodology (e.g., de-O-acylation, peeling reactions). In principle, it is possible to use the system also for fractionations at a more neutral pH. For most separations, HPAEC requires salt concentrations ranging from 50 to 400mM, making desalting procedures necessary prior to structural studies of isolated fractions. Besides conventional off-line procedures, in-line desalting possibilities using micromembranes have been developed.295 In many structural analysis strategies, HPAEC forms one of the important steps in generating pure compounds. For instance, a valuable strategy for N,O-glycan analysis comprises (1) release of the N-glycans from the N,O-glycoprotein by PNGase-F, (2) separation of the N-glycan pool and the O-glycoprotein by size-exclusion chromatography, (3) release of the O-glycans from the O-glycoprotein by alkaline borohydride, (4) fractionation of both the N- and O-glycan pool on a weak anion-exchanger (LPLC; Resource Q), (5) subfractionation of all LPLC fractions by normal phase HPLC (Lichrosorb-NH2), (6) further subfractionation of HPLC fractions by HPAEC (CarboPac-PA1).222 In such a strategy at each level of the fractionation track, investigations by monosaccharide analysis, 1H NMR spectroscopy, and MS, as well as exoglycosidase studies can be incorporated. For a comprehensive review on the HPAEC fractionation methodology, see Chapter 2.09.

2.01.7.1.2

Lectin affinity chromatography

Lectins are carbohydrate-recognizing proteins that are widely distributed in nature. They occur in plants as well as in mammalian and microbial species. Comprehensive reviews have appeared on this topic,296 and updated reviews can be found in several chapters of this book series. Due to their specificity for binding specific carbohydrate epitopes, immobilized lectins on insoluble adsorbents have been used in fractionation and purification strategies not only for glycoproteins, glycopeptides, and oligosaccharides,272–274,276 but also for evaluating changes in glycosylation patterns in health and disease, for example, changes in (a1-6)-fucosylation, (a2-3/6)-sialylation, or (b1-4)-galactosylation, or changes in antennary types. The methodology has especially been worked out for the study of N-glycans, because these chains obey structural rules that are effective in their group fractionation, and is also used in O-glycan investigations. Table 8 presents a selection of lectins that is used in (serial) lectin affinity chromatography. The a-L-Fuc-recognizing Aleuria aurantia lectin (AAL),297 immobilized on Agarose, can be used for the separation of core (a1-6)-fucosylated and core nonfucosylated N-glycans (fucosylation of the proximal GlcNAc residue of the Man3GlcNAc2 core structure). The binding is not influenced by the remaining part of the structure, including a bisected GlcNAc residue. Glycans containing Fuc(a1-2)Gal(b1-4)GlcNAc(b1-, Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-, and Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1- fragments interact weakly with AAL. This means that care should be taken in translating ‘binding’ into ‘occurring structures’. The a-D-Man/a-D-Glc-recognizing lectin Concanavalin A (Con A) is an ideal lectin for separating a pool of N-glycans into different subpools. For binding, at least two terminal [Man(a1-; Glc(a1-; GlcNAc(a1-] or 2-substituted [-2)Man(a1-; -2)Glc(a1-] Man/Glc residues must be present in the N-glycan. Oligomannose- and hybrid-type N-glycans bind strongly to Con A-Sepharose, whereas diantennary complex-type N-glycans show a somewhat weaker binding. Tri- [2,4-substituted at the Man(a1-3)-arm or 2,6-substituted at the Man(a1-6)-arm], tetra-, and pentaantennary complex-type N-glycans do not bind. The presence of a bisecting GlcNAc [GlcNAc(b1-4)Man(b1-4)] residue weakens strongly the interaction with the immobilized lectin.298 Also the presence of a Lex element [extra Fuc (a1-3) residue at GlcNAc] in the outer chains decreases considerably the affinity.299 A typical example comprises its use in a study on the site-specific glycosylation of Tamm–Horsfall glycoprotein.300 The lectin phytohaemagglutinin E4 (E4-PHA), immobilized on Sepharose, can be used to separate complex-type bisecting-GlcNAc N-glycans, having the minimal structural unit (20 C) shown in Table 8, from other N-glycans.301 Remarkably, the minimal structural unit, that causes retardation, changes when the fractionation is carried out at 4 C (Table 8); the requirement for the lower arm GlcNAc(b1-2)Man(a1-3) fragment and a bisecting GlcNAc residue, necessary at 20 C, is lost at this lower temperature.302 The lectin Datura stramonium agglutinin (DSA), immobilized on Sepharose, binds strongly to the upper arm Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-6) fragment of N-glycans as well as to poly-N-acetyllactosamine sequences (Table 8). A weaker binding is seen for the lower arm Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc (b1-4)]Man(a1-3) fragment of N-glycans.303,304 The binding is not influenced by the remaining part of the structure, including a bisected GlcNAc residue. The presence of a Gal(a1-3) or a Neu5Ac(a2-3) residue at the b-Gal unit does not influence the affinity to the lectin, but the presence of a Fuc(a1-3) residue at the b-GlcNAc unit does. It may be clear that this lectin is useful in separating diffent branching patterns of complex-type N-glycans.

46


Table 8 Structures of N-glycans which interact with immobilized plant lectin columns 276 Aleuria aurantia lectin (AAL)/mushrooms, specific for a-Fuc (recombinant in E. coli). Strong binding (bound) of N-glycans having an (a1-6)-fucosylated core fragment; elution with 1mM L-fucose. . . ...GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc Weak binding (retarded) of fucosylated outer chain fragments; elution with 0.5mM L-fucose. Fuc(a1-2)Gal(b1-4)GlcNAc(b1Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1Canavalia ensiformis lectin (Concanavalin A; Con A)/jack beans, specific for a-Man/a-Glc. Strong binding of oligomannose- and hybrid-type N-glycans; elution with 200mM methyl a-D-mannopyranoside.

Man(a1-6) [Man(a1-2)]0−4

Man(a1-6) Man(a1-3)

Man(b1-4)GlcNAc(b1-4)GlcNAc Man(a1-3)

Man(a1-6) Man(a1-6) Man(a1-3)

Man(b1-4)GlcNAc(b1-4)GlcNAc

R-Gal(b1-4)GlcNAc(b1-2)Man(a1-3) R = H or monosaccharide Weak binding of diantennary complex-type N-glycans; elution with 5mM methyl a-D-glucopyranoside.

±Fuc(a 1-6)

R⬘-4)GlcNAc(b 1-2)Man(a 1-6)

Man(b 1-4)GlcNAc(b 1- 4)GlcNAc R⬘-4)GlcNAc(b 1-2)Man(a 1-3) R0 ¼H or monosaccharide; in the case of R0 ¼H, the N-glycans show a strong binding, and are eluted with 200mM methyl a-D-mannopyranoside instead of 5mM methyl a-D-glucopyranoside (higher affinity because of terminal GlcNAc residues). Phaseolus vulgaris phytohaemagglutinin E4 (E4-PHA)/red kidney beans. Strong binding of specific complex-type N-glycans (retarded at 20 C and 4 C)

±Fuc(a 1-6)

R-3)Gal(b 1-4)GlcNAc(b 1-2)Man(a 1-6)

GlcNAc(b 1-4)Man(b 1-4)GlcNAc(b 1-4)GlcNAc R-4)GlcNAc(b 1-2)Man(a 1-3) R-4) Weak binding of specific complex-type N-glycans (retarded at 4 C only).

±Fuc(a 1-6)

R-3)Gal(b 1-4)GlcNAc(b 1-2)Man(a 1-6)

Man(b 1-4)GlcNAc(b 1-4)GlcNAc R-3) R = H or monosaccharide Datura stramonium agglutinin (DSA)/jimson weed. Strong binding (bound) of specific complex-type N-glycans and poly-N-acetyllactosamine sequences; elution with 1% N-acetyl-D-glucosamine oligomers.

Gal(b 1-4)GlcNAc(b 1-6) Gal(b 1-4)GlcNAc(b 1-2)Man(a 1-6)

±Fuc(a 1-6)

±GlcNAc(b1-4)Man(b 1-4)GlcNAc(b 1-4)GlcNAc R-)Man(a1-3) [Gal(b 1-4)GlcNAc(b 1-3)]1-nGal(b 1-4)GlcNAc(b 1-


47

Table 8 (continued) Weak binding (retarded) of specific complex-type N-glycans.

R-)Man(a 1-6)

±Fuc(a 1-6)

±GlcNAc(b1-4)Man(b 1-4)GlcNAc(b 1-4)GlcNAc Gal(b 1-4)GlcNAc(b 1-2)Man(a 1-3) Gal(b1-4)GlcNAc(b1-4)

R = H or monosaccharide

Ricinus communis agglutinin I (RCA-I)/castor beans. Strong binding of b-Gal-terminated N-glycans; elution with 10mM lactose Gal(b1-4)GlcNAc(b1Weak binding (retarded) of b-Gal-terminated fragments Gal(b1-3)GlcNAc(b1Gal(b1-3)GalNAc Gal(b1-6)Gal(b1Allomyrina dichotoma lectin-II (Allo A-II) / beetles. Strong binding of 6-substituted Gal(b1-4)GlcNAc elements; elution with 10mM lactose. Sia(a2-6)Gal(b1-4)GlcNAc(b1Gal6S(b1-4)GlcNAc(b1Weak binding (retarded) of terminal Gal(b1-4)GlcNAc elements Gal(b1-4)GlcNAc(b1Sambucus nigra agglutinin (SNA)/elderberry bark. Strong binding of (a2-6)-sialyloligosaccharides; elution with 200mM lactose. Sia(a2-6)Gal(b1-4)GlcNAc(b1Sia(a2-6)GalNAc(a1-O)Ser/Thr Maackia amurensis agglutinin (MAA) / tree seeds. Strong binding of oligovalent (a2-3)-sialyloligosaccharides; elution with 400mM lactose. [Sia(a2-3)Gal(b1-4)GlcNAc(b1-]2-4-R Weak binding (retarded) of monovalent (a2-3)-sialyloligosaccharides Sia(a2-3)Gal(b1-4)GlcNAc(b1Griffonia simplicifolia-I (GS-I)/vine seeds. Strong binding of oligovalent Gal(a1-3)-terminated glycans; elution with 100mM D-galactose. [Gal(a1-3)Gal(b1-4)GlcNAc(b1-]2-4-R Weak binding (retarded) of monovalent Gal(a1-3)-terminated glycans. Gal(a1-3)Gal(b1-4)GlcNAc(b1Wistaria floribunda agglutinin (WFA)/Wistaria seeds. Strong binding of GalNAc(b1-4)-terminated glycans; elution with 100mM N-acetyl-D-galactosamine. GalNAc(b1-4)Gal(b1-4)GlcNAc(b1GalNAc(b1-4)GlcNAc(b1Ulex europaeus I (UEA-I)/furze seeds. Strong binding of Fuc(a1-2)-terminated glycans; elution with 50mM L-fucose. Fuc(a1-2)Gal(b1-4)GlcNAc(b1Tomato lectin (TL) Strong binding of poly-N-acetyllactosamine sequences; elution with 0.2% N-acetyl-D-glucosamine oligomers. [Gal(b1-4)GlcNAc(b1-3)]2-nGal(b1-4)GlcNAc(b1-

The lectin Ricinus communis agglutinin I (RCA-I),305 immobilized on Agarose, basically recognizes terminal b-D-Gal residues, more specifically glycans containing Gal(b1-4)GlcNAc(b1-R segments (Table 8). The affinity is enhanced by the oligovalent presence of N-acetyllactosamine units. The Allomyrina dichotoma lectin-II (Allo A-II) interacts with N-acetyllactosamine units in glycan chains, with a strong preference for 6-substituted Gal residues, that is, (a2-6)-sialylated or 6-O-sulfated N-acetyllactosamine

48


units (Table 8).306,307 The interaction increases when N-acetyllactosamine is presented in an oligovalent way, that is, going from a mono- to a tetra-antennary N-glycan. (a1-3)-Fucosylation at GlcNAc or (a1-2)-fucosylation/(a1-3)galactosylation/(a2-3)-sialylation at Gal abolishes the interaction of the lectin with N-acetyllactosamine. The difference in affinity for (a2-3)- and (a2-6)-sialylated N-glycans can be used in fractionation protocols. The Neu5Ac(a2-6)-recognizing lectin Sambucus nigra agglutinin (SNA)308 and the Neu5Ac(a2-3)-recognizing lectin Maackia amurensis lectin (MAL)309 are quite useful in isolating glycans with specific sialylation patterns (Table 8), but are also applied in the separation of different charged glycans, for example, sialylated from GlcNAc-phosphorylated N-glycans.310 Contrasting reports exist about the ability of SNA to bind to Neu5Ac(a2-6)GalNAc.308,311 The a-D-Gal-recognizing lectin Griffonia simplicifolia-I (GS-I) can be applied in isolating glycans with the Gal(a1-3) Gal(b1-4)GlcNAc sequence, whereby oligovalent availability is important.312 Furthermore, the b-D-GalNAc-recognizing lectin Wistaria floribunda agglutinin (WFA),313 the a-L-Fuc-recognizing lectin Ulex europaeus I (UEA-I) (blood-group type-2 H antigen),314 and the tomato lectin (TL) (high affinity for glycans which contain three or more linear N-acetyllactosamine units; poly-N-acetyllactosamine type)315,316 are of interest in fractionation protocols (Table 8). Over the years, in several structural studies serial lectin chromatography procedures have been used.272,274,276 Typical examples describe the use of the technology in analyzing Schistosoma mansoni glycoproteins317 and blood coagulation factor VIII318 N-glycans. To give an impression of a possible sequence of lectin columns, an N-glycan pool can be fractionated as follows (see Table 8 for specificities): (1) fractionation of the glycan pool on RCA-I Agarose; (2) fractionation of the RCA-I-bound fraction on E4-PHA Agarose; (3) fractionation of the E4-PHA-unbound fraction on Con A Sepharose; (4) fractionation of both the Con A-bound and unbound fraction on AAL Sepharose; (5) fractionation of both the AAL-bound and unbound fractions on DSA Sepharose, which results in bound, weakly bound, and unbound DSA fractions.

2.01.7.2 Labeling Procedures In general, glycopeptides are analyzed as such using monosaccharide analysis, methylation analysis, MS, 1H NMR spectroscopy, and exo- and endo-glycosidase digestions, separately or in combination. However, simple labeling procedures of the peptide backbone with fluorescent compounds such as dansyl chloride319 have also been reported. A typical example comprises the 1H NMR analysis of dansyl glyco-asparagines from quail ovalbumin.320 Glycopeptides can also be labeled via N-acetylation with [3H] or [14C]acetic anhydride.321 For the analysis of free oligosaccharides or oligosaccharide-alditols, several protocols can be followed, and quite often combinations of monosaccharide analysis, methylation analysis, MS, 1H NMR spectroscopy, HPLC, and exoand endo-glycosidases are used. Their detection in fractionation procedures can be performed by monitoring the UV absorbance at 195–215 nm when one or more C¼O groups are present. Sometimes, traditional colorimetry is applied, for example, the phenol/conc. H2SO4 assay with detection at 490 nm. In addition, pulsed amperometric detection, as used in HPAEC, is a highly useful technique (see Section 2.01.7.1.1). The labeling of free oligosaccharides at the reducing end is mainly carried out with radioactive and fluorophoric compounds. A radioactive label can be introduced by reduction with NaBT4.180 Note that such a label can also be introduced in oligosaccharide-alditols in case the alkaline borohydride treatment for the release of O-glycans is performed in the presence of NaBT4.190 Nowadays, the extremely sensitive (pmol–fmol range) and selective fluorescent labeling of free glycans is widely used. Among the many compounds reported over the years, three stable probes are very popular, namely 2-aminopyridine, 2-aminobenzamide, and 2-aminobenzoic acid (anthranilic acid).322,323 In fact, all incorporations are based on a reductive amination reaction (Figure 35). The fluorescent labeling plays a major role in HPLC profiling studies in combination with exoglycosidases324 (see Sections 2.01.7.3 and 2.01.7.4), although for MS the benefits of the aromatic labeling have been noted. The earlier discussed chemical technologies (such as monosaccharide analysis, methylation analysis, partial acid hydrolysis, acetolysis, and Smith degradation) can be carried out without any problem, whereas the 1H NMR spectroscopic structural-reporter-group concept developed for glycopeptides and free oligosaccharides17,36,325 is, with some adaptations for the reducing end, directly applicable. The preparation of pyridylamino (2PA) derivatives is performed by reaction with 2-aminopyridine in the presence of dimethylaminoborohydride or sodium cyanoborohydride,326–329 and fluorescence is detected using excitation and emission wavelengths of 310 (320) nm and 380 (400) nm, respectively. For the separation of 2PA-derivatized glycans, three types of HPLC with different separation principles are in use, namely, anion-exchange, normal phase, and reversed-phase HPLC. For quantification purposes, the peak area ratios of fluorescent 2PA derivatives can be considered as molar ratios. Over the years, 2-D and 3-D mapping technologies for HPLC profiling of neutral and sialylated N-glycans have been developed (see Section 2.01.7.4). The 2PA derivatives have also been used in CE approaches.330

O OH

OH

OH

O RO HO

RO HO

OH

NH

NH

Ac

NH2

O

H+/65 ⬚C

H

Ac

O

OH N H

NH2 Schiff's base

OH RO HO NH

Ac

2-Aminopyridine

NH2

−O

SO3−

O

NH2 NH2

−O S 3

2-Aminobenzoic acid

N

NH2

NH2

Ac

2-Aminobenzamide O

NH CH2

NH2

OH

SO3− 8-Amino-1,3,6-naphthalene trisulfonic acid

3S

8-Amino-1,3,6-pyrene trisulfonic acid −O

3S

SO3−

Figure 35 Schematic presentation of the reductive amination of oligosaccharides, using fluorophore compounds.


NH

O

OH NaCNBH3

OH RO HO

NH2

NH2

49

50


The preparation of 2-aminobenzamide (2AB) derivatives is performed by reaction with 2-aminobenzamide in the presence of sodium cyanoborohydride,191–193,331–333 and fluorescence is detected using excitation and emission wavelengths of 330 (373) nm and 420 (448) nm, respectively. Also in this case, weak anion-exchange, normal phase, and reversed-phase HPLC are included in profiling protocols, whereas the fluorescent label is highly suited for quantitative analyses. For routine analysis, computerized technologies and databases have been developed (see Section 2.01.7.4). The preparation of 2-aminobenzoic acid (2AA) derivatives is performed by reaction with 2-aminobenzoic acid in the presence of sodium cyanoborohydride,334,335 and fluorescence is detected using excitation and emission wavelengths of 230nm and 425nm, respectively. The most suitable separations are obtained by normal phase and anion-exchange HPLC.322,334 Besides HPLC, gel electrophoresis has been applied.335 In addition to the three most-popular compounds to generate fluorescent derivatives as described above, several other fluorophores have been introduced in the glycoanalytical field. Two of these approaches will be further commented. The highly charged 8-amino-1,3,6-naphtalene trisulfonic acid (ANTS) has been introduced as a tag for oligosaccharides following reductive amination in the presence of sodium cyanoborohydride.336 These derivatives have been used in gel electrophoresis337,338 as well as in CE338 and HPLC.339 The three sulfonic acid groups on the fluorescent tag create glycan derivatives that, regardless of their intrinsic charge, have always a net negative charge. Well-developed protocols are available in an approach called fluorophore-assisted carbohydrate electrophoresis (FACE), a polyacrylamide slab gel electrophoresis methodology that can be used for performing routine oligosaccharide profiling/exoglycosidase-based sequence analysis. For gel imaging of the glycan banding patterns, the gel is placed on a long-wave UV lightbox with a peak excitation output at approximately 360nm, and the further handling is completely computerized.340 In CE systems, the ANTS-derivative detection is carried out by UV absorption at 226nm or by laser-induced fluorescence detection using a helium-cadmium laser (excitation at 325nm/emission at 520nm). In a similar way, the highly charged 8-amino-1,3,6-pyrene trisulfonic acid (APTS) is applied as an oligosaccharide tag in CE338,341,342 and in gel electrophoresis343,344 approaches. In CE systems, the APTS-derivative detection is performed by laser-induced fluorescence detection using an argon-ion laser (excitation at 488nm/emission at 520nm). In the polyacrylamide gel electrophoresis protocol use is made of standard DNA-sequencing equipment (excitation at 434nm/emission at 520nm).

2.01.7.3 Exoglycosidases Sequence analysis using exoglycosidases plays a very important role in the structural analysis of N- and O-glycans.345–347 These enzymes release monosaccharide units from the nonreducing end on. Initially, this technology formed a cornerstone of the original hydrazinolyis procedure (hydrazinolysis/high-voltage paper electrophoresis/Bio-Gel P-4 chromatography/sequential exoglycosidase incubations).180 When using exoglycosidases, specific attention should be paid to contaminant glycosidases, which can severely influence the data obtained.346 In nearly all cases, exoglycosidases have a strict glycon specificity toward monosaccharide units, including their absolute (D/L) and anomeric (a/b) configurations: a-D-galactosidases, b-D-galactosidases, a-L-fucosidases, etc. Their specificity toward glycosidic linkage and ‘aglycon’ types is more variable, but limited. An exception are the N-acetyl-bD-hexosaminidases, which cleave both b-D-GlcNAc and b-D-GalNAc linkages. Steric hindrance of neighboring monosaccharide residues in the glycan can influence the activity, as well as the amounts of enzyme to be used. Therefore, a negative result does not always mean the absence of the aimed terminal monosaccharide residue, and unsuccessful digestions should be interpreted with care. Table 9 presents an overview of exoglycosidases that are applied in structural studies. The a-sialidases (a-neuraminidases) from Arthrobacter ureafaciens,348 C. perfringens,349 Vibrio cholerae,350 and Salmonella typhimurium351 have similar broad substrate specificities, and in sequence analysis studies they are used to release all sialic acids (Table 9). However, a linkage preference does exist. For A. ureafaciens, the preference of hydrolysis is Neu5Ac(a2-6)Gal>Neu5Ac(a2-3)Gal>Neu5Ac(a2-8)Neu5Ac; for C. perfringens, it is Neu5Ac(a2-3)Gal>Neu5Ac (a2-6)Gal¼Neu5Ac(a2-8)Neu5Ac; for V. cholerae, it is Neu5Ac(a2-3)Gal>Neu5Ac(a2-6)Gal>Neu5Ac(a2-8)Neu5Ac; for S. typhimurium, it is Neu5Ac(a2-3)Gal>Neu5Ac(a2-6)GalNeu5Ac(a2-8/9)Neu5Ac. As the a-sialidase from Newcastle disease virus352 does not cleave the (a2-6) linkage in N-glycans, it is frequently used to discriminate between (a2-3) and (a2-6) linkages in complex- and hybrid-type N-glycans. The same holds for the a-sialidase from D. pneumoniae.192 In the case of O-glycans the Newcastle disease virus a-sialidase releases Neu5Ac, (a2-6)-linked to 2AB-labeled core GalNAc (open ring form), but the diplococcal enzyme does not.192 For the cleavage of the (a1-3) linkage in Gal(a1-3)Gal(b1-, a-galactosidase from coffee beans is mainly applied353 (Table 9). This enzyme is also active on Gal(a1-3)[Fuc(a1-2)]Gal(b1-, the blood group B determinant, and more generally on Gal(a1-4/6) linkages.346


51

Table 9 Exoglycosidases and their specificity, as used in sequence analysis studies345–347 Exoglycosidase

Source

pH

Specificity

a-Sialidase (Neu5Ac and Neu5Gc)

Arthrobacter ureafaciens

4.5–5.5

Clostridium perfringens

5.0–5.5

Vibrio cholerae

5.6

Neu5Ac(a2-3/6)Gal Neu5Ac(a2-6)GlcNAc Neu5Ac(a2-6)GalNAc Neu5Ac(a2-8)Neu5Ac Neu5Ac(a2-3/6)Gal Neu5Ac(a2-6)GlcNAc Neu5Ac(a2-6)GalNAc Neu5Ac(a2-8)Neu5Ac Neu5Ac(a2-3/6)Gal Neu5Ac(a2-6)GlcNAc Neu5Ac(a2-6)GalNAc Neu5Ac(a2-8)Neu5Ac Neu5Ac(a2-3/6)Gal Neu5Ac(a2-8/9)Neu5Ac Neu5Ac(a2-3)Gal Neu5Ac(a2-8)Neu5Ac Neu5Ac(a2-3)Gal Gal(a1-3)Gal Gal(b1-3/4/6)GlcNAc Gal(b1-3/4/6)GlcNAc Gal(b1-3/4/6)GlcNAc Gal(b1-3/4)GlcNAc Gal(b1-4)GlcNAc GlcNAc(b1-2/4/6)Man GlcNAc(b1-4)GlcNAc GlcNAc(b1-3/6)Gal GalNAc(b1-4)GlcNAc GlcNAc(b1-2)Mana GlcNAc(b1-3/6)Gal Glc(a1-2)Glc Glc(a1-3)Glc Glc(a1-3)Man Man(a1-2/3/6)Man Man(a1-2)Man Man(a1-2/3/6)Mana Man(b1-4)GlcNAc Man(b1-4)GlcNAc broad specificity for Fuc(a1-2/3/4/6)a broad specificity for Fuc(a1-2/3/4/6)a broad specificity for Fuc(a1-2/3/4/6) broad specificity for Fuc(a1-2/3/4/6) Fuc(a1-3/4)GlcNAc Fuc(a1-2)Gal Xyl(b1-2)Man GalNAc(a1-

Salmonella typhimurium Newcastle disease virus

5.5–6.5

Diplococcus pneumoniae recombinant in E. coli Coffee bean Jack bean Aspergillus niger Bovine testis Streptococcus 6646K Diplococcus pneumoniae Jack bean

7.5 6.5 3.5–4.5 4.0–4.5 5.0 5.5 6.0–6.5 5.0–6.0

Diplococcus pneumoniae

3.5–4.0 5.0–6.0

a-D-Glucosidase

Porcine liver (I) Porcine liver (II)

7.0 7.0

a-D-Mannosidase

Jack bean Aspergillus saito (I) Aspergillus saito (II) Helix pomatia Achatina fulica Turbo cornutus Charonia lampas Bovine epididymis Bovine kidney Almond emulsin I Corynebacterium sp. Charonia lampas Porcine liver

4.0–5.0 5.0 5.0 4.5

a-D-Galactosidase b-D-Galactosidase

N-Acetyl-b-Dhexosaminidase

b-D-Mannosidase a-L-Fucosidase

b-D-Xylosidase N-Acetyl-a-Dgalactosaminidase a

4.0 3.3–4.5 6.5 6.5 5.5 8.5

For details of the substrate specificity, see text.

Although the b-galactosidases from Jack bean,354 Aspergillus niger,355 and Streptococcus 6646K356 (Table 9) show a higher preference for cleaving Gal(b1-4)GlcNAc when compared to Gal(b1-3)GlcNAc, the difference in selectivity is not as such that it can routinely be used to discriminate between both linkages. Therefore, they are applied in the sequence analysis studies of N-glycans to release both Gal(b1-4) and Gal(b1-3) residues. The b-galactosidase from bovine testis has the preference Gal(b1-3)GlcNAc>Gal(b1-4)GlcNAc>Gal(b1-6)GlcNAc,357 but is also used to release both Gal(b1-4) and Gal(b1-3) residues. In order to discriminate between both linkages, the b-galactosidase from D. pneumoniae (¼S. pneumoniae) should be used:358 only Gal(b1-4)GlcNAc linkages are cleaved. However, both the bovine testis and the diplococcal b-galactosidase do not cleave the Gal(b1-4)GlcNAc linkage in a Lex determinant

52


(Fuc(a1-3) residue at the GlcNAc unit).192 The diplococcal enzyme is not applicable for the cleavage of the Gal(b1-3) GalNAc linkage in O-glycans. The bovine testis b-galactosidase does not release or releases only very slowly Gal, (b1-3)-linked to 2AB-labeled core GalNAc (open ring form).192 The most frequently used b-N-acetylhexosaminidase is that isolated from Jack bean359 (Table 9). It has a very broad activity, releasing all types of GlcNAc(b1-x) units, including the bisecting GlcNAc residue, in N-glycans: ...GlcNAc(b 1-6) ...GlcNAc(b 1-4)----------Man(a 1-6) ...GlcNAc(b 1-2)

GlcNAc(b 1-4)Man(b 1-4)GlcNAc(b 1-4)GlcNAc

...GlcNAc(b 1-4)----------Man(a 1-3) ...GlcNAc(b 1-2)

Therefore, it is an ideal enzyme in sequence analysis studies. Combined with the b-N-acetylhexosaminidase from D. pneumoniae,360,361 having a strict specificity for GlcNAc(b1-2)Man linkages under the right conditions (e.g., proper amounts of enzyme), it is even a more powerful tool in sequence analysis studies of N-glycans. When the GlcNAc(b1-2) Man element is present in tri- and tetraantennary complex-type N-glycans, a further fine-tuning of the diplococcal enzyme has been observed: When present in the branching element GlcNAc(b1-2)[GlcNAc(b1-4)]Man(a1-, the (b1-2) linkage is hydrolyzed; when present in the branching element GlcNAc(b1-2)[GlcNAc(b1-6)]Man(a1-, the (b1-2) linkage is resistant to cleavage. Using the diplococcal enzyme, the presence of a bisecting GlcNAc residue [GlcNAc(b1-4)Man (b1-] inhibits the hydrolysis of the GlcNAc(b1-2)Man linkage in the (a1-6)-arm, but not in the (a1-3)-arm.345 In the case of O-glycans, the diplococcal enzyme has been shown to release GlcNAc, (b1-6)-linked to 2AB-labeled GalNAc (open ring form);192 also GlcNAc(b1-3/6)Gal linkages are cleaved. For the hydrolysis of Glc(a1-2)Glc linkages, as present in the gluco-oligomannose-type N-glycans, a-glucosidase I from porcine liver has been reported,362 and for the hydrolysis of the Glc(a1-3)Glc and Glc(a1-3)Man linkages in these glycans, a-glucosidase II from porcine liver is used.363 The a-mannosidase from Jack bean364 hydrolyzes all a-mannosidic linkages in N-glycans, and is widely used in sequence analysis studies (Table 9). By varying the concentration of Jack bean a-mannosidase, an arm-specificity can be induced; the release of Man from the Man(a1-6)[GlcNAc(b1-2)Man(a1-3)]Man(b1-4)GlcNAc fragment needs a higher concentration of enzyme.346,365 The a-mannosidases I and II isolated from Aspergillus saito are of further interest.366,367 a-Mannosidase I specifically hydrolyzes Man(a1-2)Man linkages, which makes it an interesting enzyme in oligomannose-type N-glycan analysis. Although in principle a-mannosidase II can cleave all a-mannosidic linkages in N-glycans, there is a high ordering in effectivity, for example, the Man(a1-6)Man linkage in the Man(a1-3) [Man(a1-6)]Man element is cleaved only after the Man(a1-3) residue has been split off. For the hydrolysis of the Man(b1-4)GlcNAc linkage, b-mannosidase from the snails Helix pomatia368 and Achatina fulica369 is applied. In glycoprotein glycans, a-Fuc residues occur in several microenvironments, as is shown in the following structural elements, wherein the difucosylated structures also occur in separated monofucosylated forms: Fuc(a1-2)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1Fuc(a1-2)Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1Fuc(a1-2)[Gal(NAc)(a1-3)]Gal(b1-3/4)GlcNAc(b1GlcNAc(b1-4)[Fuc(a1-3/6)]GlcNAc(b1The a-fucosidases from Turbo cornutus,370 Charonia lampas,371 bovine epididymis,347 and bovine kidney192 show a broad specificity (Table 9) with respect to the release of Fuc(a1-2), Fuc(a1-3), Fuc(a1-4), and Fuc(a1-6) residues. For the a-fucosidases from T. cornutus and C. lampas, differences in preference of hydrolysis have been found: Fuc(a1-4) GlcNAc>Fuc(a1-2)Gal>Fuc(a1-3)GlcNAc versus Fuc(a1-2)Gal>Fuc(a1-4)GlcNAc>Fuc(a1-3)GlcNAc, respectively. In the case of C. lampas, a resistance to release Fuc in the Lex element has been reported, so that higher concentrations of enzyme are needed.346,372 Furthermore, C. lampas a-fucosidase releases very efficiently the Fuc(a1-6) residue in N-glycan-derived oligosaccharide-alditols, fucosylated at GlcNAc-ol; in the case of the Fuc(a1-3) variant, a concentration dependency has been observed.372 The a-fucosidase of Corynebacterium species345 specifically cleaves the Fuc(a1-2)Gal linkage, and that of almond emulsin (a-fucosidase I) the Fuc(a1-3)GlcNAc and Fuc(a1-4)GlcNAc linkages as present in outer chain immunodeterminants. Note that (a1-2)-fucosidase is inhibited by the presence of such elements. Furthermore, it should be noted that in polyfucosylated outer chains with Fuc(a1-3)GlcNAc elements


53

steric hindrance for almond emulsin a-fucosidase I has been observed for the inner elements. Almond emulsin a-fucosidase does not release Fuc(a1-6) and Fuc(a1-3) residues in N-glycan-derived oligosaccharide-alditols, fucosylated at GlcNAc-ol.372 Finally, the a-fucosidase from bovine epididymis has a strong preference for Fuc(a1-6)GlcNAc linkages.347 The b-xylosidase from C. lampas cleaves specifically Xyl(b1-2) residues,373 and the a-N-acetylgalactosaminidase from porcine liver GalNAc(a1- linkages.374

2.01.7.4 Profiling of Glycoprotein Glycans Nowadays, three major chromatographic technologies to profile glycoprotein glycans are widely applied: (1) HPAEC combined with pulsed amperometric detection (PAD); (2) combinations of different types of HPLC combined with fluorescent detection (FD); and (3) CE combined with FD. The approaches based on gel electrophoresis (see Section 2.01.7.2) have only partly settled. Besides these approaches to get a qualitative/semiquantitative impression of the ensembles of glycans (e.g., quality control of recombinant expression systems of glycoproteins), MALDI-TOF MS plays an important role. Although having played a dominant and highly important role in the past in setting up structural analysis strategies for N-glycans,180 convential size-exclusion chromatography on Bio-Gel P-4 is not frequently used anymore. It should be noted that the applications of profiling glycoprotein glycans are mainly found in areas wherein the knowledge of biosynthetic pathways is highly developed: higher mammalian glycoproteins, insect glycoproteins, and plant glycoproteins. In fact, quite often use is made of these pathways in assigning glycan structures. Especially, lower animals can have very unusual glycosylation patterns, which need detailed NMR and MS analysis. In the HPAEC-PAD profiling system, free oligosaccharides are separated according to their charge, that is, their numbers of sialic acid residues and their possibilities of oxyanion formation (see Section 2.01.7.1.1). The sensitivity amounts 10–100 pmol. By making use of isolated glycoprotein N-glycans from different biological sources, a database has been constructed containing over 100 N-glycans.375–377 The method has been used to follow the production of recombinant therapeutic glycoproteins in terms of lot-to-lot consistency. A disadvantage of the profiling system is that it can only be used in a semiquantitative way because of the missing knowledge of response factors. Fluorescent 2PA derivatives (see Section 2.01.7.2) are extensively used for the HPLC profiling of N-glycans, and 2-D and 3-D mapping technologies are available.378–387 The initially developed 2-D approach focused only on neutral oligosaccharides. Typically, an N-glycoprotein digest is prepared by incubation with pepsin, and the mixture of glycopeptides is de-N-glycosylated with PNGase-A. Charged components in the obtained N-glycan pool are converted into neutral compounds (e.g., desialylation with a-sialidase), and the neutral N-glycan pool is derivatized with 2-aminopyridine. Then, the mixture of 2PA derivatives is fractionated by reversed-phase HPLC on a CLC-ODS column, followed by size-exclusion HPLC fractionation of each ODS fraction on a TSKgel Amide-80 column. The separation on the ODS column depends on the fine structure of each N-glycan, wherease the separation on the amide column depends mostly on the molecular size of each N-glycan. The calibration of both columns is carried out with a mixture of 2PA (a1-6)-linked glucose oligomers, prepared from a dextran hydrolyzate, making it possible to express the elution times of the N-glycans in glucose units (GUs). Then, the GU values of the various peaks on both columns are plotted on a 2-D glycan map (Y-axis, amide-silica column; X-axis, ODS-silica column) created with standard 2PA N-glycans. The database of standards contains over 220 neutral N-glycans, making structural assignments of regular N-glycans possible in a reliable way. The computerized approach can be combined with sequential exoglycosidase digestions to reach an even higher reliability. Coupling with mass spectrometric analysis is also possible. The profiling technology makes use of a parametrization concept that includes the contribution of constituent monosaccharide units to elution volumes of oligosaccharides on both columns.380,384 The sensitivity of the detection of 2PA derivatives is in the picomole range. The 3-D mapping technique also includes charged N-glycans. To this end, the first separation of the derivatized N-glycan pool is carried out on a TSK-gel diethylaminoethyl(DEAE)-5PW column, yielding neutral, mono-, di-, tri, and tetra-charged pools, according to the sialic acid content of each fraction. Then, each pool of glycans is subjected to HPLC on the ODS column, and each individual ODS fraction is subjected to HPLC on the Amide-80 column; the GU values of the individual peaks on both columns are converted in a 2-D map, as discussed above. By layering the various 2-D maps, a 3-D map is created, which makes structural assignments possible.384a Typical examples comprise the structural analysis of the human IgG,388 recombinant human erythropoietin,389 and human serum glycoproteins385 N-glycans. A similar 2-D approach has also been developed for O-glycans.390 A full description of this technology is presented in Chapter 2.08. In a comparable approach, fluorescent 2AB derivatives (see Section 2.01.7.2) have also been used to develop a sensitive and reproducible HPLC profiling method.191–193,331–333 Typically, an N- or O-glycoprotein is deglycosylated chemically (see Section 2.01.6.2) or enzymatically (see Section 2.01.6.3.1), and the released N- or O-glycans are

54


derivatized with 2-aminobenzamide. In order to minimize desialylation, the derivatization has to be carried out with great care. Each glycan pool containing a mixture of neutral and/or acidic 2AB derivatives is fractionated by HPLC on three different phase types: a weak anion-exchange GlycoSep C/Vydac 301VHP575 column, a normal phase GlycoSep N/TSKgel Amide-80 column, and a reversed-phase GlycoSep R/Hypersil ODS C18 column. In most cases, only the GlycoSep C and N columns are used. In the case of neutral/sialo glycans, fractionation on GlycoSep C gives rise to a separation in neutral and mono-, di-, tri-, and tetra-charged components, according to the sialic acid content of each fraction. In the case of the additional presence of sulfated or (capped) phosphorylated glycans, the charged patterns can be more complex. For evaluation of the peak pattern, a PNGase-F digest of fetuin is used for calibration of the column, indicating the various areas of elution (Figure 36). Fractionation on GlycoSep N, using acetonitrile-water gradients buffered with volatile salts, yields highly resolved profiles of both N- and O-glycan pools (Figure 37). Making use of an external standard dextran hydrolyzate ladder [2AB (a1-6)-linked glucose oligomers], the column is calibrated by plotting the number of glucose residues against the retention times of the peaks, yielding a standard curve using a fifth-order polynomial line fit. Using this curve, elution times of glycans are converted into GU values. For structural assignments, the GU values of unknown glycans are compared with those of a large library of standard N- and O-glycan structures (reference HPLC database/GlycoBase),333a yielding first-line proposals for the constituting glycans.192,332 For automatic assignments, a computer program has been designed that cross-correlates experimental GU values with standard values in the database. Based on the GU values of many standard glycans, incremental values for the addition of monosaccharides to oligosaccharides could be calculated. These incremental data are of great help in additional sequential exoglycosidase studies of isolated fractions (Figure 37), yielding confirmative or corrective evidence for first-line assignments. The structural features, which contribute to the GU values, include arm specificity, linkage of the monosaccharide units, as well as monosaccharide composition. In fact, a plot can be constructed of retention times expressed in GU values (Y-axis) versus masses of labeled glycans (X-axis), showing a straight line. Analyses are carried out on the sub-picomolar level. The developed methodology has been combined with MALDI-TOF MS for composition analysis, and with LC-ES-MS and LC-ES-MS/MS for sequence analysis.391,392 It should be noted that GlycoSep R needs an arabinose ladder instead of a dextran ladder, because labeled dextran oligomers are poorly retained on the reversed-phase HPLC system. The methodology is applied in several ways, that is, structure elucidation of glycosylation patterns, monitoring glycosylation patterns of different batches of recombinant therapeutic glycoproteins as quality control, and examining glycosylation changes in health and disease. In case of complex normal phase HPLC patterns, a prefractionation can be carried out on GlycoSep C, and relevant fractions/pools can be isolated for GlycoSep N experiments. Finally, the methodology is also applicable for the sequencing of N-glycans directly from protein gels, that is, in-gel de-N-glycosylation with PNGase-F followed by normal phase HPLC and combined with MALDI-TOF MS.393–395 Typical examples comprise the structural

1

N

2

3

4

Fetuin

2AB

rEPO-P

rEPO-T

Figure 36 Anion-exchange HPLC profile of the released, 2AB-labeled N-glycans of two different recombinant erythropoietin batches rEPO-P and rEPO-T on GlycoSep C. Fetuin is included as a reference. N, neutral; 1, monosialylated; 2, disialylated; 3, trisialylated; 4, tetrasialylated.


3

4

55

6 Dextran ladder g.u. 7 8 9 10

5

2 (a) Total lgG A2 glycans G1(1,6)F

G2F G1(1,3)F

G0F

G2FS

(b) + Sialidase

G1(1,3)F

G2F

G1(1,6)F G0F

(c) G0F

+ b -Galactosidase

G0

Fluorescence

(d) + b -Hexosaminidase

G0FB

M3N2F

(e) + a -Fucosidase

M3N2

(f) M1N2

+a-Mannosidase

(g) N2

+b-Mannosidase

(h) 40

60

80

100

120

Retention time (min) © 1997 Current Opinion in Biotechnology

Figure 37 Normal phase HPLC profile of the released, 2AB-labeled N-glycans of normal IgG on GlycoSep N. A dextran ladder is included to calculate GU values (a). The pool of N-glycans (b) was sequentially incubated with a series of exoglycosidases as indicated in (c–h). Nomenclature for describing the diantennary oligosaccharides: G(0-2) indicates the number of terminal Gal residues in the structure; F, (a1-6)-linked core Fuc; N, GlcNAc; B, bisecting GlcNAc; S, sialic acid; M, Man; (1,6), (a1-6) arm; (1,3), (a1-3) arm. Rudd, P. M.; Dwek, R. A. Curr. Opin. Biotechnol. 1997, 8, 488–497.

analysis and profiling of human erythrocyte CD59 N-glycans,396 the glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation,397 and the glycan analysis of the prostate-specific antigen from prostate cancer sera.398 Going through the literature, many protocols, including those from manufacturers, have been described for exoglycosidase digestions, and different ensembles of exoglycosidases have been applied in sequence analysis studies. To give an impression of enzyme ensembles used, three examples have been selected (for enzyme specificities, see

56


Section 2.01.7.3). The first example comprises the following exoglycosidases:345 a-sialidase from A. ureafaciens; a-galactosidase from coffee bean; b-galactosidase from Jack bean; b-galactosidase from D. pneumoniae; b-galactosidase from Streptococcus 6646K; b-N-acetylhexosaminidase from Jack bean; b-N-acetylhexosaminidase from D. pneumoniae; a-mannosidase from Jack bean; a-mannosidase I from Aspergillus saitoi; a-mannosidase II from Aspergillus saitoi; b-mannosidase from Helix pomatia; and a-fucosidase from Corynebacterium sp. The second example comprises:332 a-sialidase from A. ureafaciens; b-galactosidase from bovine testis; b-N-acetylhexosaminidase from Jack bean; a-mannosidase from Jack bean; b-mannosidase from Helix pomatia; and a-fucosidase from C. lampas. The third example comprises:192 a-sialidase from A. ureafaciens; a-sialidase from Newcastle disease virus; a-sialidase from D. pneumoniae recombinant in E. coli; b-galactosidase from bovine testis; b-galactosidase from D. pneumoniae; b-Nacetylhexosaminidase from Jack bean; b-N-acetylhexosaminidase from D. pneumoniae; a-fucosidase from bovine kidney; and a-fucosidase from almond emulsin. Quite often, relatively high concentrations of enzyme are proposed to allow the reactions to go to completion, which is important for the reliability of the sequencing methodology.346 However, one should keep in mind that several exoglycosidases have a concentration-dependent specificity, which can generate high detailed information. So, a right play with exoglycosidases is very important. In general, in sequence analysis studies substrates are incubated with a series of exoglycosidases, and after each incubation the effect of the specific incubation is checked following well-documented shifts in retention times (GU values). Also suitable mixtures of exoglycosidases are chosen. A quite different approach is the reagent-array analysis method (RAAM), as has been reported for the profiling of N-glycans.365 Here, a purified oligosaccharide sample is divided into aliquots, and each aliquot is incubated with a precisely defined mixture of exoglycosidases, whereby one mixture contains all the exoglycosidases; one aliquot of the oligosaccharide sample is used as a blank, whereby no exoglycosidases are added. After the digestions are complete, the individual samples plus the original oligosaccharide substrate are pooled and subjected to a single chromatographic analysis. The obtained peak profile is compared with a database of computer-generated simulations of digestions of oligosaccharides to obtain a fingerprint match. The major drawback of this approach is the absolute requirement for noncontaminated exoglycosidases in the enzyme array. Besides HPAEC and HPLC profiling techniques, CE profiling gets more and more attention.279,283,399–401 Although only one glycan-mapping methodology has been developed for nonderivatized N-glycans, with UV detection at 190nm,402 most profiling techniques use fluorescently labeled glycans. Typical examples comprise the use of 2PA derivatives,330,403–405 ANTS derivatives,338 and APTS derivatives.338,406 These methods are also combined with sequential exoglycosidase studies. A full description of the CE technology is presented in Chapter 2.12. Finally, lectin frontal affinity chromatography (FAC) of fluorescent 2PA-derivatized glycans has been proposed as a profiling methodology.407–409

2.01.7.5 NMR Spectroscopy of Glycoprotein Glycans Over the years, NMR spectroscopy has shown its high value in analyzing primary structures of glycoprotein N- and O-glycans. The 1H NMR structural analysis studies of glycoprotein glycans played a major role in unraveling the regular mammalian biosynthetic pathways. The information that can be deduced from NMR spectra is enormous: monosaccharide details, anomeric configurations, linkage types, sequence data including branching, noncarbohydrate substituents. Highly detailed information can be generated, which is extremely useful in the total elucidation of very complex glycan chains, even on the glycoprotein level. Furthermore, the methodology is nondestructive, allowing recovery of the intact sample after analysis. This means that 1H NMR spectroscopy is also highly suitable in evaluating fractionation procedures. A disadvantage compared with HPLC/HPAEC, CE, and MS is that the relatively large amounts of material still needed, commonly at the microgram level. Two comprehensive reviews, including many 1 H NMR spectra, have appeared, one focused on the 1H NMR analysis of N-glycans17 and the other focused on the 1H NMR analysis of O-glycans.18 A general review has been published, also including a survey of previous NMR reviews.410 Each 1H NMR spectrum can be considered as a unique identity card (fingerprint) of the glycan under investigation, and based on the interpretation of many 1H NMR spectra, so-called 1H NMR structural-reporter-group concepts have been developed for both N-glycans (glycopeptides, oligosaccharides, oligosaccharide-alditols) and O-glycans (oligosaccharide-alditols). These structural reporters comprise the positions (d values) and coupling constants (J values) of signals that resonate outside the carbohydrate bulk signal, that is, H1 atoms (anomeric protons), Man H2 atoms, GalNAc-ol H2, H3, H4, and H5 atoms, sialic acid H3 atoms, Fuc H5 and H6 atoms, Gal H3 and H4 atoms, protons shifted out of the bulk region due to glycosylation shifts, protons shifted out of the bulk region due to the presence of noncarbohydrate substituents such as acyl, sulfate, and phosphate groups, and protons belonging to noncarbohydrate substituents such as O-methyl, N,O-acetyl and N-glycolyl groups. For automatic


57

interpretations, an 1H NMR database computer program, called Sugabase, has been developed.411,412,412a In Sections 2.01.2.1, 2.01.6.1, 2.01.6.2, 2.01.6.3.1, and 2.01.6.5, several references are included which illustrate the technology very well.64,69,71–73,76,90,176,177,186–188,190,198–201,221–223,225,254,300 More recent studies focusing on Man-6-Pand GlcNAc-6-P-containing N-glycans have also appeared.310,413 A full description of the use of 1H NMR spectroscopy in glycoprotein glycan analysis is presented in Chapter 2.04.

2.01.7.6 Mass Spectrometry of Glycoprotein Glycans Since the introduction of soft-ionization techniques in mass spectrometry, the possibilities for its use in the primary structural analysis of biomolecules have grown enormously. With respect to the glycoscience field, the technique has become one of the cornerstones. Nowadays, the majority of the structural studies on glycoprotein glycans contains MS data, mainly MALDI-TOF MS and ES-MS data. Because MS is dealing with masses, discrimination between enantiomers, between diastereomers, and between anomers on the monosaccharide level is not easily possible, whereas discrimination between linkage types and between positional isomers (branching) on the glycan level needs advanced MS technologies. In this context it should be noted that many studies use the general knowledge of biosynthetic pathways for the interpretation of MS data, especially in the case of mammalian glycoproteins, a strategy that should be handled with care, certainly when focusing on glycoproteins of nonmammalian origin. Apart from this comment, a typical warning for handling MS data in glycoproteomics applications has been stated recently.414 However, coupled with other technologies such as monosaccharide analysis, methylation analysis, HPLC profiling and/or CE profiling, and using exoglycosidases, the MS applications seem to be without end. Several comprehensive reviews on MS have appeared.23,279,401,415–429 Of specific interest are the strategies that have been developed for the glycan analysis of glycoproteins separated using 1-D and 2-D gel electrophoresis with cutting out gel spots or electroblotting onto membranes/release of N- and O-glycans/analysis of glycans by HPLC, and LC-ES-MS and MALDI-TOF MS,393–395,430–434 as well as the software tools for interpreting glycan MS data.435–440 A few typical examples, illustrating the possibilities, are: MALDI-TOF MS and HPLC-ES-MS studies of recombinant erythropoietin variants;441,442 an HPLC-ES-MS study on murine immunoglobulin M;443 HPLC-ES-MS/MS studies on recombinant human thrombomodulin444 and hepatocyte growth factor;445 a MALDI-TOF MS study, whereby the PNGase-F digestion is carried out on recombinant tissue-type plasminogen activator immobilized on polyvinylidene difluoride membranes;446 a MALDI-TOF MS/HPAEC-PAD study of N-glycans released from glycoprotein bands from isoelectric focusing gels;447 a chemoselective glycoblotting/MALDI-TOF MS study on IgG;448 development of a lectinaffinity technology (‘glyco-catch’) combined with PNGase-F digestion in H18 2 O and a 2-D-LC-ES-MS/MS advanced proteome strategy;409 MS analysis of N- and O-glycosylation of tissues and cells.449 A full description of the use of mass spectrometry in glycoprotein glycan analysis is presented in Chapter 2.02.

2.01.7.7 Glycan Detection and Differentiation Assays/Lectin Microarrays In order to develop simple procedures for glycan detection and differentiation, several approaches have been worked out. In one of these approaches450 aimed at glycan detection, a (glyco)protein mixture is separated by SDS-polyacrylamide gel electrophoresis, then transferred onto a nitrocellulose or polyvinylidene difluoride membrane. The blot is incubated with periodate, whereby bonds between vicinal hydroxyl functions are cleaved (see Section 2.01.5.6). Generated aldehyde functions are aminated with digoxigenin-succinyl-e-aminocaproic acid hydrazide (spacer-linked digoxigenin; DIG). The detection of DIG-labeled bands is carried out in an enzyme immuno-assay using a DIGspecific antibody conjugated with alkaline phosphatase and a substrate to trace alkaline phosphatase activity (5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium chloride). Using the same steroid hapten, information can be generated on the type of terminal glycan elements present (glycan differentiation).450 Now, the nitrocellulose blot is incubated with different DIG-labeled lectins. Bound lectins can be detected with the DIG-specific antibody conjugated with alkaline phosphatase/alkaline phosphatase substrate system. The DIG-labeled lectins included in the technology are D. stramonium agglutinin [DSA; Gal(b1-4)GlcNAc in N- and O-glycans; GlcNAc in O-glycans], Galanthus nivalis agglutinin [GNA; Man(a1-2/3/6)Man in N- and O-glycans], M. amurensis agglutinin [MAA; Neu5Ac(a2-3)Gal], S. nigra agglutinin [SNA; Neu5Ac(a2-6)Gal/GalNAc], peanut agglutinin [PNA; Gal(b1-3)GalNAc] (see also Table 8). A few reports describe the development of lectin microarrays for first-line glycan information in health and disease, that is, quality control of therapeutic recombinant glycoproteins, glycosylation patterns of glycoproteins in cellbiological studies, and changes in glycosylation in disease states. In one model study, six plant lectins (PNA, UEA,

58


TPA/Tetragonolobus purpureas agglutinin, SNA, MAA, DSA; see also Table 8) were immobilized through a biotin– neuravidin–biotin bridge on OptoDex-biotin platforms. The lectins were probed with fluorophore (Cy3 or Cy5)labeled glycoconjugates, including glycoproteins.451 In another approach nine plant lectins [Con A, GNA, GS-I, GS-II (terminal GlcNAc), MAA, SBA/Glycine max (terminal GalNAc), SNA, UEA, WGA/Tritiicum vulgare (b-GlcNAc, sialic acid, GalNAc); see also Table 8] were arrayed on aldehyde- or epoxide-derivatized glass slides. The arrays were incubated with glycoproteins labeled at lysine residues with the fluorescent dye Cy3, and scanned for fluorescent spots indicating carbohydrate–lectin interaction. The system was tested with N- and O-glycoproteins having welldescribed glycosylation patterns;452 see also the development of a lectin microarray approach for the analysis of bacterial polysaccharides/bacterial species.453 Recently, an optimized lectin array-based glycoanalysis methodology has been reported that is kit based. The technology consists of arrays of 24 plant lectins with overlapping specificities. The slides were incubated with glycoproteins, fluorescently labeled with Cy3 or Fluos, or with nonlabeled glycoproteins, subsequently followed by protein-specific nonlabeled primary rabbit IgG antibodies and secondary Cy3-labeled goat antirabbit IgG antibodies. After incubation, the slides were scanned for glycoprotein binding via fluorescence detection, resulting in a characteristic fingerprint. Since the signal intensities on the array do not correspond to the relative abundance of the respective epitopes, an automatic algorithm was constructed for calculating these abundances from the array signals. The generated array-binding pattern provided information on the proportion of the various features within a glycoform population (N-glycans, O-glycans, oligomannose type, complex type, fucosylation, sialylation, etc.), and has been validated for several N- and O-glycoproteins.454 Finally, an interesting technology has been described that makes use of an evanescent wave-excited fluorescence-assisted scanning system, which allows sensitive, real-time observation of multiple carbohydrate–lectin interactions under equilibrium conditions. Arrays of 39 immobilized lectins incubated with various Cy3-labeled glycoproteins and glycopeptides, and tetramethylrhodamine-labeled oligosaccharides were evaluated, showing promising results.455

2.01.7.8 Analysis of Sialic Acids Over the years, several methodologies have been developed for the structural analysis of naturally occurring sialic acids, a family with more than 62 members (Figure 38).85–87,456 Currently, two primary instrumental approaches are in use: (1) analysis of volatile sialic acid derivatives by GLC-EI/MS and (2) analysis of fluorescently labeled sialic acids by HPLC eventually coupled with ES-MS. The volatile sialic acid derivatives include TMS methyl ester derivatives, heptafluorobutylated methyl ester derivatives, and perTMS derivatives. The fluorescent sialic acid derivatives

8-O-Sulfate 9-O-Sulfate

7-O-Acetyl 8-O-Acetyl 9-O-Acetyl 9-O-Lactyl CH3

CH3

8-O-Methyl

O

C

CH C

HO

O OH O

CH3

S O

OR8

OH 6

9-O-Phosphate

9

R9O

4

R7O R5HN

P O

COOH 1

O

OH HO

2

7

8

5 CH3

R4O

C O

N-Acetyl

3 CH3

HOCH2

C

C O O

N-Glycolyl Figure 38 Schematic overview of the family of naturally occurring sialic acids.

4-O-Acetyl


COOH C

H2N

O

HO

N

O

H2N

O

H2C

N

O

59

+

O

CH2 H

C

OH

H

C

OH

AcHN

C

H

AcHN

C

H

HO

C

H

HO

C

H

H

C

OH

H

C

OH

H

C

OH

H

C

OH

CH2OH

CH2OH

Figure 39 Conversion of Neu5Ac into the corresponding fluorescent DMB derivative.

H

G

B O

R5HN

OR2

COOR1

H H

R4O D

R1: Me or SiMe3 R2: SiMe3 R4, R7, R8, R9: Ac, L-lactyl, Me and/or SiMe3 R5: Ac, Gc (+ SiMe3)

H

H

− R2OH; R4OH

C

F

E⬘

− NH2COR5

E

CHOR7 CHOR8

A:M

CH2OR9

B : M+ • minus •COOR1

+•

minus •CH3

C : M+ • minus •CHOR8-CH2OR9 D : C+ minus R2OH minus R4OH E:M

+•

minus •CHOR7-CHOR8-CH2OR9 minus NH2R5

+

F : R8O = CH-CH2OR9 +

•

G : R5NH = CH-CHOR4 H : M + • minus •CH2OR9 minus R4OH minus R7OH Figure 40 Structural analysis of naturally occurring sialic acids by GLC-EI/MS. Survey of the selected fragment ions A-H, worked out for the TMS methyl ester and the perTMS derivatives of N-acylneuraminic acids with O-acyl and/or O-alkyl substituents.

are prepared by reaction with 1,2-diamino-4,5-methylenedioxybenzene (excitation wavelength, 373nm; emission wavelength, 448nm) (Figure 39) or with 1,2-diaminobenzene (excitation wavelength, 230nm; emission wavelength, 420nm). Detailed GLC, HPLC, EI/MS (Figure 40), and ES-MS data have been recently reviewed.87

2.01.8 Final Remarks In this chapter, the major methodologies used nowadays in the structural analysis of polysaccharides and glycoprotein glycans have been reviewed. Similar methodologies are also applied in the structural analysis of glycolipids, proteoglycans, and glycosylphosphatidyl inositol membrane anchors. Glycolipids are analyzed as such457 and as oligosaccharides, released chemically by ozonolysis/alkaline fragmentation458 or enzymatically by endoglycoceramidase.459,460 Like for glycoprotein glycan profiling, also for glycolipid glycan profiling 2-D HPLC mapping methods, using

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fluorescently labeled oligosaccharides, released enzymatically from glycosphingolipids, have been published.461 In proteoglycan studies, fragments for structural analysis are mainly generated via (de-N-acetylation) – deamination or via lyase degradation (see Sections 2.01.5.7 and 2.01.5.9). Glycosylphosphatidyl inositol anchors are analyzed along various routes, including enzyme digestions and special hydrolysis techniques in order to prepare suitable glycans for further instrumental analysis.462,463

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Biographical Sketch

Johannis P. Kamerling (1944) studied chemistry at Utrecht University (1962–1969), and obtained his Ph.D. degree in 1972 with Prof. J. F. Arens and Prof. J. F. G. Vliegenthart. His glycoscience research is focused on (1) structural studies on polysaccharides and glycoprotein glycans (since 1969); (2) organic/enzymatic synthesis of (conjugated) carbohydrates (since 1984); and (3) carbohydrate-mediated interaction studies (since 1990). Since 1969, he is connected to the UU Department of Bio-Organic Chemistry. In 1974 he was a visiting scientist with Prof. B. Lindberg at the University of Stockholm (Sweden), and in 1981 with Prof. A. Kobata at the University of Kobe (Japan). In the period 2000–2003, he was Dean of Studies of the Faculty of Chemistry. Since 2002, he is Chairman of the Department of Bio-Organic Chemistry. In 1990, in Yokohama (Japan), he was honored with the prestigious Roy L. Whistler Award of the International Carbohydrate Organization. He is Doctor Honoris Causa of the Lajos Kossuth University of Debrecen, Hungary (1999), and Profesor Invitado of the University of Havana, Cuba (2002). Since 2003, he is the Dutch representative in the European Carbohydrate Organization (ECO) and in the International Carbohydrate Organization (ICO). Since 2007, he is secretary of the ICO. He has been/is editor and board member of several scientific journals, and acted as secretary of the XIIth International Carbohydrate Symposium (1984), the 9th European Carbohydrate Symposium (1997), and the XVIth International Symposium on Glycoconjugates (2001). He is (co-)author of over 390 scientific papers/books/ reviews, and has supervised 45 doctoral theses at Utrecht University.

In 1969, Gerrit J. Gerwig (1946) started as Chemical Laboratory Technician at the Department of BioOrganic Chemistry, Utrecht University, headed by Prof. J F. G. Vliegenthart. After his part-time study to become a Biochemical Research Technician (1969–1971) and his MO study in Physics and Chemistry at Utrecht University (1972–1976), he obtained his Ph.D. degree in 1991 with Prof. J. F. G. Vliegenthart and Prof. J. P. Kamerling. His glycoscience research program includes the structural analysis of complex carbohydrates and the development of analytical methods, using NMR spectroscopy and mass spectrometry. In the period 1987–1998, he was a board member of the Faculty of Chemistry of Utrecht University. Since 1978, he is a member of the Netherlands Society for Glycobiology. He is (co-)author of over 75 scientific papers.

2.02 Mass Spectrometry of Glycoprotein Glycans: Glycomics and Glycoproteomics A. Dell, S. Chalabi, P. G. Hitchen, J. Jang-Lee, V. Ledger, S. J. North, P.-C. Pang, S. Parry, M. Sutton-Smith, B. Tissot, H. R. Morris, M. Panico, and S. M. Haslam, Imperial College London, London, UK ß 2007 Elsevier Ltd. All rights reserved. 2.02.1 Introduction

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2.02.1.1 Scope of the Chapter

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2.02.1.2 Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)

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2.02.1.3 Electrospray Ionization Mass Spectrometry (ES-MS)

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2.02.1.4 Tandem and Ion Trap Mass Spectrometry (CAD-MS/MS)

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2.02.1.5 Fragmentation Pathways

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2.02.2 Glycomics

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2.02.2.1 Overview

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2.02.2.2 Release of Glycans from Glycoproteins

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2.02.2.3 Derivatization of Glycans

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2.02.2.4 MALDI-TOF Profiling

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2.02.2.5 Analyzing Charged Glycans

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2.02.2.6 CAD-MS/MS Sequencing

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2.02.2.6.1 2.02.2.6.2 2.02.2.6.3

ES-MS/MS using Q-TOF instrumentation ES-MS/MS using ion trap instrumentation Fragmentation using MALDI-TOF/TOF instrumentation

2.02.2.7 Chemical and Enzymatic Digestion

77 78 81

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2.02.2.8 Linkage Analysis

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2.02.2.9 Tagging Methodologies

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2.02.3 Glycoproteomics

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2.02.3.1 Overview

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2.02.3.2 Analysis of Murine CD8

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2.02.3.3 Analysis of Murine Zona Pellucida

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2.02.3.4 Analysis of Prokaryotic Glycoproteins

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2.02.4 Toward Automation

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2.02.4.1 Automation of Data Interpretation

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2.02.4.2 Automation of Sample Handling

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2.02.1 Introduction 2.02.1.1 Scope of the Chapter It is now well established that complex carbohydrates as constituent cell surface glycoconjugates are involved in the mediation of cell–cell interactions, cell adhesion, and cell migration. In order to elucidate the roles that carbohydrates play in such processes, their molecular structure requires assignment. In recent years, this has led to the development of a new analytical field of glycomics, which aims to define the entire set of glycans present in a defined system whether it be an individual cell type, tissue, or whole organism such as Caenorhabditis elegans. Knowing the glycome of a tissue or cell type is a vital first step to exploring the roles glycans play in cell communication. But determining how glycan-binding proteins engage with their specific ligands and the functional consequences of this recognition

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requires research efforts which are underpinned by more precise knowledge of the structures of individual glycoproteins and even the glycan structures present at individual glycosylation sites. This has led to the development of glycoproteomics. The basic principles of the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), electrospray mass spectrometry (ES-MS), and tandem mass spectrometry (MS/MS) methodologies, that are used to generate the data in both glycomic and glycoproteomic work, are covered in this section and illustrated by specific structural studies in later sections. An additional important methodology used in such studies, gas chromatography–mass spectrometry (GC–MS), is dealt with in greater detail elsewhere in this volume. Finally, it should be pointed out that interpretation of the generated MS glycan data, especially for mammalian glycoproteins, is heavily dependent on a prior knowledge of biosynthetic information.

2.02.1.2 Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) In the MALDI-MS experiment, the sample to be analyzed is co-crystallized on a metal plate with a large excess of a low molecular weight, UV-absorbing matrix. The plate is then introduced into the high-vacuum MALDI source and the crystal is irradiated with intense laser pulses, which excite the matrix molecules. This leads to the sputtering of analyte and matrix ions from the surface of the crystal. Depending on the type of analyte, different matrices can be chosen to optimize sensitivity. For example, 2,5-dihydroxybenzoic acid (DHB) is ideal for native and derivatized glycans, whereas a-cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid are usually used for peptides/glycopeptides and protein analysis, respectively. Most commercially available MALDI mass spectrometers have a pulsed nitrogen laser of wavelength 337 nm, although solid-state lasers (e.g., Nd-YAG), which permit smaller areas of irradiation and faster acquisition, are being incorporated into some of the newer instruments. MALDI is a ‘soft’ ionization method and yields singly charged molecular ions regardless of the molecular weight, with minimal fragmentation. Thus, it is an ideal technique for mass profiling of mixtures of glycans.

2.02.1.3 Electrospray Ionization Mass Spectrometry (ES-MS) ES-MS is a method by which a stream of liquid containing the sample of interest is introduced into the atmospheric pressure ion source of a mass spectrometer via a metal-coated capillary. By the appropriate application of voltages on the capillary, counter-electrode, and focusing optics, an aerosol of highly charged microdroplets is generated in the source, which traverses a series of skimmers, encountering a drying gas, the net effect of which is the creation of gaseous ions, devoid of solvent, whose charge distribution is proportional to the number of ionizable groups in the molecule. This ability to form multiple charged species permits the analysis of very large components by bringing their observable mass/charge ratio (m/z) to within the mass limit of the MS analyzer. The ionization process in ES-MS is very gentle resulting in little or no fragmentation, although this can be engineered to some extent by varying source voltages, particularly the ‘cone.’ The original ES technique involved flow rates of about 1–10 ml min–1. Nano-ES is a variation of this procedure, whereby the flow rate is reduced to as little as 10–40 nl min–1, resulting in far less sample being consumed, allowing adequate time for the selection of many molecular ions for MS/MS analysis in a single experiment. These flow rates are also compatible with online nano-LC-ES-MS analysis (see Section 2.02.3).

2.02.1.4 Tandem and Ion Trap Mass Spectrometry (CAD-MS/MS) As noted earlier, MALDI and ES experiments yield very few fragment ions. While this can be an advantage for mass fingerprinting (see Section 2.02.2.4), it is clearly a problem for more detailed structure analysis. Fortunately, this can be solved by mass spectrometers which have at least two analyzers in tandem or which have ion trap analyzers. In tandem instruments, the first mass analyzer is used to select a precursor ion of interest. This ion is passed into a pressurized collision cell containing an inert gas such as argon or xenon. As the ion traverses the cell, it undergoes collisonally activated decomposition (CAD) (also known as collisionally induced dissociation or CID). The resulting fragment ions are then separated in the second mass analyzer to yield a spectrum of fragment ions. In ion trap instrumentation, the trap acts as both the analyzer and the collision chamber. First, the precursor ion of interest is retained in the ion trap while all other ions are expelled. Then the trap is pressurized with an inert gas. Fragment ions produced by collisional activation are then sequentially expelled from the trap and the spectrum is recorded. An advantage of the ion trap is that individual fragment ions can be retained in the ion trap and subjected to subfragmentation by further collisional activation. This so-called MSn (multiple-stage mass spectrometry) technology may be helpful for the assignment of structural features such as linkages and branching patterns. A great variety of tandem and ion trap instruments are commercially available. Among the former, the Q-TOF (quadrupole orthogonal-acceleration time-of-flight) mass


71

spectrometers, and the more recently introduced TOF/TOF instruments, are currently the most powerful instruments for glycoproteomic and glycomic analysis, respectively.

2.02.1.5 Fragmentation Pathways The majority of fragment ions are produced by cleavage on either side of glycosidic bonds to yield nonreducing and reducing-end fragment ions, respectively. These ions provide sequence and branching information. Ring fragmentation can also occur, provided the MS/MS experiments are carried out under conditions that permit high-energy collisional activation. This type of fragmentation is helpful for assigning linkages. The most common pathways are illustrated in Schemes 1–4 and systematic nomenclature for the resulting fragment ions is shown in Figure 1.1 In summary, the main fragmentation pathways common to all classes of polysaccharides and glycoconjugates are as follows: 1. A favored cleavage in the positive ion mode occurs on the nonreducing side of the glycosidic bond to form an oxonium ion (Scheme 1), which may carry sodium if the parent ion was sodiated prior to cleavage. This type of fragmentation is often referred to as A-type cleavage. A-type cleavage is favored at HexNAc residues and is usually accompanied by secondary fragmentation involving b-elimination of the substituent at position 3 on the ring (Scheme 2). 2. Glycosidic cleavage occurs with a hydrogen transfer, with charge residing on either the reducing or nonreducing ends depending on which bond to the glycosidic oxygen is cleaved. This fragmentation is often referred to as b-cleavage and occurs in both positive and negative modes (Scheme 3). Tagging glycans with reducing-end reagents (see Section 2.02.2.9) will enhance the formation of reducing-end ions. 3. In ring cleavage, the charge can remain on either the reducing or the nonreducing end, depending on the nature of the sample and whether positive or negative ions are being detected (Scheme 4).

2.02.2 Glycomics 2.02.2.1 Overview Figure 2 depicts strategies developed in our laboratory for profiling the N- and O-glycan repertoire of cells, tissues, fluids, etc. (Chapters 3.02 and 3.03).2–4 Y2

Z2

1,5

X1

CH2OH

CH2OH

OH

0,2

OH

HO 2,5

2,4

B1

A2

C1

Z0

O

R

O O

HO

Y0

CH2OH

O

HO

A1

Z1

O

O

HO

Y1

B2

C2

A3

OH

B3

C3

Figure 1 The systematic nomenclature introduced by Domon and Costello.1 A, B, and C refer to nonreducing terminal fragments, whereas X, Y, and Z represent reducing terminal fragments.

CH2OR

CH2OR

O

O

+ O

O

CH2OR A-type cleavage O

+ O

O

H RO

OR

RO

OR

RO

OR

Oxonium ion Scheme 1 Glycosidic cleavage to form an oxonium ion.

72


m/z 825 Elimination of substituent at C3

CH2OMe + O H MeO

Type 1 -(Galb1-3GlcNAc)-

NeuAc Gal O

m/z 228 CH2OMe + O MeO

NMeAc

NMeAc

m/z 825 Elimination of substituent at C3

CH2OMe + O H NeuAc Gal O

Type 2 -(Galb1-4GlcNAc)-

MeO

m/z 793 CH2OMe + O NeuAc Gal O

NMeAc

NMeAc

Scheme 2 b-Elimination from the 3-position of oxonium ions.

+ or − CH2OR

CH2OR O

O

CH2OR + or − H+

O

O H OR

RO

+ or −

O RO

+ O + or − H O

HO

OR

RO

OR + or −

+ or − CH2OR

CH2OR O

O O

CH2OR + or −

O RO

OR

O H RO

+ O + or − H OH

H+

OR

O RO

OR

Scheme 3 Glycosidic cleavages with hydrogen transfer, charge retained on the reducing end (upper) and nonreducing end (lower); sometimes called b-cleavages. In the positive mode, the ions may be protonated as shown or sodiated.

These protocols have been adopted by the Consortium for Functional Glycomics which is assembling a resource of mouse and human glycomic data.4a Similar strategies are employed in other laboratories, although specifics may vary, for example, in the choice of derivative and/or MS methodology. Briefly, MALDI-TOF of permethylated glycans provides mass profiles at high sensitivity, while MS/MS experiments, complemented by linkage analysis and a variety of chemical and enzymatic procedures, yield detailed sequence information. In all experiments, data interpretation is facilitated by knowledge of biosynthetic pathways. The major steps are described in the following sections.

2.02.2.2 Release of Glycans from Glycoproteins Liberation of N-glycans from glycoproteins can be achieved either chemically using hydrazine5 or enzymatically with PNGase F or PNGase A. Although hydrazinolysis is an effective method to completely remove both N- and O-glycans, hydrazine is a highly explosive reagent and can be difficult to obtain. Hence enzymatic release is


+ or −

+ or −

+ or − H+ CH2OR

O

O RO

CH2OR

O

O

O

(a)

+ or − H+

CH2OR

O OR

H

O RO

O

O RO

OR

OR

+ or − + or −

O

O

O

RO

+ or −

OR

O

RO

H+

CH2OR

O

O

(b)

+ or −

H+

CH2OR

CH2OR

73

O

O

OR

RO

RO

OR

+ or − + or − + or − H+ CH2OR

CH2OR

O

CH2OR

O

O

(c)

O

O RO

+ or − H+

OR

O RO

OR

O

O RO

CH2OR

OR

Scheme 4 Examples of ring cleavages.

preferred. PNGase F is the most effective enzymatic method for removing almost all of the N-linked glycans from glycoproteins.6,7 The glycans are released as intact oligosaccharides, and the only change in the protein backbone arises from the deamination of the substituted asparagine residue to aspartic acid. Although PNGase F is suitable for most applications, it does not cleave N-glycans that have an a1!3-linked fucose attached to the reducing GlcNAc.8 Such modifications occur in glycoproteins obtained from plants and invertebrates, and release of N-glycans from these organisms is achieved by the enzyme PNGase A from almonds. In general, glycoproteins must be denatured for PNGase F and A to act efficiently, as sites of N-glycan attachment can be inaccessible when the glycoprotein is in its native form. This problem is routinely overcome by reduction/alkylation of the glycoprotein followed by digestion with trypsin. O-Glycans are most commonly released chemically using alkaline b-elimination under reducing conditions to prevent degradation of the glycans by the ‘peeling’ reaction. Although an enzyme, O-glycosidase, is available to release Oglycans, it is not as versatile as PNGase F. O-Glycosidase has a restricted specificity and it only releases the simple core type 1 sequence (Galb1!3GalNAc) from proteins. Therefore, this enzyme must be used in conjunction with additional enzymes to remove more complex O-linked structures ,9,10 making it unsuitable as a structure analysis tool.

2.02.2.3 Derivatization of Glycans Purified, intact glycans can be analyzed directly by MALDI-TOF or derivatized first and then analyzed. As carbohydrates do not ionize as efficiently as other molecules such as peptides,11 direct analysis of underivatized glycans by MS is not very sensitive. Derivatization of the glycans by permethylation or peracetylation significantly improves the sensitivity of detection and is a frequently used strategy in glycan analyses.12,13 Permethylation

74


Tissues/cells

Carboxymethylation, proteolysis, C18 chromatography

PNGase F/ PNGase A Enzymic/ chemical digestion

Red u elim ctive inat ion

Permethylation, C18 chromatography

Enzymic/ chemical digestion

Figure 2 Schematic representation of a glycomic strategy. Glycoproteins are extracted from tissues or cells by a detergent extraction buffer. Disulfide bonds are reduced and protected to increase the efficiency of subsequent enzyme digests. Trypsin is the usual protease of choice to generate glycopeptides/peptides, which can be utilized for glycoproteomic studies. N-Linked glycans are enzymatically cleaved from the peptide backbone by digestion with PNGase F and subsequently purified on a Sep-Pak C18 reverse-phase cartridge. O-Glycans are chemically released from glycopeptides by reductive elimination. Aliquots of the purified N- and O-Glycans are permethylated and initially screened by MALDI-TOF MS. Data generated from this initial screening is used to direct subsequent additional experiments such as chemical and enzymatic hydrolyses and MS/MS experiments.

catalyzed by methylsulphenyl carbanion14 or sodium hydroxide15 gives the smallest increase in molecular weight of the sample, and the greatest enhancement in sensitivity. In addition, permethylated glycans fragment very selectively resulting in a limited number of structurally diagnostic fragment ions which are relatively easy to interpret (see Section 2.02.2.6).12 Thus, permethylation is the method of choice for most glycomic investigations. Some organisms, such as the nematodes, C. elegans16 and Toxocara,17 contain a degree of natural methylation on their glycans. In these cases, permethylation masks the native methyl groups and information will be lost by derivatization. This problem can be overcome by using deuteromethyl iodide instead of methyl iodide. Under these conditions each methyl group derived from the derivatization process can be discriminated from the naturally occurring methylation based on mass difference (18 mass units for a CD3 group versus 15 mass units for a natural CH3 group (Table 1)). Perdeuteromethylation can also be used to increase the level of confidence in assignments made from a standard permethylation experiment. The MALDI-MS profiles of the ions generated by glycans derivatized with the two different reagents should be the same except that the m/z values of the perdeuteromethylated glycans will be greater by a predicted amount than that of the permethylated counterparts (Table 1).

2.02.2.4 MALDI-TOF Profiling A MALDI-MS spectrum of permethylated glycans typically consists of a series of singly charged [MþNa]þ molecular ions. Although MALDI-MS is a soft-ionization technique, fragment ions such as A-type ions12 are occasionally observed, which are useful in determining the structure of the glycan antennae (see Scheme 1). Interpretation of MALDI-MS spectra is carried out by assigning the compositions of molecular ions in terms of the number of their monosaccharide constituents (Table 1). Together with the knowledge of the glycan biosynthetic pathways, putative glycan structures can be assigned. Recently, an algorithm ‘Cartoonist’ has been developed, which provides an automated interpretation of MALDI-MS spectra (see Section 2.02.4.1).


75

Table 1 The mass of a (deutero)permethylated glycan is calculated by adding the masses of the ends (shown in (a)) to the increment mass of each sugar (shown in (b)) (a): Non-reducing (R) and reducing ends (OR) Oligosaccharide structure H

CH2OR O H H OR H

RO

H

CH2OR O H H OR H

OR

CH2OR O H OR H

O

O H

H

H

OR

n

OR

Permethylated mass of ends

Perdeuteromethylated mass of ends

R þ OR ¼ 46

R þ OR ¼ 52

H H

OR

R ¼ H for underivatized oligosaccharides R ¼ CH3 for permethylated oligosaccharides R ¼ CD3 for deuteromethylated oligosaccharides (b): Monosaccharide residues Monosaccharide Deoxyhexose Fucose (Fuc) Hexose Mannose (Man) Galactose (Gal) Glucose (Glc) N-Acetylhexosamine N-Acetylgalactosamine (GalNAc) N-Acetylglucosamine (GlcNAc) Sialic acid N-Acetylneuraminic acid (NeuAc) N-Glycolylneuraminic acid (NeuGc)

Symbol

Permethylated mass

Perdeuteromethylated mass

174

180

204 204 204

213 213 213

245 245

254 254

361 391

376 409

Sugar symbols used throughout this chapter are those employed by the Consortium for Functional Glycomics.

Partial glycomic data from human neutrophil N-glycans, which exemplify a typical MALDI-MS profiling experiment, are shown in Figure 3a (more complete data are publicly available on the Consortium for Functional Glycomics website.4a Using the monoisotopic ion (i.e., the ion corresponding to the C12 isotope peak (see Figure 3b)), putative structures can be assigned based on the usually unique glycan composition for a given molecular weight. For example, compositions of the type HexnHexNAc2 are high-mannose structures (e.g., m/z 2396.1 in Figure 3a), whereas those containing three HexNAc residues are likely to be hybrid structures or biosynthetic intermediates of complex structures. Oligosaccharides with four or more HexNAc residues are likely to be complex glycans. Since glycans are made from common biosynthetic pathways, intermediates in the same pathway are expected to be observed in the spectrum. In Figure 3a, there is a relatively intense ion at m/z 2605.0, which corresponds to a putative biantennary sialylated structure with a single fucose. Addition of another fucose (þ174) or sialic acid (þ361) to this glycan results in ions at m/z 2779.0 and 2965.9, respectively, both of which are observed in the spectrum at significant intensity. This indicates that active fucosyltransferases and sialyltransferases were present in the sample under investigation, a feature that is reflected by the high levels of fucose and sialic acid in the other putative structures annotated in the spectrum. The sample also has a significant level of GlcNAc transferase activity since there are a large number of ions corresponding to putative higher-order antenna structures. Since terminal GlcNAc residues are often quickly capped by galactose, the mass increment of the resulting HexHexNAc addition is þ449. Hence, the intense ion at m/z 3053.9 likely corresponds to the glycan at m/z 2605.0 carrying an extra HexHexNAc disaccharide. In the spectrum shown (Figure 3), this has been drawn as a tri-antennary structure, but it could also correspond to a biantennary structure with an extended antenna. Further experiments must be undertaken to distinguish these structures. Another important consideration in assigning structures to molecular ions is the species and sample under investigation. For example, the Gal-a-Gal epitope is a common terminal structure in mice but this moiety is not observed in humans. Furthermore, although NeuGc-containing glycoconjugates are found in most mammals, NeuGc is barely detectable in normal human tissues.18,19 Indeed, even within mammals, NeuGc is not detected in the brain. Diseased tissues or cell lines derived from diseased cells may contain NeuGc and other unusual structures, so it is vital that the source of the sample is considered during the assignment of structures. As described earlier, permethylation of glycans improves the sensitivity and interpretability of fragmentation. However, this process can also produce a series of ions indicating species 30 Da larger than the fully methylated

76


100

x1 x1

90 x1

2779.0

x1 x3

80

x2 x2

70

x1

2401.1 2418.1

4024.6 3414.8

% Intensity

60

x1 3040.9

50 40

3053.9

4037.6

3052.8

x1 x1

x1 x2

3676.7

3850.7

x2

2396.1

x1 x2

x1 x1

2605.0 2192.2

3863.6

x1 x1

x2 x1

30

2965.9 2592.1

3214.9

3401.8

3227.9

x1 x1

x1

20

3588.8

2244.1

2431.0

0 2160

3141.9

2867.0

2693.1

2560

x1

x1

x1 4125.6

10

(a)

x2 x1

x1

2960

3775.7

3315.8

3360

3760

3951.6

4160

Mass (m/z)

100

x1 x1

90

3141.9 100

x1

3227.9

80 70 3053.9

%Intensity

3139.8 50

% Intensity

60 50

0 3130

x2

3136

3142 3148 Mass(m/z)

3154

3160

40 30

3040.9 3214.9

20 10

(b)

0 2980

3044

3172

3108

3236

3300

Mass (m/z)

Figure 3 MALDI-TOF mass spectrum of N-glycans released from human neutrophils. The N-glycans of human neutrophils were released from tryptic glycopeptides by digestion with PNGase F, separated from peptides by Sep-Pak purification and permethylated. The derivatized glycans were purified by Sep-Pak C18 and the 50% (v/v) aqueous acetonitrile fraction was screened by MALDI-TOF (a). A region of the spectrum has been magnified to show greater detail (b). Putative structures based on knowledge of N-glycan biosynthetic pathways and glycan composition are shown. For convenience, only one branching pattern for tri-antennary structures is shown. Monosaccharide symbols are shown in Table 1.

carbohydrate molecules. These species, which are typically between 0% and 10% of the intensity of the fully methylated glycan ions, are thought to be caused by a side reaction between the permethylation reagents resulting in incorporation of a methoxymethyl derivative instead of a methyl group.20 This is particularly important to be aware of in MALDI profiling as the mass difference between a Fuc (174 mass units) and a Hex (204 mass units), and between NeuAc (361 mass units) and NeuGc (391 mass units), is 30 mass units. If the þ30 mass unit artifact is at significant levels in a sample, it should be associated with every peak as it is a nonspecific effect. MS/MS can be used to unequivocally determine whether a particular glycan contains the modification.


77

Throughout the preceding discussion it should be apparent that, although MALDI-MS spectra provide a semiquantitative glycan profile, assignment of structures based only on the compositional data derived from these spectra is essentially an educated guess. To strengthen the reliability of the interpretation, further experiments must be undertaken. MS/MS (Section 2.02.2.6) can be used to sequence specific glycans, whereas linkage analysis by GC–MS (Section 2.02.2.8) can be used to define the linkages present and the terminal monosaccharides in the glycans. Furthermore, MALDI-MS profiling after glycosidase/glycosyltransferase reactions can also be used to define structures and has the advantage that it can also distinguish between anomeric configurations (Section 2.02.2.7). Together, these data can be used to build up an accurate picture of the structure of the glycans present in a sample.

2.02.2.5 Analyzing Charged Glycans Permethylation underpins many glycomic investigations; however, it does have one drawback. Charged glycans, such as those carrying sulfate or phosphorylcholine groups, are usually lost during the cleanup step following the derivatization, meaning that only neutral and sialylated glycans are normally observed. To analyze charged glycans, several different approaches may be taken. Firstly, glycopeptides may be analyzed directly by MALDI-MS or ES-MS and the glycan component calculated (see Section 2.02.3). However, the sensitivity of analyzing glycopeptides is rarely comparable to that for permethylated glycans, so a method involving derivatization is usually preferred. Sulfated glycans can be recovered after permethylation under Hakomori conditions (see Section 2.02.2.3), provided suitable cleanup is employed,21 but these procedures are experimentally more challenging than hydroxide-catalyzed permethylation. One approach for detecting the presence of phosphorylcholine (PC) involves splitting a sample of N-glycans into two, and screening one portion for the presence of components lacking PC moieties using the standard glycomic protocols. The second portion is treated with aqueous hydrofluoric acid (HF) using conditions that are known to cleave phosphodiester linkages. The products are permethylated and analyzed by MALDI-MS. Molecular ions that are observed only after HF treatment are indicative of components in the native sample that have HFsensitive functional groups, such as PC.22 PC-modified glycans can be directly detected if peracetylation or perdeuteroacetylation is used instead of permethylation, but glycans having multiple PC groups are problematical for MS analysis due to their Zwitter-ionic nature. Thus, although many glycans carrying sulfate or other charged groups have been successfully analyzed using mass spectrometric strategies, devising high-throughput glycomic protocols that are compatible with this type of glycan remains a challenge for the future.

2.02.2.6 CAD-MS/MS Sequencing Although biosynthetic information can be employed to attribute tentative sequences to compositions arising from MALDI-TOF profiling, further experiments are required for rigorous structure assignment. CAD-MS/MS (see Section 2.02.1.4) is a powerful strategy for accessing the fine structure of an oligosaccharide by breaking it into smaller pieces and analyzing the fragments (see Section 2.02.1.5 for a summary of the most important glycan fragmentation pathways). Derivatization plays a pivotal role in MS/MS of glycans. In addition to vastly improving the sensitivity (see Section 2.02.2.3), derivatization facilitates interpretation of MS/MS data in several different ways. Thus, it has the advantage of directing fragmentation along a few well-defined fragmentation pathways, leading to a restricted number of daughter ions. Also, fragmentation of permethylated samples is reliable and predictable.12 Importantly, by taking into account the number of methyl groups on each fragment ion, unambiguous information about the branching, the sequence, and sometimes about the nature of the linkage can be obtained from a permethylated sample. In contrast, underivatized glycans frequently yield ambiguous data because of multiple cleavage events. To maximize structural information from MS/MS experiments, it is helpful to have a range of instrumentation enabling both low- and high-energy collisional activation. This is because glycosidic cleavages (Schemes 1–3) occur with low collision energies which are readily achievable with many instruments, including the widely available Q-TOF-type instrumentation (see Section 2.02.1.4), while ring cleavages (Scheme 4) require the higher collision energies associated with ion traps and TOF/TOF instruments (see Section 2.02.1.4). The power of MS/MS technology for glycomic investigations is illustrated in the following sections. The examples selected for discussion exemplify MS/MS instrumentation that is widely used for glycomic analysis.

2.02.2.6.1

ES-MS/MS using Q-TOF instrumentation

This technology is illustrated by data from a structural study of the ovarian tumor marker CA125. The full characterization of the oligosaccharides present on this mucin, which is rich in both N- and O-glycans, is described elsewhere.23

78


100

13432+ 2+

90

1213

486

2+

1111

2+

2+

1343

[M+2Na]

80 660 1562 1766 2+ 1024

70

474

% Intensity

60 50 40

227 486 474

30

660

859

20 10

891

10242+ 11112+ 2+ 1213

1562

1766

0 200

400

600

800

1000 1200 Mass (m/z)

1400

1600

1800

2000

Figure 4 CAD-MS/MS spectrun of the [M+2Na]2+ molecular ion of Fuc2Hex5HexNAc5. The major component assigned from the fragment ion data is shown in the schematic. For key to symbols, see Table 1.

ES-MS/MS sequencing of one of its N-glycans is discussed here. MALDI-TOF profiling of the total N-glycan population revealed dozens of glycans among which was a component of m/z 2664 corresponding to a sugar composition of Fuc2Hex5HexNAc5. This component was selected for MS/MS analysis using a nanospray Q-TOF instrument. The doubly charged parent ion at m/z 1343 was selected for collisional activation and the MS/MS spectrum is shown in Figure 4. The singly charged A-type ion at m/z 660 together with the doubly charged glycosidic cleavage ion at m/z 1024 (z ¼ 2) demonstrate the presence of a fucosylated antenna. The ion at m/z 474 corresponds to the reducing-end FucHexNAc and is formed by cleavage of the glycosidic bond between the two GlcNAc residues of the chitobiose core. This ion is diagnostic of core fucosylation. The doubly charged ion at m/z 1213 (loss of a nonreducing HexNAc) suggests that the structure is bisected. This interpretation is supported by the singly charged ion at m/z 1766 resulting from double cleavage of the bisecting HexNAc and the fucosylated antenna. Assignments and key signals are given in the insert to Figure 4.23

2.02.2.6.2 ES-MS/MS using ion trap instrumentation Many of the first-generation ion trap instruments are not ideal for glycomics. For example, they are often less sensitive and spectra are less well resolved than for the Q-TOF-type instruments. In addition, the mass range of the fragment ions is dictated by the size of the precursor ion and small fragment ions are thus often impossible to detect. Nevertheless, their MSn ability can be extremely useful for providing information concerning branching and also for helping in resolving isobaric mixtures. The last point is illustrated in the following example. In the determination of the structure of a permethylated oligosaccharide from chicken ovalbumin, the MSn ability of the ion trap was employed to distinguish the two isobaric structures I and II corresponding to the same Hex5HexNAc5 composition (Scheme 5). The MS/MS spectrum of the [Mþ2Na]2þ ion at m/z 1169.5 shows ions specific to each of the isobaric structures as well as ions common to both of them.24 Because both parents are fragmented at the same time, no clear structural assignments can be made from the MS/MS data. However, selecting one or more of the specific ions for further fragmentation provides unambiguous information on each of the isobaric structures. Information deduced for compound I is described here. The MS2 spectrum of ion m/z 1169.5 contains the doubly sodiated m/z 937.7 ion which comes from the loss of a terminal HexHexNAc moiety carrying no substituent which can only occur in compound I. Therefore, m/z 937.7 was chosen for MS3 analysis. Fragmentation of this ion leads to a major doubly sodiated ion at m/z 808.0 corresponding to a


79

CH2OR Y4d

O OR RO

O CH2

RO

Y3d

O

0,4A 4

RO

O

B3d

CH2OR

O

RNAc

B5

B4

O

CH2OR

RNAc

OR

OR

O Y4b

OR

OR

RNAc

C4

RNAc

O

OR

O

RO

O

O

O

RO

O

OR

O

OR

CH2OR

CH2OR

CH2OR

CH2

O

OR

CH2OR

O

RO

Y3g

O

B2b CH2OR

3,5A 6

3,5A

5

RO

Y3a

O

HexNAc5Hex5-I

O

OR

RO Y4a

RNAc

CH2OR 1,5X

5e

Y4e

O OR RO

Y3d

RO

O CH2

CH2OR 1,5

C2d

O X5d

O

A3

O

RO

OR RO

RO

0,4

RO

CH2OR Y CH2 3g O O

O Y4d

OR

O

RO

CH2OR

CH2OR

Y4b

CH2OR

CH2OR O

O C3

OR

RNAC B4

O

OR

OR

RNAC

O

O OR

O

OR

B3

3,5A 5

O

RO RNAc

3,5A 4

O

RO

HexNAc5Hex5-II

Y3a

RNAc CH2OR O OR

RO RNAc

O Y4a

Scheme 5 Structure and fragmentation scheme of permethylated (a) HexNAc5Hex5-I and (b) HexNAc5Hex5-II. Reproduced with permission from Rapid Commun. Mass Spectrom., Weiskopf, A. S.; Vouros, P.; and Harvey, D. J. 1997. Copyright John Wiley & Sons Limited.

80


Relative abundance

100

[Y4b /Y3g,4a ]2+

1169.5 > 937.7 > [M+2Na]2+ [Y4b ]2+

808.0

80 60 40 [Y4b /Y(3g,4a)2]2+ 678.5

20

Y3a 1403.0

Y4b /Y(3g,4a) 1333.7 Y4b /Y4d /Y(3g,4a)2 Y4b /Y4d /Y3g,4a 1129.8 1388.7 B5/Y4b /Y4δ/ Y(3g,4a)2 Y4b /Y3g,4a 853.2 1592.9

0 (a)

400

Relative abundance

100

600

800

1000

1169.5 > 937.7 > 1333.7 > [M+2Na]2+ [Y4b ]2+ Y4b /Y(3g,4a)

1200

3,5 B5-Y3a A5

2

1400

1600

1800

B5 1056.8

866.5 900.2

80 B3d 445.2

60

Y3d 912.9

B5/Y3d 634.4

40

3,5A /Y 5 3a

709.8

20

Y4d 1115.6

C4 830.3

Y3a 1144.5

0 400

(b)

Relative abundance

100

500

600

700

900

1000

60

0,4

B4/Y3a 621.3

B5/Y3d 634.3

A4 505.3

40

3,5

A5/Y3a 709.6

3,5 A5 B5/Y3a 899.6 866.6

830.8 6/Y3a

721.3

1200

1300

1400

3,5

A6 and/or 1.5x 5d 1143.7

Y4d 1115.8

C4

3,5A

B3d 444.9

1100

B5 1056.5

1169.5 > 1333.7 > [M+2Na]2+ Y4b /Y(3g,4a)2

80

20

800

Y3d 912.8

Y3d 1145.0 ×OCH3 1302.8

0 (c)

400

500

600

700

800

900 m/z

1000

1100

1200

1300

1400

Figure 5 MSn characterization of the HexNAc5Hex5-I isomer from chicken ovalbumin. a, MS3 – m/z 1169.5 ! 937.7; b, MS4 – m/z 1169.5 ! 937.7 ! 1333.7; c, MS3 – 1169.5 ! 1333.7. Reproduced with permission from Rapid Commun. Mass Spectrom., Weiskopf, A. S.; Vouros, P.; and Harvey, D. J. 1997. Copyright John Wiley & Sons Limited.

loss of another terminal HexNAc, and a singly charged ion at m/z 1333.7 corresponding to the loss of two terminal HexNAc residues. MS4 analysis of m/z 1333.7 affords several important ions: Y3d at m/z 912.9 and its complementary B3d at m/z 445.2 which proved the presence of an antenna comprised of two consecutive Hex residues (Figure 5). The ion at m/z 1144.5 (resulting from a loss of 189 mass units, that is, the loss of a Hex which lacks one methyl group) shows that one of the remaining Hex is doubly substituted. To summarize the data obtained for compound I, the MSn spectra suggest a structure presenting a short HexHex antenna, another short HexHexNAcHex antenna where the last subterminal Hex carries a HexNAc as branching. Also, according to the molecular composition and the number of terminal HexNAc residues lost upon fragmentation, this structure must be bisected. In recent years, there has been considerable progress in ion trap technology, and a new generation of instruments incorporating linear ion traps are showing great promise. Linear geometry provides a larger space in which the ions can be accumulated. This gain of space decreases the charge-state effect inherent to the trap and thus allows significant gain of sensitivity. The use of linear ion traps in the field of glycan structural analysis is at an early stage but several studies using this technique have been published recently and clearly demonstrate its potential.25,26


2.02.2.6.3

81

Fragmentation using MALDI-TOF/TOF instrumentation

Linear ion trap and Q-TOF analyzers are mostly associated with ES ionization. This type of ionization is, however, not always the most convenient for glycomics, bearing in mind that mass fingerprinting is best achieved by MALDI-TOF analysis (see Section 2.02.2.4). Until recently, sequencing using MALDI-TOF has relied on in-source decay (ISD) or post-source decay (PSD) fragmentation. ISD ions are produced within the source whereas PSD ions are derived from parent ions that are decomposing between the source and the analyzer. Neither process involves collisional activation, and the quality and quantity of fragment ions is very variable. In recent years, MALDI sources have been coupled to a variety of tandem and ion trap instruments. Currently the most promising of these instruments is the MALDITOF/TOF. This instrument is able to perform both PSD-type and CAD-type fragmentations, and the latter can be performed at both high and low energy. Its tandem TOF analyzers preserve the resolution and the sensitivity that can be achieved on a ‘classical’ MALDI-TOF instrument.27 The potential of MALDI-TOF/TOF for glycomics investigations is illustrated by MS/MS analysis of the lowabundance molecular ion cluster near m/z 3140 observed in MALDI-TOF screening of human neutrophils (see Section 2.02.2.4). As shown in the inset to Figure 3b, this cluster has an isotopic distribution that suggests that two sugar compositions are present. This was confirmed by selecting m/z 3143 for low-energy MALDI-TOF/TOF analysis using instrumental conditions that allow a window of several ions to be collisionally activated. The resulting data (Figure 6) not only firmly established the compositional identity of the components, but also allowed unambiguous assignments of antennae sequences and extent of core fucosylation. For example, the fact that the reducing-end residue cannot be lost without concomitant loss of fucose (giving m/z 2692) shows that all major components are core fucosylated. Therefore, the sialylated glycan of composition NeuAc2HexNAc4Hex5Fuc2 has only one sialyl Lewis X antenna, as shown. This is corroborated by similarly abundant ions for loss of sialylated LacNAc and sialyl Lewis X at m/z 2319 and 2145, respectively, and by the sodiated A-type fragment ions at m/z 847 and 1021 (see Figure 6). The second component present in the molecular ion cluster has a

Figure 6 MALDI-TOF/TOF MS/MS spectrum of the molecular ion cluster centered at m/z 3143 in Figure 3. Two glycan compositions are present in this cluster and three sequences were clearly identified upon low-energy fragmentation using air as the collision gas. Additional minor variants of the nonsialylated components are fucosylated on one of the antennae, instead of on the core. These components give the minor signals at m/z 660 and 2506.The low-mass fragment ions are sodiated A-type ions and the high-mass fragment ions are the result of b-cleavage. For key to symbols, see Table 1.

82


composition of Fuc1Hex7HexNAc6. Several structures could correspond to this composition: tetra-antennary with short antennas, tri-antennary with an extended antenna, or bi-antennary with a longer extended antenna. The MS/MS data show that only the bi- and tri-antennary arrangements exist. Thus, the three consecutive losses of 463u, corresponding to release of three terminal HexHexNAc moieties (m/z 2680, 2217, and 1754), provide evidence for a tri-antennary component. Moreover, the ion at m/z 1782, which corresponds to loss of a polyLacNAc antenna (HexHexNAcHexHexNAcHexHexNAc), is indicative of a bi-antennary structure. In summary, the structures shown in Figure 6 could be unambiguously determined by MALDI-TOF/TOF despite these glycans being only a fraction of a percent of the total neutrophil N-glycome. However, it is important to bear in mind that structural features such as linkage, stereochemistry, and antenna location require additional experimentation of the type described in the following sections, and it might not be possible (or, indeed, necessary) to rigorously characterize all minor components in a complex mixture.

2.02.2.7 Chemical and Enzymatic Digestion Chemical and enzymatic digestions are a vital adjunct to MALDI-TOF screening and MS/MS sequencing because they can provide information on structural features such as stereochemistry and linkage. MS is an ideal method for monitoring the progress and analyzing the products of these reactions. Its sensitivity allows identification of minor byproducts alongside the more major end products to provide extremely useful data for structural assignment. Progressive reactions such as hydrolyses and methanolyses can be easily monitored via a time course by mixing an aliquot of the reaction mixture with a matrix and then analyzing via MALDI-MS. It is often useful to include a permethylation step prior to MS analysis in order to optimize sensitivity and assist MS/MS sequencing. Although enzymatic digests must be carried out on native glycans, chemical hydrolyses can be performed on either native or derivatized samples depending on the information sought. Methanolysis is an excellent example of the use of MS to monitor a reaction over time. The reagents are compatible with the MALDI matrix and, once the sample is captured in the crystals and the volatile reagents have evaporated, the reaction is halted, creating a perfect ‘snapshot’ of that particular time point. This form of structural elucidation is applicable to a wide range of glycans and glycoconjugates. Mild degradation conditions can be used to follow removal of the more labile groups, such as fucose and sialic acids, whereas stronger conditions will facilitate a more complete digestion.28 Data from permethylated samples can provide evidence for branching patterns and arrangements of terminal structures. For example, if an NeuAc2HexNAc moiety is present, the two NeuAc sugars may be joined separately to the HexNAc or in tandem to one another. The former will yield a terminal HexNAc sugar with two free OH groups after methanolysis, while the latter will carry a single hydroxyl on the terminal HexNAc. Enzymatic degradation studies can yield a great deal of significant structural information from relatively simple experiments. Data from the glycomics profiling and MS/MS experiments (see Sections 1.27.2.4 and 1.27.2.6) are used to guide the choice of enzymes, some of which are specific for linkage as well as sugar type and anomeric stereochemistry. Comparison of glycan profiles before and after an enzymatic digest will show unambiguously which glycans have been digested and to what extent. In some cases, it can be useful to subject the sample to more than one enzyme prior to MS analysis.29 Some examples of enzymes frequently employed in mammalian glycomics are given below. Treatment of mixtures of glycans with sialidases provides useful information on the extent of sialylation of individual glycans and uncovers subterminal residues for further digestion. For example, all tentative sialylated glycans in the human neutrophil mixture shown in Figure 3 were predictably shifted after sialidase digestion. For example, the peak at m/z 2966 disappeared and there was an increase in the m/z 2244 ion, confirming that the former is a disialylated bi-antennary glycan (Figure 7). A variety of neuraminidases of different specificities are available for probing terminal sialylation. For example, sialidase A from Arthrobacter ureafaciens has a broad specificity and is able to remove NeuGc as well as NeuAc in a2!3, 6, and 8 positions, whereas the neuraminidase from Newcastle disease virus is specific for a2!3-linked sialic acid and therefore can be helpful in defining sialic acid linkages.30 Sialidase digestion m/z 2966

m/z 2244

Figure 7 Digestion with sialidase removes terminal sialic acids giving a mass shift to that of its unsialylated counterpart. For key to symbols, see Table 1.


83

a1→3/4

Fucosidase III

m/z 2779

m/z 2605

Figure 8 Fucosidase III removes fucose groups joined in an a1!3/4 configuration which are commonly found on the antennas of glycans. Mammalian core fucose is linked in an a1!6 configuration. The glycan m/z 2779 bears the sialyl Lewis X epitope, which can be further confirmed via the use of the appropriate sialidase enzyme. For key to symbols, see Table 1.

b-Galactosyl transferase

m/z 2489

m/z 2489 b-Galactosyl transferase

m/z 2489

m/z 2693

Figure 9 bGalT will add a galactose sugar to terminal but not bisecting GlcNAc and hence is able to distinguish the latter’s presence unambiguously. For key to symbols, see Table 1.

Fucosidase digestion is used to confirm the presence of fucose and to assist in defining sites of its attachment. There are several fucosidase enzymes available commercially, which means that the choice of enzyme can be carefully directed toward the sample in question. For example, to determine the presence of core fucose within a mammalian sample, an a-fucosidase with specificity toward (or including) the a1!6 linkage should be used, whereas fucosidase III, which is specific for a1!3/4-linked fucose, is helpful for probing antenna fucosylation (see Figure 8).31 Alternatively, fucose can be removed chemically via incubation with HF. This will readily remove a1!3-linked fucose, whereas a1!2- and 6-linked fucose are released at a slower rate. b-Galactosyltransferase (bGalT) has been shown to readily transfer b-galactose residues from UDP-Gal to nonreducing truncated GlcNAc but not to bisecting GlcNAc residues.23 Therefore, the results of an incubation of a sample containing putative bisected N-glycans with this enzyme and a source of UDP-Gal will confirm or reject their presence unambiguously. Take the example of an N-glycan at m/z 2489 (see Figure 9), which can have either the tri-antennary or bi-antennary, bisected structure shown. Following the bGalT incubation and subsequent permethylation, the tri-antennary structure would have a galactose added to the terminal GlcNAc hence increasing the m/z to 2693, but there would be no change to the bi-antennary, bisected structure. Hence, if a mass shift is seen, this is indicative of the structure being tri-antennary, and no change indicates the bisected bi-antennary structure. Digestion with endo-b-galactosidase is used to confirm the presence of polylactosamine-type structures (Galb1!4GlcNAc)n.32 This can be especially useful in cases where a glycan may be a tetra-antennary structure or a bi-/tri-antennary structure with polylactosamine repeats. The enzyme cleaves on the reducing-end side of internal galactose sugars as illustrated in Figure 10. The post-digested permethylated mixture can then be compared with the original to confirm the existence of polylactosamine chains via the appearance of diagnostic ions in the low-mass region and corresponding mass shifts in the higher-mass region. There are however some important specificities to note: the enzyme will not cleave on the nonreducing side of a GlcNAc sugar that carries fucose or on the reducing-end side of a branched galactose. Hence, the absence of diagnostic ions after digestion, while indicative, cannot completely rule out the absence of polylactosamine chains that are branched or internally fucosylated.

84


m/z 518 m/z 722 ±

±

±

endo-bGalactosidase

m/z 896

m/z 1083

Figure 10 endo-b-Galactosidase can be used to confirm the presence of polylactosamine antennas. Care must be taken with regard to its specificity as the enzyme is only able to cleave at the points indicated by the arrows. The m/z values of diagnostic ions are given. For key to symbols, see Table 1.

Exoglycosidases specific for hexose residues are useful for the determination of high-mannose-type structures (a-mannosidase digestion) and to confirm the linkage of galactose residues (a/b-galactosidase digestions).33,34 Peaks corresponding to high-mannose structures can be confirmed via their abolition following digestion with a-mannosidase, together with the appearance of a signal at m/z 763, which corresponds to the b-linked mannose attached to the chitobiose core (Manb1!4GlcNAcb1!4GlcNAc). Digestion with b-galactosidase will confirm the presence of glycans believed to carry unmodified lactosamine antennase. For example, the bi-antennary glycan at m/z 2244, which carries two terminal b-galactose linked sugars, will shift to m/z 1836 after digestion. It is, however, important to bear in mind that galactose will not be released if the subterminal GlcNAc is substituted with fucose. Subsequent to digestion with b-galactosidase, incubation of the sample with b-N-acetyl-hexosaminidase will define the number of terminal GlcNAc residues, including those exposed by the b-galactosidase.35 However, this enzyme removes bisecting GlcNAc relatively slowly, so this residue may still be present after digestion. Although some commercially available exoglycosidases are linkage specific, the majority of linkages cannot be defined using this approach. Hence the importance of linkage analysis experiments, which are described in the next section.

2.02.2.8 Linkage Analysis Linkage analysis by GC–MS, coupled with the techniques described earlier, facilitates the assignment of all aspects of the primary structure of glycans. Most importantly, it allows the identification of monosaccharide constituents and glycosidic bond positions. For instance, linkage analyses of N-linked glycans will reveal the presence of 3,6-linked mannose and 4-linked GlcNAc residues due to the conserved trimannosyl core. If N-glycans are core fucosylated, then 4,6-linked GlcNAc is observed. Core 1 and core 2 O-glycans can be detected as 3-linked and 3,6-linked GalNAc-ol, respectively. Other important structural features include bisected N-glycans, which can be identified by monitoring the presence of 3,4,6-linked GlcNAc residues, thereby complementing bGalT experiments (Section 2.02.2.7). Nonreducing structures such as Lex (Galb1!4(Fuca1!3)GlcNAc) and Lea (Galb1!3(Fuca1!4)GlcNAc) are characterized by terminal fucose, terminal galactose, and 3,4-linked GlcNAc. Sites of fucosylation can be probed by comparing the data before and after fucosidase and/or hydrofluoric acid (HF) hydrolysis experiments. Alternatively, permethylated glycans can be subjected to mild acidic methanolysis – an experiment where labile fucose residues are readily released. The free hydroxyl groups resulting from methanolytic removal of fucose are subsequently deuteromethylated, resulting in the labeling of the original fucosyl attachment sites. By comparing the linkage data before and after the methanolic removal, it is possible to determine the linkage positions by observing characteristic mass shifts of relevant fragment ions in the electron impact/ionization (EI) spectrum. For example, the fucose linkages on the antennae from the ovarian tumor marker CA12523 and core fucosylation sites from the parasite Haemonchus contortus were obtained using this method in conjunction with MS/MS experiments.36 Due to the conditions under which the sample is prepared for linkage analyses, sialic acids are destroyed and not detected. Fortunately, this is not a problem in structure analysis, because sialylated glycans are unambiguously identified by the MALDI-MS, MS/MS analyses, and enzymatic/chemical digestions described earlier. Moreover, their attachment sites can usually be revealed by comparing linkage data before and after sialidase digestion. For example, changes in 3- and/or 6-linked galactose levels upon sialidase digestion provide information on linkage to galactose.


85

2.02.2.9 Tagging Methodologies The structural analysis of glycans can be facilitated by tagging with chromophores or fluorophores at their reducing ends. Such derivatives are readily purified chromatographically, and this approach has been particularly useful for sensitive detection, purification, and quantitation of glycans from complex mixtures. Some issues to consider when choosing suitable tagging reagents are: (1) rapid and robust production of tagged oligosaccharides requiring minimum specialized equipment, (2) nonselective labeling so that the correct molar proportions are detected, (3) efficiency of purification, (4) stability, and (5) suitability for subsequent processes such as mass spectrometric analyses. Among the methods developed, fluorescent labeling coupled with high performance liquid chromatography (HPLC) has become the preferred choice for this type of analysis. Commonly used tags include fluorescent compounds such as 2-aminopyridine (2-AP), 2-aminobenzamide (2-AB), and 2-anthranillic acid (2-AA)37,38 that are incorporated via reductive amination. Depending on the tags, different elution methods such as reverse-phase and normal-phase HPLC may be applied. For example, 2-AB- and 2-AP-tagged oligosaccharides are typically fractionated on a normal-phase HPLC. Here, structures of individual glycans can be predicted from the HPLC elution time that is expressed as glucose units with reference to a dextran ladder.39 Furthermore, direct quantitation of individual glycans from the HPLC profiles is achievable. This type of method is well established and has been shown to be highly reproducible in obtaining a high resolution separation of glycans and is also compatible with MS analysis.40 Similarly, many electrophoretic techniques have also been developed to purify, quantify, and structurally analyze tagged glycans. Examples include fluorophore-assisted carbohydrate electrophoresis (FACE), where fluorescently labeled glycans are analyzed using polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis (CE).41–43 Moreover, there is considerable potential for immobilizing tagged glycans to faciltate structure–function studies. For example, biotinylated diaminopyridine (BAP) allows exploitation of both HPLC and avidin–biotin technology.44,45 Another recently developed tag is 2,6-diaminopyridine (DAP).46 Using this tag, purification, quantification, and structural characterization by HPLC and MS of individual glycans is possible, and, importantly, the availability of a primary amine in the tagged glycan allows further conjugation with molecules such as biotin, protein carriers, or covalent attachment to solid supports. Effectively, this approach could allow the exploration of glycan structures and quantification as well as the identification of their respective carbohydrate-binding proteins (CBPs).

2.02.3 Glycoproteomics 2.02.3.1 Overview The full structural analysis of glycoproteins is a complicated task due to inherent glycan complexity and glycoprotein heterogeneity. Although such studies are difficult they are important to perform as they often have particular relevance in many areas of advanced clinical and biomedical research. Indeed, it is becoming increasingly clear that many ‘biomarkers’ of health and disease are in fact glycoproteins. Such biomarkers now encompass many major diseases and illnesses ranging from diabetes, rheumatoid arthritis, cardiovascular diseases to various cancers.47–50 Recent advances in analytical instrumentation and methodologies are allowing high-sensitivity glycoprotein analysis to be performed on a routine basis although the precise methodologies vary according to the nature of the particular glycoprotein in question. Indeed, strict quality-control measures are increasingly being implemented in many product pipelines that generate therapeutic antibodies and bioactive glycoproteins, for example, erythropoietin.51 Structural glycoproteomics possess a multifaceted problem requiring the definition of several important features of the glycoprotein. These features include the following:

protein sequence; disulfide bridging; sites of glycosylation; glycans attached to each glycosylation site (can be >50); and linkage, anomeric configuration, branching and sequence of each glycan.

Accordingly, to successfully perform detailed structural glycoproteomics, a number of experiments are required. Initial experiments begin with the isolation of the glycoprotein that is typically either generated by recombinant technology, or extracted from a biological matrix such as an organ, tissue, or cell. This usually involves a variety of chromatography-based techniques. Solid-phase chromatography, lectin-based glycan capture, immunoprecipitation, and electrophoresis are among the most routinely used techniques for this task. Once isolated, preliminary information may be attained by electrophoresis to give a rough estimate of intact glycoprotein molecular weight, and

86


lectin-binding studies may be performed to provide information on glycan structure. These studies are commonly followed up by more detailed analysis using MS and/or NMR. In many circumstances sample quantities are limiting, so MS is frequently the method of choice. At this stage of analysis, it is often desirable (e.g., medical diagnostics) to get an accurate molecular mass of the intact glycoprotein in question, although this may not always be possible. Advancements in MALDI-MS and nano-ES-MS technologies have allowed this to become feasible although this analysis tends to be restricted to a specific selection of glycoproteins that have limited heterogeneity, moderate molecular weights, and good ionization properties. To establish key structural relationships, the glycoprotein is analyzed in further detail. Analysis of the predicted peptide sequences informs the strategies required to perform this type of analysis. As a first step, potential N- and O-glycosylation sites are identified. N-glycosylation site prediction involves the identification of N-glycan consensus sequences, Asn-Xa-Ser/The (Xa 6¼ Pro), and for O-glycosylation, site prediction NetOGlyc software is frequently used.52 The sequence is then theoretically cut into peptides and glycopeptides to determine the most favorable digestion strategy. Ideally, for mass spectrometric analysis, glycopeptides should be doubly/triply charged species with mass-to-charge values 300ppm and the nucleus has been utilized in carbohydrate-related work.19 However, in biomolecules in general and carbohydrates in particular, the 15N resonances are found in a more limited region, 110–130ppm.20,21 Detection of the 15N resonances are often carried out by way of 1 H,15N-HSQC-based experiments in 90% H2O/10% D2O (vide infra), due to the low natural abundance of the 15 N isotope. Chemical shift referencing is most conveniently performed from TSP based on the ratio of gN/gH,22 a procedure that can be used also for other nuclei.23

2.03.1.3 Assignments of Resonances in NMR Spectra The NMR spectra of oligo- and polysaccharides may be assigned by recognizing that each sugar residue often can be identified as a unique spin system, that is, a number of resonances can be tied together by their spin–spin coupling network and the corresponding nuclei thus identified. The spin system of nonexchangeable protons in the sugar part of a-D-Glcp-OMe contains seven nuclei (Figure 4). The resonance from the anomeric proton resides outside the bulk region at 4.8ppm. It can subsequently be used as a handle in 2-D NMR spectra to unravel the remaining part of the spin system. The 1H,1H-DQF-COSY spectrum24 of a-D-Glcp-OMe is shown in Figure 5, from which the complete spin system can be traced out to the H-6 protons. Since the spectral region of the ‘ring protons’, 3.2–4.2ppm, often becomes crowded, a series of 1H,1H-TOCSY experiments25 are usually performed with increasing mixing time of the spin-lock, for example, 10, 30, 60, and 100ms,

104

General NMR Spectroscopy of Carbohydrates and Conformational Analysis in Solution

Figure 4 The spin-system of non-exchangeable protons (in red) for a-D-Glcp-OMe.

3.2 3.4 3.6

1H

(ppm)

3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.0

4.8

4.6

4.4

4.2 4.0 1H (ppm)

3.8

3.6

3.4

3.2

Figure 5 Phase-sensitive 1H,1H-DQF-COSY NMR spectrum of a-D-Glcp-OMe in D2O.

leading to magnetization transfer throughout the spin system. In cases where the 2JH,H and 3JH,H coupling constants are sufficiently large, for example, in b-D-Glcp-OMe, the complete spin system can readily be unraveled starting from H1. In sugars like b-D-QuipNAc, that is, 2-acetamido-2,6-dideoxy-b-D-Glcp, two entrance points, H1 and H6, can be used to assign the complete spin-system. The different configurations of hexopyranoses lead to stereochemical arrangements between protons that result also in small 3JH,H values, for example, between H1 and H2 in b-Manp and between H4 and H5 in Galp, thereby limiting the magnetization transfer. Thus, the cross-peaks are absent in these 2-D NMR spectra, leading to isolated subspin systems, which need to be joined. As a remedy to the above limitations, the 2-D 1H,1H-NOESY experiment26 can be used since cross-peaks in the spectrum will be observed between protons that are close in space, up to 4A˚ in oligosaccharides when the mixing time is kept relatively short (0) and left-handed (n6) glucosidic bonds. Appl. Environ. Microbiol. 2002, 68, 4283–4291. 328. Ebisu, S.; Kato, K.; Kotani, S.; Misaki, A. Structural differences in fructans elaborated by Streptococcus mutans and Streptococcus salivarius. J. Biochem. 1975, 78, 879–887. 329. Hancock, R. A.; Marshall, K.; Weigel, H. Structure of levan elaborated by Streptococcus salivarius strain 51 – Application of chemical-ionization mass-spectrometry. Carbohydr. Res. 1976, 49, 351–360. 330. Simms, P. J.; Boyko, W. J.; Edwards, J. R. The structural analysis of a levan produced by Streptococcus salivarius SS2. Carbohydr. Res. 1990, 208, 193–198. 331. Corrigan, A. J.; Robyt, J. F. Nature of the fructan of Streptococcus mutans OMZ 176. Infect. Immun. 1979, 26, 387–389. 332. Baird, J. K.; Longyear, V. M. C.; Ellwood, D. C. Water insoluble and soluble glucans produced by extracellular glycosyltransferases from Streptococcus mutans. Microbios 1973, 8, 143–150. 333. Rosell, K. G.; Birkhed, D. An inulin-like fructan produced by Streptococcus mutans, strain JC-2. Acta Chem. Scand. 1974, 28, 589. 334. Olivares-Illana, V.; Wacher-Rodarte, C.; Le Borgne, S.; Lo´pez-Munguıá, A. Characterization of a cell-associated inulosucrase from a novel source: A Leuconostoc citreum strain isolated from pozol, a fermented corn beverage of Mayan origin. J. Ind. Microbiol. Biotechnol. 2002, 28, 112–117. 335. Monchois, V.; Willemot, R.-M.; Remaud-Simeon, M.; Croux, C.; Monsan, P. Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a(1-6) and a(1-3) linkages. Gene 1996, 182, 23–32. 336. Monchois, V.; Remaud-Simeon, M.; Monsan, P.; Willemot, R.-M. Cloning and sequencing of a gene coding for an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a a(1-6) glucan. FEMS Microbiol. Lett. 1998, 159, 307–315. 337. Bozonnet, S.; Dols-Laffargue, M.; Fabre, E.; Pizzut, S.; Remaud-Simeon, M.; Monsan, P.; Willemot, R.-M. Molecular characterization of DSR-E, an a-1,2 linkage-synthesizing dextransucrase with two catalytic domains. J. Bacteriol. 2002, 184, 5753–5761.

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338. Funane, K.; Mizuno, K.; Takahara, H.; Kobayashi, M. Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F. Biosci. Biotech. Bioch. 2000, 64, 29–38. 339. Hanada, N.; Isobe, Y.; Aizawa, Y.; Katayama, T.; Sato, S.; Inoue, M. Nucleotide sequence analysis of the gtfT gene from Streptococcus sobrinus OMZ176. Infect. Immun. 1993, 61, 2096–2103. 340. Simpson, C. L.; Cheetham, N. W. H.; Giffard, P. M.; Jacques, N. A. Four glucosyltransferases, GTFJ, GTFK, GTFL and GTFM, from Streptococcus salivarius ATCC 25975. Microbiology 1995, 141, 1451–1460. 341. Gilmore, K. S.; Russell, R. R. B.; Ferretti, J. J. Analysis of the Streptococcus downei gtfS gene, which specifies a glucosyltransferase that synthesizes soluble glucans. Infect. Immun. 1990, 58, 2452–2458. 342. Fukushima, K.; Ikeda, T.; Kuramitsu, H. K. Expression of Streptococcus mutans gtf genes in Streptococcus milleri. Infect. Immun. 1992, 60, 2815–2822. 343. Russell, R. R. B.; Gilpin, M. L.; Mukasa, H.; Dougan, G. Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli. J. Gen. Microbiol. 1987, 133, 935–944. 344. Heyer, A. G.; Schroeer, B.; Radosta, S.; Wolff, D.; Czapla, S.; Springer, J. Structure of the enzymatically synthesized fructan inulin. Carbohydr. Res. 1998, 313, 165–174. 345. Gruter, M.; Leeflang, B. R.; Kuiper, J.; Kamerling, J. P.; Vliegenthart, J. F. G. Structural characterization of the exopolysaccharide produced by Lactobacillus delbrueckii subsp bulgaricus rr grown in skimmed milk. Carbohydr. Res. 1993, 239, 209–226.

Exopolysaccharides from Lactic Acid Bacteria

519

Biographical Sketch

Luc De Vuyst (1963 – Aalst, Belgium) finished his studies in agricultural chemistry engineering at Ghent University (Ghent, Belgium) in 1986. As research assistant of the Fund for Scientific Research – Flanders (FWO-Vlaanderen) at the same university, he obtained a Ph.D. in agricultural sciences with a specialization in Industrial Microbiology in 1990. After research stints at NIZO (The Netherlands), the University of Alberta (Canada), and the NZDRI (New Zealand), he worked as a Postdoctoral Researcher of the FWO at Ghent University and the Vrije Universiteit Brussel (VUB). In 1994 he became professor at the VUB in the area of ‘Biotechnology and Agriculture’ of the ‘Applied Biological Sciences and Engineering’ educational program. Currently, he teaches industrial microbiology, industrial biotechnology, industrial food biotechnology, and dairy technology. He is head of the Research Group of Industrial Microbiology and Food Biotechnology that belongs to the Department of Applied Biological Sciences and Engineering. His research deals with the biodiversity, microbial physiology, fermentation aspects, modeling, and metabolomics of lactic acid bacteria in food ecosystems and the human colon.

Filip de Vin (1975 – Berlin, Germany) finished his studies in biochemical engineering at the Vrije Universiteit Brussel (Brussels, Belgium) in 2000. In 2001, he started a Ph.D. at the Research Group of Industrial Microbiology and Food Biotechnology (IMDO) that belongs to the Department of Applied Biological Sciences and Engineering. Also, he worked at Lund University (Lund, Sweden) as part of a Marie Curie Host Fellowship. His research deals with galactose catabolism and exopolysaccharide production in the yoghurt bacterium Streptococcus thermophilus.

2.16 Bacterial Exopolysaccharides I. W. Sutherland, Edinburgh University, Edinburgh, UK ß 2007 Elsevier Ltd. All rights reserved. 2.16.1 Introduction and Definition

522

2.16.2 Chemical Composition – Homopolysaccharides and Heteropolysaccharides

522

2.16.2.1 Homopolysaccharides

524

2.16.2.2 Heteropolysaccharides

524

2.16.2.3 Polysaccharide Families

528

2.16.3 Physical Properties and Structure/Function Relationships

531

2.16.3.1 Helix and Coil Structures

532

2.16.3.2 Gelation

532

2.16.3.3 The Relationship of Chemical and Physical Structures

535

2.16.4 Physiological Aspects of Exopolysaccharides Production

536

2.16.5 Biosynthesis

538

2.16.5.1 General Concepts of Biosynthesis

538

2.16.5.2 Dextran and Levan Synthesis

538

2.16.5.3 Heteropolysaccharide Synthesis

539

2.16.6 Genetics and Regulation of Exopolysaccharides

539

2.16.6.1 General Aspects of Genes Involved in Polysaccharide Synthesis

539

2.16.6.2 The Genetics of Repeat Unit Synthesis and Excretion

540

2.16.6.3 Genetics of Xanthan Synthesis by X. campestris

541

2.16.6.4 Genetics of Polysaccharide Synthesis in LAB

542

2.16.6.5 Genetic Engineering of Bacterial Exopolysaccharides

542

2.16.7 Natural Functions of Bacterial Exopolysaccharides

542

2.16.8 Biological Properties of Bacterial Exopolysaccharides

544

2.16.9 Enzymic Degradation of Bacterial Exopolysaccharides

545

2.16.9.1 Polysaccharide Hydrolases

545

2.16.9.2 Polysaccharide Lyases

547

2.16.9.2.1 2.16.9.2.2 2.16.9.2.3 2.16.9.2.4

Alginate lyases Emulsan lyase Gellan lyase Xanthan lyase

547 548 548 549

2.16.9.3 Glycosidases

549

2.16.10

550

Commercialization

2.16.10.1 Manufacture and Usage of Bacterial Exopolysaccharides

550

2.16.10.2 Bacterial Cellulose

551

2.16.10.3 Dextran

551

2.16.10.4 Bacterial Hyaluronic Acid

551

2.16.10.5 Xanthan

552

2.16.10.6 Emulsan

553

2.16.10.7 Gellan

553

2.16.10.8 Other Potential Exopolysaccharide Products

553

521

522

Bacterial Exopolysaccharides

2.16.1 Introduction and Definition The bacterial surface structure depends on the type of cell – the outer layers of each of the three major groups of bacteria, Gram-positive and Gram-negative bacteria and archeae, possess intrinsically different chemical composition and structure even though they are similar in their gross morphology. All possess a rigid wall which encloses the cell membrane and cytoplasm. External to the rigid wall structures, the cell may secrete further material which is normally of a polysaccharide nature. This ‘exopolysaccharide’ may either be attached to the rigid cell wall or may be excreted as a soluble macromolecule and the capacity to synthesize and excrete these polymers is widespread among bacteria. Exopolysaccharides are found outside the microbial cell, either in the form of distinguishable capsules or totally dissociated from the cells as amorphous slime; a bacterial cell may possess both forms simultaneously. In electron micrographs, some exopolysaccharides have been shown to form a network-like structure, and in others, fibrillar, macromolecular strands have been demonstrated. Such polymers are normally highly hydrated. The exact physical nature and indeed sometimes the chemical composition can depend on the substrate or substrates available to the bacteria and to the growth conditions employed. The amount of material synthesized is also greatly affected by the growth environment. Some bacteria always synthesize exopolysaccharides while others may only do so under very limited physiological conditions. These general concepts apply whether the bacteria are human, animal, or plant pathogens or are commensal or free-living types found in soil or water. Some bacteria may possess the genetic information to enable them to produce more than one type of exopolysaccharide. This may require specific physiological conditions but occasionally the microorganisms may form two exopolysaccharides simultaneously. An example of this was Streptococcus thermophilus LY03 which yielded two polysaccharides both containing D-glucose and D-galactose in the same molar ratio of 1:4, but differing in mass.1 A few bacteria may even be able to synthesize three different polysaccharides. Colanic acid provides an example of a polysaccharide formed by many of the Gramnegative Enterobacteriaceae either in addition to other exopolysaccharides or expressed under limited growth conditions including lowered incubation temperature, high salt concentrations, or other forms of nutritional stress. In some species, it has also been shown that synthesis of one polysaccharide suppresses that of another. Loss of the one through mutation, etc., can lead to its replacement by the second cryptic type. Wide variations are to be found within different genera and species of bacteria. In some bacterial genera, including many of the Gram-negative Enterobacteriaceae, a very wide range of exopolysaccharides are produced, whereas in a smaller number of bacterial groups a single polysaccharide chemotype or a very limited range may be formed. An example of the latter is the plant pathogenic group of Xanthomonas spp., the majority of which make a single type of polysaccharide – xanthan. One cannot make generalizations although it would appear that synthesis of exopolysaccharides, often in copious amounts, is perhaps more common in Gram-negative than in Gram-positive bacteria. Exopolysaccharides have also been obtained from bacteria isolated from a very wide range of environments, including some which are hostile to other biological activity.

2.16.2 Chemical Composition – Homopolysaccharides and Heteropolysaccharides A large number of exopolysaccharides from an extensive range of bacteria from many different environments have now been analyzed and an increasing number of structures of these polymers have been elucidated. These data have shown that although most contain a limited range of common hexoses and methylpentoses, exopolysaccharides are composed of a very wide range of carbohydrates. Additionally they may contain either organic or inorganic substituents as integral components of their chemical structure (Table 1). The organic, noncarbohydrate constituents include acyl groups, amino acids, and ester-linked organic acids such as succinic acid. These substituents may greatly influence the physical, biological, and immunological properties of the macromolecules. The monosaccharides which are most commonly present are those found in other polysaccharides of bacterial, plant, or animal origin such as D-glucose, D-galactose, and D-mannose and the corresponding N-acetylaminosugars and the corresponding sugar acids D-glucuronic acid, D-galacturonic acid, and D-mannuronic acid. There may also be some much rarer sugars such as L-altrose which has only been found so far in a polysaccharide from Butyrivibrio fibrisolvens.2 Indeed, a few monosaccharides are only found as components of bacterial exopolysaccharides, but, in general, exopolysaccharides lack many of the exotic sugars found in the lipopolysaccharide structures which are components of the cell walls of Gram-negative bacteria. Some bacterial genera typified by Escherichia or Klebsiella/Enterobacter species from the family Enterobacteriaceae yield a very wide range of chemotypes and serotypes. This is also true of the Gram-positive species, Streptococcus pneumoniae. Each of these three groups of bacteria produces a range of almost 100 chemotypes and corresponding serotypes. Other bacterial species have been found to form either a single polymer or a more


523

Table 1 Some monosaccharide and noncarbohydrate constituents of exopolysaccharides Constituent Monosaccharides D-Glucose D-Galactose D-Galactofuranose D-Mannose D-Fructose D-Ribose L-Altrose L-Fucose L-Rhamnose D-Rhamnose N-Acetyl-D-glucosamine N-Acetyl-D-galactosamine N-Acetyl-D-mannosamine N-Acetyl-L-fucosamine N-Acetyl-L-talosamine D-Glucosamine D-Glucuronic acid D-Galacturonic acid D-Mannuronic acid L-Guluronic acid L-Iduronic acid D-Galactosaminuronic acid D-Mannosaminuronic acid D-Glucosaminuronic acid 2-Deoxy-D-arabino-hexuronic acid D-Riburonic acid Organic acids Acetate Glycerate Hydroxybutanoate Malonate Propionate Pyruvate Succinate Taurine Amino acids L-Glutamic acid L-Serine Inorganic acids Phosphate Sulfate

Linkage

Ester Ester Ester Ester Ester Ketal Half-ester Ester

Charge conferred

Occurrence

None None None None None None None None None None None None None None None Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

Very common Very common Lactobacillus rhamnosus, LAB Very common Common Relatively uncommon Very rare (Butyrivibrio fibrisolvens) Very common Very common Rare Very common Very common Uncommon Rare Rare Rare Very common Very common In alginates, otherwise not very common In alginates, otherwise not very common Rare Pseudomonas solanacearum strain Pseudomonas fluorescens strain Haloferax denitrificans Sphingomonas paucimobilis strain Rhizobium leguminosarum

None Negative None None None Negative Negative None

Very common – e.g., Klebsiella spp.; colanic acid Sphingomonas elodea Rhizobium trifolii; R. leguminosarum, etc. Escherichia coli K10 Rare – some Escherichia coli Very common – e.g., Klebsiella spp.; colanic acid Rhizobium spp.; Agrobacterium spp. Staphylococcus aureus Klebsiella aerogenes K82 Pseudoalteromonas aliena, Escherichia coli K40

Negative Ester

Negative

Common in some genera and Gram-positive spp., including LAB Cyanobacteria; Haloferax mediterranea

limited range of chemotypes. Thus most Azotobacter vinelandii strains form a type of bacterial alginate and most Xanthomonas strains yield xanthan. Initially many structural studies on exopolysaccharides focused on polymers from Gram-negative species and some Gram-positive pathogens, but more recently the interest in lactic acid bacteria (LAB)3 has produced a large number of structures from this group. Quite a few of the extracellular polysaccharides from LAB are neutral polymers. Although some of the bacteria in this group can produce homopolymers composed of a single monosaccharide, the majority are heteropolysaccharides. Among the homopolysaccharides synthesized by LAB are glucans and fructans, several of which are produced only when the bacteria are grown in the presence of sucrose. Improved culture techniques have also permitted the isolation in pure culture of prokaryotic species previously considered difficult. Thus, examination of exopolysaccharides from species of cyanobacteria (blue-green bacteria) has shown that these tend to be much more complex than the polymers from other bacteria. They commonly contain pentoses including D-xylose and D-arabinose, monosaccharides which are not so frequently present in exopolysaccharides from other bacterial groups.

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2.16.2.1 Homopolysaccharides Most bacterial exopolysaccharides which are homopolysaccharides are composed of either D-glucose or D-fructose. Among such products are the dextrans and levans formed by many Gram-positive Streptococcus and Lactobacillus spp. There are also various other D-glucans including curdlan from Agrobacterium and Rhizobium species and bacterial cellulose from Gluconoacetobacter xylinus and others, mainly Gram-negative bacterial species. Curdlan is a neutral gelforming (1!3)-b-D-glucan of much lower molecular mass than most exopolysaccharides (74000) and, unusually, is insoluble in cold water but dissolves in alkali and dimethylsulfoxide (DMSO). It is also produced exceptionally by Cellulomonas flavigena as an extracellular storage polymer, the only bacterial exopolysaccharide known to fit this role. The structure of Curdlan [→3)-b-D-Glcp-(1→3)-b-D-Glcp-(1→3)-b-D-Glcp-(1→3)-b-D-Glcp-(1→]n

Gluconoacetobacter xylinus and some other, mainly Gram-negative bacterial species, produce cellulose as an exopolysaccharide. Like curdlan, it too is insoluble in water. In these bacteria, the cellulose is excreted into the medium where it rapidly aggregates as microfibrils. Bacteria differ from plants as the polysaccharide has no structural role and is not associated with other macromolecules. The polysaccharide is thus very pure when formed by bacteria. Dextrans are high-molecular mass (107–108 Da) exopolysaccharides composed predominantly of a-D-glucosyl(1!6)-linked residues. In some dextrans, there may be almost no other type of linkage; alternatively, they may be very highly branched. Up to 50% of the glucose residues may be linked 1!2, 1!3, or 1!4; the number and nature of the branch points depend on the bacterial strain used.4 Industrial dextran production is primarily from a strain of Leuconostoe mesenteroides, yielding a polysaccharide with about 95% 1!6 linkages and 5% 1!3 linkages and a molecular mass of about 4–5107. For many purposes, the molecular weight is reduced by mild acid hydrolysis. An oral bacterial species, Streptococcus mutans, also yields a type of dextran designated mutan. This is a linear (1!3)-aD-glucan, insoluble in water, which plays a major role in oral biofilms and in dental decay. Another a-D-glucan produced by some Le. mesenteroides strains is alternan. This polysaccharide is unusual in that 1!3- and 1!6-linkages alternate on a regular basis.5 Further types of a-D-glucans contain large amounts of 1!4-glucosidic bonds as in Lactobacillus reuteri strain 121 polymer, or large amounts of 1!2-linkages. Levans (fructans) are produced as exopolysaccharides by several bacteria including many oral isolates, some plant pathogens, and LAB.6 Some bacteria are capable of producing both levans and dextrans. One bacterial species found in soil, Azotobacter chroococcum, is unusual in that it synthesizes a levan when grown on sucrose, but an alginate when grown on glucose.7 A strain of Lactococcus lactis subsp. lactis yields a galactan. Some bacteria produce exopolysaccharides that closely resemble the alginates obtained commercially from marine algae. While these are generally linear heteropolysaccharides containing both D-mannuronic acid and L-guluronic acid, in a few bacterial strains the product is a homopolysaccharide in which the sole sugar is D-mannuronic acid, although O-acetyl residues are also present. Another homopolysaccharide containing solely uronic acid was obtained from a Sinorhizobium meliloti mutant strain,8 the only monosaccharide being D-glucuronic acid. Homopolysaccharides composed of other monosaccharides are rare, but recently a polysaccharide from Burkholderia gladioli pv. agaricicola, a bacterial species infecting the edible mushroom, was identified as a linear D-rhamnan in which a- and b-linked residues of D-rhamnose formed the structure, the first example of a homopolysaccharide with this monosaccharide from bacteria:9 [→4)-a-D-Rhap-(1→3)-a-D-Rhap-(1→3)-a-D-Rhap-(1→3)-b-D-Rhap-(1→]n

Another uncommon type of homopolysaccharide is the poly-N-acetyl-D-glucosamine secreted by some strains of Staphylococcus epidermidis described below.

2.16.2.2 Heteropolysaccharides Apart from the examples given above, bacterial homopolysaccharides are less frequently found than are heteropolysaccharides. The latter may be formed from two or more sugars, although they rarely contain more than three or four. Some are very simple structures and Table 2 lists several which contain only the two monosaccharides D-glucose and D-galactose, and, in some cases, acyl substituents. They are thus neutral polymers unless charge is conferred by the presence of pyruvate ketal groups or succinyl half esters. The variety of possible structures to be found in some of these can be seen in Table 3. Most exopolysaccharides are more complex and contain three or more monosaccharides; in some of the polymers, several residues of one of these sugars may be present. Many and perhaps the majority of bacterial heteropolysaccharides


525

Table 2 Galactogluco-polysaccharides Source

Glucose

Achromobacter sp. Agrobacterium radiobacter Burkholderia cepacia Escherichia coli K37 Lactobacillus helveticus K16 Pseudomonas fluorescens Pseudomonas marginalis Pseudomonas putida Rhizobium meliloti YE2 Zoogloea ramigera 1 Z. ramigera 2

1 0.9 1 1 2 1 1 1 1 2 11

Galactose *

1 1 1 2 1 1 1* 1 1 1 3

Pyruvate 0.81–0.99 0.83 1 1 0 0.5 1 1 1 þ 1.5þþ

Acetate

Succinate

0 0 þ 0 þ 0.9 (Ill defined)

0 0 1 0 þ

*Pyruvylated sugar.

Table 3 The structures of some bacterial galactoglucans

Rhizobium meliloti [→4)-a-D-Glcp-(1→3)-b-D-Galp-(1→]n 6 ⇑ ↑ Pyr Ac R. meliloti strains YE-2(S1), Rm1021 [→3)-b-D-Glcp-(1→ 3)-a-D-Galp-(1→]n 6 ⇑ ↑ Pyr Ac Agrobacterium radiobacter Strain II; Burkholderia cepacia [→3)-b-D-Glcp-(1→3)-a-D-Galp-(1→]n ⇑ Pyr Pseudomonas marginalis HTO41B [→3)-b-D-Glcp-(1→3)-a-D-Galp-(1→]n ⇑ ↑ Succ Pyr Escherichia coli K37 [→3)-b-D-Glcp-(1→3)-a-D-Galp-(1→]n 4 ↑ 1 Pyr ⇒ a-D-Galp

are acidic in nature due to the presence of uronic acids (in addition to pyruvate ketal groups in many such structures). Commonest is D-glucuronic acid, while D-galacturonic acid is also relatively frequently found and D-mannuronic acid less so, although it along with L-guluronic acid is found in bacterial alginates. In addition, increasing numbers of rarer or unusual sugar acids have been identified, as the range of exopolysaccharides has extended from those wellcharacterized and easily grown bacterial species first examined to uncommon types from exotic environments including deep thermal vents and hot springs. Some aminouronic acids are also found in exopolysaccharides. N-Acetylamino sugars and the corresponding sugar acids also contribute to many exopolysaccharide structures. N-Acetyl-D-glucosamine is probably the commonest while the corresponding galactose and mannose derivatives are also found. Some

526


heteropolysaccharides may even contain several N-acetylaminosugars. In the pentasaccharide repeat unit of Pseudomonas aliena strain KMM3562, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, and N-acetyl-D-quinosamine are all present.10 However, some sugars found in the walls of Gram-negative bacteria, including dideoxysugars, have not yet been found. A very few exopolysaccharides may be polycationic due to the presence of D-glucosamine lacking the normal N-acylation. This is exemplified by the adhesive polymer obtained from strains of Staphylococcus epidermidis strains associated with biofilms. These bacteria produce two polymers one of which contains both N-acetylD-glucosamine and D-glucosamine while the other is a homopolysaccharide containing N-acetyl-D-glucosamine as the only monosaccharide.11 The structure of Staphylococcus epidermidis biofilm exopolysaccharides is shown below: Exopolysaccharide I: [→6)-b-D-GlcpNAc-(1→6)-b-D-GlcpNAc-(1→6)-b-D-GlcpN-(1→6)-b-DGlcpNAc-(1→6)-b-D-GlcpNAc-(1→]n

The ratio GlcpNAc:GlcpN in this polysaccharide is c. 5:1; it is cationic, and of relatively high molecular mass (>26000). Exopolysaccharide II: [→6)-b-D-GlcpNAc-(1→6)-b-D-GlcpNAc-(1→6)-b-D-GlcpN-(1→6)-b-DGlcpNAc-(1→6)-b-D-GlcpNAc-(1→]n

In the second polymer, the ratio GlcpNAc:GlcpN is c. 16:1. As this polysaccharide also contains phosphate and succinyl half esters, it is anionic in nature. The great majority of bacterial extracellular heteropolysaccharides are composed of regular repeating units. Although these vary in size, they generally range from disaccharides to octasaccharides. The presence of 1 mole of uronic acid in the repeating structure is a common feature of polyanionic exopolysaccharides. These are most frequently D-glucuronic acid and D-galacturonic acid. One of the first exopolysaccharide structures to be determined from a Gram-positive bacterium was also among the simplest heteropolysaccharide repeat units elucidated. The polymer from Streptococcus pneumoniae type 3 was composed of b1!3-linked cellobiouronic acid units. The same structure has also been identified in the product from the unrelated Gram-negative bacterium Rhizobium sp. T1 which is symbiotic with alfalfa.12 Another example of a disaccharide repeat unit, devoid of acyl or other substituents, was found in the neutral exopolysaccharide from the Gram-negative bacterium Alcaligenes latus.13 Alcaligenes latus exopolysaccharide: [→2)-a-D-ManpA-(1→3)-a-L-Fucp-(1→]n

The proposed uniformity of the repeat units is mainly based on chemical studies, and some irregularities may exist, especially in polymers which are composed of larger and more complex repeat units. The heteropolymer repeating units commonly possess one or more short side chains. These may vary from one to four or five sugars in length, although examples with longer side chains are known. Most exopolysaccharides, which are composed of three or four monosaccharides, are formed from a uniform backbone to which side chains are attached at regular intervals. Commonly, the structure is a trisaccharide or tetrasaccharide main-chain repeat unit carrying either a single sugar or a disaccharide side chain. A more complex structure was found in the exopolysaccharide from a Burkholderia cepacia strain. The trisaccharide repeat unit of this polymer had three distinct side chains, a disaccharide, and two single 14 D-galactosyl residues. In a few polymers, the side chains may also be branched. Bacterial alginates are exceptional as they are linear macromolecules composed only of uronic acids and the only bacterial extracellular heteropolysaccharides which do not possess a regular repeat unit. They are composed of D-mannuronic and L-guluronic acids (and O-acetyl groups) in irregular unbranched structures. D-Riburonic acid is another uncommon uronic acid found in the structure of polymers from some Rhizobium leguminosarum strains. Rare aminouronic acids and other unusual acid sugars have also been found in some polysaccharides. These include mannosaminuronic acid, glucosaminuronic acid, and galactosaminuronic acid (Table 1). Very occasionally, two different uronic acids are present in a repeat unit. Whereas a repeat unit may frequently contain several residues of the common monosaccharides such as glucose, mannose, or galactose, the rarer sugars are usually only present as a single molecule in the repeat unit. A few exopolysaccharides may contain relatively large amounts of the methylpentoses rhamnose and fucose. An example of this was found in the polymer from a plant pathogenic Erwinia sp.15 This possessed a hexasaccharide repeat unit containing D-glucose, D-galactose, and D-glucuronic acid together with 3 mole of L-rhamnose. Another


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exopolysaccharide, from Erwinia chrysanthemi, was of fairly similar composition except that mannose replaced galactose.16 In the heptasaccharide of each repeat unit of the polysaccharide from Lactobacillus rhamnosus RW-9595, four rhamnose residues were present.17 Rhamnose is also very widely found as a component of the exopolysaccharide structures produced by the Gram-positive Lactococcus, Lactobacillus, and Streptococcus species among the LAB.18 Various acyl groups and other substituents may be found in exopolysaccharides. The commonest substituents are O-acetyl groups which are ester linked. Therefore, they do not confer any charge on the macromolecule. Pyruvate ketals also very frequently form components of exopolysaccharides, and they do confer a negative charge due to the presence of a free carboxylic acid group. They are usually attached to D-glucose, D-galactose, or D-mannose residues, but in Rhodococcus equi type 3, are attached to the O2 and O3 positions of D-glucuronic acid.19 Acetyl groups and pyruvate ketals are more common in heteropolysaccharide structures than homopolysaccharides and both are commonly found in the same polymer, but bacterial alginates from Az. vinelandii or Pseudomonas aeruginosa and related species provide one example of an exopolysaccharide in which O-acetyl groups are the only noncarbohydrate constituent and one, moreover, in which some of the D-mannuronic acid residues carry multiple acetyl groups as seen below. Some xanthan polysaccharides also appear to be O-acetylated, again with possible multiple acetylation of some monosaccharide residues, but devoid of pyruvate ketal groups. Pyruvylated but nonacetylated polysaccharides are exemplified by the Escherichia coli K50 linear tetrasaccharide repeat unit, the mannosyl residue of which carries pyruvate ketals while no O-acetyl groups are present although N-acetyl groups are present on the acetylaminosugars.20 The presence of acetyl and pyruvate ketals attached to the same monosaccharide is relatively uncommon, but exopolysaccharides from Pseudomonas ‘gingeri’ and Es. coli K55 carried an acetyl group on the O2 position and a pyruvate ketal on the O4 and O6 positions of the mannose residue which forms part of the trisaccharide repeat unit.21 The same carbohydrate structure with different acetylation was also found in the polymer from a Klebsiella strain. The structure of the exopolysaccharide from Ps. ‘gingeri’ strain Pf9 is shown below: Pyr 4⇓6 [→4)-b -D-GlcpA-(1→4)-b -D-Glcp-(1→3)-b -D-Manp-(1→]n 2 ↑ Ac

This exopolysaccharide has the same structure as that of Es. coli K55 EPS; Klebsiella K5EPS (below) also has the same carbohydrate structure but is acetylated on C2 of the glucopyranose residue. Pyr 4⇓6 [→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→3)-b-D-Manp-(1→]n 2

↑

Ac

The structure of bacterial alginates depends on the bacterium studied with marked differences between those from Az. vinelandii and those from Ps. aeruginosa. Az. vinelandii Ac ↓ [→4)-a-L-GulpA-(1→4)-b-D-ManpA-(1→4)-b-D-ManpA-(1→4)-b-D-ManpA-(1 →4)-a-L-GulpA-(1→4)-a-L-GulpA-(1→4)-a-L-GulpA-(1→ ]n

Ps. aeruginosa Ac

Ac

Ac

→

→

→

[→4)-b-D-ManpA-(1→4)-b-D-ManpA-(1→4)-b-D-ManpA-(1→4)-b-D-ManpA-(1 →4)-b-D-ManpA-(1→4)-b-D-ManpA-(1→4)-a-L-GulpA-(1→]n

In the bacterial alginates, there is no regular structure and only D-mannuronosyl residues carry O-acetyl groups; some of these monosaccharides are multiply acetylated. In Pseudomonas polymers, there are only single L-guluronosyl

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residues, whereas Azotobacter has block structures (contiguous sequences) of either type. The polysaccharides from Az. vinelandii and Ps. aeruginosa are all normally high molecular mass products, but some alginates from plant pathogenic Pseudomonas species are of much lower mass. As well as O-acetylation, succinyl half esters are found in many of the polymers from plant symbiotic Rhizobium species, as are hydroxybutyryl groups. Monosaccharides esterified with lactic acid have also been reported (Table 1). A few exopolysaccharides carry amino acid substituents. Two major types of inorganic substituents are also found. Although phosphate is absent from most of the Gram-negative bacterial polysaccharides investigated so far, including those from genera such as Klebsiella, Rhizobium, Xanthomonas, and Pseudomonas, it has been identified in the products of a number of E. coli strains. The phosphate-containing polysaccharides from these bacteria resemble the teichoic acids found in the walls of Gram-positive bacteria and the teichoic acid-like exopolysaccharides from Streptococcus (Diplococcus) pneumoniae strains. Phosphate groups are relatively common among polysaccharides from Gram-positive bacteria, but are also present in some exopolysaccharides from Gram-negative bacteria including several E. coli serotypes. One, E. coli K24, contained phosphate together with ketodeoxyoctonic acid, a monosaccharide more commonly associated with the lipopolysaccharides of Gram-negative bacterial cell walls.22 Although pentoses are not present in the majority of exopolysaccharide structures which have been published so far, the polymer from Aeromonas nichidenii 5797 is composed of a trisaccharide main chain repeat consisting of the sequence a-D-mannose-bD-xylose-a-D-glucuronic acid, to which single residues of glucose and mannose are attached as side chains on the mannose and uronic acid, respectively.23 The main chain mannose is also O-acetylated. Sulfate groups have been found in exopolysaccharide products of cyanobacteria and of halophilic species from the primitive archeae as well as isolates obtained from sampling close to deep hydrothermal vents.24 The halophile Haloferax mediterranea yielded a viscous, high molecular weight polysaccharide containing almost 6% sulfate,25 while the polyanionic exopolysaccharides from strains of the cyanobacteria, Synechocystis, contained 8% sulfate in addition to uronic acids and neutral monosaccharides.26 The exopolysaccharide from H. mediterranea strain R4 proved to be composed of trisaccharide repeat units to which ester-linked sulfate was attached at the 3-position of N-acetyl-D-glucosaminuronic acid.27 Both phosphate and sulfate groups add to the polyanionic nature of exopolysaccharides. The structure of H. mediterrane exopolysaccharide is shown below: [→4)-b-D-GlcpNAcA-(1→6)-a-D-Manp-(1→4)-b-D-GlcpNAcA-(1→]n 3 ↑ − O-SO3

In a relatively short space of time, structural studies on bacterial exopolysaccharides have provided a rapidly increasing list of chemically well-characterized macromolecules, although accurate determination of their molecular mass is less easy.

2.16.2.3 Polysaccharide Families From the extensive studies on bacterial exopolysaccharide structure, it is now clear that within some bacterial groups and sometimes from different species, there exist polysaccharides which are structurally very similar indeed. These can be regarded as families of polymers. Two widely studied examples of extracellular heteropolysaccharide ‘families’ from Gram-negative bacterial species are xanthan and colanic acid. Due to its commercialization, the polysaccharide xanthan from Xanthomonas campestris is undoubtedly one of the best-known and most-studied exopolysaccharide structures.28,29 It is composed of pentasaccharide repeat units which represent trisaccharide side chains attached to a cellulose backbone. Attached to the side chains are O-acetyl and pyruvate ketal groups. While the monosaccharide composition remains constant, the acyl groups vary depending on the bacterial strain used (and possibly also on the physiological conditions used for their production). Some lack acetyl groups and others are nonpyruvylated. Mutant X. campestris strains have also been shown to form polysaccharides lacking the terminal side chain monosaccharides and their attached acyl substituents (vide infra). These add further to the group of closely related structures which can provide information on structure/function relationships.30 Xanthan is not the only bacterial exopolysaccharide formed from a cellulosic main chain. Some strains of G. xylinus are capable of producing a polymer which has been named acetan. This resembles xanthan in having a main chain composed of cellobiose repeat units. However, instead of the trisaccharide side chain found in xanthan, a pentasaccharide composed of D-mannose, L-rhamnose, D-glucose, and 31 D-glucuronic acid is present. Acetan does not contain pyruvate ketal groups, but, as is the case with xanthan, is also O-acetylated.


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The structure of the exopolysaccharide from X. campestris (xanthan) is shown below: [→4)-b-D-Glcp-(1→4)-b-D-Glcp-(1→]n 3 ↑ 1 a-D-ManpAc 2 ↑ 1 b-D-GlcpA 4 ↑ 1 b-D-ManpPyr

Typically, the internal a-mannosyl residue of xanthan is fully acetylated but only c. 30% of the b-mannosyl termini are ketalated. In preparations from X. campestris pv. oryzae, the internal mannosyl residue carries two O-acetyl groups, while pyruvate ketals are absent. The structure of the exopolysaccharide from strains of G. xylinum (acetan) is shown below: [→4)-b-D-Glcp-(1→4)-b-D-Glcp-(1→]n 3 ↑ 1 a-D-Manp 2 ↑ 1 b-D-GlcpA 4 ↑ 1 a-D-Glcp 6 ↑ 1 b-D-Glcp 4 ↑ 1 a-L-Rhap

Normally the acetan polysaccharide carries two O-acetyl groups on each repeating unit, one of which is possibly on a main-chain glucose residue. Colanic acid, as already mentioned, is a product of many strains of Enterobacteriaceae when grown under suitable conditions. Some variants of the E. coli K12 strain, which were used in the early studies on bacterial genetics, proved to be stable producers of large amounts of colanic acid. The polymer is formed from hexasaccharide repeat units from four monosaccharides L-fucose, D-glucose, D-galactose, and D-glucuronic acid but is additionally acetylated and carries pyruvate ketal groups.32 The structure of the exopolysaccharide colanic acid from strains of E. coli, Salmonella typhimurium, and Enterobacter cloacae is shown below: [→3)-b-D-Glcp-(1→3)-b-L-Fucp-(1→4)-a-L-Fucp-(1→]n 4 ↑ 1 b-D-Galp 3 ↑ 1 b-D-GlcpA 4 ↑ 1 b -D-Galp3,4Pyr

This polymer typically carries an O-acetyl group on the 2- or 3-position of the unsubstituted fucose residue of each hexasaccharide repeating unit. Although the same carbohydrate structure is found in colanic acid from different bacterial species, the acyl substituents may vary. The pyruvate ketal may be linked to C4 and C6 instead of C3 and C4 as shown above, or may be replaced by other acetal groups. Recently, Cescutti et al.33 have shown that the structure of an exopolysaccharide produced by Enterobacter amnigenus is also formed from hexasaccharide repeat units resembling colonic acid, in which the terminal D-galactosyl residue is

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replaced by D-mannose. Although five of the monosaccharides are found in the same sequences, there are however differences in a number of the linkages as can be seen below. It is also relatively unusual to find exopolysaccharides containing both L-fucose and D-mannose, as mannose is an intermediate in fucose biosynthesis at the sugar nucleotide level, although another example was seen in the disaccharide repeat of Alcaligenes latus cxopolysaccharide.13 Similarities in composition to colanic acid are also seen in a polysaccharide from Er. chrysanthemi Ech6, but the trisaccharide side chain is attached to a glucose residue rather than fucose. The structure of the En. amnigenus polysaccharide is shown below: [→3)-b-D-Glcp-(1→4)-a-L-Fucp-(1→4)-a-L-Fucp-(1→]n 4 ↑ 1 a-D-Galp 3 ↑ 1 a-D-GlcpA 4 ↑ 1 a-D-Manp4,6Pyr

Another group of polysaccharides showing many common features in composition and structure have been found in the plant symbiont bacteria genus Rhizobium. These polymers, termed succinoglycans, are mainly formed from octasaccharides which carry O-acetyl, pyruvate ketal, and succinyl half ester substituents.34 They are also atypical in that most are formed of neutral sugars, although in some D-riburonic acid,35 D-glucuronic acid, or methylglucuronic acid may be present as the terminal monosaccharide in the side chain of the repeat unit. Most are formed from a highly conserved tetrasaccharide backbone which carries a tetrasaccharide side chain. Although this latter unit is generally conserved, in some species, variants are found including those in which the uronic acids replace neutral monosaccharides. A typical succinoglycan structure is seen in the exopolysaccharide which can be isolated from Si. meliloti, Agrobacterium radiobacter, and other related bacterial species. Ac ↓ 6 [→4)-b-D-Glcp-(1→4)-b-D-Glcp-(1→3)-b-D-Galp-(1→4)-b-D-Glcp-(1→]n 6 ↑ 1 b-D-Glcp-(1→3)-b-D-Glcp-(1→3)-b-D-Glcp-(1→6)-b-D-Glcp 6 4⇑6 ↑ Pyr Succ

Sharing of structure is also seen in a group of three polysaccharides with tetrasaccharide repeat units from Klebsiella aerogenes type 54, Enterobacter XM6, and E. coli K27.36–38 The carbohydrate structures of the first two polymers are identical, but the K. aerogenes products are either acetylated with either acetyl groups on each repeat unit or on alternative repeat units (i.e., on each octasaccharide). In the E. coli exopolysaccharide, a D-galactosyl residue replaces the D-glucosyl side chain. A further group includes the commercial product gellan which is considered below. The ability of different bacterial strains and species to synthesize exopolysaccharides structures with considerable similarities to one another is not limited to the examples given above. Sialic acid-type polymers have been identified from various pathogenic and nonpathogenic bacterial species. Some bacteria only secrete sialic acid, while others include it among a range of heteropolysaccharides. All serotypes of Streptococcus agalactiae form a polymer in which a common structural feature was N-acetylneuraminic acid a2!3-linked to D-galactose.39 Although mannans are a common feature of fungal walls and are formed by various eukaryotic microorganisms, they are not normally products of bacteria. However, an exopolysaccharide product from P. syringae pv. ciccaronei is an a-linked D-mannan which was both highly branched and phosphorylated.40 It thus resembled some of the fungal products but differed in also carrying a small number of glucose residues. Some of the most complex repeat units have been found in exopolysaccharides from cyanobacteria. One such, from Cyanospira capsulata, was composed of octasaccharide repeat units, containing seven monosaccharides including L-arabinose and 4-O-(1-carboxyethyl)mannose.41 The structure had three branches attached to the main chain. An even more complex pentadecasaccharide repeat unit structure was proposed for the exopolysaccharide from Mastigocladus laminosus, a thermophilic cyanobacterium.42 No doubt, as this group is examined further, it will yield other very complex polysaccharides, perhaps with novel or rare monosaccharide components. Another group of bacterial exopolysaccharides currently yielding some unusual monosaccharides and complex structures is the one isolated from marine isolates, especially from hypobaric or


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hypothermal environments. In one such polysaccharide, Perepelov et al.43 reported the presence of 5,7-diacetamido3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid. Another from the deep sea isolate Alteromonas infernus has a repeat unit with nine monosaccharides and is sulfated.44 The sulfated trisaccharide main chain has a branched hexasaccharide side chain in which a tetrasaccharide contains two residues of D-glucuronic acid each of which carries an a-D-glucose. More complicated structures are increasingly being found as improved procedures for structural analysis have become available. Klebsielle pneumoniae SK1 provides an example of a heteropolysaccharide of relatively simple composition but possessing two side chains on each repeat unit. Another highly branched polymer with a heptasaccharide repeating unit was identified as a product of strains of Burkholderia cepacia, a pathogen frequently associated with cystic fibrosis patients.14 In this polymer, the trisaccharide backbone carried three side chains, two of which were attached to the same D-glucuronosyl monosaccharide in the main chain. Structure of the K. pneumoniae SK1 exopolysaccharide: [→3)-b-D-Galp-(1→3)-b-D-Galp-(1→3)-a-D-Glcp-(1→]n 4 2 ↑ ↑ 1 1 b-D-GlcpA a-D-Galp

Burkholderia cepacia polysaccharide structure: b-D-Galp-(1→2)-a-D-Rhap 1 ↓ 4 [→3)-b-D-Glcp-(1→3)-a-D-GlcpA-(1→3)-a-D-Manp-(1→]n 2 6 ↑ ↑ 1 1 a-D-Galp b-D-Galp

Increasingly, unusual sugars are also being found in polysaccharide structures which otherwise relate to recognized families such as succinoglycan or gellan. One monosaccharide found in polymers from each of these groups is a 2-deoxy-D-arabino-hexuronic acid.45

2.16.3 Physical Properties and Structure/Function Relationships Because of the large number of monosaccharides present in exopolysaccharides, the diversity of linkage types, and the possible presence of noncarbohydrate constituents, bacterial exopolysaccharides present a very wide potential range of physical properties. They differ from plant polysaccharides in that they are regular, linear structures lacking extensive branching of side chains. Any side chains which are present are attached at regular intervals along the macromolecule and are of constant length and composition. Atkins46 pointed out that the variety of condensation linkages in polysaccharides, coupled with variations in monomer sequence, produced a wide range of possible shapes and architectures. The effects which certain polysaccharide components confer on their physical properties can be seen in Table 4. The extensive range of primary structures which have now been found in bacterial polysaccharides ensures that the number of possible conformations or secondary structures adopted is on the increase. There are considerable

Table 4 Effect of polysaccharide composition on physical properties Polysaccharide component

Effect

Properties affected

Example

Neutral sugars Uronic acids Pyruvate Methylpentoses Acetylation Side chains 1!3- or 1!4-linkages 1!2-linkages

Uncharged polymer Polyanionic Polyanionic Lipophilicity Solubility Various Rigidity Flexibility

Insoluble Solubility, ion binding Ion binding, transition Solubility Gelation, reduced ion binding Solubility (In)Solubility Solubility, stability

Cellulose, biofilms Xanthan, alginates Xanthan, galactoglucans Biofilm exopolysaccharides Alginates, gellan, etc. Xanthan, gellan Curdlan, cellulose Dextrans

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differences in solubility and in other physical properties of these polysaccharide solutions and gels even when they are derived from closely related chemical structures. If the rheological properties of bacterial exopolysaccharides are to be understood, a thorough knowledge of the tertiary structure and of the potential for intermolecular interactions is required. The association of the polymer chains, which are frequently of very high molecular mass of the order of 0.5–2M, results in complex entanglements which contribute greatly to the physical properties that have been observed. Studies on families of structurally related exopolysaccharides, together with the extensive examination of the physical properties of a more limited number of polymers, have provided considerable information on structure/function relationships. The commercial development of xanthan and gellan has also stimulated extensive physical studies on these and other bacterial exopolysaccharides, especially those which bear structural similarities to the commercialized polymers. Information is now available on various physical properties including tertiary structures, viscosity, crystallinity, and gel-forming capacity. It is clear that a wide range of physical attributes can be determined and that these relate to the chemical composition and to the structural niceties of the polysaccharides. Due to their high mass, many yield viscous aqueous solutions, while others are capable of gelation, either per se or in the presence of specific cations, or when associated with certain other polysaccharides. The very significant role played by acyl groups in determining or modifying the physical properties of some exopolysaccharides has also become clear. Cations and water molecules were found to promote the association of helices, while the tendency toward helix formation was a feature of both the solid and the liquid state. These and other physical properties were determined by the shape of the helix, the interactions between the helices, and the interactions with cations and with ordered water molecules.47

2.16.3.1 Helix and Coil Structures The primary conformation of the bacterial exopolysaccharides, as with all polysaccharides, is determined by their composition and structure. In some of these bacterial polymers, the backbone composition of sequences of b1!4- or b1!3-linkages may confer considerable rigidity as is seen in the cellulosic backbone of xanthan from X. campestris. Where no side chains are present to influence the rigid structure, as in the a1!3-linkages of mutan or the b1!3linkages of curdlan, the polysaccharide can be effectively insoluble even though these particular exopolysaccharides are of lower mass than most. Other linkages in polysaccharides may yield more flexible structures. These can be exemplified by the a1!2- or a1!6-linkages found in many dextrans. Further, ordered secondary configuration frequently takes the form of aggregated helices. The transition in solution from random coil to ordered helical aggregates is often greatly influenced by the presence or absence of acyl substituents such as O-acetyl or O-succinyl esters or pyruvate ketals.48 In those polymers such as acetan and xanthan which have cellulosic main chains, the presence of regular side chains renders soluble macromolecules which would normally be insoluble. This may be the case even when, as in the products of mutant strains, the side chains have been abbreviated to a single monosaccharide (mannose) in place of the normal trisaccharide or pentasaccharide. The original structures also yield highly viscous aqueous solutions with good salt stability and compatibility with other macromolecules. Normally, in most natural and experimental environments, the exopolysaccharides are present in the ordered configurations which are found at lower temperatures and in the presence of salts. The majority of exopolysaccharides are essentially very long thin molecular chains with mass of the order of 0.5–2 million Da, but they can associate in a number of different ways. Several polysaccharide preparations have been visualized as fine strands which are attached to the bacterial cell surface and form a complex network surrounding the cell. Mayer et al.49 suggested that electrostatic and hydrogen bonds are the dominant forces implicated. Ionic interactions may be involved, but more subtle chain–chain complex formation in which one macromolecule ‘fits’ into the other may result in either floc formation or networks which are very poorly soluble in aqueous solvents. Another result may be the formation of strong or weak gels.

2.16.3.2 Gelation A number of water-soluble bacterial exopolysaccharides can, under appropriate conditions, form gels. This may involve coil-to-helix transition of the type seen on cooling a heated random coil transition.50 The gel which is formed may be either weak, brittle, or strong. Comparison of bacterial and algal alginates gives a clear indication of one aspect of the relationship of polysaccharide structure and function. Gelation and significant changes in viscosity are usually transitional effects. Extensive studies on several groups of exopolysaccharides have provided information on the exact conditions necessary for transition from order to disorder and vice versa. This transition may be very sharp as exemplified by the Enterobacter XM6 polysaccharide. This polymer is composed of tetrasaccharide repeat units composed of D-glucose, L-fucose, and D-glucuronic acid in the molar ratio 2:1:1.38 In aqueous solution, it provides


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high viscosity, but it gels in the presence of several monovalent or divalent cations, gelation depending on the ion concentration. Much higher concentrations of monovalent ions were required compared to divalent ions. The ionic radius was also important with Naþ and Ca2þ giving optimal gelation. There was a very sharp transition from the ordered gel form at about 28 C and gelation on cooling at a slightly lower temperature. In the case of xanthan, transition is more gradual, occurring over a temperature range of 35–55 C and is greatly influenced by the presence or absence of the acyl substituents (acetyl and pyruvate ketal groups) and by the ionic environment.51 Comparison of the polysaccharides from K. aerogenes K54 and Enterobacter aerogenes XM6 indicated the effects of acetyl groups. The nonacetylated XM6 polymer presented a highly crystalline structure recognizable by X-ray fiber diffraction,52 whereas the K54 polymers carrying either 0.5 or 1 acetate group per repeat unit were amorphous. When the K54 polymers were deacetylated by treatment with mild alkali, the fiber diffraction patterns changed to ones similar to XM6. XM6 and deacctylated K54 formed gels in the presence of various ions while native K54 did not.53 Algal alginates lack any acylation and readily form rigid, nondeformable gels as a result of the highly specific interaction with either Ca2þ or Sr2þ, a property which is widely used in biotechnology for the immobilization of cells and enzymes. This is not seen in bacterial alginates from Az. vinelandii, even though these exopolysaccharides closely resemble the algal polymers in possessing sequences of polyguluronic acid blocks producing the characteristic egg-box structure, in addition to polymannuronic acid and mixed sequences.54 The bacterial polysaccharides are acetylated and the acetyl groups strongly inhibit the interaction between polymer chains and cations and resultant gel formation. Some binding of cations does occur, and there is also some limited specificity toward Ca2þ. Pseudomonas aeruginosa alginates totally lack sequences of guluronosyl residues; they are normally incapable of gelling with divalent cations but do still bind them to a more limited extent.55 The Ps. aeruginosa polysaccharides yield highly viscous aqueous solutions. In both types of bacterial alginates, chemical removal of the O-acetyl residues significantly alters their physical properties, and leads to increased binding of divalent cations. This modification also has the effect that the aqueous solubility of the macromolecules is very much reduced or else, as is the case with high molecular mass Az. vinelandii alginates, yields a totally insoluble product. Many bacterial exopolysaccharides possess backbone structures in which sequences of b1!3- or b1!4-linked hexose residues predominate. When such sequences are present, the polymers tend to be much more rigid in structure, less deformable, and, in the case of neutral polysaccharides such as mutan from Streptococcus mutans or those from some strains of Enterobacter agglomerans, either poorly soluble or effectively insoluble. These exopolysaccharide molecules may be very robust; stiffer chains are also found in some of the polysaccharides from LAB, such as that from Lactococcus lactis subsp. cremoris B40. The long chains of stiff macromolecules may be present as gels due to the entanglements found within the long chains and also due to the ionic environment in some polymers.56,57 The stability of the gel state will depend on the effective polysaccharide concentration, the ionic status, and the other macromolecules present. Those exopolysaccharide molecules which are effectively in solution may well dissolve with dilution or as ions are gradually removed. In natural environments, this may thus account in part for the observed ‘sloughing off’ of biofilm material. Another aspect, which has received relatively little study, is the possibility of interaction of exopolysaccharides in the gel state with proteins and other excreted or surface-associated macromolecules. Either association or segregation may occur. The conformation and the tendency to form gels may be very greatly affected by any substituents present. This is clearly seen in the commercial product ‘gellan’ from a Sphingomonas elodea strain when compared with the properties of structurally related polysaccharides (Table 5). The native, acylated gellan formed weak gels, whereas the deacylated material yielded brittle, rigid gels. The polysaccharide adopts a double helical structure in which the duplex is stabilized by interchain hydrogen bonds.58 It has been suggested that the O-acetyl groups on the native polysaccharide only have a weak effect on aggregation of gullan molecules, whereas the L-glyceryl residues are detrimental to crystal packing.59 This result agrees with the observation by Crescenzi et al.60 that through hydrogen bonding, the short side chains of L-mannose or L-rhamnose mask the uronic acid residues in the conformation adopted in aqueous solutions. Further confirmation of this interpretation was obtained by Lee and Chandrasekaran61 in an X-ray and computer modeling study of gellan and three structurally related polysaccharides, in which it was concluded that although all have the same double-helical conformations, the side chains shielded the carboxylate groups to varying degrees. The shielding was considerable in welan and S-657 polysaccharides, but much less in rhamsan. In aqueous solutions of welan, the polysaccharide is very highly ordered and, even on heating, fails to show the normal order–disorder transition.62 It was suggested that the complete removal of the side chains would be required to expose the carboxylate groups entirely, as is seen in gellan. Picullel50 pointed out that a common type of gelation among exopolysaccharides involved a coil-to-helix transition. This could yield various types of gel – the exact type depended on the nature of the polysaccharide and on the solvent present. Xanthan from X. campestris formed very weak gels which dissolved in excess solvent, whereas other bacterial

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Table 5 The structures of gellan and related polysaccharides

(a) Gellan gum [→3)-b-D-Glcp-(1→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→4)-a-L-Rhap-(1→]n

(b) Welan gum (S130) [→3)-b-D-Glcp-(1→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→4)-a-L-Rhap-(1→]n 3 ↑ ↑ Ac 1 a-L-Rhap or a-L-Manp

(c) Rhamsan gum (S194) [→3)-b-D-Glcp-(1→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→4)-a-L-Rhap-(1→]n 6 ↑ 1 b-D-Glcp-(1→6)-a-D-Glcp

(d) Sphingomonas S657 [→3)-b-D-Glcp-(1→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→4)-a-L-Rhap-(1→]n 3 ↑ 1 a-L-Rhap-(1→4)-a-L-Rhap

(e) Sphingomonas S88 [→3)-b-D-Glcp-(1→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→4)-a-L-Rhap-(1→]n 3 or a-L-Manp ↑ 1 a-L-Rhap

(f) Sphingomonas S198 [→3)-b-D-Glcp-(1→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→4)-a-L-Rhap-(1→]n 3 or a-L-Manp ↑ 1 a-L-Rhap

(g) Sphingomonas NW11 [→3)-b-D-Glcp-(1→4)-b-D-GlcpA-(1→4)-b-D-Glcp-(1→4)-a-L-Manp-(1→]n

polysaccharides formed brittle gels unaffected in shape by the presence of excess solvent. In the case of gellan, the nature of the gel has been shown to be highly dependent on the extent of acylation present on the polymer. Alterations in acylation produced a range of weak or strong gels, the latter favored by removal of acyl groups. A feature of xanthan which has been used commercially is its ability to form gels with various plant glucomannans and galactomannans, the so-called synergistic gels. These are formed when the mixed polysaccharides are heated above their transition temperatures, then allowed to cool. Neither the bacterial nor the plant polysaccharide can form stable gels on their own. The role of acyl and other groups in this type of complex formation has now been widely studied. Acetyl groups greatly affect the interactions between the different polysaccharides, as was demonstrated for mixtures of xanthans and plant polysaccharides.63 Acetyl groups in several of these systems weakened the gel network and reduced gelation, while the removal of the acetyl groups reversed the effect. Although native acetan fails to form gels with either locust bean gum (LBG) or konjac mannan (glucomannan), both these plant polysaccharides will form synergistic gels with deacetylated acetan, thus providing another example of the effect of acetyl groups.64 X-ray fiber diffraction patterns of mixtures of deacetylated acetan and glucomannans indicated that the side chains were


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sufficiently flexible to permit association of the chains with the same or opposite polarity yielding a weak gel.65 Xanthan in aqueous solution normally undergoes thermoreversible helix–coil transition. A similar transition was also found for solutions of acetan despite the longer side chains and lack of pyruvylation in this exopolysaccharide.66 For both polysaccharides, the ionic strength played an important role in determining the transition temperature for the ordered form. Acetylation did not inhibit the formation of the helices. It was also clear that the pattern of acylation on the xanthan macromolecules only had a relatively slight influence on its inherent stiffness.67 It is possible that synergistic gelation may also be found between different bacterial exopolysaccharides as well as between these polymers and plant or animal polysaccharides. Most xanthan preparations formed strong thermoreversible gel networks with LBG but interact more weakly with guar gum. The strength of the gels depended on the polysaccharide concentrations and on the degree of acylation of the xanthan.68 Ross-Murphy et al.63 used a series of different xanthan preparations to demonstrate that the acyl groups of xanthan played a significant role in the interactions with guar gum, LBG, and konjac mannan. Removal of the acetyl groups from xanthan enhanced gelation. Most of the xanthan preparations tested formed relatively strong gel networks with LBG. An exception was xanthan with high acetate and low pyruvate content. When mixed with konjac mannan, the xanthans showed similar behavior to the interaction with LBG but higher polysaccharide concentrations were needed. The acetylated, nonpyruvylated xanthan failed to gel, whereas the deacetylated material formed a strong gel. This again demonstrated the inhibitory effect of the O-acetyl groups. Initially, mixtures of xanthan and LBG showed areas enriched in xanthan. After heating above the transition temperature, these disappeared, probably due to more uniform distribution of the two component polysaccharides and disappearance of the liquid crystal mesophases.69 Variations in the ratio of mannose to galactose in LBG also affected the properties of the mixed gels. A difference in gelation temperature of almost 13 C was observed by Lundin and Hermansson70 when comparing mixtures of xanthan and LBG with high and low mannose:galactose ratios. It has been suggested that within mixed gels there is coexistence of both LBG–LBG and LBG–xanthan junction zones.71 Interaction of plant glycommannans with partially degraded xanthan in which the side chains had been removed by mild acid hydrolysis has also been examined. Removal of the terminal b-D-mannosyl residue did not affect the transition temperature; the modified exopolysaccharide showed order–disorder properties very similar to the native polymer and also adopted a doublestranded state. It has now been found that although native acetan does not form gels with LBG or konjac mannan, deacetylation of this bacterial polysaccharide promotes synergistic interactions with both.72 Acetan resembled xanthan in that it adopted a similar conformation in the solid state and showed the same thermally reversible transition form order (helical form)-to-disordered coil in solution. The failure of the native, acetylated acetan to form mixed gels was attributed to the solubility promoted by the presence of the O-acetyl groups and the resultant inhibition of intermolecular association.

2.16.3.3 The Relationship of Chemical and Physical Structures Now that many exopolysaccharide structures have been fully determined, some attempt to relate chemical structure to function and their physical attributes can be made. This is seen both in homopolysaccharides with relatively simple structures and more markedly when greater structural differences in heteropolysaccharides are assessed. The existence of ‘families’ of polysaccharides with closely related structures also indicates the specific role played by certain structural components and substituents. In particular, the influence of noncarbohydrate substituents has now been widely studied for some of the bacterial exopolysaccharide families and assists in interpreting the relationship of structure to function. The existence within ‘families’ of polysaccharides in which single monosaccharides are altered within a specific structure, or with varying side chains, also gives an indication of the way in which specific substituents affect the physical properties of the polymers in aqueous solution and in other physical properties. Comparison of the properties of bacterial exopolysaccharides with those of similar polymers from eukaryotes may also prove useful. Curdlan is one of a group of exopolysaccharides which is capable of forming triple helices in which the strands are very firmly held together by hydrogen bonds. Such tight bonding together of the linear molecules may effectively exclude water molecules and consequently render the polymer insoluble.73 Heating of curdlan to 120 C or higher temperatures alters the configuration from a single helix to the triple helical form and yields gels melting between 140 and 160 C. These gels dissolved in M NaOH compared to the 0.1M NaOH required to solubilize the original gels.74 The corresponding a-D-glucan, mutan, may behave similarly as it adopts a highly crystalline structure.75 The conformation was stabilized by intramolecular hydrogen bonding yielding a sheet-like structure, within which there was alternating polarity of the chain directions. At the molecular level, the physical properties of the polysaccharides are dependent on inter- and intramolecular interactions and may be very greatly influenced by the presence or absence of free anionic groups derived from uronic

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acids, phosphate groups, pyruvate ketals, or succinyl half esters. Exposed hydroxyl groups can also cause significant hydrogen bonding. Localized hydrophobic regions may also exert considerable influence; these may be derived from either O-acetyl groups, methyl groups, or 6-deoxyhexoses such as L-rhamnose or L-fucose. The availability of improved imaging techniques, such as atomic force microscopy, has shown that a number of exopolysaccharides, again including gellan, can form a dense network of thin fibers.76 This has also been observed in some electron micrographs of individual, capsulated bacterial cells. X-ray fiber diffraction analysis has revealed the three-dimensional structures of a number of exopolysaccharides. Chemical deacetylation of the polymer was sometimes necessary to obtain oriented crystalline films. Beijeran, a polymer with a trisaccharide repeat unit synthesized by a nitrogen-fixing soil organism, adopted an extended twofold helical structure.78 The two chains were tightly packed in an antiparallel manner by hydrogen bonding and water molecules to form thick sheets. These sheets then associated in a network with water molecules and sodium ions. The structure of beijeran, the exopolysaccharide from Azotobacter beijerinkia YNM1, is shown below: [→3)-a-D-GalpA-(1→3)-b-L-Rhap-(1→3)-a-D-Glcp-(1→]n 6 ↑ Ac

From the studies of Chandrasekaran et al.79,80 gellan was shown to adopt a double-helical conformation. Each chain was translated half a pitch (p¼5–64nm) relative to the other with interchain hydrogen bonds stabilizing the structure adopted by the polymer. Examination of other members of the gellan family of exopolysaccharides (Table 4) indicated that they too adopted a similar double-helical conformation.81 This demonstrates that the range of side chains present in these polysaccharides do not affect the ordered conformation. Welan, another polysaccharide structurally related to gellan, formed half-staggered, parallel doublehelical chains similar to gellan. In the calcium salt of this polymer, the side chains folded back onto the main chain and hydrogen bonds were formed with the carboxyl groups. This enhanced the stability of the double helix. Clearly, a wide range of possible conformations, flexibility, and configurations can be expected among the different polysaccharides, but all do not adopt an ordered conformation. This was revealed for two Klebsiella pneumoniae exopolysaccharides. The product from strain K40 was relatively flexible in solution and adopted a random coil conformation, while that from strain SK1 showed a similar lack of order.82,83 Several exopolysaccharides form effective emulsifying agents. Two groups of biosurfactants are represented by ‘emulsan’ and ‘alasan’ from Acinetobacter lwofii (formerly designated calcoaceticus) and other related bacteria, respectively. Some cyanobacteria, exemplified by Phormidium sp., also yield polymers with emulsifying activity. Some of these macromolecules are similar in their structures to lipopolysaccharides and are amphipathic molecules capable of stabilizing oil-in-water emulsions.84

2.16.4 Physiological Aspects of Exopolysaccharides Production Most of the bacteria from which exopolysaccharides have been isolated are either aerobes or facultative anaerobes. There has been little intensive investigation of such products from strict anaerobes. Studies on a number of bacterial isolates from deep marine environments have been shown to produce exopolysaccharides in good yields,85 but the effects of hypobaric conditions on growth and polymer production by these bacteria have apparently not been investigated. The formation and excretion of exopolysaccharides is a process requiring considerable amounts of energy. Consequently, it is favored by growth under conditions of high aeration and plentiful, readily utilizable energy sources such as glucose or sucrose. Most exopolysaccharide-producing bacteria can synthesize polysaccharides using a wide range of carbon substrates with the final composition being independent of the substrate used; exceptions are the dextran- and levan-forming bacteria which require the specific substrate sucrose for polymer production. In early studies on production by bacteria from the facultatively anaerobic family Enterobacteriaceae, media containing high ratios of C:N sources or of carbon sources such as glucose to limiting nutrients including phosphorus, magnesium, or potassium yielded large amounts of polymer under highly aerobic conditions. These growth conditions also favor the diversion of substrate into the synthesis of intracellular storage materials such as glycogen and poly-bhydroxyalkanoic acids but at least in the enteric bacterium K. aerogenes, conversion of substrate to exopolysaccharides appeared to be favored rather than glycogen synthesis.86 Many nitrogen-fixing bacteria from different taxonomic


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groups are capable of forming considerable amounts of exopolysaccharides and in these species grown with high carbohydrate substrate concentrations, the effective limitation of available nitrogen again favors polysaccharides synthesis. Incubation of bacteria at suboptimal temperatures, leading to slower growth rates or subjection to alternative stresses such as increased osmotic pressure, may also enhance exopolysaccharide production. These factors may also increase the availability of the specific sugar nucleotide precursors required for polysaccharide synthesis in preference to their diversion to formation of structural polymers. Some bacteria only produce exopolysaccharides either late in the exponential phase of growth or after cell growth and division has effectively ceased. This may be due to utilization of shared precursors for cell growth in preference to exopolysaccharide synthesis. In other species, production may occur throughout the growth cycle. As an example of this, xanthan is produced throughout growth of X. campestris as well as in the stationary phase. The specific rate of xanthan synthesis by X. campestris is closely related to the growth rate in batch culture; it is maximal during exponential growth and minimal during the stationary phase. Although in this respect xanthan production resembles the synthesis of several other bacterial exopolysaccharides, in some other bacteria polysaccharide synthesis is entirely growth related. In further groups of bacteria, production may only occur extensively after cessation of cell growth and division. The yield of an exopolysaccharide may be affected by production of other macromolecules by the bacteria. Thus, many form intracellular storage polymers such as glycogen or poly-b-hydroxyalkanoic acids and these utilize substrate that might otherwise be converted to exopolysaccharides. Nevertheless, Linton87 suggested that exopolysaccharide production by bacteria was a very efficient process. Xanthomonas campestris is unusual in that a very high proportion of the carbohydrate substrate supplied during growth of the bacteria is converted to the exopolysaccharides xanthan and there is negligible formation of intracellular storage materials. Typically, media for laboratory synthesis of xanthan contain 0.1–0.2% ammonium salt and 2–3% glucose or sucrose. The quality and the final yield of xanthan may be enhanced by the addition of small amounts of organic acids or of citric acid cycle intermediates such as a-ketoglutaric acid. Their presence may lead to an improved metabolic balance between carbon flow from carbohydrate substrate through the hexose monophosphate and Entner–Doudoroff pathways and the oxidation of the substrate through the tricarboxylic acid cycle. The energy needed for xanthan synthesis is obtained from the oxidation of the carbon substrate, either through complete oxidation to carbon dioxide or through partial oxidation to products such as acetate or pyruvate which are components of normal xanthan. According to Jarman and Pace,88 the proportion of the energy derived from partial oxidation of the carbon substrate depends both on the composition of the xanthan and on the ATP/O quotient. As X. campestris is a strict aerobe, oxygen is required both for growth and for xanthan production. This efficient production of a marketable product from simple substrates such as glucose or sucrose has contributed to its commercial success. In the laboratory, pure substrates such as glucose or sucrose are used whereas for commercial production of xanthan or other polysaccharides different substrates are employed. These are plentiful and cheap carbon sources which include starch, starch hydrolysates, corn syrup, molasses, glucose, and sucrose (derived from either sugarbeet or from sugarcane). If less pure products are acceptable, cruder, cheaper substrates can be employed. Some may be waste products from other industries. These include whey, cereal grain hydrolysates, and dry milled corn starch. As the optimal synthesis of xanthan requires a balance between the utilizable carbon and nitrogen sources, care must be taken to obtain consistency in yield and product quality when using cruder substrates which may contain nitrogen in addition to carbohydrate. The nutritional versatility of X. campestris is clearly a major factor in favor of its use for commercial xanthan production. However, the quality of the polymer produced from different substrates may vary considerably – the molecular weight and hence the rheological characteristics of xanthan synthesized from glucose or starch may differ from that formed when whey or other proteinaceous material is employed. The nitrogen sources that have been used for industrial production of xanthan include distillers’ solubles, yeast hydrolysate, soybean meal, cottonseed flour, and casein hydrolysate. Industrial production uses batch culture with careful control of pH and of the aeration rate; because of the viscosity of the culture fluids, adequate oxygen transfer rates may be difficult to achieve unless the fermentation vessel has been carefully designed to ensure that mixing is optimal. The conditions utilized for polymer production and processing are carefully standardized to ensure that product yield and quality are consistent.89,90 In the LAB there is a markedly different situation. Yields of exopolysaccharides are much lower than in many aerobic bacteria, reaching 1.5 g l1 as a maximum. In some strains polysaccharide production is entirely growth associated, while in others greatest yields are found under conditions which are not optimal for bacterial growth.91 The reason for this is the conversion of a large proportion of their carbon substrates to lactic acid.3 Only a small amount is therefore available for conversion to exopolysaccharide. The LAB are normally in environments in which lactose is the major carbon and energy source but they are able to use other sugars.92 LAB also have more complex growth requirements than do the Gram-negative exopolysaccharide-producing bacteria, and polysaccharide production in

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synthetic media is much less easily achieved than in enteric bacteria or X. campestris. Production of exopolysaccharides in some bacteria, including LAB, is unstable and may be readily lost.

2.16.5 Biosynthesis 2.16.5.1 General Concepts of Biosynthesis Biosynthesis of bacterial exopolysaccharides is a complex process, requiring considerable amounts of energy, available substrates, and a large number of enzymes with very different functions. Some of the gene products are needed to ensure substrate acquisition and entry of that substrate into the bacterial cell and finally to the cytoplasm. These proteins are essential for cell growth and division. They are not specific to polysaccharide synthesis as their substrates are needed for many other aspects of cell metabolism. They may also provide energy to the substrate through phosphorylation during entry to the cytoplasm, the first step in the formation of the activated sugar nucleotides. Other enzymes yield precursors common to both cell wall and exopolysaccharide synthesis and thus are essential for maintaining cell viability. Finally, another group of enzymes is specifically involved in exopolysaccharide biosynthesis and excretion from the cell, and its loss is not critical for cell viability under laboratory conditions. Three mechanisms for exopolysaccharides’ biosynthesis have been demonstrated. Most bacterial exopolysaccharides are excreted from the cell surface following intracellular assembly of the sugars and other components such as acyl groups, on a specialized lipid precursor. This precursor is also involved in the biosynthesis of the polysaccharides which form integral components of the prokaryotic wall structure. The second mechanism is that for synthesis of bacterial alginates. Although it also involves a similar intracellular process, the excreted product is subjected to extracellular postpolymerization modification. The third mechanism is entirely extracellular but unlike the other two, requires the presence of a specific substrate and does not utilize any intracellular enzymes or activated, energy-rich precursor products. This yields dextrans which are produced by an extracellular process involving lipoprotein enzymes secreted at the cell surface of Gram-positive bacteria such as Le. mesenteroides. Sucrose is required as a specific substrate together with an acceptor in the form of low molecular weight dextrans. The enzyme dextransucrase, synthesized in sucrosecontaining media, cleaves the substrate molecule and catalyzes transfer of the glucosyl moiety to the acceptor. Fructose is simultaneously released from the substrate and assimilated and metabolized intracellularly by the bacteria.

2.16.5.2 Dextran and Levan Synthesis Dextransucrases are extracellular proteins which have a dual function as they both form the initial polymer and introduce the various types of branch point – no other enzymes are involved. This group of glucansucrases are large molecules of average mass 160000Da.93 The energy needed for the biosynthesis is provided by the hydrolysis of the sucrose molecule. Levans (fructans) are formed by a similar process in which the fructose residues are polymerized and the glucose assimilated. The enzyme catalyzing their formation is levansucrase. Some bacteria are capable of both levan and dextran synthesis and in the absence of sucrose may also produce other polysaccharides. The major difference from synthesis of bacterial heteropolysaccharides (and other homopolysaccharides) is the lack of involvement of sugar nucleotides and the extracellular nature of the biosynthetic process. Glucansucrases can synthesize exopolysaccharides (glucans) with different linkage types which also differ in their degree of branching and their mass. Most of the dextransucrases are produced by LAB and related bacterial species and some strains may produce more than one glucansucrase. The enzymes may be produced either constitutively or may be induced in the presence of sucrose. Synthesis of the polysaccharide involves the transfer of the glucose to form a glucosyl enzyme intermediate from which the monosaccharide can be transferred to a series of acceptors. This can elongate the dextran chain by one glucose residue or it may be transferred to an alternative acceptor to form an oligosaccharide.94 The exact mechanism involving the enzyme complex in growth of the polysaccharide chain remains unclear. There may be elongation at either the nonreducing or the reducing terminal.95 Effectively, the glucansucrase group of enzymes in cleaving the sucrose molecule and forming the poly-D-glucan product therefore have three major functions – transglycosylation of glucose units, hydrolysis, and acceptor function.93 Depending on the type of polymer formed they can be regarded as dextransucrases, mutansucrases, alternansucrase, or, if the polysaccharide is mainly composed of a1!4 glucosidic linkages, reuteransucrases.96 Levans are formed in a very similar manner. Levansucrase catalyzes the transfer of D-fructosyl residues from sucrose to form the characteristic b2!6-bonds of the polysaccharide.94 Although levansucrases are found in both Grampositive Bacillus subtilis and in oral Streptococcus species, differences are found in respect of the two types of enzyme. The group recognized as levansucrases is widely distributed but the one termed inulosucrase is only found in LAB.93 Leuconostoc mesenteroides strains may possess both levansucrase and dextransucrase.


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2.16.5.3 Heteropolysaccharide Synthesis The synthesis of the great majority of bacterial homopolysaccharides and of all heteropolysaccharides is a complex intracellular process in which sugar nucleotides (normally nucleoside diphosphate sugars) provide activated forms of the monosaccharides. It also enables the bacterial cell to channel substrate either toward an intracellular storage product glycogen which is synthesized from ADP-glucose, or to other carbohydrate-containing polymers formed from UDP-glucose. The sugar nucleotides provide monosaccharides or, in the polysaccharides containing phosphorylated sugars, sugar phosphates for the repeat unit structures. They also provide the bacterial cells with a means of interconversion or synthesis of various monosaccharides, as the bacterium is often unable to utilize sugars found in the exopolysaccharides as carbon or energy sources. Through epimerization, dehydrogenation, and decarboxylation reactions, respectively, at the sugar nucleotide level, sugars such as D-glucose or D-mannose can be converted to galactose, uronic acids, or pentoses. D-Mannose can also be converted to L-fucose. Acyl adornments such as acetyl and ketal groups also require activated precursors in the form of acetyl-CoA and phosphoenolpyruvate, respectively. The biosynthetic process comprises five distinct steps: (1) activation of sugars through the formation of specific sugar nucleotides; (2) assembly of the repeat units on an isoprenoid lipid-phosphate (bactoprenol, C55-isoprenoid lipid) through the sequential addition of the monomers to this carrier or scaffold; (3) addition of any acyl groups to the oligosaccharide attached to its lipid carrier;97 (4) polymerization of the repeat units; and (5) excretion of the polysaccharide from the cell membrane into the extracellular environment.89 The isoprenoid lipid acceptor molecule is located in the cytoplasm membrane of prokaryotes. Thus, the repeating units of heteropolysaccharides are synthesized as lipid-soluble components to which any acyl groups are also attached. The repeating units are then polymerized and the polysaccharides are excreted into the extracellular environment where they may be found either attached to the cell surface or as cell-free colloidal masses, or both. The products are usually of fairly discrete molecular weight. Although the method by which this is controlled is unknown, genes controlling chain length have been identified in many exopolysaccharide-synthesizing bacteria. Mutants yielding polysaccharide of increased viscosity and thus assumedly greater mass have been obtained in some bacterial genera.98 Various possible polymerization and secretion mechanisms have been proposed. One involves the isoprenoid-linked intermediates being flipped across the cytoplasmic (inner) membrane by a mechanism using the wzx gene product. Polymerization then requires the action of the wzy gene product. In a second mechanism, the nascent polymer is exported across the cytoplasmic membrane by an ATP-binding cassette (ABC)-transporter. The second mechanism appears to be that involved in some E. coli exopolysaccharides but colanic acid synthesis involves a wzx/wzy-dependent pathway. Genes responsible for exopolysaccharide export have been widely found (Figure 1).99 What is still uncertain is whether other genes are responsible for binding mechanisms in those bacteria where the exopolysaccharide is mainly found as capsular material firmly attached to the cell surface. While the sequential addition of monosaccharides is a highly specific process requiring the correct sugar nucleotide and the appropriate transferase enzyme, the actual polymerization process is much less specific. This was clearly demonstrated in studies on xanthan biosynthesis. It proved possible to synthesize a series of polymers which were reduced in the length of the side chains, lacking the terminal sugar or sugars and the acyl adornments which they would normally carry.100 A similar series of truncated polysaccharides has been prepared from acetan-synthesizing Acetobacter xylimun strains.101 In other work using enteric bacteria, it also proved possible to alter the polymers formed at the level of polymerization. A modification to this type of biosynthetic mechanism is found in the production of bacterial alginates. The primary product is thought to be an acetylated homopolymer, poly-D-mannuronic acid. This is modified in an extracellular postpolymerization reaction by the enzymes classed as ‘polymannuronic acid epimerases’, which converts some of the nonacetylated D-mannuronosyl residues to L-guluronic acid. Acetylation prevents this conversion and thus the extent of the initial acetylation will determine the amount of epimerization that is possible. Acetylation may also protect adjacent mannuronosyl residues from epimerization. The resultant exopolysaccharides may either contain single residues of guluronic acid or sequences of varying length alternating with sequences of mannuronosyl residues. In bacterial alginate from Pseudomonas spp., only single isolated residues of L-guluronic acid have been detected. In contrast, Az. vinelandii possesses a family of seven Ca2þ-dependent polymer-level epimerases,102 and these yield an alginate in which both single guluronosyl residues and blocks of contiguous residues are detectable.

2.16.6 Genetics and Regulation of Exopolysaccharides 2.16.6.1 General Aspects of Genes Involved in Polysaccharide Synthesis Various bacteria have been used to reveal detailed knowledge of the genetics of exopolysaccharide synthesis. Initial studies used enteric bacteria, but much interest has centered on xanthan as an industrial product from X. campestris, while interest in bacterial plant and animal pathogens has prompted study of a wide range of bacteria from these groups. Rhizobium

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8 Wzi

Wza complex Outer membrane 7

Other cellular proteins?

Peptldoglycan P

2 1 WbaP

P

Wzc oligomer

P

P

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3

Inner membrance Wzx

Wzy

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P

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P 6 Wzb

ATP Pl Pl

Figure 1 Possible mechanism for exopolysaccharide export in E. coli. (1) Glycosyltransferase reactions yield lipid-linked repeat units of the exopolysaccharide at the cytoplasmic face of the inner membrane. (2) These lipid-linked repeat units are flipped across the inner membrane by a process which involves Wzx. (3) The repeat units are then polymerized by a reaction involving Wzy. (4) Wzc is essential for high-level polymerization. (5) Wzc functions in autophosphorylation and subsequent transphosphorylation between proteins in an oligomeric form. (6) Dephosphorylation by the Wzb phosphatase is essential for exopolysaccharide synthesis. (7) Export of the polymer requires the outer-membrane complex which may play the role of an export channel. (8) The nascent polysaccharide is assembled on the cell surface and Wzi is required if the exopolysaccharide is attached as a capsule. Adapted from Whitfield, C.; Poiment, A. Carbohydr. Res. 2003, 338, 2491–2502.

species as well as producing some polysaccharides of potential industrial interest have received attention because of their symbiotic relationship with leguminous plants. Alginate production by Ps. aeruginosa has been studied because of the correlation between polysaccharide secretion and the infection of cystic fibrosis patients. The information from these diverse polysaccharides enables us to note some common aspects in the genetic control and regulation of exopolysaccharide synthesis. While most early work on the genetics of exopolysaccharide synthesis used Gram-negative systems, recent interest in LAB has yielded much relevant information and shown many common features. Genes controlling the enzymes in the early stages of polysaccharide biosynthesis and those common to other aspects of bacterial metabolism are widely separated on the chromosome or are found in small groups with related activities. The availability of precursors synthesized by these genes may control the amount of exopolysaccharide formed and excreted by the bacteria. The discovery of a cassette or cluster of biosynthesis genes unique for each polysaccharide, first conceived in E. coli, is now known to be at least partly applicable to many exopolysaccharide-synthesizing bacteria including Gram-positive species from the LAB group. Other components which are responsible for assembly and excretion are conserved within closely related bacterial species and permit genetic manipulation of the polymer formed. The cassette of genes appears to be very similar in size in many bacteria, even though in some of these the exopolysaccharide is much simpler than in others.

2.16.6.2 The Genetics of Repeat Unit Synthesis and Excretion The cassette or cluster of genes required for exopolysaccharide repeat unit and polymer formation regulates a number of functions. These are the synthesis of the repeat units, the assembly of these to form the polymer, and finally the transfer of that polysaccharide from the cytoplasmic membrane to the extracellular surface. In E. coli strains, similar organization of the gene clusters controlling K antigen (exopolysaccharide) synthesis was observed in several species.


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A region of approximately 17kb of DNA formed three functional segments. A region extending to about 9kb appeared to code for genes functioning in the translocation of the completed polysaccharide to the bacterial surface. Transposon insertions in this region yielded cells in which the polysaccharide appeared in the periplasmic space rather than extracellularly. A second region of 5kb provided genes responsible for the enzymes involved in biosynthesis of the specific sugar nucleotide precursors of the polymer, as well as specific transferases and a polymerase. Mutants in this region failed to produce any exopolysaccharide. The functions of the third region were less clearly defined. They appeared to be involved in modification of the exopolysaccharide after it had reached the cell surface. This might include attachment of the polymer to the cell surface through a terminal linkage including the sugar ketodeoxyoctonic acid. Transposon mutagenesis in this region caused intracellular accumulation of the polysaccharide. Analysis of the genes from various E. coli strains revealed sequence homology; analysis of the proteins encoded by the homologous DNA sequence revealed sets of similar polypeptides.103 The linkage of gene clusters for different exopolysaccharides to other markers on the bacterial chromosome in a bacterium such as E. coli was also found to be independent of the polysaccharide composition and structure.104 On the other hand, the central cassette of genes responsible for biosynthesis was unique to each bacterium and varied in sequence and size depending on the size of the repeat unit and the quantity of genetic information required. In other polysaccharide-synthesizing bacteria, the genetic organization may be different, although some homology has been demonstrated between E. coli and a related member of the family Enterobacteriaceae, K. aerogenes. Common features were also involved in the translocation of some of the E. coli polysaccharides and colanic acid to the bacterial surface. Genetic analysis has also made clear that while some E. coli strains can synthesize both colanic acid and another exopolysaccharide in the form of a capsule, in other strains of this bacterium the two types of polymer are mutually exclusive.99 There is also much in common between some of the systems found for exopolysaccharide synthesis and excretion in E. coli strains, xanthan biosynthesis in X. campestris, and the production of polymers by Gram-positive bacteria such as Streptococcus species.105 Whitfield105 points out that in E. coli exopolysaccharide assembly apparently utilizes a multiprotein complex which spans the cell envelope and that this may facilitate, spatially and temporally, the coupling of the three aspects of polysaccharide biosyntheis, export, and translocation. This mechanism operates for polysaccharides differing structurally. As Agrobacterium tumefaciens and Si. meliloti are both capable of synthesizing succinoglycan, some common features might be expected in the genetic control of this group of exopolysaccharides. In Si. meliloti, three loci involved in polysaccharide production (exoA, exoB, and exoF) are carried on a plasmid, whereas in Ag. tumefaciens all the loci identified as having roles in exopolysaccharide synthesis appear to be chromosomal. Curing (i.e., removal) of the Ti plasmid carried in Ag. tumefaciens leaves the cells exoþ. ExoC mutants from each bacterial species were similar, each being pleiotropic, slow growing, and partially defective in exopolysaccharide synthesis; the locus may code for a cell surface structure affecting many surface-associated phenotypes. Despite the difference in the genotypic distribution of the loci concerned with exopolysaccharide synthesis in the two genera, analogous complementation groups are found in any non-exopolysaccharide-forming mutants.106

2.16.6.3 Genetics of Xanthan Synthesis by X. campestris The xanthan system has revealed much of interest as well as providing mechanisms for the production of increased rates and yields of polysaccharide. It has also proved possible to obtain xanthan altered in its acetylation, pyruvylation, and side-chain oligosaccharides. In X. campestris, mutations have been found in a 13kb DNA sequence which appears to control polysaccharide biosynthesis. Among the genes linked to this cluster are one involved in pyruvylation and two in O-acetylation of xanthan. Plasmids containing this gene could be used to enhance the pyruvylation of xanthan by 45%, while other genes could increase polysaccharide synthesis by about 10%. Vanderslice et al.100 have demonstrated a cluster of 12 genes involved in xanthan production in a 16kb region of DNA. The function of most of the gene products has been identified and the Bam HI restriction map indicates the order and approximate size of the fragments produced by the restriction enzyme. All seven transferase activities needed for the production of a fully acylated repeat unit were identified, as was a polymerase and three enzymes possibly involved in later stages of the synthetic process. Contrary to what might have been expected, the strains defective in adding either the side-chain terminal b-D-mannosyl residue or the terminal disaccharide (b-D-mannosyl-b-D-glucuronic acid) yielded polymeric products designated ‘polytetramer’ and ‘polytrimer’, respectively. This indicated that the later stages of polymerization were of relatively low specificity. Mutants producing nonacylated and nonketalated xanthan were also obtained. Through the use of recombinant DNA technology, strains with multiple defects were also constructed. These formed nonacetylated trimer or nonacetylated tetramer. The yield of polytetramer was 50% of that from wild-type bacteria, while that of polytrimer was only 1–3%. As well as the cluster of genes related to the synthesis of the repeat unit of

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xanthan, unlinked genes were found, concerned with the synthesis of the sugar nucleotides needed for xanthan production. One gene of the main cluster (gumJ) provided a lethal mutant; it was inferred that this mutant was unable to export the polysaccharide and that its accumulation inside the cell proved to be lethal.

2.16.6.4 Genetics of Polysaccharide Synthesis in LAB Studies on genetic control of exopolysaccharide production in LAB have shown that the genes encoding synthesis of the polymers may be located either on plasmids as in Lactobacillus lactis and Lactobacillus casei, or on the chromosome as in thermophilic LAB species.3,107 As with the earlier studies on genes of Gram-negative bacteria, the loci controlling exopolysaccharide synthesis proved to be sequences of genes with considerable homology to genes with similar function in Es. coli. All the information essential for exopolysaccharide synthesis in Lactobacillus lactis strain NIZO B40 was coded in a 12kb gene cluster on a 40kb plasmid.108 Close similarity has also been found between the gene clusters of other LAB with the functional sequences of the clusters controlling regulation and chain-length determination in addition to biosynthesis of the repeating units and their polymerization and export.109 Thus, as pointed out by Laws et al.,3 both plasmid-controlled and chromosomal gene sequences in LAB contained similar contiguous groups. In Lactobacillus lactis, these were responsible for regulation, polymerization/export and chain-length determination, glycosyltransferases, and polymerization/export. The corresponding sequence in Streptococcus thermophilus was regulation, chain-length determination, biosynthesis of the repeating unit (glycosyltransferases), and polymerization/export.107 De Vuyst et al.92 have pointed out that the four functional regions of the polysaccharide synthetic cluster in LAB are highly conserved in their general organization, direction of transcription, and gene function. The central region contains the genes for glycosyltransferases and is flanked by two regions, each involved in chain-length determination and export. Finally, a regulatory region is present at one end of the gene cluster.

2.16.6.5 Genetic Engineering of Bacterial Exopolysaccharides From all these studies, it is clear that the genes specifically involved in exopolysaccharide synthesis are normally found as discrete regions on the bacterial chromosome or on plasmids. These regions each control certain stages of polymer synthesis and are separate from genes involved in mechanisms common to other carbohydrate-containing bacterial macromolecules. A surprising observation has been the lack of specificity at the level of polymerization as opposed to the highly specific addition of each individual monosaccharide to form the repeat unit structure. This latter part of the biosynthetic process requires both enzyme and substrate specificity. Another feature has been the discovery of cryptic genes for exopolysaccharide synthesis. This was exemplified by the discovery of the potential for cellulose synthesis among species of Enterobacteriaceae.110 In other species which have been studied, loss of one polysaccharide leads to the formation of another using some or all of the same sugar nucleotide precursors. Sinorhizobium meliloti strains no longer able to form succinoglycan may produce a galactoglucan.111 The results from xanthan clearly show that it is possible to modify polysaccharide structure and hence the physical properties of at least some polysaccharides through mutation. The earlier work on enteric and other species indicated the possibility of increasing mass and viscosity. The lack of specificity of the polymerase enzymes also presents the possibility of introducing new monosaccharides into the repeat unit or changing the oligosaccharide. It should, however, be pointed out that in the studies on xanthan and acetan, although the respective polysaccharide polymerases synthesized incomplete repeat units, no new sugars were introduced.112,113 The products from genetically engineered strains may also be low in yield and lack the properties anticipated. Attempts have also been made to alter bioemulsifying polysaccharides.

2.16.7 Natural Functions of Bacterial Exopolysaccharides Why do bacteria produce exopolysaccharides, sometimes in very large amounts? In the case of human and animal pathogens, they form part of the repertory of pathogenic mechanisms by which they achieve infection of the host. This may involve masking the bacterial cell surface from the defense mechanisms of the host. They certainly protect some bacteria which are pathogenic for humans and animals, against phagocytosis. In one of the earliest studies on the pathogenicity of Streptococcus spp. in experimental animals, the noncapsulate strains were nonpathogenic and only regained pathogenicity when they received genetic material responsible for capsule synthesis. The exopolysaccharides are often poorly antigenic, or, as is the case with hyaluronic acid in some Streptococcus spp. and other bacteria, the polymer mimics a natural component of the host cell and thus appears to be ‘self’ as opposed to a foreign antigen. This can also be seen in several dangerous bacterial pathogens such as E. coli K1 and Neisseria meningitides which are responsible for many cases of meningitis. These bacteria produce capsular exopolysaccharides composed of sialic acid, varying in the


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linkages and in the presence or absence of O-acetylation and of other sugars. More than 20 pathogenic bacterial species possess the ability to add sialyl groups to their surface polysaccharides,114 and a sialic acid O-acetyltransferase has been demonstrated to be a conserved gene within the gene clusters responsible for sialic acid biosynthesis in 18 bacterial species.115 Very many plant pathogenic bacteria are capable of forming large amounts of exopolysaccharides and in situ preventing the normal flow of water within the plant structure, causing wilting and eventually in combination with other factors such as secretion of enzymes and toxins, resulting in death of the plant host. Leguminous plants are widely distributed throughout the world in a range of climatic habitats. Many species are of considerable, economic significance and also play a significant ecological role because of their nitrogen-fixing symbiosis with Sinorhizobium or Bradyrhizobium spp. Leguminous trees, shrubs, and fodder plants are widely found in temperate, tropical, and subtropical regions where many are used for human or animal nutrition. The symbiotic nitrogen-fixing bacteria of the Rhizobial group generally produce large amounts of exopolysaccharides although the polysaccharides found in the bacterial species can vary very widely in their composition and structure. The role of exopolysaccharide in the interactions involved in plant–bacterium symbiosis has been widely discussed and shown to be crucial for the invasion of plant root nodules.116 Some exopolysaccharides may even be able to replace others of different structure in this function.111 The polysaccharides from species of the genus Rhizobium and closely related strains, although similar in their function of establishing plant–bacterium symbiosis, possess different chemical structures and physical properties: they comprise a gel-forming, neutral capsular polymer and a range of other polyanionic structures. As well as the specific interactions of the type described above, a levan from Bacillus polymyxa has been shown to be essential for soil aggregation in the plant rhizosphere.117 In soil and other environments in which there are either diurnal or other changes in water availability, the polysaccharides may both assist the bacterial cell in retaining water and form a hygroscopic layer surrounding the cells. Biofilms probably comprise the normal environment for the majority of bacterial cells in many natural and artificial habitats. These biofilms are complex associations of cells, extracellular products, and detritus either trapped within the biofilm or released from cells which have lysed as the biofilm ages. The main ‘cement’ for all these products, maintaining a constantly changing structure, is the mixture of polysaccharides which are secreted by the cells established within the biofilm. In natural conditions, monospecies biofilms are relatively rare; thus, most biofilms are composed of mixtures of microorganisms. This adds to the interspecies and intraspecies interactions and the general complexity of the macromolecular mixture present. In the biofilms, the bacteria are found as aggregates of microbial cells of differing species and their extracellular products, attached to surfaces.118,119 In some examples, adhesion can be very firm and the amount of material accumulated may be large. In other locations, attachment may be more ephemeral and less significant. The exopolysaccharides assist in the protection of bacterial cells within the biofilm from desiccation and from injurious biological and chemical agents. The chemical composition and the tertiary structures of the exopolysaccharides will determine whether they form effective adhesives and they will also affect the hydrophilic or hydrophobic nature of the surface. They may also bind essential nutrients including cations and create a local, nutritionally rich environment in which growth of specific types of bacteria is favored. The correlation between exopolysaccharide synthesis and biofilm formation is not absolute. Porphyromonas gingivalis is an anaerobic bacterium associated with severe dental disease. Loss of capsule synthesis from this bacterium caused enhanced biofilm formation.120 As exopolysaccharides are mainly highly hydrated and in some examples may absorb many times their own weight of water, they may function in protecting free-living bacteria against desiccation in natural environments in which there is either great variability in the presence or availability of water or water is essentially absent for long periods. They may also protect against attack by viruses and protozoa although, as is indicated below, bacterial viruses (bacteriophage) provide a wide range of exopolysaccharide-degrading enzymes. When biofilms or flocs are established, the polysaccharide components of microbial origin may exhibit phenotypic differences from free-living bacteria of the same species, and it is more probable that the bacteria secrete exopolysaccharides identical in composition (and probably also in physical properties) with those formed by the same bacteria when grown in pure culture. Another possibility is that the polymers formed may be of identical composition to those formed by the freeliving bacteria but, due to minor structural differences such as the degree of acylation or the molecular mass, differ in their physical properties. These differences may result in altered viscosity or gel-forming capacity. The polysaccharides may act as receptors for cell-induced adhesins through coating the cells which synthesize them as well as adjacent microbial cells and inert surfaces, as is probably the case for the oral bacterial flora. The impetus of intercellular signals generated within a complex biofilm or floc habitat and adaptation to some of the unique physiological conditions prevailing might stimulate the bacteria to synthesize polymers of novel composition and physical properties totally dissimilar from those found in the free-living state, or more probably, derepress synthesis of a polymer found only in highly specialized environmental conditions. In wastewater treatment and purification, the flocs and other polysaccharides formed by bacteria play a significant role.121 This may involve multivalent cations, but the hydrophobicity of some of the polymers also plays a major role acting as a focus for the formation and propagation of flocculent particles.

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Surprisingly, a pyruvylated polysaccharide with a tetrasaccharide repeat unit, lacking any unusual monosaccharides, was found to play an important role in the resistance of Rhodococcus sp. 33 to benzene, a substance which is normally toxic to bacterial and other cells.122 The structure of the exopolysaccharide from benzene-tolerant Rhodococcus sp. 33 is shown below: Pyr 4⇓6 [→4)-b-D-Galp-(1→4)-b-D-Glcp-(1→3)-b-D-Manp-(1→4)-b-D-GlcpA-(1→]n

Some bacteria exhibit gliding motility on solid surfaces, and the extracellular polymers appear to form an essential lubricant for movement across such surfaces. In some types of Gram-negative bacteria exemplified by Azotobacter spp. and the Myxobacteriaceae, differentiation cycles involve the formation of desiccation-resistant resting cells termed microcysts. In Azotobacter spp., the microcysts are surrounded by large amounts of bacterial alginate, while the same polysaccharide forms an essential part of the outer coat of the resting cells. In the second group, the process is more complicated. They exhibit gliding motility and require the secretion of polysaccharide as an adhesive. Additionally, fruiting bodies of varying complexity are formed enclosing the microcysts (or myxospores). In this morphogenetic process, polysaccharide synthesis is essential. The fruiting bodies contain their myxospores surrounded by a matrix of exopolysaccharides which presumably acts as a protective coating for the cells within. A few bacteria, including Hyphomonas and other prosthecate species, possess small discrete patches of adhesive polysaccharides which aid attachment of the stalked cells.123 It is even suggested that the polysaccharide adhesive formed by one of this group of bacteria, Caulobacter crescentus, is one of the strongest natural adhesives. In one bacterial species only, Ce. flavigena, exopolysaccharide has been clearly shown to function as a carbon and energy reserve.124 Bacterial species do not normally degrade their own exopolysaccharides and the polymers do not have a structural role (even in cellulose-forming species). Bacteria that have lost the ability to form their exopolysaccharides are not at a disadvantage under laboratory conditions although the situation may be different in natural environments.

2.16.8 Biological Properties of Bacterial Exopolysaccharides Now that the structures of a large number of bacterial exopolysaccharides have been determined, many clearly show sufficient sharing of at least part of the structure to provide immunological cross-reactions. This can be significant in bacterial species that are pathogenic for humans and animals. There may even be cross-reactions with components of the lipopolysaccharide structures found in the cell walls of Gram-negative bacteria. As mentioned above, pathogenic bacteria often owe some or all of their pathogenicity to the presence of exopolysaccharides at their surface. Bacterial exopolysaccharides have not been shown to suppress tumor formation or stimulate immune responses in a manner similar to several fungal b-D-glucans, but a polymer from Ba. polymyxa S4 containing both D-glucuronic and 125 D-mannuronic acids appeared to reduce cholesterol levels in experimental animals. Other bacterial exopolysaccharides which do not in themselves show any biological activities may do so when subjected to chemical modification. Sulfation and depolymerization of a heteropolysaccharide from Alteromonas infermus yielded fragments with anticoagulant activity.126 Similarly, the polymer from E. coli serotype K5, which resembles the structure of desulfatoheparin, has been used to prepare analogs of heparin.127 This polymer is one of the several relatively simple structures showing some similarity to hyaluronic acid or chondroitin, including one recently characterized from Moraxella bovis, the cause of bovine keratoconjunctivitis.128 The Mo. bovis polymer is composed of a disaccharide repeat unit analogous to unmodified chondroitin. The structures of the exopolysaccharides from E. coli strains K4 and K5, Mo. bovis, and of bacterial hyaluronic acid are shown below. E. coli K4: [→3)-b-D-GalpNAc-(1→4)-b-D-GlcpA-(1→]n 3 ↑ 2 b-D-Fru Mo. bovis: [→3)-b-D-GalpNAc-(1→4)-b-D-GlcpA-(1→]n E. coli K5: [→4)-a-D-GlcpNAc-(1→4)-b-D-GlcpA-(1→]n Hyaluronic acid from Streptococcus equi and other bacteria: [→4)-b-D-GlcpNAc-(1→4)-b-D-GlcpA-(1→]n


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2.16.9 Enzymic Degradation of Bacterial Exopolysaccharides Two major types of enzyme degrade bacterial exopolysaccharides: polysaccharide hydrolases and polysaccharide lyases.129,130 Both are found as intracellular products in polysaccharide-synthesizing bacteria and as extracellular enzymes from heterologous microorganisms. Bacteriophage active against exopolysaccharide-producing bacteria supply a wide range of both types of enzyme. As with other polysaccharide-degrading enzymes, the action of all these enzymes may be either endolytic or exolytic leading to a rapid or relatively gradual release of reducing material, respectively. Although exopolysaccharides may be degraded by a wide range of enzymes from bacteria, fungi, protozoa, and other sources, commercial enzymes with such activity are rarely available and a specific search for novel enzymes usually has to be made. It has also proved relatively rare to find a pure bacterial or other microbial culture which is capable of degrading an exopolysaccharide following normal enrichment procedures. One successful example of this approach was the isolation of the succinoglycan-degrading bacterial species Cytophaga arvensicola.131 More commonly, enrichment culture has led to the production of a complex mixture of polysaccharide-degrading microorganisms. Generally speaking, such mixed cultures produce a range of both polysaccharases and glycosidases with the result that any products are highly degraded. Another problem encountered when using the exopolysaccharide as the carbon source for enrichment and isolation of degradative bacteria is the low activity of some of the enzymes from these cultures. It may then be difficult to separate and purify any enzyme or enzymes from undegraded polysaccharide. One of the most prolific sources of exopolysaccharide-degrading enzymes has proved to be virulent bacteriophage (bacterial viruses) capable of lysing their host bacteria. The phages with such activity vary greatly in their structures. Some have small icosahedral heads and small, barely visible tails. Others possess long slightly curved tails resembling the coliphages T1 and T5. Enzyme activity from the bacterial lysates is usually found both associated with mature phage particles and in soluble form in phage-free filtrates from the lysates. In many of the examples which have been studied, the phage-associated enzyme has been found to be associated with small spikes attached to the base-plate of the viral particles. Although many exopolysaccharide-producing bacteria have proved to be hosts for polysaccharase-inducing bacteriophage, others, including X. campestris, have proved recalcitrant and viruses of this type have been difficult to isolate. The advantage of phage-induced enzymes is that even when not completely purified, they are usually free from other glycosidases or polysaccharide-degrading enzymes. While very few commercial enzyme preparations show any activity against bacterial exopolysaccharides, they may possess enzymes that can degrade the oligosaccharides produced from exopolysaccharides by other degradative enzymes. Polysaccharide hydrolases or other polysaccharide-degrading enzymes must be sought from more specific sources. The polysaccharides are only rarely degraded by enzymes formed by the same bacteria which synthesize the polymer. The reason for this is that such degradative enzymes, although found as gene products from an increasing number of bacteria, play a role in the destruction of unwanted intracellular polymer. The amounts of such enzymes are very small and are not released into the extracellular environment where they could act on the exopolysaccharide substrates. Microbial exopolysaccharides may be degraded either by polysaccharide hydrolases or by polysaccharide lyases. The latter cleave the linkage between a neutral monosaccharide and the C4 of a uronic acid with simultaneous introduction of a double bond at the C4 and C5 of the uronic acid. Both types of enzymes are commonly found to degrade exopolysaccharides as well as eukaryotic polymers.130 It is very rare for a microbial species to use its exopolysaccharide as a source of carbon and energy; secretion of large amounts of enzymes or enzymes degrading their own polymers is seldom found. An exception is the curdlan-synthesizing bacterium Ce. flavigena, which does produce an extracellular enzyme capable of degrading the exopolysaccharide to utilizable products.124

2.16.9.1 Polysaccharide Hydrolases Polysaccharide hydrolase enzymes may be endo- or exo-acting, leading to rapid or slow breakdown of the polymer chain, respectively, and corresponding rapid or slow loss of solution viscosity and release of reducing material. Homopolysaccharides are degraded by a wide range of enzymes. Mishra and Robbins132 have pointed out that b-D-glucanases with a variety of specificities can be used to elucidate the structures of this group of betaglucans. The linear (1!3)-b-D-glucan curdlan may be degraded by four different types of enzyme. Two distinct exoglucanases release D-glucose and laminaribiose, respectively, while two endoglucanases differ in their modes of action. One attacks randomly, releasing glucose, laminaribiose, laminaritriose, and other oligosaccharides in the b1!3-linked series. The other yields the pentasaccharide laminaripentaose. Most rapid hydrolysis of homopolysaccharides is observed when there is concerted action of endo- and exo-b-D-glucanases. In contrast to these systems, the hydrolysis of many microbial heteropolysaccharides is commonly achieved by a single, highly specific endoacting polysaccharase, which yields a series of oligosaccharides representing either the repeating unit structure or multiples of it. The a-D-glucans such as dextran are also substrates for a wide range of different dextran-hydrolyzing enzymes which have been isolated

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from eukaryotic and prokaryotic microorganisms.133 These dextranases can be either endo- or exoglucanases. Some yield relatively large oligosaccharides together with glucose, while others yield only oligosaccharides. They also vary in the linkages cleaved. Enzymes from Flavobacterium species as well as Streptococcus mutans hydrolyze 1!2-a-D-glucose linkages.134,135 Some of the exodextranases release glucose as the sole product, while others release isomaltose from the nonreducing ends of the dextran chains. The polysaccharide hydrolases from bacteriophage normally yield the repeat unit of the polysaccharide substrate or oligomers of it and other enzymes degrading these products further are usually absent. Highly active preparations of such enzymes can thus easily be obtained from phage lysates of the host bacteria and most appear to act randomly on the polymer chains. They have therefore proved extremely useful in the characterization of the repeat units of bacterial exopolysaccharides by procedures such as fast atom bombardment spectroscopy, as acyl groups and other labile substituents are not removed. In terms of the linkages cleaved, phage-induced enzymes provide a very wide spectrum (Table 6). Although several ‘endoglucosidases’, ‘endogalactosidases’, or ‘endorhamnosidases’ are listed, each is distinct in its specificity. While some of the polysaccharide hydrolases act either on native, acylated substrate or on chemically deacylated material, a few appear to be more specific and fail to act on polysaccharides from which acyl groups have been removed or vice versa. Side chains and charged groups appear to play an important role in determining the specificity of the phage enzymes and it is perhaps of interest that many of the phage-induced enzymes appear to attack either a1!3- or b1!3-bonds preferentially. The residue targeted is very often adjacent to an anionic residue such as glucuronic acid, which may be part of the main chain or attached as a side chain. Among the polysaccharide-degrading enzymes from heterologous bacteria, one example is the b1!4-fucoside hydrolasedegrading colanic acid which was reported by Verhoef et al.136 This endo-acting enzyme was obtained from a mixed

Table 6 Examples of phage-associated polysaccharide depolymerases Bacterial host/polysaccharide

Enzyme action (Endoglycanases)

Klebsiella type K3 Klebsiella type K8 Klebsiella type K13 Klebsiella type K18 Klebsiella type K25 Klebsiella type K26 Klebsiella type K34 Klebsiella type K36 Klebsiella type K51 Klebsiella type K74 Erwinia amylovora Escherichia coli K103 Klebsiella serotype K11 Klebsiella serotype K25 Klebsiella serotype K39 Klebsiella serotype K60 Klebsiella serotype K63 E. coli serotype 29 Klebsiella pneumoniae SK1 Klebsiella serotype K2 E. coli type 8 Klebsiella serotype K30 E. coli type 44 Acetobacter methanolicus Klebsiella serotype K17 Klebsiella serotype K19 Pseudomonas syringae pv. morsprunorum E. coli E. coli E. coli K1 phage E E. coli

Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endogalactosidase Endoglucosidase Endoglucosidase Endoglucosidase Endoglucosidase Endoglucosidase Endoglucosidase Endoglucanase (endoglucosidase) Endoglucosidase Endomannosidase Endomannosidase Endo-N-acetyl-b-D-galactosaminidase Endorhamnosidase Endorhamnosidase Endorhamnosidase Endorhamnosidase Neuraminidase Endo-N-acetylneuraminidase Endosialidase Kdo-Kdo glycanase

Linkage cleaved

1!4-b-D-Gal

1!2-b-D-GlcA 1!3-b-L-Rha 1!3-a-D-Gal 1!2-b-D-Man 1!3-b-D-Gal 1!4-a-D-Gal 1!3-b-D-GlcA 1!3-b-D-Glc

1!3-b-D-GlcA 1!3-b-D-Glc 1!4-b-D-Glc

1!4-b-D-Glc 1!2-a-D-Glc

Almost all enzymes listed are highly specific and only degrade one exopolysaccharide structure or a small number of closel structures. Adapted from reference ( ).


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Sgase ⇓ b -D-Glcp-(1→4)-b-D-Glcp-(1→3)-b-D-Galp-(1→4)-a-D-Glcp -(1→4)↑ b -D-Glcp-(1→3)-b-D-Glcp-(1→3)-b-D-Glcp-(1→6)-b-D-Glcp 6 ⇑ ↑ Pyr CH2CO.O. | CH2.COOH Sgase = succinoglycanase → octasaccharide repeat unit

⇐ Glucanase

Glucanase = 1,6-endo-b-D-glucanase → one neutral and one charged tetrasaccharide Figure 2 Degradation of succinoglycan exopolysaccharides by the enzyme activities from Cytophuga arvensicola.125

culture grown by enrichment on colanic acid as carbon source and extensively purified. It liberated repeating units of the E. coli polysaccharide with varying degrees of acetylation. Removal of the O-acetyl groups caused greatly increased catalytic activity from the enzyme although the affinity for colanic acid modified through removal of pyruvate was lower than for the native polysaccharide. The enzymes from heterologous microorganisms tend to provide a complex mix of activities, degrading the polymer substrate either to its component monosaccharides or at least to a spectrum of different-sized and partially degraded products rather than the repeat units and oligomers of them. Thus, they tend to be of less value in structural studies unless they are extensively purified, as was the case for this fucosidase. It also provides an example of an exopolysaccharide for which more than one type of enzyme with differing degradative patterns has been isolated. In the case of the succinoglycan polysaccharide secreted by Agrobacterium and Rhizobium spp., two distinct enzymes from the same bacterial isolate have contributed to elucidation of the structures. An enzyme preparation from Cytophaga arvensicola yielded both succinoglycan depolymerase (endo-b-D-glucanase) and 1,6-endo-b-D-glucanase activities.131 It was thus capable of degrading the exopolysaccharide to its repeating unit (and oligomers of it) and then degrading the octasaccharides further to two distinct tetrasaccharides, one derived from the main chain of the polymer and the other from the side chains, without loss of the acyl substituents (Figure 2). These enzymes also appeared to be largely unaffected by the presence or absence of the various acyl adornments found on this group of exopolysaccharides.

2.16.9.2 Polysaccharide Lyases Polysaccharide lyases all cleave anionic polysaccharide possessing a . . .hexose-1!4-a- or b-uronic acid. . . sequence by base-catalyzed b-elimination yielding products in which the nonreducing terminus is modified to form an unsaturated uronic acid. This action can be easily followed through the increase in UV absorption at 230nm on product release, or through the use of the thiobarbituric acid assay following periodate oxidation of the double bond. Very few members of this group of enzymes have been characterized at the molecular level; a small number of amino acid sequences have been published, primarily for enzymes acting on eukaryotic polymers. The lyases are in some examples the only known type of enzymes degrading their bacterial exopolysaccharide substrates. This is the case for bacterial alginates. Although a few of the bacterial exopolysaccharides are degraded by both hydrolases and lyases, this has only been noted relatively rarely. Most of the lyases are randomly endolytic in their action and, with the exception of xanthan lyases, cleave the main chain of polysaccharide structures. In xanthan, the side chain is cleaved at the subterminal uronic acid residue by a xanthan lyase, while the main chain is cleaved by enzymes with endoglucanase activity. Despite the cellulosic nature of its main chain, xanthan is not normally degraded by cellulases. Three groups of bacteria which synthesize polyanionic exopolysaccharides also yield lyases as constitutive enzymes. These are alginate-, gellan-, and hyaluronan-synthesizing bacterial species. Alginases (alginate lyases) are widely distributed in nature, and most of the bacterial species which form alginates, including Azotobacter and Pseudomonas spp., also produce lyases. Apparent exceptions are some plant pathogenic Pseudomonas spp.

2.16.9.2.1

Alginate lyases

Alginases (alginate lyases) are of widespread occurrence, having been isolated from marine gastropods, bacteriophage, various marine prokaryotic and eukaryotic microorganisms, and some soil Bacillus spp. When compared to other

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polysaccharide lyases, the mode of action of enzymes degrading alginate is complicated by the differences in distribution of monosaccharide residues in different substrates. These polysaccharides are not of uniform composition. Thus, alginates with the same or very similar mannuronate: guluronate ratios can have very great differences in monosaccharide sequence and in the frequency of adjacent residues. Bacterial alginates used as substrates provide further complexity due to the random distribution of O-acetyl groups on the 2- and 3-positions of mannuronate residues. The products of alginate lyase action have generally been characterized as oligosaccharides of degree of polymerization (DP) 2 or 3, in which the terminal nonreducing residue has been converted to O-(4-deoxy-a-L-erythrohex-4-enolpyranosyluronic acid. This unsaturated uronic acid is formed from cleavage of either guluronic or mannuronic acid sequences. Although few of these enzymes have been fully purified, they have been shown to be endo- or exo-enzymes with specificity toward b-D-mannuronosyl or a-L-guluronosyl linkages. ‘Guluronic-specific’ enzymes appear to be less common. But some have been described from K. aerogenes and from Pseudomonas alginovora. The acetyl groups present on bacterial alginates greatly influence the susceptibility of these polysaccharides to the action of alginases, and the majority of the alginate lyases, which have been described, show relatively slight activity against native acetylated alginates from either Az. vinelandii or Pseudomonas spp. An exception was the enzyme from Pseudomonas maltophilia which was active against acetylated and nonacetylated substrates.137 The alginate lyases from strains of Az. vinelandii and Az. chroococcum were located in the periplasm. Although several bacterial and algal alginate preparations were degraded by the enzymes, highest activity was found on poly-D-mannuronate or on algal alginates with high mannuronate content. Deacetylation of a preparation of Ps. aeruginosa alginate increased its susceptibility to enzyme action 15-fold. The mass of alginates from Pseudomonas fluorescens and Pseudomonas putida fell approximately 50% in shaken cultures through the action of endogenous poly-D-mannuronate-specific lyases after cell growth had ceased. This might be due to release of the enzymes following bacterial lysis. In the case of some of the alginate-degrading enzymes, it is also unclear whether some of the activities reported are only found after oligosaccharides have been released from the initial polysaccharide. An example of this is an oligoalginate lyase enzyme from Haliotis, releasing disaccharides from alginate.138 The same marine organism also yielded an alginase showing preferential activity against mannuronic-rich alginate substrates.132 Whether these two enzymes are also active against bacterial exopolysaccharide alginates and their degradation products is not clear.

2.16.9.2.2 Emulsan lyase Emulsan, the exopolysaccharide with emulsifying activity obtained from a strain of Acinetobacter lwoffii, is the substrate for a lyase from another bacterium. The enzyme was inducible and its endolytic action rapidly reduced both the viscosity and the emulsifying activity of emulsan. Cleavage of 0.5% of the glycosidic linkages reduced emulsifying activity by 75% and yielded oligosaccharides of 2000–6000Da, eluting as four major peaks by size-exclusion chromatography. The enzyme was less active against the de-esterified polymer than against the native material.140 It appeared that the specificity of the enzyme was related both to the degree of esterification of the polymer and to the distribution of the ester groups. Although, as indicated in Table 1, exopolysaccharides may have various organic or nonorganic substituents, enzymic removal of these has been seldom demonstrated. Shabtai and Gutnick141 did demonstrate esterase activity against emulsan, the surface-active lipopolysaccharide-like exopolysaccharide synthesized by Acinetobacter lwoffii strain RAG-1. The emulsan esterase appeared to show specificity for this unusual polymer but also cleaved nitrophenyl esters. Further studies may yet reveal more esterases acting on exopolysaccharide substrates.

2.16.9.2.3 Gellan lyase A number of bacterial strains capable of degrading the bacterial exopolysaccharide gellan have been isolated by standard enrichment procedures. They include several pink-pigmented Gram-negative rod-shaped bacteria. A redpigmented Gram-positive bacillus earlier found to degrade the exopolysaccharide xanthan from X. campestris also showed slight gellanase activity. All the Gram-negative bacteria are nonfermentative, motile, and amylase producing. The gellan degradation in each case is due to eliminase-type enzymes (lyases) which appear to be extracellular enzymes cleaving the sequence . . .b-D-glucosyl-(1! 4)-b-D-glucuronosyl. . . in the tetrasaccharide repeat unit of the substrate polysaccharides. Although in some isolates these enzymes appeared to be exo-acting, it appeared from the loss in viscosity of the alternative substrate deacetylated rhamsan that they are predominantly endo-enzymes. The enzyme activity was inducible and was almost absent from glucose-grown cells. Associated with the ‘gellanase’ activity all the Gram-negative bacterial isolates possess intracellular a-L-rhamnosidase and b-D-glucosidase activities apparently located in the periplasm.142 The enzymes were highly specific and failed to cause significant degradation of most of the other bacterial exopolysaccharides which have been shown to be structurally related to gellan (Table 5).


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The related polysaccharides from these bacterial strains, all now considered to be Sphingomonas paucimobilis isolates, possess similar linear structures in which there is at least an identical-(D-glucose-D-glucuronic acid-D-glucose)trisaccharide sequence with the same anomeric configurations as gellan. However, the differing configurations adopted and the presence of other acyl or glycosyl residues appear to inhibit enzyme activity. As well as their lyase activity on gellan, the enzymes only exert similar degradative activity against the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a form of gellan carrying gentiobiosyl substituents. The action of the lyases on deacetylated rhamsan, in which there is a gentiobiosyl side chain attached to the glucose residue distal to the uronic acid, indicated that the ordered structure resulting from this substituent was still available for enzyme attack provided all acyl groups had been removed. However, polymers in which the glucosyl residue attached to the reducing terminus of the uronic acid was substituted by a side chain were not attacked. The side chains clearly inhibited enzyme–substrate binding. The binding or cleavage site must therefore extend to the glucose residues on either side of the glucuronic acid. Crescenzi et al.143 reported that short side chains of L-mannose or L-rhamnose in these exopolysaccharides mask the uronic acid residues in the conformation adopted in aqueous solutions through hydrogen bonding and this would also appear to affect the enzyme action. While the enzymes only have relatively slight activity against the natural, acylated gellan-like polysaccharides from the bacteria now designated as strains of Sp. paucimobilis, their specificity is unusual in that only the polysaccharides which have been modified by chemical deacetylation are substrates. Even the native gellan is not degraded. Hashimoto et al.144,145 also isolated enzymes degrading gellan from a Bacillus species. In addition to the polymer-degrading lyase, an enzyme degrading the oligosaccharides containing unsaturated glucuronosyl residues was present in the crude mixture. It has been suggested that the use of such specific enzymes in combination with other methods might be suitable for determination of the concentration of gellan in food or other complex mixtures.146

2.16.9.2.4

Xanthan lyase

Despite its cellulosic main-chain structure, commercial cellulase preparations generally lack activity against xanthan. Most enzyme preparations capable of degrading xanthan are impure preparations containing several different enzyme activities. Many have been prepared from mixed bacterial cultures. Usually the enzyme activities include both 1,4-b-D-glucanases (cellulases) which cleave the cellulosic main chain and xanthan lyases which are unusual in that unlike the other lyases mentioned above, they cleave a side-chain sequence of sugars and the linkage targeted is not on the exopolysaccharide backbone. Also, because the xanthan lyases which have been studied are always associated with 1,4-endo-b-D-glucanases, the same problem arises as with other mixed enzyme preparations acting on exopolysaccharides in that glycosidases may also be present and cause degradation of the initial oligosaccharides liberated. It is not certain to what extent the polymer or products from endoglucanase action are the primary targets for xanthanase action. Most xanthan lyases act on xanthan whether or not either acetyl or pyruvate ketal groups are present, although acetan, a polysaccharide with a pentasaccharide side chain and structural similarities to xanthan, was not degraded unless the acetyl groups present on the main-chain glucose residues were first removed. A xanthan lyase from a heatstable, salt-tolerant bacterium, purified to homogeneity,147 was a protein consisting of a single subunit which had an Mr of 33kDa and was specific for mannose residues carrying the pyruvate ketal. It apparently lacked activity against unsubstituted residues and thus differed in its substrate specificity from most of the other xanthan lyases which have been described. Another xanthan lyase from the Gram-positive bacterium Paenobacillus alginolyticus also showed specificity for pyruvylated mannose side chains.148 A lyase isolated from a Bacillus sp. had a similar molecular mass (30–33kDa), but was capable of cleaving either pyruvylated or nonacylated mannose residues on the xanthan side chains and was not affected by the presence or absence of O-acetyl residues on the internal a-D-mannosyl group. The optimal substrates appeared to be oligosaccharides of DP 25–35, representing five to seven repeat units of the polysaccharide, but fragments of DP>90 were still degraded with high efficiency. This indicated that the bacterial polysaccharide must first be cleaved by endo-b-glucanases to yield lower-molecular mass material, which then acts as substrate for this particular lyase. The enzyme did not degrade Klebsiella K5 exopolysaccharide in which the same linkage is present in the main chain of the structure.149

2.16.9.3 Glycosidases Very few glycosidases show activity against exopolysaccharides. However, a polymer from a Rhizobium species has been shown to be susceptible to an a-D-galactosidase.150 The glycosidic linkage cleaved is found on the terminus of a side chain and loss of the galactosyl residue altered the physical properties of the polymer. Prolonged incubation is usually necessary and removal of the monosaccharide residues is seldom complete.

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2.16.10 Commercialization 2.16.10.1 Manufacture and Usage of Bacterial Exopolysaccharides Despite the large number which have now been chemically characterized, the physical properties of some of which have also been determined, only a small number of bacterial exopolysaccharides have found commercial applications (Table 7). The bacteria may be pathogenic, yields may be low, costs of production may be high, maintenance of product quality may be difficult, or there may simply be no appropriate market niche. Several of these which have been developed including dextran, gellan, and xanthan have been marketed as commercial products of considerable importance and are produced on a large scale. Xanthan in particular is manufactured on a scale of several thousand tonnes per annum. The amount of other polysaccharides marketed commercially is difficult to determine. Usage for some other polysaccharides which at one time appeared to be suitable candidates for commercialization has now lapsed. Bacterial exopolysaccharides have been developed either because of their unique properties or because they provide a cheaper and superior alternative to other materials derived from plant, animal, or synthetic sources. The range of industries using bacterial exopolysaccharides is diverse.89,151 It includes oil extraction, printing, food processing, cosmetics, and healthcare. The polysaccharides represent valuable renewable resources but their development and evaluation requires considerable investment in time, money, and technology. Many bacterial exopolysaccharides for which potential industrial applications have been claimed have not proved to be of commercial value. They have to compete against established plant and algal polysaccharides and against synthetic materials which may have longestablished acceptability and uses as well as possible economic advantages. Xanthan and gellan were originally developed as potential ingredients for processed foods but they have now found many other applications. The acceptability of all these microbial products depends on their properties, quality, and price. The products intended for food use must conform to the appropriate regulatory requirements in respect of the substrates, equipment, and processing procedures used. A small number of other microbial polysaccharide products have also been commercialized. These usually have very specific properties and applications and include bacterial cellulose and also bacterial hyaluronic acid, both of which depend for their commercial value on their very high quality. Some are employed either because of their unique physical properties or because their physical properties are superior to other agents which are currently used. Some of the exopolysaccharides must be modified chemically before they find applications. The uses and value of the polysaccharides vary widely; one represents a very high value product while others are essentially bulk chemicals. The usage of the polysaccharides may be based on either their biological or physical properties and falls into three distinct areas: food applications, nonfood applications, and medical or other

Table 7 Some applications of bacterial exopolysaccharides

Biological properties

Usage

Polymer

Eye and joint surgery Heparin analogs Wound dressings

Hyaluronic acid (Streptococcus EPS) E. coli K5 EPS Bacterial cellulose

Enzyme substrates Oligosaccharide preparation

E. coli K4 and K5 EPS Curdlan

Foods, thixotrophic paints Acoustic membranes Water clarification, ore extraction Beer, fire-fighting fluids Cell and enzyme technology Foods Oil recovery (blockage of permeable zones) Cosmetics, pharmaceuticals Frozen foods, pastilles, and sugar syrups Oil-drilling ‘muds’ Food Paper coatings Agrochemical pesticides and sprays Jet printing

Xanthan Bacterial cellulose Various Xanthan Gellan Curdlan, gellan Xanthan Hyaluronic acid Xanthan Xanthan Xanthan Various Xanthan Xanthan

Chemical properties

Physical properties Emulsion stabilization Fiber strength Flocculant Foam stabilization Gelling agents

Hydrating agent Inhibitor (crystal formation) Shear thinning/viscosity control Suspending agent

Viscosity control


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biological purposes. Among those found in the third group, some are used in very small quantities as components of antibacterial vaccines. Thus, several polysaccharides from strains of Streptococcus pneumoniae are incorporated into vaccines used to prevent diseases caused by these bacteria in the very young and the elderly. As the polymers are poorly antigenic, they are also coupled to proteins to form protective vaccines against some of the causal agents of meningitis and related infections. As this type of usage is very specialized and only small amounts of material are manufactured, it will not be discussed further.

2.16.10.2 Bacterial Cellulose Bacterial cellulose has proved to be a high-value exopolysaccharides product. This is perhaps surprising as plant cellulose is so widely available and inexpensive. Cellulose is produced as an exopolysaccharide by G. xylinum and other, mainly Gram-negative, bacterial species, and selected strains have been used for industrial production. The polymer is excreted into the medium where it rapidly aggregates as microfibrils and forms a surface pellicle in shallow unshaken cultures. The design of the fermenter and the degree of aeration are important factors in optimizing cellulose yield. The bacterial cellulose is essentially a high-value, speciality chemical with highly specific applications and usage. It can be produced commercially as a source of highly pure polymer. This is in the so-called cellulose I form (60% Ia:40% Ib) and is free from lignin and other noncellulosic material. The fibrils form a unique ribbon 3–8nm thick and c. 100nm wide, which differs in morphology from other native celluloses.152 A commercial product named BioFill has various potential uses and has been marketed in Brazil in the form of wound dressings for patients with burns, chronic skin ulcers, or other extensive loss of tissue. The bacterial cellulose acts as a temporary skin substitute; it possesses high mechanical strength in the wet state, and the high water capacity (c. 150gg1) of the oxygen-permeable film appears to stimulate regrowth of the skin tissue. It also appears to assist in preventing infection.153 Bacterial cellulose has also been used to form high-quality, acoustic diaphragm membranes. In these, the distribution of the fibrils containing parallel orientation of the glucan chains results in fibers possessing high tensile strength. In addition to these technical applications using highly purified material, bacterial cellulose forms part of the traditional fermented food ‘Nata de Coco’ to which it gives texture and structure.

2.16.10.3 Dextran Industrial dextran production is mainly from a strain of Le. mesenteroides, which yields a polysaccharide with about 95% 1!6 linkages and 5% 1!3 linkages and has a molecular mass of about 4–5107. For the production of dextrans of medium molecular weight, the initial high molecular weight product must be hydrolyzed with acid or with enzymes and fractionated under carefully controlled conditions. Dextran was originally approved for food use, but this application has lapsed. It is, however, the base material for the preparation of the range of Sephadex® gel filtration and ion-exchange materials which are widely used in the separation and purification of biological macromolecules. For these applications, the dextran must be chemically modified to introduce the active groups responsible for their ionexchange properties, while they must be also cross-linked chemically to provide gels which swell in aqueous media to yield the required macromolecular permeability. Another modified form of dextran, coupled to a blue dye, provides a convenient test material for determination of void volume of gel permeation columns. Dextrans are of low antigenicity and saline solutions are of low viscosity. Consequently, dextran solutions also have potential use as a plasma substitute but again require to be partially hydrolyzed to ensure the correct mass. Further applications may be found in photography and in cosmetic preparations.

2.16.10.4 Bacterial Hyaluronic Acid Bacterial hyaluronic acid154 has two very useful properties – its very high capacity for regaining water and its compatibility with the human immune systems. It can be used as a replacement for hyaluronic acid in human fluids during joint replacement or eye surgery or as an effective moisturizing agent in high-quality cosmetics. The bacterial hyaluronic acid, apparently identical in chemical structure to that obtained from eukaryotic material, can be isolated from cultures of group A and group C streptococci. The polysaccharide is composed of repeating units of b1!4-linked disaccharides of D-glucuronosyl-(1!3)-b-N-acetyl-D-glucosamine. One group C streptococcal strain forms both a high molecular mass, cell-bound product and a soluble exopolysaccharide of average molecular mass 2106. A potential problem with the bacterial hyaluronic acid can be the variability in molecular mass due to enzymic degradation by

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endogenous hyaluronidases during bacterial culture and product isolation. Despite this, strains of Streptococcus equi and Streptococcus zooepidemicus have been used in the commercial production of hyaluronic acid yielding a high-quality (and high-cost) polysaccharide for use in surgery as well as in cosmetic preparations. This provides an alternative material from renewable resources to replace the use of material from human or animal sources. Mutant strains lacking hyaluronidase have also been developed to ensure that no degradation of the high molecular weight polymer occurs. The bacterial exopolysaccharide offers advantages over the traditional sources in terms of cost, purity, and molecular mass. Aqueous solutions of hyaluronic acid molecules display very limited configurational flexibility and thus show very marked stiffness. The two major applications for bacterial hyaluronic acid are very different. Its use to replace the natural product in humans in surgery of the eye or of joints simply replaces one natural product with another which is nonantigenic in the human body. The polymer has the additional unique property of very high water-binding and water-retention capacity. For this reason, it has been incorporated into cosmetic preparations. Water-binding capacity correlates with the molecular weight. Products with very high molecular mass can retain up to 6l of water per gram of polysaccharide. Hyaluronic acid is also used in veterinary applications and as a biocompatible coating material for prostheses. It is nonpyrogenic, thus making it ideal as a sheath for such implants. For therapeutic applications, the hyaluronic acid has to be of high purity and high molecular mass if its benefit is to be long-lasting. Other potential use of hyaluronic acid lies in the development of cross-linked polymer and its application as viscosurgical implants to control scarification and to prevent postoperative adhesions. Ester derivatives of hyaluronic acid in the form of microspheres have also been proposed as vehicles for drug delivery.155

2.16.10.5 Xanthan A very small number of bacterial exopolysaccharides have unique properties which have led to their commercial production. One product in particular, xanthan from X. campestris, is widely available commercially from various producers and has found an extensive range of applications, especially in the food field. It provides an example of a bacterial exopolysaccharide which is now manufactured in large quantities and can be found in an extensive range of food and other products.89 Xanthan was granted approval for food use by regulatory agencies a number of years ago, and is to be seen on the contents lists of many processed and semiprocessed foods found on supermarket shelves. Most industrial applications of xanthan derive from its ability to dissolve in hot or cold water to yield high-viscosity, pseudoplastic solutions, even at low polysaccharide concentrations. Most foodstuffs already contain polysaccharides such as starch or pectin as well as proteins and lipids and it is important that when a polysaccharide such as xanthan is added, it should be totally compatible with them. Some of the foodstuffs, including salad dressings, relishes, and yoghurts, are of low pH. There is therefore an added requirement that any polysaccharide which is incorporated into foods of these types must be acid stable. The xanthan solutions are compatible with acids, bases, and salts and can remain stable for long periods at ambient temperatures. Xanthan also withstands higher temperatures used in food processing. In nonfood applications, such as oil-drilling muds, it must also withstand high temperatures and be compatible with the other chemicals used in the oil-extraction process. As detailed above, xanthan shows synergistic interaction with plant galactomannans and glucomannans such as guar gum and LBG which are widely used in the food processing industry. The mixed polysaccharides show enhanced viscosity or gelation and these properties are widely employed in food processing. Although neither of the components forms gels on its own, when xanthan and LBG are mixed they produce elastic, cohesive gels above total polysaccharide concentrations of 0.03%. As the interactions between the bacterial and plant polysaccharides are highly dependent both on the acylation of the xanthan molecules and on the extent and distribution of D-galactosyl side chains present on the galactomannans, xanthan for food use employs a polysaccharide with 1 mole of acetate per repeat unit and approximately 0.3mole of pyruvate. The mixed gels are opaque but clarity is not necessarily needed for many food uses. Synergistic gels of this type are employed in a range of foods including spreads, cream cheeses, etc. Xanthan has many other industrial applications unrelated to foods and beverages. It is of value in the pharmaceutical and cosmetic industries where its applications employ the same physical attributes that are used as in foods. An example is in the formulation of toothpastes, where the pseudoplastic behavior of xanthan allows the product to flow freely when pressure is applied, but viscosity is regained immediately after the shear stress is removed. The role played by acyl groups in polysaccharide–polysaccharide interactions and the frequent need for their removal to reveal useful properties49 suggests that genetic manipulation of several bacterial strains to delete the polysaccharide acetylase genes might prove a useful approach although such modification might not be permissible for food applications.


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2.16.10.6 Emulsan Several exopolysaccharides possess emulsifying activity and one of these, designated emulsan, was obtained from a strain of Acinetobacter lwoffii. It has been proposed for use as a biological surfactant with applications in cleanup of oil spills or cleaning of tanks contaminated with oil.

2.16.10.7 Gellan Gellan, is the exopolysaccharide produced commercially as a chemically deacylated gelling agent and product from a bacterium designated originally as Auromonas (Pseudomonas) elodea, but now termed Sp. paucimobilis. The polysaccharide is one of a series of eight, structurally closely related bacterial products listed in Table 5. These polymers share much of their backbone structure but differ in the nature and location of their side chains and in the presence or absence of acyl groups. In its native form, gellan carries both O-acetyl and glyceryl substituents on a linear polymer and these are chemically removed by mild alkaline treatment to yield the commercial product. After polysaccharide production is complete in submerged culture, the viscous broth is pasteurized then treated with mild alkali to remove acyl groups. It is then hot filtered to remove bacterial cell debris. The native, acylated gellan polysaccharide does not form gels, whereas after chemical deacylation, gellan forms gels with divalent cations. The gellan gels are thermoreversible and form at concentrations as low as 0.75%. They provide high gel strength. Marketed as Kelcogel® or Gelrite® gellan has received approval in the US and EU for food use as a gelling, stabilizing, and suspending agent for a wide range of foods, either on its own or in combination with other hydrocolloids. Gels prepared from gellan resemble xanthan in that they give good flavor release. The gellan gels are stable over the wide pH range found in food products. This property has found commercial applications as a gelling agent in food and in plant cell biotechnology. The gels have very good clarity when compared with many other gels formed from plant or algal polysaccharides. The strong, optically clear gels have a difference between melting and setting temperatures of 45–60 C.157 Gellan also has the advantage that the concentrations required to provide a specific gel strength are much lower than those needed for many other polysaccharides. The other polymers in the gellan series yield highly viscous aqueous solutions some of which show a high degree of thermal stability, but, as yet, none appear to have been developed commercially. As well as its approval for food use, gellan has found applications in plant biotechnology. It is marketed as Phytogel®. It has some intriguing gelation properties, as Donor and Douds158 found that after removal of multivalent cations from the commercial product, solutions of gellan in monovalent cation form such as Naþ, Liþ, or NHþ 4 could be used to form beads with calcium ions in a manner similar to alginate bead preparation. In the form of the pure potassium salt, gellan also forms gels at a much lower concentration.159 Among potential industrial applications for gellan are its use as a gelling agent in dental and personal-care toiletries. Deodorant gel products can also use much lower levels of gellan than those of the polysaccharides (carrageenan/LBG mixtures) currently used. Gellan can be incorporated into microbiological and cell culture media as a replacement for agar. It has been suggested that it may even lead to some growth enhancement when compared to the corresponding agar-based bacterial culture media. Tests on the growth of a wide range of bacterial species have indicated that media solidified with gellan compare favorably with agar-based preparations, while the high clarity of the gels may have some distinct advantages. The high thermostability of gellan also makes it potentially useful for the culture of thermophilic microbial species. Similar gellan-based plant cell culture media may also avoid the problems caused by impurities in agar.

2.16.10.8 Other Potential Exopolysaccharide Products A few other products have been developed, some specifically replacing plant or animal products. Additionally, some bacterial exopolysaccharides are incorporated as components of conjugate vaccines to protect against a range of bacterial infections. Until recently, no bacteria were known to produce heparin but since investigation of several strains of Pasteurella multocida showed that they yielded several different exopolysaccharides this may provide a bacterial source of the product.160 Types D and F strains synthesized unmodified heparin and chondroitin, respectively, while the type A strain formed hyaluronic acid. A strain of E. coli serotype K5 forms a capsular polysaccharide in which the disaccharide repeat unit is essentially a form of desulphatoheparin.161 The polymer, composed of a repeating unit of !4)-b-D-glucuronosyl-(1!4)-a-N-acetyl-D-glucosamine-(1!, is similar to N-acetylheparosan, a biosynthetic precursor of heparin. As it is structurally similar to heparin, this bacterial polysaccharide may prove useful in medical research and in determination of the specificity of heparinases and related enzymes. It is of relatively low molecular mass, 50kDa. Another polysaccharide of this general type from E. coli K4 possesses a chondroitin backbone to which b-D-fructofuranosyl residues are attached at the C-3 position of the D-glucuronic acid. After removal

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of the fructosyl residues by mild acid treatment, the polysaccharide is a substrate for both hyaluronidase and chondroitinase. Although neither of these polysaccharides is used commercially, they can provide sources of oligosaccharide fragments for use in the study of the biological activities of heparin and chondroitin. Although many other uses for bacterial exopolysaccharides have been suggested in preliminary papers, few of these have been realized yet. It should be remembered that any bacterial product has to undergo stringent safety testing as well as possessing suitable rheological or other properties. It must also be cost effective in competition against established polymers from other natural sources and against those produced synthetically. The bacterial products always have to compete against other natural or synthetic polymers which may be inferior in their physical or ecological properties but are nevertheless much cheaper to produce and market! It is therefore unlikely that, despite the search in ever more exotic environments for polysaccharide-producing bacteria, more products will be developed from such sources. Research and development costs are high and there are probably few market niches waiting to be filled. It is possible that some effective new gelling agents for nonfood use might be discovered and the poly162 D-glucuronic acid from a Rhizobium strain may have potential uses in this field. It resembles alginate in forming 2þ thermostable gels in the presence of Ca . Alteration of the chemical properties of the original exopolysaccharide as exemplified by the dextran-derived Sephadex products may greatly enhance the value and extend the range of applications for other bacterial exopolysaccharides. As opposed to chemical modifications, biological treatments may become available. One possibility might be the use of polymer-level mannuron epimerases to enhance the guluronic acid content of alginates and thus modify their characteristics.77 Although we now have a much better idea of the relationships that exist between structure and function, it is still difficult to predict which bacterial exopolysaccharides may justify the expense of development. Many initial reports in the literature have proved wildly overoptimistic. If there is a clear market niche for which no satisfactory product is currently available, the time and effort might be rewarded.

Glossary archae Primitive bacteria many of which are found in extreme environments such as deep thermal vents, acid springs, etc. cyanobacteria Previously termed blue green algae, a group of photosynthetic Gram-negative bacteria. exopolysaccharides Microbial polysaccharides secreted outside the cell wall. Gram-negative bacteria Prokaryotic cells in which the cytoplasmic contents are bounded by an inner and an outer membrane between which lies the periplasm. Gram-positive bacteria Prokaryotic cells in which a single membrane surrounds the cytoplasm and is protected by a rigid wall complex. LAB Lactic acid bacteria belonging to the Gram-positive genera Lactobacillus, Lactococcus, or Streptococcus. phage A bacterial virus which may be either virulent, resulting in death of the bacterial host cell, or temperate, integrating its genetic material with that of the host. prokaryote Microorganisms belonging to one of the three major groups of bacteria. xanthan The exopolysaccharide produced by the Gram-negative bacterial species Xanthomonas campestris.

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Rougeaux, H.; Pichon, R.; Kervarec, N.; Raguenes, G. H. C.; Guezennec, J. G. Carbohydr. Polym. 1996, 31, 237–242. de Souza, A. M.; Sutherland, I. W. J. Appl. Bacteriol. 1994, 76, 463–468. Linton, J. D. FEMS Microbiol. Rev. 1990, 75, 1–18. Jarman, T. R.; Pace, G. W. Arch. Microbiol. 1984, 137, 231–235. Sutherland, I. W. Biotechnology of Microbial Exopolysaccharides; Cambridge University Press: Cambridge, UK, 1990. Garcia-Ochoa, F.; Santos, V. E.; Casas, J. A.; Go´mez, E. Biotechnol. Adv. 2002, 18, 549–579. Degeest, B.; Vaningelgem, F.; De Vuyst, L. Int. Dairy J. 2001, 11, 747–757. De Vuyst, L.; De Vin, F.; Vaningelgem, F.; Degeest, B. Int. Dairy J. 2001, 11, 687–707. van Hijum, A. F. T.; Kralj, S.; Ozimek, L. K.; Dijkhuizen, L.; van Geel-Schutten, I. G. H. Microbiol. Mol. Biol. Rev. 2006, 70, 157–176. Monsan, P.; Bozonnet, S.; Albenne, C.; Joucla, G.; Willemot, R.-M.; Remaud-Simeón, M. Int. Dairy J. 2001, 11, 678–685. Mochois, V.; Willemot, R.-M.; Monsan, P. FEMS Microbiol. Rev. 1999, 23, 131–151. Kralj, S.; van Geel-Schutten, I. G. H.; van der Maarel, M. J. E. C.; Dijkhuizen, L. Microbiol. 2004, 150, 2099–2112. Ielpi, L.; Couso, R. O.; Dankert, M. Biochem. Int. 1983, 6, 323–333. Sutherland, I. W. J. Appl. Biochem. 1979, 1, 60–70. Whitfield, C.; Paiment, A. Carbohydr. Res. 2003, 338, 2491–2502. Vanderslice, R. W.; Doherty, D. H.; Capage, M.; Betlach, M. R.; Hassler, R. A.; Henderson, N. M.; Ryan-Graniero, J.; Tecklenberg, M. In Biomedical and Biotechnological Advances in Industrial Polysaccharides; Crescenzi, V., Dea, I. C. M., Paoletti, S., Stivala, S. S., Sutherland, I. W., Eds.; Gordon and Breach: New York, 1989; pp 145–156. MacCormick, C. A.; Harris, J. E.; Gunning, A. P.; Morris, V. J. J. Appl. Bacteriol. 1993, 74, 196–199. Svanem, B. I. G.; Strand, W.; Ertesva˚g, H.; Skja˚k-Braek, G.; Hartmann, M.; Barbeyron, T.; Valla, S. J. Biol. Chem. 2001, 276, 31542–31550. Whitfield, C.; Roberts, I. S. Mol. Microbiol. 1999, 31, 1307–1319. Pearce, R.; Roberts, I. S. J. Bacteriol. 1995, 177, 3992–3997. Whitfield, C. Annu. Rev. Biochem. 2006, 75, 39–68. Glucksman, M. A.; Reuber, T. L.; Walker, G. C. J. Bacteriol. 1993, 175, 7045–7055. Welman, A. D.; Maddox, I. S. Trends Biotechnol. 2003, 21, 269–274. Van Kranenburg, R.; Marugg, J. D.; Van Swam, I. I.; Willem, N. J.; de Vos, W. M. Mol. Microbiol. 1997, 24, 387–397. Jolly, L.; Stingele, F. Int. Dairy J. 2001, 11, 723–732. Zogaj, X.; Nimtz, M.; Rohde, M.; Bokranz, W.; Romling, U. Mol. Microbiol. 2001, 39, 1452–1463. Glazebrook, J.; Walker, G. C. Cell 1989, 36, 661–672. Edwards, K. J.; Jay, A. J.; Colquhoun, I. J.; Morris, V. J.; Gasson, M. J.; Griffin, A. M. Microbiol. 1999, 145, 1499–1506. Betlach, M. R.; Capage, M. A.; Doherty, D. H.; Hassler, R. A.; Henderson, N. M.; Vanderslice, R. W.; Marelli, J. D.; Ward, M. B. In Industrial Polysaccharides; Yalpani, M., Ed.; Elsevier: Amsterdam, The Netherlands, 1987; pp 145–156. Vimr, E.; Lichtensteiger, C. Trends Microbiol. 2002, 10, 254–257. Lewis, A. L.; Hensler, M. E.; Varki, A.; Nizet, V. J. Biol. Chem. 2006, 281, 1186–1192. York, G. M.; Gonzalez, J. E.; Walker, G. C. In Biology of Plant-Microbe Interactions; Stacev, G., Mullin, B., Gresshof, P. M., Eds.; International Society of Plant/Microbe Interactions: St. Paul, MN, 1996; pp 325–330. Bezzate, S.; Aymerich, S.; Chambert, R.; Czarnes, S.; Berge, O.; Heulin, T. Environ. Microbiol. 2000, 2, 333–342. Sutherland, I. W. Trends Microbiol. 2001, 9, 222–227. Sutherland, I. W. Water Sci. Technol. 2001, 43, 77–86. Davey, M. E.; Duncan, M. J. J. Bacteriol. 2006, 188, 5510–5523. Langille, S. E.; Weiner, R. M. Appl. Env. Microbiol. 1998, 64, 2906–2913. Urai, M.; Aizawa, T. Carbohydr. Res. 2006, 341, 616–623. Houghton, J. I.; Quarmby, J. Curr. Opin. Biotechnol. 1999, 2, 259–261. Voepel, K. C.; Buller, C. S. J. Ind. Microbiol. 1990, 5, 131–138. Fukui, H.; Tanaka, M.; Misaki, A. Agr. Biol. Chem. 1985, 49, 2343–2349. Joualt, S. C.; Chevolet, L.; Helley, D.; Ratiskol, J.; Bros, A.; Sinquin, C.; Roger, O.; Fischer, A. M. Biochim. Biophys. Acta 2001, 1528, 141–151. Casu, B.; Grazioli, G.; Razi, N.; Guerrini, M.; Naggi, A.; Torri, G.; Oreste, P.; Tursi, F.; Zoppetti, G.; Lindahl, U. Carbohydr. Res. 1994, 263, 271–284. Wilson, J. C.; Hitchen, P. G.; Frank, M.; Peak, I. R.; Collins, P. M.; Morris, H. R.; Dell, A.; Grice, I. D. Carbohydr. Res. 2005, 340, 765–769. Sutherland, I. W. Carbohydr. Polym. 1999, 38, 319–328. Sutherland, I. W. FEMS Microbiol. Rev. 1995, 16, 323–347. Oyaizu, H.; Komagata, K.; Amemura, A.; Harada, T. J. Gen. Appl. Microbiol. 1982, 28, 369–388. Mishra, C.; Robbins, P. W. Glycobiol. 1995, 3, 643–654. Khalikova, E.; Susi, P.; Korpela, T. Microbiol. Mol. Biol. Rev. 2005, 69, 306–325. Mitsuishi, Y.; Kobayashi, M.; Matsuda, K. Carbohydr. Res. 1980, 83, 303–313. Pulkownik, A.; Walker, G. J. Carbohydr. Res. 1977, 54, 237–251. Verhoef, R.; Beldman, G.; Schols, H. A.; Siika-aho, M.; Ra¨tto¨, M.; Buchert, J.; Voragen, A. G. J. Carbohydr. Res. 2005, 340, 1780–1788. Sutherland, I. W.; Keen, G. A. J. Appl. Biochem. 1981, 3, 48–57. Suzuki, H.; Suzuki, K.; Inoue, A.; Ojima, T. Carbohydr. Res. 2006, 341, 1809–1819. Suzuki, H.; Suzuki, K.; Inoue, A.; Ojima, T. Carbohydr. Res. 2003, 338, 2841–2852. Shoham, Y.; Rosenberg, E. J. Bacteriol. 1983, 156, 161–167. Shabtai, Y.; Gutnick, D. L. J. Bacteriol. 1985, 161, 1176–1181. Kennedy, L.; Sutherland, I. W. Microbiology 1994, 140, 3007–3013. Crescenzi, V.; Dentini, M.; Dea, I. C. M. Carbohydr. Res. 1987, 160, 283–302. Hashimoto, W.; Maesaka, K.; Sato, N.; Kimura, S.; Yamamoto, K.; Kumagai, H.; Murata, K. Arch. Biochem. Biophys. 1997, 339, 17–23. Hashimoto, W.; Kobayashi, E.; Nankai, H.; Sato, N.; Miya, T.; Kawai, S.; Murata, K. Arch. Biochem. Biophys. 1999, 368, 367–374. Craston, D. H.; Farnell, P.; Francis, J. M.; Gabriac, S.; Matthews, W.; Saeed, M.; Sutherland, I. W. Food Chem. 2001, 73, 103–110. 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Biographical Sketch

Ian Sutherland is emeritus professor of microbial physiology in the University of Edinburgh. He is the author of a number of papers and reviews on bacterial exopolysaccharides. After undergraduate and postgraduate study in Edinburgh University, he worked with Dr. Otto Lu¨deritz in the Max Planck Institute fu¨r Immunbiologie in Freiburg, Germany, on the elucidation of the core structure of Salmonella lipopolysaccharide. Subsequently, he and colleagues in Edinburgh published the structure of colanic acid, an exopolysaccharide produced by a range of enteric bacteria. From further work it became clear that enzymes induced by virulent bacteriophage provided a valuable source of highly specific enzymes applicable to structural studies. Isolation of mutants yielding highly viscous colanic acid and other bacterial polysaccharides formed the start of a series of studies attempting to correlate chemical structure to the physical properties of this group of biopolymers and to their possible applications. The recent emphasis on the biofilm mode of growth by many pathogenic and environmental bacterial isolates provided an interesting field in which the roles of bacterial exopolysaccharides and enzymes degrading them could be experimentally tested.

2.17 Yeast and Fungal Polysaccharides N. Ohno, Tokyo University of Pharmacy and Life Science, Tokyo, Japan ß 2007 Elsevier Ltd. All rights reserved. 2.17.1 Taxonomy of Eukaryotic Microorganisms

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2.17.2 Fungal/Yeast Polysaccharides

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2.17.3 b-Glucan

561

2.17.3.1 BG of Candida albicans

561

2.17.3.2 BG of Edible and Medicinal Mushroom

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2.17.3.3 Higher-Order Structures of BGs

563

2.17.3.4 Molecular Biology of BG Biosynthesis

564

2.17.3.5 Early Diagnosis of Deep Mycosis and BG

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2.17.3.6 Immunopharmacological Action and BG

565

2.17.3.7 Action of BGs toward Mucosal Immune System

565

2.17.3.8 Metabolism of BG

566

2.17.3.9 Progress Related to the Recognition Mechanism of BGs

567

2.17.3.10 Antibody to BG

567

2.17.4 a-Glucan

568

2.17.4.1 Glycans of Aspergillus

568

2.17.4.2 Glycans of Schizosaccharomyces pombe

568

2.17.5 Yeast Mannan

569

2.17.5.1 Effect of Mannan on Host Defense System

570

2.17.6 Chitin

570

2.17.7 Cryptococcal Capsular Polysaccharide

571

2.17.8 Summary

571

2.17.1 Taxonomy of Eukaryotic Microorganisms The present microorganism classification originates in the ‘five kingdoms’ concept proposed by Whittaker in 1969, in which the difference between prokaryotic and eukaryotic cells is considered to be important.1 Yeast and mushroom belong to ‘eukaryotic cell’ and are called ‘fungi’ or ‘true fungi’ generically. Although they are microorganisms, their basic cellular structures are similar to those of animal or plant. The fungus classification system was revised by Ainsworth in 1973 based on the ‘five kingdoms’.2 Further, with the rapid advances in molecular biology, molecular systematics was recently established, which was distinct from the classification system based on traditional ‘morphological characteristics’. The analysis of various microorganisms with molecular systematics showed that the eukaryote domain consisted of several tens of families that branched simultaneously (many kingdoms).3–5 Molecular phylogeny is progressing rapidly, and the number of the microorganisms whose genome structures were elucidated is increasing rapidly year after year. More time is needed to realize a comprehensive classification system based on molecular systematics and other approaches. Currently, there are approximately 80000 known fungal species in the world.6–10 It is surmised that approximately 1500000 species exist, including undiscovered species. These fungi are classified by kingdom, phylum/division, class, genus, and species. Many fungi are classified into Basidiomycota or Ascomycota. It is supposed that some fungi are classified also into the kingdom Protozoa or the kingdom Chromista. Fungi include mushrooms, molds, and yeasts, which have significantly different appearance and sizes. As mushrooms are too large to be considered microorganisms, they are referred to as ‘macrofungi’. Lichens with which two or more microorganisms live in a symbiotic relationship are also included.

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Yeast and Fungal Polysaccharides

Fungi exhibit both the sexual form (e.g., morphology of mushroom) and the asexual form for regeneration (e.g., morphology of mycelium), and either form is used depending on surrounding environmental changes. However, the existence of both forms (holomorph) is not known for all fungi. Their nomenclature is also characteristic. The background of the discovery of a fungus is reflected in its name, and different names may be given depending on whether the fungus exhibits the sexual form (teleomorph) or the asexual form (anamorph) of regeneration. Some natural fruit bodies weigh as much as several tens of kilograms per piece. These fruit bodies are formed by the accumulation of mycelia. Considering the whole fruit bodies and how deep mycelia grow in the ground, they can be thought of as infinite living things. Eukaryotic microorganisms in the form of mycelia have cell walls that consist mainly of polysaccharides. Cell wall degradation is necessary for fungal growth, and many polysaccharide-degrading enzymes exist for this purpose. Fungi take up various nutrients for growth and efficiently utilize dead trees or dry grass, as exemplified by mushrooms. Many kinds of enzymes, including polysaccharide-degrading enzymes, are also produced in the process. Thus, fungal species vary widely. Much remains to be analyzed and there is great possibility that various structures and activities will be discovered in the future.

2.17.2 Fungal/Yeast Polysaccharides Fungal polysaccharides not only demonstrate particular biological functions but also play various roles in organisms, such as stimulating the biodefense system. They are also industrially important substances.11–22 There are many scientific papers, reports, and patents on them in various fields. Although many of their structures were analyzed, the structural details differed slightly. They are derived from microorganisms, and it cannot be easily concluded how significant these differences really are when one takes into consideration the variation in microbial strains, culture conditions, individual differences, differences in extraction methods (including partial degradation during the extraction process), and analytical methods with their accuracy. In the case of yeast with an egg-shaped morphology, constructed by a delicately controlled network, it is difficult to imagine that the structure of polysaccharide (one of the representative cell wall components) is only a repetition of sugar units. Yeast reproduces by budding, and a bud scar is known to remain after growth. Therefore, the structure of this portion is also expected to be characteristic. Assuming that the morphology of microorganisms itself is a result of network formation of the primary structures of polysaccharides, changes might occur related to the specific conditions of the extraction and purification process that are characteristic of the polysaccharides. For example, b-glucan (BG), one of the main cell wall components, forms a functionally and physically strong cell wall, but has low solubility in water. Therefore, partial degradation is necessary prior to purification, and this gives rise to subtle differences in its structure. Moreover, in crude or low-purity commercial polysaccharides, the measured biological activity can be sometimes due to impurities. It is dangerous to accept all results only because they are published in major journals. Compared with low molecular weight natural organic compounds, such as enzymes and lipids, it is difficult to estimate the uniformity and homogeneity of polysaccharides. Compared to other substances, large variations in results can therefore be expected. On the other hand, some soluble polysaccharides are secreted by fungi. Similar to the above-mentioned compounds, they are difficult to purify as well because their molecular weights could not be determined accurately. Nevertheless, it seems that they are simple compared with insoluble cell wall components because they are secreted as soluble substances. However, there are still many unknown factors for polysaccharides. The analysis of fungal polysaccharides, which has hitherto been performed by conventional polysaccharide chemistry, seems not to have profited from the advances in molecular biology. This is because the polysaccharides are secondary metabolites with large and complicated structures, their solubilities are low, and the progress of the analytical methods was slow compared with other fields. Meanwhile, the number of young researchers has significantly decreased. As a result, we strongly feel that the progress in this field has been significantly delayed. In the twenty-first century, as various receptors involved in natural immunity were analyzed and the receptors for fungal polysaccharides were found, the number of researchers rapidly increased recently. It is expected that the progress in receptor research will contribute to the development of polysaccharide chemistry itself. However, the structures of polysaccharides are very complicated and special techniques are needed, as mentioned above. Nevertheless, changes have begun. In addition, whole genome analysis of a variety of eukaryotic microorganisms has been completed or is in progress. Such information is quite useful for analyzing fungal polysaccharides, especially in relation to morphology of fungi/ yeast, that is, induction of phenotypic changes by targeting genes gives various clues. Recently, many of the diseaserelated eukaryotic microorganisms and their metabolites are being analyzed by these modern technologies.


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Table 1 Classification and properties of yeast and fungal polysaccharides Name

Note

b-Glucan

b1-3- and b1-6-linked glucan present in cell walls of yeast and fungi b1-3- and b1-6-linked glucan produced as soluble form Branching as well as length of side-chain glucose vary significantly Applied to medical use for immunostimulation. Also present in cell wall of pathogenic fungi, Candida, Aspergillus, Pneumocystis, and so on, and released into patients’ sera Lentinan from Lentinus edodes, sonifilan from Schizophyllum commune are used for cancer therapy. Similar glucans were obtained from various fungi, such as sclerotan, isosclerotan, pachyman, Grifolan, CSBG, ASBG, OX-CA, SSG, OL-2, BBG, and BGG Pustulan from Umbilicaria, Lasallia, Gyrophora is b1-6-glucan. O-Acetylation is mentioned Islandic acid and luteic acid from Penicillium sp. is b1-6-glucan associated with malonate b1-6-Glucan was also prepared from Agaricus braziliensis (Agaricus blazei) Lichenan from lichen was composed of b1-3 and b1-4-linkages a1-3-Glucan was present as major cell wall component of fungal cell wall Pullulan from Aureobasidium pullulans having structure of a1-4- and a1-6-linkages Nigeran from Aspergillus niger is a1-3- and a1-4-glucan Isolichenan from lichen was composed of a1-3- and a1-4-linkages b1-5-Galactan, galactocarolose, was from Penicillium charlesii Heterogalactan compsed of galactose, mannose, and fucose was extracted from Armillaria mellea Heterogalactan composed of galactose and glucuronic acid was extracted from Inonotus levis Malonogalactan was extracted from Penicillium citrinum Phospho-a1-6-; a1-3-galactan was from Sparebolomyces sp. Mannoprotein is major antigenic glycan on pathogenic yeasts, Candida sp. Linkage of mannan region significantly varied among species. Structure is also significantly regulated by culture conditions Mannocarolose from Penicillium charlesii is a1-6-mannan with a1-4-branches b1-3 and b1-4-linked mannan was from Rhodotorula glutinis Phospho-a1-6-; b1-2-; a1-2-mannan was from Hansenula capsulate and Hansenula hostii Product of pathogenic Aspergillus and Penicillium. Main chain was composed of a1-6-mannan. Side chain was composed of 1-2- or 1-3-linked galactose Galactomannan was also extracted from Schizosaccharomyces pombe Present in cell wall of Trichosporon cutaneum and Cryptococcus neoformans Partially N-acetylated a1-4-linked polygalactosamine was from the culture filtrate of Aspergillus parasiticus b1-4-Linked N-acetylated glucosamine present in animals, plants, as well as fungi. Chitin and chitosan were present as components of fungal cell wall Mucoric acid is polymer of b1-4-linked glucuronic adic present in Mucor species Protuberic acid is a polymer of glucuronic acid and iduronic acid present in Kobayasia nipponica

a-Glucan

Galactan

Mannan

Galactomannan

Xylomannan Polygalactosaminide Chitin, chitosan Polyuronide

Structure of fungal/yeast polysaccharides vary significantly (Table 1). Analysis of these polysaccharides was started over 50years ago. It is impossible to review all this information. Our laboratory is mainly working on fungal BGs and mannans from edible/medicinal mushrooms and pathogenic fungi. The focus here is mainly on these glycans. Glycans with pathogenic interest, such as a-glucan, chitin, and capsular polysaccharides, are also included.

2.17.3 b-Glucan 2.17.3.1 BG of Candida albicans Zymosan, which is prepared from baker’s yeast (Saccharomyces cerevisiae), is a reagent that has been widely used for many years in inflammation and immunology researches.23–25 Many parameters concerning those functions have been clarified by these studies. Since the main component of yeast cell wall is BG, it was thought that the activity of zymosan might be due to the receptor of BG, or to the activation of the complement system by BG. After zymocel, which is pure BG derived from zymosan, was marketed in the 1980s, researchers were even more convinced that BG plays a role in the activity of the crude zymosan.26 Since rapid progress was made with the analyses of natural immunity, such as the discovery of Toll-like receptors (TLRs), in the late 1990s, it was logical that the receptor of zymosan received attention in major journals.27–31 However, it should be noted that some of the data showing in these

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major journals contain overestimation of the molecular mechanism of zymosan that all of the activities were induced by the major component, BG. There are soluble and insoluble (particulate) BGs, both of which show significantly different biological activities. One notable example is the production of reactive oxygen species from neutrophil and macrophage.32,33 Although particulate BG showed this activity, soluble BG showed no such (or weak) activity. The similar tendency appeared in some other activities tested. However, these conclusions were conducted by analyzing the data using substances having different primary structures. An attempt was made to confirm the concept by comparing soluble and particulate BGs with the same primary structure. After various trials, it was found that oxidation with hypochlorous acid was the most appropriate method to remove and/or degrade impurities (Figure 1). Hypochlorous acid is a strong oxidant that can oxidize and decompose all substances, including proteins, lipids, and nucleic acids. Fortunately, BG is relatively resistant to such treatment. As a result, by applying this method, purified, particulate BG was obtained and its dissolution in alkali or DMSO yielded soluble BG (hereafter referred to as the hypochlorous acid–DMSO method); that is, soluble (solubilized Candida b-glucan, CSBG) and particulate (hypochlorous acid-oxidized Candida albicans, refered to OX-CA). BGs with the same primary structure were prepared by using this method. Although this is a classic method, it can be widely used and so we applied it to baker’s yeast, Candida, and Aspergillus.34 CSBG prepared from Candida using this method had a b1-6-chain with a degree of polymerization of approximately 10–50, which was attached to a long b1-3-backbone. The b1-6-chain was slightly branched (Figure 2). This structure does not contradict the biosynthesis route of yeast cell wall.35–37 Candida shows dimorphism and mycelium-like growth. When BG was prepared from mycelium-like Candida, its basic structure was similar.38 However, the most characteristic result was its yield. As the content of BG in myceliumlike Candida cell wall was low, the amount of BG prepared was also quite low. BG has a high molecular weight, which was estimated to be approximately 1 million by size-exclusion chromatography and a large molecular weight distribution. One reason for this is that CSBG was obtained by hypochlorous acid oxidation, which is a severe treatment, and partially disrupts cell wall to enable solubilization followed by an alkali treatment step for the extraction process which may cause partial chain scission. Once CSBG is dissolved in alkaline solution, it is water soluble even after neutralization. From these procedures, it is clear that CSBG was obtained by partial decomposition of an originally large and insoluble substance. Sizofiran (SPG) derived from Schizophyllum commune is also used as a drug. It is obtained by an ultrasonication treatment during the manufacturing process which causes a decrease in the molecular weight and the formation of a rod-like triple helix. Without such a treatment, the molecular weight distribution is expected to be quite large.

2.17.3.2 BG of Edible and Medicinal Mushroom The analysis of the polysaccharides of mushrooms that form fruit bodies is complicated. The procedure and the fundamental structure will be discussed using Grifola frondosa as an example.39–41 In G. frondosa, the fruit body, the Acetone-dried yeast Suspended in 0.1 M NaOH Added, NaClO solution (various conc.) 4 ⬚C overnight Collect ppt. Dried, EtOH and acetone NaClO-oxidized yeast (particle glucan:OX-CA) Suspended in DMSO Occasional sonication Collect extract Dried, EtOH and acetone Soluble glucan (CSBG) Figure 1 Outline of NaClO–DMSO method.


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(b)

(a) b1-3-gluco b1-6-gluco b1-3;1-6-branch

(c)

(d)

Figure 2 Outline of the structure of fungal/yeast BGs. a, b1-6-glucan chains attached to backbone of b1-3-glucan. This type of structure was found in a medicinal fungus, Agaricus brasiliensis. b, highly branched b1-6-glucan segment. This type of segment was found in the cell wall of fission yeast, Schizosaccharomyces pombe. c, long b1-6-glucan linked to b1-3-glucan backbone. This type of structure was found in the cell wall of Saccharomyces cerevisiae and Candida albicans (OX-CA, CSBG). d, 6-branched b1-3-glucan. Ratio of branch varies depend on species and strains. This type of structure was found in various medicinal mushrooms, that is, Grifola frondosa (grifolan, GRN), Lentinus edodes (lentinan), Hypsizigus marmoreus, Sparassis crispa (SCG), Peziza vesiculosa (PVG), Omphalia lapidescens (OL-2), and Sclerotinia sclerotiorum (SSG).

surface pad of the fungus (root portion of fruit body), and the fraction that was released by the fungus upon fermentation of mycelium were analyzed. For the fruit body and the surface pad of the fungus, hot water extraction, cold alkali extraction, and hot alkali extraction were performed in this order, and the polysaccharide fractions obtained were purified. Six branched b1-3-glucans having similar basic structures were obtained and named grifolan (GRN) (Figure 2). Due to the simplicity of purification, GRN obtained by the fermentation process is mainly in use. Its structure was identified as a b1-3-glucan carrying single b-glucopyranose residues as side chains. This basic structure is similar to that of Lentinus edodes, named lentinan (LNT) and SPG. BGs having this basic structure were also found in Sparassis crispa,42–45 Sclerotinia sclerotiorum,46–48 Omphalia lapidescens,49,50 Hypsizigus marmoreus, and Peziza vesiculosa.51–53 However, the degree of branching and the ratio of side chains and main chains differed from fungus to fungus. OL-2 derived from O. lapidescens Schroet showed two branched points per three residues of the main chain, which was the highest degree of branching, whereas PVG derived from P. vesiculosa showed approximately one branched point per five residues. On the other hand, the main BG of Agaricus contained a large amount of b1-6-chains and a small amount of b1-3-chains.54 Since this structure was resistant to enzymolysis, it is presumed to be highly branched. The fruit bodies of edible and medicinal mushrooms have characteristic morphologies and BG seems to participate in the morphogenesis as a representative framework material. As the detailed structures are expected to differ in each mushroom, careful analysis should be conducted in the future.

2.17.3.3 Higher-Order Structures of BGs Neutral aqueous solutions of BGs form a gel. Concentrated mushroom extracts may also become agar-like when stored for a long period of time. This is evidence of their molecular interaction; the presence of a triple-helix structure has been suggested by X-ray analysis and other methods. In reality, however, all BGs in nature do not always exist in the form of triple helices; some form a single helix by coordinating with water molecules, whereas others exist in the form of random coils. These higher-order structures have considerable effects on the biological activity. We examined this aspect in detail by using a coagulation factor (factor G) found in horseshoe crab, and clearly demonstrated that BGs are active in the form of single helices or random coils.55–57 The dependence on the higher-order structure has also been observed in analyses using immunocompetent cells, and it is necessary to control the higher-order structure in order to evaluate the activity of BGs.58–60

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2.17.3.4 Molecular Biology of BG Biosynthesis In line with the advances in genome analysis in various microorganisms, eukaryotic microorganisms, such as Sa. cerevisiae, Ca. albicans, and Aspergillus fumigatus, were analyzed. In the early 1980s, research on the biosynthesis of cell wall BG was conducted intensively. Cabib et al. suggested that BG was synthesized inside the cell membrane using UDPglucose as the starting material by the b1-3-glucan synthase gene (FKS), then gradually transported through the cell membrane with the progress of the synthesis, and finally reaching the cell wall.61–63 It is known that Fksp, which has a catalytic site of FKS, and Rho1p, which is a regulatory factor of FKS having low guanosine triphosphatase activity, are involved in this reaction.64,65 FKS-related genes were found in Candida, Cryptococcus, Paracoccidioides, Pneumocystis, Aspergillus, Coccidioides, etc. Thus, BG biosynthesis appears to take place in these fungi by a relative similar fundamental mechanism. However, as mentioned above, an extremely wide variety of structures are formed, and, therefore, detailed analysis should be conducted in the future.66–70 Recently, echinocandins, antifungal drugs having BG synthetase inhibitory activity, were marketed for the treatment of mycosis.71–74 We expect that research in this field will be performed from a new viewpoint and increasingly accelerate.

2.17.3.5 Early Diagnosis of Deep Mycosis and BG The number of deep mycosis patients tends to increase with time. It seems that several high-risk factors, such as an increase in the number of patients with an impaired immune system accompanying aging and the spread of highly advanced medical care, are involved. Therefore, the availability of early diagnostic methods is important. The typical causes of deep mycoses are Candida and Aspergillus. Since b1-3-glucan is present in their cell walls, it is useful for early diagnosis to measure trace amounts of BG in the blood. BG can be measured at trace level by the limulus test, in which the biodefense mechanism in limulus specifically reacts with b1-3-glucan in the patient’s blood (Figure 3).75–80 A positive reaction in the limulus test indicates that b1-3-glucan is actually released into the blood. However, as the substance released could not be isolated, its real structure is unknown. When Ca. albicans was grown in a complete synthetic medium, the soluble macromolecular fraction, named CAWS, was released into the medium. CAWS was subjected to the limulus test, and a positive result was obtained.81 Interestingly, this fraction consisted not only of BG; rather, its main component was a mannoprotein complex. Further analysis revealed that CAWS has a large molecular weight distribution and was not a single population. When the biological activity of CAWS was examined, various activities were found, including acute lethal toxicity and angiitis-inducing effect. The question arises then if this is indeed the substance present in blood. No conclusions have been drawn yet at this stage.

b1-3-Glucan

Endotoxin

Factor C ===

Activated factor C

Factor B ===

Activated factor G

=== Factor G

Activated factor B

Proclotting enzyme ===

Clotting enzyme

Coagulogen ===

Coagulin === Gel

Boc-Leu-Gly-Arg-CONH-pNA pNA (A405) Figure 3 Outline of coagulation cascades of endotoxin and b1-3-glucan in limulus lysate. Limulus lysate contained specific recognition proteins for bacterial endotoxin (factor C) and b1-3-glucan (factor G). Binding of endotoxin or b1-3glucan to these proteins activates coagulation cascade of the lysate and produces ‘clot’. The coagulation cascade include several specific proteinases showing very restricted substrate specificity. For the quantitative determination of clinical materials, synthetic substates are applied, as shown in this figure.


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2.17.3.6 Immunopharmacological Action and BG In the beginning, BG attracted attention as an ‘antitumor polysaccharide’, and various evaluation systems were examined. The most interesting result we obtained was the relationship between the degree of branching and activity. The in vivo antitumor activity of highly branched OL-2 (degree of branching: 2/3) against Sarcoma 180 solid tumor was weak; the activity was strong in BG having an intermediate degree of branching and weak in BG having no branching. An appropriate degree of branching seems to be needed to induce the antitumor activity. However, when OL-2 was administered with a chemotherapeutic drug, it exhibited antitumor activity and accelerated recovery from leukopenia. Therefore, OL-2 seems to exhibit some degree of activity, although the mode of action may be slightly different from that by the other immunomodulating BGs. It was reported that the cytokine mRNA expression pattern was also different. In addition, a linear b1-3-glucan, named curdlan (CRD), showed antitumor activity when a carboxymethyl or a hydroxyethyl group was chemically introduced to free hydroxyl groups, or when CRD was oxidized by hydrogen peroxide or by hypochlorous acid. At a moderate degree of substitution, lower than c. 0.5 substitution per glucose residue, CRD still showed the activity. Although it had been suggested that the triple-helix structure was necessary for activity expression, the result obtained by such chemical modification did not agree with this idea. On the other hand, there are reports that the optimum concentration for activity expression differed significantly in SPG when its higher-order structure was changed. That the triple-helix structure is important for expressing the antitumor activity is a well-accepted fact. However, the results mentioned here are in contradiction with this. We have been studying the effects of higher-order structure, such as triple helix, single helix, and random coil, through in vivo and in vitro analyses of laboratory animal and human peripheral blood leukocytes and cell strains. It was clarified that various activities, such as NO production of macrophage and interleukin-8 (IL-8) production, depended on the higher-order structure and produced higher concentration of mediators by the single helical conformation. Further, as mentioned above, we developed the hypochlorous acid–DMSO method for the preparation of Candida cell wall BG, and particulate (OX-CA) and soluble (CSBG) BGs having the same primary structure were obtained using this method. When their activities were compared using human peripheral white blood cells, activity of CSBG was strongly dependent on the presence of complement activation. Furthermore, when gene expression in stimulated human peripheral blood mononuclear cells was compared using the DNA micro-array method, it was found that there was not only a difference in the intensity of activity, but also a qualitative difference, between soluble and particulate BGs. For activity evaluation, the evaluation systems themselves, such as those related to species or strain difference, are interesting. Type II collagen-induced arthritis, which is known as a rheumatism model, is mostly limited to the DBA/1 strain.82 In addition, the adjuvant activity of BG against antibody production differs significantly depending on the strain. Not only the strength but also the class of the antibody produced was different. Furthermore, high cytokine production by BG in an in vitro culture system of spleen cells was noted in the DBA/2 strain. It was clarified recently that GM-CSF production played a key role in this reaction.42,44 Significant individual difference was also observed in humans. When we administered a branched b1-3-glucan, named SCG, derived from Sp. crispa to leukocytes from several volunteers and measured cytokine production, a significant difference in quantity was observed.83 Thus, in activity evaluation, it is necessary to consider not only the structural factor but also information of the host. Comparison of in vivo and in vitro biological activities indicated that the extent of activity differed considerably from individual to individual and from fungus to fungus. As we made sure that all samples had the same level of purity prior to analysis, we realized the difficulty of generalizing the structures and activities of BG. In vivo activity was built up by adding the various factors, such as the absorption, distribution, metabolic, and excretory systems, and the histocompatibility antigen. Since all the elements involved in each activity evaluation system have not been analyzed at the molecular level, further development of these systems in future is required.

2.17.3.7 Action of BGs toward Mucosal Immune System There are high expectations regarding the utility of orally administered BGs, particularly in the mushroom-processing industry. Strictly speaking, as mushrooms contain various biologically active compounds in addition to BGs, for making an evaluation, it is necessary to evaluate the effects by using purified BGs. This seems logical; however, there are several obstacles in achieving this. One question is of what is meant by useful effects and the other question is whether the structures of the BGs having a repeat structure which demonstrate effects can be completely specified. An obstacle is the extraction efficiency. We reported in 1986 the isolation of a highly branched BG, named SSG, from the culture medium of the Ascomycetes strain Sclerotinia sclerotiorum IF09395. During the course of analyzing SSG, we found that it demonstrated antitumor activity even when administered orally. Therefore a systematic study of the oral immunomodulation by SSG was

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conducted in the mid-1990s, which clearly showed that (1) SSG is effective against multiple solid tumors of the allogeneic as well as syngeneic systems, (2) SSG demonstrates metastasis inhibitory effect, (3) SSG increases the responsiveness of spleen cells to ConA and LPS, (4) SSG causes an increase in NK activity, (5) SSG exhibits peritoneal and alveolar macrophage-activating action (acid phosphatase, phagocytosis, bactericidal action, hydrogen peroxide, IL-1), and (6) SSG enhances IgA production. Moreover, these effects were clearly demonstrated to be involved with an increase in Peyer’s patch function.84–88 Although these effects appear to be similar to the effects of intraperitoneal or intravenous administration, the optimum dosage is a factor of 10 higher than that of intraperitoneal administration, and there may be considerable differences in the mechanisms underlying the expression of the pharmacological effects between oral and nonoral administration. In addition, the oral administration of SCG from Sp. crispa clearly produced hematopoiesis-promoting effects in a leukopenia model.89 Also, this research demonstrated that BGs are able to exhibit useful effects even when orally administered. Moreover, orally administered SSG was not absorbed at a detectable level; namely, it is unlikely that BGs demonstrate activity as a result of their being absorbed. Although it is presumed that BGs exert multiple actions that are mediated by the activation of immunocompetent cells in the digestive tract mucosa, further analyses will be required to examine the details of this.

2.17.3.8 Metabolism of BG It is difficult to comprehend that BG is absorbed by the body under normal physiological conditions and there is also little knowledge of its metabolism and accumulation. Its metabolism and accumulation should be taken into consideration when a large amount of fungi grow inside the body of a deep mycosis patient, or when LNT and SPG, which are known as antitumor agents, are administered. Further, it is not known if and to what extent various drugs or medical devices are contaminated by BG. If its toxicity was as well known as that of endotoxin, measurement and proof of no contamination for all products and devices would be required. However, the toxicity of BG is not emphasized strongly. Because of this, there is little knowledge on the accumulation and metabolism of BG in the body. To understand the mechanism of action of BG, we conducted an analysis from two viewpoints: one is the elimination of BG from blood, and the other is the accumulation of BG in organs. The former was mainly performed using the limulus test. The half-life differed significantly depending on the BGs used. It was as long as approximately 6h for SPG. By contrast, the elimination was relatively rapid for CSBG. Moreover, the elimination rate depended on the higher-order structure; the half-life for the triple-helix structure appeared to be longer than that for the single-helix structure. Accumulation kinetics were investigated by using the limulus reagent for detection of BG and isotope-labeled BG administered to mice.90–96 It was found that BG injected into mice was accumulated mainly in the liver and spleen for an extremely long time with little decomposition (Figure 4). Here, ‘extremely long’ indicates a period as long as half a year or 1year, which corresponds to the survival time of mice. 50 1 week

40

% to total

2 weeks 3 weeks

30

4 weeks 20

Excrement

Feces

PEC

Sup

Lung

Intestine

Stomach

Spleen

Heart

Kidney

Liver

0

Blood

10

Figure 4 Organ/tissue distribution of metabolically 3H-labeled SSG (a highly branched 1-3-glucan from Sclerotinia sclerotiorum) in mice. Metabolically tritium-labeled SSG (3H-SSG) was administered intraperitoneally and after 1, 2, 3, and 4 weeks, mice were sacrificed and 3H-SSG was quantified by monitoring 3H content. sup; soluble fraction of the peritoneal exudate.


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We can conclude that BG administered orally is hardly absorbed. If a large amount of BG were absorbed in the body, the risk of its accumulation inside the body without being metabolized throughout life would emerge. Although conclusions cannot be drawn, detailed analyses of the absorption, accumulation, metabolism, and excretion of orally administered BG should be improved and continued.

2.17.3.9 Progress Related to the Recognition Mechanism of BGs TLRs have been determined to play a central role in natural immunity, and various ligand molecules have been identified for these TLRs.97–101 Although the involvement of TLRs cannot be ruled out for BGs as well, analyses of BG-specific receptors, such as surfactant protein D, lactosylceramide, scavenger receptor, and dectin-1, are under way.102 Starting in the latter half of the 1980s, reports indicating that the complement receptor CR3 is one of these receptors have been systematically published by Ross et al.103–107 In addition, in the end of the 1990s, dectin-1 began attracting attention as a new receptor.108–112 We have also prepared a recombinant dectin-1 and determined that BG binds to it.113 Analysis of the signal transduction mechanisms mediated by these receptors is attracting attention throughout the world, and it is likely that the details of the activation mechanism will be elucidated in the near future.

2.17.3.10 Antibody to BG The production of antibodies against BGs is being studied. BGs have previously been associated with low antigenicity, making it unlikely for them to induce antibody production. It is quite true that production of monoclonal antibodies against SPG was difficult. Similar attempts have been made to produce monoclonal antibodies against GRN obtained from G. frondosa, but they have met with little success. However, anti-BG antibody was produced when mice were administered yeast cells.114 These findings suggest that not all BGs have difficulty in inducing antibody production but it is particularly difficult for SPG-type polysaccharides. When antibody titers against CSBG obtained from Candida were actually measured using human globulin preparations, an IgG class antibody was determined to be present.115,116 Moreover, these antibodies were specific for the side-chain portion of the glucan. When the antibody titers were measured in healthy volunteers, the antibodies were confirmed to be present in all the volunteers. Moreover, when the anti-CSBG antibody titers in blood were measured in cancer patients, autoimmune disease patients, and mycosis patients, the antibody titers were observed to fluctuate depending on the disease (Figure 5).117 Similar antibodies were also observed in pigs and cows. In addition, antibodies against BGs were observed in mice albeit only in specific strains.118 These findings indicate that humans and animals are spontaneously sensitized to BGs which occur in food

5000

Unit of anti-BG antibody

4000

3000

2000

**

1000

0

A

B

**

C

**

D

** E

F

G

Figure 5 Anti-BG antibody in human sera. CSBG-coated enzyme-linked immunosorbent assay (ELISA) plate was used for determination. A, normal human (n¼22); B, rheumatoid arthritis patients (N¼77); C, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis patients before therapy (n¼14); D, ANCA-associated vasculitis patients after therapy, early phase (n¼24); E, ANCA-associated vasculitis patients after therapy long phase (n¼11); F, patients with fungal infection (n¼5); and G, cancer patients (N¼26). **, P1).53 AXs isolated from rice endosperm and highly substituted at O3 exhibited an X-ray diffraction pattern compatible with the threefold helix found for pure xylans.97 The conformation of WE-AX from wheat exhibiting different A/X ratios was examined in aqueous solution.35,100 As shown on Figure 7, Lp is not significantly affected by A/X ratio within the range observed, so that it can be concluded that arabinose substitution had no real influence on the conformational behavior of AX in solution.

2.20.4 Interactions of AX with Cell Wall Components Cell walls of the cereal grains are mainly constituted of AX and mixed-linked b-glucans in the endosperm and AX, cellulose, and a variable amount of lignin in the outer tissues. The close association of the different polymers in

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Plant Cell Wall Polysaccharides in Storage Organs: Xylans (Food Applications)

Figure 7 Persistence length (Lp) of the xylan backbone of WE-AX as a function of the arabinose/xylose ratio (A/X). Values are from Dervilly-Pinel et al.35

Figure 8 Double labeling of AX and b-glucans in aleurone cell walls from wheat grain. Polyclonal antixylan106 and monoclonal anti-beta-glucan105 antibodies were used with a second-stage goat antirabbit (orange-red fluorescence) and a second-stage goat antimouse (green fluorescence) antibody, respectively. Yellow fluorescence indicates the presence of both b-glucans and AX.

the wall is well shown in Figure 8. The aleurone cell wall was specifically labeled with antibodies directed against mixed-linked beta-glucans (green in Figure 8)105 and AX (red in Figure 8).95,106 The wall appeared as a multilayered system, AXs are more abundant at the interface between cells and cell corners, whereas mixed-linked b-glucans are concentrated close to the plasma membrane. However, both polymers are distributed across the wall giving the yellow color and suggesting close interactions. Similar co-localization of the polymers is also observed in central endosperm cell walls. Interactions of AX with the different polymers of the wall are likely to occur through hydrogen bonds, covalent bonds, or mechanical entanglement.

2.20.4.1 Interaction with Mixed-Linked b-Glucans and Cellulose The two main polysaccharides associated with AX are mixed-linked b-glucans in endosperm tissues and cellulose in outer tissues. Although both AX and beta-glucans are co-localized in the wall of grain and in cereals in general,107 the nature of the interactions had never been studied in model systems using WE-AX and soluble b-glucans. Indirect evidence of possible noncovalent interaction between AX and b-glucan was reported in barley for the fraction

Adsorbed polymer (µg/mg cellulose)


667

3.5 3 2.5 2 1.5 1 0.5 0 0

15 20 25 5 10 Polysaccharide equilibrium concentration (µg/ml) XG

AX (DS 0.5)

AX (DS 0.3)

30

HX (DS 0.8)

Figure 9 Adsorption isotherms of xyloglucan (XG), water-extractable arabinoxylan (AX) from wheat endosperm of low and average degree of substitution (DS) and highly substituted heteroxylan (HX) isolated from maize bran. Cellulose Avicel (20mg) was used and 1ml of polymer solution (25mg ml--1) in acetate buffer pH 5. After 6h of incubation at 40 C, the samples were centrifuged (1min, 20000g), and the supernatants were analyzed for their neutral sugar content, from which the adsorbed amount was calculated. Adapted from Dervilly-Pinel, G. Ph.D. thesis, Universite´s de Paris VII, XI, ENSIA et INA-PG, Paris, France, 2001.

recovered after alkaline extraction.108 A xylan/b-glucan complex with unusual low substitution for AX and high proportion of b1!4 linkages for b-glucan was recovered, suggesting possible chain--chain interactions. However, alkaline extraction used to isolated AX and b-glucans from the cell walls creates serious artifacts by removing alkali labile groups such as hydroxycinnamic acids but also acetyl groups, which are generally present on lowsubstituted AXs. As a matter of fact, the presence of acetyl groups strongly affects the crystallization behavior of hardwood xylans.109 Therefore, this fraction might also reflect a co-precipitation rather than real chain--chain association in the wall. Cellulose--hemicellulose interactions through hydrogen bonding are well demonstrated in the case of xyloglucan,110 but such interactions are less clear for xylans. The xylan retention phenomenon on to cellulose has been explained by co-crystallization of xylan segments with cellulose and by the formation of strong xylan--cellulose hydrogen bonds.111 However, the conformation adopted by xylans and cellulose in the crystalline state is not favorable to establish hydrogen bonding. A different mechanism that is not based on cellulose--xylan chain interaction has been proposed to explain the retention of low-substituted glucuronoxylans from birchwood on cellulose surfaces under conditions similar to those found in wood pulping:111 the xylan molecules could associate together through interactions between the unsubstituted, linear regions of the chains or hydrophobic interaction promoted by lignin residues covalently associated to xylans. Preformed xylan aggregate structures could then diffuse and interact with cellulose surfaces. Furthermore, hydrogen bonding is clearly restricted by the presence of arabinose side chains that hamper chain-chain interactions in the case of highly substituted AXs found in endosperm or outer pericarp cell walls of cereal grains. This is experimentally evidenced by coating xyloglucan and AX with different degrees of substitution to cellulose microfibrils in the same conditions.94 As shown in Figure 9, the binding of AX to cellulose is very limited compared to xyloglucan. Little difference is obtained between WE-AXs exhibiting substitution degrees varying from 25% (A/X¼0.4) up to 50% (A/X¼0.8), attesting that large blocks of unsubstituted xylose, which are supposed to facilitate chain--chain association, are not likely to occur even in low-substituted WE-AXs. Very highly substituted AXs isolated from maize pericarp did not bind at all to cellulose in the same conditions, showing that hydrogen bonding of AX to cellulose is not likely to occur in pericarp tissue as previously reported.72

2.20.4.2 AX--AX Interactions through Phenolic Acid Bridge Phenolic acids are incorporated into plant cell walls of various monocots.45 Ferulic acid is most predominant within species of the Gramineae, where it is ester-linked at the O5 position to a-L-arabinosyl residues of AX. Because they can act as cross-linking agents between polysaccharides, or between polysaccharides and lignin, ferulic acids are not

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HO

HO

OH HO

O

O OH

O OMe

O

O

HO

O

OMe H

O

H

OMe MeO OH

OMe

HO

O

OMe

OH

OH 8-O-4⬘

8-5⬘ OH

HO

O

O

OH

MeO HO

O

OH MeO

OH O

O

OMe OH OH

OH

OMe

OMe

OH

5-5⬘

8-8⬘

O HO HO

O OMe MeO OH 4-O-8⬘5⬘-5⬘⬘

O

Figure 10 Structure of dehydrodimers and dehydrotrimers of ferulic acid identified in cereal grains.

only contributing to wall assembly, promoting tissue cohesion, restricting cell expansion,112 but also controlling the mechanical properties of mature tissues.50 The different dehydrodimers113 and -trimers82,114,115 found in plant tissues (see Figure 10) indicate that the cross-linking reaction is an oxidative mechanism probably mediated by peroxidase in vivo.116 In vitro, numerous studies have shown peroxidase gelation of WE-AX from different cereals (see Section 2.20.6.4)17,77,117 and the effect of oxidative enzymes on the mechanical properties of outer tissues of wheat grain.118 Besides, the feruloylation degree of AX has also been shown to control the properties of AX gels.119 Feruloylation of AX is therefore an essential aspect of cell wall development and tissue properties. The amount and type of hydroxycinnamic acids vary greatly according to the different tissues in cereal grains. Clearly the outer tissues of the kernel and the aleurone layer are very rich in ferulic acid and dehydrodimers.50,51 Whatever the tissue, 8-O-40 and 5-80 benzofuran dehydrodimers were mainly encountered, but the 5-50 , 8-50 , and 8-80 forms were also detected in durum wheat and bread wheat,66,118 maize pericarp,79 rye,120 barley,121,122 rice, and oat.122 Coumaric acid is mainly located in aleurone while dehydrotrimer has been detected only in the pericarp tissue of wheat and maize.52,82,115 Most of the studies are limited to the identification of dehydrodiferulic acids released following saponification. The structural evidence that dehydrodimers cross-link polysaccharide chains in the cell wall polysaccharides are more limited and provided by the isolation of 5-50 and 8-O-40 saccharide fragments following acidic hydrolysis of cell walls from maize bran.80,81 The formation of phenolic cyclobutane dicarboxylic acids (truxillic and truxinic acids) under ultraviolet (UV) irradiation is also a coupling mechanism of AX in plant cell walls.123,124 However, such dimers have never been detected in grain tissues.

2.20.4.3 Other Covalent Interactions Dehydrodiferulic bridges are likely to be the dominant parameter that explains differences between WE-AXs and WU-AXs in endosperm and aleurone cell walls of cereal grains. Rhodes et al.47 have suggested that such associations occur to only a very limited extent in wheat aleurone walls; however, they have clearly underestimated the content in dehydrodimers by measuring only the 5-50 isomer, which represents 25--30% of total dehydrodimers. However, the same authors have proposed that protein--polysaccharide cross-linking through tyrosine-hydroxycinnamic acid dimerization could occur in aleurone cell wall.125 As a matter of fact, alkaline treatment used to cut ester linkage between


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hydroxycinnamic acids and AX are generally not able to release completely the polysaccharide from the cell walls, especially in outer tissues;72 protein--polysaccharide linkages have been suggested in maize pericarp to explain heteroxylan extraction.72 Dehydrodiferulic acid-tyrosine has been isolated in rye and wheat flour dough,126 but this cross-link between AX and proteins might be the result of the processing conditions. In addition, the outer tissues of the grain generally contain lignin. Different cross-linkings of AX to lignin have been suggested.112 First, hydroxycinnamic acid is known to be directly esterified or etherified to lignin surface,127,128 and it is likely that all ferulic acid etherified to lignin is also esterified to AX.112 Direct ester links between uronic acid on glucuronoxylans and hydroxyl groups of lignin surfaces and direct ether linkage between AX and lignin involving, for example, primary hydroxyl of arabinose side chains are also possible.112 However, such linkage has never been directly demonstrated. In the case of cereal grains, a tight association of typical lignin structure was evidenced for maize pericarp heteroxylan extracted by alkali and extensively purified.129

2.20.5 Biosynthesis of AX The biosynthesis of feruloylated AX in cereal cell wall grain requires the orchestration in defined order of several enzymes in the presence of the right substrates. The spatial and temporal heterogeneity of AX will probably imply an heterogeneity of biosynthetic machinery (i.e., different set of enzymes could be implicated for the synthesis of a poorly substituted AX and/or a highly substituted one).

2.20.5.1 The Synthesis of the Building Blocks of Feruloylated AX Preliminarily to the biosynthesis of AX, the presence of the right building blocks is required, that is, specific nucleotide sugars and ferulic acid. These molecules must be synthesized and channeled to the right location within the cell for polymer assembly. Two pathways are required, part of the nucleotide sugar interconversion pathway (for review, see Seifert),130 and part of the lignin pathway that leads to ferulic acid (for review, see Barrie`re and Ralph).131 The two major nucleotide sugars are UDP-D-xylose (pyranose form) and UDP-L-arabinose (furanose form). The UDP-D-xylose units are made from UDP-D-glucuronic acid by possibly two types of decarboxylase, the UDP-xylosesynthase (UXS) or UDP-apiose/UDP-xylose-synthase (AXS). The UXS gene family (six members in arabidopsis) encodes both soluble and membrane-bound enzymes with synthesis of UDP-xylose occurring both in the cytosol and in the endomembrane system.132 Two Arabidopsis genes distantly related to the UXS genes encode AXS.130 The UDP-L-arabinose is made from UDP-D-xylose by a UDP-D-xylose-4-epimerase in the endomembrane system.130 An epimerase could be necessary for the catalysis of the furanose form (less stable than the pyranose). In Arabidopsis thaliana, the mur4 mutant has enabled identification of a small gene family of three other potential xyloseepimerase genes.133 Several other nucleotide sugars might be required for AX biosynthesis but in minor amounts, such as UDP-Dglucuronic acid (UDP-D-GlcA), and UDP-D-Galactose (UDP-D-Gal).9 The lignin pathway has been mostly studied in maize and in Arabidopsis.134,135 The part of the pathway that leads to ferulic acid within the cell is still controversial. However, the biosynthesis of ferulic acid in wheat seedlings seems to be correlated with the activity of O-methyltransferases.136 The work done on maize mutant131 suggests the presence of a CCoAOMT hub that leads to Feruloyl CoA (Figure 11). The transfer of ferulic acid from Feruloyl-CoA to AX is also supported by work on rice suspension cells.137 However, another group suggested that feruloyl-glucose (1-O-feruloylb-glucose) was the precursor for intracellular feruloylation of AX in wheat suspension culture, and that Feruloyl CoA was required for feruloylation of proteins.138 Another pathway described using an Arabidopsis mutant suggests that accumulation of soluble ferulic acid could come from the oxidation of coniferaldehyde by coniferaldehyde-dehydrogenase.139

2.20.5.2 The formation of the linkages found in feruloylated AX The variety of linkages found in AX (see Section 2.20.3.2 and Figure 2) requires at least six different enzyme activities to be formed. Four classes of enzyme are needed for AX assembly: glycosyltransferases (for the glycosidic bonds), feruloyltransferase (ferulic acid), oxidative enzymes (ferulic acid dimerization), and acetylesterases (xylose acetylation). In plant, glycosyltransferases represent a very large multigene family with several hundred members found in monocots and dicots (for review, see Coutinho et al.).140 The presence of motifs and the conservation of these genes allow the classifications in subfamilies, some of which have been extensively studied and had their function assigned.141

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UDP-D-Gal GAE UDP-D-Glc

UGD

UDP-D-GlcA

UDP-D-GlcA

AXS

UXS

UXS UDP-D-Api UDP-D-Xyl

UDP-D-Xyl Phenylalanine or tyrosine

Caffeoyl CoA Lignin pathway

CCoAOMT

Feruloyl CoA

XS

UXE UDP-L-Ara ?

Xylan

AT

Feruloyl Glc

AX

? FCoApSFt CCR

Peroxidases ME

Coniferaldehyde

Coniferaldehyde dehydrogenase

? Ferulic acid

F-AX

Golgi

F-AX Cell wall

Cytoplasm

Figure 11 Schematic representation of AX biosynthesis. The building blocks are synthesized or channeled in the Golgi apparatus to be assembled. The xylan backbone is synthesized by a xylan synthase (XS), then substituted by arabinose residues by possibly several arabinosyltransferases (ATs). Others transferases add in minor amounts residues and side chains made of galactose (Gal) and glucuronic acid (GlcA). Ferulic acid molecules are linked to some arabinosyl residue to form feruloylated AXs (F-AX) that are then exported to the wall. The ferulic acid is further reticulated in the wall by peroxidases. Other modifying enzymes (MEs) are possibly involved to obtain the final AX found in muro. The precursors and the different enzymes (circular boxes) involved in AX biosynthesis are indicated. Sugar and ferulic acid transferases are indicated in red. The hypothesis for ferulic acid synthesis and incorporation in AX are also shown. The sugar building blocks: UDP-D-Glc (glucose); UDP-D-GlcA (glucuronic acid); UDP-D-Xyl (xylose); UDP-D-Api (apiose); UDP-L-Ara (arabinose); and UDP-D-Gal (galactose). The enzymes: UGD, UDP-D-glucose dehydrogenase; UXS, UDP-D-xylose synthase; AXS, UDP-D-apiose/UDP-D-xylose synthase; UXE, UDP-D-xylose 4-epimerase; GAE, UDP-D-glucuronic acid 4-epimerase; FcoApSFt, feruloyl-CoA:polysaccharide feruloyltransferase; CCR, cinnamoyl CoA reductase; CCoAOMT, caffeoyl coenzyme A O-methyltransferase; XS, xylan synthase; AT, arabinosyltransferases; and ?, unknown enzymes.

Although most of the glycosyltransferases have been identified by motifs and sequence homology or other bioinformatics tools142 and inserted in the CAZy database,143 relatively few of them have been assigned to a particular function, especially for glycosyltransferases involved in cell wall polysaccharide biosynthesis. Different glycosyltransferase activities are required for the formation of the three main glycosidic bonds found in AX, namely the b1!4-linked xylose backbone and the a1!2- and a1!3-linked arabinose side chains.

2.20.5.2.1 b-1,4-Xylosyltransferase or xylan synthase EC 2.4.2.24 The biosynthesis of (1!4)-b-xylan backbones of AX is catalyzed by UDP-D-xylose:1,4-b-D-xylan 4-b-D-xylosyltransferase (EC 2.4.2.24 ), commonly named xylan synthase, using uridine 50 -diphosphoxylose (UDP-Xyl) as the donor substrate.144 The evidence that the xylan backbone of AX is made first comes from the fact that arabinosyltransferase activity is dependent on the synthesis of unsubstituted xylan as an acceptor molecule,145 but the size of these acceptor molecules is not known. Thus it is possible that small xylan molecules are linked end-to-end to form the final xylan backbone that could be up to several thousand residues long.9 The enzyme activity for xylan biosynthesis has been detected in tissues undergoing secondary wall formation in various plants,146 but also in wheat seedlings147,148 and most interestingly in barley endosperm.144


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Two potential xylosyltransferases were partially purified and characterized (Mr 38000 and 40000) from French bean.149 The presence of one of these enzymes seems to correlate with a peak of activity during the period of maximum secondary wall synthesis.149 However, the structural features of the synthesized xylan have not been characterized in detail. Studies in wheat seedlings indicate that b-1,4-xylosyltransferase activity can be detected with exogenous acceptor substrates such as xylooligosaccharides148 or with endogenous acceptors, in which case xylan molecules (>500kDa or more than 3300 residues) are synthesized.147 In the last conditions, UDP-arabinose had no effect on the incorporation of xylose into xylan (in wheat seedlings) and this suggests that xylosyltransferase is independent of the availability of UDP-arabinose.147 Recent work in developing barley endosperm shows that the maximal activity for a b-1,4-xylosyltransferase occurred between 13 and 14 days after flowering,144 a stage at which deposition of AX is very active. However, the increased deposition of AX continued after the xylosyltransferase activity ceased (after 25 days), suggesting the presence of other b-1,4-xylosyltransferase not detected in this assay.144 As yet, only an a-xylosyltransferase activity has been identified at the molecular level. A small gene family of glycosyltransferase encodes this enzyme (seven members in Arabidopsis, GT34). Some of these members may be implicated in xyloglucan biosynthesis.150 We could speculate that xylan-synthase belongs to a multigene family with several isoforms implicated in the biosynthesis of different types of xylans (arabinoxylans, heteroxylans, and xylan) and in the different walls (primary, secondary, type I, type II). This multigene family may share features with other backbone synthesizing enzymes such as cellulose synthase (chain of (1!4)-b-glucosyl residues, CesA, GT2), mannose synthase (chain of (1!4)-b-mannosyl residues, CslA, GT2), or callose synthase (chain of (1!3)-b-glucosyl residues, GT48). These enzymes have high Mw (>500 amino acids) with several predicted membrane spanning domains (4--17). However, neither the enzyme nor its gene have been characterized at the molecular level and so they remain to be identified. The catalytic mechanism for b-1,4-xylosyltransferase is presumably, by analogy with glycosidases, the inverting type (as opposed to retaining) and most likely follows a single-displacement mechanism where the acceptor performs a nucleophilic attack at carbon C1 of the sugar donor.151 Xylan synthase is likely to be a processive enzyme, meaning that it remains attached to its substrate and performs multiple rounds of catalysis before dissociating, and it may form a complex with arabinosyltransferase.145

2.20.5.2.2

Arabinosyltransferase (a-1,2- and/or a-1,3)

The xylan backbone of cereal AX is decorated with mainly mono- and disubstitution of a-L-arabinosyl residue attached through 1!3 and 1!2 linkages.9 As for xylan activity, arabinosyltransferase activity has been detected in secondary wall-forming tissues of several plants,149 and in monocot seedlings.145 A putative arabinosyltransferase of Mw 70000 (corresponding to about 630 amino acids) was partially purified from the Golgi-bound fraction in French bean.149 The arabinosyltransferase activity identified in the microsomal fraction of wheat seedlings is described, from the product characterization, as an AX arabinosyltransferase.145 Interestingly an Arabidopsis mutant ARAD1 has recently been characterized and presents a reduced amount of arabinose in the wall of leaves and stems, and thus could be the first arabinosyltransferase gene identified.152 This mutant is part of a large glycosyltransferase family in plant (GT47) with 39 and 25 members in Arabidopsis and rice, respectively. Three members of this family involved in cell wall biosynthesis have been previously identified. There are two galactosyltransferases for xyloglucan biosynthesis153 and glucuronosyltransferase for pectic rhamnogalacturonan II biosynthesis.154

2.20.5.2.3

Other glycosyltransferases

Other enzymes are required for adding the minor components of AX (short side chains, galactose and glucuronic acid). Several other glycosyltransferase activities are probably implicated for the minor substitutions found on the xylan backbone. These activities have not been studied for AX biosynthesis but galactosyltransferases, for example, have been identified for xyloglucan biosynthesis,153 that could be used to identify related enzymes.

2.20.5.2.4

AX feruloylation

The feruloylation of arabinosyl residues of AX has been described in monocot wheat and rice cell suspension culture.137,138 An enzyme (Feruloyl-CoA: arabinoxylan-trisaccharide O-hydroxcinnamoyltransferase) activity has been isolated from rice that suggest the implication of feruloyl-CoA as a substrate.137 The enzymatic nature of AX

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feruloylation has been demonstrated.137 However, another group suggests that AX feruloylation uses feruloylglucose as substrate.138 In any case, the enzyme implicated in the feruloylation and its gene remains to be identified clearly. The oxidative coupling could be catalyzed by peroxidases, oxidases, or a nonenzymic system involving active oxygen species. Recently, a group using maize suspension cells has suggested that the coupling feruloyl residues was done by peroxidases rather than oxidases.155

2.20.5.3 The Different Location of AX Biosynthesis Like other noncellulosic polysaccharides, AX biosynthesis takes place in the Golgi apparatus and is then exported to the wall by vesicular transport. Direct evidence of this comes from immunolocalization of AX in Golgi vesicle156 and the localization of xylan synthase 146,147,157 and arabinosyltransferase145 activites in the Golgi. The feruloylation of AX has not been localized to such a level but seems to occurs inside the cell,116,137,138 and therefore most likely in the Golgi apparatus. Experiments using labeled 3H arabinose and 14C ferulic acid have indicated the intracellular location of AX feruloylation, in cell suspension cultures of Festuca, maize, and wheat.116,138,158 Dimerization of ferulic acid has long been believed to only take place in the cell wall but the most recent studies116,138 support that dimerization also takes place intracellularly at least for some of the 8-50 dehydrodimers.138 One of the limits of the study of AX biosynthesis comes from the fact that the harvest of the AX from the wall might not entirely reflect its biosynthesis inside the cell. Indeed, important structural modifications of the polymer could occur in muro and that would also be part of its biosynthesis, that is, (1) reticulation by laccases and peroxidases of the ferulic acid residues; and (2) removal of certain arabinose residue by an arabinosylfuranosidase. The presence in the Golgi or in the wall of glycosylhydrolase (CAZy, GH) implicated in the biosynthesis of AX is not excluded. In cellulose, for instance, an endoglucanase (Korrigan) has been found to be involved in its biosynthesis, together with the glycosyltransferases known as the cellulose synthases (CesA) from the GT2 family.159 The biochemical characterization of only half a dozen enzymes implicated in cell wall biosynthesis has been carried out among the hundreds of plant glycosyltransferases. To date, less than 20 genes identified in mutant screens are likely to play a direct role in polysaccharide synthesis. The number of enzymes identified by mutant screen compared with biochemical data shows the importance of genetics for unraveling complex biosynthetic pathways such as polysaccharide biosynthesis. It is also not surprising that all the enzymes implicated in AX biosynthesis remain to be identified and characterized at the molecular level. A synthetic view of the biosynthetic pathways for AX is proposed in Figure 11.

2.20.6 Physicochemical Properties of AX AX exhibits different physicochemical characteristics such as water solubility, viscosity, gelling, and hydration properties, which are the basis of its functional properties in different processes and food systems.

2.20.6.1 Water Solubility The water solubility of AX and of polysaccharide in general depends on the delicate balance between chain--chain and chain--solvent interactions. Structural factors such as chain length, presence of side-chain groups, and their distribution will modify this balance and the solubility behavior of the polymers. Generally, the presence of side chains that prevent chain--chain interactions favors water solubility of the polymers. In the case of AX, water solubility is not only related to structural features of the polymer chain but also to covalent linkage to other cell wall polymers. As an example in endosperm, WU-AXs have an higher A/X ratio than WE-Axs, which should lead to a better water solubility, but the presence of a high proportion of chain--chain cross-linking through covalent ‘diferulic bridges’ renders WUAXs insoluble. Similar trends are observed for highly branched HXs found in pericarp tissues. Cereal grains AX are generally soluble in water at neutral pH, naturally or after alkaline extraction, provided that substitution degree is higher than 30%. Small changes in ionic strength or ionic composition, and limited shifts of temperature, can induce a change in the physical nature of polysaccharide system, for example, a transition from the state of a macromolecular solution to that of a gel.91 Such transitions are not observed for AX, and, for example, gel formation is the result of the formation of covalent linkage under the action of oxidative systems that couple ferulic acid residues. However, arabinose side chains are an important parameter of AX solubility. Different studies have


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shown that removal of arabinose residues by controlled acid hydrolysis160 or using arabinofuranosidase104 changes the solubility behavior of AX and gives rise to aggregation and precipitation of the polymer. However, the mechanism of the aggregation is not clear, although generally described as an interaction between regular zones of the polymer chain.

2.20.6.2 Viscosity The viscosity of polymer solution is directly related to the fundamental molecular properties (molecular conformation, molecular weight, and molecular weight distribution) and concentration of the polymer. The main information that can be extracted from viscosity measurements on dilute macromolecular solutions is contained in the intrinsic viscosity of the macromolecule. Intrinsic viscosity is not a viscosity at all, but actually a measure of the hydrodynamic volume of the coil in the case of noncharged polymeric chains, or of the asymmetry of the particle in the case of rigid macromolecules.91 Intrinsic viscosities of WE-AX from cereal endosperm were determined in a large number of studies. Large variations exist between studies, due to different extraction procedures including inactivation or not of endogenous enzymes, but clearly variations of the intrinsic viscosities are observed between varieties; for example, intrinsic viscosities reported for WE-AX are in the range 200--600mlg--1 for wheat with an average value of 400mlg--1.16,161 Rye WE-AX intrinsic viscosity also exhibits variation between 400 and 1000mlg--1, but values are generally larger than those observed for wheat.17,24 Values reported for WE-AXs of barley, oat, and triticale are generally similar to those reported for wheat.17 The relation between intrinsic viscosity and molecular weight of polymer-like macromolecules is usually expressed in the form of the empirical Mark--Houwink--Sakurada equation: []¼K.Mwa, where K and a are empirical parameters which depend on the polymer--solvent pair and on the temperature, and are both related to chain stiffness. The value of the exponent a gives indication about the general conformation of the polymer. For flexible linear chains 0.5 a 1, whereas stiff chains display larger values of a (values as high as 1.8 are reported for rod-like chain conformations).91 The values of coefficient a determined for wheat (a¼0.74)35 and rye (a¼0.94)162 WE-AXs are well within the range of flexible chain although in the upper limit for rye AX, which suggest a slightly different behavior compared to wheat AX. The conformational behavior of AX in solution is in fact remarkably similar to that of galactomannans and is characteristic of semiflexible polysaccharides.35,100 As pointed out earlier, the conformation of AX chain is not affected by the degree of branching of the xylan backbone. The viscosity of AX solution is therefore mainly dependent on change in concentration and Mw of the polymer, and contrary to the generally admitted assumption11 structural features such as A/X ratio have probably very limited effect on the viscosity of AX solution. However, the formation of dehydrodiferulic bridges that have been detected in WE-AX17 might strongly affect the viscosity behavior of AX solution by increasing dramatically the Mw of AX. The viscosity generated by WE-AX has a strong influence on the functional, technological, and nutritional properties of AX. Udy163 had shown very early that 95% of the intrinsic viscosity of wheat flour water extract was due to WE-AX, and he pointed out that the average size of the soluble molecules was a specific varietal characteristic as well as the amount of water-soluble polysaccharide.

2.20.6.3 Hydration Properties The components of wheat flour (starch, AX, gluten proteins, gliadins) are incompatible with one another and therefore compete for the available water forming separate aqueous phases, each with a peculiar composition.164 The hydration properties (water absorption or retention properties) of insoluble AX have a strong impact on functional properties of AX, as fiber can modify the distribution of water among the different components of food which is very critical in cereal products and especially during bread-making. Insoluble fiber can absorb, swell, and entrap water within its porous matrix. Definition of hydration properties has been proposed for dietary fiber:165 waterretention capacity (water holding, water binding) is defined as the amount of water retained by a known weight of fiber under the conditions used, for example, application of centrifugation. Besides ‘water-retention’ capacity, hydration properties can be also assessed by ‘swelling’ (the volume occupied by a known weight of fiber under the conditions used) and ‘water absorption’ (kinetics of water movement under defined conditions). It is important to keep in mind that the hydration properties of an insoluble fiber depends on the chemical (polymers nature) and physical (porosity, particle size) structure and are influenced by a number of extrinsic parameters such as pH, T , ionic strength, and by the history of the product (drying, etc.). The literature concerning hydration properties of AX is rather confusing and all the results reported as ‘waterbinding capacity’ do not actually reflect the same measurements. On one hand, WU-AX can be assimilated to dietary

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fiber; the water-holding capacity of water-unextractable solids (WUSs) containing about 50% (w/w) of WU-AX is about 7.6g/g, and treatment of WUS with a xylanase reduces it.166 The swelling property of WUS with similar composition was evaluated to 26g/g.44 On the other hand, many studies have used the Farinograph as an indirect tool to evaluate the hydration properties of AX by measuring changes in dough consistency after addition of WE-AX or WU-AX to flour. Based on this method, researchers determined that WU-AX can hold 6.7 times their weight of water,167 compared to 6.3 for WE-AX. Using the same method, more pronounced differences were observed between WUAX and WE-AX, with values of 9.0 and 3.5 times their weight of water.168 Based on studies concerning the water distribution in dough, it was calculated that approximately 22.5% of water in dough was associated with AX.169 Finally, a ‘water binding capacity’ has been determined for WE-AX, which actually is the amount of nonfreezable water. This amount of water has been determined by differential scanning calorimetry170 or NMR,104 and it should be pointed out that the conditions of measurement, such as speed of cooling, should can greatly affect the values obtained. A value of about 0.40g/g was measured whatever the origin of WE-AX (wheat, rye, barley) or the A/X ratio.

2.20.6.4 Gelation 2.20.6.4.1 Mechanisms The capacity of flour water extracts to thicken and gel in certain oxidizing conditions has been recognized for a long time.171 This unique property is related to the reaction of the solution of feruloylated AX with free radical-generating agents like chemicals (ferric chloride, ammonium persulfate) or enzymatic systems (hydrogen peroxide/peroxidase, linoleic acid/lipoxygenase, laccase/oxygen).78,172--176 The gelation primarily results from the formation of threedimensional networks of AX chains anchored by dehydrodimers of ferulic acids, as showed by the report of the presence of 5-50 dehydrodimers in gelled AX fractions.172 The aqueous phase is retained in the network. The dimerization of ferulic acids linked to AX derives from a radical mechanism and occurs in two steps. First, the parent radical is generated from the attack of the hydrogen of the hydroxyl group at the phenolic position and results in a phenoxy radical. This radical is stabilized by delocalization of the unpaired electron onto the whole molecule. In a second step, the radical monomers are coupled, two unpaired electrons bore by two different monomers forming a covalent linkage,113 which will then strongly connect two AX chains. The structures of the dehydrodimers that are formed during this coupling step depend on the distribution of the unpaired electrons. Coupling will occur at the positions of highest electron density. In the case of ferulic acid, these positions are at C4-O and C5 on the ring and at C8 on the propenoic side chain.177 In AX gels, the 8-50 (normal and benzofuran forms), 8-O-40 , 5-50 , and 8-80 dehydrodiferulates have been detected, the 8-50 and 8-O-40 being the most abundant. Higher oligomers of ferulic acids have been found in maize pericarp cell walls.82,83 They are coupling products of further oxidized dimers and monomers. The 4-O-80 , 50 -500 dehydrotrimer was also detected in gelled wheat AX but in lower concentrations than dehydrodimers.119

2.20.6.4.2 Properties of AX gels Apart from the observation of solidification in test tubes or the measurement of the viscosity of solution, a relevant method to study the gelation of AX is small-amplitude shear oscillatory rheometry. Before addition of a suitable oxidizing agent, the mechanical spectrum of the sample is typical of a true viscous solution with the loss modulus (G00 dominating over the storage modulus (G0 ). After addition of the oxidant, G0 values rise rapidly over G00 and then reach a plateau region. The mechanical spectrum at the plateau is typical of a solid-like material with high G0 values independent of frequency and much lower G00 values dependent on frequency. Upon a few days of storage, enzymatically induced AX gels exhibit a significant decrease of their G0 values. This effect was the result of a loss of ferulic acid coupling products and a depolymerization of AX chains by cleavage of some glycosidic bonds. It was attributed to radical reactions catalyzed by the still-active enzymatic system and can be overcome by thermal inactivation of the enzyme.178 The structural characteristics of AX play a determinant role in their gelation ability. The comparison of AX from different sources has shown that a high content in ferulic acid, a high molecular weight/hydrodynamic volume, and a lowly substituted xylan backbone are favorable features to form strong gels.11,17,161,179 A positive correlation has been thus established between gel rigidity and intrinsic viscosity of AX. The better gelling ability of poorly arabinosesubstituted chains would be due to easier contacts between adjacent feruloyl groups of neighboring chains. The distribution of the feruloyl groups along the xylan chain could also influence the gelling ability.119,179 During the gelation process, the monomers of ferulic acid esters disappear since they are converted into dimers or higher oligomers. The apparition of dimers and trimers is however far from compensating monomer consumption.


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The formation of higher oligomers has been suggested although they have not been detected so far. The superimposition of G0 and ferulate compound evolutions along the gelation process shows that detectable ferulate crosslinking was terminated while the gel stiffness still continued to increase. This suggested that additional linkages might be responsible for gel formation. The combination of swelling and rheological measurements has shown that weak linkages (hydrogen bonding, Van der Waals interactions, chain entanglements) play a role in the gel structure. However, covalent linkages were found dominant but involved probably ferulate oligomers higher than trimer. From the swelling experiments, average mesh sizes between 200 and 400 nm have been calculated for 1% AX gels, which are in the same range as for ‘physical’ (noncovalent) gels like alginate, amylose, or gellan.119

2.20.6.4.3

Applications

AX gels are neutral, transparent, and odorless. Due to their covalent nature, they resist thermal, pH, and ionic stresses. AX gels can be dried once formed, then rehydrated. They exhibit high water absorption and retention capacity (more than 100g of water per gram of polymer) and this property appears insensitive to electrolytes. The capacity to absorb water increases with cross-linking density up to an optimum level. Beyond, the possibility to swell is impeded and the absorption decreases. The use of AX gel-based wound dressing for burns has been proposed.180 The cross-linking of AX was reported to occur and to play a determinant role during flour dough processing, especially in the case of oxidant supplementation, or in other processes involving grains of flours. This aspect is discussed further on. With the aim to create gels with novel and modulable properties, attempts have been made to cross-link covalently feruloylated arabinoxylans and proteins. The potential sites of proteins for connections to a ferulate are the phenolic amino acid tyrosine and the thiol group of cystein. The direct bonding of ferulate to cysteinyl residues by laccase or peroxidase cannot be obtained because the initially generated feruloyl radicals transferred their electrons onto the amino acids which preferentially self-di(poly)merized. Only once all of the amino acids were di(poly)merized, ferulate started to di(ploy)merize.181 With polysaccharides and protein, this mechanism resulted in the formation of two distinct networks, interpenetrated but not covalently connected. However, model studies have proved that low molecular weight adducts of tyrosine or tyrosine-rich peptides and ferulate can be produced in specific conditions with regard to enzymes or substrates.181--183 AX/casein adducts have thus been obtained.184 The use of AX gels has been suggested for the transport of macromolecules or cells and their controlled release in the gastrointestinal tract. Model studies have shown that bovine serum albumin embedded in an AX gel can be protected from pepsin proteolysis.185 Other experiments have demonstrated that high concentrations of proteins can be charged in the gels and that their rate of release can be modulated by playing on gel characteristics through ferulic acid content and AX chain concentrations.186 Due to their interesting properties, cross-linked AXs could certainly be used as food additives in a number of preparations where texturization and stabilization are desired. They could be used also in nonfood applications where high water absorption, protection, and stability are required. The main limitating factor for their use is the absence of an economically viable source of soluble high molecular weight feruloylated AX. Cereal milling by-products are rich in AX but they are generally hardly extractable by mild processes and become mostly ungellable after alkaline extraction, which is the only quantitative extraction method. Effluents from gluten/starch separation contain appreciable levels of soluble AX but they are generally depolymerized during the process and have therefore lost their gelling ability.

2.20.7 AX in Food Applications 2.20.7.1 Milling Milling is a generic designation for the first processing stage of cereal (wheat) grains. The general aim of milling is to dissociate and separate the starchy endosperm (rich in starch and gluten proteins) from the peripheral grain layers (rich in cell wall material) and to recover it as flour (common wheat) or semolina (durum wheat) with the highest possible yield and purity. Flour and semolina are mostly used for the manufacture of mass-market cereal food products like bread, biscuits, cakes, pastas, and couscous. The main by-product of flour or semolina is called bran and comprises the aleurone layer, the seed coats (testa and hyaline layer), and the pericarp. Apart from some specialty food products for human consumption, bran is generally used for animal feeding. There exists an important variability for the milling or semolina value character within grain varieties. The milling flour or semolina values depend on three main properties of the grains: endosperm friability, endosperm/envelopes ratio, and endosperm/bran separability. The latter property is governed both by the extent of endosperm/aleurone layer adhesion (in relation with yield) and

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the bran friability (in relation with purity).187 Recently, the debranning technology based on peeling or abrasion of the grain has attracted a renewed attention from technologists for the production of flours with lower microbial contamination and with higher incorporation of nutritionally beneficial compounds (fibers, micronutrients, phytochemicals) from the peripheral layers. Also, the possibility to crack the miller’s bran as a source of nutritional and functional ingredients has stimulated research on the bases of grain fractionability. AXs are the main cell wall components in cereal grains and as such play a determinant role in the mechanical properties of the grain tissues that in turn determine the fractionability behavior. AX amounts and structures vary in the different tissues of the grain (see Section 2.20.3.2) but keep the presence of a (1!4)-b-xylan main chain and phenolic ester side chains as a general structural feature. Total AXs and ferulic acid sharply increase in milling products for extraction rates above 60%. This is a reason why methods based on ferulic acid concentration or on its autofluorescence properties have been proposed to assess flour or semolina purity instead of the traditional ash content method.188 The complex distribution patterns of phenolic acids within grain tissues and substructures constitute a promising starting point for their use as biochemical markers. Thus, sinapic acid and diferulic acid were used as markers of aleurone layer and pericarp, respectively, in durum grains.189 A dehydrotrimer of ferulic acid was identified as specific marker of the outer pericarp in common wheat, and p-coumaric acid was also taken as a marker of aleurone layer cell walls.52 These markers allowed following the fate of the different parts of the grains upon fractionation processes. The natural extent of AX cross-linking by ferulate dehydrodimers in aleurone cell wall was found correlated to durum wheat bran extensibility and related also to semolina purity.190 In situ oxidation of durum bran or of its constitutive tissues induced by peroxidases/hydrogen peroxide systems or UV light resulted in the formation of covalent linkages involving phenolic moieties in cell walls or at tissue interfaces.118,191 These structural changes corresponded with dramatic modifications of the mechanical properties of the tissues: induced AX ester-linked ferulic acid dehydrodimers in aleurone layer cell walls and increased bran extensibility, whereas the establishment of linkages involving phenolic monomers and dimers resistant to mild saponification were related to increases of stress and strain to rupture, resulting in an increased friability during grinding, that is, a decrease in the size of generated bran particles.118,191 In common wheat, among the different bran tissues, it was shown that the higher the degree of AX cross-linking by ferulate dehydodimers, the lower the extensibility, that is, a decreasing extensiblity from aleurone layer through intermediate layer (including testa) up to outer pericarp. It must be kept in mind, however, that these different tissues have different microstructures and different cell wall polymer compositions (beta-glucan, cellulose, lignin). These results point out that grain tissue mechanical properties result in a complex manner not only from the polymer cross-linking but also from their relative abundance, individual properties, interactions and environment in the walls. Wheat hardness is an important milling factor that affects the energy required to reduce the endosperm kernel into a flour of suitable particle size.192 Although not totally elucidated, the hardness character seems to be related primarily to the quality of adhesion between starch granules and the endosperm protein matrix.193 However, the behavior of hard and soft wheat at the first break suggests that cell walls and hence AX could play a role in kernel hardness: fissures follow cell walls in hard varieties but propagate across the cells in soft varieties. Positive correlations have been obtained between AX content and wheat hardness.194,195 Differences in the endosperm/aleurone layer interface separation between hard and soft varieties have been observed at milling and resulted in differences in flour compositions.196 The subaleurone layer of soft varieties appeared more concentrated in soluble AX-like polysaccharides than hard wheat as revealed by FT-IR imaging.62 This could be related to the higher levels of contamination of soft wheat brans by residual endosperm and the difficulty to ‘finish’ (purify) these brans. Debranning significantly reduces the level of xylanase activities in flours (endogenous and/or microbial xylanases) but has much less effect on xylanase inhibitor levels in flours. As a result, flours obtained after debranning give frozen doughs that are much less sensitive to the ‘syruping phenomenon’, a release of water due to a slow hydrolysis of AX.197,198 AXs are clearly involved in the mechanical properties of grain tissues and therefore contribute undeniably to their fractionability. Further investigations will be necessary, however, to better understand their exact contributions to kernel and hull mechanical properties. In particular, emphasis should be put on the mechanisms of cohesion/adhesion at the levels of tissue and cellular interfaces. To this aim, the use and development of methods for the determination of local compositions/structures/properties will be necessary.

2.20.7.2 Bread Making Bread and pasta are the major food products made from wheat, with many recipes existing around the world. In Western countries, yeast-leavened and sourdough-developed breads are the most widely consumed. Although breads


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can be produced with flours of high extraction rates, including bran fractions, white (endosperm) wheat flour is generally the main ingredient of common breads. The bread-making process includes the essential steps of mixing, fermentation, and baking. Pentosans, the main nonstarch polysaccharides of wheat flours, have been reported to have a marked influence on dough and bread qualities. AXs constitute the major components of this so-called pentosan fraction that many older studies refer to. In the past, the use of ill-defined fractions in certain studies has made difficult the comparison of the observed results and has impeded the access to a synthetic view on the exact role of AX in bread making. Several reviews on AX with descriptions of their role in the bread-making process and the impact of added xylanases have been published.11,199,200 It appears that both WU-AX and WE-AX and the enzymes that modify them play an important role at different steps of the dough and bread processing. WU-AXs may be regarded as discrete particles with irregular edges resulting from cell wall disruption and breakdown at the milling stage and with high water-absorption capacity. Addition of WU-AXs leads to higher dough consistencies or increased dough water absorptions and mixing times, at constant consistency.167,201,202 A negative correlation has been observed between dough quality and native WU-AX ratio to total AX content. Both bread loaf volume and crumb texture are also negatively correlated to the amount of added WU-AXs.203 WU-AXs are thought to destabilize dough structure. They would play a role of physical barrier, limiting glutenin aggregation during gluten formation and development. They also compete with gluten for water that they retain punctually in swollen particles, which is an additional factor of dough foam destabilization. During fermentation and oven rise, WU-AXs intrude in gas cells and provoke their coalescence and gas release.204 Breads with coarser and stiffer crumbs are obtained together with lower loaf volumes. WE-AXs also increase dough consistencies or baking absorptions at given consistencies; they tend to enhance dough resistance to extension.167,202,205 The importance of WE-AX molecular weight has been noted, with high molecular weight fractions being the more active.206 The ratio of WE-AXs to total AXs in flours is positively correlated to dough characteristics.203 During fermentation, WE-AXs reduce the rate of carbon dioxide diffusion, thereby improving gas retention. WE-AXs were reported to increase dough foam stability because of their viscosity-forming properties in the dough aqueous phase and their ability to stabilize the film surrounding the gas cells.207 This stabilization of dough allows to prolonge the oven rise and results in breads with larger loaf volumes and thinner and softer crumbs when compared to doughs with low WE-AX contents or hydrolyzed WE-AX. The occurrence of WE-AX gelation during bread-making has been hypothesized, with development of a secondary network superimposed to gluten, playing a role in dough rheology.208 The extensive setting up of such a network has not been demonstrated in normal dough.209 The addition of oxidizing enzymes generating free radicals in the dough may result however in partial AX polymerization by diferulate cross-linking, which plays at least a role in enhancing dough liquid-phase viscosity. Interactive effects between added xylanases and oxidizing enzymes were observed on bread doughs.210,211 AXs interfere also with bread staling. Staling primarily results from amylopectin retrogradation, that is, a partial reorganization of starch into pseudo-crystal structures. This phenomenon is related to molecular mobility and hence much depends on water content and water distribution in breads. Although AXs tend to decrease the amount of available water due to their high water-binding capacity, in fact it has been observed that staling rate was increased in AX fortified breads.205,212,213 This seems to be related to the higher water content in these breads with corresponding higher starch mobility. Other results suggest that AXs lower retrogradation by sterically interfering with starch intermolecular associations.168 A full comprehension of the role of AX during bread aging, including mechanisms of water distribution, water losses, molecular mobility, and interactions, remains to be reached. Microbial endoxylanases are widely used as processing aids in bread making. Their effects depend on their concentration, specificity (preferences for specific structural features of AX chain), and substrate selectivity (preferences for WU-AX or for WE-AX).199,200 Endoxylanases that are beneficial in bread making (in general from family 11) preferentially attack WU-AX from which they solubilize high molecular weight AX. The positive effects are related to both increase in dough liquid-phase viscosity and degradation of WU-AX with concomitant reduction in water-holding capacity, allowing water to be redistributed toward gluten which gains in extensibility.203,214 When excessive levels of enzymes are added, the extensive release of water from hydrolyzed WU-AX and the depolymerization of WE-AX and solubilized AX led to slack and sticky dough. This effect can be counteracted by lower water addition at the mixing step. Endoxylanases that prefer WE-AX may be initially slightly positive but turn to negative as the fermentation proceeds, in relation to extensive hydrolysis of liquid-phase AX. Bread-making positive endoxylanases allow the production of breads with improved loaf volumes and crumb structure. Overdosages of these enzymes or uses of WEAX degrading endoxylanases may lead to underdeveloped or collapsed breads. Low endogenous endoxylanase activities exist in flours.215 The use of xylanase inhibitors has suggested that they could have a positive effect on bread loaf volume.216

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The sensitivity of endoxylanases toward xylanases inhibitors217,218 is susceptible to deeply impact their functionality.219 The levels of triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP) are generally high in flours and can vary appreciably depending on varieties.220 They are in concentrations that can reduce significantly the activity of sensitive added endoxylanases, and a mutant endoxylanase resistant to inhibition exhibits improved bread-making functionality.221 It has also been suggested that inhibitors can influence enzyme selectivity in a positive manner for bread-making, by selective adsorption to WU-AX which then favors WU-AX solubilization and preservation of WEAX/solubilized AX.222,223 AXs, in spite of their low concentrations in wheat flours, are major actors in a bread-making process together with the enzymes able to transform them and inhibitors able in turn to modify enzyme action. The effects of AXs, as they are described in literature dealing with bread-making, appear to be mostly indirect and result from viscosity-forming, gelling, water-holding, and mechanical properties. However, other types of mechanisms implying AX-bound ferulate esters and reactive groups in gluten cannot be excluded, in particular in the case of oxidoreductase additions. Such mechanisms would be similar to those described for gluten agglomeration.

2.20.7.3 Biscuits, Pasta, Beer Production 2.20.7.3.1 Biscuits In contrast with other cereal foodstuffs, biscuit recipes are characterized by a low water ratio and the presence of numerous ingredients, with flour, fat, sugar, and baking powder as the major ones. Flours from soft wheat cultivars are preferred for biscuit manufacture for their low level of starch damage and weaker gluten. Dough is generally mixed, laminated, cut up, then baked. One of the major processing quality attributes of biscuits is their dimensional stability, with retraction and excessive spreading being the two opposite main flaws.224 WU-AXs have been recognized for a long time as detrimental to normal biscuit spreading because of their high water-absorption capacity.225,226 The role of WE-AXs has been more disputed.195,225,227 The extensive degradation of AXs leads to excessive dough spreading but the controlled enzymatic solubilization of WU-AXs appears beneficial, provided that solubilized AXs were sufficiently depolymerized to avoid dough retraction. Therefore, the characteristics of endoxylanases used for biscuit-making should differ from those used for bread-making. Water absorption and retention and viscosity-forming effects appear as the main properties of arabinoxylans which impact biscuit processing. These properties related to water availability in the dough will strongly interfere with the level of gluten network development that is the primary determinant parameter of biscuit dough processability.224

2.20.7.3.2 Pasta Durum wheat semolina serves commonly as the starting material for pasta manufacture. Pasta processing includes the steps of mixing, extrusion, and drying. The hydration level at the mixing and extrusion steps is much lower than in a bread-making process. AXs are partly solubilized during pasta production, leading to higher levels of WE-AXs in the final product than in semolina.228--230 This solubilization seems to result more from the processing mechanical constraints (shear stress) than from endogenous enzymes. Experimental studies have shown that the addition of microbial endoxylanases has profound effects in pasta processing.230 Despite the low water level, endoxylanases convert efficiently WU-AX into WE-AX during the process, with a concomitant redistribution of WU-AX-bound water. As a consequence, extrusion pressure is decreased, or, reciprocally, a lower amount of water is needed with xylanase addition to reach the extrusion pressure of the control. However, the expected energy savings at the drying step were not observed in this case. Sometimes, the use of endoxylanases provoked checking problems on the endproduct. The main benefit of endoxylanase addition seems to lie rather in the increase of soluble dietary fiber content of pasta than in processing advantages. Solubilized AX fragments, even of low molecular weight, are particularly well retained within the pasta and hence not lost in the cooking water.231

2.20.7.3.3 Beer Owing to their lower concentration than b-glucans in barley grain cell walls (20% AX, 75% b-glucan in barley cell wall polysaccharides), AXs have received in the past minor attention in relation to malting/brewing industry. The developing use of cereals with higher AX content than barley for brewing, such as wheat and rye, together with the observation that AX concentration is generally higher than b-glucan concentration in beers produced from barley,232 have stimulated the research on the influence of AXs in brewing. The enzymes that degrade AXs are produced at late stages in the germination process.233,234 Therefore, appreciable amounts of AXs survive the malting step better than b-glucans do.235 It appears that several problems formerly attributed to the presence of b-glucans (reduced filtration efficiency, haze formation) could in fact result from the presence of AXs.87 Both WU-AXs


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(as substrate for the release of WE-AX) and WE-AXs are detrimental in brewing technology. These effects could be corrected for by addition of exogenous microbial xylanases. However, brewers are not very favorable to the use of additives in the process, in relation with possible consumer concerns. They would prefer the development of adapted varieties for brewing, with low levels of soluble arabinoxylans or high levels of endogenous endoxylanases. Another reason to avoid the use of added xylanases is the role that arabinoxylans play in the quality of the final product. AXs were reported indeed to determine in part beer texture and mouth feel and also foam stability, which represents a crucial attribute for consumer’s satisfaction.236 On the other hand, unwanted flavors may develop, in particular in beers made with wheat, from the thermal or enzymatic conversion of ferulic acid.237 The main by-product of brewing is called the brewer’s spent grains (BSG; 20kg/100 l beer produced). AXs, mostly WU-AXs, represent more than 20% of BSG weight. Due to their high nutritional value (protein, minerals, vitamins, fibers, etc.) and low cost, BSG could be used in human or animal nutrition. Other applications have been suggested, such as energy production, charcoal production, brick component, paper manufacture, absorbent, and substrate for biotechnological processes.238

2.20.7.4 Starch and Gluten Production Wheat is being increasingly employed as a source of gluten and starch, which are used as-is or as derivatives for food and nonfood applications. The processes of starch/gluten separation are numerous and start from wheat grains or wheat flours. The processes starting from grains follow generally the steps of steeping, milling of wet grains, then separation of the different fractions including prime starch, gluten, and fibers. The processes starting from flour are classified on the basis of degree of gluten protein agglomeration as dough, dough-batter, and batter methods.239 Whereas dough, dough-batter, and batter systems are of widespread use all over the world, in Europe, processes based on diluted batter limiting gluten protein agglomeration before prime starch separation are generally preferred. Starch is recovered by dough lixiviation and centrifugation or decantation from batters. Nonstarch polysaccharides and in particular AXs are detrimental to protein agglomeration in starch/gluten separation.166,240--247 The high water-binding capacity of AXs makes them competitors of the proteins for gluten formation when a limited amount of water is available during a dough-making step. Both WU-AXs and WE-AXs were reported to play a negative role in gluten/starch separation. WUSs, whose major part is WU-AXs, exist as discrete particles which can act as a physical barrier limiting contacts and interactions between protein particles.166,243--247 WE-AXs or water-extractable pentosans (WEPs) have been reported to exert their negative effects through three different mechanisms:246,247 (1) viscosity, (2) depletion/attraction, and (3) ferulic acid-mediated reactions. The high viscosity related to the concentration and molecular weight of AX is a nonspecific effect that limits protein agglomeration rate and gluten yield. Similar effects can be obtained by addition of other viscous hydrocolloids. A partial crosslinking of AX through oxidative gelation will amplify this phenomenon. This effect can be corrected for by dilution (water addition) or by addition of xylanase that degrades the viscosity-forming capacity of WE-AX. A striking effect of WE-AX and WUS is to impair gluten agglomeration while favoring the formation of larger glutenin macropolymer (GMP) insoluble in sodium dodecylsulfate solutions. A depletion/attraction mechanism would account for this phenomenon.246 AX macromolecules would be excluded from the shell of glutenin particles having a larger size (by 1 order of magnitude), forming an AX-free region, resulting in an uncompensated osmotic AX pressure pushing the gluten particles together.166,245--248 Again, the degradation of AX with xylanases can correct this effect. A third effect was suggested, qualified of ‘direct effect’ and related to the ferulic acid bound to AX. On the one hand, cross-linking of WE-AX could occur and reinforce the viscosity (indirect) effect. On the other hand, direct linkages between ferulate groups of AX and gluten proteins could form, modifying the aggregation capacity of particles and decreasing gluten extensibility. This effect, which could also occur with WU-AX or AX solubilized from WU-AX, could not be corrected by dilution but by free ferulic acid addition which should thus compete with AX-bound ferulic acid. However, the latter effect is still being discussed. Although tyrosine-rich peptides/ferulic acid adducts and AX/ casein complexes have been obtained in vitro in selected enzyme and substrate conditions,183,184 covalent AX--gluten complexes failed to be detected in overmixed dough or WE-AX/gluten/starch mixtures.249 Alternative explanations have been proposed on the role of soluble bound ferulic acid where it acts as a redox compound able to shift thiol/ disulfide interchanges between gluten proteins.250 In this respect, the redox status of the medium, the production of free radicals by homolytic disruptions of glutenin disulfide bonds during mixing, the reactivity of the different forms of ferulate (free, soluble bound, insoluble bound) and other flour phenolics and the presence of oxidizing enzymatic activities acting on ferulate, tyrosine, or thiol would be determinant factors. Further work is clearly needed to fully understand the role of phenolics in dough and gluten formation and properties. Gluten yields, protein recovery, and gluten protein agglomeration index of wheat varieties with low processing quality can be increased using xylanase.251,252 The use of xylanase with different selectivities toward WU-AX and

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WE-AX allowed to demonstrate the negative impact of both of them.242,244 The enzyme that degrades WE-AX but had very low effect on WU-AX improved gluten protein agglomeration. On the other hand, moderate dosages of the enzyme that had high selectivity toward WU-AX increased strongly batter viscosity in liberating high-Mw AX from WU-AX and had a clear negative effect on the agglomeration process. Finally, high dosages of the latter enzyme further depolymerized AX in the batter solution, counteracting the viscosity effect. An additional positive effect could result from the partial degradation of WU-AX particles by this enzyme. The amount of water retained in recovered gluten depends to a large extent on AX content.253 Therefore the residual AX concentration in gluten will determine not only the elastic properties but also the energy required for drying.

2.20.8 AX in Human Nutrition and Health 2.20.8.1 AX as Dietary Fiber Cereal grains represent a main component of our diet. It is a natural source of dietary fiber. They comprise both insoluble and soluble fibers, the proportion of which will depend on the cereal, cultivar, origin, and processing. To date, most attention has been paid to wheat, barley, rye, and oat.

2.20.8.1.1 Dietary fiber in the small intestine In the small intestine, the effects of dietary fibers are predominantly due to their intrinsic properties that unbalance nutrient absorption in the intestinal lumen. Several mechanisms that can be cumulated could be involved, including the ability of soluble fiber to delay gastric emptying, to impair peristaltic mixing process, to retard transport of digestive enzymes to their substrates, and to delay the diffusion of nutrient across the unstirred layer. The properties of some fibers able to bind or trap bile acids may have an important role in the interference with the absorption of dietary cholesterol and lipids or possibly may alter their rate and site of absorption.254--256 A part form viscous soluble fiber, nonfractionated food with intact cell walls, preserved during cooking and chewing, can protect starch/lipid from digestion until physical action of stomach or even microbial activity in the large intestine. In cereal, the soluble fiber effect has been mainly reported for b-glucans (particularly in oat bran; see review by Ma¨lkki and Virtanen).257 However, AX soluble fibers may have the potential to reduce glucose and lipid absorption. It will depend on their molecular weight and concentration (in some way related to the amount ingested), two parameters that influence viscosity. AX-rich fibers extracted as by-product from wheat flour processing with a ratio of soluble to insoluble fiber equal to 1.6 were shown to be effective in improving metabolic control in people with type 2 diabetes when ingested at a level of 15g/day.258 No effect was observed on plasma lipid concentration but the subjects were normolipidemic and had a habitual diet relatively low in total and saturated fat. In another study with AX fiber derived from maize, ingested at a level of 10g/day for 6 months, the effect was less obvious and depended on the status of the subject.259 AX fiber was efficient to decline HbA1c levels in obese subjects with impaired glucose tolerance but not in nonobese ones. In rats fed a diet containing cholesterol, soluble AX extracted from maize bran was found to reduce the cholesterol absorption.260 They were effective in lowering plasma cholesterol and triglyceride-rich lipoprotein. Soluble AXs decreased the accumulation of cholesterol in the liver. Among cereals, rye has a higher content of soluble AXs. In pigs, diets based on rye breads (DF; 230--235g/kg dry matter), although they introduced a significantly higher luminal viscosity in the small intestine than did the wheat diets, have no influence on starch digestibility and/or the plasma concentration of glucose and insulin.261 For Finnish women with elevated serum cholesterol, the consumption of rye bread was found to decrease serum total and LDL cholesterol. An alternative way by which cereal fiber can ameliorate or prevent the development of atherosclerosis is the antioxidant activity of phenolic compounds. Feruloylated oligosaccharides prepared from wheat flour AX showed potent antioxidant activity and inhibit LDL oxidation.262 Antioxidant property in the LDL oxidation system was also demonstrated for chemically hydrolyzed corn bran cell wall fragments.263

2.20.8.1.2 Dietary fiber in the large intestine In the large intestine, nondigestible carbohydrates, which to a large extent consist of dietary fibers, are a major source of energy for bacteria inhabiting the colon.264 They are fermented to short-chain fatty acids, mainly acetic, propionic, and n-butyric, and gases. Short-chain fatty acids can have effects on colonic mucosa and colonic function as well as postabsorptive action on the liver and other tissues. The availability of fermentable carbohydrate in the large intestine


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can have a positive effect of health colon through decreasing pH and diluting the colonic content of potentially carcinogenic substances. Insoluble fiber such as in cereal bran has direct in the colon by increasing fecal bulk, decreasing transit time, and binding bile acids and carcinogens. In vitro studies using human/pig fecal inoculates and in vivo studies in rats or pigs agree with the fact that digestibility of outer bran AXs is low whereas endosperm AXs are extensively degraded.261,265--271 In pigs, starchy endosperm AXs are degraded more proximally than aleurone AXs, whose degradation continues between cecum and proximal colon.267 The A/X ratio of aleurone and endosperm AXs increases on the course of fermentation, suggesting a faster fermentation of AXs of low branching degree.269,271 Soluble AXs, native or alkali extracted, are fermented more rapidly than their unsoluble counterpart found in cell walls,33 and ferulic acid cross-linking reduces the rate of AX fermentation.272 It should be stressed that degradability of AX is not determined solely by their structure. Other factors such as physical entrapment or cross-linking to lignin, for example, are also believed to be important. The genus Bacteroides has been shown to be particularly active in AX metabolism.273--275 AXs are fermented to shortchain fatty acids, acetic, propionic, n-butyric, and gases. Acetate is produced in the highest amount, but propionate and butyrate proportions seem to be slightly above average compared to other DF sources.269,271,272,276 Of the short chain fatty acid, butyrate has been attracting the most attention; it is the principal source of energy for colonic mucosa and exhibits chemopreventive activity.277,278 A range of probiotic bacteria were examined for their ability to ferment AXs, and derived oligosaccharides.273,275,279,280 Bifidobacteria species are able to grow well using AX-derived oligosaccharides and xylo-oligosaccharides as the sole source of carbon. Feruloyl oligosaccharides are also well used, suggesting that ferulic acid moiety does not inhibit bifidobacteria growth.279 In contrast, growth on unsubstituted xylans is rare among bifidobacteria.275,281 However, further studies will be needed to evaluate whether arabinoxylo-oligosaccharides selectively stimulate the growth and activity of some beneficial bacteria in the colon.

2.20.8.2 Antitumoral Properties Only a few heteroxylans occur among the numerous polysaccharides exhibiting immunomodulatory and antitumor activities in various biological tests. Glucuronic acid-containing (acidic) xylans isolated from annual plant residues such as bamboo leaves, corn stalks, corn cobs, and wheat straw have been reported to inhibit markedly the growth of sarcoma-180 and other tumors, probably due to the indirect stimulation of the nonspecific immunological host defense.282--284 However, the structural principle responsible for the biological response of the different acidic xylan types remains unknown.284 Ghoneum reported that modified AX from rice bran, of about 3--5kDa molecular weight, enhances anti-HIV activity.285 Furthermore, it was shown that the AX from rice bran has a potent ability to activate human NK cell function in vivo and in vitro.286 AX extracted by alkaline treatment from corn husk and partially hydrolyzed with endoxylanase has been shown to have significant immunopotentiating activity after oral administration in mice.287 This suggests that besides their role as dietary fiber, some AXs or AX fractions derived from cereal grains, and especially from rice and maize bran, might have other potential benefits for human health.

2.20.9 Conclusion Cereals are the core of human nutrition and their incorporation into a wide range of products is of great economic importance. The major components of the grain are starch (60--70% of grain, 70--80% of flour) and protein (10-15%), with nonstarch polysaccharides derived from the cell walls only accounting for about 3--8% of the total. Nevertheless, these components have major effects on grain processing, due to the their viscosity and hydration properties, and on nutrition, due to their role as dietary fiber. AXs are the major polymers of grain cell walls and their structures show large differences according to tissue but also to cereal species. Wheat, rye, and barley are very largely used in human food (bread, beer, biscuits, etc.) and their AXs have been extensively examined. Although rice and maize are also largely used in human foods (tortillas, popcorn, noodles, etc.), very little work has been dedicated to the possible role of AXs or cell walls in their processing. Obviously, these differences lie in the lower content of AXs in the endosperm of rice and maize grain compared to wheat, barley, and rye, and to the lack of WE-AX fractions that induce high viscosity in solution. As a matter of fact, the impact of WE-AX on the viscosity of aqueous solution is without any doubt one of the major factors of AX action in different uses of cereal grain, as it modifies the rheology of solutions and of complex system (dough, intestinal tract, etc.). Besides, the hydration properties of WU-AX can affect the water distribution and have important impact on food system. The role of WE-AX and WU-AX as individual components in different food systems

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and processes is better understood nowadays, which allows, for example, to better control the use of enzymes to rebalance the ratio between WE-AX and WU-AX in bread-making. The structural diversity of AXs has been very extensively studied with the aim to relate the structure to specific functional properties. Obviously, major differences occur in the structure of AXs according to their tissue of origin, especially in the outer part of the grain. The endosperm also exhibits relatively large variation in the amount and the structure of AXs, not only within the tissue but also between different varieties. However, the relation between these structural variation and the so-called ‘functional properties’ of AXs is not straightforward, especially for the level of substitution by arabinose side chains. The presence of ferulic acid is better related to the formation of firmer gels upon oxidative conditions and to the modification of mechanical properties of outer layers. The biological significance of this structural diversity is not better understood. AXs and cell walls are involved in water transport or diffusion during the different physiological states of the grain: development, desiccation, germination. One might speculate that structural variation might be involved in the modulation of the hydration properties of the cells walls that might affect the water content of the grain. Similarly, arabinose substitution might play a role in controlling possible interactions with other cell wall components, but up to now the mechanisms of xylan aggregation are poorly understood and need further study. The structural diversity of AXs observed in cereal endosperm among wheat, rye, and barley varieties, although not fully understood or explained in terms of functional properties, indicates that plant breeding could be used to produce new types of wheat with potentially improved nutritional quality and processing properties. The elucidation of the mechanisms that control their synthesis in the endosperm is a challenge for the future that will need to better understand the deposition of AXs during grain development, the local diversity of AXs at the endosperm level, the identification of main enzymes and genes involved in the biosynthesis mechanisms. The development of a new screening method based on enzymatic fingerprinting64 and IR and Raman microspectroscopy,61,62,288 together with genetic and molecular biology tools, should allow significant progress in this area. The use of endoxylanases in many fields of cereal usage has become essential, not only because they have a positive impact on processing, but also because they improve the quality of the final product. Up to now, relatively little research has focused on the interaction between the enzyme and its substrate, that, owing to the structural diversity of AX in cereal grains, is very important for not only the development of new enzymes (endoxylanases and other enzymes modifying AX) for food applications but also other use of the grain such as production of bio-ethanol. Both approaches based on control of biosynthesis and uses of exogenous enzymes will probably open new opportunities of product development and quality enhancement for cereal grains in the future.

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Charun, E.; Morel, M. H. Industries des Ce´reáles 2001, 125, 2--16. 225. Yamazaki, W. T.; Donelson, J. R.; Kwalek, W. F. Cereal Chem. 1977, 54, 352--360. 226. Abboud, A. M.; Hoseney, R. C.; Rubenthaler, G. L. Cereal Chem. 1985, 62, 130--133. 227. Kaldy, M. S.; Rubenthaler, G. I.; Kereliuk, G. R.; Berhow, M. A.; Vandercook, C. E. Cereal Chem. 1991, 68, 508--512. 228. Neukom, H.; Kuendig, W.; Deuel, H. Cereal Sci. Today 1962, 7, 112--125. 229. Lintas, C.; D’Appolonia, B. L. Cereal Chem. 1973, 50, 563--570. 230. Ingelbrecht, J. A.; Verwimp, T.; Delcour, J. A. J. Agric. Food Chem. 2000, 48, 2017--2022. 231. Ingelbrecht, J. A.; Verwimp, T.; Grobet, P. J.; Delcour, J. A. J. Agric. Food Chem. 2001, 49, 1783--1789. 232. Schwarz, P. B.; Han, J. Y. J. Am. Soc. Brew. Chem. 1995, 53, 157--159. 233. Banik, M.; Garrett, T. P. J.; Fincher, G. B. Plant Mol. Biol. 1996, 31, 1163--1172. 234. Sungurtas, J.; Swanston, J. S.; Davies, H. V.; McDougall, G. J. J. Cereal Sci. 2004, 39, 273--281. 235. Coote, N.; Kirsop, B. H. J. Inst. Brew. 1976, 82, 34--44. 236. Evans, D. E.; Sheehan, M. C. J. Am. Soc. Brew. Chem. 2002, 60, 47--57. 237. Coghe, S.; Benoot, K.; Delvaux, F.; Vanderhaegen, B.; Devaux, F. R. J. Agric. Food Chem. 2004, 52, 602--608. 238. Mussato, S. I.; Dragone, G.; Roberto, I. C. J. Cereal Sci. 2005, 43, 1--14. 239. Van Der Borght, A.; Goesaert, H.; Veraverbeke, W. S.; Delcour, J. A. J. Cereal Sci. 2005, 41, 221--237. 240. Roels, S. P.; Cleemput, G.; Vandewalle, X.; Nys, M.; Delcour, J. A. Cereal Chem. 1993, 70, 318--323. 241. Christophersen, C.; Andersen, E.; Jakobsen, T. S.; Wagner, P. Starch-Starke 1997, 49, 5--12. 242. Frederix, S. A.; Courtin, C. M.; Delcour, J. A. J. Agric. Food Chem. 2003, 51, 7338--7345. 243. Frederix, S. A.; Courtin, C. M.; Delcour, J. A. J. Cereal Sci. 2004, 40, 41--49. 244. Frederix, S. A.; Van Hoeymissen, K. E.; Courtin, C. M.; Delcour, J. A. J. Agric. Food Chem. 2004, 52, 7950--7956. 245. Wang, M.; Hamer, R. J.; van Vliet, T.; Oudgenoeg, G. J. Cereal Sci. 2002, 36, 25--37. 246. Wang, M.; Van Vliet, T.; Hamer, R. J. J. Cereal Sci. 2004, 39, 395--402. 247. Wang, M.; Van Vliet, T.; Hamer, R. J. J. Cereal Sci. 2004, 39, 341--349. 248. Wang, M. W.; van Vliet, T.; Hamer, R. J. J. Cereal Sci. 2005, 41, 251--258. 249. Labat, E.; Rouau, X.; Morel, M.-H. Lebensmittel-Wissenschaft und-Technologie 2002, 35, 185--189. 250. Labat, E.; Morel, M. H.; Rouau, X. Cereal Chem. 2000, 77, 823--828. 251. Hamer, R. J.; Weegels, P. L.; Marseille, J. P.; Kelfkens, M. Wheat Is Unique: Structure, Composition, Processing, End-Use Properties and Products; Pomeranz, Y., Ed.; AACC: St Paul, MN; 1989, pp 467--477. 252. Weegels, P. L.; Marseille, J. P.; Hamer, R. J. Starch-Starke 1992, 44, 44--48. 253. Roels, S. P.; Grobet, P. J.; Delcour, J. A. J. Agric. Food Chem. 1998, 46, 1334--1343. 254. Mathers, J. C.; Daly, M. E. Advanced Dietary Fibre Technology; McCleary, B. V., Prosky, L., Eds.; Blackwell: Oxford, UK, 2001; pp 186--195. 255. Kahlon, T. S. Advanced Dietary Fibre Technology; McCleary, B. V., Prosky, L., Eds.; Blackwell: Oxford, UK, 2001; pp 206--220. 256. Lairon, D. Advanced Dietary Fibre Technology; McCleary, B. V., Prosky, L., Eds.; Blackwell: Oxford, UK, 2001; pp 178--185. 257. Ma¨lkki, Y.; Virtanen, E. Lebensmittel-Wissenschaft und-Technologie 2001, 34, 337--347. 258. Lu, Z. X.; Walker, K. Z.; Muir, J. G.; O’Dea, K. Eur. J. Clin. Nutr. 2004, 58, 621--628. 259. Hanai, H.; Ikuma, M.; Sato, Y.; Iida, T.; Hosoda, Y.; Matsushita, I.; Nogaki, A.; Yamada, M.; Kaneko, E. Biosci. Biotechnol. Biochem. 1997, 61, 1358--1361. 260. Lopez, H. W.; Levrat, M. A.; Guy, C.; Messager, A.; Demigne, C.; Remesy, C. J. Nutr. Biochem. 1999, 10, 500--509. 261. Knudsen, K. E. B.; Serena, A.; Kjaer, A. K. B.; Jorgensen, H.; Engberg, R. J. Nutr. 2005, 135, 1696--1704. 262. Katapodis, P.; Vardakou, M.; Kalogeris, E.; Kekos, D.; Macris, B. J.; Christakopoulos, P. Eur. J. Nutr. 2003, 42, 55--60. 263. Ohta, T.; Nakano, T.; Egashira, Y.; Sanada, H. Biosci. Biotechnol. Biochem. 1997, 61, 1942--1943. 264. Rowland, I. Advanced Dietary Fibre Technology; McCleary, B. V., Prosky, L., Eds.; Blackwell: Oxford, UK, 2001; pp 226--231. 265. Stevens, B. J. H.; Selvendran, R. R.; Bayliss, C. E.; Turner, R. J. J. Sci. Food Agric. 1988, 44, 151--166. 266. Glitso, L. V.; Brunsgaard, G.; Hojsgaard, S.; Sandstrom, B.; Knudsen, K. E. B. Br. J. Nutr. 1998, 80, 457--468. 267. Glitso, L. V.; Gruppen, H.; Schols, H. A.; Hojsgaard, S.; Sandstrom, B.; Knudsen, K. E. B. J. Sci. Food Agric. 1999, 79, 961--969. 268. Glitso, L. V.; Jensen, B. B.; Knudsen, K. E. B. J. Sci. Food Agric. 2000, 80, 1211--1218. 269. Amrein, T. M.; Granicher, P.; Arrigoni, E.; Amado, R. Lebensmittel-Wissenschaft und-Technologie 2003, 36, 451--460. 270. Harris, P. J.; Chavan, R. R.; Ferguson, L. R. Mol. Nutr. Food Res. 2005, 49, 536--545. 271. Salvador, V.; Cherbut, C.; Barry, J.-L.; Bertrand, D.; Bonnet, C.; Delort-Laval, J. Br. J. Nutr. 1993, 70, 189--197. 272. Hopkins, M. J.; Englyst, H. N.; Macfarlane, S.; Furrie, E.; Macfarlane, G. T.; McBain, A. J. Appl. Environ. Microbiol. 2003, 69, 6354--6360. 273. Van Laere, K. M. J.; Hartemink, R.; Bosveld, M.; Schols, H. A.; Voragen, A. G. J. J. Agric. Food Chem. 2000, 48, 1644--1652. 274. Saylers, A. A.; Gherardini, F.; O’Brien, M. Appl. Environ. Microbiol. 1981, 41, 1065--1068. 275. Crittenden, R.; Karppinen, S.; Ojanen, S.; Tenkanen, M.; Fagerstrom, R.; Matto, J.; Saarela, M.; Mattila-Sandholm, T.; Poutanen, K. J. Sci. Food Agric. 2002, 82, 781--789. 276. Grasten, S.; Liukkonen, K. H.; Chrevatidis, A.; El-Nezami, H.; Poutanen, K.; Mykkanen, H. Nutr. Res. 2003, 23, 1503--1514. 277. McIntosh, G. H.; Royle, P.; Pointing, G. J. Nutr. 2001, 131, 127--131. 278. Young, G. P.; Hu, Y.; Le Leu, R. K.; Nyskohus, L. Mol. Nutr. Food Res. 2005, 49, 571--584.


279. 280. 281. 282. 283. 284. 285. 286. 287. 288.

Yuan, X.; Wang, J.; Yao, H. Food Chem. 2005, 90, 759--764. Laere, K. M. J. V.; Beldman, G.; Voragen, A. G. J. Appl. Microbiol. Biotechnol. 1997, 47, 231--235. Hespell, R. B.; Whitehead, T. R. J. Dairy Sci. 1990, 73, 3013--3022. Whistler, R. L.; Bushway, A. A.; Singh, P. P. Adv. Carbohydr. Chem. Biochem. 1976, 32, 235--257. Ebringerova, A.; Hromadkova, Z.; Alfodi, J.; Hribalova, V. Carbohydr. Polym. 1998, 37, 231--239. Ebringerova, A.; Kardosova, A.; Hromadkova, Z.; Malovikova, A.; Hribalova, V. Int. J. Biol. Macromol. 2002, 30, 1--6. Ghoneum, M. Biochem. Biophys. Res. Commun. 1998, 243, 25--29. Ghoneum, M.; Abedi, S. J. Pharm. Pharmacol. 2004, 56, 1581--1588. Ogawa, K.; Takeuchi, M.; Nakamura, N. Biosci. Biotechnol. Biochem. 2005, 69, 19--25. Mills, E. N. C.; Parker, M. L.; Wellner, N.; Toole, G.; Feeney, K.; Shewry, P. R. J. Cereal Sci. 2005, 41, 193--201.

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Biographical Sketch

Dr. Luc Saulnier was born in 1959. He received his Ph.D. degree in plant physiology in 1987 from Paul Sabatier University (Toulouse, France). He is is research scientist at INRA since 1988 and is currently working at the Research Unit Biopolymers, Interactions Assemblies (Nantes, France). His research activity is mainly focused on the structure and functional properties of cell wall polysaccharides from plant and especially cereal grains. He is author of about 70 original peer-reviewed international publications and 2 patent applications.

Dr. Fabienne Guillon was born in 1961. She received her Ph.D. degree in Food Science in 1987 from the University of Nantes (France). She is research scientist at INRA since 1989 and is currently working at the Research Unit Biopolymers, Interactions, Assemblies (Nantes, France). She has several years of experience in the area of dietary fiber and gastrointestinal functions. Her research is now focused on structure and composition of plant cell walls and how these different architectures might influence the end-use properties of plant-derived food. She is author of about 40 original peer-reviewed international publications.


Dr. Pierre-Etienne Sado was born in 1974. He received his Ph.D. in cellular biology in 2003 working on secondary cell wall formation in the John Innes Centre (Norwich, UK). He is author of 4 original peerreviewed international publications and has several years of experience in forward and reverse genetics in plant research. He holds a postdoctoral position at the Research Unit Biopolymers, Interactions Assemblies (Nantes, France), and is currently working on a biointegrative project on wheat grain cell wall enzymes.

689

Dr. Xavier Rouau was born in 1956. He received his Ph.D. degree in food science in 1985 from Paris VII University. Xavier Rouau is research scientist at INRA since 1985. He is currently Research Director at the Joint Research Unit on Agropolymer Engineering and Emerging Technologies (Montpellier, France), which focuses on cereal chemistry and technology. He has 20 years experience in the field of plant and food chemistry and technology and is author of 70 original peer-reviewed international publications.

2.21 Seaweed Polysaccharides M. Rinaudo, Centre de Recherches sur les Macromolećules Ve´ge´tales, CNRS, Grenoble, France ß 2007 Elsevier Ltd. All rights reserved. 2.21.1 Introduction

692

2.21.1.1 Generalities on Seaweeds

692

2.21.1.2 Classification and Production

692

2.21.1.3 Algae Composition

694

2.21.1.4 Uses of Algae

694

2.21.1.5 Polysaccharides of Industrial Importance

695

2.21.2 Polysaccharides from Brown Seaweeds

695

2.21.2.1 Alginates

695

2.21.2.1.1 2.21.2.1.2 2.21.2.1.3 2.21.2.1.4 2.21.2.1.5 2.21.2.1.6 2.21.2.1.7 2.21.2.1.8 2.21.2.1.9 2.21.2.1.10

Extraction of alginates Molecular structure Methods for characterization Stiffness of alginates Mechanism of gelation in relation with the chemical structure Physical properties in solution Polyelectrolyte complex formation Ion-exchange properties Chemical modifications Uses of alginate

2.21.2.2 Fucans/Fucoidans 2.21.2.2.1 2.21.2.2.2 2.21.2.2.3

696 697 699 700 702 703 704 704 706 706

707

Extraction of fucoidans Molecular structure Uses of fucoidans

708 708 709

2.21.2.3 Food Reserve in Brown Algae: Laminaran

709

2.21.2.3.1 2.21.2.3.2

Molecular structure Uses of laminaran

709 710

2.21.3 Polysaccharides from Red Seaweeds

710

2.21.3.1 Agar/Agarose

710

2.21.3.1.1 2.21.3.1.2 2.21.3.1.3 2.21.3.1.4 2.21.3.1.5 2.21.3.1.6 2.21.3.1.7

Extraction of agar Molecular structure Characterization and properties Conformation and mechanism of gelation Polymeric complexes Chemical modification Uses of agar

2.21.3.2 Sulfated Polysaccharides: Carrageenans 2.21.3.2.1 2.21.3.2.2 2.21.3.2.3 2.21.3.2.4 2.21.3.2.5 2.21.3.2.6 2.21.3.2.7 2.21.3.2.8

711 712 712 713 716 716 717

718

Extraction of carrageenans Molecular structure Methods of characterization Conformation of k-carrageenan and ionic selectivity Properties in solution Mechansim of gelation Polymer complexes Uses of carrageenans

718 718 719 720 722 723 725 726

2.21.3.3 Other Sulfated Polysaccharides from Red Seaweeds

727

2.21.3.3.1 2.21.3.3.2 2.21.3.3.3

Hypneans Porphyran Furcellaran

727 727 728

691

692

Seaweed Polysaccharides

2.21.3.3.4 2.21.3.3.5 2.21.3.3.6

2.21.4

Funoran Dulsan Iridophycan

Conclusion

728 730 730

730

2.21.1 Introduction Polysaccharides are now recognized as valuable polymers for industrial applications and for their biological properties. They are renewable materials with a very large variety of chemical structure and as a consequence a variety of physical and biological properties. Polysaccharides have some general characteristics which are important: they are rich in –OH groups that make them hydrophilic; they are often water soluble; this allows one to establish an intra H-bond network implying the local stiffness of the molecules; from this rigidity, they get a high thickener character. The regularity of the structure also favors cooperative interactions with counterions or interchain H-bonds involved in physical gelation. In general, plants consist of a large source of polymeric materials; among plants, the seaweeds are interesting to investigate. This chapter presents the different seaweeds usually exploited over the world and describes the structure and properties of the main polysaccharides extracted from algae.

2.21.1.1 Generalities on Seaweeds Phycologists consider seaweed as any of a large number of marine benthic algae that are multicellular and macrothallic (large body). Seaweeds are marine algae included in the general category of ‘plants’. Most of them are green (1200 species), brown (2000 species), or red (6000 species). They are very important ecologically: they dominated the rocky intertidal in most oceans, and in temperate and polar regions dominate rocky surfaces in the shallow subtidal. Some are found to depths of 250m in particularly clear waters.1a,1b Algae, which are the sources of many important polysaccharides from the point of view of applications, are among the oldest known living organisms.2 Morphologically, they are very primitive and the evolution was very slow over the ages. The blue-green algae (Cyanophyta) appeared first, then, the red algae (Rhodophyta), the green algae (Chlorophyta), and finally the brown algae (Phaeophyta).

2.21.1.2 Classification and Production Different classifications were proposed for seaweeds, but the most relevant follows the biochemical character based on pigmentation and food reserve.3 A classification of the different algae is given in Table 1, in which the most important species used for extraction of the commercially valuable polysaccharides and studied in this chapter are indicated in bold. In Table 2, the main types of algae with their morphology, food reserve, and other producing polysaccharides are given.2 Food reserve components are mainly floridean glycogen, starch, and laminaran; cell walls and envelopes have cellulose and other usual glycans as found in plants; especially from blue-green algae, a slimy gel develops as a capsule around unicellular forms or as intercellular substance in multicellular forms which has been described as pectin-like. In red algae (Rhodophyta), the glycans mentioned are glycogen-like food, a primitive form of cellulose, mannans, and xylans looking like hemicelluloses in terrestrial plants, and also sulfated glycans together with a variety of O-methylated sugar residues from which carrageenans and agars are described in this chapter. They are pigmented with chlorophyll and various proteins called phycobilins giving the color. In brown algae (Phaephyta), the color comes from the dominance of the xanthophyll pigment fucoxanthin, which masks the other pigments chlorophyll a and c, b-carotene, and other xanthophylls. The glycans are cellulose, laminaran, lichenan, fucoidan, sulfated hexouronoxylofucans, and alginates; laminaran, fucoidan, and alginates are described in here. Concerning the structure, there is also evidence that many algal glycans in their native state exist, at least partly as proteoglycans; an example mentioned should be fucoidan extracted from Fucales. The seaweeds were for a long time first used for food in Eastern countries: they can be traced back to the fourth century in Japan and the sixth century in China. These two countries and Korea are the largest consumers of seaweeds: the harvest is 6106 ton of wet seaweeds per year; with the demand increasing continuously, the development of


693

Table 1 Classification of algae Division Cyanophyta (blue-green algae) Order Chroococcales Order Nostocales Order Stigonematales Division Rhodophyta (red algae) Class Bangiophyceae Order Bangiales Family Bangiaceae: Porphyra Class Florideophyceae Order Nemalionales Order Bonnemaisoniales Order Gelidiales Family Gelidiaceae: Acanthopeltis, Gelidium, Gelidiella, Pterocladia Order Cryptonemiales Family Endocladiaceae Order Gigartinales Family Gracilariaceae: Gracilaria, Family Furcellariaceae: Furcellaria, Halachnion Family Solieriaceae: Euchema Family Hypneaceae: Hypnea Family Gigartinaceae: Chondrus, Girgatina, Iridea, (¼Iridophycus), Rhodoglossum Family Phyllophoraceae: Phyllophora Order Rhodymeniales Order Ceramiales Family Rhodomelaceae: Laurencia, Polysiphonia Division Cryptophyta Division Pyrrophyta (predominantly dinoflagellates) Division Bacillariophyta (diatoms) Division Phaephyta (brown algae) Order Ectocarpales: Pylaiella Order Sphacelariales: Sphacelaria bipinnata Order Dictyotales: Dictyota dichoma Order Chordariales: Chordaria flagelliformis Order Desmarestiales: Desmarestia aculeata Order Dictyosiphonales: Dictyosiphon lomentaria Order Laminariales Family Chordaceae Family Laminariaceae: Costaria, Laminaria Family Lessoniaceae: Macrocystis, Nereocystis, Pelagophycus Family Alariaceae: Ecklonia, Undaria Order Fucales Family Notheiaceae Family Fucaceae: Ascophyllum, Fucus, Pelvetia, Durvillaea Family Himanthaliaceae Family Cystoseiraceae Family Sargassaceae: Sargassum, Coccophora Division Chrysophyta Division Xanthophyta Division Euglenophyta Division Chlorophyta (green algae)

aquacultures represents now more than 90% of the market demand. In Asia, cultivation of Porphyra (red algae) and Laminaria and Undaria (brown algae) has become a major industry, especially in China, Korea, and Japan.4 China is the first producer (5106 tonyr–1) of seaweeds (more than 50% of the world production) growing on suspended ropes in the ocean; they produce mainly Laminaria japonica (brown seaweed); Korea produces 8105 tonyr–1 from Undaria pinnatifida (brown seaweed); Japan produces 6105 tonyr–1 from Porphyra species (red seaweed). Between 1981 and 1994, world production of seaweed increased from 3.2 million ton (fresh weight) to nearly 7 million ton, with the brown algae representing 75% followed by red algae (25%); a very small amount is green algae (0.5%).1a,1b,5 In Table 3, the relative world production of seaweeds is given, as established for the year 1997.

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Table 2 Habitat, morphology and principal food reserve and other glycans of the recognized algal phyla2 Phylum

Food reserve

Habitat

Morphology

Other glycans

Rhodophyta (red)

Floridean glycogen

Mostly SW, benthic

OCT, unicells (rare)

Chlorophyta and Charophyta (green)

Starch, inulin, possibly laminaran Laminaran

FW (90%), SW (10%)

Unicells, colonies, OCT OCT

True cellulose doubtful; mannans, xylans, sulfated galactans, complex extracellular mucilages True cellulose, modified cellulose, mannans, glucomannans, xylans, pectic acid, complex hemicelluloses and sulfated mucilages Cellulose, lichenan, alginate, fucoidan, sulfated hexuronoxylofucans, complex sulfated heteroglycans

Phaeophyta (brown)

SW, mostly benthic

FW, fresh water; SW, salt or brackish water. OCT, organized cellular tissue implying intercellular connection and dependence. Reproduced from Painter, T.J. Algal polysaccharides. In The Polysaccharides; Aspinall, G.O., Eds.; Academic Press: New York, 1983; Vol. 2, pp 195–285, with permission from Elsevier.

Table 3 Seaweed world productiona Countries

Percent share

Growth rate per year (1993–97)

China Korea DP Rep. Korea Rep. Japan Philippines Chile Norway Indonesia USA India Others

52.23 10.22 8.94 8.66 6.56 3.27 2.58 1.61 1.16 1.13 3.64

99.37 99.75 96.57 94.18 92 96.03 100 91.29 99.99 95.06 97.84

a

www.da.gov.ph/agribiz/seaweeds.html from FAO statistics.

2.21.1.3 Algae Composition The composition of algae and especially the structure of polysaccharides extracted is under the control of enzymes and therefore often depends on the phase of their life cycle at the point of harvesting, on the environmental conditions, seasons, irradiation, etc. Algae are in fact rich in nonessential and essential amino acids such as proline, glycine, and lysine in addition to many other molecules. The following composition is described for two examples. Fucus is also called antifat herb; it contains vitamin C, provitamin A (fucoxanthin, carotenoids), and trace elements, mainly iodine partially bound to proteins, polyphenols, sterols, polar lipids, sulfated C18–C30 aliphatic alcohols, etc. Dried seaweeds still contain 10–12% water, 15% mineral salts (iodine, potassium), 1–2% fatty acids and fucosterol, 4–5% proteins, and 65% condensed carbohydrates (basically algin, and a small amount of cellulose in Fucus vesiculosus). Laminaria digitata contains calcium, potassium, iodine, mannitol, proteins, carbohydrates, vitamins E, C, B12, B6, B3, A, and Zn, F, Cr, Co, Mn, Na, Fe, P, and Mg. Dried Laminaria seaweed contains less than 1% lipids, 5% proteins, 65% sugars: mannitol (12–15%), soluble condensed glycosides (15–40%), and particularly fucoidin and laminaran (up to 35% depending on the time of the year they are collected); algin 15–40%.

2.21.1.4 Uses of Algae The main use, especially in Eastern countries, is in foods representing 90% of the total production.4 The three most important seaweeds used for human food are species of Porphyra (nori in Japan), Laminaria (kombu in China), and Undaria (wakame in Korea) for salad-type preparations, soups, pickles, or tea sold on local markets. A commercial product is used in Western countries as food additive known as E 407 in the EU; it is processed Eucheuma seaweed (PES), containing carrageenan and 8–15% of insoluble materials. It is used for human food and pet food.


695

Algae (Fucus and Laminaria) are used in cosmetics because of certain peptides they contain which have good interaction with skin and keep the skin hydrated. Fucose acts as a hydrating agent, and ursolic acid (a pentacyclic triterpenoid compound) helps to form a protective barrier for the skin. They are used also in preparations against cellulite and obesity; the iodine stimulates the general metabolism. Fucus vesiculosus and Laminaria digitata are used in ‘herbal cellulite gels’. After the use as food, it was recognized that algae can be extracted to give three main polysaccharides (agar, alginate, and carrageenan) which can be used for their thickening and gelling properties. They will be further described in this chapter. Today, approximately 1106 ton of wet seaweeds are harvested to extract these polymers and produce an amount in the range of 55000ton. The main groups exploited are based on their color, the brown algae (Phaephyceae), and the red algae (Rhodophyceae). The brown algae are usually large and thick while the red algae are smaller (few centimeters to 1m in length).4 Seaweeds have been used extensively in European agriculture, particularly as animal fodder and fertilizer. But these applications have been declining. Nevertheless, seaweeds are still employed as additives for animal feeds in Europe and Canada. Now, new applications of algae develop as for the production of drugs and pharmaceuticals;6a–8b genetically engineered Laminaria have been employed for specific products. The development of pharmaceuticals and other high added value products requires a high-quality macrophyte supply, which is only possible when the efficiency of seaweed farming methods is improved. India has an active research program for selective breeding for higher agar yields. The use of seaweeds as a means of processing polluted and effluent waters has been tested; the integrated culture of fish, mollusks, and seaweeds has been proposed as a means of minimizing environmental impacts of aquaculture.9 Also, ethanol production, using different microorganisms, by fermentation of mannitol and laminaran from seaweeds harvested in autumn has been explored.10

2.21.1.5 Polysaccharides of Industrial Importance The cell walls of seaweeds contain polysaccharides which give the flexibility to the algae and allow them to adapt to a variety of water movements in which they grow. They also swell in seawater and preserve algae hydration. They are usually extracted from these algae by water in different conditions of temperature and have relatively high molecular weight. These polymers were often named phycocolloids; being water soluble, they are also called ‘hydrocolloids’, as they have dimensions in the colloidal range. The three main commercial polysaccharides are agars, alginates, and carrageenans;11–14 they are used mainly for their thickening and gelling properties depending on the thermodynamic conditions and on their molecular structures; this behavior is discussed further on. Red seaweeds contain agar, carrageenans, and few other polysaccharides belonging to the category of sulfated polysaccharides. Brown algae produce alginates, a carboxylic polymer, initially under different ionic forms and few other polysaccharides like fucoidans (Table 4).

2.21.2 Polysaccharides from Brown Seaweeds 2.21.2.1 Alginates Brown algae contain large amounts of anionic polysaccharides in their cell walls. These are principally the alginates and heterogeneous glycans rich in sulfated L-fucose, the fucans. They are described later. Alginates, also named alginic acid or algin, were discovered in 1880; they are extracted from brown seaweeds, nearly all of which are harvested in their wild state; cultivation is too expensive to provide alginates at a reasonable price for industrial applications. The quantity and quality of the alginates extracted depend on the algae species and on the season of harvest. For feasible commercial exploitation, the seaweed needs to contain at least 20% alginate based on dry weight. It was shown that brown algae growing in cold water produce generally higher molecular weight alginates than those growing in temperate to tropical waters. About 26000ton of the brown algae Macrocystis pyrifera are harvested each year off the coasts of California, Mexico, and Chile to give alginates. About 16000ton of Ascophyllum nodosum is harvested in Ireland. Laminaria hyperborea stipes are harvested in Norway, Scotland, and Ireland. These seaweeds of the cold waters mainly belong to the species in the orders Laminariales and Fucales. Alginic contents of some algae based on dry weight are: As. nodosum 22–30%, Laminaria digitata fronds 25–44%, Laminaria digita stipes 35–47%, Laminaria hyperborea fronds 17–33%, Laminaria hyperborea stipes 25–38%. They are

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Table 4 Main sources used for the production of polysaccharides Polymers and production

Algae

Production Country (Ireland) Europe

From red algae Agar E 406a (25000tyr1)

Ascophyllum nodosum Ecklonia Durvillea Macrocystis pyrifera Fucus serratus Fucus vesiculosus Laminaria japonica Undaria pinnatifida Laminaria hyperborea Laminaria digitata Porphyra Gelidium

Carrageenan E 407a (30000tyr1)

Hypnea Gracilaria Pterocladia Petocladiella Gelidiella Chondrus crispus

a

1

From brown algae Alginate E 400–405 (40000tyr )

Kappaphycus alvarezzi (cottonii) Euchema denticulatum (spinosum) Porphyra yezirgatina Girgatina pistillata Hypnea Soleria Furcellaria a

South America South Africa Australia and Chile California, Mexico, Chile China Korea Norway, Scotland, Ireland Japan Spain, Portugal, Morocco, Japan, Republic of Korea, China, Chile, South Africa Chile, Argentina, South Africa, Japan, Indonesia, Philippines, China, India Azores, New Zealand India, Egypt, Madagascar Ireland, France, Canada (Nova Scotia), Spain, Portugal, Republic of Korea Philippines, Indonesia, Canada, Republic of Tanzania, India, Africa, Pacific islands, Chile, Spain Philippines Japan

Food additive reference in the EU.

Table 5 Composition of alginate treated by the epimerase16 Alginate

FG

Acetyl (%)

Native bacterial Epimerized native Epimerase deacetylated

0.45 0.51 0.67

22 22 0

mainly produced in California, British Isles, Norway, France, Chile, Japan, and China. The total production of alginate is around 40000tonyr–1, from which 30% are used in the food industry. Alginates are also produced by soil bacteria (Azotobacter vinelandii and Pseudomonas species), and they have been investigated particularly by Skja˚k-Braek.15a,15b These alginates have O-acetyl groups on HO2 and/or HO3 of M units. Azotobacter vinelandii also produces the important enzyme, mannuronan C5 epimerase, which is responsible for the in-chain conversion of M (mannuronic acid) into G (guluronic acid) residues. The enzyme has been isolated and was found to be able to transform bacterial as well as algal alginates.16 It was shown that O-acetyl substituents inhibit the epimerase activity. Some results are given in Table 5; it is shown that FG, the molar ratio of guluronic acid units, increases upon epimerase action.

2.21.2.1.1 Extraction of alginates Brown algae are washed, macerated, and then extracted by sodium carbonate. The extract is then filtrated and, to the filtrate, sodium or calcium chloride is added, which forms a fibrous precipitate of sodium or calcium alginate.17 This


697

alginate salt can be transformed to alginic acid by treatment with dilute HCl. After a series of purifications, the alginate is dried and powdered. It can be isolated in different ionic forms and used especially for foods as acid (E 400), sodium (E 401), potassium (E 402), ammonium (E 403), or calcium form (E 404) (E is the code for food additives in the EU regulation).

2.21.2.1.2

Molecular structure

Alginates are linear block co-polymers composed of (1-4)-linked b-D-mannuronic acid (M) with 4C1 ring conformation and a-L-guluronic acid (G) with 1C4 conformation, both in the pyranosic conformation and present in varying proportions. After partial hydrolysis, Haug et al.18–21 established that they are formed of three types of blocks: blocks composed of alternating M and G resistant to acid hydrolysis and the most flexible part of the chain, blocks of GG and blocks of MM with a degree of polymerization (DP)20 (Figures 1 and 2). In the solid state, on extended fibers, Atkins performed X-ray analysis and proposed helical conformation for the different types of blocks.22–24 The conformation of the two types of blocks are given in Figure 3: a twofold helix is obtained for the mannuronic block (Figure 3a), a threefold helix for guluronic blocks when present as sodium salt (Figure 3b), and a twofold helix for the guluronic block when present as calcium salt (Figure 3c) giving a specific ion complex formation.23 It was demonstrated that the physical properties in aqueous medium for these polymers depend not only on the M/G ratio but also on the distribution of M and G units along the chain. The GG blocks in which axial–axial linkage is CO−2

CO−2

OH

OH

O

O

O

(a) CO−2 O

CO−2

O

(b)

CO−2

O

O

HO CO−2

HO O

HO

CO−2

O O OH

O

O

OH −

CO−2

O O

(a1-4)

COONa

O OH

M(4C1)

(b1-4)

COONa O

OH O

OH

(a1-4)

G(1C4)

O

OH

OH

O COONa

D-mannuronic

M(4C1)

(b1-4)

O

acid (M); c, alternated

G(1C4)

COONa

HO

HO

HO

O

CO−2

Figure 1 Structure of alginate-constituting blocks: a, L-guluronic acid (G); b, L-guluronic and D-mannuronic acids.

G(1C4)

HO O

OH

O HO

CO−2

(c)

O

HO O

HO

O

CO2

O HO

HO

OH O HO

O

OH

CO−2

O HO

HO

O

OH

O

OH

OH

OH

O

HO

O COONa

O

OH

O OH

Figure 2 Schematic representation of an alginate chain constituted of different arrangements of M and G units. Reproduced from Heyraud, A.; Rinaudo, M.; Rochas, C. Physical and chemical properties of phycocolloids. In Introduction to Applied Phycology; Akatsuka, I., Ed.; SPB Academic Publishing BV: The Hague, The Netherlands, 1990; pp 151–176, with permission from Backhuys Publishers B.U.

698


0.87 nm 1.51 nm 1.04 nm

(a)

(b)

(c)

Figure 3 Computer-drawn chain conformation of: (a) the twofold poly(mannuronic acid) helix, in directions parallel and perpendicular to the chain axis; (b) threefold sodium salt form; and (c) twofold poly(guluronic acid) helix. Reproduced from Atkins, E. D. T.; Isaac, D. H.; Nieduszynski, I. A.; Phelps, C. F. The polyuronides: Their molecular architecture. Polymer 1974, 15, 263–271, with permission from Elsevier.

Table 6 Ratio of D-mannuronic acid (M) to L-guluronic acid (G) in some commercial alginates Species

M/G

Macrocystis pyrifera Ascophyllum nodosum Laminaria digitata Laminaria longicruris Laminaria hyperborea (fronds) Laminaria hyperborea (stipes) Ecklonia cava and Eisenia bicyclis Fucus serratus Pelvetia canaliculata Pylaiella Sphacelaria bipinnata Dictyota dichotoma Desmarestia aculeata Chordaria flagelliformis Dictyosiphon lomentaria

1.56 1.0–1.85a 1.16–1.63a 2.03 1.28–1.35a 0.37–0.65a 1.60 1.06 1.28 0.60 0.40 1.05 0.58 0.63 0.90

a

It depends on the culture conditions.

involved are more rigid than the diequatorially linked MM blocks; then, the stiffness of the alginate chains as well as calcium complex formation depend on the composition (M/G ratio) and distribution of M and G units in the chains as discussed by Smidsrd.25 The average composition of some commercial alginates is given in Table 6. G-blocks of more than 6–10 residues each form stable cross-linked junctions (and gels) with divalent counterions (Ca, Ba, Sr, etc.) but not with Mg as was also found with pectins with low degree of methylation; in Figure 4, it is shown that the relative viscosity increases rapidly over a critical amount of divalent counterions (Ca, Ba, Sr) when the chains interact.26,27 At low pH, alginates form acidic gels stabilized by H bonds. The homopolymeric blocks form the junctions, and the stability of the gels is determined by the relative content and length of the G-blocks. The gelation and properties of alginates are described in this chapter.


699

1.5

1.2

Ba2+

Sr 2+

Ca2+

0.9 h h0 0.6

Mg2+ 0.3

0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

q Figure 4 Evolution of the relative viscosity of an alginate solution (0.6gl –1 ) for progressive additions of divalent salts (q ¼[X2þ]/2[–COO– ].26 Reproduced from Rinaudo, M. In Kirk-Othmer Encyclopedia of Chemical Technology; Kirk-Othmer, Ed.; Wiley: Hoboken, NJ, 2006; Vol. 20, p 549, with permission from John Wiley & Sons.

H5 GG H1 G

H1 M + H5 GM

5.0

4.5 ppm

Figure 5 Anomeric region of 1H NMR spectum of an alginate allowing the determination of M/G ratio. The signal H1 G corresponds to the total G units, the signal H5 GG to the G groups included in a block, the intermediate region corresponds to H1 M þ H5 of isolated G units. This signal allows to determine the M content.

2.21.2.1.3

Methods for characterization

2.21.2.1.3.1 (i) NMR spectroscopy Before their characterization, alginates have to be purified and isolated in the sodium form; the method used was described previously.28 The heterogeneity of alginates is well described after partial hydrolysis as developed by the Trondheim team.18,20,29,30 Nuclear magnetic resonance (NMR) is the most powerful technique to characterize the chemical composition and the microstructure of the alginate.31–34 For this analysis, the conditions are usually the following: 5mg of the purified alginate in the sodium salt form is dissolved in D2O; 1H NMR spectrum is obtained on a 300MHz spectrometer at 85 C. A statistical model was developed to analyze the NMR spectra, and the two monad frequencies (FG, FM), the four diads (FGG, FMM, FMG, FGM), and eight triads (FGGG, FGGM, FGMM, FGMG, FMGG, FMGM, FMMM, FMMG) were calculated and deduced from 1H and 13C NMR. Figure 5 gives a proton NMR spectrum allowing to determine the M/G composition of alginates.35,36

700


The following relations for diads can be used to establish the proportions of the different sequences: FGG þ FMM þ FMG þ FGM ¼ 1 FGG þ FGM ¼ FG FMM þ FMG ¼ FM This model was extended by Grasdalen37 and also used to demonstrate the influence of epimerase on the microstructure of different alginates.38 From these analyses, they deduce M/G ratio and the average length of the G blocks NG>1. Some values are given in Table 7. The ratio M/G depends on the location and harvest time in the year.

2.21.2.1.3.2 (ii) Size-exclusion chromatography Purified alginates isolated in the sodium salt form were characterized by size-exclusion chromatography (SEC) using a Waters Alliance GPCV2000 (USA) equipped with three detectors in line: a differential refractometer, a viscometric detector, and a multi-angle laser light scattering (MALLS) detector from Wyatt (USA). The concentration injected was in the range of 0.5–5gl–1 (depending on the range of molecular weights), with an injection volume of 108ml using two columns in series (Shodex OH-pack 805 and 806 for the native commercial purified alginates). All the samples were filtrated on a 0.2mm pore membrane (Sartorius AG; cellulose acetate filter) before injection, in order to retain large aggregates. The eluent used was 0.1M NaNO3, elution temperature was 30 C and the flow rate of 0.5mlmin–1; the molecular weight distribution, weight-average molecular weight (Mw), polydispersity index (Mw/Mn, where Mn is the number-average molecular weight), and intrinsic viscosity of the eluted polymers were obtained as characteristics of the biopolymers (Figure 6). For this example, Mw ¼275000 and []¼650mlg–1 in 0.1M NaNO3 at 30 C using a specific refractive index increment, dn/dc¼0.165. For commercial products, molecular weight may range between 32000 and 400000. Purified alginates in sodium form are also characterized by viscometry allowing to determine their intrinsic viscosity and molecular weight using the Mark Houwink relation: ½1

½ ¼ KM a

with K¼210–3 and a¼0.97 in 0.1M NaCl solvent at 25 C.40 Previously, Smidsrd et al. gave the following parameters K¼2.4410–3 and a¼0.982 from a series of light scattering experiments on Laminaria digitata (38.5% G units).41 Mackie et al.42 showed that the parameters K and a depend on the M/G, a increasing and K decreasing when G content increases. This dependence is discussed later.43

2.21.2.1.4 Stiffness of alginates The dimensions of alginate chains (radius of gyration and intrinsic viscosity) in aqueous solutions depend on the external salt concentration; the expansion is directly related to the thickening performance of a polymer. But it seems that the expansion of polysaccharides, even in external salt excess, is larger than that of a random coil in absence of electrostatic repulsion. Smidsrd first introduced a procedure to characterize the stiffness of polysaccharides and especially that of alginates. The analysis is as follows:44,45 the intrinsic viscosity of alginate in solution is determined at different monovalent salt concentrations (Cs); the values of [] are plotted as a function of Cs–1/2 giving a straight line with a slope S. S is proportional to the molecular weight Mw and in a log–log plot of S–Mw, a linear curve is obtained with a slope 1.37. S is then related to B, a characteristic of the stiffness as proposed by Smidsrd. Synthetic polymers were compared with polysaccharides and DNA, and the following expression was considered: S ¼ Bð½0:1 Þn

½2

Table 7 Compositional characteristics of some alginates39

L. hyperborea (outer cortex) L. hyperborea (LF-10/60) L. hyperborea (leaf) L. digitata (whole plant) M. pyrifera Durvillea antarctica (whole plant)

Monad frequency (FG)

Diad frequency (FGG)

Trial frequency (FGGG)

Mean blocklength NG>1

0.75 0.70 0.55 0.41 0.43 0.34

0.66 0.60 0.38 0.25 0.21 0.13

0.62 0.56 0.32 0.20 0.16 0.10

17.5 15.0 7.3 6.0 6.3 5.3

Reproduced from Martinsen, A.; Skja˚k-Braek, G.; Smidsrd, O. Biotechnol. Bioeng. 1989, 33, 79–89, Copyright © (1989, Wiley). Reprinted with permission of John Wiley & Sons, Inc.


701

LS # 11, AUX1, AUX2

0.06 LS # 11 AUX1 AUX2

0.04

0.02

0.00

−0.02

0

5

10

15

20

25

Differential weight fraction

Volume (ml) 1.2

0.8

0.4

0.0 10 104

10 106 10 105 Molar mass (g mol−1)

10 107

Figure 6 SEC chromatogram and molecular weight distribution on purified alginate using a three-detector equipment. T¼30 C; solvent¼0.1M NaNO3. a, The three trace chromatograms: red, light scattering; green, viscometer; blue, differential refractometer. b, Differential molecular weight distribution obtained from the previous chromatogram.

Here, n is nearly independent of the polymer stiffness (S¼1.2 for Na polyphosphate and 1.4 for DNA); an average value of 1.3 is adopted and B calculated for each polysaccharide from relation [2] after determination of S and the intrinsic viscosity at 0.1M monovalent salt. The lower the B value, the larger is the stiffness; in Table 8, B values are given and in a few cases the values for the intrinsic persistence length Lp are added. The relative extension of the three types of blocks increases in the following order:45 MG < MM < GG Later, the worm-like chain model was applied to analyze the behavior of polysaccharides and their local stiffness characterized by the intrinsic persistence length Lp; Lp corresponds to the value of the total persistence length Lt determined at various ionic concentrations and extrapolated to infinite salt concentration to screen the electrostatic contribution (Le is the electrostatic persistence length; it decreases when the ionic concentration increases). In these conditions, it is assumed that the y-conditions are approached.46 The Lp value was also calculated for few polysaccharides by molecular modeling and found to be in good agreement with the experimental values.47–50 For alginates rich in G units, Lp was found experimentally to be 9nm while for a sample rich in M units, Lp¼4nm.46,51 The proposed treatment allows one to predict the dimensions of the chain (and particularly the radius of gyration, Rg) and the intrinsic viscosity ([]).The main relation to analyze the radius of gyration at a given ionic concentration is the following: Rg2 ¼ a2s ðLLtÞ=3;

Lt ¼ Lp þ Le

½3

This relation is valid when the contour length L (proportional to M) is large compared with Lt; otherwise, it is necessary to use the development proposed for the y-state by Benoit and Doty based on the assumption that in salt excess the y-conditions are approached.52 as is the excluded volume which contains a large contribution of the electrostatic interaction. The model of worm-like chain polyelectrolyte is exposed in details by Reed.53

702


Table 8 Charasteristic B parameter for different types of polymer Polymers

B parameter44

Polyphosphate Polyacrylate Carboxymethylamylose Carboxymethylcellulose Alginate

0.44 0.23 0.20 0.065 0.04

DNA

0.0055

Lp (nm) 1 5 4 (M/G¼1.92) 9 (M/G¼0.28) 90

Reproduced from Smidsrd, O.; Haug, A. Biopolymers 1971, 10, 1213–1227, Copyright © (1971, Wiley). Reprinted with permission of John Wiley & Sons, Inc.

08

g Ca2+

CaCl2 06

04 1 2

02 3 0 1

20

40

60

81 91

4000

DP Figure 7 Activity coefficient of calcium counterions as a function of the degree of polymerization. (1) b-D-mannuronic block; (2) a-L-guluronic block; (3) a-D-galacturonic block. Reproduced from Kohn, R. Ion binding on polyuronates. Alginate and pectin. Pure Appl. Chem. 1975, 30, 371–397. With permission from IUPAC.

The local stiffness of the polysaccharides explains the high viscosity obtained at a given molecular weight compared to flexible polymers and the relative low sensitivity to external salt concentration. This value is obtained experimentally from the curve Rg(M) established from size-exclusion chromatography (SEC) experiments using a multidetection equipment in which three detectors are on line: a differential refractometer, a multiangle light scattering detector, and a viscometer, as described before. These data allow one to determine Lt when the ionic concentration of elution is known, that is, 0.1M in our work, which gives a negligible value for Le (around 5A˚) and from which Lp can be determined.50

2.21.2.1.5 Mechanism of gelation in relation with the chemical structure Alginates are able to form gels in the presence of divalent counterions such as Ba, Sr, Ca, while Mg does not form a gel. There are two methods to form gels: dialysis against a CaCl2 solution which give some heterogeneity in calcium and alginate distributions inside the gel volume or homogeneous formation by slow hydrolysis of D-glucono-d-lactone in the presence of Ca-EDTA, resulting in a mixed Hþ, Ca2þ gel (pH3.5), which is then converted to the calcium form by dialysis against a calcium chloride solution (EDTA¼ethylenediamine tetraacetic acid).26,43,54,57 A specific cooperative Ca interaction forms on G blocks, which is the basis of the formation of junction zones in an ionic network as soon as DP is larger than 20 (Figure 7); this interaction is not observed for M unit blocks.56,57–60 The conformation of acidic polysaccharides and interactions with calcium was approached by molecular simulation.61,62 The authors demonstrated the specific calcium binding with poly-a-L-guluronate. From Figure 4, it is seen that the viscosity of alginate in dilute solution first decreases due to the reduction of the net charge of the polymer and then increases strongly when dilute solutions of Ca, Ba, and Sr chlorides are progressively added (q¼[X2þ]/2[–COO–]) due


703

O

O −O2C

OH

HO O

O Ca2+

O

O −O2C HO

HO O

O

Figure 8 Specific interaction of calcium with a-L-guluronic–box block. Dark circles represent the oxygen atoms involved in the coordination of the calcium ion. Reprinted with permission from Braccini, I.; Perez, S. Molecular basis of Ca2þ-induced gelation in alginates and pectins: The egg-box model revisited. Biomacromolecules 2001, 2, 1089–1096. Copyright 2006, American Chemical Society.

to the formation of interchain junctions.26 It can be seen that Mg does not induce cross-linking, just as observed for sodium polygalacturonate.27 This ionic selectivity was used to fractionate alginates: the precipitate obtained with Ca in the presence of MgCl2 is enriched in guluronic acid.63 The selectivity observed, Sr>Ca, is related to the larger dimension of Sr which fits better than Ca in the cavity.64 The mechanism of complex formation involves calcium interaction with different oxygen atoms of two adjacent guluronic acid units in both chains involved in the interchain binding of Ca as it is adopted in the egg-box model (Figure 8). The mechanism of gelation is described in Figure 9: the first step is a dimer formation followed by precipitation for small chains or gelation for long chains formed with different types of blocks. The properties of the gels obtained depend on the molecular characteristics of the alginates; the stability of the gels and their physical properties depend directly on the G content and on the length of the G blocks; in Figures 10 and 11, the role of the composition is shown.39 From these curves, it is clear that the stiffness of the gels increases with the G content (FG) and with the length of the G blocks reflected by the value of NG>1. The gel strength is determined in this work by measuring the load that is required to compress small gel beads (diameter 3–4mm) by 1mm at a constant rate of 0.2mms–1. The contraction observed when gels are formed in the presence of CaCl2 is lower for the stiffer gel. In Figure 12, it is shown that the gel strength increases for a given molar structure when the molecular weight increases up to a limit around M¼3105.39 It is a behavior which seems similar to the behavior obtained with k-carrageenan. In a separate work, the gel strength was determined by compression of an alginate gel (M/G¼0.56) formed at 10gl–1 against 0.1M CaCl2; the elastic modulus was found equal to 4.74104 Pa in the same order of magnitude as found for k-carrageenan.26

2.21.2.1.6

Physical properties in solution

The most important technical properties of alginates are their thickening character (increase of the solvent viscosity upon dissolution), their ionic exchange properties, and their gel-forming ability in the presence of multivalent counterions. An alginate molecule is a polyelectrolyte; alginates in monovalent salt form are water soluble whatever the temperature. Its ionic properties (electrostatic short- and long-range interactions) as well as their conformation and molecular weight are important and control the viscometric behavior. The viscosity of aqueous alginate solution depends as usually for polyelectrolytes on the polymer concentration, molecular weight, and external salt concentration (due to screening effect on electrostatic interactions); this behavior is typical.28 In addition, the viscosity of alginate solution is nearly constant between pH 6 and 8 but, in moderate concentration, after a slow decrease below pH 6, it increases below pH 4.5 and reaches a maximum around 3–3.5 and then decreases; in fact, alginic acids form gels. In this range of pH, H-bond attractions overdominate the electrostatic repulsions, as was observed recently on hyaluronan.65 In dilute solution, the viscosity decreases when pH decreases from pH6. In the range of pH 1–4,

704


'Egg box' Ca2+

Ca2+

(a)

Ca2+

Ca2+

(b) Figure 9 Mechanism of cooperative interaction between calcium and alginate. a, Egg-box model:calcium interaction with a-L-guluronic–box block. b, Chain association going to gelation induced by calcium involving specific a-L-guluronic– box block.

alginic acid is hydrolyzed. Then, the solutions are stable in the range of pH 5–10. The intrinsic pK for alginic acid is around 3 as found for many polyuronic acids.66 The characteristics of alginates are their ability to retain water, in addition to gelling and stabilizing properties. Because of the linear structure and high molecular weight, alginates form strong films and good fibers in the solid state.

2.21.2.1.7 Polyelectrolyte complex formation When an anionic polymer, such as alginate, is mixed with a cationic polymer (like chitosan in acidic condition), an electrostatic complex is formed whose stability depends on the pH and salt concentration.66–68 The mechanism of complex formation was derived from conductimetric measurements; it can be expressed by the following equilibrium: COO Naþ þ Cl þ NH3 $ COOþ NH3 þ Naþ þ Cl

½4

At stoichiometric ratio, the complex is usually insoluble and allows one to obtain fibers, films, or capsules. Chitosan–alginate complex formation was examined for alginate with different M/G ratios.69 In fact, many applications are proposed based on this chitosan–alginate complex, which is the polyelectrolyte complex mostly investigated, especially for biomedical applications.70,71

2.21.2.1.8 Ion-exchange properties Alginates have interesting ion-exchange properties; most monovalent counterions (except Agþ) form soluble alginate salts, while the divalent and multivalent (except Mg2þ) ones form gels or precipitates. The affinity was found following the order:43,72–74 Mn < Zn; Co; Ni < Ca < Sr < Ba < Cd < Cu < Pb


705

30

Load (g)

20

10

0 0

0.02

0.04

0.08 0.10 0.06 CaCl2 concentration (M)

0.12

0.14

Figure 10 Gel strength as a function of calcium concentration for two different alginates. Filled circles, Laminaria digitata, FG ¼0.41, NG>1 ¼6.0; open circles, Laminaria hyperborea, FG ¼0.70, NG>1 ¼17.5. Reproduced from Martinsen, A.; Skja˚kBraek, G.; Smidsrd, O. Alginate as immobilization material. I: Correlation between chemical and physical properties of alginate gel beads. Biotechnol. Bioeng. 1989, 33, 79–89. Copyright © (2006, Wiley). Reprinted with permission of John Wiley & Sons, Inc.

50

Volume reduction (%)

40

30

20

10

0 0

0.02

0.04

0.06

0.08

0.10

0.12

0.14

CaCl2 concentration (M) Figure 11 Volume reduction as a function of the CaCl2 concentration used for gelation for Laminaria digitata, FG ¼0.41, NG>1 ¼6.0 (solid circles) and Laminaria hyperborea, FG ¼0.70, NG>1 ¼17.5 (open circles). Reproduced from Martinsen, A.; Skja˚k-Braek, G.; Smidsrd, O. Alginate as immobilization material. I: Correlation between chemical and physical properties of alginate gel beads. Biotechnol. Bioeng. 1989, 33, 79–89. Copyright © (2006, Wiley). Reprinted with permission of John Wiley & Sons, Inc.

The amount of salt to induce gelation was found with a selectivity:75,76 Ba < Pb < Cu < Sr < Cd < Ca < Zn < Ni < Co < Mn; Fe < Mg The efficiency of a divalent ion as a precipitant for alginate depends not only upon its affinity for the alginate, but also upon the amount of ion which must be bound to the alginate for gel formation. So the sequences are not exactly the same.

706


5

Load (corrected) (g)

4

3

2

1

0 0

2

4

6 [x], [100 ml g−1]

8

10

12

0

1

2

3 MW = 105

4

5

6

Figure 12 Influence of the molar mass, intrinsic viscosity, and polymer concentration on the gel strength. Alginate from Laminaria hyperborea (outer cortex), FG ¼0.75, NG>1 ¼17.5. Concentration: 1% (solid circles), 0.8% (open circles), 0.6% (solid squares), 0.4% (open squares). Reproduced from Martinsen, A.; Skja˚k-Braek, G.; Smidsrd, O. Alginate as immobilization material. I: Correlation between chemical and physical properties of alginate gel beads. Biotechnol. Bioeng. 1989, 33, 79–89. Copyright © (2006, Wiley). Reprinted with permission of John Wiley & Sons, Inc.

2.21.2.1.9 Chemical modifications 2.21.2.1.9.1 (i) Periodate oxidation Upon oxidation of Laminaria digitata alginate with sodium metaperiodate and reduction with sodium borohydride, the cyclic osidic unit structure turns to segments of single bonds after ring opening between C2 and C3 positions which carry vicinal secondary hydroxyl groups.77 The mechanism of chain modification due to oxidation of a block of mannuronic units is shown in Figure 13; in this reaction, the relatively rigid pyranoid ring is converted into a segment of the polymer chain containing three continuous single bonds; some values obtained for the stiffness parameter B are given in Table 9 for progressive oxidation. This reaction decreases considerably the stiffness of the chain; this was confirmed recently on M. pyrifera alginate.78 At the same time, it was demonstrated that a large effect of depolymerization occurs and that G units were modified first. The evolution of stiffness of polyguluronate during oxidation has also been described.78,79 2.21.2.1.9.2 (ii) Propylene glycol alginic acid This derivative has been produced already for many years in large amounts. The ester is prepared by reaction with propylene oxide under moderate pressure on a partially neutralized alginic acid in fibrous form. Due to its amphilic character, it is a good emulsifier with thickening properties associated with protective-colloid properties. The advantage of this derivative is that it remains perfectly soluble in a large range of acidic pH; it is hydrolyzed at pH < 2. The reference of this derivative is E 405 as food additive.

2.21.2.1.10 Uses of alginate Purified alginates are produced in different salt forms: Na, K, NH4, Ca, and/or propylene glycol derivative as food additives for thickening soups and jellies. They are used as antiacid preparation such as Gaviscon®, for making molds


O

CO− 2

CO2− O OH

O

HO

CO− 2 5 C

O

H H

HO

H 4

1 O 2 CH2OH

O OH

CO2− C 5 3 H CH2OH

CO− 2 O

O OH

707

O

HO

CO− 2

H 4

O

O

C

1

O

H 2

CH2OH

H CH2OH 3

Figure 13 Periodate oxidation of mannuronic unit block. Reproduced from Smidsrd, O.; Painter, T. Effect of periodate oxidation upon the stiffness of the alginate molecule in solution. Carbohydr. Res. 1973, 26, 125–132, with permission from Elsevier.

Table 9 B parameter as a function of the oxidation degree on alginate77 Percent oxidation

B parameter

0 5 10 20 30 44 88

0.04 0.075 0.087 0.14 0.19 0.23 0.23

in dentistry (in the presence of slow release calcium salt to control the delay of gelation), prosthetics, and in textiles. Different food applications, pharmaceutical, cosmetics, and other industrial domains are cited in previous reviews on alginates.17,29,30,80 In the following, only few examples are given due to the large number of applications found in the literature. Alginates can be processed as fibers or films which are commercialized as hemostatic material or also as wound dressing. An example, AlgiDERM® or Sorbsan® are calcium alginate dressings, made of sterile purified alginate fibers.81 It absorbs on contact with exudate into a web-like gel up to 20 times its weight. It is removed easily without damaging healthy tissue. The films resist oil penetration due to the presence of –OH and carboxylic groups. Calcium gels are often used in bead form as an immobilization matrix for animal cells or plant protoplast.82a–82c It was claimed that encapsulation enhanced the stability of plasmids and enzymatic expression.83 Microparticles were also obtained by emulsion; this method allows one to get beads with smaller pores.84,85 Calcium alginate gels have been used to encapsulate enzymes;86 in these conditions, the enzyme remained active in organic solvents.87 The polyelectrolyte complexes are often used in biomedical applications; chitosan–alginate beads were evaluated as an immobilization matrix for lipase-catalyzed hydrolysis of olive oil.88 The complex is also convenient in tissue engineering89 and wound dressing.90 It was demonstrated that beads of alginates encapsulated in chitosan are superior to drug-loaded chitosan as well as alginate beads for sustained release.91

2.21.2.2 Fucans/Fucoidans Fucans or fucoidans constitute a complex series of sulfated polysaccharides found in the cell wall matrix of brown algae (often together with alginate); the molecular weight was determined in the range of 100000.92 Fucoidan is mainly intercellular but it seems that a fraction may be in the cell walls.2,92 Fucoidans are present in all brown algae that grow in the intertidal zone but mainly in Fucales. They play a role in the algal cell organization and

708


may be involved in the cross-linkage of alginate and cellulose.93 Sulfated a-L-fucans (also known as fucoidan or fucoidin) have complex and heterogeneous structures, but recent studies reveal the occurrence of ordered repeat units in the sulfated fucans from several species. But the presence of highly branched regions and the complex distributions of sulfates and acetyl groups still highlight the heterogeneity of algal fucans.94 A recent review clarifies the nomenclature and the chemical structure of fucoidans;95only a small amount of fucoidan is extracted from brown algae – in the range of 0.8gper1kg of brown seaweed. Extraction processes are designed to preserve the mineral content and also the content in antioxidant (fucoxanthin, a natural carotenoid). Fucoidan or fucan has recognized physiological and biological activities as an anticoagulant, antithrombin, antitumor agent, and anti-human immunodeficiency virus (HIV) infection agent.96 As already reported 90 years ago, these are polysaccharides composed of L-fucose residues.97 Products range from essentially pure fucan sulfate in Fucus distichus to ascophyllan from As. nodosum. Fucose-containing polysaccharides comprise a spectrum of molecules ranging from molecules rich in uronic acid and poor in fucose sulfate to relatively pure sulfated fucans containing little uronic acid. The principal sources of fucoidans are mentioned in Table 1.95 The complexity of the fucan sulfate varies with the algal source. Fucans were arbitrarily divided into three main families: homofucans, xylofucoglycuronans, and glycuronofucoglycans.98a–98d Homofucans are primarily composed of (a1-2)-linked units of 4-sulfuryl-Lfucose with branching or sulfate at position 3. The first fucoidan studied was ascophyllan or xylofucoglycuronan, which has a backbone of uronic acid (mainly poly-(b1-4)-D-mannuronic acid) with fucose-containing branches (3-O-Dxylosyl-L-fucose 4-sulfate). The other family isolated was sargassan or glycuronofucoglycan with a linear chain of (1-4)-linked D-galactose with branches of L-fucose 3-sulfate at C5 or occasionally uronic acid (probably D-glucuronic acid).95 This type of structure is in agreement with findings from Larsen et al.98a–98d for one fraction of As. nodosum extracts where they found L-fucose, D-xylose, uronic acid (mainly D-glucuronic), and sulfate half ester; a polypeptide is also attached to the side chain in an unknown manner. Fucoidans, extracted from F. vesiculosus, are often used as crude extract; they can also be extracted from many orders in the brown algae (Phaeophyceae): Fucales, Laminariales, Chordariales, Dictyotales, Dictyosiphonales, and Ectocarpales.95,99 The extract from Laminaria japonica, after fractionation and mild acid hydrolysis, demonstrated antioxidative properties against low-density lipoprotein oxidation.100 The only other source of fucoidans are marine invertebrates; they are obtained from eggs of sea urchin and from the body wall of sea cucumber Ludwigothurea grisea. Their structure is usually more regular than that of the fucoidans from algae. It has been proposed to reserve the name ‘fucoidan’, the fucans from algae, while the name ‘sulfated fucan’ (sulfated L-fucose with less than 10% other monosaccharides) should be used for the marine invertebrates. We will limit here to the algal fucans containing 20–60% fucose. These fucoidans are usually heterogeneous, branched, and contain other sugars and O-acetyl groups; in addition, the sulfation pattern is irregular.99 Fucoidans are water-soluble polysaccharide made of units of L-fucose (fucopyranoside) with one to two links and with groups of sulfuric esters at C4. In some studies, a backbone in (a1-3) instead of (a1-2) is proposed and also (a1-4) links have been found; in As. nodosum, a repeat unit of alternating (a1-3) and (a1-4) glycosidic bonds was found. Two models representing fucoidans are shown in Figure 14.101a,101b

2.21.2.2.1 Extraction of fucoidans Fucoidans are extracted from algae in dilute acid conditions; at least partly, they consist of proteoglycans. They are fractionated by DEAE ion-exchange chromatography. They contain mainly L-fucose residues, but some fractions also contain galactose, xylose, hexouronic acids, and mannose as well. A result of a fractionation of fucoidans extracted from Fucus evanescens is given in Table 10.99

2.21.2.2.2 Molecular structure The fucoidans extracted from As. nodosum, F. vesiculosus, and F. evanescens have the repeat unit given in Figure 14. Fucoidan is made of fucose in the backbone with branches and a large amounts of acetyl groups.101a,101b Another repeat unit has been proposed:101a ½!3Þ-a-L-Fucpð2SO 3 Þ-ð1!4Þ-a-L-Fucpð2; 3diSO3 Þ-ð1!n

An acetyl fucoidan was isolated from Cladosiphon okamuramus (together with alginate); it is commercially cultured at 18500tyr–1 off Okinawa Islands (Japan in 2000);96 the yield is 2.3% based on wet algae. The composition was estimated to be L-fucose:D-xylose:D-glucuronic acid:acetic acid:sulfuric acid 4:0.03:1:2:2.96 This alga is widespread in Japan. A glucuronoxylofucan was also found in As. nodosum.102a,102b


709

a-L-Fucp-4(SO3−) 1

2)-a-L-Fucp-(1 4

2)-a-L-Fucp-(1 4

SO3−

3 2)-a-L-Fucp-(1 4

SO3−

2)-a-L-Fucp-(1 4

SO3−

SO3−

Model 1 a-L-Fucp 1

3)-a-L-Fucp-(1 4

3)-a-L-Fucp-(1

3)-a-L-Fucp-(1 4

3)-a-L-Fucp-(1

SO3−

1 a-L-Fucp

2 3)-a-L-Fucp-(1 4 SO3−

Model 2 Figure 14 Two structural models proposed for fucoidans. Reproduced from Chevelot, L.; Foucault, A.; Chaubet, F.; Kervarec, N.; Sinquin, C.; Fisher, A.-M.; Boisson-Vidal, C. Further data on the structure of brown seaweed fucans: Relationship with anticoagulant activity. Carbohydr. Res. 1999, 319, 154–165, with permission from Elsevier. Table 10 Yields and composition of fucoidan fractions from Fucus evanescens obtained by ion-exchange chromatography of crude fucoidan (F)99 Neutral monosaccharides Fraction

Yield (% of F)

Fuc

Xyl

Gal

F1 F2 F3 F4 F5

3.9 2.6 21.4 47.4 4.5

35.4 10.7 33.2 58.7 34.0

6.1 7.4 8.1 1.6 3.8

3.0 4.5 1.6 5.4

2.21.2.2.3

Man

Glc

0.8 3.7 3.5

4.0 1.1

SO3Na (%)

Uronic acids (%)

n.d. 19.6 28.9 46.5 32.5

n.d. 15.6 11.4

Uses of fucoidans

Fucoidan is a water-soluble polysaccharide; it gives highly viscous solutions.103 The most important application of fucoidan is in food – fucoidan containing food and supplements are very popular on the Japanese market. Raw fucoidan materials are also used for cosmetics because the product is directly adsorbed by the human skin with the following effects: whitening, preserving moisture, removing freckles (e.g., Fucoidan Premium Gold®, Arabino fucoidan®, Sea fucoidan® from Natureza Co.). Fucoidan has also important medical applications. The higher the sulfation, the higher its therapeutic potential. The role of fucoidan is as modulator of coagulation, as an alternative to the anticoagulant heparin,104,105 and many other biological activities such as anti-inflammation, anticell proliferation, and antiadhesion and antiviral infection, as fertilizer,95 anticoagulant, antitumor, antiviral agents.106a,106b In addition, they act as ligands for selectins, protect gastric mucosa against proteolytic activity of gastric juice, block mammalian fertilization,107 etc.

2.21.2.3 Food Reserve in Brown Algae: Laminaran 2.21.2.3.1

Molecular structure

Laminaran is a glucan which exists in two forms, one soluble in cold form and the other insoluble; it is a neutral polysaccharide with a relatively small size (it contains approximately 20–30 glucosyl residues). The two forms are made up of (1-3)-linked D-glucose with a proportion of (1-6)-linked-glucose and mannitol. It may represent 35% of the dried weight of the algae.108 Its solubility increases when the degree of branching increases. Laminaran (also named laminarin) was first discovered in Laminaria species. It appears to be the food reserve of all brown algae; it is often present in the same sources as alginates and fucoidans.109a,109b It is absent during the period of

710


fast growth in spring, but in autumn and winter it may represent up to 35% of the dried weight of the fronds. Laminarans from several species of Laminaria are water insoluble and contain only linear (b1-3)-linked D-glucose. Laminaran from Laminaria digitata is water soluble. Its structure as established by NMR spectroscopy is a linear backbone of (b1-3)-linked D-glucose with (b1-6)-linked side chains.110 There are two types of terminal units: one with mannitol (M-series with a nonreducing 1-linked D-mannitol residue) and the other terminated by a reducing glucosyl unit (G-series) present in about a 3:1 ratio.111 The number of glycosyl units in the side chains is also heterogeneous.110 The degree of branching was given as 0.07111 and 0.05:112 75% of the branches were single glucosyl residues. M-series molecules show an average of 1.3 branches per molecule. Forty to seventy-five percent of the reducing end groups are linked to one of the primary hydroxyl groups of D-mannitol in Laminaria and Fucus species. Laminaran from Eisenia bicyclis is a linear chain of (1-3) and (1-6) linkage in a ratio 2:1.

2.21.2.3.2 Uses of laminaran (b1-3,1-6)-linked D-glucans have the ability of enhancing and stimulating the immune system of humans and are thus called biological response modifiers;113a it has an inhibitory effect against putrefactive compounds produced by intestinal microflora in vitro and in rats;112 laminaran from Alaria praelonga or Laminaria coriacea promote adhesion of human skin fibroblast cells, and enhance proliferation of human osteoblast cells.113b Also, antitumor and hypolipid effects of different fractions of laminaran were claimed.114 Laminaran was proposed as elicitors (with a larger activity than pectic acid) of phytoalexins and flavonoids in alfalfa cotyledons.115a,115b

2.21.3 Polysaccharides from Red Seaweeds Sulfated polysaccharides occur in the structural elements of animals and plants (limited to algae polysaccharides) where they may constitute up to 70% of the dry matter of some red seaweeds. Usually, ester sulfates are associated with galactans in the Rhodophyceae and fucans in the Phaeophyceae.11,13,116 Carrageenans are linear sulfated polysaccharides extracted from red algae; the color results in the dominance of phycoerythrin and phycocyanin pigments which mask the other pigments. Other main polysaccharides present in these algae are floridean starch as a storage polysaccharide and, in their walls, cellulose. Agar and carrageenans were first produced from wild-type species, an especially Irish moss growing in cold water. It was first extracted in 1844 but it lasted until after 1970, when its production developed rapidly through the availability of other seaweeds cultivated in warm-water countries with low labor costs. In Red algae, agar and carrageenans are the most important (Table 4). Only recently has agar been fractionated in neutral and anionic fractions; it appeared that there is a continuous spectrum of molecules. It was proposed for Laurencia pinnatifida and Polysiphonia lanosa to classify them as agars, porphyran, funoran, and sulfated galactans.11 Many rhodophycean galactans from different species have been described. With a few exceptions, the structure is a linear chain of b-D-galactopyranose residues linked through positions 1 and 3 (A units) and a-D/L-galactopyranose residue linked through positions 1 and 4 (B units) arranged in an alternated sequence (AB)n:

Units A may carry methyl ether groups on position 6, sulfate hemiester groups on position 2, 4, or 6; few A units may carry pyruvic acid, linked as a cyclic ketal bridging O4 and O6 (1-carboxyethylidene groups). Units B can occur in either D or L form, they can carry methyl groups on position 2, 4-O-methyl-a-L-galactopyranosyl groups on position 6, and sulfate hemiester groups on position 2 or 6 or both; B units can be wholly or partly converted to 3,6-anhydro forms by elimination of sulfate from 6-position (by enzymatic elimination or alkaline treatment).2 The different structures for agar and carrageenans are given in Tables 11 and 12, respectively. The ideal composition of the polysaccharides obtained depends on the species, habitat, season of harvesting, and the extraction conditions.

2.21.3.1 Agar/Agarose Agar, also previously named agar-agar, is extracted from red seaweed. It is known from the seventeenth century in Japan for its gelling properties. Farming now develops in Indonesia and the Caribbean. The total production of agar is around 25000tyr–1. Agar is extracted for commercial production mainly from Gelidium, Pterocladia, Petrocladiella (giving the best quality), and Gracilaria and Hypnea species; Gelidium gives the better commercial quality of agar (especially the higher gel strength), only available from wild type. Gracilaria is cultivated and produces 65% of the produced agar. Gelidium cartaligineum is harvested in Mexico, and Gelidium spinulosum and Gelidium sesquipedale in


711

Table 11 Compositionsa of typical agaroses2 A units

B units

Species

D-Gal

6-O-Me-D-Ga

L-Gal

3,6-anhydro-L-Gal

Sulfate

Gelidium amansii Gelidium subcostatum Gelidium japonicum Pterocladia tenuis Acanthopeltis japonica Campylaephora hypnaeoides Gracilaria verrucosa Ceramium boydenii

49 44 49 50 47 48 35 31

1.2 6.5 1.4 0.7 2.8 0.7 14.6 18.8

1.9 1.6 1.9 1.3 1.9 3.9 2.0 1.0

48 48 48 48 48 47 48 49

0.05 0.06 0.05 0.06 0.05 0.06 0.05 0.03

a

Expressed as mole percent. Reproduced from Painter, T. J. Algal polysaccharides. In The Polysaccharides; Aspinall, G. O., Eds.; Academic Press: New York, 1983; Vol. 2, pp 195–285, with permission from Elsevier. Table 12 Idealized repeating units in carrageenans2 Identifying letter

A units (b-D-Galp)

B units (a-D-Galp)

k l

3,6-Anhydro 2,6-Disulfate

m n i

4-Sulfate 2-Sulfate (70%) Unsulfated (30%) 4-Sulfate 4-Sulfate 4-Sulfate

x

2-Sulfate

6-Sulfate 2,6-Disulfate 3,6-Anhydro 2-Sulfate 2-Sulfate

Reproduced from Painter, T. J. Algal polysaccharides. In The Polysaccharides; Aspinall, G. O., Eds.; Academic Press: New York, 1983; Vol. 2, pp 195–285, with permission from Elsevier. Table 13 Chemical structure and physical parameters of agars from Chinese Gracilaria spp.13

Species

Gel strength (gcm2)

Gelling temp. ( C)

Melting temp. ( C)

3.6-AG (%)

SO4 (%)

Gracilaria asiatica Gracilaria tenuistipitata Gracilaria blodgettii Gracilaria sjeostedtii Gracilaria chorda Gracilaria eucheumatoides

187 36 258 153 15 12

35.3 29.3 42.0 29.5 29.4 34.5

84.5 76.5 94.5 88.8 76.2 57.0

32.5 23.9 28.2 31.7 21.9 20.7

4.5 10.1 4.2 5.3 7.1 8.8

Spain.13,116 Temperature of gelation, gel strength, composition, and the yield of polysaccharide extracted depend on the season, solar irradiation, etc., and on the algae species (Table 13). The best quality is harvested in Spain, Portugal, Morocco, the Azores, California, Mexico, New Zealand, South Africa, India, Chile, and Japan; at present, it is not yet cultured and still harvested by hand which makes it an expensive polysaccharide. At present, 50% of all agar is produced for food grade and extracted from Gracillaria gracilis (Atlantic) and Gracillaria chilensis (Chile). The other qualities are produced also by Canada, USA, China, Peru, Thailand, Madagascar, and Argentina.

2.21.3.1.1

Extraction of agar

Agar is soluble in boiling water but insoluble in cold water. The extraction of agar is performed from boiled seaweed; the extract is then frozen and thawed. During this last step, water separates from agar, carrying with it soluble impurities. Washing, bleaching, sterilization, and drying are performed depending on the applications. Treatment in the presence of alkali increases the gel strength by formation of the 3,6-anhydrogalactose.80,117a–117c

712


2.21.3.1.2 Molecular structure Agar is built up of alternating D- and L-galactopyranose units; the name comes from a Malaysian name ‘agar-agar’, which means ‘seaweed’. The neutral fraction is named agarose (Figure 15); it is obtained by precipitation of the anionic agaropectins by quaternary ammonium salt and separation by centrifugation. Agaropectin is used for polysaccharides having the same backbone as agarose but containing many acid groups such as sulfate, pyruvate, and glycuronate. The structure of the different types of molecules (agaropectins) is difficult to establish due to the presence of substituents such as methyl ether and 1-carboxyethylidene groups on the A unit and the B units are 118a,118b,119 L enantiomers. The better gel-forming polysaccharide is agarose: the B unit is a 3,6-anhydro-L-galactose with low sulfate content but methyl groups may be present at up to 20% of the A units (Table 11). Three extreme types of agar are recognized: agarose (also named agaran), pyruvated agarose with low sulfate and high 3,6-anhydrogalactose content, and galactan with high sulfate and low pyruvate content.

2.21.3.1.3 Characterization and properties Agar melts c. 85 C and solidifies in the range from 30 to 40 C (Table 13); the hysteresis observed between melting temperature, a gel–sol transition observed on heating, and the gelling temperature corresponding to a sol–gel phase transition observed on cooling, is characteristic of the chemical structure (nature and quantity of substituents). The chemical structure of the different fractions of agaroids can be established by NMR in D2O. Agarose is also soluble in DMSO under the coil conformation and allows easy analysis with not too high viscosity.

2.21.3.1.3.1 (i) Chemical structure Complete analysis of 13C NMR spectrum measured at 80–90 C could be obtained with the help of oligomers prepared by enzymic degradation and dissolved in D2O.120a,120b,121 An NMR spectrum for pure agarose is given in Figure 16 and the chemical shifts are given in Table 14a; the anomeric C1 signals for agarose and carrageenans identification are given in Figure 17. The spectra obtained for oligomers with different types of substituents were also analyzed.122 The 1H NMR is also interesting to identify substituents and compare agarose and carrageenan, as seen in Figure 18 (Table 14b). Chemical methods for analysis of sulfated galactans were also reviewed and they include methylation, hydrolysis, and alditol acetate preparation for gas chromatography (GC) analysis.123 Infrared spectroscopy of dried films is also useful; agars are characterized by the presence of a peak at approximately 936cm–1 due to 3,6-anhydrogalactose, a peak at 890cm–1 due to unsubstituted galactose; diminution or absence of sulfate peaks at 805, 820, 830, 845, and 1250cm–1 is also characteristic. 2.21.3.1.3.2 (ii) Molecular weight determination Different agaroses were analyzed by size-exclusion chromatography using a differential refractometer and a low-angle laser light scattering detector; the samples are first heated at 90–95 C (to get the coil conformation for agarose) and injected in the equipment stabilized at 45 C. The eluent was 0.1M NaNO3 at the flow rate 1mlmin–1. The dn/dc adopted was 0.140.124 The intrinsic viscosity and the Mark–Houwink parameters were determined in 0.75M NaSCN at 35 C; it was demonstrated that under these conditions agarose is in the coil conformation.124 The following relation was established: ½ ¼ 0:07 M 0:72

½5

From this relation, the viscometric average molecular weight can be determined for other agaroses.

O

HO

O

O

OH

HO O

O

n (a)

(b)

→3)-b-D-galactose- (1→4)-3,6-anhydro-a -L-galactose- (1→ Figure 15 Structure of agarose.


100.0 Figure 16

90.0

80.0

70.0

713

60.0 ppm

C NMR spectrum for agarose in D2O at 85 C recorded at 300MHz. The signals are identified in Table 14a.

13

Table 14a NMR signal attributions in D2O at 80 C for agarose and carrageenans (reference TMS): chemical shifts of 13 C signals

k G unit k AG unit i G unit i AG unit Agarose G unit Agarose AG unit

2.21.3.1.4

C1

C2

C3

C4

C5

C6

Reference

102.2 94.98 102.20 92.10 102.44 98.24

69.35 69.69 69.30 74.90 70.27 69.92

78.64 78.90 77.40 77.80 82.24 80.13

73.83 77.97 72.20 78.20 68.79 77.36

74.50 76.50 74.90 77.90 75.55 74.67

60.99 69.21 61.30 69.70 61.43 69.43

144 144 144 144 121 121

Conformation and mechanism of gelation

Agarose adopts a double-helix conformation in the solid state;125a,125b different types of helical structure were predicted by molecular modeling.126 It was shown that the double helix is the basis of gel formation, the main characteristic of agarose. Different techniques can be used for investigating this, among which optical rotation is one.127a,127b The gelation occurs from aggregation of double helices. The gelation temperature is related to the methoxyl and sulfate contents which can prevent gelation. At 1.5% weight in aqueous solution, agar gels melt between 60 and 97 C. Agarose is the neutral fraction which gives the stronger gels. The three-dimensional (3-D) network based on association of double helices is a physical gel stabilized by cooperative H bonds. In addition, these gels undergo a long time syneresis phenomenon. When agarose is solubilized over the melting temperature TF, the agarose turns to the coil conformation; the solution remains stable when it is cooled down to a temperature in the range of the gelling temperature TG. At that temperature, double helices are formed and gelation occurs. Some evidence for a sol–sol transition (spinodal decomposition) depending on the temperature (temperature fixed in the interval TF – TG) was discussed based on photon correlation spectroscopy in a range of concentrations of 0.005–1%. This phenomenon is observed at low polymer concentration and seems to precede the gelation.128 A more recent study interpreted the behavior near the sol–gel transition in terms of percolation.129 The role of alkaline treatment on the reinforcement of the gel strength of agar is clear from Table 15; it decreases the yield in sulfate, increases the 3,6-anhydrogalactose, but does not change significantly the O-methyl and pyruvic acid contents. The conditions for the alkaline treatment are on the average: NaOH 6–10% concentration, temperature 50–90 C, time of treatment 0.5–1h. The mechanism of gel formation and essential properties are described in comparison with the gelation of k- and i-carrageenans. The basis of gelation is the formation of double helices which phase-separate to form a gel; the gels are turbid due to the high degree of helix aggregation and the width of the hysteresis. Stronger gel corresponds with purer agarose; these gels show syneresis (separation of water on aging) corresponding to the slow organization of the double

714


99.2 CH2OH

O HO

O CH2

HO

O

103.4

O

O (a)

OH

O

96.2

CH2OH

CH2 O

RO

O

O

103.4

O

OH

OH R = SO3− (b)

CH2OH

CH2 O

O

RO

O

103.6 96.1

O

95.6

O

OH

OH R = SO3− H (c)

CH2OH

CH2 O

O O

RO

103.2 93.1

O O

OR

OH

R = R = SO3−

105

95

85

(d) Figure 17 13C NMR spectra of anomeric carbons in agarose and carrageenans in D2O at 90 C; chemical shifts in ppm. a, agar; b, k-carrageenan; c, partially desulfated k-carrageenan; d, i-carrageenan. Reproduced from Bhattacharjee, S. S.; Yaphe, W. 13C-NMR spectroscopic analysis of agar, k-carrageenan and i-carrageenan. Carbohydr. Res. 1978, 60, C1–C3, with permission from Elsevier.

helices. This gelation and conformational change may be characterized by calorimetry (Table 16).130 The enthalpy for conformational change (and dissociation of helix aggregates), DH, was found to be nearly independent of the polymer concentration. Water plays an important role in structure stabilization, as was examined by NMR and also by swelling–deswelling experiments in hydro-organic solvents (Figure 19).131,132 These results showed that gels of agarose formed in water do not shrink in acetone or methanol (two nonsolvents for polysaccharides) in contradiction to covalently chemically cross-linked synthetic gels.133a,133b This behavior is in direct relation with the stiffness of the junction zones. Agarose gels are nearly uncharged but there is some sensitivity to external salts following the Hofmeister series for lyotropic effects. The effect occurs in the 0.01–1M range of salt concentration; NaCl weakly stabilizes the gel (increase of TF) while the others destabilize in the order: NaBr NaNO3 < NaSCN There is no selective effect of cation, but anions seem to be preferentially adsorbed.134a,134b,135


5.0

4.5

4.0

3.5

5.0

4.5

4.0

3.5

5.0

4.5

4.0

3.5

ppm

715

3.0

(a)

ppm

3.0

(b)

ppm

3.0

(c) Figure 18 1H NMR spectra in D2O at 85 C for (a) agarose, (b) k-carrageenan, and (c) i-carrageenan (chemical shifts of anomeric protons are given in Table 14b).

716


Table 14b NMR signal attributions in D2O at 80 C for agarose and carrageenans (reference TMS): chemical shifts of H anomeric signals

1

Agarose k-Carrageenan i-Carrageeanan

H1 A unit

H1 B unit

4.53 4.69–4.68 4.70–4.75

5.00 4.96 5.18–5.21

Table 15 Influence of alkali treatment on chemical compositiona and gel properties of Gracilaria agars13 3,6-anhydroAlga

Gel strength

Before treatment with alkali G. damaecornis 10 G. compressa 41 G. debilis 140 G. foliifera 20 After treatment with alkali G. damaecornis 50 G. compressa 80 G. debilis 315 G. foliifera 220 a

Total D-galactose (%)

Pyruvic acid (%)

L-galactose

(%)

Sulfate SO42 (%)

Total L-galactose (%)

42.7 43.5 45.6 42.5

0.17 2.92 0.18 0.13

35.6 37.0 36.1 37.6

6.0 4.1 3.4 2.5

47.6 45.2 42.9 42.6

36.7 38.5 42.9 39.0

0.15 2.50 0.12 0.10

37.5 40.6 47.3 40.8

5.5 1.3 0.8 1.1

48.5 43.2 48.9 43.0

Weight % in the polysaccharide.

Table 16 Enthalpy of helix–coil transition of galactans DH (kJ/dimer) Agarose i-Carrageenan k-Carrageenan (K form) k-Carrageenan (K form)

5.5 0.5 4 0.2 5 0.3 12.1 15.9 12.6 0.5

Conc. (g l l )

Solvent

Reference

4.6-35 24

Water 0.1M NO3K 0.1M KCl Water Water 0.1M KCl

130 130 159 158 158 159

4 32 40.4

2.21.3.1.5 Polymeric complexes Different blends of polysaccharides were tested for compatibility. Synergy was observed with galactomannans (locust bean gum); carboxymethylcellulose has a smaller effect but sodium alginate and starch were found to decrease the gel strength.117a–117c Agar, near neutrality, is compatible with most proteins and other polysaccharides.136 When agarose is mixed with k-carrageenan, it was shown that the two polymers form independent gels (interpenetrated network); the gelling temperatures of the two polymers observed by differential scanning calorimetry (DSC) are perfectly separated on cooling (see Figure 20) and correspond to those of the isolated polymers.137

2.21.3.1.6 Chemical modification The main modification of agar is the alkaline treatment to increase the 3,6-anhydrogalactose content, which enhances the gel properties and increases the melting temperature (Table 15). When other chemical modifications are performed on this linear (AB) co-polymer, there is a disruption of the homogeneous microstructure with a loss of regularity; the ability to form double helices is rapidly lost and the performance of the polymer as thickening polymer decreases. This was demonstrated for the progressive acetylation of agarose under heterogeneous conditions.138


717

100

H2O (%)

80

60

40

20

0 2

4

6

8

10

12

14

16

18

V/V * Figure 19 Dependence of the ratio (V/V*) with the solvent composition at equilibrium. Agarose gel formed at 5gl–1 in water open circles, stars), k-carrageenan formed at 5gl–1 in 0.1M KCl (solid squares, solid triangles), i-carrageenan formed at 20gl –1 in 0.25M KNO3 (open triangle). V is the initial gel volume and V* the volume after equilibrium in hydro-organic solvent mixtures. Reproduced from Rinaudo, M.; Landry, S. On the volume change on noncovalent gels in solvent–nonsolvent mixtures. Polym. Bull. 1987, 17, 563–565. Copyright 2006, with kind permission of Springer Science and Business Media.

dQ dT

2

1

0

30

50

70 T (C)

Figure 20 Calorimetric data: derivative of heat (dQ/dT) in arbitrary units vs temperature for cooling of an agarose/ k-carrageenan mixture (50/50; 10gl–1 of each in 0.1M KNO3). Peak 1 is the gelation of agarose and peak 2 that of k-carrageenan. Mixture data superimposed with the signals obtained for the isolated polysaccharides in the same thermodynamic conditions. Reproduced from Zhang, J.; Rochas, C. Interactions between agarose and k-carrageenans in aqueous solutions. Carbohydr. Polym. 1990, 13, 257–271, with permission from Elsevier.

2.21.3.1.7

Uses of agar

Many applications of agarose and agar are described in the literature:13,117a–117c pharmaceutics, separation and purification media (gel electrophoresis, gel-bead chromatography, size-exclusion chromatography) for fine chemicals, hormones, enzymes, vaccines, suspending barium meals for X-ray of the gastrointestinal tract, sustained-release

718


devices, production of intricate casts used in dentistry, criminology, dye making, vegetarian gelatin substitute, thickener for soups, additive for jellies, ice cream. As food additive, agar has the reference E 406 in the EU. Agarose is also widely used as growth medium for microorganisms (bacteria and fungi) and for biotechnological applications. Agarose is used as macroporous support for gel electrophoresis and immobilization of enzymes and cells. In food industry, soft gels are preferred (agar with some substituents), while pharmaceutical and microbiological industries prefer a harder gel (best agarose).

2.21.3.2 Sulfated Polysaccharides: Carrageenans Irish moss (Chondrus crispus) was the original source of carrageenan until 1960. Then, Kappaphycus alvarezzi (also named cottonii), Betaphycus, and Euchema denticulatum (also spinosum) were cultivated and became the main raw material used for carrageenan production (85% of the production). It is estimated that 50% of all carrageenans are derived from cultivated sources and principally from the genus Euchema produced by the Philippines, the major producer (around 80% of the world production), followed by Indonesia and Zanzibar. The most commonly used are cottonii (Eucheuma cottonii) and spinosum (Eucheuma spinosum), representing three-fourths of the world production. Cottonii variety has been reclassified as Kappaphycus cottonii introducing the genus Kappaphycus, on the basis of the production of kappa carrageenans. The main species used are of Gigartina, Chondrus, Euchema, and Furcellaria; the producers are the Philippines, Indonesia, Canada, Denmark, Chile, Spain, Japan, and France (Tables 3 and 4). General papers are available giving an overview on carrageenans covering topics ranging from production to characterization and properties.2,139–141 Two families can be recognized: the k-family esterified at the 4-position and those which are not (the l-family); the k-family includes i- and k-carrageenans which contain as B unit a 3,6-anhydroþ D-galactose forming gels in the presence of K (see later) as well as the m- and n-carrageenans (without the anhydrogalactose, also named the precursors) (Figure 21); the l-family includes l- and x-carrageenans and the pyruvate containing p-carrageenans, with no anhydrogalactose or gelling properties (Table 12). In this chapter, the properties and characterization of i- and k-carrageenans are mainly described and compared with agarose, because they are the three main gelling polysaccharides from red seaweeds.

2.21.3.2.1 Extraction of carrageenans Extraction of commercial products is normally performed under strong alkaline conditions (to form the 3,6-anhydrobridge) at a temperature near the boiling point for several hours; the extract is filtrered to get a clear solution directly precipitated by addition of isopropanol. To obtain k-carrageenans, another method can be used, the ‘gel press technology’, in which the carrageenan solution is extruded in a concentrated KCl solution followed by removing the liquid phase. The k-carrageenans form the gel phase.

2.21.3.2.2 Molecular structure Carrageenans are composed of alternating units of modified galactose, as given in Table 12. The structures of k- and i-carrageenans are given in Figure 21. l-Carrageenan has no anhydrogalactose but sulfate in the 2- and 6-positions of the B unit and sulfate on the 2-position of the A unit. The different idealized structures were named by Rees.142 In the algae, m and n forms appear as ‘precursors’ of k- and i-structures, respectively, and have no anhydrogalactose. The 3,6-anhydrogalactose is important for the gelling properties of carrageenans.

−O 3

SO CH2OH O OH

O O O OR

(a)

n

(b)

(k) →3)-b -D-galactose-4-sulfate-(1→4)-3,6-anhydro-a -D-galactose-(1→ (i) →3)-b -D-galactose-4-sulfate-(1→4)-3,6-anhydro-a -D-galactose-2-sulfate-(1→ Figure 21 Structure of k-carrageenan (R= –OH) and i-carrageenan (R= –SO–3).


719

There are three classes of carrageenans based on their physical properties:

kappa – strong and rigid gels (from K. cottonii, Chondrus, Hypnea, Furcellaria); iota – soft gels (Euchema spinosum, Hypnea, Gigartina); and lambda – thickening polymer (Girgatina pistilata, Ch. crispus). They are soluble in hot water but form gels on cooling; only lambda remains water soluble. Recently, an intermediate structure between k- and i-carrageenan, named kappa-2 carrageenan, was suggested.143a–143c

2.21.3.2.3

Methods of characterization

2.21.3.2.3.1 (i) NMR spectroscopy This technique is the most powerful to identify the type of carrageenan, but preferentially the samples need purification and transformation to the sodium salt form to be sure that the polymer is in the coiled conformation in dilute solution at ambient temperature. 1H NMR spectra for purified samples of k- and i-carrageenans dissolved in D2O, at 85 C, are given in Figure 18 and the assignment of the different signals for these AB co-polymers is given in Table 14b. All the protons were identified previously taking into account the work of Welti.144,145 Usually, in the spectrum, traces of impurities corresponding to some heterogeneity of the polymers are present (small signals corresponding to H1 of k form appear in the i spectrum and vice versa). This indicates some heterogeneities in the microstructure, which is difficult to avoid even after serious purification. The 13C NMR is also very suitable to identify the type of carrageenans, as shown in Figure 17 (Table 14a).146a–151 In the research described in these papers, oligomers were prepared using specific enzymes able to split the oligomers corresponding to different n values for (AB)n or (BA)n series allowing better data and better interpretation of results.146a,146b,151 The assignment of the signals obtained by 13C NMR of the different carrageenans together with infrared identification are reported in Tables 1 and 2.141 13C NMR is also very useful to identify substituents in the polysaccharides. To conclude, 1H NMR allows getting rapidly a quantitative determination of the different substituents when they exist and also gives information on the degree of purity of the sample tested. This point is important, because it also controls the physical properties, as discussed later. 2.21.3.2.3.2 (ii) Infrared spectroscopy Infrared spectroscopy is also often used for the characterization of carrageenans, and the spectra obtained allow the identification of the structure as well as the presence of substituents. The spectra are realized on thin films casted from solution. The anhydrogalactose and sulfation are clearly identified.142,152 2.21.3.2.3.3 (iii) Size-exclusion chromatography The application of steric exclusion chromatography was adopted to determine the molecular weight distribution. The equipment used was the same as described for alginate (Section 2.1.3(ii)) and includes a Waters chromatograph GPCV Alliance 2000 connected to three detectors on line (differential refractometer giving the polymer concentration; viscometer giving the relative viscosity; MALLS detector giving the molecular weight and the radius of gyration). The molecular weight distribution can be calculated from these data without the necessity of any standard for calibration, as well as the average molecular weights, the intrinsic viscosity, and the Mark–Houwink parameters. An example of results is given in Figure 22 for a k-carrageenan purified and isolated in the sodium form. From these results, the intrinsic viscosity was calculated to be []¼319mlg–1 in 0.1M NaNO3 and the weight-average molecular weight Mw ¼215000. This technique has been used before by various authors;154–157 dn/dc as determined by the different teams was found to be ranging between 0.095 and 0.150mlg–1 for k-carrageenan and between 0.113 and 0.154mlg–1 for i-carrageenan. In pure water, the following values were obtained: 0.127 for iota- and 0.140 for kappa-carrageenan.156 In 0.1M NaCl, dn/dc¼0.123 is obtained for kappa-carrageenan, and in 0.05M LiCl 0.129 for iota144 at 30 C, values of 0.113 and 0.118 were obtained for i-carrageenans in 0.1M LiCl at 60 C,154 and 0.126 in 510–3 M NaCl and KCl at 25 C for k-carrageenan.155 For k-carrageenan at 25 C in 0.1M NaCl, the intrinsic viscosity allows the determination of the molecular weight using the following relation: ½ ¼ 3:1 103 M 0:95 This relation was obtained on fractions obtained by preparative gel chromatography.154

½6

720


Peak ID - 21mr11-02_01

LS #11, AUX1, AUX2

0.06

LS # 11 AUX1 AUX2

0.04 0.02 0.00

−0.02

0

10

Differential weight fraction

(a)

(b)

20 Volume (ml)

30

21mr11-02_01 Norm = log 1st order

Differential molar mass

0.16

40

0.12 0.08 0.04 0.00 1.0 103

1.0 104

1.0 105 Molar mass (g mol−1)

1.0 106

1.0 107

Figure 22 SEC chromatography on a k-carrageenan at 30 C; solvent 0.1M NaNO3 with three detectors on line. a, The chromatogram with light scattering detector (in red), the viscometer (in green), and the differential refractometer (in blue). b, Differential molecular weight distribution for a purified k-carrageenan.

2.21.3.2.4 Conformation of k-carrageenan and ionic selectivity For stereoregular charged polysaccharides, the formation of a helical conformation and eventually gelation depends on the ionic concentration, nature of electrolyte, and temperature. The helix–coil transition for k-carrageenan was demonstrated by different techniques: conductivity, optical rotation, NMR, and DSC.157–159 First, it was demonstrated that the stability of the ordered conformation depends on the DP of k-carrageenan. Samples with different DPs were obtained from enzymic hydrolysis and the optical rotation (then, the conformation) of aqueous solution in 0.1 M KCl goes to the same limit at low temperature but the melting temperature (corresponding to the helix–coil transition) increases when the DP increases up to 45; the behavior is then the same as for a light molecular weight polymer.149 This seems to indicate that the helical structure involves all the polymeric chains without any disorder.149 From optical rotation as shown in Figure 23, an ordered conformation is stabilized at low temperature and goes to a disordered conformation when temperature increases. This conformational transition is characterized by Tm, the temperature for conformational helix–coil change determined at half transition. In fact, the helix–coil transition is perfectly reversible (no hysteresis) at low ionic concentration (Figure 23a), but hysteresis appears when the ionic concentration increases. The hysteresis modifies after aging corresponding to an increase of the degree of aggregation, which is fact related to the syneresis phenomenon discussed later (Figure 23b), but the temperature for coil–helix transition on the cooling part remains unchanged. At the same time, at lower temperature, the molecular weight is doubled and the activity coefficient g of monovalent counterions is changed. This is clearly demonstrated in Figure 24. In the figure, the activity of sodium and potassium counterions were determined in salt-free solution but as a function of the polymer concentration (corresponding to an increase of the ionic concentration causing screening of electrostatic repulsions). At 15 and 35 C, the activity of Naþ is nearly not influenced by temperature or polymer concentration, and it tends to a value of 0.71 at infinite dilution. This value is in good agreement with the prediction from the polyelectrolyte theories and corresponds to a single linear chain. The same values are obtained for Naþ and Kþ at 35 C, but at 15 C, a transition is observed in the Kþ activity coefficient which goes to 0.37 and corresponds to a double charge parameter l.


721

a 300

500

400

300 (a)

10

20

30

T (C)

a 300

500

450 A, B, C 400

350

(b)

10

20

30

T (C)

Figure 23 Temperature dependence of the specific optical rotation at 300 nm for k-carrageenan under K salt form (a) in absence of external salt. Concentration 1.0510–2 equiv. l–1; b, in the presence of KCl. Concentration 0.17910–2 equiv.l–1; salt concentration 0.910–2 equiv.l–1 with A, the cooling curve, followed directly by B, the heating curve; C depicts the heating curve after 15h aging at 21 C. Reprinted with permission from Rinaudo, M.; Rochas, C. Investigations on aqueous solution properties of kappa carrageenans. In Solution Properties of Polysaccharides; Brant, D. A., Ed.; ACS Symposium Series 150; American Chemical Society: Washington, DC, 1981; pp 367–378. Copyright 2006, American Chemical Society.

The interpretation of these data is unambiguous; particularly, in the presence of Kþ, two chains associate (molecular weight and l double) to form a double-helical conformation (shown by optical rotation).160,161 Kinetic studies also allow one to conclude for the dimer formation.162 On i-carrageenan, a doubling of the molecular weight was also found in nonaggregating conditions.156 In the solid state, X-ray diffraction also demonstrated that double helices are formed in k- and i-carrageenans.163a–163c The transition in k-carrageenans was characterized by an enthalpy of conformational change DH much larger than for agarose or i-carrageenan (Table 16).57,159 The experimental values were compared with predicted values obtained by application of the Manning treatment for polyelectrolyte without good agreement in fact.164,165 This point was also discussed from a more theoretical approach.166 The different values of Tm, TF, and TG can be determined from heating and cooling curves obtained by optical rotation or DSC on aqueous solutions of k-carrageenan and plotted in a log–log representation of these characteristic

722


g 0.7

0.6

35 C Na+, K+ 15 C Na+

0.5 0.4

15 C K+

0.3

0.05

0.10

0.15

0.5 Cp

eq/I

Figure 24 Activity coefficient of counterions as a function of the root square of the polymer concentration (with Cp in equiv.l–1) in absence of external salt at 15 and 35 C. Reprinted with permission from Rinaudo, M.; Rochas, C. Investigations on aqueous solution properties of kappa carrageenans. In Solution Properties of Polysaccharides; Brant, D. A., Ed.; ACS Symposium Series 150; American Chemical Society: Washington, DC, 1981; pp 367–378. Copyright 2006, American Chemical Society.

temperatures (T –1), as a function of the total ionic concentration CT. The helix–coil transition is perfectly reversible (no hysteresis) at low ionic concentration (CT lower than a critical value CT* around 7.510–3 M in KCl and 210–1 M in NaCl), but hysteresis appears when the ionic concentration increases as shown in Figure 23.160 Figure 25 gives the phase diagram for k-carrageenan in presence of Kþ and Naþ counterions and salt excess. Figure 25 introduces the evidence of ionic selectivity which appears in the coil conformation first and this was a little surprising because, according to the Manning model, it was expected that selectivity occurs when the charge parameter, a characteristic for a polyelectrolyte, is larger than 1, as was found previously for carboxymethylcellulose.167 The ionic selectivity was associated with ion pair formation, and this could be demonstrated using ultrasound absorption.168 With k-carrageenan, under coil conformation, the charge parameter l is equal to 0.68. Nevertheless, ultrasound absorption showed an ionic selectivity with potassium forming more ion pairs (at least more dehydration) than sodium.169 The ion pair formation with Kþ reduces the net charge of the polymer and favors the helical dimer formation, as shown in Figure 25. The phase diagram relating the total ionic concentration and the inverse of the temperature for conformational (helix– coil) or phase (sol–gel) transition was established for the different monovalent and divalent counterions. For k-carrageenan, the ionic selectivity among monovalent cations is very important while it is low among divalent counterions; the Tm values were determined at a constant ionic concentration (0.1M) and Tm was varying following the sequence:161 þ Rbþ > Kþ ; Csþ > Naþ > Liþ ; NHþ 4 > R4 N

For divalent counterions, a small selectivity is observed with the sequence: Ba2þ > Sr2þ Ca2þ > Mg2þ Zn2þ > Co2þ No selectivity was observed among the monovalent anions with the exception of I–, which stabilizes the helix but prevents gelation, as discussed later.170–174 The phase diagrams for the helix–coil transition of k- and i-carrageenans in different ionic salt forms have been established, and the stability of the different conformation discussed by Rinaudo et al.175

2.21.3.2.5 Properties in solution k-, i-, and l-carrageenans are polyelectrolytes characterized by a charge parameter (l), which depends on the conformation (helix formation). For such polymers, l controls the activity coefficient of counterions (g). The


723

CT eq/I

C T* Na+ 10−1

K+ 10−2 C T*

2.85

3.05

3.25

3.45

Tm−1 (K−1) 103

Figure 25 Phase diagram obtained by plotting the inverse of the temperature for conformational change Tm (crosses, open circles) when CT CT* with the logarithm of the total concentration of counterions CT (including external salt and free counterions from the polymer). The phase diagram is established for potassium and sodium salt. Reprinted with permission from Rinaudo, M.; Rochas, C. Investigations on aqueous solution properties of kappa carrageenans. In Solution Properties of Polysaccharides; Brant, D. A., Ed.; ACS Symposium Series 150; American Chemical Society: Washington, DC, 1981; pp 367–378. Copyright 2006, American Chemical Society.

properties of these polysaccharides can be related to their chemical structure, as can be deduced from their sequence in solubility, Agarose < k-carrageenan < i-carrageenan < l-carrageenan and the order of sulfate density (and charge density), No sulfate < 1 sulfate < 2 sulfates < 3 sulfates With the exception of l-carrageenan, these polysaccharides involve 3,6-anhydrogalactose units and form physical gels at moderate temperature with some hysteresis between heating and cooling, as discussed later; only l-carrageenan remains water soluble in all conditions and increases the viscosity of the solvent.

2.21.3.2.6

Mechansim of gelation

Agarose, k-carrageenans, and i-carrageenans are recognized as gelling polymers but their properties depend on the chemical structure; the conditions of gelation as well as the mechanism of gelation have been abundantly discussed in the literature.

724


For these polymers, the physical thermoreversible gel formed is stabilized by H bonds between double helices. From Figure 25, it can be seen that when the ionic concentration increases over CT*, the helix–coil transition is no more reversible, and that some hysteresis in relation with aggregation of double helices occurs. The aggregates are more stable when temperature increases than the isolated helices. Then, the hysteresis is characterized by two temperatures: TF, the melting temperature of the gel, and TG, the temperature for gel formation (with TF >TG). It must be pointed out that Tm, the temperature for helix formation, is identical to TG over CT*; this implies that the helices form in the same time as the gel is formed on cooling. The aggregates or junction zones are the basis of the network formation: gelation proceeds in a two-step process as it has been clearly demonstrated for thermodynamic conditions around CT* and a polymer concentration larger than the overlap concentration (see Figure 23b).160 The width of the hysteresis in temperature is directly related to the degree of aggregation of double helices and the charge density of the polymers (width of this hysteresis decreases from agarose to k-carrageenan to i-carrageenan). The related rigidity of the gels follows the same trend. The stiffness of the gels formed in presence of Kþ counterions varies in the following order: Agarose > k-carrageenan > i-carrageenan For k-carrageenan, the mechanism was described in detail and related to the solution behavior. The ionic selectivity observed for the double-helix induction is also recognized for the gelation; the ion pairs which reduce the net charge of the single coil play a role in the double-helix stabilization but also in the aggregation of the double helix.176 In this process, the electrostatic repulsion is counterbalanced by cooperative H-bond attractions. The mechanical properties of the gels formed in the presence of different counterions follow the same order as for the stability of the double helices: potassium-k-carrageenan gels form at a lower polymer and ionic concentrations and have a higher modulus and a higher melting temperature than tetramethylammonium-k-carrageenan gels, which only form at much higher ionic concentrations and have a much lower modulus as determined in dynamic rheological experiments. For example, in 0.1M salt, at 0.5Hz and 20 C, 1gl–1 potassium-k-carrageenan gives a strong gel with an elastic modulus G0 ¼57.45Pa, while the tetramethylammonium form even at 10 gl–1 gives no gel at 20 C. It was shown that i-carrageenan exhibits only very low ionic selectivity and when gelation occurs it may be due to k-type impurities. The role of I– is very important;170,172–174 this anion stabilizes the double helix but prevents gelation; the mechanism of its interaction with k-carrageenan is not clearly established. Nevertheless, the stiff double helices are dispersed in solution, and in absence of aggregation they form a cholesteric liquid crystalline phase.174 The mechanical properties of the k-carrageenan gels formed were investigated by compression measurements, and it was demonstrated that during gel formation at a given KCl concentration the elastic modulus increases when the molecular weight increases up to M 300000 and that the modulus is directly related to the polymer concentration (Figure 26).153 For the same polymer concentration, the modulus depends on the molecular weight but also on the ionic KCl concentration (causing the screening of the electrostatic repulsion) and goes to a limit for the same range of molecular weight (Figure 27). The yield stress for gel rupture however increases linearly with the molecular weight in the range covered. When the gels are formed from k-carrageenan, they present the syneresis phenomenon corresponding to a slow evolution with reorganization of the gel and water expulsion; at the same salt concentration, this mechanism is stronger at lower polymer concentration (Figure 28).144 The gels from i-carrageenan are less strong with less syneresis and need larger salt excess to be formed; in direct relation with these differences, a polymer fractionation can be performed by gel-phase separation in the presence of KCl based on the different gel points for the k- and i-polymers.177 The properties of different samples of i-carrageenan often depend on the presence of k impurities.178 The elastic modulus increases when the ionic concentration increases up to 0.25M and, at higher concentrations, it decreases due to a salting out effect. A 10gl–1 gel formed in 0.25M KCl has an elastic modulus of 0.32104 Pa, while for a k-carrageenan gel in 0.25M KCl it is 6.6104 Pa. To conclude, as also displayed in Figure 19, the stiffness of i-carrageenan is low due to shorter junction zones with lower stability (larger charge density) and they shrink strongly in nonsolvents. From a general point of view, for all these physical gels, it was found that the elastic modulus varies according to the following equation:179 E ¼ KC 2 0:1 C is expressed in gl–1; in 0.25 M KCl, K¼745 and 43 for k- and i-carrageenans, respectively.144

½7


725

E

12

10

8

6

4

2 *

0

*

*

*

0

2

*

*

*

4

MW

6

105

Figure 26 Elastic modulus (104 Pa) obtained in compression measurement as a function of the molecular weight of different fractions of k-carrageenan in 0.1M KCl for 5gl–1 (stars), 10gl–1 (open squares), and 20gl–1 (solid squares). Reproduced from Rochas, C.; Rinaudo, M.; Landry, S. Role of the molecular weight on the mechanical properties of kappa carrageenan gels. Carbohydr. Polym. 1990, 12, 255–266, with permission from Elsevier.

E

6

4

2

0 0

2

4

6

MW 105

Figure 27 Elastic modulus (104 Pa) obtained in compression measurement as a function of the molecular weight of different fractions of k-carrageenan at 10gl–1 in different KCl concentrations: 0.05M (solid squares); 0.1M (open triangles); 0.5M (solid triangles). Reproduced from Rochas, C.; Rinaudo, M.; Landry, S. Role of the molecular weight on the mechanical properties of kappa carrageenan gels. Carbohydr. Polym. 1990, 12, 255–266, with permission from Elsevier.

The gels are more or less stable in the presence of organic solvent (Figure 19); nevertheless, the nonsolvents are more efficient when the charge density increases and the stiffness and the degree of helicity decrease.

2.21.3.2.7

Polymer complexes

Synergy exists when k-carrageenan is mixed with locust bean gum (galactomannan);180 the resulting gel is reinforced but the syneresis effect decreases. Different mixtures were investigated with other galactomannans136,181,182 or other

726


Syncresis (%)

30

20

10

(a)

50

100

150

200

250 Time (h)

E 13

12 3.6 3.3 0.4

0.3 (b)

5

10

15

20

25 Time (h)

Figure 28 Syneresis (in % of volume change) and elastic modulus (10 4 Pa) as a function of time obtained on a k-carrageenan prepared at different concentrations in 0.1M KCl. (a) Polymer concentrations: 5gl–1 (solid squares); 2.5gl–1 (solid triangles); 1gl–1 (solid stars). (b) Polymer concentrations: 20 gl –1 (solid squares); 10gl –1 (solid triangles); 5gl–1 (open triangles.)144

polysaccharides.137,183–187 Mixtures of k- and i-carrageenans were also investigated within a large range of compositions.144,147 Lambda carrageenans form gels when mixed with proteins; many papers are also concerned with the role of carrageenans in milk.188–190 Carrageenans, negatively charged polymers, interact with proteins (especially with the milk components); at pH lower than the isoelectic point of the protein, the protein is positively charged and interacts strongly with carrageenan and the complex precipitates. Above the isoelectric point, the two polymers are negatively charged and Ca2þ ions make a bridge between the two polyelectrolytes.

2.21.3.2.8 Uses of carrageenans Industrial applications are the most important uses; 30000tyr–1 of carrageenan of the three types are produced.13,80,117c,139–141 When used for food products, carrageenans have an EU additive number E 407. Their essential characteristic induces their use as thickening and stabilizing agents. Carrageenan is also used to solidify and emulsify solutions. Carrageenans are used to prevent chocolate milk from creaming and sedimenting and to permit solidification of flans, custards, yoghurts, chocolate milk, ice creams, and milk puddings. Toothpastes and canned and frozen pet foods contains carrageenans to allow solidification. Beers are clarified with carrageenans used to complex and precipitate proteinaceous impurities. As agarose and alginates, carrageenans are used for encapsulation.191 The behavior observed depends directly on the composition of the systems in which carrageenans are incorporated. It seems that it is recognized now, that degraded carrageenans might cause ulcerations in the gastrointestinal tract and gastrointestinal cancer.


727

2.21.3.3 Other Sulfated Polysaccharides from Red Seaweeds Agar, alginates, and carrageenans constitute certainly the more developed production of algae polysaccharides. There are also several other less-known seaweed species with potential applications. Many different names were introduced in the past related to the name of the algae source.117c Hypnean was suggested for agar-like extract from Hypnea musciformis and other Hypnea; iridophycan is extracted from Iridaea (previously named Iridophycus), porphyran is extracted from Porphyra, and dulsan from Rhodymenia.

2.21.3.3.1

Hypneans

2.21.3.3.1.1 (i) Structure and properties Hypneans are produced from Hypnea seaweed species; their main value is a strong ability to form gels.117c They are extracted in water between 50 and 121 C; KCl is added to reduce the viscosity of this extract. The extract is filtrated and the polysaccharide is recovered by freeze–thawing, which separates KCl and other water-soluble carbohydrates. The chemical structure of this polysaccharide looks like that of agar and carrageenan with a high percentage of 3,6-anhydrogalactose forming strong thermoreversible gels. The gel strength depends on the presence of electrolytes with some ionic selectivity (lyotropic series) mainly among the cations; for chloride salts, the sequence in decreasing order is þ Csþ > Rbþ > Kþ > NHþ 4 Na

and for potassium salt, the anions rank as follows: 2 AcO > Cl > SO2 4 > Br > HPO4 > NO3

Just as for k-carrageenan, KCl is known to influence the gel strength, the gelling temperature, and the melting temperature. Hypnean gels also show syneresis depending on the salt used.

2.21.3.3.1.2 (ii) Uses of hypneans These polysaccharides are mainly used for their gelling properties for food applications; they are known as a vermifuge in Indonesia, Greece, and Turkey. In the north of Brazil, they use the algae (H. musciformis) as fertilizer for young coconut palms in the arid, sandy soil (the palm roots are covered with seaweeds to keep them humid).

2.21.3.3.2

Porphyran

2.21.3.3.2.1 (i) Structure and properties Porphyran is a highly substituted agarose-type polysaccharide from the red alga of the Porphyra genus such as Porphyra umbilicalis mainly produced in Japan. It is constituted of 4-linked 6-O-sulfo-a-L-galactopyranose residues (the biological precursor of the 3,6-anhydro residue) and 3-linked 6-O-methyl-b-D-galactopyranose residue.192 After enzymatic fragmentation, NMR studies on oligosaccharides demonstrated that a neutral fraction is constituted of 6(3)-O-methyl-neoagarotetraose and 6(3),6(5)-di-O-methyl-neoagarohexaose; the anionic fraction reveals 6-O-sulfatoa-L-galactopyranosyl-(1!3)-b-D-galactopyranosyl-(1!4)-3,6-anhydro-a-L-galactopyranosyl-(1!3)-D-galactopyranose and its 6(3)-O-methylated derivative. The polymer was described as being composed for 49% of sulfated disaccharide units. The extract from Porphyra capensis (Rhodophyta) has a linear backbone of alternating 3-linked b-D-galactose and 4-linked a-L-galactose 6-sulfate or 3,6-anhydro-a-L-galactose units. The ratio of a-L-galactose 6-sulfate and the 3,6anhydrogalactose is 1.2:1 (from NMR data). A high degree of sulfation occurred at the C6 position of D-galactose units. The degree of methylation of the galactose residues was 0.64.193 The polymer extracted from Porphyra haitanensis shows a typical porphyran structure with a linear backbone made of alternating 3-linked b-D-galactopyranosyl units and 4-linked a-L-galactosyl 6-sulfate and a minor amount of 3,6anhydrogalactosyl units. Partial methylation occurs at C6 position of the D-galactosyl unit and at C2 position of the 3,6-anhydro-a-L-galactosyl units.194a,194b Similar types of polysaccharides were also extracted from different species of Laurencia (Laurencia papillosa, Laurencia cruciata, Laurencia pedicularioides, Laurencia majuscula).195 From Porphyra yezoensis, a polysaccharide with the following constituent units was found: galactose, 3,6-anhydrogalactose, 6-OSO3-galactose, xylose in ratio 25:15:10:1; the proposed repeat unit is [(1!3)-b-D-Gal-(1!4)-3,6-anhydro-a-L-Gal-]3-[(1!3)-b-D-Gal-(1!4)-a-L-6-OSO3-Gal-]2

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with a xylose moiety at the C6 of 1 of every 25 b-D-Gal residues. The authors claimed that it is the first porphyran possessing occasional xylose branches.196 Porphyran can be treated by alkali to increase the gelling ability.197 After alkaline treatment, the polysaccharides have the properties of agarose.

2.21.3.3.2.2 (ii) Uses of porphyran Porphyran is a gelling polysaccharide;117c it was fractionated and two fractions were tested for their ability to activate macrophages in vitro and in vivo.198 It was also used as a nutrition complement; it is claimed to improve immunity of humans with stress. After fractionation, it was shown that porphyran has a high emulsifying activity index and high emulsion stability over a wide pH and temperature range.199 Fractions of porphyran were proved to have antioxidant activities. They have a strong effect on the superoxide radical and/or hydroxyl radical and lipid peroxide.194a,194b

2.21.3.3.3 Furcellaran Furcellaran is produced by Furcellaria lumbricalis, from the class of Rhodophyceae, a red seaweed.200 Its most important property is to form gels used in the food and pharmaceutical industry to obtain water- or milk-based gels; it is produced in Denmark and Estonia.201 It is currently considered to be a type of k-carrageenan. The chemical structure is based on (1-3)-linked b-D-galactopyranose, (1-4)-linked 3,6-anhydro-a-D-galactopyranose, and (1-3)-linked b-D-galactopyranose 4-sulfate.202 The 13C NMR characteristics are given in Table 17. Alkaline treatment removed the sulfate precursor sequences with formation of 3,6-anhydrogalactose. This transformation increases the gelling ability of furcellaran. Its composition for native, processed, and commercial preparations are given in Table 18.202 Furcellaran can be considered as a low-sulfated polysaccharide of the carrageenan family. A coil–helix transition is promoted by potassium and rubidium counterions, followed by gelation. Under comparable conditions, the conformational transition temperature for furcellaran was found to be higher than that of k-carrageenans.203

2.21.3.3.4 Funoran Funoran is extracted with water at c. 100 C from red algae such as Gloiopeltis complanata or Gloiopeltis furcata (Postels and Ruprecht) (Cryptonemiales, Rhodophyta).204,205 They consist of a heterogeneous series of polysaccharides, Table 17 Chemical shift assignments for 13C NMR spectra of furcellaran (G: (1-3)-linked b-D-galactopyranose; AG: (1-4)linked 3,6-anhydro-a-D-galactopyranose; G4S: (1-3)-linked b-D-galactopyranose 4-sulfate202 Chemical shifts (ppm) Unit

C1

C2

C3

C4

C5

C6

G AG G4S

102.5 94.6 102.5

69.5 70.2 69.5

80.4 79.4 78.8

66.4 78.1 74.1

75.3 76.8 74.8

61.3 69.5 61.3

Reproduced from Laos, K.; Ring, S. G. J. Appl. Phycol. 2005, 17, 461–464, Copyright 2005, with kind permission of Springer Science and Business Media. Table 18 Characteristics of the furcellarans202 Neutral sugar a composition (% w/w carbohydrate) Sample

Gal

3,6-AGal

6-O-MeGal

Glc

Xyl

Man

Sulfate (%w/w)

Native Alkali modified Commercial

64–65 59–60 55–57

25–26 31–32 36–37

3.9–4.4 4.7–4.8 4.7–4.8

2.7–2.8 1.8–1.9 1.8–1.9

1.1–1.7 1.1–1.2 0.52–0.53

1.1–1.7 1.1–1.2 0.52–0.53

17.5–18.3 16.2–17 17.6–18.3

a Gal, galactose; 3,6-AGal, 3,6-anhydrogalactose, 6-O-MeGal, 6-O-methylgalactose; Glc, glucose; Xyl, xylose, Man, mannose. Reproduced from Laos, K.; Ring, S. G. J. Appl. Phycol. 2005, 17, 461–464, Copyright 2005, with kind permission of Springer Science and Business Media.

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sulfated galactans, with a basic structure similar to sulfated agarose, a precursor moiety, and carrageenan-type molecules.117c Two fractions at least are separated from G. complanata; one forms gels (PS2) in the presence of KCl and the other does not (PS1).204 The fraction PS2 was again separated in two fractions, PS2G and PS2S; each of them was alkali-treated; their compositions are given in Table 19. From G. furcata, three fractions were isolated in four different KCl concentrations at room temperature. Eighty-six percent of the material was in PS4 extracted with 4M KCl at 100 C.205This fraction is constituted of 6-sulfated agarose (also named agaroid) recognized by a negative optical rotation, while the other fractions showed positive optical rotation. The compostion of the fraction PS3 was examined and is given in Table 20. Table 21 represents the main di- and trisaccharides obtained by hydrolysis. The main properties described in the literature for funoran from G. furcata is that it inhibits the adherence and colonization of oral bacteria;206 it was found to decrease caries in rat studies. It was also mentioned to reduce the blood pressure and lower cholesterol.207 The funoran from Gloiopeltis tenax inhibits the growth of Ehrlich ascites carcinoma and other solid tumors in mice. It is suggested that the antitumor effect of funoran is related to the augmentation of T-helper, T-cytotoxic, and NK cells.208

Table 19 Yield and compositiona of funoran fractions PS1, PS2G, and PS2S from the red algae, Gloiopeltis complanata and their derivatives after alkali treatment (-A)204 Yield (%) b PS1 PS2G PS2G-A PS2S PS2S-A

0.9 4.8 1.4

D-Gal

6M-D-Gal c

L-Gal

2M-L-Gal c

L-AGal

87.7 97.4 98.0 95.8 96.5

12.3 2.6 2.0 4.2 3.5

4.3 8.9 0.1 54.5 25.5

1.7 1.3 0 2.0 0

3.6 76.0 85.9 33.4 52.4

d

SO 3 158.8 109.0 89.0 139.7 77.5

a

Molar ratio. Total of D-series galactoses is taken as 100. Based on the dry seaweed. c 6M-D-Gal¼6-O-methyl-D-galactose, etc. d 3,6-Anhydro-L-galactose. b

Table 20 Compositionsa of funoran fraction PS3 before and after alkali treatment (PS3A) from Gloiopeltis furcata205

D-Gal

6-O-Methyl-D-Gal 3,6-Anhydro-D-Gal L-Gal 3,6-Anhydro-L-Gal Sulfate (as SO3K)

PS3

PS3A

30.1 (100) 2.3 (7) 0 (0) 5.4 (18) 2.7 (10) 39.7 (179)

30.7 (100) 2.9 (8) 6.1 (22) 0 (0) 10.0 (36) 34.9 (155)

a

Expressed as wt.%. The calculated values for the sugars are based on molecular weight of anhydro sugar unit. The numbers in the parentheses express molar ratio (the value for D-Gal is taken as 100).

Table 21 Disaccharides and trisaccharides isolated from partial hydrolysis product of funoran PS3 fraction205 1 2 3 4 5 6 7 8 9 10

b-D-Gal-(l!4)-L-Gal b-D-Gal-(l!4)-D-Gal a-L-Gal-(l!3)-D-Gal a-D-Gal-(l!3)-D-Gal b-D-Gal-(l!4)-3,6-anhydro-L-Gal 6-O-Methyl-D-Gal-(1!3)-D-Gal a-L-Gal-(l!3)-b-Gal-(1!4)-L-Gal a-D-Gal-(l!3)-b-D-Gal-(1!4)-D-Gal b-D-Gal-(l!4)-a-L-Gal-(1!3)-D-Gal b-D-Gal-(l!4)-a-D-Gal-(1!3)-D-Gal

730


2.21.3.3.5 Dulsan Dulsan is extracted from Rhodymenia palmata, a red alga known as dulse; this alga is completely digestible by man and was mainly used for food. It does not form gels and it is water soluble; acid hydrolysis gives principally D-xylose, but also galactose and glucose with (1-3) and (1-4) linkages.117c

2.21.3.3.6 Iridophycan Iridophycan is extracted from Iridaea or Iridophycus genera. It is extracted with boiling water and filtrated before isolation. It was shown that it is a sulfated galactan giving only D-galactose after hydrolysis. Some authors consider that the extract from alga is a mixture of fucan and galactan. In general, the properties look like that of carrageenan or funoran, but no studies of these properties are published. It is used as a cold-mix stabilizer, for its adhesive properties, and it prevents blood coagulation.117c

2.21.4 Conclusion The main seaweeds exploited for industrial applications are described. They are directly consumed, especially in the Eastern countries. Seaweeds are a source of many valuable molecules, and today more and more pharmaceuticals are isolated from them. Seaweeds are recognized to have many biomedical applications and these developments are in progress in many countries. The industrially important species are more and more cultivated. The polysaccharides extracted from some of the selected seaweeds are important from a commercial point of view. These polymers are often water soluble and have very different chemical structures, which also means different physical properties and interactions; they are good thickeners but also depending on the thermodynamic conditions they can form physical gels; two main types of gelation mechanisms are recognized and discussed in this chapter: alginates form gel in the presence of divalent counterions (Ba, Sr, Ca) in relation with the existence of guluronic acid blocks. k- and i-carrageenans are sulfated galactans forming gels by association of double helices when they contain anhydrogalactose units; the same mechanism is valid for agarose. Ionic selectivity is discussed, and it is shown that the nature of the counterions (and also the co-ion) is important in all these polysaccharide systems; it plays a role not only on the conformation of the polymers even in dilute solution (helical or coiled conformation) but also on the net charge of the polymers and intermolecular interactions going to gelation.

Glossary copolymer A polymer formed with a least two different monomeric units included in the same polymeric chain. DPn number-average polymerization degree = Mn/m0. homopolymer A polymer formed with a single type of monomeric unit. intrinsic viscosity [] Represents the specific hydrodynamic volume of a polymer in solution; it is related to the molecular weight of the polymer (in mlg–1). m0 The molecular weight of the repeat unit. persistence length Characterizes the local stiffness of a polymeric chain (in nm). polyelectrolyte A polymer with ionic sites regularly disposed along the backbone. SEC Size-exclusion chromatography; fractionates the polymeric chains following their hydrodynamic volume. syneresis Aging of a gel which shrinks with expulsion of free solvent. u-state Characterizes a polymer–solvent system at a given temperature y in which the solution is ideal; for a given polymer, it includes well-defined solvent and temperature.

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Biographical Sketch

Marguerite Rinaudo has a master’s degree (1964) and a Ph.D. (1966) from the University of Grenoble (France) on physicochemical properties of polyelectrolytes; her work was devoted to the synthesis and solution properties of carboxymethylcelluloses. From that time, she continued to develop her research work on water-soluble polymers and especially on polysaccharides (cellulose, starch and chitosan derivatives, bacterial polysaccharides, seaweed polysaccharides); in her laboratory, she set up a series of equipment and methodologies to investigate the specific behavior of polysaccharides, which have to be considered as semirigid polymers. She started as full professor in 1968 at Joseph Fourier University (Grenoble, France); she has been emeritus professor since January 2002. For 12 years (1984–96), she has been the director of a CNRS institute named Centre de Recherches sur les Macromolećules Ve´ge´tales, working on plant oligo- and polysaccharides. She has received important awards from the French government (Chevalier dans l’Ordre National du Me´rite (1984) and dans l’Ordre de la Le´gion d’Honneur (1998)); she is also an associate member of the Chilean Academy of Sciences (2005) and of the Brazilian Academy of Sciences (1991). She has published more than 400 original papers and delivered more than 250 main lectures in international meetings. She has also supervised more than 50 Ph.D. theses, and she is a member of the editorial board of 5 international journals (including Biomacromolecules and Food Hydrocolloids).

2.22 Biosynthesis of Cellulose H. Ho¨fte, M. Gonneau, and S. Vernhettes, INRA Versailles-Grignon, Versailles, France ß 2007 Elsevier Ltd. All rights reserved. 2.22.1 Cellulose, A Sophisticated Macromolecule

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2.22.1.1 Cellulose Is Ubiquitous

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2.22.1.2 Cellulose Structure

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2.22.1.3 Degree of Polymerization

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2.22.1.4 The Cellulose Synthesizing Complex

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2.22.1.5 Orientation of Cellulose Microfibrils

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2.22.1.6 Critical Role of Orientation of Microfibrils in Growth Control

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2.22.2 Discovery and Evolution of the Cellulose Synthesis Machinery

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2.22.2.1 Cellulose Biosynthesis in Bacteria

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2.22.2.2 Structure–Function, Characterization of the Cellulose Synthases

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2.22.2.3 Possible Enzymatic Mechanisms for Synthesis of Cellulose

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2.22.2.4 Plant Cellulose Synthases

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2.22.3 Lessons from Cellulose-Deficient Mutants

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2.22.3.1 A New Strategy to Identify Cellulose-Deficient Mutants: FTIR Spectroscopy

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2.22.3.2 Developmental Function of CESA Genes

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2.22.3.3 The CSL Superfamily

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2.22.3.4 The Role of a Cellulase in Cellulose Synthesis

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2.22.3.4.1 2.22.3.4.2 2.22.3.4.3 2.22.3.4.4

Structure and post-translational modifications of KOR1 Substrate specificity of KOR1 Developmental role of KOR1 Other KOR1-related genes

2.22.3.5 Other Actors Involved in Cellulose Synthesis

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2.22.3.6 Identification of Novel Genes Involved in Cell Wall Formation through Expression Profiling

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2.22.4 Mechanism of Cellulose Synthesis

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2.22.4.1 Organization of the Terminal Complex

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2.22.4.2 A Sitosterol-Glucoside Primer for Cellulose Synthesis?

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2.22.5 Regulation of Cellulose Deposition

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2.22.6 Redox-Regulated Dimerization of CESA Subunits

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2.22.6.1 The Role of Phosphorylation in Cellulose Synthesis

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2.22.6.2 Glycosylation and Quality Control in the ER

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2.22.6.3 Regulated Intracellular Trafficking of CESA6 and KOR1

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2.22.6.4 Role of Microtubules in Cellulose Synthesis

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2.22.6.5 Role of Lipids in Intracellular Trafficking

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2.22.7 Future Prospects

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738

Biosynthesis of Cellulose

2.22.1 Cellulose, A Sophisticated Macromolecule 2.22.1.1 Cellulose Is Ubiquitous Cellulose is the most abundant biopolymer. Cellulose is found in both prokaryotes (Acetobacter xylinus, Agrobacterium tumefaciens, Rhizobium spp., Escherichia coli, cyanobacteria, etc.) and eukaryotes (oomycetes, algae, terrestrial plants and animals, such as tunicates).1Photosynthetic organisms such as plants, algae, and certain bacteria produce more than 100 billion tons of organic matter each year coming from the fixation of carbon nitrogen and half of this biomass is cellulose. Cellulose is of enormous economic interest for the wood, paper, and textile industry and will also be the major source for biofuel in the future.

2.22.1.2 Cellulose Structure The primary structure of cellulose is a linear homopolymer of (b1-4)-linked D-glucose residues (Figure 1a). The repeating unit of the glucan chain is cellobiose since every glucose residue is rotated 180 with respect to its neighbors. In nature, cellulose never occurs as a single chain but as a paracrystalline microfibril composed of many glucan chains. The free hydroxyl groups present in the cellulose macromolecule are involved in a number of intra- and intermolecular hydrogen bonds that may give rise to various ordered crystalline arrangements. The conformation of the cellulose chain can be determined by various physical methods and in particular X-ray diffraction. Four cellulose allomorphs with characteristic X-ray diffraction patterns have been identified (I, II, III, and IV).1a Cellulose I, also referred to as native cellulose, is the natural allomorph. X-ray diffraction analysis along individual microfibrils showed that cellulose I is in most cases a mixture of two crystalline allomorphs a and b (Figure 1b). Ia and Ib have indistinguishable backbone conformations but that differ in their hydrogen-bonding patterns.2,3 Ia/Ib ratios can vary depending on the origin of the cellulose. For instance, cellulose from bacterial and algal origin is mainly of type Ia. Ib predominates in terrestrial plants (Ia/Ib2000 anthracycline derivatives have been synthesized to date, the precise mechanism of anthracycline action, and the corresponding glycosyl substituents, in vivo is still poorly understood. In addition to the role that anthracycline carbohydrates play in DNA affinity and the formation of covalent drug-DNAenzyme ternary complexes described above, sugar alterations have been used to reduce toxicity (e.g., C40 -doxorubicin sugar epimer epirubicin), broaden antitumor spectrum (e.g., MEN-10755) and reduce MDR efflux (as with 6 and C30 N,N-dimethyl-4).24 Finally, anthracycline deglycosidation (hydrolysis of the sugar) is a significant contributing factor to the cardiac toxicity of anthracyclines, and the carbohydrate-DNA recognition has also been implicated in the catalytic redox-cycling activity of anthracyclines which leads to free radical-based cellular damage.25 Less is known about the biological activity, and the corresponding role of glycosylation, of the aromatic polyketide models mentioned in this chapter – specifically, the angucyclines (urdamycin A, Figure 1, 7, Streptomyces fradiae Tu¨2717) and naphthacenequinones (elloramycin, Figure 1, 8, S. olivaceus Tu¨2353). The angucyclines display a broad

376

Complementary Routes to Natural Product Glycodiversification

range of in vivo and in vitro activities such as antibacterial and antitumor activity, inhibition of oxidative enzymes, and blood platelet aggregation.26 Yet, while a number of analogs have been generated via the methods described herein, information is sparse regarding specific 7 structure–activity relationships and/or mechanistic studies. Similarly, 8 has been noted for its antitumor and antibiotic activities and likely operates via topoisomerase poisoning in a manner similar to nonglycosylated members of this family (e.g., the tetracenomycins and saintopins).27 As with 7, a set of differentially glycosylated 8 have been generated, none of which outperformed the parent natural product based upon the limited set of studies published.28

3.20.2.3 Macrolides Macrolides terminate protein synthesis by inhibiting the 50S ribosome via specific binding with the 23S ribosomal subunit and various proteins.29–31 The 16-member macrolides (e.g., tylosin, Figure 2, 9) are generally found to bind 23S rRNA and inhibit peptidyltransferase activity while the 14-member macrolides (e.g., erythromycin and pikromycin, 10 and 11, respectively) generally inhibit the translocation of peptidyl-tRNA. Extensive SAR has established that the attached sugars are essential to bioactivity, consistent with the plethora of recently elucidated crystal structures of macrolide-target complexes.29–31 From these studies, the sugars clearly make up the main specific contacts with the 50S subunit tunnel wall and lead to aborted growth of the nascent peptide chain. In general, bulky macrolides tend to block peptides as small as two amino acids while inhibition by smaller macrolides results in truncated peptides of six to eight amino acids in length. Furthermore, extended saccharide moieties on certain larger macrolides such as 9 reach as far as the peptidyl-transferase center and thereby directly interfere with peptide-bond formation. Macrolides have also been shown to inhibit the assembly of new 50S ribosomal subunits by binding the 50S rRNA and protein precursors.32 Interestingly, the addition of a single sugar to 10, as in natural product megalomicin, results in a molecule with markedly distinct antiparasitic and antiviral activities (via blocking intra-Golgi transport).33–36 Cummulatively, the work highlighted above suggests that not only do sugars influence the antibiotic acitivities of this important drug class, but differential glycosylation of macrolides may also present the opportunity to convert typical antibiotics into molecules capable of interacting with completely new therapeutic targets. The first macrolide antibiotic (11, also a naturally occurring ketolide) was discovered in 1950 and interest in 10 derivatization toward compounds with improved pharmacological properties began in the 1960s. With respect to macrolide development, the first-generation macrolides developed for clinical use were the natural products themselves, the second-generation modifications focused primarily upon improved acid stability (e.g., azithromycin), and the recent third generation ‘ketolides’ (e.g., telithromycin) were developed to counteract macrolide resistance.

3.20.2.4 Glycopeptides The glycopeptide antibiotics are structurally defined by a heptapeptide backbone appended by a variety of carbohydrates.37–44 vancomycin (Vancomycin®, Eli Lilly & Co., Figure 2, 12) was isolated from Streptomyces (Amycolatopsis) orientalis in the early 1950s, introduced into practice in 1958, elucidated structurally in 1983, and was only sparsely used during its first 30 years in the clinic.43 However, by the early 1990s, vancomycin had become known as the treatment of last resort for severe Gram-positive infections and has since remained a critical weapon within the physicians’ dwindling antibiotic arsenal. Other notable ‘first-generation’ glycopeptides include teicoplanin (Targocid®, Lepetit, 13), in clinical use in Europe since 1988; ristocetin A (Ristocetin®, Abott), withdrawn due to a high incidence of thrombocytopenia; and avoparcin (Avotan®, Wyeth), used for a brief period in the past as an agricultural feed additive to stimulate growth.43 Glycopeptides kill bacteria by binding to the N-acyl-D-Ala-D-Ala termini of uncross-linked lipid-PP-disaccharide-pentapeptides. This binding inhibits the transglycosylase/transpeptidase activity required for cross-linking and thus renders the bacteria susceptible to lysis by osmotic pressure. The glycopeptide carbohydrates (e.g., the L-vancosaminyl-D-glucose disaccharide of 12) are not directly involved in binding D-Ala-D-Ala, but three lines of evidence implicate the critical role of these carbohydrates in bioactivity. First, removal of the disaccharide provides an aglycon with markedly reduced antibacterial activity. Second, as described later in this chapter, monoglycosylated glycopeptide variants derived from ‘glycorandomized’ libraries of the parent vancomycin aglycon also show some activity against resistant pathogens.8 Third, as described in the next paragraph, N-alkylation via reductive amination of naturally appended glycosamines of the chloroeremomycins or teichoplanins with variant hydrophobic groups present the ability to circumvent resistant organisms.41,43–45 In VanA and VanB glycopeptide-resistant organisms, five tandemly arranged genes encode for proteins which reprogram the peptidoglycan termini from N-acyl-D-Ala-D-Ala to N-acyl-D-Ala-D-lactate. This switch from D-Ala-D-Ala dipeptide to D-Ala-D-lactate depsipeptide effects a thousand-fold decrease in the binding constant for vanco-

O

O

O

CHO OH O

HO

O

O

OMe

MeO

O

O

HO

NMe2 O O

OH

OH

HO O

OH

O

O

OH

O

OH

O

O O

Cl

O

OH

OH

O

OH OH

HO HO

O AcHN

O HN HO O

N H

Cl H N

O

O OH OH

O

Pikromycin 11

N H O

OH O

H N O

NH2

N H

H N

O O O

HN HO O HO

N H

O

Cl H N

O

O HO

Vancomycin 12

N H

Cl

O

H N

O NH O

O

HO

Figure 2 Representative glycosylated macrolides (9–11), glycopeptides (12, 13) and cardiotonic steroids (14).

OH

O

O

OH

Teicoplanin 13

O

NH2

O

OH HO OH

O

OH

HO

OH OH

O

O

O

OH

OH

H N

OH

O

HO

OH

Digitoxin 14


O

O

O

O

Erythromycin 10

2

O

O

O

Tylosin 9

HO

O

OMe

NMe2

HO O

O

O

O

HO NH

HO

NMe2

HO O

377

378


mycin and is attributed to the loss of a single hydrogen bond and potential repulsion presented by the ester oxygen of the terminal lactate. The VanB operon has a comparable five-gene cluster; however, while both vancomycin 12 or teicoplanin 13 induce the transcription of the five genes of the VanA operon, only vancomycin 12 induces the VanB operon. As described above, hydrophobic modifications of glycopeptide-appended carbohydrates have proven beneficial against glycopeptide-resistant pathogens – the more successful analogs via this approach include Ortivancin® (LY-333328, Eli Lilly & Co.), Dalbavancin (BI-397, Vicuron Pharmaceuticals), and TD-6424 (Theravance, Inc.).41,43–45 The precise mechanism of action of these hydrophobic derivatives remains under active investigation and, based upon these ongoing studies, a number of formal hypotheses have been put forth.41,43–45 The first suggests these compounds to bind the D-Ala-D-lactate termini via cooperative binding where the weak affinity of the antibiotic-depsipeptide complex is increased by dimerization and membrane anchoring (afforded via the hydrophobic substitution). The second suggests that these molecules may be selectively inhibiting an alternative enzyme (the transglycosylase), redirecting these drug candidates from transpeptidase inhibition to new targets. Third, these modifications may also be preventing recognition by the sensor kinase (VanS/VanSB) and thereby preventing the initial induction of glycopeptide resistance. Importantly, while glycopeptide hydrophobic substitutions favor activity against resistant organisms, this is in delicate balance with a typical reduction of activity against glycopeptide-sensitive organisms and unfavorable pharmacokinetics attributed to the same substitutions.

3.20.2.5 Cardiotonic Steroids Beginning well before William Withering’s 1785 book, An Account of the Foxglove and Some of Its Medical Uses, the cardiac glycoside digitalis (also known as digitoxin, Figure 2, 14) remains the best treatment for myocardial dysfunction. Cardiac glycosides are noncompetitive allosteric inhibitors of the a-subunit of the membrane-bound Naþ/Kþ-ATPase a,b-dimer and inhibit the exchange of intracellular Naþ for extracellular Kþ to provide for a Ca2þ influx, which boosts the contractility of the myofibrils of the heart muscle.46–48 In the case of digitoxin, the trisacharide or aglycon shows little pharmacological effect and the activity dramatically increases upon the addition of the first sugar and then is slightly reduced upon each successive sugar addition. The delicate balance between enhanced aqueous solubility and oral bioavailability is also predominately dictated by the carbohydrates appended to cardiotonic steroids such as digitoxin. In addition to their well-known cardiac activity, cardiac glycosides have been shown to also induce signaling pathways via the Naþ/Kþ-ATPase and have validated anticancer properties.49–54 Cardiac glycosides were also recently noted to inhibit the expression of certain genes overexpressed in prostate cancer cells, to provide protective effects against polyglutamine-based diseases and to inhibit TNF-a/NF-kB signaling. Cumulatively, these fascinating molecules render a vast range of therapeutically important biological activities, the mechanisms of which remain under active investigation by many groups.

3.20.3 Pathway Engineering With the advent of modern molecular genetics techniques and genome sequencing projects, scientists now have unprecedented access to sequence information from diverse organisms, and this has fueled the burgeoning field of pathway engineering (also referred to as combinatorial biosynthesis). The principle objective of combinatorial biosynthesis is to vary the structure of natural products made by a particular organism through the replacement or alteration of the genes normally present. Such combinatorial or engineered generation of novel polyketide skeletons through manipulation of polyketide synthase (PKS) genes has proven fruitful and has been extensively reviewed.55–64 With respect to glycosylation, Solenberg and Baltz were the first to accomplish differentially glycosylated natural products by combining the genes from various glycopeptide-producing hosts.65 Shortly thereafter, Hutchinson and co-workers were the first to advance this approach to specific molecular engineering by replacing the native daunosamine 4-ketosugar reductase gene with an inverting 4-ketosugar reductase gene to ultimately convert the daunorubicin producer into an epirubicin-producing host.66 These landmark experiments paved the way for the contemporary pathway engineering applications highlighted below.

3.20.3.1 Macrolides The macrolide antibiotics, produced mainly by Actinomycetales bacteria, provide an excellent system for the development of combinatorial approaches to novel drug discovery. All macrolide antibiotics are composed of two essential


379

portions, a polyketide-derived aglycon scaffold appended with deoxysugar(s).67 Streptomyces venzuelae can produce both 12- and 14-membered macrolactone rings with a single set of PKS genes. These aglycones (15 and 16, Scheme 1) can be glycosylated with TDP-D-desosamine 17 by DesVII/DesVIII to give 10-deoxymethmycin 18 and narbomycin 19. Subsequent hydroxylation by a P450 enzyme (PikC), generates methmycin 20, neomethmycin 21, and pikromycin (11 – the first characterized member of the macrolide family). In an attempt to assign functions for the desosamine biosynthetic genes, several des gene deletion/disruption mutants (KdesI, KdesII, KdesV, and KdesVI) were constructed, and in each case, a new deoxysugar was found to be incorporated into the macrolide products at the position normally occupied by D-desosamine (Scheme 2, 22–25).68–72 In these mutants, the endogenous GT (DesVII), with

O O

O

O

O

R1 O

O

OH

R2

O

DesVII DesVIII

O HO 18

HO O

HO O

17

O

O

O

O NMe2

OH

O

20: R1 = OH; R2 = H 21: R1 = H; R2 = OH

PikC

O

OTDP

O HO

NMe2 NMe2

15 O

O

O

O

O

O HO

O

Me O

NMe2

O

O 19

16

O HO

O NMe2

11

Scheme 1 The final stages of methymycin (20), neomethymycin (21) and pikromycin (11) biosynthesis.

OH O

HO HO

DesIII

OH O

HO HO

19–21, 11

HO OPO = 3 30

HO OTDP 31

DesVII DesVIII 15 or 16

O

Me2N

HO OTDP 17

DesIV O

DesI

O

HO

HO 32 KdesI

DesVI

HO OTDP

O

KdesII

KdesV

33

Ketoreductase DesVII DesVIII

DesV

O

O H2N

OTDP

15 or 16

DesII

O

H2N HO

15 or 16

DesVII DesVIII

HO OTDP 34

HO OTDP

35

KdesVI

Ketoreductase

15 or 16

DesVII DesVIII

15 or 16

N-Acetylase

26 O

O HO

OH

O HO

NHAc OH

OH 22

27 O

23

DesVII DesVIII N-Acetylase

28 O

O HO

OH

29 O

O HO

24

Scheme 2 Examples of macrolide pathway engineering via disruption of native biosynthetic enzymes.

NHAc 25

380


the help of its auxiliary protein (DesVIII), is able to couple these new sugar donors to its normal aglycon acceptors 15 and 16. As shown in Scheme 2, the new macrolide derivatives formed include 22 carrying a 6-deoxyglucose (26),70 23 carrying a 4-N-acetylamino-4,6-dideoxyglucose (27),71 24 carrying a 4,6-dideoxglucose (28),69 and 25 carrying a 3-N-acetylamino-3,4,6-trideoxyglucose (29).68 The acetylation of the sugar amino group observed in 27 and 29 is believed to be due to acetylase activities not encoded in the pik gene cluster. This modification probably occurs after the coupling of the amino sugar to the aglycone, and likely represents a self-defense mechanism in Streptomyces venezuelae.68 Also, the KdesI and KdesV mutants should have accumulated 32 and 34, respectively. It is believed that the reduction of the 4- and 3-keto groups to generate the reduced sugars (in 26 and 28, respectively) seen in the products is catalyzed by pathway-independent reductase(s), and this reduction likely occurs prior to the DesVII/ DesVIII-catalyzed coupling of the sugar to the aglycone.70 Not only did these experiments facilitate the functional assignment of the des genes, but they also revealed the remarkable flexibility of DesVII toward its deoxysugar substrates. To further test the substrate flexibility of DesVII, the strM/L genes from Streptomyces griseus were heterologously expressed in the KdesI mutant.72 As shown in Scheme 3, these two genes are normally involved in streptomycin (36) biosynthesis, where StrM is proposed to be a 3,5-epimerase (32 ! 37) and StrL is the streptose synthase catalyzing the ring contraction of 37 to give TDP-L-dihydrostreptose (37 ! 38).73,74 The substrate for StrM (32) is the same intermediate that accumulates in the KdesI mutant. When the strM gene was introduced into the KdesI mutant, the new macrolide derivative 22, identical to that obtained from the KdesI mutant, was generated. Apparently, DesVII/ DesVIII failed to accept the StrM product (Scheme 3, 37) as a substrate. When both strM and strL were expressed in KdesI, four new macrolide derivatives 40–43 were obtained, each containing a L-rhamnose 44 moiety. Formation of TDP-L-rhamnose in this mutant likely proceeds via 32 ! 37 ! 39, requiring the presence of both StrM and StrL enzymes. Thus, while the proposed role of StrL as streptose synthase (37 ! 38) remains to be verified, the above observation reveals a new role for StrL as a TDP-4-keto-6-deoxy-L-glucose reductase (37 ! 39). It is also clear that DesVII and DesVIII are capable of coupling a L-deoxysugar to their natural aglycones. More importantly, these experiments showed that targeted gene disruption in combination with heterologous gene incorporation is a feasible approach to synthesize hybrid macrolide antibiotics in vivo. A recent important discovery is that efficient glycosyl transfer catalyzed by DesVII requires an auxiliary protein, DesVIII.75 In a separate experiment, a KdesI/KdesVII S. venezuelae strain, carrying a plasmid containing the genes (tyl1a, tylB, tylM2, and tylM3) involved in D-mycaminose biosynthesis in S. fradiae, was constructed to produce methynolide and neomethynolide derivatives carrying a D-mycaminose 50 moiety (Scheme 4, 48 and 49).76 This experiment was designed to test the ability of this S. venezuelae mutant to make D-mycaminose, to determine the competence of the mycaminosyl GT (TylM2) to transfer its natural sugar donor, TDP-mycaminose 47, to S. venezuelae aglycones (15 and 16), and to ascertain the possible dependence of this glycosyltransfer on an auxiliary protein (TylM3). The desI disruption resulted in the accumulation of 32 in this strain, which could be converted by the heterologously expressed Tyl1a and TylB to 46. Subsequent N,N-dimethylation of 46, an established property of DesVI,77 led to the production of 47. Because this S. venezuelae mutant also has a disrupted desVII gene, any glycosylation capability of this strain would be an indication of functional expression of the S. fradiae mycaminosyl transferase TylM2, and its putative auxiliary protein, TylM3. In fact, when all four S. fradiae genes (tyl1A, tylB, tylM2, and tylM3) were present, the mutant S. venezuelae cells produced 3-O-mycaminosyl-methynolide 48 and 3-O-mycaminosyl-neomethynolide 49. In contrast, when tylM3 was absent, no mycaminosylated aglycone could be detected. Also, when tylactone (the natural aglycone substrate for TylM2/M3) was fed to the S. venezuelae cultures, the strains expressing both TylM2 and TylM3 produced 40-fold more 5-O-mycaminosyl-tylactone than the strain lacking TylM3. Similar results were also obtained when the desosaminyltransferase MycB, from the mycinamicin pathway of Micromonospora griseorubida,78 was expressed with or without its putative auxiliary protein, MydC, in an S. venezuela KdesVII mutant.76 Here, all of the desosamine biosynthetic genes are intact, but the glycosyltransferase gene, desVII, is disrupted. It was found that only in the presence of MydC could MycB effect efficient coupling of 17 onto 15 and 16. Since 15 and 16 differ markedly form the 16-membered aglycone substrate used by MycB and MydC in mycinamicin biosynthesis, the fact that 18 and 19 were produced by this mutant clearly demonstrated the flexibility of MydC/ MycB toward their aglycone substrate. Thus far, a total of 16 desVIII homologs have been identified in various deoxysugar biosynthetic gene clusters.76 They are predicted to be the auxiliary protein for the corresponding glycosyltransferase in each pathway. The discovery of the dependence of GTs on an auxiliary protein should facilitate the use of these glycosyltransferase/auxiliary protein pairs toward the combinatorial generation of new macrolide compounds carrying rationally designed sugar moieties.76


381

NH HN

H H2N

N

NH2 OH OH

HO O

NH

HO O

HO

OTDP OH

O HO 38

O

HO

StrL

O

HO HO

OTDP

O

NHMe O

36

OH

OH 37 StrM

DesIII (StrD)

OH O

HO HO

HO

OH O

HO HO

HO

OPO = 3

DesIV (StrE)

O HO

HO

OTDP

OTDP

32

31

30

KdesI or KdesI/StrM or KdesI/StrL

O

Me KdesI/StrM

O HO

R1

O

O R2

O

O

OH OH

O

41: R1 = H; R2 = OH 15 or 16

O O

OH OH

22

OTDP

O HO

OH 39

HO

O

O

StrL/NAD(P)H HO

O O

OH 37

DesVII DesVIII

42: R1 = H; R2 = H

O

OH

44

40: R1 = OH; R2 = H

OTDP

O

26 O HO

OH OH 44

43 Scheme 3 Examples of macrolide pathway engineering via insertion of heterologous sugar biosynthetic genes.

3.20.3.2 Aromatic Polyketides Urdamycin A (Figure 1 and Scheme 5, 7) is an angucycline-type antibiotic and anticancer agent produced by S. fradiae Tu¨2717.79–81 Structurally, it is derived from a tetracyclic polyketide carrying two L-rhodinose 51 and two 82,83 D-olivose 52 residues. One L-rhodinose is attached to the hydroxyl group at C12b, and a trisaccharide composed of alternating D-olivose, L-rhodinose, and D-olivose residues is attached to C9 through a C-glycosidic linkage to the first olivosyl residue. Sequencing of the urdamycin biosynthetic gene cluster from S. fradiae Tu¨271784–87 led to the identification of four GTs responsible for the addition of 51 and 52 to the aglycone (UrdGT2, UrdGT1a, UrdGT1b, and UrdGT1c).85,86 The first GT characterized was UrdGT2, the inactivation of which resulted in the production of

382


OH O

HO HO

HO

DesIII

HO HO

OH O

30

O

KdesI Tyl1a

O HO

OTDP

31

O

HO

HO HO

OPO3=

DesIV

HO OTDP

O

OTDP

45

32

O TylB R1

KdesVII TylM2/TylM3

O R2

O

O HO

O 50

OH NMe2

O

HO Me2N

DesVI

O

HO H2N

HO

HO OTDP 47

OTDP

46

48: R1 = OH; R2 = H 49: R1 = H; R2 = OH Scheme 4 Examples of macrolide pathway engineering via insertion of heterologous sugar biosynthetic genes.

three new urdamycin derivatives, none of which contain the C-glycosidic deoxysugar moiety at C9.85 Hence, it was concluded that UrdGT2 catalyzes the C-glycosyl transfer of NDP-D-olivose (65, Scheme 6), which is most likely the first glycosylation performed on the aglycon. Interestingly, one of the shunt products generated by the udrGT2 knockout shows higher anticancer activity than the parent compound urdamycin A,85 demonstrating that targeted gene disruption experiments can lead to new compounds with improved activities. To characterize the functions of the other GTs in the urd gene cluster, a mutant S. fradiae Tu¨2717 strain with chromosomal deletions of the urdGT1a, urdGT1b, and urdGT1c genes (termed mutant Ax) was constructed.86 By expressing each of the deleted GTs individually and in combinations in this mutant, the order of deoxysugar addition in urdamycin A biosynthesis was determined (Scheme 5, 53 ! 54 ! 55 ! 7). An impressive array of new urdamycin derivatives was also generated in this study. It was observed that UrdGT1b can add D-olivose to 55 and 58, and UrdGT1c can attach L-rhodinose to both 54 and 57.86 In addition, the overexpression of UrdGT1c in the Ax mutant revealed that it can add a second L-rhodinose to 58 to complete the trisaccharide chain, forming urdamycin N 59.87 If a functional UrdGT1a is present, UrdGT1c can also add L-rhodinose to 55, generating urdamycin O 56.87 The substrate flexibility of the urdamycin GTs was further explored through mutation of individual urd deoxysugar biosynthetic genes.87 The proposed pathways for NDP-D-olivose (65) and NDP-L-rhodinose (68) biosynthesis are shown in Scheme 6. Inactivation of urdQ, urdZ1, or urdZ3 led to the accumulation of mainly urdamycinone B 57. The absence of L-rhodinose in any of the isolated products suggests that these three genes are needed for biosynthesis of 68, but not 65. The inactivation of urdR, a putative 4-ketoreductase needed solely for synthesis of 65, led to the production of urdamycin M 70, which carries a C-glycosidically linked D-rhodinose at the C9 position, and no D-olivose moieties.87 The production of D-rhodinose in this mutant (as opposed to the L-rhodinose that is normally seen) suggests that the 4-ketoreductase UrdZ3 may be able to reduce both 66 and 67 to produce 69 and 68, respectively. Most importantly, these studies demonstrated that UrdGT2 can also catalyze the C-glycosylation at C9 with 69, which is quite different than its natural substrate, 65.87 As another recent aromatic polyketide model, ellaromycin (Figure 3, 8) is an anthracycline-like antitumor drug produced by S. olivaceus Tu¨2353. It is composed of a tetracyclic aromatic polyketide decorated with a permethylated 88 L-rhamnose 71 moiety. Expression of a cosmid (16F4)-containing part of the ellaromycin (elm) gene cluster in the urdamycin-producing organism, S. fradiae Tu¨2717, resulted in the production of a hybrid compound 73. This compound contains the elm-derived aglycone, 8-demethyltetracenomycin C (8-DMTC, 72), and an urd-derived sugar, D-olivose 52.89 Although cosmid 16F4 contains all genes necessary for 8-DMTC biosynthesis, some of the Lrhamnose biosynthetic genes and the 12-O-methyltransferase needed for ellaromycin production are absent.89,90 When 16F4 was expressed in an S. fradiae Tu¨2717/DPKS mutant, not only was 73 formed, but an L-rhodinosyltetracenomycin 74 was also produced.91 Interestingly, by deleting the urdamycin PKS genes, it was possible to increase the yield of hybrid macrolides by freeing up the urdamycin sugars for attachment to the ellaromycin aglycon. In the same study, 16F4 was also expressed in the mithramycin 75 producer, Streptomyces argillaceus, whose mtm gene cluster encodes enzymes involved in the production of three deoxysugars, D-olivose (52), D-oliose 76, and D-mycarose 77


383

Precursor aglycon UrdGT2

UrdGT2

O O HO

O

O

OH

OH

OH

O

HO HO

O

HO HO

OH

O

O 53

57

O

O O OO

O HO HO

OH

O OH

OH

UrdGT1c

O

O

58

OH UrdGT1c

O

HO O

OH OH

O UrdGT1b

O

O

O OO

59

O O

HO O

OH O

O

O

O

UrdGT1c OH

UrdGT1b

O

O

O OO

HO O L-Rhodinose (51) O

OH

55

OH

O

60

O

OH

O

O

OH

OH

OH OH

D-Olivose

HO HO

O

O

O O

O 54

O

OH

HO O

OH

OH

O

O

HO HO

OH

UrdGT1a

UrdGT1c

HO O

OH

(52) O

O OO

OH

OH

12b

OH

9

OH

HO O

O

O

7

O

OH

O OH

O

56

O OH Scheme 5 The final stages of the biosynthesis of the urdamycins.

(Figure 3). In this experiment, three hybrid macrolides were produced: 73, D-mycarosyltetracenomycin 78 and 80, a derivative with a diolivosyl moiety attached to 8-DMTC 72.91 To determine whether the GT responsible for the coupling of these sugars to 72 was encoded in the elm cluster of 16F4, or by the host organisms, the S. fradiae Tu¨2717/DPKS and S. argillaceus strains were fed 72 in the absence of cosmid 16F4.91 The failure to detect any glycosylated tetracenomycins in these experiments provided the first evidence that the GT responsible for the glycosylations of 8-DMTC 72 was encoded by one of the elm genes in the 16F4

384

HO HO


OH O HO

UrdG

HO HO

OH O

O

O

UrdS

O

O

HO HO

OPO3=

30

UrdH

HO

ONDP

HO

O

ONDP

62

61

ONDP

63 UrdT

HO O

UrdZ3

O

O

UrdQ

O

O

HO OTDP

ONDP

ONDP 64

66

NDP-D-rhodinose (69)

UrdR

UrdZ1

UrdGT2

UrdZ3

ONDP

O

O

ONDP

O

OH NDP-L-rhodinose (68)

O

HO HO

ONDP NDP-D-olivose (65)

67 OH

UrdGT1a

UrdGT2

O O OO

O

O

OH

OH

HO OH

O OH

O

Urdamycin M (70) Main compound produced in urdR mutant

HO HO

O OH

O

Urdamycinone B (57) Main compound produced in urdQ, urdZ1, and urdZ3 mutants

Scheme 6 Examples of urdamycin pathway engineering via disruption of native sugar biosynthetic genes.

cosmid. This result was later confirmed by expression of 16F4 in an S. argillaceus mutant that lacked the mitramycin GTs,92 and by co-expression of 16F4 and the mithramycin sugar biosynthetic genes (without the mtm GTs) in Streptomyces lividans, a nonantibiotic producing strain.91 In both cases, only 73 and 78 were obtained, providing compelling evidence that an ellaromycin GT couples the mithramycin sugars, 52 and 77, to the ellaromycin aglycon, 72. The sugar-flexible glycosyltransferase (named ElmGT) in cosmid 16F4 was subsequently identified, sequenced, and integrated into the Streptomyces albus chromosome.93 When selected deoxysugar genes were heterologously expressed in this ElmGT-producing S. albus strain, which was then fed a variety of aglycons, it was shown that ElmGT can attach L-rhamnose 44 and L-olivose 81 moieties to 72, forming hybrid compounds 82 and 83, respectively (Figure 3). This study also showed that two mithramycin GTs, MtmGI and MtmGII, are responsible for synthesizing the D-diolivosyl unit 79, which is then added as a disaccharide to 72 by ElmGT. In fact, ElmGT is one of the more sugar-flexible GTs characterized thus far, being able to transfer L-rhodinose 51, D-olivose 52, D-mycarose 77, 93 L-rhamnose 44, L-olivose 81, and a D-diolivosyl disaccharide 79, to 8-DMTC. It holds promise as a useful tool for future combinatorial biosynthetic applications.

3.20.3.3 Indolocarbazoles Indolocarbazole alkaloids are a group of compounds that exhibit many biological activities including antitumor and neuroprotective properties. Structurally, most of these compounds contain an indolo[2,3-a]pyrrolo[3,4-c]carbazole core

385


D-Olivose

Mithramycin (75)

(52)

OMe OH O

O HO O

HO HO

O H

Ellaromycin (8) HO

O

R1O O

OMe O

D-Mycarose

MeO

O

D-Oliose

OH

OH

OH

O

OH

O O

(76)

OH

MeO MeO

O

OMe O

OH O HO O

O

HO O

HO

(77)

O

OMe 8-DMTC (72)

71

O OHO

OH

O MeO R1O

OMe O OHOH

73:

16F4 expressed in S. fradiae ∆PKS

16F4 expressed in S. argillaceus

ElmGT and sugar biosynthetic gene cassettes expressed in S. albus cultures fed 8-DMTC

R1=

R1=

R1=

O

HO HO

73:

O

HO HO

82:

O HO HO

52

52

O

74:

78:

O

HO Me

83:

51

77 80:

HO HO

O HO O

O HO HO

OH

OH

OH

44

81 O

79 Figure 3 Examples of aromatic polyketide glycodiversification via bioconversion in heterologous hosts expressing variant sugar biosynthetic genes.

(derived from two molecules of tryptophan, Scheme 7, 84) and an attached sugar. Among the indolocarbazoles, the biosynthesis of rebaccamycin (Scheme 7, 94) from Saccharotrix aerocolonigenes and staurosporine (Scheme 8, 105) found in several Streptomyces species has been the most extensively studied. The similarity between the indolopyrrolocarbazole cores of these two compounds (differing only by an extra carbonyl group in rebeccamycin) has allowed the generation of novel, hybrid products by combinatorial biosynthetic methods. During their efforts to reconstitute rebeccamycin biosynthesis in S. albus, Sanchez et al. were able to generate several rebeccamycin biosynthetic intermediates through heterologous expression of different combinations of reb genes (86–93, Scheme 7).94 Furthermore,

386


NH O

O NH O

O

RebG RebM NH R1

N H

NH2

92: R1 = R2 = H 93: R1 = Cl; R2 = H 94: R1 = R2 = Cl

84 HOOC

NH

OH

OH OMe

RebC RebP

RebO RebD

O HO

R2

89: = =H 90: R1 = Cl; R2 = H 91: R1 = R2 = Cl

OH

R2

R R2

R1

O

N

NH 1

NH

COOH

RebH NH

NH R2

R1

RebO RebD

O OH

86: R1 = R2 = H 87: R1 = Cl; R2 = H 88: R1 = R2 = Cl StaC RebP

NH2 Cl 85

NH O

N

NH

NH O

N H

RebG RebM

R1

O

R2 OH

HO NH

NH R2

R1

95: R1 = R2 = H 96: R1 = Cl; R2 = H 97: R1 = R2 = Cl

OH OMe 98: R1 = R2 = H 99: R1 = Cl; R2 = H 100: R1 = R2 = Cl

Scheme 7 The key steps in indolocarbazole biosynthesis.

they discovered that replacement of a single rebeccamycin monooxygenase gene (rebC) with its staurosporine homolog (staC) was sufficient to confer the ability of the recombinant strain to produce the staurosporine indolopyrrolocarbazole 95. Using this finding, they were able to generate an array of novel derivatives 96–100 containing the staurosporine indolopyrrolocarbazole core through expression of staC along with various combinations of reb genes. Further structural diversity was obtained by expressing the tryptophan halogenases PyrH from Streptomyces rugosporus LL-42D005 and Tha1 from Streptomyces albogriseolus, which chlorinate the 5- and 6-positions of tryptophan, respectively. It was also found that RebH can incorporate Br onto position 7 of tryptophan if the cultures are grown in media containing KBr. In total, over 30 rebeccamycin- and staurosporine-like compounds were generated in this study, some of which contained biological activity. In a separate study, Salas et al. were able to fully reconstitute staurosporine biosynthesis in S. albus.95 After constructing a strain harboring both the genes necessary to make 95 (rebODP and staC ) and staG (the putative glycosyltransferase), the L-ristosamine (Scheme 8, 101) biosynthetic genes were introduced on a separate plasmid. This experiment demonstrated that StaG was responsible for the attachment of 101 to the indolocarbazole core through a C–N glycosidic linkage to generate 102. A second gene, staN, which encodes a cytochrome P450


H N

95

H N

O

H N

O

102

O

103 StaN

StaG N H

387

N H

N

N H

N H

O

H N 105

NH2 O

101 HO

N

HO

H2N StaN

O

H N

104

O

StaMA StaMB N

N NHMe

NH2

O MeO

N

N

O HO

Scheme 8 The final steps in staurosporine biosynthesis.

enzyme, was required in order to generate 104, in which a second C–N linkage is formed between C50 of the sugar and N13 of the indolocarbazole. Subsequent sugar methylations by StaMA and StaMB give the final product, staurosporine 105. Further investigation into the glycosylation event revealed that both StaG and StaN are substrate flexible. Heterologous expression of plasmids encoding the production of different deoxysugars in the S. albus strain expressing rebODP and staCGN demonstrated that StaG can transfer a variety of sugars (L-rhamnose 44, L-olivose 81, L-digitoxose 106, and D-olivose 52) to 95 through C–N glycosidic linkages (Scheme 9, 107–110). After glycosyl transfer, StaN can form the second C–N linkage with L-sugars to generate 111–113. From these results, it was proposed that in order to establish this second C–N linkage, the sugar needs to ‘flip’ from a 4C1-into a 1C4-conformation (Scheme 8, 102 ! 103) – a feat which is more easily achieved with L-sugars. These studies illustrate the promising future in combinatorial engineering of the indolocarbazole alkaloids.

3.20.3.4 Other Recent Pathway Engineering Studies The examples detailed above represent some of the more extensively characterized systems for combinatorial generation of hybrid macrolide antibiotics. However, other substrate-flexible antibiotic glycosyltransferases are known, and similar combinatorial biosynthetic studies have been performed in a number of other systems. For example, GTs from the oleandomycin96,97 and tylosin96 biosynthetic pathways were used to generate new erythromycin derivatives. Heterologous expression of selected Streptomyces avermitilis L-oleandrose biosynthetic genes in S. lividans yielded new avermectin derivatives when cultures were fed the avermectin aglycone.98 Here, it is evident that the GT (AvrB) is capable of coupling several alternative sugars onto the avermectin aglycone.98 As further highlighted in the next section, a number of the glycopeptide glycosyltransferases have been shown to be flexible to both alternative NDP-sugars and cyclic heptapeptides, and these substrate flexible GTs have been used to generate hybrid glycopeptide antibiotics both in vivo and in vitro.9,65 Together, the combinatorial biosynthetic studies described here have revealed the inherent substrate flexibility of several glycosyltransferases (DesVII, TylM2, MydB, UrdGT2, UrdGT1b, UrdGT1c, ElmGT and StaG). Although only a few of these substrate-flexible GTs have been characterized in much detail, GTs with similar flexibility most likely exist in the deoxysugar biosynthetic pathways of other antibiotics. As more of these GTs are discovered, their utility for the generation of new hybrid antibiotics in future in vivo or in vitro studies should be facilitated. Clearly, the

388


H N

95

N H StaG

H N

107

N H

N H

OH OH

HO

H N

108

N

44

HO

H N

113

N

N

N O

OH OH

StaN

O

112

O

52

106 HO

H N

N

N H

O

StaN

O

N

N

O OH

H N

O 110

N H

81

StaG

109

N

StaN

111

StaG

H N

O

HO

H N

N H

StaG

O

O

O

O

N

N OH

O

HO HO

OH

O

HO HO

HO

O

Scheme 9 An example of engineering variant indolocarbazoles in a heterologous host.

successful implementation of this approach will continue to rely upon the discovery and characterization of new deoxysugar biosynthetic pathways and the inherent or engineered promiscuity of the corresponding glycosyltransferases. Furthermore, these studies have also demonstrated that engineering novel, hybrid compounds through gene disruption and heterologous expression techniques is a feasible method for expanding the diversity of nature’s bioactive metabolites, and similar experimental approaches should continue to fuel the use of combinatorial biosynthesis in the search for new drugs.

3.20.4 Glycorandomization Efforts to alter the glycosylation of natural products have relied upon conventional strategies ranging from total/ semisynthesis to in vivo pathway engineering. In a complementary fashion, glycorandomization attempts to capitalize upon the strengths of these existing strategies to provide robust methods to specifically generate compound libraries which solely differ via their glycosyl substituents (i.e., glycorandomized libraries).9 This last section highlights recent advances relevant to the two existing complementary glycorandomization strategies (Scheme 10): (1) chemoenzymatic glycorandomization – a biocatalytic approach dependent upon the substrate promiscuity of enzymes to activate and attach sugars to natural products, and (2) neoglycorandomization – an efficient one-step chemical sugar ligation reaction that does not require prior sugar protection or activation. Both strategies allow for further diversification via

389


Neoglycorandomization Aglycon

Sugar library

Neoglycorandomized libraries

1

R2 R3 R6 R7 R10 R11 R14 R15

R

O 4

R R5

OH

R5

R6 R7

O

N

R2 Y1 O R3 R4

O R4

N

O

R5

Y2 R7

O

O R8

N

R

OH

R13

R14 R15

O

R16

R10 R11

DMF/AcOH (3:1), 40 ⬚C

R9

R16

N

9 R10 R O Y3 R12

O

R12

O

N

O

R13

R14 R15

O

O

Y4

OH

N

O

O

CSL O 12

R13

R

O R8

R8 OH R9

R2 R3

O

HN

1

N

N

O

O

Chemoenzymatic E1 R2 R3

R1

glycorandomization

O

R4 OPO2− 3

5 R6 R O 7 R R8 OPO23− 9 10 R R O R11 R12 OPO23− 13 R O R14 R15 R16 OPO2− 3

Sugar-1-P library

E2

1 R2 R O 3 R R4 NDP R5 6 R O R7 R8 NDP 9 R10 R O R11 R12 NDP 14

R R15

R13

6

R R7

R

O R8

R1

O R4

Y6 R7

X

X

GlyT

O

R16

R2 R3

5

XH

14

R R15

R13

R10 R11

O

R16

X

R9 R

R5

R2 R3

O

R8

Y5

O R4

X

X

CSL O 12

X

14

R R15

R13

R10 Y7

O

Y8

R9

O 12

R

X

X

NDP

NDP-sugar library aglycon

Glycorandomized libraries

Scheme 10 The two routes to natural product glycorandomization. CSL, Chemoselective ligation chemistries; E1, flexible sugar anomeric kinase; E2, flexible sugar-1-phosphate nucleotidylyltransferase; GlyT, natural product glycosyltransferase.

chemoselective ligation reactions (designated CSLs in Scheme 10). In contrast to combinatorial biosynthesis or pathway engineering, glycorandomization can extend beyond the monosaccharide building blocks found in nature and, given that glycorandomization can be performed either in vitro or in vivo, is not restricted by genetically amenable hosts and/or lethal ‘unnatural’ product toxicity.

3.20.4.1 Glycopeptides (Chemoenzymatic Glycorandomization Proof of Concept) The inherent promiscuity of many natural product glycosyltransferases (GlyT) as described in the preceding ‘Pathway Engineering’ section (Section 3.20.3) is central to the concept to chemoenzymatic glycorandomization. Conceptually, the basis of chemoenzymatic glycorandomization derives from presenting libraries of natural and ‘unnatural’ NDPsugars to these promiscuous GlyT’s as a means to rapidly glycodiversify natural product-based scaffolds. As previously described, combinatorial biosynthesis or pathway engineering can require up to 10 different enzyme combinations to generate a single unique NDP-sugar for glycodiversification. In contrast, chemoenzymatic glycorandomization employs a single flexible anomeric kinase (E1) and a single flexible nucleotidylyltransferase (E2) to provide robust short activation pathways to nucleotide diphosphosugar (NDP-sugar) donor libraries from chemically synthesized or commercially available monosaccharides. The initial design objective for chemoenzymatic glycorandomization was to provide a robust three-enzyme (E1, E2, and GlyT) in vitro single-vessel reaction. The key factors to overcome for this design were primarily the limited flexibility of known anomeric kinases (E1) and sugar-1-phosphate nucleotidylyltransferases (E2). For E1, the directed evolution of Escherichia coli galactokinase (GalK), coupled with a high-throughput multi-sugar colorimetric assay, presented a GalK mutant (Y371H, 20A˚ from the active site) with a remarkable degree of kinase activity toward

390


sugars as diverse as D-galacturonic acid, D-talose, L-altrose, and L-glucose – all of which failed as wild-type GalK substrates.99,100 Subsequent structure-based engineering of this variant presented a double mutant (M173L–Y371H) with far superior substrate flexibility.101,102 The initial demonstration of M173L–Y371H GalK-E. coli strain to generate ‘unnatural’ sugar-1-phosphates via in vivo bioconversion has also been reported,102 and these bioconversion experiments have since been expanded toward the in vivo production of >25 sugar-1-phosphate analogs (Yang and Thorson, unpublished). For E2, wild-type Salmonella enterica LT2 a-D-glucopyranosyl phosphate thymidylyltransferase (also known as RmlA or Ep) displayed significant flexibility toward both both nucleotide triphosphate (dTTP and UTP) and sugar-1-phosphate substrates.103 This E2 flexibility was further enhanced via structure-based engineering to give an enzyme capable of providing >40 NDP-sugars for glycorandomization.103–105 More recently, E2 was coupled with E1 in vivo to generate >20 ‘unnatural’ NDP-sugars via bioconversion (Yang and Thorson, unpublished) and, under certain in vitro conditions, was also demonstrated to efficiently utilize ATP, dATP, CTP, dCTP, GTP, and dGTP (Moretti and Thorson, unpublished). The first proof of concept for chemoenzymatic glycorandomization focused upon the glycopeptide vancomycin. In this demonstration, E1–E2 were utilized to provide 33 natural and unnatural NDP-sugars to GtfE, the first of the two vancomycin glycosyltransferases which assemble the L-vancosaminyl-1,2-D-glucosyl disaccharide upon Phegly4 hydroxyl of the heptapeptide aglycon (Scheme 11, 114). In this study, GtfE accepted 31 of the 33 NDP-sugar substrates tested to provide the first demonstrated glycorandomized library which included derivatives (115) poised for downstream chemoselective ligation.106 Chemoselective diversification of monoglycosylated derivative 115 via Huisgen 1,3-dipolar cycloaddition (Scheme 12) led to 39 additional novel vancomycin library members bearing diverse functionality, albeit in varying cycloaddition yields.106,107 Screening representatives from this library against methicillin-resistant Staphylococcus aureus, vancomycin-sensitive Enterococcus faecalis, and vancomycin-sensitive Enterococcus faecium, revealed two analogs slightly more potent, and a third with equal potency, to the parent antibiotic vancomycin. Thus, this work both highlights the complete proof of concept for chemoenzymatic glycorandomization and also presents biological validation of glycorandomization. Chemoenzymatic glycorandomization has also been successfully extended toward indolocarbazoles, enediynes, and avermectins (Zhang and Thorson, unpublished). In contrast, attempts to glycorandomize the natural product novobiocin led to only four new analogs from >40 NDPsugars tested – a reminder of how restrictive enzymes, such as the novobiocin glycosyltransferase NovM, can negatively impact the outcome of glycorandomization.108

OR

Library, R =

O

OH O

HO

OH HOO

HO

OH HO

OH

HOOH HO

HO HOOH HO O HO

HO O

HO HO

O N H

HN HO

OH O

HO O

O

Cl H N

O N H O

O

O HO

N3 HO

OH OH

HO

HO

114, R = H

HO HO

HO OH HO O

HO

R

GtfE

HO

O

O

HO H2N

HO OH

O HO

O

HO HO

HO OH

NH2

O

O

O

HO HO

HO

HO

NDP

HOOH HO O OH

HO N3

O

O

HO

HO

HO

OH

O

O

O H2N

OH O

H2N HO

H2 N HO

NH2

HO

HO

HO

SH

OH O

H N

N H

O

OH HO HO

OH O

H N

OH

O

HO

Cl

O 4

O HO

NHAc O HO HO HO

O

HO HO

HO

HO AcHN

O HO

OH HO H2N

O HO

O

HO AcHN

HO

OH N3 HO HO

O HO 115

Scheme 11 The enzymatic glycorandomization of vancomycin.

HO HO

O AcHN

OH

OH HO HO

O

HO HO

O H2N

391


Library (R2 = H unless otherwise noted), R1 =

115 Huisgen 1,3-dipolar cycloaddition

R1

H N

R

HO O

2

O

O O

R1

N H

OH

Cl

HO O

O

O

N H

O

O S

O N

HN HO

O

O

H N

O

H N

O O

HN NH2

H N

R1 =

O

H N O O

H O N N O

Cl

O O

N

H O N N O Ph H

S

O O

OH

O

O O

CF3

R1 , R2 =

O NH2

OH OH

O O

R2 =

HO

O

H N

OH H N

H S N N

O

O N N

O

COOH

O H O N N O Ph H

N H

O

O

OH

HO

O

OH

O

O

O H N

H N

N H

O

OH O

OH

R1 , R2 =

O

OH O

O

O

O

R2 OH N Cl N N

OH

HN H N O

H N

N S

O

H O N S 5 O

OH N

O

N

Cl Cl H N O

H N O

O

H N O

N H

H N O

H N

O OH N H O

O

O

N

H N N H

N

O

Scheme 12 An example of further vancomycin diversification via chemoselective ligation chemistries.

3.20.4.2 Cardiotonic Steroids (Neoglycorandomization Proof of Concept) The restrictions imposed by certain glycosyltransferases (such as NovM highlighted in Section 3.20.4.1) served as inspiration to seek alternative glycorandomization approaches. Neoglycorandomization is based upon the chemoselective formation of a neoglycosidic bond between a reducing sugar and a secondary alkoxylamine-containing aglycon. In contrast to the traditional chemical glycosylation reactions which rely upon tedious sugar donor protection and activation schemes, the ‘neoglycosylation’ reaction advantageously utilizes unprotected and nonactivated reducing sugar donors under mild conditions. Early examples of this chemoselective reaction revealed that, unlike primary alkoxylamines which provide open-chain oxime isomers, secondary alkoxylamines react to form closed-ring neoglycosides.109,110 The natural glycoside digitoxin was selected as a model platform to test the concept of neoglycorandomization (Scheme 13).111 A library of 78 digitoxin derivatives was synthesized and purified in parallel from 39 reducing sugars and aglycons 116a/116b, which were easily obtained from digitoxin in three simple chemical steps. The reducing sugars employed included L-sugars, deoxy sugars, dideoxy sugars, disaccharides, uronic acids, and sugars containing reactive handles, all of which reacted successfully to provide the first neoglycorandomized library. The digitoxin neoglycosides showed only slight degradation under acidic conditions (t1/2 1 month, pH 5.0), and crystallographic studies revealed structural similarities to O-glycosides. High-throughput cytotoxicity assay of this library against nine human cancer cell lines representing a broad range of carcinomas including breast, colon, CNS, liver, lung, and ovary, and a mouse mammary normal epithelial control line led to the discovery of analogs with striking potency and tumor selectivity with respect to the parent natural product. Interestingly, all hits from this study contained sugars with a common S-configured C(2’) sugar stereocenter and were also demonstrated to be significantly less potent Naþ/Kþ-ATPase inhibitors in HEK-239 human embryonic kidney cells than digitoxin. Cummulatively, this study presented clear validation of the glycorandomization concept and highlighted a mechanistic divergence between cardiac glycoside cytotoxicity and Naþ/Kþ-ATPase inhibition. Alkoxylamines have been also recently incorporated into natural glycosides such as nonribosomal peptides (Griffth and Thorson, unpublished), macrolides (Ahmed and Thorson, unpublished), and anthracyclines (Liao and Thorson, unpublished), as well as nonglycosylated natural products such as colchicine (Ahmed and Thorson, unpublished). In each case, the corresponding neoglycorandomized libraries have been generated, further illustrating the broad applicability of this spectacular chemistry.

392

O N

OH OH O

HO HO

OH

HO HO

O OH

O N

HO HO O O HO N

HO HO O O N HO

O N

HO HO

O

O N

HO

OH N3 HO HO

HO HO HO

OH O

OH

HO HO

F O N

O O

HO

O OH

O N

O HO OH

O

O N

OH HO

O N

O O O N OH

O HO OH

F

HO O N

HO

OH OH O

O N

O HO OH OH

O N

HO OH

HO HO HO HO

O N

OH

O OH

HO HO O

O N

HO OH

HO

HO OH

HO HO

OH OH O O N

O OH

O N

HO

OH O N NHAc

O

HO

OH

O OH

OH

O N

HO

OH HO OH O O N

O N

HO

O N

HO

HO HO O HO

O N

HO HO HO

O N

HO O O N HO OH

O

OH

OH

O

HO

HO OH

O

HO

HO OH HO HO O

O N

HO

OH

O N

O HO OH

O N

OH O N

O

HO HO

F

HO OH O N

HO

O

HO

OH

OH

O N

OH HO O

HO

HO

O OH

O HO OH

O HO OH OH

HO O

HO

O OH HO O O HO N OH

O N

O N

O OH

O

HO HO

OH

O OH

HO HO

HO OH

O OH

O N

HO

O O HO

OH

OH

OH O OH

O N

Scheme 13 The chemical neoglycorandomization of digitoxin.

HO HO

O OH

OH O HO

O OH

O N

HO HO

HO O

O N

HO HO O

HO

HO OH

HO HO

O OH

O N

HO

O HO OH

O N

O N

OH N3 O N

OH O

HO HO

O N

OH

HO

O HO O

OH O N

OH

HO HO O

O N

OH O HO

O

HO

HO OH

O N

OH

OH HO

OH HO HO

O N

O N

O N

HO

O O O N OH

OH O HO

O N

HO

HO OH O O N HO OH

OH O N

O

HO

O

OH

O

HO HO

HO

HO O N

HO

O N NHAc O

OH O

HO

HO O N

OH O N

F

HO O

HO

OH

HO

HO O N

O

O N

HO HO

HO OH

O

HO

HO

OH HO

O N

HO O N

O

HO

O

HO

OH

HO O N

OH HO

HO

OH O N

O

HO HO

HO

OH

HO O N

OH OH O O HO N HO OH HO O HO HO O HO HO

OH

HO

OH HO HO

O

O N

OH HO O

HO O N

O

HO OH

HO OH

HO O

HO

OH O

OH

Acid-catalyzed

OH OH O O N OH

OH

OH

(2 eq.) HO OH

O

HO

O N

O

HO

HO O N

OH

HO

O

R

HO O

HO HO

116a / 116b

HO

O N

OH

OH

OH OH O O N OH

O

O

HO HO O O HO N OH

O O N OH

HO HO

OH O

HO O O N HO

HO HO

O HN

O N

HO HO

O

O N


O HO O

HO


393

3.20.5 Conclusions The extensive work highlighted within this chapter reveals that scientists have clearly developed clever and potentially practical methods for natural product-based glycodiversification. Many of the world’s anti-infective and anticancer drug leads directly derive from, or are inspired by, glycosylated natural products. The sugars attached to these metabolites greatly enhance the natural product chemical diversity and wield remarkable, but poorly understood, influence upon pharmacology, pharmacokinetic properties, and/or target specificity of the parent compound. Thus, the emerging glycodiversification strategies described herein hold spectacular future promise to understand and exploit the role of sugars in the context of bioactive metabolites, drug candidates, drugs, and even macromolecules.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

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Microbiol. 2005, 3, 925–936. Yoon, Y. J.; Beck, B. J.; Kim, B. S.; Kang, H.-Y.; Reynolds, K. A.; Sherman, D. H. Chem. Biol. 2002, 9, 203–214. Staunton, J.; Wilkinson, B. Curr. Opin. Chem. Biol. 2001, 5, 159–164. Solenberg, P.; Matsushima, P.; Stack, D. R.; Wilkie, S. C.; Thompson, R. C.; Baltz, R. H. Chem. Biol. 1997, 4, 195–202. Madduri, K.; Kennedy, J.; Rivola, G.; Inventi-Solari, A.; Filippini, S.; Zanuso, G.; Colombo, A. L.; Gewain, K. M.; Occi, J. L.; MacNeil, D. J.; Hutchinson, C. R. Nat. Biotechnol. 1998, 16, 69–74. Omura, S. Macrolide Antibiotics, Chemistry, Biology, and Practice; Academic Press: New York, 1984. Zhao, L.; Sherman, D. H.; Liu, H.-w. J. Am. Chem. Soc. 1998, 120, 10256–10257. Zhao, L.; Que, N. L. S.; Xue, Y.; Sherman, D. H.; Liu, H.-w. J. Am. Chem. Soc. 1998, 120, 10259–10260. Borisova, S. A.; Zhao, L.; Sherman, D. H.; Liu, H.-w. Org. Lett. 1999, 1, 133–136. Zhao, L.; Borisova, S.; Yeung, S.-m.; Liu, H.-w. J. Am. Chem. Soc. 2001, 123, 7909–7910. 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Rohr, J.; Schonewolf, M.; Udvarnoki, G.; Eckardt, K.; Schumann, G.; Wagner, C.; Beale, J. M.; Sorey, S. D. J. Org. Chem. 1993, 58, 2547–2551. Decker, H.; Haag, S. J. Bacteriol. 1995, 177, 6126–6136. Kunzel, E.; Faust, B.; Oelkers, C.; Weissbach, U.; Bearden, D. W.; Weitnauer, G.; Westrich, L.; Bechtold, A.; Rohr, J. J. Am. Chem. Soc. 1999, 121, 11058–11062. Trefzer, A.; Hoffmeister, D.; Kunzel, E.; Stockert, S.; Weitnauer, G.; Westrich, L.; Rix, U.; Fuchser, J.; Bindseil, K. U.; Rohr, J.; Bechtold, A. Chem. Biol. 2000, 7, 133–142. Hoffmeister, D.; Ichinose, K.; Domann, S.; Faust, B.; Trefzer, A.; Drager, G.; Kirschning, A.; Fischer, C.; Kunzel, E.; Bearden, D. W.; Rohr, J.; Bechtold, A. Chem. Biol. 2000, 7, 821–831. Drautz, H.; Reuschenbach, P.; Zahner, H.; Rohr, J.; Zeeck, A. J. Antibiot. 1985, 38, 1291–1301. Decker, H.; Haag, S.; Udvarnoki, G.; Rohr, J. Angew. Chem., Int. Ed. Engl. 1995, 34, 1107–1110. Decker, H.; Rohr, J.; Motamedi, H.; Zahner, H.; Hutchinson, C. R. Gene 1995, 166, 121–126. Wohlert, S.-E.; Blanco, G.; Lombo, F.; Fernandez, E.; Brana, A. F.; Reich, S.; Udvarnoki, G.; Mendez, C.; Decker, H.; Frevert, J.; Salas, J. A.; Rohr, J. J. Am. Chem. Soc. 1998, 120, 10596–10601. Prado, L.; Fernandez, E.; Weissbach, U.; Blanco, G.; Quiros, L. M.; Brana, A. F.; Mendez, C.; Rohr, J.; Salas, J. A. Chem. Biol. 1999, 6, 19–30. Blanco, G.; Patallo, E. P.; Brana, A. F.; Trefzer, A.; Bechtold, A.; Rohr, J.; Mendez, C.; Salas, J. A. Chem. Biol. 2001, 8, 253–263. Sanchez, C.; Zhu, L.; Brana, A. F.; Salas, A. P.; Rohr, J.; Mendez, C.; Salas, J. A. Proc. Natl. Acad. Sci. USA 2005, 102, 461–466. Salas, A. P.; Zhu, L.; Sanchez, C.; Brana, A. F.; Rohr, J.; Mendez, C.; Salas, J. A. Mol. Microbiol. 2005, 58, 17–27. Gaisser, S.; Reather, J.; Wirtz, G.; Kellenberger, L.; Staunton, J.; Leadlay, P. F. Mol. Microbiol. 2000, 36, 391–401. Doumith, M.; Legrand, R.; Lang, C.; Salas, J. A.; Raynal, M. C. Mol. Microbiol. 1999, 34, 1039–1048. Wohlert, S.-E.; Lomovskaya, N.; Kulowski, K.; Fonstein, L.; Occi, J. L.; Gewain, K. M.; MacNeil, D. J.; Hutchinson, C. R. Chem. Biol. 2001, 8, 681–700. Yang, J.; Fu, X.; Jia, Q.; Shen, J.; Biggins, J. B.; Jiang, J. Q.; Zhao, J. J.; Schmidt, J. J.; Wang, P. G.; Thorson, J. S. Org. Lett. 2003, 5, 2223–2226. Hoffmeister, D.; Yang, J.; Liu, L.; Thorson, J. S. Proc. Natl. Acad. Sci. USA 2003, 100, 13184–13189. Yang, J.; Liu, L.; Thorson, J. S. Chem. Biochem. 2004, 5, 992–996. Yang, J.; Fu, X.; Liao, J.; Liu, L.; Thorson, J. S. Chem. Biol. 2005, 12, 657–664. Thorson, J. S.; Barton, W. A.; Hoffmeister, D.; Albermann, C.; Nikolov, D. B. Chem. Biochem. 2004, 5, 16–25. Barton, W. A.; Lesniak, J.; Biggins, J. B.; Jeffrey, P. D.; Jiang, J. Q.; Rajashankar, K. R.; Thorson, J. S.; Nikolov, D. B. Nat. Struct. Biol. 2001, 8, 545–551. Barton, W. A.; Biggins, J. B.; Jiang, J. Q.; Thorson, J. S.; Nikolov, D. B. Proc. Natl. Acad. Sci. USA 2002, 99, 13397–13402. Fu, X.; Albermann, C.; Jiang, J. 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Biographical Sketch

Christopher J. Thibodeaux was born in Baton Rouge, Louisiana in 1980. He completed his Bachelor of Science degree at Louisiana State University in 2002, earning undergraduate majors in both Biochemistry and Plant Biology and a minor in Chemistry. He joined the research group of Prof. Hung-wen Liu as a graduate student at the University of Texas, Austin, in the spring of 2004. Since then, the main focus of his work has been enzyme kinetics and the elucidation of novel enzyme-catalyzed reactions. In his spare time, he enjoys traveling, hunting, fishing, camping, watching LSU Tiger football games, visiting his family, and spending time with his girlfriend, Ms. Gabrielle Moreno.

Hung-wen (Ben) Liu was born in Taipei, Taiwan, in 1952. He graduated with a Bachelor of Science degree in Chemistry from Tunghai University, Taichung, in 1974. After two years of military service, he began his graduate study at Columbia University where he carried out research under Professor Koji Nakanishi. His work on the additivity relation in exciton-split circular dichroism curves and its application to the structural studies of oligosaccharides earned him a Ph.D. in 1981. He then joined the laboratory of Professor Christopher Walsh at Massachusetts Institute of Technology as a postdoctoral fellow where he was introduced to the field of mechanistic enzymology. In 1984, he joined the faculty of Chemistry at the University of Minnesota, Minneapolis, where he was promoted to the rank of Full Professor in 1994, and to Distinguished McKnight University Professor in 1999. In 2000, he moved to the University of Texas, Austin, where he is now the George H. Hitchings Regents Chair in Drug Design and Professor of Medicinal Chemistry, Chemistry, and Biochemistry. His research lies at the crossroads of organic and biological chemistry, with particular emphasis on enzymatic reaction mechanisms, protein function regulation, inhibitor design and synthesis, natural product biosynthesis, and metabolic pathway engineering.

396


Jon Thorson was born in Brainerd, Minnesota in 1964. He received his B.A. degree in chemistry (1986) from Augsburg College and a Ph.D. degree in organic chemistry (1993) from the University of Minnesota with Professor Hung-wen (Ben) Liu. He held a postdoctoral appointment as a Merck Postdoctoral Fellow of the Helen Hay Whitney Foundation (1993–1996) at the University of California, Berkeley with Professor Peter Schultz. From 1996–2001, Jon held appointments as an assistant member of the Memorial Sloan-Kettering Cancer Center and assistant professor of Sloan-Kettering Division, Joan and Sanford I. Weill Graduate School of Medical Sciences, Cornell University, during which he was named a Rita Allen Foundation Scholar (1998–2002) and Alfred P. Sloan Fellow (2000–2002). Professor Thorson joined the School of Pharmacy in the summer of 2001 and since moving to UW has been designated an American Society of Pharmacognosy Matt Suffness Awardee (2004) and a UW H. I. Romnes Fellow (2004). His research interests include understanding and exploiting biosynthetic pathways and enzyme mechanisms, bioorganic and chemoselective ligation chemistries, enzyme engineering and evolution. Professor Thorson has also been credited with establishing the general area of natural product glycorandomization.

3.21 Fundamentals of Lectin–Carbohydrate Interactions T. K. Dam and C. F. Brewer, Albert Einstein College of Medicine, New York, NY, USA ß 2007 Elsevier Ltd. All rights reserved. 3.21.1 Introduction

397

3.21.2 Monovalent Carbohydrate Binding

398

3.21.2.1 Physical Forces

398

3.21.2.1.1 3.21.2.1.2 3.21.2.1.3 3.21.2.1.4 3.21.2.1.5 3.21.2.1.6

Hydrogen bond Hydrophobic interactions Divalent metal ions Water molecules Ionic interactions Carbohydrate conformation

398 404 404 407 411 412

3.21.2.2 Carbohydrate-binding Mechanisms 3.21.2.2.1 3.21.2.2.2 3.21.2.2.3 3.21.2.2.4 3.21.2.2.5 3.21.2.2.6 3.21.2.2.7 3.21.2.2.8 3.21.2.2.9

413

Influenza virus HA Legume lectins Cereal lectin Bulb lectins Galectins C-type lectins P-type lectins Fucose-binding lectins I-type lectins

413 414 416 416 419 419 422 425 427

3.21.3 Multivalent Carbohydrate Binding

430

3.21.3.1 Intramolecular Binding

431

3.21.3.1.1 3.21.3.1.2

Asialoglycoprotein receptor Shiga-like toxin and cholera toxin

431 432

3.21.3.2 Intermolecular Binding 3.21.3.2.1

434

Legume lectins and galectins

434

3.21.3.3 Some Other Examples of Multivalent Binding 3.21.3.3.1 3.21.3.3.2 3.21.3.3.3 3.21.3.3.4 3.21.3.3.5 3.21.3.3.6

Interaction of lectins with multivalent polymeric ligands Multivalent inhibitors of influenza virus HA Xenopus laevis lectin XL35 Interaction of cyanovirin-N with high-mannose oligosaccharides Photoswitchable cluster glycosides Multivalent binding by garlic lectin

3.21.3.4 Carbohydrate–Lectin Cross-linking Interactions 3.21.3.4.1 3.21.3.4.2 3.21.3.4.3

Type 1 and type 2 cross-linked complexes A multivalent carbohydrate can form a unique cross-linked complex with a lectin in the presence of other carbohydrates The structures of the carbohydrates and lectins determine their cross-linking properties

3.21.4 Conclusions

442 442 443 443 444 444 444

444 444 444 447

448

3.21.1 Introduction Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin. This evolutionarily conserved group of multi-subunit and therefore multivalent proteins has been implicated in diverse biological functions that include cell adhesion, cell recruitment, intracellular trafficking, and immune recognition.1–5 Recognition and binding of

397

398

Fundamentals of Lectin–Carbohydrate Interactions

carbohydrate ligands by lectins is crucial for their biological functions. Therefore, elucidating the physical nature of these interactions is important for understanding the functions of lectins and their carbohydrate receptors in biological systems. The production of recombinant lectins (both native and mutant forms) and synthesis of diverse arrays of mono- and multivalent carbohydrate analogs, together with the application of biochemical and biophysical techniques, have provided important insights into the physical basis of the specificity of lectin–carbohydrate interactions. The carbohydrate-binding sites of lectins are generally shallow structures on the surface of the protein that do not undergo significant conformational changes upon ligand binding.1,6 Mannose 6-phosphate receptors are among a few exceptions that possess relatively deep binding pockets, reminiscent of carbohydrate-binding bacterial periplasmic receptors.7 The carbohydrate-binding sites of lectins are usually composed of amino acids from a single polypeptide chain with certain exceptions, such as wheat germ agglutinin (WGA) where amino acids from different polypeptide chains are involved in ligand binding.1,8 Binding of a monosaccharide or oligosaccharide possessing a single carbohydrate binding epitope to a lectin is defined as monovalent binding. The interactions of branched chain oligosaccharides, polymeric glycans, and glycoproteins possessing multiple carbohydrate epitopes with lectins result in multivalent binding. The first section of this chapter describes the molecular mechanisms of monovalent carbohydrate–lectin interactions while the second section focuses on multivalent interactions. Other carbohydrate-binding proteins including sugar transporters, anticarbohydrate antibodies, and carbohydrate-processing enzymes are not covered in this chapter.

3.21.2 Monovalent Carbohydrate Binding 3.21.2.1 Physical Forces Pauling and Delbru¨ck9 in 1940 postulated the importance of intermolecular hydrogen bonding, van der Waals, and electrostatic interactions in molecular complex formation. This postulate is relevant to carbohydrate–lectin interactions since these physical forces help to stabilize complexes between lectins and carbohydrates. Solvent reorganization of the hydrated surfaces of the lectin and carbohydrate as well as conformational changes of the carbohydrate also contribute to stabilization of the complexes. A brief description of these physical forces (Figure 1) is given below. The mechanisms of binding of different lectin groups that include viral lectins, legume lectins, cereal lectin, bulb lectins, galectins, C-type lectins, P-type lectins, I-type lectins, fucose (Fuc)-binding lectins, and bacterial toxins have been reported. While the physical mechanisms of carbohydrate binding are similar, lectins have evolved to recognize a vast array of carbohydrate structures that are crucial for their diverse biological functions.

3.21.2.1.1 Hydrogen bond The presence of multiple hydroxyl groups (OH) on a carbohydrate allows formation of multiple hydrogen bonds with different amino acids in the binding site of a lectin. OH groups of the carbohydrate can donate and accept hydrogen bonds simultaneously (cooperative hydrogen bonding). The OH group can potentially accept two hydrogen bonds and donate a single hydrogen bond. Often, one acidic side chain acts as a hydrogen bond acceptor whereas main-chain

1

Sugar

2 O Lectin

3 +

− 4

Figure 1 Physical forces that stabilize a lectin–carbohydrate complex. These forces include hydrophobic interaction (1), Ca2þ (red circle) coordination (2), direct and water molecule-mediated hydrogen bonding (3), and ionic interactions (in limited instances) (4). Reorganization of water molecules (yellow circles) at the binding site of the lectin also contributes to binding.


399

amide groups and side-chain amide group of asparagine (and to a lesser extent glutamine) are main hydrogen bond donors. Charged side chains of the protein are often found as hydrogen bond donors. The side-chain OH groups of serine, threonine, and tyrosine residues appear less frequently involved in hydrogen bonding with sugar OH groups.6 One common type of bond found in lectin–carbohydrate interaction is a bidentate hydrogen bond. Such bonds involve two adjacent OH groups of a sugar ring and different atoms of the same amino acid residue.1,10 Compared to the fixed hydrogen bond geometry that arises between planar donors and acceptors, OH groups enjoys much more rotational freedom in establishing hydrogen bonds with adjacent groups. This rotational freedom, however, comes with some entropic cost. Formation of hydrogen bonds between protein OHs and sugar OHs may be limited in part due to high entropic cost.6 Hydrogen bonds between amino acids and sugar OHs may exist in a lower dielectric environment and may be stronger than those formed in water. Hydrogen bonds between donor and acceptors with fixed planar geometry play important roles in the determination of specificities. Freely rotating OH groups of amino acids such as serine show plasticity in hydrogen bond formation with sugar OH and may not distinguish between epimeric OHs.6 The ring oxygen of the sugar can serve as hydrogen bond acceptor and generally shares a donor amino acid with another OH of the sugar. Lectins often bind to the acetamido moiety of GlcNAc, GalNAc, and N-acetylneuraminic acid (Neu5Ac) residues. Generally, the amide group donates hydrogen bond to planar carboxyl or carboxylate oxygen whereas the acetamido oxygen serves as an acceptor of hydrogen bonds from a serine residue. The carbonyl oxygen of the acetamido substituent and the amide group display fixed planar geometry.6 The pattern of recognition of specific OH groups on sugars is lectin and lectin group specific. For example, D-mannose (Man)/Glc-specific legume lectins generally bind with the 3-, 4-, and 6-OHs of the carbohydrates. Therefore, these lectins do not discriminate between Man and Glc. However, the Man-specific bulb lectins interact with the 2-OH in addition to other OH groups of Man, and therefore do discriminate between Man and Glc binding. Gal specific legume lectins form hydrogen bonds with the 3-, 4-, and 6-OH of Gal and often tolerate a bulky group at the C2 position as in GalNAc. The hydrogen bond geometry with axial 4-OH of Gal is different from that of equatorial 4-OH of Man and Glc. The specificity of galectins for Gal results from the recognition of the 4- and 6-OHs of the sugar.6 Structurally unrelated monosaccharides that possess similar topographical features can sometimes bind to the same lectin (Figure 2). For example, WGA recognizes N-acetylglucosamine, Neu5Ac, and N-acetylgalactosamine.1 The three-dimensional structures of these monosaccharides reveals similarity at positions C2 (acetamide group) and C3 (hydroxyl group) of the pyranose ring of the two hexosamines with those of C5 and C4 on Neu5Ac, respectively. These

4 HO

O

CH2OH

HO

OH

HO 3

COOH HO

2

HO 3 L-Fucose

(a)

HNAc H

N-Acetylneuraminic acid

O

OH

OH 4

CH2OH

HO CH3

HO

H

C

OH

D-Mannose

2 HO

O

OH

C

CH2OH

OH

O

HNAc OH

N-Acetyl-D-glucosamine (b)

Figure 2 Common structural features of Man and Fuc (a) and of Neu5Ac and N-acetylglucosamine (b). Groups that occupy the same position in space are underlined. (a) Rotation of the Fuc molecule by 180 allows superimposition of its ring oxygen, 4-OH, 3-OH, and 2-OH with the ring oxygen, 2-OH, 3-OH, and 4-OH of Man, respectively. (b) Conformational similarity of N-acetylglucosamine and Neu5Ac at the underlined positions (acetamide and hydroxyl) of the pyranose rings is observed when the sialic acid molecule is suitably rotated. The conformation of N-acetylgalactosamine (not shown) at the relevant positions is identical with that of N-acetylglucosamine.1 Reprinted with permission from Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. Copyright (1998) American Chemical Society.

400


Group 1 Mannose

Group 2 Glucose

Galactose 4-Epimers

N-Acetylglucosamine

N-Acetylgalactosamine

Figure 3 In pyranose form, monosaccharides such as Man, Glc, GlcNAc (shown as group 1) and Gal and GalNAc (shown as group 2) share a common arrangement of the 3-OH group, the ring oxygen, and the carbons at positions 5 and 6. The two groups are distinguished by the orientation of the 4-OH groups. Within each group, the orientation and substitution at position 2 of the ring differ. Reproduced from Drickamer, K. Structure 1997, 5, 465–468, Copyright (1997), with permission from Elsevier.

are the positions critical for productive contact with the binding site of the lectin. Also, Man-specific animal lectins (e.g., the rat Man-binding proteins, MBPs) bind to Fuc because some of its structural features are similar to Man (Figure 2).1 Homologous lectins with different specificities can recognize specific sugars with the same conserved amino acids. In contrast, structurally different lectins with identical specificities may bind to the same ligand through different sets of residues.8 Drickamer11 classified common monosaccharrides into two groups based on the orientation of the OH groups (Figure 3). He proposed four principles after analyzing Man/Glc/GlcNAc-specific and Gal/GalNAc-specific animal lectins. First, specificity for one group of sugars over others requires specific interaction with the hydroxyl group, or groups, that distinguish the favored from the unfavored ligands. The orientation of these hydroxyl groups must be established by reference to common structural features. Interactions with these key structural features serve to orientate the sugar in the binding site. Second, once an orientation is established, additional selectivity can arise by steric exclusion of groups that might be common to bound and unbound sugars but which do not interfere with binding of the favored ligands in the established orientation. Third, positive interactions with common aspects of the sugars generally contribute to stability of the bound complex but not to selectivity. Fourth, when lectins can bind to multiple different sugars, the hydroxyl group or other substituents that differ among these ligands cannot contribute substantially to the binding interaction. A growing number of crystal structures of C-type lectins with the MBP-A-like (‘asymmetrical’) distribution of hydrogen bond donors and acceptors have shown that the core binding site is compatible not only with any two equatorial hydroxyl (3- and 4-OH of Man and Glc, 2- and 3-OH of Fuc), but also with a combination of axial and equatorial hydroxyl groups (3- and 4-OH of Fuc, as in E- and P-selectin structures).12 By analyzing several known structures of complexes of Gal- and Man/Glc-binding lectins, Elgavish and Shaanan13 suggested that a crucial level of ligand selection or specificity of the binding site is achieved by matching the C4 epimer (equatorial/axial 4-OH) with the given constellation of 4-OH donors/acceptors. When a match is established, the binding energy is enhanced by contributions from other ligand- and protein-dependent factors, such as additional hydrogen bonds, van der Waals interactions, and aromatic stacking. Poget et al.14 confirmed this observation and also noted that in C-type lectins the same rule is also true for the 3-OH proton.

3.21.2.1.1.1 Contribution of hydrogen bonding to the thermodynamics of binding Isothermal titration calorimetry (ITC) and crystallographic studies of lectin–carbohydrate complexes have demonstrated the contribution of hydrogen bonds to the binding free energy and enthalpy.15–18 These studies also show that in certain instances, additional hydrogen bonding in the extended binding sites of lectins is consistent with more negative values of DH.19 3.21.2.1.1.1.1 Involvement of additional hydrogen bonding in extended site interaction The Ka (4.9105 M–1) and DH (–14.4 kcal mol–1) values for concanavalin A (ConA) binding trimannoside 1 (Figure 4) are greater than those of


401

Me aMan (Ka ¼ 8.2103 M–1; DH¼–8.2kcalmol–1) and constituent disaccharides, Man(a1-3)Man and Man(a1-6)Man, which represent the two arms of the 3,6-trimannoside. These results provided direct evidence that ConA possessed an extended binding site which is responsible for the high affinity of the trimannoside.15,20,21 Indeed, crystallographic data clearly show that the trimannoside combining site is extended beyond the monosaccharide binding site.17 While the Man(a1-6) residue of trimannoside occupies the monosaccharide binding site, the Man(a1-3) and ‘core’ Man residues make additional hydrogen bonds with the extended area residues. Increased number of hydrogen bonds contributes to the higher Ka and DH values. As observed from the ITC studies with a series of deoxy analogs of trimannoside, disruption of any of these bonds adversely affect the affinity and the enthalpy of binding. The increased –DH value of the trimannoside relative to the monosaccharide was found to be consistent with an extended binding site. A view of the H-bonding interactions between the hydroxyl groups of the trimannoside and the binding site of ConA, derived from the X-ray data, is shown in Figure 5. ITC studies using a complete set of deoxy analogs (Figure 4) as well as di- and trideoxy analogs of the trimannoside15 showed excellent agreement with the X-ray data.17 The crystal structure shows that the 3-, 4-, and 6-OH groups of the Man(a1-6) residue of the trimannoside are hydrogen-bonded in the same manner as Me aMan in its crystalline complex with ConA. These results agree with the ITC data for the 3dMan(a1-6) 7, 4dMan(a1-6) 8, and 6dMan(a1-6) 9 trimannoside analogs.15 All these analogs showed reduced Ka and DH values compared to trimannoside, indicating the contribution of the hydrogen bonds. The X-ray data also show binding of the 3-OH of the Man(a1-3) residue to the N-H and side-chain O of Thr15 and the 4-OH of the Man(a1-3) residue to the side-chain OH of Thr15 (Figure 5). 2-OH and 4-OH of the central Man residue were also found hydrogen-bonded to the lectin. These observations were also in agreement with the thermodynamic

Man(a1-6) 1

Man(a1-OMe)

4dMan(a1-6)

Man(a1-3)

Man(a1-3) Man(a1-6) 2

Man(a1-OMe)

6dMan(a1-6)

Man(a1-3) Man(a1-6)

Man(a1-6) Man(a1-OMe)

Man(a1-3) Man(a1-6)


Man(a1-3) Man(a1-6)


3,4dMan(a1-3) Man(a1-6)

2dMan(a1-6) Man(a1-OMe)

2,4dMan(a1-OMe)

12

6dMan(a1-3)

6

4dMan(a1-OMe)

11

4dMan(a1-3)

5

2dMan(a1-OMe)

10

3dMan(a1-3)

4

Man(a1-OMe)

9

2dMan(a1-3)

3

Man(a1-OMe)

8

13

Man(a1-6) Man(a1-3)

Man(a1-3)

Man~OH

Man(a1-3) 3dMan(a1-6) 7

GlcNAc(b1-2)Man(a1-6)

Man(a1-OMe) Man(a1-3)

14

Man~OH GlcNAc(b1-2)Man(a1-3)

Figure 4 Structures of core trimannoside 1, deoxy analogs 2–12, Man 5 oligomannose carbohydrate 13, and biantennary complex carbohydrate 14. Man, GlcNAc, 2dMan, 3dMan, 4dMan, and 6dMan represent mannose, N-acetylglucosamine, 2-deoxymannose, 3-deoxymannose, 4-deoxymannose, and 6-deoxymannose residues, respectively.

402


O Tyr12

CH3

N O

H

H

Thr15

H

O 3 HO

Tyr100

H O

Asp16 O _ O

OH OH

O

N H O

H O

N H Leu99 O

H OO

C O

O

O

6

OH O H

O O HO HO

H

H

H

O

H

H2N + NH2

N H

Asn14

NH

O _ O

Arg228

Asp208 H N O

Figure 5 View of the X-ray crystal structure of trimannoside 1 (no anomeric methoxy group) bound to ConA. The trimannoside is shown with the central Man indicated by C, the Man(a1-6) by 6, and the Man(a1-3) by 3. Reproduced from Naismith, J. H.; Field, R. A. J. Biol. Chem. 1996, 271, 972–976, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 1996 by ASBMB.

data. The Ka and DH values for the 3dMan(a1-3) 3, 4dMan(a1-3) 4, ‘core’ 2dMan 10, and ‘core’ 4dMan 11 analogs were all less than the parent trimannoside because of the removal of the corresponding hydrogen bonds.15 3.21.2.1.1.1.2 Nonlinearity of the DDH and DDG values of the individual hydroxyl groups of 1 Thermodynamic data indicate that the DDH values for the monodeoxy analogs are nonlinear.15 For example, the combined DDH value for the 3-OH and 4-OH of the Man(a1-3) residue of trimannoside obtained from 3 and 4, respectively, and the 2-OH and 4-OH of the central Man residue obtained from 10 and 11, respectively, was c. –8.8 kcal mol–1. This could be compared to the difference in DH between 1 and Me aMan of –6.2 kcal mol–1, which reflects binding of the Man(a1-3) and the central Man residues of 1. Furthermore, the sum of the DDH values for the 3-, 4-, and 6-OH of the Man(a1-6) residue (7–9), the 3- and 4-OH of the Man(a1-3) residue (3 and 4), and the 2- and 4-OH of the central Man residue (10 and 11) was –17.5 kcal mol–1, which was greater than the DH for 1 of –14.4 kcal mol–1. Thus, the sum of the DDH values for the hydroxyl groups of 1 obtained from the monodeoxy analogs did not correspond to the measured DH of 1. In all of the above cases, the sum of the DDH values for specific hydroxyl groups on certain Man residues of 1 obtained from the corresponding monodeoxy analogs was greater than the measured DH for that residue(s). This nonlinear relationship in DDH was also present in the di- and trideoxy analogs. The same nonlinearity was also present in the DDG values of the monodeoxy analogs. The DDH and DDG values for each monodeoxy analog of 1 also did not scale with the number of H bonds at each position as determined from X-ray crystallography. The DDH values for the monodeoxy analogs are not proportional to the number or type of H bonds involved at specific hydroxyl groups of 1. This is of particular interest since it has been suggested that the free energy associated with elimination of a H bond between an uncharged donor/acceptor pair is 0.5–1.5kcal/mol and between a neutral-charged pair is 3.5–4.5kcalmol–1.22 The data, however, indicate no such relationship in the free energy difference (DDG) of monodeoxy analogs that represent the loss of one or more H bonds such as 7 versus 8 and 9. The presence of nonlinear relationships in the DDH and DDG values for the deoxy analogs indicates other contributions to these terms such as solvent and protein effects. Thus, the magnitude of the DDH and DDG values represents not only the loss of the H bond(s) involved, but also differences in the solvent and protein contributions to binding of 1 and the deoxy analogs.


403

Contributions of solvent to the DH of sugar binding to a lectin have been shown experimentally and presented in Section 3.21.2.1.4. 3.21.2.1.1.1.3 Homologous lectins with conserved binding sites interact with the same ligand through the same set of hydrogen bonds but with different binding thermodynamics Thermodynamic data in combination with structural data clearly show that homologous Diocleinae lectins interact with the same set of hydroxyl groups of the trimannoside. The X-ray crystal structures of two members of this group namely ConA and Dioclea grangiflora lectin (DGL) complexed with the core trimannoside17,18 show conserved contact residues for both proteins (Figure 5). Three other Diocleinae lectins from Dioclea guianensis, Canavalia floribunda, and Canavalia brasiliensis also have these same contact residues for the trimannoside. 23 However, the ITC data in Figure 6 also show a range of DDH values for certain deoxy analogs which have corresponding hydroxyl groups involved in binding to the nine Diocleinae lectins. These include DDH values ranging from 6.5 to 7.7 kcal mol–1 for 7–9 binding to Dioclea rostrata to the much lower values of 3 kcal mol–1 for ConA.15 Thus, there is a wide variation in the DDH values of the nine Diocleinae lectins in Figure 6. This is true not only for the same deoxy analog with different lectins, but also for different deoxy analogs that have corresponding hydroxyl groups that bind to the same lectin. For example, analogs 3, 7, and 11 possess different DDH values in binding to ConA and DGL, respectively, even though their respective hydroxyl groups of 1 show hydrogen bonds to both lectins (Figures 5 and 15). Another example comes from the comparison of ConA and C. brasiliensis. The X-ray crystal structure of the lectin from C. brasiliensis shows only two amino acid changes relative to ConA. 24 Gly58 and Gly70 in C. brasiliensis are replaced by Asp and Ala, respectively, in ConA. Neither of the residues is near the carbohydrate-binding sites in both lectins, and only small changes in the quaternary structures of the two lectins were noted. However, these two amino acid changes result in significant differences in the DDH values of both lectins’ binding to analogs 7–9 (5 kcal mol1 for C. brasiliensis vs 3.0 kcal mol–1 for ConA). In addition, although the Ka values of the two lectins for 1 are comparable (3.7 105 M–1 for C. brasiliensis and 4.9 105 M–1 for ConA), the lectin from C. brasiliensis possesses a DH of –12.4 kcal mol–1 for 1 while ConA possesses a DH of 14.4 kcal mol–1 for 1. It is also interesting that ConA and the C. brasiliensis lectin are reported to have different lectin-induced nitric oxide production in murine peritoneal cells in vitro.25 Differences in the binding thermodynamics in a homologous group of lectins where the binding residues are conserved indicate the indirect but important roles of the nonconserved residues away from the carbohydrate-binding site. Subtle changes in the hydration of the lectins or subtle differences in their conformation due to the minor alteration of amino acid residues away from the carbohydrate-binding sites may be responsible for their thermodynamic binding differences.19 Effects of single-site mutations on the conformational features of lectins have been explored by Siebert et al.26 Similar changes in the thermodynamics of binding of lectins with amino acid substitutions away from the carbohydrate binding site have been reported. For example, galectin-1

8

C. brasiliensis

7 C. floribunda D. rostrata

5

4d core(11)

D. grandiflora

2d core(10)

0 6d(1-6)(9)

Concanavalin A 4d(1-6)(8)

1

3d(1-6)(7)

D. virgata

2d(1-6)(6)

2

6d(1-3)(5)

D. violacea

4d(1-3)(4)

3

3d(1-3)(3)

D. guianensis

2d(1-3)(2)

4

Trimannoside(1)

∆∆H (kcal/mol)

6

C. grandiflora

Carbohydrates Figure 6 Bar graph showing DDH values of the seven Diocleinae lectins as well as ConA and DGL for deoxy analogs 2–11.19

404


from Chinese hamster ovary cells was reported to undergo significant changes in DH and TDS but not DG in binding to LacNAc when single or multiple mutations were introduced in the N-terminal region of the protein.27 Binding affinity of influenza virus hemagglutinin (HA) is affected when certain residues remote from the binding sites are changed.28

3.21.2.1.2 Hydrophobic interactions The presence of OH groups and the ring oxygen make carbohydrates highly polar and solvated molecules. However, the aliphatic protons and carbons at the various epimeric centers that extend out to the exocyclic 6 positions of hexoses and the glycerol moiety of sialic acids create nonpolar faces on sugar residues. This nonpolar face usually stacks on one or more side chains of the aromatic amino acids such as phenylalanine, tyrosine, and tryptophan and provide considerable driving force for complex formation. Unlike Gal, Man does not always stack on an aromatic side chain. The equatorial position of the 4-OH of Man, compared to the axial 4-OH of Gal, gives rise to a less extensive nonpolar surface than that of Gal (Figure 7). In Man-specific legume lectins, a conserved phenylalanine ring stacks against the Man C5 and C6; however, no aromatic interactions have been recorded in MBPs and Galanthus nivalic lectin (GNA).6 The carbon backbone of the glycerol moiety of sialic acid may interact with aromatic amino acid as observed in WGA. Hydrophobic interaction is also mediated through the methyl group of the acetamido moiety of GalNAc, GlcNAc, and Neu5Ac (WGA, HA)6 and the methyl group of Fuc. Side chains of certain aliphatic amino acids (e.g., valine or leucine) may also be involved in hydrophobic interactions.8 Binding of blood group H type II [Fuc(a1-2)Gal(b1-4)GlcNAc] to some legume lectins is partially hydrophobic in nature. When this trisaccharide binds to Erythrina corallodendron lectin (ECorL), the Fuc residue is involved in a strong hydrophobic interaction, most likely with Trp135 of the lectin.29 Combining structural and thermodynamic studies, Surolia and co-workers have shown that the binding of blood group H type-II trisaccharide to winged bean agglutinin II (WBA II) is predominantly hydrophobic in nature (Figure 8). The GlcNAc residue and the CH3 group of Fuc show strong hydrophobic interaction with Tyr215 (Figure 9).30,31 Hydrophobic interaction is also demonstrated to occur between the methyl group of GalNAc of T-antigen and WBA I.32

3.21.2.1.3 Divalent metal ions For legume lectins, the divalent cations Ca2þ and Mn2þ are not directly involved in sugar binding but are important for maintaining the integrity of carbohydrate-binding sites. They are essential for proper spatial orientation of the residues that directly interact with the carbohydrate ligands and thus stabilize the binding site (Figure 10).

1 1

a-Man 2

5

2

6

5 3

3

6

B

4

A

4

1 1

b-Gal 2

5

2

6

5

B 3 3

4

4

6

A

Figure 7 Nonpolar surface on Man and Gal. The axial 4-OH of galactose provides a more continuous aliphatic patch formed by the 3-, 4-, 5-, and 6-carbon relative to Man.6 Reprinted, with permission, from the Annual Review of Biochemistry, Volume 65, Copyright 1996 by Annual Reviews.


405

NHAc OH H3C

HO

O

O

* OH O b

*

HO

a c

OCH3 O OH) OH

O

OH

OH

* Fuc(a1-2)Gal(b1-4)GlcNAc-OMe c b a Figure 8 Binding of blood group H-type-II-OMe trisaccharide to WBA II. The continuous line depicts the surface of the saccharide molecule that interacts hydrophobically with the lectin. The asterisks show the hydroxyl groups that are potentially involved in direct hydrogen bonding with WBA II. Reprinted by permission of Federation of the European Biochemical Societies from Srinivas, V. R.; Reddy, G. V.; Surolia, A. FEBS Lettres, 450, 181–185, Copyright (1999).

W131

W131

Fuc

O7

O2

Fuc

NAG

N129

O3

O7

Gal

O6 Q216

N129

O3

O3 G106

Gal

O6 Q216

O4

F127

Y215 D88

O2

NAG

O4 Y215

O3 G106 F127 D88

Figure 9 Stereo view of the interactions of H-type II trisaccharide in conformation I with protein atoms in WBA II. Loop D of EcorL is shown in gray. Reproduced from Manoj, N.; Srinivas, V. R.; Surolia, A.; Vijayan, M.; Suguna, K. J. Mol. Biol. 2000, 302, 1129–1137, copyright (2000), with permission from Elsevier.

Figure 10 A monomer of ConA with metal ions. Metal ions are shown as gray spheres. Bound ligand (trimannoside Man (a1-3)[Man(a1-6)]Man) is shown in green ball-and-stick representation. Reproduced from Loris, R. Biochim. Biophys. Acta 2002, 1572, 198–208, copyright (2002), with permission from Elsevier.

The Ca2þ coordinates with the side-chain carbonyl oxygen of a conserved asparagine residue. The side chain NH2 of the same residue forms a hydrogen bond with the sugar ligand. One carboxylate oxygen of an acidic amino acid is connected to a water molecule that in turn is coordinated with Ca2þ; the other oxygen makes a hydrogen bond with the carbohydrate. In this way, the side chains are spatially adjusted by Ca2þ coordination shell for optimal carbohydratebinding interactions. The Mn2þ, on the other hand, fixes the Ca2þ position.6

406


Brewer and co-workers33 studied the role of metal ion binding in legume lectins using nuclear magnetic relaxation dispersion measurements. They reported the detailed molecular mechanism of Mn2þ and Ca2þ binding and activation of the saccharide-binding activity of ConA. It was shown that ConA existed as mixture of two conformational states: a ‘locked’ form and an ‘unlocked’ form. The unlocked form of the protein weakly binds metal ions and saccharide and is the predominant conformation of demetalized ConA (apo-ConA) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide-binding activity. The group measured the kinetics of the transition of the unlocked form to the fully metalized locked conformation containing Mn2þ and Ca2þ. It was also demonstrated that Mn2þ alone could form a locked ternary complex with ConA, and that rapid removal of the ions resulted in a metastable form of apo-ConA in the locked conformation which slowly reverted back to the unlocked conformation. These studies showed the relative contributions of the metal ions and protein to the affinity of the lectin for sugars and suggested a cis–trans-isomerization of the backbone of the protein as a metal ion-dependent mechanism of activation of ConA, which was subsequently confirmed by X-ray crystallography. A variety of paramagnetic transition metal ion derivatives of ConA and other lectins were prepared, and electron spin resonance, nuclear magnetic resonance (NMR), and circular dichroism studies were used to characterize the metal ion-binding sites of the lectins. Ca2þ participates in sugar binding in C-type lectins and helps to stabilize the protein structure. There are four different Ca2þ-binding sites in C-type lectins (Figure 11); however, the occupancy of those sites depends on the lectin involved. Among the four sites, Ca2þ at site 2 is involved in carbohydrate binding.12 As shown in the MBP, one lone pair of electrons from each of two OHs of Man (3-OH and 4-OH) establishes a coordination bond with the Ca2þ, the other lone pair accepts a hydrogen bond from a side-chain amide group, and the proton is donated to an acidic oxygen in a hydrogen bond (Figures 12 and 29). This bonding pattern is common for all C-type lectins. The extent of hydrogen bonding and van der Waals interactions in monosaccharide binding by other non-C-type lectins has not been observed in MBP–Man interaction. This suggests that the direct coordination bonds between Ca2þ and sugar ligand significantly contribute to the affinity of the monosaccharide.6 The other notable factor that supports Man binding is the hydrophobic interaction from His189. Crystallographic and mutational analysis showed that the Ca2þ binding site geometry did not change with the sugar specificity of the lectin. Specificity is determined through hydrogenbonding patterns of respective monosaccharides, for example, from Man-type asymmetrical to Gal-type symmetrical (Figure 12).12

Ca1 Ca2

Ca2

Ca3 Ca1

Ca4

Human ASGPR-I

Rat MBP-A

Figure 11 Ca2þ-binding sites in C-type lectins. Ribbon diagrams of rat MBP-A and human ASGPR-I, demonstrating the four typical locations of calcium ions in the lectins. Ca2þ ions are shown as black spheres, and numbers indicating different Ca2þ-binding sites are shown next to the arrows. Reproduced with permission from Zelensky, A. N.; Gready, J. E. FEBS J. 2005, 272, 6179–6217. Copyright (2005) Blackwell Publishing.


H bond (don acc)

407

Coordination bond

Figure 12 Ca2þ-mediated monosaccharide binding by C-type lectins. A schematic representation of a Ca2þ-hexoseC-type lectin complex. Two hydroxyl oxygens and the ring of the hexose are shown. The Ca2þ atom is shown as a large gray sphere, and oxygens as empty circles and ovals. Protein groups that act as hydrogen donors and acceptors are not shown. Arrows show the direction of hydrogen bonds in mannose-specific C-type lectins, while light gray arrows indicate changed directions in Gal-specific C-type lectins. Reproduced with permission from Zelensky, A. N.; Gready, J. E. FEBS J. 2005, 272, 6179–6217. Copyright (2005) Blackwell Publishing.

Carbonyl side-chain-containing residues that simultaneously coordinate with site 2 Ca2þ and recognize monosaccharides constitute two characteristic motifs in C-type lectin-like domain (CTLD) sequence. These motifs are known as EPN motif (E185, P186, N187) and WND motif (W204, N205, D206). It should be mentioned that some C-type lectin domains that possess these motifs do not show any carbohydrate-binding activity, some CTLDs (e.g., Dectin-1) do not contain this motif and do not require Ca2þ for carbohydrate binding, and in some CTLDs (e.g., antifreeze protein) the Ca2þ at site 2 is involved in noncarbohydrate ligand binding.12 Removal of Ca2þ makes some CTLD susceptible to proteolytic degradation and alters some other physical properties. However, the tunicate C-type lectin is able to maintain its structural integrity in the absence of calcium ions. In lysosomes, pH-induced Ca2þ loss destabilizes the binding site loop, and thus asialoglycoprotein receptors and macrophage Man receptor release their bound ligands.

3.21.2.1.4

Water molecules

Water on the biomolecular surface occupies less space than the polar side chains of a protein, causes some steric constraints on bond formation, yet can participate in multiple hydrogen bonding by serving both as hydrogen bond donor and acceptor. For all these properties, water contributes significantly to biomolecular complex formation and affinity and specificity. The entropic cost of surface-bound highly mobile water is so large it would adversely influence the binding affinity; however, the enthalpic gain from additional water-mediated hydrogen bonding is greater than the entropic cost. The location of a water molecule in a binding site determines the number and nature of hydrogen bonds it can form with other vicinal determinants.34 It has been shown with a glycogen phosphorylase inhibitor that the strength of water-mediated hydrogen bonds between protein and sugar is comparable to direct protein sugar hydrogen bonds.6,35 Hydrogen bonds between a saccharide and water are reported to be stronger than water–water hydrogen bonds.36 Three water molecules have been shown to mediate Gal binding to enterotoxin and cholera toxin (CT) as well as lactose and Gal binding to enterotoxin. Sometimes water molecules behave as fixed structural entities of the binding site and contribute to hydrogen-bonding interactions like other participating groups of amino acids in the vicinity. In situation like this, water molecules are considered as the extension of the binding site (e.g., arabinose-binding protein and others). Gal interacts with one such water molecule when it binds to CT. The interactions of the water molecules that are observed in biomolecular complexes in high-resolution crystallographic studies are energetically more favorable than bulk solvent interactions.37 Buried water molecules have a long residence time in protein structures.34

408


Before complex formation, the polar groups of both the ligand and the receptor site are expected to be extensively hydrogen-bonded to water molecules.38 Ordered water molecules form hydrogen bonds with unligated lectins in a way that resembles the hydrogen bonding by the sugar hydroxyl groups. Hydrogen-bonded water molecules are displaced by sugar ligands from the binding site as seen in pea lectin and MBP-C.6 Displacement of these water molecules is a prerequisite for complex formation. Release of surface-bound water molecules into the bulk results in entropic gain; therefore, in many instances, removal of water molecules from a binding site shows favorable effect. Several reports show that removal of surface water generates the driving force required for ligand–receptor complex formation.34 Surface-bound water possesses higher energy content than those in bulk. Water molecules form an organized layer on nonpolar surfaces. Release of these molecules to bulk solvent raises the entropy. On the other hand, removal of less-ordered perturbed water molecules, characteristically associated with polyamphiphilic surfaces, decreases the entropy and DH. The return of high-energy surface water to bulk would cause heat liberation and decrease the free energy of the system.38 A favorable enthalpic contribution to binding is expected when the weakly bound disordered water molecules are displaced by a carbohydrate ligand and return to bulk where it may form stronger hydrogen bonds.39 Involvement of water molecules in carbohydrate–lectin interactions in aqueous solution has been shown by several studies as described below.

3.21.2.1.4.1 Role of the ordered water molecule in the binding site of ConA Binding of ConA to trimannoside and its derivative (in which the 2-OH of central Man was replaced with a hydroxyethyl moiety (Figure 13) that displaced a conserved water molecule from the binding site of ConA) shows the importance of the conserved water in ConA–trimannoside complex formation.39 Dunitz40 has reported that the release of highly ordered water may be associated with a favorable entropy of 2 kcal mol–1 at 300 K. Analog 16 released a highly ordered water molecule from the binding site and therefore showed more favorable entropy of binding compared to 15. This favorable entropic effect was compensated for by a relatively large unfavorable enthalpic term resulting in reduced affinity compared with 15. As a matter of fact, binding enthalpy of trimannoside 15 was more favorable than analog 16. The authors proposed that indirect interaction mediated by the conserved water molecule at the binding site contributed to the favorable enthalpic effect observed with 15 and to the stability of the ConA–trimannoside complex (Figure 14). 3.21.2.1.4.2 Relative contribution of solvent to the enthalpy of binding of saccharides to ConA and DGL Chervenak and Toone41 obtained thermodynamic binding data from different ligand–receptor interactions (including lectin–carbohydrate) in H2O and D2O. The enthalpy of binding in D2O was 400–1800calmol–1 less negative than the enthalpy in H2O. Binding free energy remained unchanged due to the offsetting change in the entropy. A strong correlation between the differential enthalpy of binding and DCp, the change in heat capacity, for binding was observed, with a slope of 5K. The authors concluded that solvent reorganization provided 25–100% of the observed enthalpy of binding. 3.21.2.1.4.3 ITC measurements of carbohydrate binding to ConA under osmotic stress The number of water molecules involved in the binding of ConA to trimannoside 1, Man(a1-3)Man, Man(a1-6)Man, and Man was determined by ITC under osmotic stress using glycerol and ethylene glycol.42 The osmotic release of water molecules from the binding sites caused a concomitant decrease in the binding free energy. This observation, as

HO HO HO

HO HO HO OH O

OH O

O OH

HO

O

O 15

HO HO HO

OH HO O HO HO OH HO O O HO

O

O OMe

16

OMe

Figure 13 Trimannoside 15 and its modified analog 16 used by Clarke et al. (2001). Reprinted with permission from Clarke, C.; Woods, R. J.; Gluska, J.; Cooper, A.; Nutley, M. A.; Boons, G.-J. J. Am. Chem. Soc. 2001, 123, 12238–12247. Copyright (2001) American Chemical Society. 39


409

Binding of 15 to ConA

H 2O

H2O

H2O

H2O H2O

+ n H2O

H2O

(a) Binding of 16 to ConA H2O

H2O

H2O H 2O

H2O

+ (n+1)H2O

H2O

(b) n = number of water molecules released or taken up during binding 15 = conserved water molecule = hydroxyl H2O = solvation water molecules = hydrogen bond Figure 14 Binding of 15 and 16 to ConA.39 Reprinted with permission from Clarke, C.; Woods, R. J.; Gluska, J.; Cooper, A.; Nutley, M. A.; Boons, G.-J. J. Am. Chem. Soc. 2001, 123, 12238–12247. Copyright (2001) American Chemical Society.

the authors suggested, indicated the importance of water mediation in sugar binding by the lectin. The number of solute-excluding water molecules coupled to the binding of sugars to ConA as a function of osmotic stress was found to be 5, 3, 3, and 1 for Man, Man(a1-3)Man, Man(a1-6)Man, and trimannoside Man(a1-3)[Man(a1-6)]Man, respectively. The slope of the enthalpy–entropy compensation plot was greater than unity, both in the presence and absence of osmolyte.

3.21.2.1.4.4 Role of water molecules in the extended binding sites of ConA and DGL The effects of solvation in carbohydrate binding have been studied with ConA and DGL.43 Binding of trimannoside 1 and its deoxy analogs 2–11 (Figure 4) to ConA and DGL in H2O and D2O showed primary solvent isotope effects in the DH values. The solvent isotope effect in DH for DGL binding to 1 is greater than that for ConA. DG values for the two lectins binding to 1 do not significantly change in H2O and D2O. The X-ray crystal structures of ConA17 and DGL18 complexed with the trimannoside 1 are similar, and the location and conformation of the bound trimannoside as well as its hydrogen-bonding interactions are nearly identical in both lectin complexes. However, differences exist in the location of two loops outside of the respective binding sites containing residues 114–125 and residues 222–227. The latter residues affect the location of a network of hydrogen-bonded water molecules that interact with the trisaccharide (Figure 15). Differences in the arrangement of ordered water molecules in the binding site of the two lectins may account for their differences in the DDH (H2O – D2O) values.43 3.21.2.1.4.4.1 Correlation of the DDH (H2O – D2O) data for analogs 2–11 with differences in the location of ordered water in the DGL and ConA complexes with trimannoside 1 The X-ray structures (Figure 15) of ConA17 and DGL18 with 1 reveal differences in the location of ordered water in the following three regions of the two complexes. The first region is associated with the 2-OH group on the Man(a1-3) unit of 1. The DDH (H2O – D2O) data for the dMan(a1-3) analogs of 1 show that only the 2-deoxy analog 2 exhibits a large variation in the DDH (H2O – D2O) for

410


Dioclea grandiflora lectin

Concanavalin A Ser21

Pro13 HO

HO

O NH 3.3

3.2

w w 69 58 3.7

O

Asp16 3.1

2.9

O

2.9 2.8 3.0

2.7 3.5

NH

Thr12 O

OH

w 58

w 70 3.6

2.7

H N 2

Asn21

O 3.8 3.1 3.7 O w 3.0 68

Thr15 O

NH

w 59

3.2 HO 2.9

w 69

3.0

3.0

2.6

O

Asp16

2.9 2.9

NH

OH O OH 2.9 (4) HO Asn14 2.9 w 2.9 HO (3) OH (6) (2) 41 O H N 3.1 (5) 2 w 3.2 H C O 1.7 OH 3 39 O(1,3) 2.8 3.5 (1) 3.7 w 2.0 O 2.7 2.8 HO Ca2+ 1 OH (2)OH 3.2 2.8 O (4) O 3.2 NH (5) w 3.3 85 H2N (1,6) (4) Leu99 O 2.9 NH 2.9 (5) OH 3.0 3.6 NH O O 2.9 O Asp208 NH OH 3.0 OH (6) (2) OH (3) 3.0 3.3 O HO 3.2 Try100 2.8 2.9 3.1 w 2.9 NH 3.4 w w 60 Arg228 87 4.1 3.6 66 w H2N NH 86 3.6 O 3.0 w w 3.2 Gly224 2.7 67 89 O 2.9 3.0 NH 2.9 Asn168 O 2.8 HN Gly226 w 37 O Gly227

OH 3.3 (4) HO Asn14 3.1 OH HO (3) (2) O (6) H2N (5) H3C w OH 2.8 O 2.4 O (1,3) 2.6 39 3.5 (1) O 2.4 w 2.8 2+ HO Ca 2.9 1 OH OH 3.0 (2) 2.8 O (4) O 2.9 NH w (5) 85 3.5 (1,6) H 2N (4) Leu99 O 2.7 NH 2.6 2.9 (5) OH 3.0 NH O 3.1 O O Asp208 NH 3.1 (2) OH OH (6)OH 3.0 2.9 (3) O HO 2.8 Tyr100 2.8 3.5 2.7 2.9 w NH Arg228 4.2 2.7 w 60 w w 87 66 w 4.0 86 NH 3.3 2.4 w 4.2 3.9 2.8 Gly224 67 OH OH 3.0 2.7 O O NH Ser168 3.4

2.8

Thr226

(a)

NH

Glu205

O

NH

Pro13

N

Tyr12

HO

NH

His205

Thr15

4.1

3.1

(b)

Figure 15 Schematic representation of the hydrogen bond interactions between the trimannoside ligand and the surrounding amino acid residues and ordered water molecules in ConA and in DGL. The dashed lines represent hydrogen bonds, and the distances are labeled in angstroms. Water 39 has a strong electron density, is held by the side chains of Asn 14, Asp 16, and Arg 228, and makes a direct hydrogen bond with the trimannoside ligand, and its position is strictly conserved between DGL and ConA. The remaining ordered water molecules possess weaker electron density, which may indicate a decrease in binding strength between themselves and the protein. Reproduced from Dam, T. K.; Oscarson, S.; Sacchettini, J. C.; Brewer, C. F. J. Biol. Chem. 1998, 273, 32826–32832, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 1998 by ASBMB.

DGL and ConA (Figure 16).43 The second region with altered order water in the two complexes is that associated with the 4-OH group of the central Man residue of 1. The DDH (H2O – D2O) data for the two deoxy analogs of the central Man residue of 1 show that the 4-deoxy analog 11 exhibits a significant difference in this parameter for DGL relative to that for ConA (Figure 16). The third area of the X-ray crystal structures of the DGL and ConA complexes which differ in ordered water structures is near the 2-OH on the (a1-6)-arm of 1. The DDH (H2O – D2O) data for DGL and ConA binding to the deoxy analogs of the (a1-6)-arm of 1 show that the 2-deoxy derivative 6 possesses the largest difference in their respective values (Figure 16). 3.21.1.4.4.2 Correlation of DDH (H2O – D2O) values of deoxy analogs of 1 with the number and strength of solvent hydrogen bonds to hydroxyl groups of trimannoside 1 in DGL and ConA The relative magnitude of the DDH (H2O – D2O) values of DGL is greater than that of ConA for deoxy analogs 2 (2dMan on (a1-3)-arm), 6 (2dMan on (a1-6)-arm), and 11 (core 4dMan) (Figure 16). The numbers of water molecules and corresponding hydrogen bonds connected to the 2-OH group of Man(a1-3), the 4-OH group of core Man, and the 2-OH group of Man(a1-6) are more in the DGL complex than those in ConA complex (Figure 15). This shows a correlation with the numbers and strength of water molecules interacting with the corresponding hydroxyl groups of the trimannoside in the respective complexes and the magnitude of the DDH (H2O – D2O) values. 3.21.2.1.4.4.3 Correlation of the DDH (H2O – D2O) data for Me aMan and Me aGlc with differences in the location of ordered water in the DGL and ConA complexes with trimannoside 1 Me aMan and Me aGlc (Figure 16) also show a correlation with the altered ordered water structures observed in the binding site regions of the DGL and ConA complexed with the trimannoside (Figure 15). Since Me aMan occupies the same site as the Man(a1-6) residue of 1 and makes similar contacts with ConA (2), it is reasonable to assume that the altered ordered water near the 2-OH of


411

ConA ∆∆H (H2O − D2O) DGL ∆∆H (H2O − D2O) 3.5

∆∆H (kcal mol−1)

3.0 2.5 2.0 1.5 1.0 0.5

Me aGlc

Me aMan

Core 4dMan

Core 2dMan

6dMan(a1-6)

4dMan(a1-6)

3dMan(a1-6)

2dMan(a1-6)

6dMan(a1-3)

4dMan(a1-3)

3dMan(a1-3)

2dMan(a1-3)

Trimannoside

0

Figure 16 Plot of DDH (H2O – D2O) (kcal mol–1) data for DGL and ConA with trimannoside 1, analogs 2–11, Me aMan, and Me aGlc.43 Reproduced from Dam, T. K.; Oscarson, S.; Sacchettini, J. C.; Brewer, C. F. J. Biol. Chem. 1998, 273, 32826– 32832, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 1998 by ASBMB.

the Man(a1-6) residue of 1 in the DGL and ConA complexes is present in their respective complexes with the monosaccharide. The DDH (H2O – D2O) value for DGL binding to Me aMan is higher than that for ConA, which is consistent with altered solvation of these two lectin complexes. Furthermore, the DDH (H2O – D2O) values for DGL binding to Me aMan and Me aGlc are substantially different, while the corresponding values for ConA binding to the two monosaccharides are almost similar (Figure 16). Since the two sugars differ in the orientation of their 2-OH groups (axial and equatorial, respectively), these results are consistent with altered solvation of the two monosaccharide complexes in both lectins, specifically at the 2-axial hydroxyl group of Man in both lectins. 3.21.2.1.4.4.4 Lack of correlation of altered water structures in the DGL and ConA complexes with the core trimannoside and DDH values in H2O for binding of both lectins to the deoxy analogs of trimannoside 1 The ITC solvent isotope data with analogs 2, 6, and 11 confirm that differences in the ordered water structures observed in the X-ray crystal complexes of ConA and DGL with the trimannoside exist in their corresponding solution complexes. However, a lack of correlation was found between the DDH values of the deoxy analogs of 1 with both lectins and the altered water structure of the two complexes with 1. The DDH for the loss of 2-, 3-, 4-, or 6-OH groups at the Man(a1-6) residue is 3kcalmol–1 in H2O and 2.0kcalmol–1 in D2O greater in DGL than ConA.43 However, as shown in Figure 15, no significant difference in the water structure in the region of 3-, 4-, and 6-OH groups of the Man(a1-6) residue was found in either complexes. The 3-OH of the Man(a1-6) unit of 1 is in contact with W60 in both complexes, while the 4- and 6-hydroxyl groups are not directly bonded to water molecules. Thus, the altered water structure near the a(1-6) Man of 1 does not appear to account for the higher DDH (relative to 1) values of deoxy analogs 7–9 (both in H2O and D2O) for DGL compared with ConA. On the other hand, significant differences in ordered water exist between DGL and ConA at the 2-OH group of Man(a1-3) and the 4-OH group of core Man, yet the DDH values of 2dMan(a1-3) analog 2 and 4dMan ‘core’ analog 11 (relative to 1) in H2O are almost similar for both the lectins. Thus, the results indicate that altered structural water in these two regions of DGL and ConA complexes with 1 do not correlate with the DDH values in H2O of both lectins for 2 and 11.

3.21.2.1.5

Ionic interactions

With the exception of sialic acids and glycosaminoglycans, carbohydrates in general do not contain charged groups. Proteins that interact with glycosaminoglycans are not discussed in this chapter. Information regarding the recognition of charged groups on sugars by lectins has been obtained from several sialic acid binding lectins such as WGA, HA,

412


Figure 17 The structure of Neu5Ac with the nine carbon atoms numbered. Reprinted by permission from Macmillan Publishers Ltd: Varki, A.; Angata, T. Glycobiology, 16, 1R–27R, copyright (2006).

(a)

(b)

Figure 18 Molecular dynamics results on Man9GlcNAc2. (a) Overlay of individual snapshots taken at 100 ps intervals. The glycan arms are color-coded (dark blue: Man3GlcNAc2 core; light blue: (a1-3)-arm; green and yellow: two (a1-6)-arms). The overall topology of the molecule is well preserved, given the high degree of flexibility for each individual linkage. (b) Two structures of Man9GlcNAc2 showing the range of conformations that could be adopted by the (a1-3)-arm and the core on the basis of the flexibility of the individual linkages.45,211 Reprinted with permission from Wormald, M. R.; Petrescu, A. J.; Pao, Y.-L.; Glithero, A.; Elliott, T.; Dwek, R. A. Chem. Rev. 2002, 102, 371–386. Copyright (2002) American Chemical Society.

selectins, and sialic acid-recognizing Ig-superfamily lectins (Siglecs). Sialic acids possess a carboxylate at the carbon-1 position (Figure 17) that is ionized at physiological pH. The carboxylate can potentially form salt bridge with positively charged amino acids. However, occurrence of salt bridge in sialic acid-binding lectins is rather restricted to few reported lectins such as the Siglecs. The carboxylate moiety is important in the recognition processes but as seen in CT and influenza virus HA, it bonds with main-chain amide groups, polar side chains (preferably of serine), and ordered water molecules rather than fully charged side chains.6 Presence of acidic residues at or near the binding sites may prevent sialic acid binding. This is documented in WGA where sialic acid cannot bind to the S site because of the presence of acidic residues in the lattice contact near the S site.6,44

3.21.2.1.6 Carbohydrate conformation Monosaccharides are relatively rigid compared with oligosaccharides since the latter possess two or three torsion angles at each glycosidic linkage that provides some degree of rotation about the bond (Figure 18).1,45 This has been shown by molecular modeling and high-resolution NMR studies of oligosaccharides in solution.45


413

Different types of glycosidic linkages may result in one or more conformers and different degrees of flexibility of an oligosaccharide. The behavior of a particular linkage can also vary in different environments. Neighboring residues often restrict the conformational freedom of an oligosaccharide. Molecular modeling studies indicate that the flexibility of the overall structure is less than would be predicted from the flexibilities of all individual linkages.45 Although the freedom of rotation around the glycosidic bonds results in conformational heterogeneity in oligosaccharide structures, lectins recognize and bind to a single conformation.46–50 Due to their flexibility, different oligosaccharides may possess common topographic features and bind to the same lectin. On the other hand, different lectins may recognize different regions of the same oligosaccharide.1 The conformation of a glycan does not generally change when linked to a peptide, but the flexibility of the peptide backbone around the glycosylation site may be reduced.45

3.21.2.2 Carbohydrate-binding Mechanisms 3.21.2.2.1

Influenza virus HA

The hemagglutinin from flu virus is a membrane glycoprotein that binds to the sialic acid residues of cell surface receptors during viral invasion. X-ray crystallography has provided important insight into the mechanism of sialic binding by this viral lectin (Figure 19). One face of the pyranose ring of sialic acid is bound to the binding site while the other face of the ring is exposed to solution. One oxygen from the carboxylate, the acetamido nitrogen, and the 8-OH and 9-OH of the glycerol are hydrogen-bonded with conserved side-chain and main-chain polar atoms. The acetamido methyl group is in van der Waals contact with Trp153. A nonpolar surface formed by Gly134, Leu194, His183, and Trp153 interacts with a nonpolar surface on sialic acid constituted with C9, C7, and the acetamido methyl

Tyr195 His183

HO

* 2.4

2.9

N

HN

3.7

Glu190 Ser186 3.3 OH OH

NH

3.1 O

C

3.1 2.7 3.1

* O

Tyr98

3.0

3.4

CH

CH

OH

NH

Ca

Gly134

C

Gly135

HO

Ser136

*

3.3 O

*

O HO C

CH3

OH

C

OH 3.3

*

Thr155 CH3

3.9

CH3

O

HO HC 3.6

3.2

Leu226

3.8

Trp153

3.3

4.3 CH2

*OH

HN 3.8 3.8 3.7 3.7 3.5 3.5

CH3

H3C

OH

2.9

2.7

4.3

2.6

Ser228

*

Leu194

*

O

O

2.8

*

2.8

3.1

CH3

* HN

Asn137

3.4 HN

*

Ala138 CH3

Figure 19 Interaction of a-Neu5Ac with wild type influenza virus HA. Reprinted by permission from Macmillan Publishers Ltd: Weis, W.; Brown, J. H.; Cusack, S.; Paulson, J. C.; Skehel, J. J.; Wiley, D. C. Nature, 333, 426–431, copyright (1988).

414


group. The 4-OH, 7-OH, and the acetamido carbonyl oxygen face toward solution. About two-thirds of the solventaccessible surface of the sialic acid is buried after binding to the lectin. All the sugar atoms that make hydrogen bonds with the lectin are inaccessible to solvent. As areas segregated from bulk solvent tend to have smaller dielectric constant, these sugar atoms may form stronger hydrogen bonds to the protein than to water. The carboxylate interacts with a main-chain amide proton (Asn137) instead of bonding to a positively charged residue.28

3.21.2.2.2 Legume lectins Legume lectins in general interact with carbohydrates through the side chains of three invariant amino acids: an aspartic acid, an aparagine, and an aromatic amino acid51,52 or leucine.53 The aspartic acid and asparagine also coordinate with the calcium ion present in all legume lectins and this coordination indirectly supports carbohydrate binding. Another characteristic of the combining site of the legume lectins is the presence of a cis-peptide bond between the critical asparagine and the preceding amino acid. This bond is required for proper orientation of the asparagines.1 Although the key amino acids involved in carbohydrate binding are highly conserved, legume lectins possess the ability to distinguish between subtle differences in monosaccharide structures. This is achieved through the differential orientation of monosaccharides in the binding sites (Figure 20). For example, in Man-specific lectins such as ConA,54,55 lentil lectin,56 and Lathyrus ochrus lectin (LOL),57 the Od1 and Od2 of asparagine accept hydrogen bonds from 6-OH and 4-OH of Man, whereas Nd2 of asparagine donates a hydrogen bond to the 4-OH of the sugar. The main-chain amide of glycine, which is conserved in all legume lectins except ConA, forms an additional hydrogen bond with the 3-OH of the monosaccharide (Figure 21). In contrast, Od1 and Od2 of asparagine of Gal-specific lectins (such as EcorL,58 peanut agglutinin (PNA),59 and soybean agglutinin (SBA)60) accept hydrogen bonds from the 4-OH and 3-OH, while the Nd2 of asparagine and the NH of glycine donate hydrogen bonds to the 3-OH (Figure 22). In this way, highly conserved binding site residues interact with different monosaccharide structures. Binding specificity apparently originates from the variability of other amino acid residues in the binding site (Figure 23). Four loop regions (known as A, B, C, and D) of the polypeptide chain of legume lectin monomer constitute the carbohydrate-binding site. The conserved aspartic acid and glycine belong to loops A and B, respectively, whereas the asparagine and the hydrophobic residue reside in loop C. Additional interactions are usually provided by the backbone atoms of amino acids in loop D. There is a clear correlation between the lengths of loop D and the monosaccharide specificity of the legume lectins. Thus, in all Man-specific lectins, this loop is identical in size, consisting of 18 amino acids of variable sequence, and it is different in size from loop D found in lectins specific for Gal/GalNAc or for oligosaccharides only.1,61 During its interaction with an oligosaccharides, the primary site (monosaccharide-binding site) of a legume lectin receives the nonreducing residue, while other residues of the oligosaccharide make additional contacts with the

06 04

Asp 04 03 Phe

Asn

ECorL – galactose LOLI – glucose

Figure 20 Hydrogen bonds between side chains of conserved amino acids that ligate Glc to LOL I and Gal to ECorL.1 Reprinted with permission from Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. Copyright (1998) American Chemical Society. Courtesy Nathan Sharon.


Tyr100

Asp208 Ca C OH2 O

Asp10

Mn2+

H OH

O

C

Ca2+ O

O

N H

C C O

2

O

N H O

5

C H N

O

O

1

CH3

Arg228

C Tyr12

Asp19

H

H O NH2 H O3

Asn14

C

Leu99

H O6

O 4

O

O 4.3 Å

415

8.2 Å Figure 21 Methyl a-mannoside bound to ConA.1 Reproduced from Naismith, J. H.; Emmerich, C.; Habash, J.; Harrop, S. J.; Helliwell, J. R.; Hunter, W. N.; Raftery, J.; Kalb-Gilboa, A. J.; Yariv, J. Acta Crystallogr. D 1994, 50, 847–858, Copyright (1994), with permission from International Union of Crystallography.

Q219 A218 Y106 O* A88

D89

F131

G107

Y108

N133 P134

W135

Figure 22 N-Acetyllactosamine bound to ECorL.1 Reprinted with permission from Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. Copyright (1998) American Chemical Society.

extended binding site of the lectin.1 Interactions of N-acetyllactosamine, Gal(b1-3)GalNAc, and trimannoside Man(a1-3)[Man(a1-6)]Man, by EcorL,52 PNA,62,63 and ConA,17 respectively, highlight this feature. Structural and thermodynamic studies with ConA and the trimannoside have demonstrated that extended site interactions contribute favorably to the binding affinity.15,16 These studies also revealed the role of individual hydroxyl group of the trimannoside in binding (discussed in Section 3.21.2.1.1). A single water molecule plays important role in the trisaccharide-conA interaction. 17 Binding of the linear trisaccharide Man(a1-3)Man(b1-4) GlcNAc to LOL I involves 20 water molecules.64

416


Gly209 Thr208

Gly217

Ala218 Ala210

Gln219

Glu211

Thr216

Phe212

Ala223

Ala222

Arg220

Asp221

Figure 23 Superimposition of the variable regions of the combining sites of ECorL (residues 216–223, thin lines) and LOL I (residues 208–212, thick lines).1 Reprinted with permission from Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. Copyright (1998) American Chemical Society.

3.21.2.2.3 Cereal lectin The crystal structure of WGA, a cereal lectin, bound to sialyllactose provides important information regarding sialic acid recognition by a lectin. A number of hydrogen bonds stabilize the complex. Unlike legume lectins, aspartic acid or asparagine do not participate in hydrogen bonding. In addition, nonpolar contacts with aromatic amino acids65 have also been observed (Figure 24). The contact amino acids belong to two different subunits of the lectin, a feature not usually observed in lectins. The carbonyl and amide groups of Neu5Ac are hydrogen-bonded to Ser62 and Glu115, respectively, of WGA. Several van der Waals contacts are observed with the phenyl ring of Tyr73. OH-4 of sialic acid is hydrogen-bonded to the hydroxyl group of the same tyrosine and to an ordered water molecule. The water molecule is in turn hydrogenbonded to Ser114 and Ser43. The carboxylate group of sialic acid is within hydrogen-bonding distance of the hydroxyl of Ser114. Several van der Waals contacts stabilize the orientation of the sugar ring through nonpolar stacking interactions with the aromatic side chain of Tyr66. A third aromatic side chain, that of Tyr64, interacts through nonpolar contacts with the glycerol tail of Neu5Ac.1 The carboxylate at the carbon-1 position of sialic acid is generally ionized at physiological pH. This negatively charged group plays critical role in binding in many other sialic acidspecific lectins such as Siglecs (described below). WGA possesses four functional carbohydrate-binding sites, although two of them do not show detectable affinity in solution. Quantitative estimates for polar, nonpolar, and ionic interactions revealed that hydrogen bonding makes the largest contribution to complex stabilization, in agreement with thermodynamic data.66 Structures of WGA complexed with a branched sialoglycopeptide that possesses both (a2-3)- and (a2-6)-linked terminal Neu5Ac moieties show that the glycopeptide cross-links two crystallographically related dimers (Figure 25). This is a direct structural demonstration of cross-linking activities that occur during the binding of multi-subunit lectins and multivalent carbohydrate ligands.67,68

3.21.2.2.4 Bulb lectins Monocotyledonous plants produce a well-conserved family of bulb lectins that show excusive specificity for Man (Figure 26).69–73 Garlic lectin belongs to this family. This dimeric lectin binds to high Man oligosaccharides,74,75 and like other bulb lectins, contains three equally active Man-binding sites pre subunit (Figure 27).76–79 A highly conserved sequence motif QXDXNXVXY in all bulb lectins is essential for Man binding.80 This motif has been attributed to the unique Man specificity of bulb lectins. Exclusive specificity of garlic lectin for Man can be explained by the number of strong hydrogen bonds made by the O2 and O4 hydroxyls of the sugar with the protein side-chain atoms, all of which happen to be conserved in all similar proteins. The same hydrogen-bonding pattern cannot be achieved with Glc or Gal; therefore, these lectins cannot recognize Glc and Gal.80 Ramachandraiah and Chandra80 found six unique mannose binding protein structures out of 97 entries in the Protein Data Bank. These six include three legume lectins, one C-type animal lectin, and two bulb lectins. Man


417

Tyr66 OH Tyr64

OH

HO Ser62 OH

C

OH C

H

C

C H

H H

C

H O

O

O H

OH O H

HO N H

CH3

HO H

H H H O

Tyr73

OH HN

OH O

O

Ser114

OH

C Ser43

Glu115 Figure 24 Interaction of Neu5Ac with WGA. The carbonyl group is hydrogen-bonded to the hydroxyl of Ser62 and its amide to the carbonyl of Glu115. In addition, it forms five to seven van der Waals contacts with the phenyl ring of Tyr73. The adjacent ring hydroxyl (4-OH) is hydrogen-bonded to the hydroxyl of the same tyrosine and an ordered water molecule. The carboxylate group of the Neu5Ac is within hydrogen-bonding distance of the hydroxyl of Ser114. Several van der Waals contacts stabilize the orientation of the sugar ring through nonpolar stacking interactions with the aromatic side chain of Tyr66. A third aromatic side chain, that of Tyr64, interacts through nonpolar contacts with the glycerol tail of the Neu5Ac. Only one water molecule appears to be involved in stabilizing the lectin–sugar complex. It is tetrahedrally coordinated by hydrogen bonds with the 4-OH of Neu5Ac, the backbone amide of Ser114, and the hydroxyl of Ser43.1,65 Reproduced from Sharon, N. Trends Biochem. Sci. 1993, 18, 221–226, copyright (1993), with permission from Elsevier.

residue interacts with legume lectins (such as ConA, pea lectin, and lentil lectin) through conserved amino acids and water molecules while metal ions support the integrity of the binding site. The 2-OH and 3-OH of Man hydrogen bond only to water molecules or main-chain nitrogens or oxygens in these structures. The 4-OH hydroxyl, however, hydrogen-bonds to conserved asparagine and aspartic acid residues. The asparagine side chain is held in position by a strong coordination by a calcium ion. In contrast, sugar is directly coordinated to calcium ions through the 3-OH and 4-OH in Man-binding C-type lectin. The 3-OH also hydrogen-bonds with conserved glutamic acid and asparagine side chains, while the 4-OH hydrogen-bonds with another set of glutamic acid and asparagine side chains. It was found80 that some features were common to all six unique Man-binding lectins despite the absence of any sequence or structural homology in the three lectin families. For example, hydrogen-bonding patterns have many features in common in all the proteins irrespective of sequence, fold, and topology of the individual lectins. Every structure also has a hydrophobic residue (either a valine or an alanine) capable of van der Waals interactions with one or the other carbon atoms of the sugar. This particular arrangement of hydrogen-bonding atoms in conjunction with correctly oriented hydrophobic residue provided the framework necessary for Man binding. Assignment of contact residues is very different in these three different types of lectin families. Residues involved in ligand binding belong to a single segment in bulb lectins, to four different loops in legume lectins, and to a loop and a b-sheet in C-type lectins. In bulb lectins, the signature sequence or motif provides the required topology and geometry at the binding. In legume lectins, metal ion coordination results in the appropriate positioning of the Asp/Asn side chains for Man binding. Whereas, in C-type lectins, a calcium ion directly coordinates with the hydroxyl groups of the Man residue. While Asp/Glu and Asn side chains are common in all of them, the hydrogen bonds they make with Man appear to be with different sugar hydroxyls in different lectins. Similarly, van der Waals interactions with Val/Ala may involve different carbon atoms of the sugar in different lectins. These subtle differences are expected to influence the

418


C2

b -ThrC2

B1

Neu5Ac(a 2-6)GalNAc(b

3-1)Gal(a 3-2)Neu5Ac

C2

-Thr-

-Thr-

-3)G Ac(a 2

B1

Ac

Neu5 (a 6-2)

Ac

3)GalN

al(b 1-

C2

B1

Neu5Ac(a2-3)Gal(b1-3)GalNAc(a6-2)Neu5Ac

Neu5

C2

eu5Ac

al(a 3-2)N

B1

(b 3-1)G )GalNAc

(a 2-6

Neu5Ac

B1

-Thr-

Figure 25 Schematic representation of WGA cross-linked with a branched sialoglycopeptide from glycophorin.1,67 Reproduced from Wright, C. S. J. Mol. Biol. 1990, 215, 635–651, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 1992 by ASBMB.

Asp91 Asn93

H2O C

C O Tyr97

Gln89

OH

NH2

O NH2

O

OH O

O H HO

OH

C O

Figure 26 Mannose binding by the first subdomain of Galantis nivalis lectin. Gln89 interacts with the 3-OH and the hydroxyl of Tyr97 with the 4-OH of the bound Man, which together with the interactions of the 2-OH with the side chains of Asp91 and Asn93 provide four H-bonds in the complex. Additionally, Val95 makes hydrophobic contacts with C3 and C4. Three water molecules form a network of hydrogen bonds with the ring oxygen, the 6-OH, and the nitrogen of the Asn93 amide. The binding is enhanced by participation of residues from neighboring subunits: His107 from a subunit in the same tetramer in the combining site of subdomain 1 and Leu48 from another tetramer in the site of subdomain 3.1,212 Reproduced from Hester, G.; Kaku, H.; Goldstein, I. J.; Wright, C. S. Nat. Struct. Biol. 1995, 2, 472–478, with permission of Nature Publishing Group. Copyright (1995) by Nature Publishing Group.

orientation of carbohydrate ligand in the binding site. Differential ligand orientation may help achieve different carbohydrate specificities. C-type lectins can tolerate other sugars such as Gal and Glc in their binding pockets; however, bulb lectins show very specific interaction with Man. This specificity results primarily through the recognition of 2-OH of Man by conserved side chains in the bulb lectins. The orientation patterns in legume and C-type lectins for the O2 hydroxyl mainly involve main-chain nitrogens and water molecules, which do not contribute to specificity.80


419

Figure 27 Crystal structure of garlic lectin showing the six Man-binding sites in a lectin dimer. E, F, and G correspond to the first, second, and third sites in the first subunit; I, J, and K refer to the three binding sites in the second subunit. The Man molecules are shown in a CPK representation. Reprinted from Ramachandraiah, G.; Chandra, N. R.; Surolia, A.; Vijayan, M. Glycobiology 2003, 13, 765–775, by permission of Oxford University Press.

Asn C

O

NH Arg

C

HN H

O Asn

Arg

C

C HN H

NH

OH

NH H

H O

6

HO NH2

O

5 4

Gal 3

N

O

O

H

C

1 2

OH 2

3

O

Glc

4

b(1-4)

5

HO OH

HN

Glu

1

O

O

6

OH His Figure 28 Bovine galectin complexed with lactose.1 Reprinted with permission from Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. Copyright (1998) American Chemical Society.

3.21.2.2.5

Galectins

Galectins are b-galactoside specific animal lectins that possess highly homologous carbohydrate recognition domains (CRDs). Galectins bind to lactose, LacNAc, or related carbohydrate structures.81–85 Crystallographic studies revealed that the structural bases of carbohydrate binding by galectin-1,-2,-3, and -7 are essentially similar.86–90 Amino acid residues of galectins involved in carbohydrate binding belong to four adjacent b-strands. These strands are contiguous in the primary sequences of the galectins and are conserved among the different members of this family. The 4-OH of Gal in LacNAc or lactose is hydrogen-bonded to the side chains of three invariant amino acids – histidine, asparagine, and arginine. The 6OH of Gal is also hydrogen-bonded with the lectin. A conserved tryptophan is stacked against the sugar ring. In the galectin/lactose complex (Figure 28), the Glc moiety contributes to binding via its 2-OH and 3-OH, while in the galectin/N-acetyllactosamine complex, only the 3-OH of N-acetylglucosamine is hydrogen-bonded to the lectin.1

3.21.2.2.6

C-type lectins

3.21.2.2.6.1 Mannose-binding proteins MBPs are soluble serum collectins that are involved in immunoglobulin-independent host defense. Crystal structures of MBP-A complexed with an oligomannose oligosaccharide and the structures of rat liver MBP (MBP-C) with Man, Glc, and Fuc have provided insights into the binding mechanism of C-type lectins.91–93 A protein-bound calcium ion

420


Asn

G lu

C

C

OH

NH H

O O

6

5

O 4

O Asn

Ca2+

HO O H

C

O 1

HO 3

2

O

NH H O

O C Glu

Figure 29 Mannose-binding protein C (MBP-C) complexed Man. Reprinted with permission from Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. Copyright (1998) American Chemical Society. 1

interacts with the 3-OH and 4-OH of Man (Figure 29). Direct involvement of a metal ion in ligand binding is unique to C-type lectins. However, the recently characterized PA-IIL has two calcium ions directly participating in Fuc binding (Figure 37).94–96 The single calcium ion in Man-specific C-type lectins is coordinated to the side chains of two glutamic acids and two asparagines that are also hydrogen-bonded to the same 3-OH and 4-OH of Man. These four amino acids are conserved in all Man-specific C-type lectins; two of them are displayed in the sequence Glu-Pro-Asn (positions 185–187 in MBP-A). In Gal-specific C-type lectins, the glutamic acid is replaced by glutamine and the asparagine by aspartic acid. This observation suggested that the three amino acids at these positions are the primary determinant of specificity of the C-type lectins.1 Mutant MBP-A, in which Glu185 and Asn187 were replaced by glutamine and aspartic acid, respectively, indeed showed a three times higher affinity for Gal over Man.97 Affinity and specificity of mutant MBP-A for Gal was further increased by replacing its His189 with a tryptophan and by inserting a glycine-rich segment of five amino acids. The crystal structures of the Gal-specific mutant MBP-A complexed with methyl b-galactopyranoside and N-acetylgalactosamine revealed that the 3-OH and 4-OH groups of Gal were directly coordinated to Ca2þ and formed hydrogen bonds with amino acids that also serve as Ca2þ ligands.98 A similar binding mechanism was observed with Man.98 However, the orientations of bound Man in the wild type and Gal in the mutant were different due to differences in the stereochemistry of the 4-OH in Man and Gal and the fixed Ca2þ coordination geometry. Moreover, the apolar patch, formed by the 3, 4, 5, and 6 carbons of Gal or N-acetylgalactosamine, stacked against the side chain of Trp189. Such stacking of the monosaccharide against an aromatic amino acid, a general feature of Gal-specific lectins, is not observed in MBP–Man complex.1

3.21.2.2.6.2 Selectins The selectins are a group of cell surface C-type lectins that participate in the recruitment of leukocytes into the sites of inflammation and their emigration into lymphatic tissues. E-selectin and P-selectin appear on vascular endothelium in response to inflammation while L-selectin is expressed on leukocytes. Selectins recognize the tetrasaccharide, sialylLewisx (SLex), and its isomer sialyl-Lewisa.99–101 Selectins show relatively weak affinities (micromolar to millimolar) for SLex; however, certain glycoproteins (P-selectin glycoprotein ligand-1 (PSGL-1), GLyCAM-1, LFA-1, MAdCAM-1,CD34) interact with selectins with enhanced affinities.99,100,102 The mucin like PSGL-1 molecule is recognized by all three selectins. O-Linked SLex epitopes and one or more tyrosine sulfate residues at the N-terminus of PSGL-1 polypeptide are essential components for high-affinity interaction with P-selectin. L-selectin displays comparable structural requirements for high-affinity binding. On the other hand, E-selectin interacts with PSGL-1 in a tyrosine sulfate-independent manner.102–104 The affinity of P-selectin for SLex-modified PSGL-1 is in the nanomolar range, while E-selectin possesses significantly lower affinity for the same ligand. The Kd of P-selectin/SLex was determined as 7.8mM, whereas E-selectin binds the same tetrasaccharide with 10-fold higher affinity.99


421

Figure 30 Stereo view of sialyl LewisX (SLeX) bound to the CRD of E-selectin (E-LE) focusing on the Fuc interaction. E-LE is shown in green, SLeX residues are shown in purple, and the calcium ion is shown as a yellow sphere. A bound water molecule is shown as a red sphere. Dashed lines represent calcium ligation and hydrogen bonds. Reproduced from Somers, W. S.; Tang, J.; Shaaw, G. D.; Camphausen, R. T. Cell 2000, 103, 467–479, copyright (2000), with permission from Elsevier.

Figure 31 Stereo view of SLeX bound to the CRD of E-selectin (E-LE) focusing on the Gal and Neu5Ac interactions. Reproduced from Somers, W. S.; Tang, J.; Shaaw, G. D.; Camphausen, R. T. Cell 2000, 103, 467–479, copyright (2000), with permission from Elsevier.

The tetrasaccharide (SLex) binding sites of E- and P-selectins are comparable and the conformation of SLex remains identical in both structures.99 The interactions are predominantly electrostatic in nature. In both structures, 3- and 4-hydroxyl groups of the Fuc residue of SLex interact with the selectin-bound calcium ion (Figure 30). In homologous MBP-C binding site, 2-OH and 3-OH of Fuc bind to the calcium ion.92 Apart from ligating to the bound calcium ion, the Fuc hydroxyl groups make hydrogen bonds with residues of selectins which are also involved in calcium coordination. The 4-OH of Fuc replaces a calcium-coordinated water molecule and makes hydrogen bonds to Asn82 and Glu80. Similarly, 3-OH of Fuc hydrogen-bonds with Asn105, displacing another calcium-ligated water molecule. The Gal residue of the SLex tetrasaccharide makes hydrogen bonds with Tyr94 and Glu92. The carboxylate group of sialic acid residue (Neu5Ac) makes hydrogen bonds with Tyr48 (Figure 31). The number of additional

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Figure 32 (a) Electrostatic potential surface representation of parts of the CRD of P-selectin with bound SGP-3. The peptide portion of SGP-3 is shown in green (sulfur is shown as a yellow atom) and the carbohydrate portion is blue. Selected amino acid residues of P-selectin important for the interactions with SGP-3 are labeled in white. (b) Ball-and-stick view of the Fuc residue of the SLex glycan of SGP-3 and its interactions with the CRD of P-selectin. P-selectin is shown in purple, the Fuc residue in orange, and the strontium ion is shown as a green sphere. The oxygen, nitrogen, and carbon atoms are shown as red, blue, and black spheres, respectively. The oxygen of a bound water molecule is shown as a red sphere (asterisked). Brown lines represent metal ligation and hydrogen bonds. Reproduced from Kogelberg, H.; Feizi, T. Curr. Opin. Struct. Biol. 2001, 11, 635–643, copyright (2001), with permission from Elsevier.

contacts made by Neu5Ac depends on the selectin involved. In P-selectin, Ser99 is hydrogen-bonded to 4-OH of Neu5Ac, and its C4 packs against Pro98. A different conformation of Neu5Ac residue in E-selectin binding site results in more extensive interactions.99 In order to avoid an unfavorable contact with Arg99, the positioning of Neu5Ac is changed in E-selectin where the carboxylate group and the glycosidic oxygen is hydrogen-bonded to Arg97. The difference in Neu5Ac binding and a lack of productive interaction involving Asn83 were cited as the reasons for lower affinity of P-selectin/SLex interaction.99 The X-ray structure of the P-selectin complex with glycopeptide SPG-3 (N-terminal domain of the glycoprotein PSGL-1) shows that the peptide adopts a hairpin-like conformation, facilitating simultaneous interactions of Pselectin with two sulfate groups and the carbohydrate residues of SPG-3 (Figure 32). The sulfate group of Tyr7 is known as a high-affinity binding determinant and it is involved in numerous electrostatic interactions. Binding of the sulfate group of Tyr10 to Arg85 of P-selectin results in translocation of the Asn82–Asp89 loop and subsequent slight rearrangement of the Fuc-binding mode compared with the E- and P-selectin–SLex complexes (Figure 32). A large surface area is buried in the P-selectin–SGP-3 complex. The anionic sulfotyrosine region binds to a region of positive electrostatic potential on P-selectin (this area may also interact with anionic glycoconjugates such as heparin sulfate, sulfatides, and fucoidan).100 However, the carbohydrate moieties (as in the P-selectin–SLex complex) interact with a region of neutral and negative electrostatic potential of P-selectin (Figure 32). Arg85 and His114 of P-selectin make important contacts with the sulfate groups on Tyr7 and Tyr10 of PSGL-1.99,105 This interaction may be missing in E-selectin, because the corresponding residues are Gln85 and Leu114. L-selectin shows lower affinity for SGP-3 compared with P-selectin. The absence of a basic residue at position 114 that interacts with the second tyrosine sulfate is attributed to the lower affinity of L-selectin for SGP-3.99,105

3.21.2.2.7 P-type lectins The P-type family of animal lectins is comprised of two type I integral membrane receptor glycoproteins, namely, the 46kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 300 kDa insulin-like growth factor II/ cation-independent MPR (IGF-II/CI-MPR). These receptors interact with the mannose 6-phosphate (M6P) moieties of N-linked oligosaccharides on lysosomal enzymes and help transport the enzymes from the trans-Golgi network to prelysosomal compartments. P-type lectins do not show any sequence homologies with other lectins.106,107


423

The crystal structures of the extracytoplasmic domain of CD-MPR complexed with M6P and pentamannosyl phosphate106,108 provide important insights into the molecular basis of carbohydrate recognition by CD-MPR. Structural data clearly establish the terminal M6P moiety as the major determinant of ligand binding and demonstrate how the binding site interacts with a pentamannosyl phosphate. As discussed above, the loop region residues of legume lectins and the amino acid side chains in the b-strand of galectins exclusively interact with carbohydrates. In contrast, residues from both the loop regions and the b-strand of CD-MPR are involved in protein–carbohydrate interactions 108 The carbohydrate-binding site of CD-MPR is deep enough to completely surround the terminal M6P residue and the penultimate sugar ring of bound pentamannosyl phosphate (Figure 33).108 This is in contrast with the structures of the binding sites of other lectins that are comparatively shallow in nature. The depth of the binding pocket provides multiple interactions between CDMPR and its carbohydrate ligands and probably contributes to the relatively high affinity (6–8mM) observed with the monosaccharide, M6P. This affinity is orders of magnitude higher than those generally observed in lectin– monosaccharide interactions. Stacking carbohydrate ligands with aromatic amino acid side chains, a common feature of lectin–carbohydrate complex formation, is not observed in the CD-MPR/M6P complex. Instead, an intricate network of hydrogen bonds and ionic interactions stabilizes CD-MPR/carbohydrate complexes. For example, nine amino acids (Tyr45, Gln66, Asp103, Asn104, His105, Arg111, Glu133, Arg135, and Tyr143) make hydrogen bonds with the terminal M6P moiety, three amino acids (Asp43, Tyr45, Gln68) are in contact with the penultimate Man, and Gln68 is also hydrogen-bonded with the adjacent sugar.107 (Figure 34). Interactions between the 2-OH group of the terminal M6P and the side chains of Gln66 and Arg111 play a key role in conferring the binding specificity of CD-MPR for M6P.107 His105 is hydrogen-bonded to one of the phosphate oxygen atoms and has been implicated in the pH-dependent binding properties of CD-MPR, a feature essential for releasing ‘cargo’ in low-pH prelysosomal compartments. The phosphate group contributes significantly to the enhanced affinity of M6P. This is consistent with the observation of several hydrogen bonds and ionic interactions involving the phosphate moiety, including its interactions with the main chains of Asp103 and Asn104. Additional contacts to the phosphate group are made by a water molecule and a Mn2þ ion.107 Analysis of structural data indicates that the presence of the Mn2þ cation enhances binding of the phosphate group by shielding it from the negatively charged binding site residue, Asp103. However, the divalent cation does not play any critical role in ligand binding as reported in some other lectins including C-type lectins. Lectins in general do not show significant conformational changes upon ligand binding. However, ligand-bound and free forms of CD-MPR show dramatic structural differences. During the ‘free-to-bound’ transition, the receptor monomers move significantly relative to each other in order to open up the ligand-binding sites (Figure 35).107 In the the ligand-free form, loop D bends into the unoccupied binding cleft and helps to maintain the correct orientation of the binding site residues. In other lectins, such as mannan-binding lectins and galectins, unbound sites are generally occupied by water molecules that fill the void otherwise held by the hydroxyl groups of sugar molecules in their bound states.107,109

Figure 33 Ligand at the binding site of CD-MPR. The terminal three sugar residues of pentamannosyl phosphate (solid yellow) are bound to CD-MPR (red mesh).108 Reproduced from Olson, L. J.; Zhang, J.; Lee, Y. C.; Dahms, N. M.; Kim, J.-J. P. J. Biol. Chem. 1999, 274, 29889–29896, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 1999 by ASBMB.

424

D103 3.1 3.0 2.4

WAT Mn

2.8

2.8

H105 2.8

O1 O2

O3

WAT 2.7 Mn

Y143

I

2.5

II

3.2 3.2 2.8

2.8 2.8

3.1

2.8 2.9

R135

3.2

2.7 2.7

2.8

2.8

E133

2.8

D43

3.2

2.7 2.7

R135

2.8

3.1

Q66 Y45

Y143

O2

3.1 2.9

H105

2.7 3.2

III 3.0

3.1 3.0 3.1

2.9 O1 O3

Q68

D103

2.6

2.8

E133 Y45

R111

Q66 R111 (a)

(b)

Figure 34 Interactions between the ligand (dark gray) and the residues of the binding site of CD-MPR. Potential hydrogen bond distances in angstroms are shown. (a) CD-MPR complexed to pentamannosyl phosphate. The terminal (I), penultimate (II), and prepenultimate (III) Man residues are indicated. (b) CD-MPR complexed to Man6P.108 Reproduced from Olson, L. J.; Zhang, J.; Lee, Y. C.; Dahms, N. M.; Kim, J.-J. P. J. Biol. Chem. 1999, 274, 29889–29896, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 1999 by ASBMB.


N104 N104


(a)

425

(b)

Figure 35 Ligand-induced conformational change of CD-MPR. (a) Crystal structure of the extracytoplasmic region of the bovine CD-MPR in the presence of pentamannosyl phosphate. (b) Crystal structure of the unbound form of the CD-MPR at pH 6.5. As compared with the ligand-bound form, the unbound CD-MPR exhibits several significant conformational changes, particularly in the positions of loops C and D.107 Reproduced from Dahms, N. M.; Hancock, M. K. Biochim. Biophys. Acta 2002, 1572, 317–340, with permission from Elsevier.

Figure 36 AAA binding site complexed with Fuc.110 Reprinted by permission from Macmillan Publishers Ltd: Nature Structural and Molecular Biology (Bianchet, M. A.; Odom, E. W.; Vasta, G. R.; Amzel, M. Nat. Struct. Biol. 2002, 9, 628–634), copyright (2002).

3.21.2.2.8

Fucose-binding lectins

3.21.2.2.8.1 Anguilla anguilla agglutinin The serum lectin of European eel (Anguilla anguilla) (Anguilla anguilla agglutinin (AAA)) is specific for Fuc and binds to blood group H and Lewisa antigens. The structure of AAA complexed with a-L-Fuc110 revealed a new CRD sequence motif as well as a novel animal lectin fold. The fold comprises a b-jellyroll sandwich composed of three- and five-stranded b sheets. The O5, 3-OH, and 4-OH of a-L-Fuc interact with Arg79, Arg86, and His52. The sugar makes van der Waals contacts with a unique disulfide bridge formed by contiguous cysteines (Figure 36). The 6-deoxy methyl moiety docks in a hydrophobic cavity stacking against the rings of His27 and Phe45. Modeling experiments show that binding of H type 1 and Lea trisaccharides involve additional interactions with residues in the loops surrounding the binding pocket.110 These interactions apparently fine-tune the binding specificities of the lectin for glycoconjugate ligands. Multiple isoforms of the Japanese eel fucolectin show variability of critical residues in the binding pocket and surrounding loops. This variability may cause alternative interactions with terminal and subterminal sugar units resulting in diverse oligosaccharide specificities. This observation supports the role of AAA as an innate immunity

426


protein. Moreover, the collectin-like trimeric structure of AAA suggests potential multivalent interactions with glycoconjugates displayed on microbial surfaces. The Fuc-binding site on the AAA trimer could potentially crosslink Fuc-containing groups that are 23A˚ apart on a pathogen surface. The Man-binding lectin trimer can cross-link epitopes that are 45A˚ apart. Unlike C-type lectins, calcium does not participate in ligand binding; however, it is essential for the stabilization of the AAA structure.110,111

3.21.2.2.8.2 Bacterial and fungal lectins A soluble lectin, PA-IIL, isolated from the Gram-negative bacterium Pseudomonas aeruginosa binds preferentially to Fuc. PA-IIL is a tetrameric lectin and its carbohydrate-binding site contains two calcium ions (Figure 37).95 A unique feature observed in the PA-IIL/Fuc complex is the direct involvement of both calcium ions in carbohydrate binding. The 2-OH, 3-OH, and 4-OH of Fuc participate in the coordination of calcium ions. The same OH groups also make hydrogen bonds with three amino acids in the calcium-binding site. The Fuc ring oxygen is hydrogen-bonded with main-chain nitrogen of Ser23. The methyl group of Fuc is involved in hydrophobic interaction with Ser23 and Thr45, and this interaction has been shown to contribute to the binding affinity.96,112 Experimental and theoretical docking experiments suggest that the Lewisa series of oligosaccharides are probably the natural ligands of this lectin. PA-IIL shows unusually high affinity for Fuc. The authors reasoned that extensive delocalization of charges between the calcium ions, the side chains of the protein-binding site, and the carbohydrate ligand is responsible for the high enthalpy of binding and therefore for the unusually high affinity (micromolar) observed in this unique mode of carbohydrate recognition. The relatively favorable entropic term also contributes to the enhanced affinity.94 Favorable entropic effect observed for Gal binding by ECorL113 may result from the release of two tightly bound water molecules upon sugar binding.114 Three tightly bound water molecules are released from the calcium ion of PA-IIL upon binding Fuc that may contribute to the observed relatively favorable binding entropy.115,116 Carbohydrate ligand binding by C-type lectins requires one calcium ion. The pentraxin family of lectins that include serum amyloid P (SAP) component and C-reactive protein (CRP) contains two closely spaced calcium ions that are involved in ligand binding. However, they differ from PA-IIL since they only bind to negatively charged carbohydrates.117

S23 T45 W2 O5 N21

D96 O1

O4 D104

O2

W1

G114 CA2

O3 CAT

D99

N103

T98 E95

D101

Figure 37 Interactions of PAIIL with calcium ions and Fuc with modeled hydrogen atoms. Coordination contacts are indicated by solid lines and hydrogen bonds by dashed lines.95 Reprinted from Mitchell, E. P.; Sabin, C.; Sˇnajdrova, L.; Pokorna´, M.; Perret, S.; Gautier, C.; Hofr, C.; Gilboa-Garber, N.; Kocˇa, J.; Wimmerova´, M.; et al. Proteins: Struct. Funct. Bioinform. 2005, 58, 735–746, with permission of John Wiley & Sons Inc., copyright (2005).


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The Fuc-binding lectin, RSL, from the bacterium Ralstonia solanacearum is a trimeric b-propeller protein with six binding sites per trimer. RSL is structurally similar to the Fuc-binding fungal lectin from Aleuria aurantia (AAL). The ligand-binding sites of AAL and RSL are very similar, and are characterized by numerous hydrogen bonds to the side chains of polar amino acids and by strong hydrophobic interactions between aromatic residues (Figure 38). Unlike PA-IIL, binding of carbohydrate ligand by RSL does not involve a calcium ion; nevertheless, its affinity for Fuc is comparable to that of the former. As observed with PA-IIL, a comparatively favorable entropic effect contributes to the high-affinity binding.118 The GlcNAc-binding lectin from the mushroom Psathyrella velutina is a seven-bladed b-propeller protein. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups and the hydrophobic interactions with a conserved tyrosine and histidine mediate the binding of GlcNAc by this lectin. However, unlike the other b-propeller lectin RSL, it shows very weak affinity for its monosaccharide ligand, GlcNAc.119 Another well-characterized b-propeller protein is tachylectin-2 from Japanese horseshoe crab. Tachylectin-2 is toroidal in shape, with five carbohydrate-binding sites found in a plane on one face.120 The structures of b-propeller lectins and the nature of distribution of their carbohydrate-binding sites are very favorable for multivalent interaction with sugar ligands.88

3.21.2.2.9

I-type lectins

The Siglecs (sialic acid-binding Ig-like lectins) are a subset of I-type lectins that are expressed in the hemopoietic, immune, and the nervous system. They are type I membrane proteins with an N-terminal sialic acid binding V-set Ig domain and varying numbers of C2-set Ig domains. Siglecs recognize sialic acid-containing glycoconjugates and participate in signaling and adhesive functions.121–126 The X-ray crystal structure of the N-terminal domain of sialoadhesin in complex with 30 -sialyllactose reveals the dominant roles of sialic acid in complex formation (Figure 39).122 One face of the sialic acid pyranose ring lies toward the protein surface, while the other face is exposed to solvent. Each of the ring substituents of sialic acid interacts with the protein. The carboxylate forms a salt bridge with the guanidino group of Arg97. The acetamido methyl group is in van der Waals contact with the indole ring of Trp2, and the terminal carbon of the glycerol side chain (C9) contacts the aromatic side chain of Trp106. The 8- and 9-OH groups of the glycerol side chain form hydrogen bonds with the main-chain amide and carbonyl of Leu107, respectively. The amide nitrogen of the N-acetyl group makes a hydrogen bond with the main chain carbonyl of Arg105, and the 4-OH interacted with the main chain carbonyl of Ser103. Interactions of sialic acid with the side chains of Trp2, Arg97, and Trp106 appear to provide the basic sialic acid recognition template in all Siglecs, as these residues are highly conserved in their sequences.122 The Gal moiety of 30 -sialyllactose binds via a lone hydrogen bond between its 6-OH group and the phenolic hydroxyl group of Tyr44 of sialoadhesin (Figure 39).122 The conformation of bound 30 -sialyllactose is similar to that of its complex(es) with influenza hemagglutinin127 and WGA.65 Sialic acids are generally found as the terminal sugar residues of N- and O-linked glycans and glycolipids. Among the limited number of reported sialic acid-binding mammalian proteins, Siglecs and selectins are best characterized. The selectins interact with sialylated and sulfated Lewisx and Lewisa, whereas Siglecs recognize oligosaccharides with terminal sialic acid. Although they are required for binding by both groups of lectins, sialic acid shows much more dominant binding interactions with Siglecs. However, the affinity of Siglecs for sialic acid is generally weak. For example, the affinity of sialoadhesin for methyl a-Neu5Ac is 1–3mM.128,129 The sialic acid-binding sites of Siglec-7 and sialoadhesin show the conserved nature of some of the residues important in the interaction with sialic acid.123 An arginine (Arg124), conserved in all Siglecs, interacts with the carboxyl group on the terminal sialic acid (Figure 40). Trp132 (equivalent of Trp106 of sialoadhesin) provides a hydrophobic interaction with the glycerol moiety. Protein backbone hydrogen bonding with the glycerol and N-acetyl groups observed in the sialoadhesin complex may also be formed in the Siglec-7-binding site. Nevertheless, the following differences have also been noted: Trp2 of sialoadhesin forms a hydrophobic contact with the N-acetyl methyl group. This tryptophan is substituted by Tyr26 in Siglec-7, which has the potential to form hydrogen bonds with the N-acetyl carbonyl group. In Siglec-9, this residue is absent. Mutational studies with sialoadhesin showed that the Trp2mediated hydrophobic interaction was important for Neu5Ac recognition.122 Sialoadhesin cannot interact with N-glycolylneuraminic acid, since presence of the additional oxygen atom in the latter form of sialic acid would be expected to result in a steric clash (Figure 40). All Siglecs, except sialoadhesin and MAG, can bind both N-acetyl- and N-glycolylneuraminic acid, suggesting that the hydrophobic contact with the Neu5Ac may not be important for sialic acid recognition in all cases. This is probably the reason why Siglec-9 shows significant sialic acid binding in the absence of an equivalent aromatic residue.123

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(a)

(b)

Figure 38 RSL trimer complexed with Fuc. (a) Ribbon diagram of the complex with Fuc (shown as sticks). (b) Fuc in binding site 1 with hydrogen bonds represented by dashed lines. Reproduced from Kostlanova´, N.; Mitchell, E. P.; Lortat-Jacob, H.; Oscarson, S.; Lahmann, M.; Gilboa-Garber, N.; Chambat, G.; Wimmerova´, M.; Imberty, A. J. Biol. Chem. 2005, 280, 27839– 27849, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 2005 by ASBMB.

N

Trp2

Ser103 O 2.79

O

11

OH

10

Arg105

O

NH

Leu107

2.71

2.80

Arg97

HN

4 5

3

6

2

O HO 7

O

3.72

HO

9

O−

O

8

2.93

2.78

O

OH

HN

1

2.72

2.73

OH

2.73

Wat

3.01

Wat

Wat

3

2.94 3.07

N

HO

Wat

4 5

2 1

Wat

O

6

OH

2.96

2.57

O

OH

HO

Wat

OH

4

6

O

Tyr44

3.89 Wat

3

5

Wat

2.70

2.79

Typ106

N H

H2N

2 1

OH

OH Hydrophobic interaction 2.77

Hydrogen bond

Figure 39 Interactions between the B molecule of recombinant N-terminal domain of sialoadhesin (SnD1) and 30 -sialyllactose. Solvent molecules are shown as ovals. 30 -Sialyllactose is illustrated in thick black lines. Reproduced from May, A. P.; Robinson, R. C.; Vinson, M.; Crocker, P. R.; Jones, E. Y. Mol. Cell 1998, 1, 719–728, copyright (1998), with permission from Elsevier.

Y26

W2

R105

K131

W132

S103

N133

W106

N130 R124

L107

K128

W129

R121

R97

N70

R48

A66 K75 I72

Sialoadhesin

D71 T68

Siglec-7

Siglec-9

Figure 40 Ligand-binding sites of sialoadhesin, Siglec-7 and Siglec-9. The backbone structures of sialoadhesin, Siglec-7, and Siglec-9 are shown as ribbons. Key residues interacting with the terminal pyranose sugars are shown for sialoadhesin (sticks with orange carbons) and compared with the equivalent residues in the Siglec-7 structure and the Siglec-9 model. The terminal sialic acid of the sialoadhesin sialyllactose ligand is shown as sticks with green carbons. Models for (a2-8)-linked disialic acid in Siglec-7 and Siglec-9 are shown as sticks with green carbons.123 Reproduced from Alphey, M. S.; Attrill, H.; Crocker, P. R.; van Aalten, D. M. F. J. Biol. Chem. 2003, 278, 3372–3377, with permission of American Society for Biochemistry & Molecular Biology (ASBMB). Copyright 2003 by ASBMB.


Y44

429

430


The members of the Siglec family possess differential linkage specificities that have been implicated to the structures of C–C loop. Alignment of different Siglec sequences demonstrates the variability of the C–C loop region. Interaction of subterminal sugars with the side chains of variable C–C loop may contribute to the differential linkage specificities.123 This is reminiscent of the loop-derived specificities of legume lectin.

3.21.3 Multivalent Carbohydrate Binding The affinities of lectins for monovalent carbohydrates are generally weak, in the millimolar to micromolar range. However, multivalent binding interactions between carbohydrates and lectins can result in substantially higher affinities, often in the nanomolar range. This is due, in part, to multivalency of the carbohydrate (Figure 41), and, in some cases, to the multivalent binding properties of lectins. The multivalent binding properties of lectins often result from their multi-subunit structures (Figure 42). Examples include the mammalian C-type lectins including selectins that oligomerize to form clusters;130 the Siglecs that exist as monomer, dimer, and higher oligomers;126 and galectins that are dimeric (e.g., galectin-1), two-site (e.g., galectin-4),81 or higher oligomeric proteins (e.g., galectin-3).131 The ligands of selectins (PSGL-1, GlyCAM-1, CD34, MAdCAM-1),102 Siglecs (CD43, PSGL-1, CD45, Muc-1),126 and galectin-1 (CD45, CD43)132 possess multiple glycan structures including mucin domains and are therefore highly multivalent (Figure 43). It has also recently been shown that a monomeric lectin can bind with high affinity (avidity) to a multivalent glycoprotein.133

Proteoglycans P Heparan sulfate 6S

6S

S

4S

4S

4S

4S

Ser-O

6S

6S

6S

Chondroitin sulfate O-Ser

NS

NS 2S NS 2S NS

NS

3S

O-Linked glycans

N-Linked glycans

sLeA/X

Glycosphingolipids GD3 GM3

GD2 STn

GM1 Tn

O O O Ser/Thr Ser Ser

N Asn

Cytoplasm O Ser O-Linked GlcNAc Glc Gal

Man GlcNAc

GalNAc GlcA

IdoA Fuc

Xyl Sia

Figure 41 Branched or polymeric structures of natural glycans. Reproduced with permission from Fuster, M. M.; Esko, J. D. Nature Reviews Cancer 2005, 5, 526–542, copyright (2005) Macmillan Magazines Ltd.


431

7

NNN 1

C 2

C

NNN 4

3

5

6

Figure 42 Schematic representation of hepatic lectin (1), selectin (2), macrophage mannose receptor (3), collectins (4), DC-SIGN (5), galectin (6), legume lectin (7). Modified after Drickamer, K. Curr. Opin. Struct. Biol. 1993, 3, 393–400, copyright (1993), with permission from Elsevier.

P-selectin

PSGL-1 −

SO3-Tyr −

SO3-Tyr

Endothelial cell or platelet

O-Glycans

Leukocyte

Figure 43 Interactions between P-selectin and PSGL-1. P-selectin molecule is depicted binding to each of the two subunits of PSGL-1. The CRD of P-selectin makes contacts with an NH2-terminal SLeX-containing, core-2 O-glycan and a nearby tyrosine sulfate residue of PSGL-1. The EGF domain and the nine short consensus repeats in the extracellular domain of P-selectin, and the 16 decameric consensus repeats in the extracellular domain of PSGL-1 are also shown. The monosaccharides on the O-glycans of PSGL-1 are: sialic acid (filled diamonds), Gal (open circles), GlcNAc (filled boxes), and Fuc (filled triangles). Reproduced from McEver, R. P.; Cummings, R. D. J. Clin. Invest. 1997, 100, 485–492, with permission of American Society for Clinical Investigation. Copyright 1997 by American Society for Clinical Investigation.

The importance and occurrence of multivalecy in carbohydrate–lectin interactions have been described in many publications.130,134–142 These reports suggest that the number of carbohydrate epitopes and their spatial distribution, as well as the number and spatial distribution of binding sites on the lectin, guide multivalent interactions which fall into two general classes: intramolecular and intermolecular binding mechanisms. Figure 44 shows illustrations of these two types of mechanisms.

3.21.3.1 Intramolecular Binding 3.21.3.1.1

Asialoglycoprotein receptor

Ashwell and co-workers143,144 first demonstrated the magnitude of affinity increase and the importance of multivalency through their studies of the rabbit hepatic lectin. The C-type Gal/GalNAc-binding mammalian hepatic lectin (Figure 42) (known as asialoglycoprotein receptor, ASGP-R), and the GlcNAc-binding avian hepatic lectin, trimerize through their stalk, and further dimerize to stable hexamers. On the surface of hepatocytes, the hexamers form an array of binding sites that bind carbohydrate ligands with high affinity. Both hepatic lectins bind to their multivalent ligand so tightly that the bound complex remains intact during endocytosis and passage to the endosomes. Studies with C-type lectins by Lee and colleagues145–152 unequivocally established the role of multivalency in affinity

432


R

L

(a)

R R

R

R

L

(b) Figure 44 Schematic representation of (a) intra-(e.g., SLT, CT, C-type hepatic lectin) and (b) intermolecular (e.g., galectins, ConA, DGL) multivalent binding.

enhancement. They demonstrated that configurational complemetarity between the binding sites and carbohydrate ligand is the main requisite for the high affinities. Using mono- or disaccharide containing bovine serum albumin (BSA)-based neoglycoproteins, they showed that a linear increase in the number of terminal Gal/GalNAc residues causes an exponential increase in binding affinity. Even smaller bi- and trivalent oligosaccharides showed high affinity. The authors suggested that the binding unit of rat hepatic lectin on the hepatocyte surface is composed of three binding sites that are configurationally complementary to a trivalent oligosaccharide. The authors also showed that when the lectin subunits are not organized, the same bi- and trivalent oligosaccharides do not bind with high affinity. Similarly, rigidly organized hepatic lectin on hepatocyte surface lost affinity when it interacted with multivalent ligands that were more flexible compared to bi- and trivalent oligosaccharides. The degree of affinity enhancement decreased when both lectin and ligands were flexible. Lee and colleagues also studied the multivalent binding properties of another C-type lectin known as the MBP. MBP is a trimeric lectin that can form an aggregate of up to six trimeric units.130 Neoglycoproteins with increasing numbers of Man residue showed exponential enhancement of affinity; however, the slope value for MBP was smaller than that for the mammalian hepatic lectin, and the maximum affinity leveled off around 30nM when more than 24 Man residues per molecule of BSA were present. The relatively restricted affinity enhancement observed with MBP is probably due to the binding site arrangements of the MBP trimer, which is more widely separated and flexible than those of the hepatic lectins.

3.21.3.1.2 Shiga-like toxin and cholera toxin Shiga-like toxin (SLT) and CT belong to bacterial AB5 toxin family that contains an enzymatically active A subunit and a doughnut-shaped pentameric B-subunit with 15 carbohydrate binding sites (3 sites per monomer). The sites are arranged on one surface of the pentamer, while the A-subunit is attached to the opposite surface. Of the three binding sites present on each monomer of B-subunit, site 1 and site 2 are more important in terms of ligand binding.153 SLT and CT have been shown to follow similar mechanisms of multivalent binding. Bundle and colleagues153,154 synthesized a decavalent ligand named STARFISH (Figure 45) designed to bind 10 binding sites (site 1 and site 2 on each monomer) of the B-pentamer. STARFISH is composed of five spacer arms radiating from a central Glc. Two properly spaced Pk trisaccharides were connected to the outer end of each arm. STARFISH showed over a millionfold higher activity than the monovalent Pk trisaccharide. Crystal structures of SLT–STARFISH complexes show that instead of binding site 1 and site 2 of the same B-subunit monomer, two Pk trisaccharides on each arm of the

R O R HO HO

O

O

OH O HO

O

O R

O

R

3 OH

HO

HO

O

HO O

HO HO

OMe

HO

O OH

OH O HO

O OH O HO O

1

HO OH O HO HO O OH HO O HO OMe O HO O O O OH O

HO

OH O

OH

HO O

OH O

HO

O

O

HO O

O O

2

OMe O OH

N H

O OH

OMe

O

(CH2)8

HO

HO

O

HO

NH

O HO

HO

N H O

HO O

OH O

HO O

HO

HO

O

O OH

NH

O

NHCH2CH2NH

O

O

OMe R=

CH2CH2CH2SCH2

O N H

N H

O

Figure 45 Structures of monomeric and oligomeric Pk inhibitors of SLTs. 1, Pk trisaccharide methyl glycoside; 2, dimeric bridged Pk trisaccharide; 3, decameric STARFISH. Reprinted by permission from Macmillan Publishers Ltd: Kitov, P. I.; Sadowska, J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.; Pannu, N. S.; Read, R. J.; Bundle, D. R. Nature 403, 669–672, copyright (2000).


O HO

R

O

433

434


(a)

(b)

Figure 46 Mode of binding of STARFISH to SLT-1. (a) Stereo diagram of the crystallographic dimer of B-subunit pentamers of SLT-I linked by the STARFISH ligand. For the protein, b-strands are illustrated as broad arrows and a-helices as coiled ribbons. The ligand is shown in a ball-and-stick representation, with gray bonds connecting atoms of the carbohydrate component and magenta bonds connecting atoms of the linker. (b) Diagram of half of the STARFISH: SLT-1 B5 sandwich. The representation is as in (a), except that dashed magenta lines show a possible conformation for the central component of the linker, which could not be seen clearly in the electron density. Reprinted by permission from Macmillan Publishers Ltd: Kitov, P. I.; Sadowska, J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.; Pannu, N. S.; Read, R. J.; Bundle, D. R. Nature 403, 669–672, copyright (2000).

STARFISH interacted with site 2 of two different B-subunits forming a 1:2 STARFISH/B-subunit sandwiched complex (Figure 46). As seen in the hepatic lectin model, this mode of multivalent interactions results in enormous increases in binding affinity. In subsequent publications, this group proposed a thermodynamic model that describes this system.155 Hol and colleagues156 designed and synthesized nonbranched pentavalent and branched decavalent ligands (Figure 47) that interact with CT with enhanced affinity. The pentavalent ligand forms a 1:1 complex with CT and the decavalent ligand gives rise to a 1:2 ligand/CT complex. Like the SLT model, reduction of entropic cost through preordering is the main factor for high-affinity binding. During decavalent ligand binding, association of the second B-pentamer is less favorable than the first B-pentamer. This was evident from the rapid shift of the 1:2 to 1:1 complex. The high-affinity interaction between ganglioside GM1 pentasaccharide and CT was suggested to originate from the conformational preorganization of the oligosaccharide.157 The large affinity enhancements described above are due to configurational complementarity between the lectinbinding sites and multivalent carbohydrates. The overall binding energy is close to the sum of the binding energy at individual sites, suggesting that the overall binding energy experiences little entropic penalty. This is elegantly shown from thermodynamic binding studies with vancomycin by Whitesides and co-workers.158 Vancomycin (V) is a glycopeptide antibiotic that is active against Gram-positive bacteria. It binds to the C-terminal D-Ala-D-Ala (DADA) of the bacterial cell wall mucopeptide precursors and disrupts the structure of the cell wall resulting in lysis. Whitesides and co-workers used V and DADA as models to study multivalent binding interactions.158,159 Although this system does not involve carbohydrate–protein interactions, it provides important insights into the thermodynamics of multivalent ligand–receptor interactions described in the above systems. The authors studied a trivalent system of receptor and ligand derived from vancomycin (RtV3) and DADA (R0 tL0 3), respectively (Figure 48), where the trivalent receptor is engaged in intramolecular binding with the trivalent ligand. This trivalent interaction showed exceptionally high affinity compared to monovalent binding.158 Although the enhanced affinity was too high to determine by ITC (1017 M–1; the affinity of the corresponding monovalent analogs was 106 M–1), they reported an ITC-derived DH for the interaction of RtV3 and R0 tL0 3. DH and TDS for the trivalent binding were approximately three times greater than that of the monovalent ligand. The increased enthalpy without a large compensatory loss in entropy was the origin of enhanced affinity and was attributed to the complementary intramolecular binding of the trivalent receptor by the trivalent ligand. This appears to be a very important thermodynamic model of intramolecular multivalent binding, applicable to the work of Bundle and colleagues as well as that of Lee and colleagues in their respective lectin–carbohydrate-binding systems.130,153

3.21.3.2 Intermolecular Binding 3.21.3.2.1 Legume lectins and galectins The carbohydrate-binding sites of legume lectins and galectins are located at the opposite ends of the protein subunits and therefore are separated by relatively large distances (Figure 42). This arrangement of binding sites is different from those in the C-type lectins, SLT, and CT where the sites are organized in the same general direction, and in


435

Nonbranched pentavalent design

Core Linkers

Fingers (a)

CT B pentamer

Pentavalent ligand

1:1 Complex

Branched pentavalent design

Core Linkers

Branched fingers (b)

CT B pentamer

Decavalent ligand

1:2 Complex

Figure 47 Structure-based design of pentavalent and decavalent ligands of CT.156 Reprinted with permission from Zhang, Z.; Merritt, E. A.; Ahn, M.; Roach, C.; Hou, Z.; Verlinde, C. L. M. J.; Hol, W. G. J.; Fan, E. J. Am. Chem. Soc. 2002, 124, 12991–12998. Copyright (2002) American Chemical Society.

closer proximities. In SLT and CT, a multivalent carbohydrate could interact with multiple sites of the molecule. This contrasts with binding of a multivalent carbohydrate to the binding sites of legume galectins that are separated from each other (Figure 44). As a consequence, the binding mechanism of carbohydrates to legume lectins, such as ConA and DGL, and galectins is very different from that valent carbohydrate to C-type lectins, SLT, and CT.

same lectin lectins and multivalent of a multi-

3.21.3.2.1.1 Multivalent binding by legume lectins To gain insight into the mechanisms of intermolecular multivalency, binding of synthetic multivalent carbohydrates to ConA and DGL was studied using ITC, which directly determines the association constant (Ka), enthalpy of binding (DH), and the number of binding sites of the protein (n value). TDS and DG were calculated from the Gibbs free energy equation DG¼DH – TDS¼–Rtln Ka.160 ConA and DGL are Man/Glc-specific lectins with similar binding specificities and possess relatively high affinities for the monovalent trisaccharide methyl 3,6-di-O-(a-D-mannopyranosyl)a-D-mannopyranoside (trimannoside Figure 49) as compared with Man.16 Synthetic multivalent clustered glycosides bearing multiple terminal trimannoside residues (Figure 49) showed increased affinities for ConA and DGL in the ITC data relative to monovalent trimannoside.161 The enhanced affinities were not as dramatic as those observed above with STARFISH.153 The bi-, tri-, and tetravalent analogs show 6-, 11-, and 35-fold higher Ka values for ConA, respectively, and 5-, 8-, and 53-fold higher Ka values, respectively, for DGL relative to the monovalent trimannoside161 (Table 1). The thermodynamic bases for these enhancements are described below. 3.21.3.2.1.1.1 ITC-determined n values are inversely proportional to the functional valency of multivalent carbohydrates The ITC data (Table 1) indicate that the number of binding sites (n) per monomer of ConA and DGL for trimannoside is close to 1.0.16 This agrees with X-ray crystal data for a single binding site on each monomer of ConA

436


OH Me

O

NH3+ HO HO

O

O

O

HO

RtV3

Me

O

Cl

O

Cl HO V

R O

H N

HN V

O H

O

H N H

OH H

H N

H

O

H N

N H

O

N H

H

N H

NH2+

H O

O

C

O HO

R

Me

O

NH2 HO O H

R Vancomycin (V) R⬘t L⬘3 H3C L⬘

Me

O HN

O

L⬘

O

N H

O−

N

N H

H

O

O Me

L⬘

Figure 48 Structures of the trivalent derivatives of vancomycin, RtV3, and DADA, R0 tL0 3 158. Reprinted with permission from Rao, J.; Lahiri, J.; Isaacs, L.; Weis, R. M.; Whitesides, G. M. Science 1998, 280, 708–711. Copyright (1998) AAAS.

and DGL18 and confirms that trimannoside is a monovalent ligand for ConA and DGL. The theoretical values of n for binding of bi-, tri-, and tetravalent carbohydrates to ConA and DGL are 0.5 (1.0/2), 0.33 (1.0/3), and 0.25 (1.0/4), respectively. The values obtained from ITC experiment are consistent with the theoretical values except for the trivalent analog.161 The n values obtained with the trivalent analog are 0.51 with ConA and 0.40 with DGL. These values show that the structurally trivalent ligand is functionally bivalent for ConA and its interaction with DGL is a mixture of bi- and trivalent binding. These data indicate that structural and functional valency of a multivalent ligand may sometimes differ. 3.21.3.2.1.1.2 DH increases in direct proportion to the valency of multivalent carbohydrates binding to ConA and DGL The results by Dam et al.161 demonstrate (Table 1) that for higher-affinity multivalent analogs, the observed DH is approximately the sum of the DH values of the individual epitopes. Similar observations, as discussed in previous section, have been made for the binding of a trivalent ligand and to a trivalent receptor derived from D-Ala-DAla and vancomycin and for the interaction of divalent C-glycosides to ConA.162 3.21.3.2.1.1.3 TDS does not directly increase in proportion to the valency of multivalent carbohydrates binding to ConA and DGL The ITC data of Dam et al.161 for binding of multivalent carbohydrate analogs to ConA or DGL show that DH scales proportionally to the number of carbohydrate epitopes but TDS does not. Data for DH and TDS for ConA and DGL, respectively, binding to multivalent analogs 17–19 are shown in Table 1. While DH scales proportionally, TDS is much more negative than if it proportionally scaled to the number epitopes in the carbohydrates. For example, the observed TDS value for tetravalent 19 is –43.3 kcal mol–1, not –28.4 kcal mol–1, if it scaled with the TDS value of –7.1 kcal mol–1 for trimannoside (Table 1). The resulting DG value of 19 would also be much greater if TDS scaled with valency, since the difference between DH and TDS would be greater. Due to the greater negative TDS values, DG is less negative (affinity is less enhanced) in this system compared with intramolecular multivalent binding.


HO

HO

O

HO HO

HO

HO

HO HO

O

HO

O HO O O

H

O

HO

1

O

O OH

NH

CO2R

HO

S

HO

O HO

NH

S

HO O

HO

O OH

HO

O

O

HO

HO

HO HO

HO

O

HO O

HO

O

HO HO

NH

NH

O

O O

O OH

HO

17 HO O

HO HO

HO

O

HO O

HO

HO

OH

O

HO HO

O

O OH

HO

HO NH

NH HN

N H

OH O

O O

HO O

O

NH

O

OH

O

O

S

O

HO HO HO

O

S

S HO

OH

O

OH OH

O HO

NH

OH

O HO O

HO HO

18

O OH

HO HO HO HO

OH O

HO

OH O

O

HO

OH OH

O

O HO HO

OH

HO

O

HO

OH O

OH OH

O

O OH

O O

S

S

NH

NH NH

S

NH

O

O

O

O C NH (CH2)6 NH C O

S NH

OH

NH

OH O

O

O HO O

O O

O HO HO

19

O

O O OH

OH OH OH

O

HO HO O OH HO HO

OH

NH

NH

HO O

OH

OH

Figure 49 Bi-(17), tri-(18), and tetravalent (19) analogs of trimannoside.

O HO

OH OH OH

437

438


Table 1 Thermodynamic binding parameters for concanavalin A and Dioclea grandiflora lectin with multivalent sugars at 27 C161 Sugar

Ka (M1 104)

Concanavalin A Trimannoside (1) 17 18 19

39 250 420 1350

Dioclea grandiflora lectin (DGL) Trimannoside (1) 17 18 19

122 590 1000 6500

DG (kcalmol 1)

DH (kcalmol 1)

TDS (kcalmol 1)

n

7.6 8.7 9.0 9.7

14.7 26.2 29.0 53.0

7.1 17.5 20.0 43.3

1.0 0.53 0.51 0.26

8.3 9.2 9.6 10.6

16.2 27.5 32.2 58.7

7.9 18.3 22.6 48.1

1.0 0.51 0.40 0.25

These results demonstrate the thermodynamics of binding of a multivalent ligand to separate receptors not possessing clustered binding sites (ConA and DGL) in solution. In this case, the distances between binding sites on ConA and DGL are too great to be spanned by a single multivalent carbohydrate. 3.21.3.2.1.1.4 Thermodynamic basis for enhanced affinities of multivalent analogs for ConA and DGL The enhanced affinities of the multivalent carbohydrates for ConA and DGL are associated with their epitopes binding to separate lectin molecules. The observed Ka value for the tetravalent analog 19 (Figure 49), for example, is the average of the four microscopic Ka values of its four epitopes, because each of the four epitopes is involved in binding to a separate lectin molecule. It follows that since DH is constant at each epitope and is approximately the same as for monovalent trimannoside (Table 1), then increases in the overall macroscopic Ka values (DG) of the four epitopes require more favorable TDS contributions of the individual four epitopes of the tetravalent analog compared with the trimannoside.161 This was directly shown in reverse ITC experiments for di- and trivalent Man analogs binding to ConA.163 3.21.3.2.1.1.5 The epitopes of a multivalent carbohydrate possess a gradient of decreasing microscopic affinity constants ITC-derived n value and other thermodynamic binding parameters clearly indicate that each epitope of the multivalent analogs possesses microscopic binding parameters, such as microscopic Ka, DH, and TDS values. The tetravalent analog 19 (Figure 49) has a total of four microscopic Ka values (Ka1, Ka2, Ka3, and Ka4) for its four epitopes. The macroscopic Ka of this analog determined by ITC is an average of the four microscopic Ka. Hill plot analysis of ITC-generated raw data reveals that the values of the microscopic Ka’s are not equal and that Ka1 > Ka2 > Ka3 > Ka4. This indicates that the multivalent analog binds to the lectin with decreasing affinity (negative cooperativity).164 The physical basis for the decreasing Ka values of epitopes of multivalent ligands can be explained by the reduction in the functional valency of the analogs as they bind an increasing number of lectin molecules. For example, Figure 50 shows the various microequilibrium constants for 19 as its four epitopes sequentially bind one, two, three, and four molecules of ConA (or DGL). The functional valency of unbound 19 (species A) is four, the functional valency of 19 with one bound lectin molecule (species B) is three, the functional valency of 19 with two bound lectin molecules (species C) is two, and the functional valency of 19 with three bound lectin molecules (species D) is one. Sequential occupancy of the four epitopes of the analog would result in a gradual decrease in valency and binding affinity. Increasing negative cooperativity as documented by the curvilinear Hill plots is consistent with the decreasing binding affinity. Another physical factor that may play a role in the negative cooperativity shown by multivalent analogs with ConA and DGL is the formation of noncovalent cross-linked complexes between lectin molecules and multivalent carbohydrates. Figure 50 is overly simplified in that each lectin molecule, represented as a monomer in the scheme, is actually a dimer under the conditions of the experiment (pH 5.2 and low ionic strength). Hence, each lectin molecule (ConA or DGL) is capable of binding and cross-linking the multivalent carbohydrates in the present study. It should also be mentioned that the multivalent glycoprotein asialofetuin (ASF) shows similar negative cooperative effects during its interaction with monomeric galectins, which are not expected to show cross-linking activities.133 3.21.3.2.1.1.6 Range of microscopic affinity constants for multivalent carbohydrates binding to ConA and DGL Based on Figure 50, eqn [1] was derived to describe the relationship between the observed macroscopic free energy of binding and the microscopic free energies of binding of the various epitopes of a multivalent carbohydrate binding to a lectin.164


439

= Tetraantennary analog 19 = ConA or DGL Ka1 + A Ka2 + B Ka3 + C Ka4 + D

E Ka1 > Ka2 > Ka3 > Ka4

Figure 50 Four microequilibrium constants of the tetravalent analog 19 can be represented by Ka1, Ka2, Ka3, and Ka4, for binding of a dimeric ConA molecule to the first arm of 19 (species A), to the second arm of 19 (species B), and so on. Hence, the observed (macroscopic) DG values of 19 (DG(obs)) for ConA are the average of the four microscopic DG terms, or DG(obs)¼(DG1 þ DG2 þ DG3 þ DG4)/4. The relative values of DG1, DG2, DG3, and DG4 must decrease on the basis of the decreasing valencies of A, B, C, and D (which have the same valencies as tetra-, tri, bi-, and monovalent analogs). Thus, it is expected that Ka1 >Ka2 >Ka3 >Ka4 for 19 binding to ConA, as shown. An increasing level of cross-linking with the progression of binding will also contribute to the decreasing microscopic binding constants.201 Reprinted with permission from Dam, T. K.; Roy, R.; Page´, D.; Brewer, C. F. Biochemistry 2002, 41, 1351–1358. Copyright (2002) American Chemical Society.

DGðobsÞ ¼ fDG1 þ . . . þ DGn g=n

½1

Equation [1] states that the observed macroscopic DG value (DG(obs)), determined by ITC, of a multivalent carbohydrate is the average of the microscopic DG values of the individual epitopes, where n is the number of epitopes of the multivalent ligand.164 This equation correctly estimates the difference in microscopic DG values of the two epitopes of bivalent analog 17 binding to ConA.163 In this case, eqn [2] for the bivalent carbohydrate, DGðobsÞ ¼ fDG1 þ DG2 g=2

½2

shows that DG(obs) from a normal ITC experiment allows calculation of DG1, the first epitope of the divalent carbohydrate, assuming that DG2 for the second epitope is the same as that of a monovalent ligand. This latter assumption was shown to be true from a reverse ITC experiment that allows direct determination of DG1 and DG2. 163 The difference between DG1 and DG2 calculated from eqn [2] using DG(obs) from a normal ITC experiment agreed well with that determined from the reverse ITC.163 Equation [1] can also be used to estimate the spread in microscopic DG values for the tetraantennary carbohydrates that bound to ConA and DGL. Equation [3] describes the relationship between the macroscopic DG(obs) and four microscopic DG values for binding of the tetraantennary analog 19 to DGL. DGðobsÞ ¼ fDG1 þ DG2 þ DG3 þ DG4 g=4

½3

DG1 in eqn [3] is associated with the binding of the first carbohydrate epitope of tetraantennary analog 19, DG2 with the second carbohydrate epitope, DG3 with the third carbohydrate epitope, and DG4 with the fourth epitope. The

440


ITC-measured macroscopic DG(obs) for binding of the 19 to DGL is –10.6 kcal mol–1,161 while DG4 in eqn [3] can be taken as the DG(obs) for binding monovalent trimannoside to DGL which is –8.3 kcal mol–1.161 Since DG(obs) is the average of the four microscopic DG values, then DG1 DGðobsÞ ¼ DGðobsÞDG4

½4

assuming that DG(obs) – DG2 DG3 – DG(obs) (i.e., there is a symmetrical distribution of microscopic DG values on either side of DG(obs)). The numerical value of DG1 calculated from eqn [4] is –12.9 kcal mol–1, which is 4.6 kcal mol–1 greater than DG4. This difference between DG1 and DG4 translates to a difference in microscopic Ka values, Ka1 and Ka4, of approximately 2800 times. In absolute terms, Ka1 is approximately 0.3 nM, while Ka4 is approximately 0.8 mM. Thus, the microscopic Ka1 of the first unbound epitope of tetrantennary analog 19 binding to DGL is 2800-fold greater than Ka4 for binding of the fourth epitope. For 19 binding to ConA, this difference between Ka1 and Ka4 is nearly 1200fold.161 This indicates a decreasing gradient of microscopic binding constants of the four epitopes of 19 binding to ConA and DGL. These differences have been postulated to be due to kinetic effects on the off-rates of the various fractionally bound complexes of the multivalent carbohydrates.161 The microscopic off-rate (k–1) for Ka1 in Figure 50 (Ka1 ¼ k1/k–1) would be expected to be slower than the microscopic off-rate for Ka2, etc., due to binding and recapture of the first bound lectin molecule by the remaining unbound trimannoside residues of the tetravalent analog before full dissociation of the complex. The literature often presents ‘valency-corrected’ binding data in studies involving multivalency. Instead of using the concentration on the basis of whole multivalent molecule (molar concentration), these ‘valency corrections’ are made on the basis of per site or per branch (equivalent concentrations) of the multivalent molecules. Two groups155,164 have independently demonstrated the validity and necessity of using the concentration on the basis of whole multivalent molecule (molar concentration). Valid analysis of thermodynamic binding data can only be achieved by expressing concentrations on a molar basis, since the units in the thermodynamic binding equations are molar.155,164,165

3.21.3.2.1.2 Binding of ASF to galectins Binding of ASF, a naturally occurring 48 kDa glycoprotein that possesses nine LacNAc epitopes (Figure 51), to galectin-1,-2,-3,-4,-5, and -7, and truncated, monomer versions of galectin-3 and -5 has been studied by ITC.133 The observed Ka values for ASF binding to the galectins and two truncated forms are 50- to 80-fold greater than that of LacNAc, the monovalent counterpart. Hill plot analysis showed that the nine LacNAc epitopes of ASF interacted with galectins with negative cooperativity that resulted into a gradient of microscopic Ka. Noncovalent cross-linking does not significantly contribute to negative cooperativity, since this effect was observed with both dimeric galectins such as galectin-1,-2, and -7 as well as with monomeric galectin-3 and truncated galectin-3 and -5. 3.21.3.2.1.2.1 Range of microscopic Ka values for ASF binding to galectins The LacNAc chains of ASF possess nine microequilibrium constants represented by Ka1, Ka2, . . ., Ka9 for binding to the galectins (Figure 51). Hill plot analysis of the ASF/galectin binding data133 suggests that the multivalent binding mechanisms are similar to those described above for ConA and DGL.164 Therefore, a similar analysis was used to determine the range of microscopic binding constants. Since the ITC-derived DG(obs) value, DGðobsÞ ¼ fDG1 þ DG2 þ DG3 þ DG4 þ þDG9 g=9

½5

is the average of the nine microscopic DG values in eqn [5], and assuming that there is a symmetrical distribution of decreasing microscopic DG values on either side of the average DG value (that is, DG8 – DG7 DG3 – DG2, etc.), then the value of DG(obs) is nearly equal to DG5 in eqn [5]. It follows then that if DG9, which represents binding of the last free epitope of ASF to a galectin, is nearly equal to DG for LacNAc binding to a galectin, then the difference between DG(obs) and DG9 is half the difference between DG1 and DG9. Thus, the 2.8kcalmol–1 increase in DG(obs) for ASF binding to galectin-3 versus DG for LacNAc binding133 indicates that the difference between DG1 and DG9 for ASF binding to galectin-3 is 5.6kcalmol–1. Since 2.8kcalmol–1 is a 78-fold increase in affinity of ASF for galectin3 over LacNAc, the difference in affinity of the first unbound LacNAc epitope of ASF for galectin-3 is 7878 or 6000-fold increase in affinity over LacNAc, with a decreasing gradient of affinities down to that of LacNAc for the last (ninth) unbound epitope of ASF. In fact, all of the galectins including truncated galectin-5 show Ka(obs) values that are 50–78-fold greater than that of LacNAc for the respective galectin.133 This indicates that the first unbound epitope of ASF binds to all of the


441

= Asialofetuin

ASF

= Galectin Ka1 ASF

+

ASF

Ka2 + ASF

ASF Ka9 + ASF

ASF

Ka1 > Ka2 > Ka3 > Ka4 > Ka5 > Ka6 > Ka7 > Ka8 > Ka9 Figure 51 Schematic representation of galectin binding to ASF.133 Reprinted with permission from Dam, T. K.; Gabius, H.-J.; Andre, S.; Kaltner, H.; Lensch, M.; Brewer, C. F. Biochemistry 2005, 44, 12564–12571. Copyright (2005) American Chemical Society.

galectins with 3000- to 6000-fold higher affinity than LacNAc and the last unbound epitope of ASF, and, therefore, that there is a gradient of decreasing affinities for the remaining epitopes of ASF for all of the galectins. In terms of numerical affinity constants, a 6000-fold increase in affinity of galectin-3 for the first unbound epitope of ASF is eqivalent to a 10nM affinity constant, using the Ka value for LacNAc as an estimate for Ka9 in Figure 51. This estimated range of nanomolar affinity constants for Ka1 is typical for all of the galectins binding to the first unbound epitope of ASF. It is important to point out that the above estimates of the range of affinity constants for ASF binding to the galectins depend, in part, on the assumed affinity of the galectins binding to the last unbound epitope of ASF. In the above calculations, that affinity constant was estimated to be similar to that of the corresponding monovalent ligand, LacNAc. However, if the affinity of binding the galectins to the last unbound epitope of ASF is lower than LacNAc due to steric crowding or other mechanisms, then the estimated range of enhanced affinities sites on ASF (the first unbound LacNAc epitopes) would be even greater. Similarly, a nonsymmetrical distribution of decreasing binding contants associated with the nine LacNAc epitopes of ASF would also affect the range of estimated affinity constants for the epitopes. Nevertheless, a large range of decreasing microaffinity constants exists for the nine epitopes of ASF binding to the galectins. The implications for a gradient of decreasing affinity constants of ASF for the galectins are important. For example, relatively low concentrations of the galectins can be expected to bind to only a few high-affinity sites on glycoprotein receptors like ASF. In this regard, as few as three galectin-1 molecules bound to ASF have been observed to lead to homogeneous cross-linking of the molecules into large insoluble aggregates.166 This contrasts with the requirement of binding to all of the epitopes of lower-valency molecules such as bi-, tri-, and tetravalent carbohydrates for crosslinking which requires much higher concentrations of lectin to occupy the higher- and lower-affinity epitopes in such molecules. In addition, binding and cross-linking of glycoprotein receptors like ASF on the surface of a cell by a dimeric galectin such as galectin-1 with individual affinity sites on each glycoprotein of 10 nM would result in an overall avidity of the dimeric galectin-1 of 1016 M–1. Hence, cross-linking by a dimeric galectin would be essentially irreversible under these conditions.

442


The same observations also apply to other types of lectins interacting with clustered glycan receptors. Fractional high-affinity binding of lectins to multivalent receptors could lead to supramolecular assemblies of homogeneous cross-linked receptors167 or heterogeneous cross-linked receptors.131 Such assemblies, in turn, can trigger cell surface signal transduction mechanisms similar to those observed for galectin-1.168 The concept of a gradient of microscopic binding affinity also has general implications for other types of clustered receptor systems. The concept of ‘spare receptors’ is well documented in the pharmacology literature.169 Maximum dose-activity responses are often observed at relatively low fractional occupancy of the receptors. The present results suggest that such ‘spare receptor’ systems may exhibit enhanced affinity for a specific ligand through a clustering mechanism similar to that observed for ASF binding to the galectins in the present study. The enhancement in affinity of the ligand could be as much as 1000–10 000-fold by clustering of receptors. Occupancy of a portion of the clustered or ‘spare receptors’ by antagonist would diminish the total number of unbound receptors, but may not reduce the ‘avidity’ of the remaining clustered receptors.169 The ‘efficacy’ of the agonist effect may be related to cross-linking or activating in some manner a fraction of the receptors that is necessary for full pharmacology effect. In the present study, fractional occupancy by galectin molecules of a few epitopes of an ASF-like receptor molecule could lead to cross-linking interactions and subsequent signal transduction effects such as apoptosis, as observed in the binding of galectin-1 to human T-cell glycoprotein receptors.132

3.21.3.3 Some Other Examples of Multivalent Binding 3.21.3.3.1 Interaction of lectins with multivalent polymeric ligands Kiessling and colleagues synthesized a series of ring-opening metathesis polymerization (ROMP)-derived multivalent sugar ligands of different length and valency and studied their binding properties with lectins (Figure 52).138,170,171 Depending on the valency, Man-containing polymers show up to several thousandfold higher affinity (relative to the monosaccharide Man) for ConA in hemagglutination inhibition assay.172 The enhancements, however, were found to be modest when measured with surface plasmon resonance technology.173 The number of bound ConA tetramers changed with the length of the polymer. For example, a shorter polymer of 21 Man residues (degree of polymerization (DP)¼21) bound two ConA tetramers, whereas a polymer of 65 residues (DP¼65) complexed four ConA tetramers.172,174 When the density of the Man epitopes in the polymer is high, greater numbers of lectin molecules interact with the polymer and the rate of clustering is faster. If the density of Man is reduced to a certain level, the binding efficiency per Man residue goes up.175 The distance between two sugar-binding sites of a ConA tetramer is 65A˚. The authors showed that a polymer with 25–35 residues and more could span two binding sites of ConA and that polymers that could simultaneously bind two binding sites of a ConA molecule were the most potent ligands (Figure 52). 173 It was

HO HO HO

HO

O

O HN

O 20 Ph n HO HO HO

(a)

HO

O

O HN

O 21: n = 21 22: n = 38 23: n = 65 24: n = 142

(b)

Figure 52 (a) Chemical Structures of ROMP-derived Man-bearing compounds. The average valency (n) was calculated by using 1H NMR integration. (b) Proposed complex between ConA and a multivalent ROMP-derived polymer of 50 monomer units. A complex was assembled between this polymer and ConA tetramers. The stoichiometry was chosen on the basis of the results from transmission electron microscopy experiments and the quantitative precipitation and FRET results. Reproduced from Gestwicki, J. E.; Strong, L. E.; Cairo, C. W.; Boehm, F. J.; Kiessling, L. L. Chem. Biol. 2002, 9, 163–169, copyright (2002), with permission from Elsevier.


443

Ph n O

O HN NH HO OH HO OH O O HO O HO O OH OH 25 26: n = 10 27: n = 25

Angular velocity (deg frame−1)

45 40 35 30 None Gal 1 2 3

25 20 15 0

(a)

(b)

20

40

60 80 Time (s)

100

120

Angular velocity (deg frame−1)

50 Gal 1 2 3

45 40 35 30 25 20 15 0

(c)

0.1

1 10 100 Galactose (µM)

1000

(d)

Figure 53 Multivalent Gal-containing polymers as chemoattractant molecules. (a) Structures of ROMP-derived Galbearing monovalent 25 and multivalent chemoattractants 26 and 27. (b) Plot of average angular velocity vs time for Escherichia coli AW405 treated with chemoattractant (Gal or ligands 25–27). The ligand concentration (100mM) is given as the total concentration of Gal or Gal residues. (c) Angular velocity of E. coli treated with various concentrations of chemoattractant. (d) Fluorescence micrographs of cheW E. coli RP1078 cells treated with Gal-bearing monovalent ligand 25 (left) or multivalent ligand 27 (right). The multivalent ligand clustered the cell surface receptor as visualized with anti-MCP antibodies. The arrow points to a sample cluster. Reprinted by permission from Macmillan Publishers Ltd: Gestwicki, J. E.; Kiessling, L. L. Nature 415, 81–84. Copyright (2002).

concluded that the high binding affinities obtained with these ligands were due to a combination of inter- and intramolecular binding effects. According to the authors, rebinding due to higher epitope density and subsequent slower dissociation rates are key factors for the observed high affinity.174 Kiessling and co-workers also employed ROMP technique for synthesizing multivalent ligands for L-selectin. These ligands were found to be very efficient in clustering and subsequent releasing of L-selectin molecules from cell surface.176–178 The same group also demonstrated that ROMP-derived ligands with multiple Gal residues could act as effective chemoattractants for E. coli.179 They synthesized several multivalent ligands with an objective to cluster the Gal-sensing chemoreceptor, Trg. Indeed, multivalent ligand-mediated chemoreceptor clustering was shown by fluorescence microscopy. The potency of the ligands was found to be directly proportional with the valency of the ligands (Figure 53).180

3.21.3.3.2

Multivalent inhibitors of influenza virus HA

Binding of multivalent analogs of sialic acid to the influenza virus HA has been studied extensively.141 Depending on the structures, the analogs showed 103–106 times higher affinities than a monovalent sialic acid. The enhancements in affinities were attributed to steric stabilization.141,181–183

3.21.3.3.3

Xenopus laevis lectin XL35

Boons and colleagues studied the interactions of Xenopus laevis lectin XL35 with jelly coat protein-derived oligosaccharides.184 Multivalent analogs showed broad specificity and moderate affinity enhancement, compared with

444


corresponding monovalent sugars. The interactions between XL35 and multivalent sugars failed to reach true equilibrium and showed unusually slow dissociation rates. The authors suggested that this mode of multivalent interactions between jelly coat protein and XL35 would help create a stable protective layer in order to prevent polyspermy.

3.21.3.3.4 Interaction of cyanovirin-N with high-mannose oligosaccharides Cyanovirin-N (CV-N) is an 11kDa protein isolated from an extract of the cyanobacterium Nostoc ellipsosporum. CV-N potently interacts with high-mannose oligosaccharide structures on gp120 and gp41 and thus is capable of inactivating various strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). 185 CV-N possesses two carbohydratebinding sites,186–189 and the protein shows highest affinity for oligomannose oligosaccharides such as Man8 and Man9.187,190 NMR and ITC studies revealed that binding of a nonamannoside to CV-N was multivalent in nature. Nonamannoside was found to cross-link CV-N molecules through this multivalent binding.191

3.21.3.3.5 Photoswitchable cluster glycosides Srinivas et al. synthesized several Man- and lactose-containing glycoclusters attached to photoswitchable azobenzenoid cores and their binding to PNA and ConA was studied by ITC.192 Binding affinities were enhanced with increased valency of the glycoclusters. A few analogs showed biphasic binding profiles in ITC plots, indicating cooperativity in the binding process. An important outcome of the study was that in addition to inherent clustering of the sugar units as a molecular feature, an induced clustering emanated because of the isomerization of the trans-form of the azobenzene scaffold to the cis-isomeric form.

3.21.3.3.6 Multivalent binding by garlic lectin The dimeric garlic lectin possesses six binding sites (three sites per monomer). Computational analysis revealed that a given oligosaccharide could not bind to more than one site on a dimer.193 Trimannoside and higher oligosaccharides of Man could potentially cross-link the lectin dimer. It was also shown that a given dimer pair could even be crosslinked by two oligosaccharides. The number of such double cross-linked structures including certain tetrameric structures increased with the size of the oligosaccharides (Figure 54).193

3.21.3.4 Carbohydrate–Lectin Cross-linking Interactions Binding of oligomeric lectins to cell surface glycoprotein and glycolipid receptors often leads to cross-linking and aggregation with concomitant biological responses. For example, cross-linking of glycoconjugates on the surface of cells has been implicated in the mitogenic activities of lectins including ConA,194 in the arrest of bulk transport in ganglion cell axons,195 in the molecular sorting of glycoproteins in the secretory pathways of cells,196 and in the apoptosis of activated human T-cells.197 Furthermore, lectin-induced cross-linking of transmembrane glycoproteins leads to changes in their interactions with cytoskeletal proteins and concomitant alterations in the mobility and aggregation of other surface receptors.198 A number of mammalian lectins are involved in receptor-mediated endocytosis of glycoproteins,199 whereas others have been implicated in cellular recognition processes including apoptosis197 and metastasis.200

3.21.3.4.1 Type 1 and type 2 cross-linked complexes Under the appropriate stoichiometric conditions, lectins undergo two general types of intermolecular cross-linking interactions with multivalent carbohydrates, designated as type 1 and type 2 complexes (Figure 55).168 In a type 1 complex, binding between a divalent lectin and a divalent carbohydrate results in one-dimensional cross-linking.168 In a type 2 complex, binding between a multivalent lectin and multivalent carbohydrate, where the valency of either the lectin or carbohydrate is greater than two, results in two-dimensional (planar or tubular) or three-dimensional crosslinking (crystalline, in some cases). Therefore, type 2 interactions can be an important source of binding specificity between lectins and glycoconjugate receptors.201 There are many reports of the X-ray crystal structures of lectin–carbohydrate cross-linked complexes.60,69,92,202,203

3.21.3.4.2 A multivalent carbohydrate can form a unique cross-linked complex with a lectin in the presence of other carbohydrates The cross-linking properties of a variety of plant and animal lectins with multivalent carbohydrates and glycoproteins have recently been reviewed.201 Importantly, type 2 interactions can lead to the formation of homogeneous or heterogeneous cross-linked complexes, even in the presence of mixtures of the molecules.204 Studies show that a


445

Figure 54 A lattice-like structure of garlic lectin formed through the binding of multivalent mannooligosaccharides. Reprinted from Ramachandraiah, G.; Chandra, N. R.; Surolia, A.; Vijayan, M. Glycobiology 2003, 13, 765–775, by permission of Oxford University Press.

Type I

Type II

Figure 55 Lectin–carbohydrate cross-linked complexes. Type I complexes are composed of bivalent lectins and bivalent carbohydrates. Type II complexes are composed of lectins and carbohydrates, one of which has a valency >2. On the left, a tetravalent lectin is complexed with a bivalent carbohydrate, whereas on the right a bivalent lectin is complexed with a tetravalent carbohydrate. Reproduced from Brewer, C. F.; Miceli, M. C.; Baum, L. G. Curr. Opin. Struct. Biol. 2002, 12, 616–623, copyright (2002), with permission from Elsevier.

446


number of lectins form homogeneous cross-linked complexes with branched-chain oligosaccharides and glycoproteins. For example, quantitative precipitation experiments with the Man/Glc-specific lectin ConA in the presence of binary mixtures of a series of bivalent N-linked oligomannose glycopeptides indicate that each glycopeptide forms its own unique cross-linked complex with the lectin.205 Subsequent X-ray crystallographic studies have demonstrated different lattice structures of crystalline cross-linked complexes of the SBA with four different divalent carbohydrates.203 The different lattice structures are due to differences in the structures of the cross-linking carbohydrates.203 The ability to form unique cross-linked complexes with glycoconjugates and to separate different counter-receptors into homogeneous cross-linked aggregates has recently been implicated in the apoptotic activity of galectin-1, a member of the b-galactosidase-specific animal lectin family (Figure 56).132 Recently, galectin-3, another member of the galectin family, has been shown to form disorganized, heterogeneous cross-linked complexes with multivalent carbohydrates.131 The biological properties of galectin-3, including its anti-apoptotic activities206 and ability to antagonize the growth inhibitory activity of galectin-1 in neuroblastoma cells,207 may relate to its ability to randomly cross-link glycoconjugates and prevent separation of different receptors. Hence, the ability of lectins to form organized or disorganized cross-linked complexes with multivalent glycoconjugate receptors, such as galectin-1 and -3, respectively, may relate to their biological activities.

(a)

(b)

CD43 CD45

CD7 CD3

P P

P P

P P P P

Radixin

Fodrin

(c) CD45 and CD3

Galectin-1

CD43 and CD7

Figure 56 Homogeneous cross-linking by galectin-1. Redistribution and segregation of cell surface glycoprotein receptors of galectin-1 following binding. (a) Random distribution of units of CD45 and CD3 (circles), and units of CD43 and CD7 (squares) on the cell surface, in the absence of galectin-1. (b) Galectin-1 binding results in homotypic lattice formation of repeating units of CD45 and CD3 (circles), and of CD43 and CD7 (squares). (c) Cross-linked complexes of galectin-1 receptors with associated cytoskeletal proteins.132


3.21.3.4.3

447

The structures of the carbohydrates and lectins determine their cross-linking properties

Studies with a series of bivalent analogs and two homologous lectins, ConA and DGL, showed unique lectin- and ligand-specific cross-linking properties.208 Bivalent ligands with different spacer length and flexibilities produce different cross-linking kinetics and cross-linked lattice patterns with a single lectin. On the other hand, a single bivalent ligand demonstrated completely different lattice organization and cross-linking kinetics with two different lectins (Figure 57).208 Kiessling and co-workers have shown that the architecture of a multivalent ligand (Figure 58) determines the rate of receptor clustering, the number of receptors in the clusters, and the average inter-receptor distance.209,210

ConA

DGL 28 29

NOL

30 31 32 33

NOL

34 35 36 NOL

37

NOL

38

Figure 57 Negative stain electron micrograph of ConA and DGL cross-linked with different bivalent carbohydrates (28–38). NOL, no observable lattice. 208

Low molecular weight compounds

Dendrimers (PAMAM)

Size (Da)

Small (600− 700)

Small (1500− 7500)

Large (−250 000)

Medium (3000− 34 000)

Large (−100 000)

Maximum valency

Low (2−3)

Variable (4−16)

Intermediate (15−20)

Variable (5−100)

High (700)

Globular proteins (BSA)

Polydisperse Linear polymers polymers (from ROMP) (PEMA)

Figure 58 Structures of multivalent ligands used by Kiessling and colleagues.209 Reprinted with permission from Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124, 14922–14933. Copyright (2002) American Chemical Society.

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3.21.4 Conclusions Lectins are evolutionarily conserved proteins found in all living forms. Interactions with carbohydrate ligands are essential for the diverse biological functions of lectins. Although carbohydrate binding is driven by essentially the same physical forces, lectins show remarkable diversities in their carbohydrate-binding specificities. This diversity, in part, is caused by structural variations in the carbohydrate-binding sites of the lectins as well as the structures of the carbohydrate ligands. The weak affinity of monovalent carbohydrates for most lectins is offset by the higher avidity of multivalent carbohydrates. The number and spatial distribution of the binding sites of the lectin and the number and spatial distribution of epitopes of a carbohydrate determine the nature of multivalent interactions. These interactions often lead to the formation of noncovalent cross-linked complexes, a phenomenon important for receptor clustering in biological systems.

Glossary functional valency Actual number of epitopes of a ligand that actively participate in binding interaction. heterogeneous or disorganized crosslinking Cross-linking of structurally dissimilar molecule leading to the formation of disordered lattice. homogeneous or organized crosslinking Cross-linking of structurally similar molecule to form ordered lattice. intermolecular binding When a multivalent ligand interacts with the binding sites of different molecules. intramolecular binding When a multivalent ligand binds to multiple binding sites on a same molecule. lectin Carbohydrate-binding protein of nonimmune origin. macroscopic binding parameters Binding parameters of the whole multivalent molecule. microscopic binding parameters Binding parameters of an individual epitope of a multivalent ligand. monovalent binding Binding limited to a single combining site. structural valency Number of binding epitopes present on a ligand.

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Biographical Sketch

Tarun Kanti Dam received his Ph.D. degree from the University of Calcutta, India, under the supervision of Professor Amalesh Choudhury. He was introduced to carbohydrate binding proteins (lectins) by Dr. Manju Sarkar of the Indian Institute of Chemical Biology and was trained in the laboratory of Professor Avadhesha Surolia at the Indian Institute of Science, Bangalore. Dr. Dam joined the Albert Einstein College of Medicine, New York, as a postdoctoral fellow to work with Professor Fred Brewer, where he focused on structural and functional glycobiology. Using plant and mammalian lectins (galectins), he studied fundamental aspects of carbohydrate-lectin interactions. He was given the Young Scientist Award at the Biocalorimetry 2001 conference for his microcalorimetric investigations of lectin-carbohydrate interactions. Dr. Dam joined the faculty of the Albert Einstein College of Medicine as an instructor after completing his postdoctoral training. His research interests include thermodynamic and structural basis of ligand-receptor interaction and structure-function relationship of carbohydrate binding proteins.

Fred Brewer did his graduate studies with Dan Santi in bioorganic chemistry at the University of California, Santa Barbara leading to a Ph.D. degree in 1971. He did postdoctoral research with Arthur Grollman and Don Marcus at the Albert Einstein College of Medicine and Himan Sternlicht at Bell Laboratories on 13C NMR studies of lectin-carbohydrate interactions. Dr. Brewer then joined the faculty of Albert Einstein College of Medicine in 1974, where he is currently Professor of Molecular Pharmacology, and Microbiology and Immunology. Professor Brewer is on the Editorial Boards of the Journal of Biological Chemistry and Glycobiology and an Associate Editor of Trends in Glycosiences and Glycotechnology. He has been on the Awards Committee of the Society for Glycobiology and is past Chairman of the Division of Carbohydrate Chemistry of the American Chemical Society. Among his awards is the 2005 Melville L. Wolfrom Award from the Division of Carbohydrate Chemistry, American Chemical Society. Professor Brewer’s interests include carbohydrate enzymology, immunochemistry, and lectin biochemistry, thermodynamics, and structural biology.

3.22 Glycopolymers S.-I. Nishimura, Hokkaido University, Sapporo, Japan N. Nagahori, Hokkaido University, Sapporo, Japan ß 2007 Elsevier Ltd. All rights reserved. 3.22.1 Introduction

453

3.22.1.1 Multivalent Display of Carbohydrates

453

3.22.1.2 Glycoside Cluster Effect

454

3.22.2 Synthetic Multivalent Glycopolymers

454

3.22.2.1 Water-Soluble Linear Polymers

454

3.22.2.2 Molecularly Imprinted Glycopolymers

456

3.22.3 Highly Efficient Enzymatic Sugar Elongation Reaction on Glycopolymers

457

3.22.4 Combinatorial Synthesis of Glycopeptides Using a Molecular Transporter

458

3.22.5 Multivalent Carbohydrate Presentation on a Peptide Backbone to Control the Topology of Carbohydrates

461

3.22.6 Sequential Glycopeptides with Both Designated Density and Desired Orientation of Carbohydrate Side Chains

463

3.22.7 Multivalent Enzyme Inhibitor

466

3.22.8 Glycopolymer Regulating Cellular Responses

466

3.22.9 Multivalent Glycopolymer Nanoparticles for the Detection of Molecular Recognition

466

3.22.10

Two-Dimensional Glycolipid Polymers for the Multivalent Presentation of Proteins

468

3.22.11

Using a Two-Dimensional Glycopolymer as a Sensing Device

469

3.22.12

Protein Glycomics by Chemoselective Glycoblotting on a Polymer Nanoparticle

471

3.22.13

Summary

472

3.22.1 Introduction 3.22.1.1 Multivalent Display of Carbohydrates The specific recognition of biomolecules is critical to a variety of biological events and is typically the first step in the phenomena of cell–cell interaction. In particular, carbohydrates on cell surfaces are recognized by specific partner molecules, such as proteins and other carbohydrates, to regulate various biological processes, that is, fertilization, cell migration, cell growth regulation, immune defense, aging, and cancer metastasis. They also serve as attachment sites for bacteria, viruses, toxins, and hormones. Among carbohydrate-involving recognition events, carbohydrate–protein interactions are the most studied since they are involved in a variety of biological processes. The specificity between carbohydrates and protein is precisely controlled by molecular recognition based on the elaborate structure of glycoconjugates. On the other hand, the intensity of the specific interaction seems to be controlled by a more complicated mechanism. The individual carbohydrate–protein interaction is based on a weak hydrogen bond in aqueous solution. Furthermore, the binding affinities are quite low; Kd generally ranges from 1 to 0.1mM. Furthermore, they often have broad specificity. Many carbohydrate-binding proteins (CBPs) contain only shallow binding pockets and have a small number of direct contacts with target carbohydrates.1 Nevertheless, the carbohydrate recognition by proteins is relatively strong in naturally occurring biological systems. Therefore, there seems to be a unique mechanism for the specific and strong carbohydrate–protein recognitions that control biological events in vivo. Carbohydrates that naturally occur on cell surfaces usually conjugate with other biomolecules to form glycoproteins, glycolipids, and proteoglycans. Most glycoproteins have branched carbohydrate chains within multiple glycosylation

453

454

Glycopolymers

sites, and proteoglycans are heavily O-glycosylated proteins containing as many as a hundred carbohydrate residues. Glycolipids assemble to form a glycosphingolipid microdomain, where multivalent carbohydrates are presented toward binding partners. In addition to the multivalent display of carbohydrates in a living organism, CBPs have multiple carbohydrate-binding sites and often aggregate into higher oligomeric structures such as viral envelope proteins. Thus it is reasonable that multiple carbohydrate–protein interactions could cooperate to strengthen binding to achieve the necessary affinity for biological functions.

3.22.1.2 Glycoside Cluster Effect Lee et al. verified the importance of multivalency in carbohydrate–protein interactions using synthetic monoantennary, diantennary, and triantennary galactosylated ligands toward hepatic lectin.2 As the sugar valency increases in the order of mono-, di-, and tri-, the affinity of ligand to the lectin is exponentially amplified 103–106 times. This phenomenon is more than could be accounted for by the statistical increase in the sugar concentration and is named the ‘glycoside cluster effect’.3–5 This phenomenon can be a means for amplification of individually weak carbohydrate–protein interactions. The power of multivalency was also observed in an antibody selection experiment. A dimeric antibody has greater affinity for O-polysaccharide than a monomeric single-chain antibody.6 This observation is presumably the result of the interaction between the dimeric antibodies with adjacent carbohydrate sites. These findings have gathered considerable attention and have prompted extensive efforts by synthetic chemists to design a variety of multivalent glycoligands. Polymers,7–9 dendrimers,10 cyclodextrin,11 calyxarenes,12 proteins,13–15 peptides,16–18 and metal nanopartices19–21 are useful scaffolds for the construction of multivalent carbohydrate presentation. They have been applied to characterize multivalent carbohydrate binding events, modulate carbohydrate-mediated cell or virus binding, immobilize specific cell types, and induce cell responses through selective binding with cell surface receptors. The glycopolymer and the glycoside cluster effect have been reviewed extensively.3–5,7–9,22–30 In this chapter, the synthesis and application of the multivalent glycoconjugates are described.

3.22.2 Synthetic Multivalent Glycopolymers 3.22.2.1 Water-Soluble Linear Polymers Glycopolymer synthesis was first reported by Horejsi et al. in 1978.31 They co-polymerized acrylamide and ally glycosides of various sugars in water using ammonium persulfate as an initiator and tetramethylethylenediamine (TEMED) as a catalyst. The resulting glycopolymer exhibits similar binding activity toward lectin compared with natural polysaccharide. Later, a new method of the synthesis of acrylamide monomers containing carbohydrate was reported by Roy et al., in which the glycosylbromide was reacted with p-nitrophenol using phase transfer catalyst.32 They reported biomimetic polymers containing GM3 trisaccharide, 30 -sulfo-Lewisx, and lectin-binding efficiency was evaluated.33,34 Whitesides and co-workers have reported a variety of pendant glycopolymers having sialic acid, which exhibited inhibition activity of influenza virus-mediated hemagglutination.35,36 These works demonstrated the high potency of glycopolymers as an inhibitor of pathogens.37,38 The biotin and fluorescence-tagged acrylamide polymer bearing a-sialic acid was synthesized using two methods: copolymerization of acrylamide with a-sialoside acryalmide and grafting a-sialoside to a polyacrylamide backbone. The binding of the glycopolymer to influenza virus A-X31 was measured by enzyme-linked immunosorbent assay (ELISA) and inhibition of hemagglutination assay. Virus binding activity was 102–106 times greater than the monomeric sialic acid,36 proving that steric stabilization plays a role in the enhancement of binding affinity for the viral lectin. A series of cluster glycopolymers was synthesized by Nishimura and co-workers using ammonium persulfate and the TEMED initiating system.39–43 Glycomonomers differing in the structure and length of aglycon (Figure 1) were homopolymerized or co-polymerized with acrylamide to obtain a polymer with various carbohydrate densities. The apparent binding constant of the glycopolymers with wheat germ agglutinin (WGA) was determined by measuring the changes in fluorescence intensity and the blue shift of the emission maximum. Compared with the low GlcNAc density polymer, the high-density polymer resulted in a much higher binding constant, suggesting that this polymer binds the maximum of binding sites (3 and 4). The interaction of LacNAc-bearing polymers (1 and 2) with Erythrina corallodendron lectin was also studied using a similar method. Unlike the previous study, high-density LacNAc polymer exhibits a lower binding constant than the low-density polymer owing to the sterically hindered sugar branches ( negative cluster effect). These results suggest that the optimum spatial distance between two LacNAc residues on the macromolecular ligand must exist for successful binding to the two subunits of E. corL. Thus, the

Glycopolymers

OH

OH

HO

O HO

HO OH

H N

O

NHAc

1

O HO

HO OH

HO HO

H N

O

NHAc

O

HO HO

O

3 OH

OH O HO NHAc 6

OH

O HO

O HO

O 5

7

OH O

O O O OH O HO

HO

O

NHAc

OH

OH

8

O

OH

O

OH

OH

HO

HO O

HO

HO

OH

O

OH O

O

O

NHAc

OH

HO OH

N H

4

HO

O

O

HO HO

N H

NHAc

O

O

NHAc

O

O

2

OH O

HO

O

O O

OH

HO

OH

OH

HO

O

O

455

OH

O

NHAc 9

Me O

HO

OH

HO

OH HO

O

OH

HO O

HO HO 10

O

O O HO

HO

NHAc

OH

O

O

NHAc 11

Figure 1 Structure of glycomonomers for the preparation of various cluster glycopolymers.

HO

OH

HO AcHN

COOH HO

O HO

HO

OH

HO AcHN

O COOH HO

O O

HO HO HO AcHN

OH O

OH HOOC O

HO

O

O

O

HO

H N

NHAc

OH HO O HO

O HO

HO

NHAc

HO O

OH

HO O

O HO

O

O

H2N O

O OH

x

O

NHAc

O y

HO 12

Figure 2 Polymer-type glycocluster with high anti-influenza virus activity.

intensity of the carbohydrate–protein interaction deeply depends on the sugar density on the polymer support. The stronger affinity of the multipoint interaction of the multivalent glycopolymer suggests that the carbohydrate density in the microscopic environment affects the ON/OFF regulation of biomolecular interactions. Molecular recognition of carbohydrates seems to be regulated by the three-dimensional (3-D) topology of binding elements with proper orientation and spacing. Glycopolymer with branched triantennary sialooligosaccharide 12 (Figure 2) was synthesized as a glycoprotein model based on the cluster effect concept and was expected to show potent anti-influenza virus activity.44 The inhibitory effect of the sialopolymers on hemagglutination by three types of influenza viruses was examined. The results revealed that the potency of the sialopolymers as an inhibitor of influenza and the preference of the virus type are controlled by the structure and density of the carbohydrates.

456

Glycopolymers

Furthermore, the antiviral activity of sialoglycopolymers were evaluated using the mouse model infected with human influenza virus by Bovin et al.45 It was found that polyacrylamide bearing 60 -sialylLacNAc (60 SLN) protected mice from the H1N1 influenza virus. So far, all human influenza viruses are known to have an affinity to 60 SLN; thus the 60 SLN-containing multivalent glycopolymers could afford protection from all human epidemic strains containing newly emerging pandemic strains.

3.22.2.2 Molecularly Imprinted Glycopolymers These water-soluble highly flexible multivalent glycopolymers exhibit high affinity for target proteins; however, these polymers often share binding specificity with a variety of CBPs. To prepare a tailor-made glycopolymer with tight binding specificity as well as high affinity for a guest protein, we applied a molecular imprinting technique, where the glycomonomers are polymerized in the presence of template guest protein.46 The concept employed for displaying and immobilizing functional glycotags with appropriate spacing and flexibility in the sugar-CBP adducts is illustrated in Figure 3. The combination of mannose and mannose-binding protein was selected as a model experiment since mannosemediated cellular recognition seems to be one of the most important processes for controlling immunological systems.3 A polymerizable mannose monomer and Concanavalin A (ConA), the template protein, were incubated in a buffer solution and co-polymerized with acrylamide and cross-linker using APS and TEMED as a promoter. Subsequently, the template was denatured by heat and a high concentration of salt and the polymer was purified by gel filtration. It is expected that the glycoligands are immobilized in the polymer network with appropriate orientation and spacing toward the template proteins. The binding constants of glycopolymers with lectins were determined by a fluorescence

O

N H

CO

NH

2

O

O

N H

H N

Template protein

O

H N O

NH 2 CO

H

O

Cross-linker

O

N

O

Polymerization

N H

Glycomonomer

Removing template

Tailor-made glycopolymer

Figure 3 Tailor-made glycopolymer by means of molecular imprinting method.

Glycopolymers

457

spectroscopic method. The tailor-made glycopolymer retained a similar binding constant with the linear glycopolymer that was not cross-linked. In addition, the tailor-made polymer exhibited 100–300 times higher affinity to the polymer prepared in the absence of the template. This result suggests that specific cavities for the guest proteins were constructed in the synthetic polymer network by the ‘template effect’ during the polymerization reactions. The tailor-made glycopolymers with specific binding and high affinity were obtained by molecular imprinting.

3.22.3 Highly Efficient Enzymatic Sugar Elongation Reaction on Glycopolymers Besides their high affinity to various CBPs, multivalent water-soluble glycopolymers are excellent acceptor substrates. The feasibility of the enzymatic modification of synthetic glycopolymers was first demonstrated by synthesizing a water-soluble polymer with 30 -sialyl-N-acetyllactosamine using bovine milk b-1,4-galactosyltransferase and transsialidase from Trypanosoma cruzi.47 The success of the enzymatic assembly of oligosaccharides on soluble polymer support is critically dependent on satisfactory water solubility and a flexible spacer-arm suitably distanced from the backbone, providing good access to the binding pocket of the enzymes. Here, the sugar elongation reaction on a polymer support could mimic the glycoconjugate biosynthetic pathway in nature, where the carbohydrates are introduced to the protein in a stepwise fashion at the Golgi apparatus by glycosyltransferases displayed on the Golgi membrane in the presence of a suitable sugar nucleotide as a donor substrate and a protein as an acceptor substrate. The use of a water-soluble polymer also enables easy isolation from the reaction mixtures and the monitoring of the reaction by conventional spectroscopic techniques such as NMR, fluorescence spectroscopy, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Thus the enzymatic elongation of carbohydrates on a water-soluble polymer support was expected to provide an efficient and practical methodology for oligosaccharide synthesis, which was urgently required to understand the critical and functional roles of glycoconjugates in cellular biology. We previously developed several functional linkers that could be specifically cleaved by a chemical or enzyme to release the oligosaccharide products from the polymers (Figure 4). For example, compound 13 contains a benzyl ether moiety that can be cleaved by conventional mild conditions of hydrogenolysis.48 Compound 14 has an L-phenylalanine residue in the middle of the linker, suitable for cleaving by

OH O

HO HO

O

H N

O

N H

O

NHAc

n

O

H2N

13

m OH O

HO HO

O O

N H H C 2

NHAc

O

H N

N H

O

H2N

14

HO

n

O

m

OH O

HO OH

OH

HO O

O OH

O O

N H HN O

15

O N H

O

n

H2N m

Figure 4 Structure of polymer support having functional linker.

458

Glycopolymers

HO HO

OH O

HO

OH O

O OH

NHAc 16

O

OH

HO O

N H

O

O

HN

OH

O N H

O

i

PAAm

ii iii iv HO

HO AcHN

OH

HO

OH HOOC HO O O O O OH O H3C

O

HO

OH O

O

OH

O OH

OH

OH

HO O

O OH

O

12

HN

NHAc O OH

14

17

HO HO Figure 5 Synthesis of a sphingolipid, IV3NeuAca, III Fuca-nLc4Cer 17. I, Compound 16 (7mg), b4GalT (1.0unit), UDP-Gal (1.2equiv., 2.6mg), a-lactalbumin (0.26mg), 50mM [4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) buffer (pH6.0), 37 C, 24h; ii, a3SiaT (0.03unit), cytidine 50 -monophosphate-N-acetylneuraminic acid (CMP-NeuAc) (1.4equiv., 3.27mg), 50mM sodium cacodylate buffer (pH 7.4), 10mM MnCl2, 1mM NaN3, calf intestinal alkaline phosphatase (CIAP) (20unit), Triton CF-54 (0.5% v/v), 37 C, 72h; iii, a3FucT (0.08unit), GDP-Fuc (1.4equiv., 3.14mg), 50mM sodium cacodylate buffer (pH 6.5), 10mM MnCl2, 1mM NaN3, 37 C, 72h; iv, ceramide glycanase (0.03unit), ceramide (5.0equiv., 9.52mg), 50mM sodium citrate buffer (pH. 6.0), Triton CF-54 (0.5% v/v), 37 C, 17h.

treatment with a-chymotrypsin to supply oligosaccharide products as o-aminoalkyl glycosides.49 The ceramide mimetic linker in compound 15 can be recognized by ceramide glycanase, and the oligosaccharide synthesized on the polymer is transferred to natural ceramide or sphingosine analogs by the enzymatic transglycosylation reaction. Recently a large number of glycosyltransferases have been cloned and some of their recombinant forms have been produced. For example, sphingolipid IV3NeuAca, III Fuca-nLc4Cer was synthesized on a water-soluble polymer support by four recombinant glycosyltransferases. The product was transferred from polymer support to ceramide by treatment with ceramide glycanase to afford the target sohingolipid in 40% yield (Figure 5).50 In addition, GM3 1851 and some non-natural sphingoglycolipids 19 and 2052 were prepared by the same procedure as shown in Figure 6, demonstrating the feasibility of the present method for the construction of carbohydrate-related compounds.

3.22.4 Combinatorial Synthesis of Glycopeptides Using a Molecular Transporter In the course of our studies of enzymatic synthesis of oligosaccharide on water-soluble polymer supports, our interest has focused on the efficient synthesis of glycopeptides as important signal molecules in cellular recognition, where the variety and specificity of recognition are determined by both the carbohydrate and peptide moieties. To achieve an efficient glycopeptide synthesis with defined carbohydrate and peptide structures, we employed a combinatorial chemical and enzymatic strategy based on solid-phase peptide synthesis and liquid-phase carbohydrate synthesis.53 Our synthetic strategy was as follows (Figure 7): (1) solid-phase synthesis of a photosensitive O-GlcNAc peptide terminated by the molecular transporter 21, (2) deprotection and release of the transporter from the resin, (3) chemoselective blotting of the molecular transporter that carries glycopeptide primers to a water-soluble polymer with alkoxyamino functional groups, (4) one-pot sugar elongation with glycosyltransferases, and (5) release of fulllength glycopeptides by a photosensitive cleavage reaction. The feasibility of the present method is demonstrated by constructing the glycopeptide with a sialyl LewisX tetrasaccharide residue as a model compound. In this strategy, only the full-length glycopeptides successfully elongated by solid-phase synthesis were blotted to polymer 22, thus they did not require purification. After a one-pot sugar elongation reaction by b-1,4-galactosyltransferase, a-2,3-sialyltransferase, and a-1,3-fucosyltransferase, selective

Glycopolymers

HO

OH

HO AcHN

OH

HOOC HO O

O

O

OH

HO

OH

OH HO O

O

O

459

12 HN

OH

18

14

O i

HO

OH

OH HO O

O HO OH

O N H

O

O

O

HN

OH

N H

O

PAAm

OH OH COOH HO AcHN

ii

O HO

O HO O HO OH 19

O

iii

OH

OH

HO O

O

12

HN

OH

O HO

HO AcHN

OH

14

HOOC HO O

OH O

O OH

HO 20

OH

OH

HO O

O OH

O

12 O

HN S O

NMe2 Figure 6 i, CMP-NeuAc (1.2equiv.), bovine serum albumin (BSA), CIAP (20units), 50mMsodium cacodylate buffer (pH 7.40), MnCl2, Triton CF-54, a-2,3-sialyltransferase (0.3units, 8.0milliunits (mU) per 1.0mmol acceptor), 37 C, 72h; C16 ceramide (5.0equiv.), 50mM, sodium citrate buffer (pH 6.0), Triton CF-54, ceramide glycanase from leech (0.005units), 37 C, 17h, 60%. ii, CMP-NeuAc (1.2equiv.), BSA, CIAP, (20units), 50mM sodium cacodylate buffer (pH 7.40), MnCl2, NaN3, a-2,6-sialyltransferase (0.1units, 2.7mU per 1.0mmol acceptor), 37 C, 48h; C16 ceramide (5.0equiv.), 50mM sodium citrate buffer (pH 6.0), Triton CF-54, ceramide glycanase (0.005units), 37 C, 17h, 60%. iii, CMP-NeuAc (1.2equiv. for lactose), BSA, CIAP (20units), 50mM sodium cacodylate buffer (pH 7.40), MnCl2, Triton CF-54, a-2,3-sialyltransferase (0.3units, 8.0mU per 1.0mmol acceptor), 37 C, 72h; D-sphingosine (5.0equiv.), 50mM sodium citrate buffer (pH 6.0), Triton CF-54, ceramide glycanase (0.005units), 37 C, 17h; dansyl chloride (5.0equiv.), Et3N (5.0equiv.), CHCl3, 25 C, 2h, 50%.

cleavage by photoirradiation at 365 nm proceeded smoothly and the target product 23 was obtained in 12% overall yield from the initial solid-phase synthesis. The precise structural characterization of product 23 was carried out by matrix-dependent selective fragmentation in MALDI-TOF/TOF MS in the presence of DHB or a-cyano-4-hydroxycinnamic acid (CHCA) as a matrix.54 Next we employed the synthetic strategy described above to the preparation of MUC1 mucin glycopeptides, important molecules for in vivo cellular regulation.55 In breast, ovarian, and other carcinomas, MUC1 mucin is aberrantly glycosylated in comparison with mucin from corresponding normal tissues.56 Although the characteristics of sugar moieties of MUC1 are interesting in terms of cancer-related immunogenecity, the functional role of the oligosaccharide structure in MUC1 has not been revealed yet because of its complexity and diverse glycosylated patterns. Taking this into consideration, MUC1 glycopeptides with various mucin core carbohydrates are critical in the fulfillment of their essential biological roles.

460

Glycopolymers

(1)

(2)

(3)

(4)

(5)

Target glycopeptides

NO2 O

O O

H N

CO2H N

:Amino acid

:Sugar

:Sugar amino acid

O O

21

OMe

(a) O m n

NH2

Pro-Ser-Val-Pro-Val-Ser-Gly-Ser-Ala-Pro-Gly-Arg

O

N O H 22

NH2 HO

OH O OH HOOC

HO AcHN (b)

O O

O

O

O

OH OH

HO

O

HO

H3C

O

NHAc OH

OH HO

23

Figure 7 Chemical and enzymatic glycopeptide synthesis using a molecular transporter 21. a, (1) Solid-phase glycopeptide synthesis, (2) deprotection and releasing, (3) blotting, (4) sugar elongation on water-soluble polymer, (5) glycopeptide release; b, structure of blotting polymer 22 and Sialyl Lewisx-carrying glycopeptide 23. Reproduced from Tachibana, Y.; Fletcher, G. L.; Fujitani, N.; Tsuda, S.; Monde, K.; Nishimura, S. Angew. Chem., Int. Ed. Engl. 2004, 43, 856–862, with permission from Wiley-VCH Verlag GmbH & Co KG.

Water-soluble polymer

BLase cleavage site O

O Phe-Glu

AA

Glycopeptide

Heterobifunctional linker Figure 8 Heterobifunctional linker sensitive to BLase.

For this purpose, heterobifunctional linkers with a protease-sensitive moiety and a reactive ketone group were designed for accelerating the chemical and enzymatic syntheses of a MUC1 library (Figure 8).57 Dipeptide Phe-Glu is selectively digested by Bacillus licheniformis glutamic acid-specific protease (BLase) at the C-terminus of the glutamic acid. These primer polymers were prepared by solid-phase synthesis. One-pot enzymatic sugar elongation reaction by b-1,4-galactosyltransferase, a-2,3-(O)-sialyltransferase, and a-2,3-(N)sialyltransferase was performed on the primer polymer, and the product was released by BLase to obtain 42 possible kinds of MUC1 glycopeptides (Figure 9). All products were purified by ultrafiltration (UF) separation and high-performance liquid chromatography (HPLC) to yield 1.0–2.0mmol (approximately 200mg to 4mg) glycopeptides. MALDI-TOF/MS analysis supported complete construction of all glycopeptides. The present 2-D synthetic scheme using a versatile ‘polymer blotting’ strategy will greatly accelerate both practical enzymatic synthesis using immobilized glycosyltransferases and functional identification of biologically important glycopeptides.

461

Glycopolymers

R1

R2

R3

O

O

O

Ala–His–Gly–Val–Thr–Ser–Ala–Pro–Asp–Thr–Arg–NH2

GP7–12, 19–24 ; R1 = GP25–36;

R2

OH O

HO

=

OH O

GP1,3; R1 = HO HO

HO

AcNH

AcNH

GP1–6, 37–42; R3 =

GP25–30, 37–24; R1 = H

O

GP7–18; R2 = H HO

O

GP19–24, 31–36; R3 = H

HO AcNH OH O

HO HO

GP13,31; R1 = GP1,19,37; R2 = GP7,25; R3 =

O

HO

OH O

HO

AcNH HO OH O O

HO

HO

GP2,4; R1 =

OH O O HO

HO

O

O

AcNH HO

O

HO AcNH

AcNH

HO

HO

GP14,32; R1 = HO GP2,20,38; R2 = GP8,26; R3 =

OH O

OH O

OH O

O HO

HO HO

O

GP15,33; R1 =

OH

GP3,21,39; R2 =

AcNH HO OH O O

HO

HO HO HO

O

GP9,27; R3 =

HOOC HO

HO AcHN

O

O

HO

HO

GP2,4; R1 = HO

OH O

HO

OH O O HO

HO

GP16,34; R1 = GP4,22,40; R2 = GP10,28; R3 =

O

AcNH HO

O

HO AcNH

OH O

OH

HOOC HO OH O HO O O AcHN HO HO

O HO

O

AcNH HO

GP12,30; R3 = GP18,36; R1 =

OH

HOOC HO HO O O AcHN HO HO OH HO AcHN

OH O

O HO

HO HOOC HO O

OH

HO AcHN

HO

O

HO HOOC O

HO

OH O

O

AcNH HO OH O O

O

O AcNH

HO

O HO

HO HO

OH O

OH O

HO

O AcNH

HO O

AcHN OH HO O O

O

O

AcNH OH HO O O

HO

AcNH

GP6,24,42; R2 =

HO

HO GP17,35; R1 = OH HOOC HO OH GP5,23,41; R2 = O HO O O GP11,29; R3 = AcHN

HO HO

HO

O HO

HO

GP5,6; R1 = HO

OH O

O AcNH

HO

HO

AcNH

O

AcNH OH HO O O

O AcHN

HO

Figure 9 MUC1 glycopeptides synthesized in combinatorial manner.

3.22.5 Multivalent Carbohydrate Presentation on a Peptide Backbone to Control the Topology of Carbohydrates Although many glycopolymers exhibit high affinity for target proteins, their structure is not uniform as they contain polymers of a broad molecular weight. Since the cluster effect can be defined as a strengthening of binding affinity by multipoint interaction, it is possible to obtain a multivalent glycocluster with a low molecular weight by rationally

462

Glycopolymers

designing the ligand for proteins with multiple binding sites. As a novel inhibitor of verotoxin, the STARFISH dendrimer, designed to bind topologically to the toxin, was synthesized using glucose as a scaffold. This dendrimer exhibited 106–107 higher inhibition activity than the monomeric glycoligand.16 We created cyclic peptide scaffolds containing tridendric carbohydrate units as a potential influenza virus hemagglutinin (HA) inhibitor using the chemoenzymatic method.17 HA is a trimeric protein and each subunit contains a sialooligosaccharide binding site located on the apices of an equilateral triangle, in which each binding pocket is separated by approximately 40–50A˚. We designed a cyclic glycopeptide as a novel designer glycocluster that topologically and simultaneously binds the three sugar binding sites of HA (Figure 10). Cyclic peptide is a versatile scaffold for multivalent carbohydrate display, since the molecular size, number, and position of the sugar can be controlled by amino acids. The cyclic peptide sequence was determined so that the

Virus surface

Hemagglutinin

Simultaneous blocking of three binding sits Cyclic glycopeptide

OH

OH HO CO

O

HO

HO

O

O

OH

O

O

O OH

OH

O OH

HO

HO

HO

O

O

HO

OH O

O

OH

HO

O

O

OH O

HOOC OH

O

OH

O

OH

HO

OH OH

OH

OH H

ACN

OC HO OH O HO HO O N AC H H

OH OH NA H c

(a)

O

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(Ser-Gly-Gly-GIn-Ser-His-Asp)3 24 (Gly-Ser-GIn-Ser-Ser-Gly)3 (b)

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Figure 10 a, Design of the HA blocker; b, chemical structure of the cyclic glycopeptide. Reproduced from Niikura, K.; Nagahori, N.; Nishimura, S.-I. Kagaku Frontier 13: Nanobioengineering; Kagaku-dojin: Tokyo, 2004; pp 24–36, with permission from Kagaku-dojin.

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Figure 11 Conformation of cyclic peptide in aqueous solution calculated from NMR spectra. Reproduced from Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C.T.; Suzuki, T.; Suzuki, Y., Monde; K.; Nishimura, S. Angew. Chem., Int. Ed. Engl. 2003, 42, 5186–5189, with permission from Wiley-VCH.

separation between sialic acid residues was 50–70 A˚ based on the molecular modeling study. Cyclic glycopeptides (24 and 25) presenting sialooligosaccharide via a glutamine residue were synthesized (Figure 10b), and the binding of cyclic glycopeptide and HA was evaluated by SPR and hemagglutination inhibition assays. The assays revealed that glycopeptide 24 is a potent inhibitor of HA; however, glycopeptide 25 is not. The 3-D structures of cyclic peptides in solution were calculated by 2-D nuclear Overhauser enhancement spectroscopy (NOESY) NMR, and it was revealed that each glutamic residue of glycopeptide 24 was directed outward from the cyclic peptide ring, while glutamines of glycopeptide 25 were directed inward (Figure 11). Thus, glycopeptide 24 is a tight scaffold that displays multivalent carbohydrates with spatially controlled orientation. These results indicate that controlling the topology of carbohydrates by a tight scaffold is an efficient strategy to obtain a designer glycocluster in which the special arrangement of multivalent sugar ligands is directed to a target protein, offering new insight into the design of a carbohydrate-based drug.

3.22.6 Sequential Glycopeptides with Both Designated Density and Desired Orientation of Carbohydrate Side Chains Among the cell surface glycoconjugates, mucins are very large molecules with a high density of O-linked oligosaccharide chains attached to serine or threonine.58,59 The biological roles of O-glycans might be deeply dependent on the multivalent structures of carbohydrate branches bound to the peptide main chains. It is also known that tandem repeating peptides bearing O-glycans have been found in a variety of mucin-type glycoproteins, and they seem to have crucial roles in cellular adhesion. However, detailed studies on the biological functions of mucins have not been achieved because of difficulties in the preparation of homogeneously pure glycoproteins that have a strictly defined primary structure. Here we will describe the efficient methodology for the construction of mucin-like glycoprotein mimics of high molecular weights with sequential (tandem repeating) structures.18,60,61 Practical synthesis of mucin-type glycoprotein analogs was performed by direct polymerization of unprotected glycopeptide macromers in the presence of diphenylphosphorylazide (DPPA) as a promoter. Figure 12 shows compounds synthesized in the present work, all containing a simple model of antifreeze glycoproteins (AFGPs) as a typical functional mucin (compound 26, syAFGPpoly). AFGPs are essential to the survival of many marine teleost fish that reside in polar and subpolar waters where temperatures decline below the colligative freezing points of their body fluids.62–65 The AFGP isolated from fish blood plasma consist of repeating tripeptide units (Ala-Thr-Ala)n with a disaccharide (Galb1-3GalNAca1-) attached to each threonyl residue.66 The weight-averaged molecular weight of the synthesized glycoproteins was estimated to range from 6600 to 11650Da by gel permeation chromatography. Sialylated glycoprotein 28 was prepared with sialyltransferase. It was suggested that both the periodateresorcinol method (98%) and the integration data of the 1H-NMR spectrum (100%) exhibit a quantitative sialylation

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Figure 12 Structure and antifreeze activity of AFGP and analogs. A, Structure and molecular weight of the compounds. B, Ice crystal morphology in the presence of (a) syAFGPpoly, (b) Galb1-3Gal analog 27, (c) sialylated syAFGPpoly 28, (d) natural AFGP in water (10mgml1). Photos were taken at 0.2 C (a and d) or 0.0 C (b and c). Reproduced from Tachibana, Y.; Fletcher, G. L.; Fujitani, N.; Tsuda, S.; Monde, K.; Nishimura, S. Angew. Chem., Int. Ed. Engl. 2004, 43, 856–862, with permission from Wiley-VCH Verlag GmbH & Co KG.

reaction. This compound as well as enzymatically modified Tn-antigenic mucin mimics67,68 may be valuable tools for investigating the immunological and biological significance of oligosaccharide sequences of O-linked-type glycoproteins. We evaluated the antifreeze activity of these AFGP analogs by measuring the inhibitory effect on ice crystal growth. It is known that the natural AFGPs generate freezing point depression by adsorption to ice surfaces and consequent inhibition of ice crystal growth. The formation of the characteristic bipyramidal ice crystals is one of the most important steps to inhibit the growth of ice crystals. Figure 12B clearly shows that both syAFGPpoly 26 (a) and natural AFGP (d) induce the formation of the typical hexagonal bipyramidal ice crystals over a certain range of temperatures (approximately 0 to 0.5 C). On the other hand, compound 27 (b) exhibited no significant inhibitory effects and sialylated glycoprotein 28 (c) showed no capacity to form the bipyramidal crystal. To determine the minimun number of tripeptide repeats necessary for antifreeze activity, low molecular weight syAFGPs were prepared in a similar manner to syAFGPpoly, but at a lower temperature and with a shorter reaction time. These syAFGPs were then separated into homogeneous fractions of precise chain lengths. The relationship between ice crystal morphology and the chain length of syAFGPs is presented in Figure 13. Although high concentrations of monomer syAFGP1 exhibited no thermal hysteresis, it did produce hexagonal ice crystals (Figure 13B, a). This strongly suggests that the glycopeptide monomer is capable of interaction with ice.

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OH HO OH O HO

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Figure 13 Relationship between repeating number of AFGP and antifreeze activity. A, Structure of syAFGPs. B, The ice crystal morphologies in the presence of (a) syAFGP1 (40mgml1), (b) syAFGP2 (40mgml1), (c) syAFGP3 (10mgml1), (d) syAFGP4 (10mgml1), (e) syAFGP5 (10mgml1), (f) syAFGP6 (10mgml1), (g) syAFGP7 (10mgml1) in water were observed. Photos were taken at 0.1 C (b–g) or 0.0 C (a). Reproduced from Tachibana, Y.; Fletcher, G. L.; Fujitani, N.; Tsuda, S.; Monde, K.; Nishimura, S. Angew. Chem., Int. Ed. Engl. 2004, 43, 856–862, with permission from Wiley-VCH.

All of the tripeptide syAFGP polymers (n¼2–7) displayed concentration-dependent thermal hysteresis and produced hexagonal bipyramidal ice crystals. In addition, there was a positive correlation between thermal hysteresis and chain length between two and five tripeptide repeating units. There was no increase in thermal hysteresis activity when the chain length increased from five to seven repeats, suggesting that the antifreeze activity of the syAFGP was maximal at five repeats. These results clearly demonstrate that a two-tripeptide syAFGP2 is sufficient to control ice crystal growth. A series of several AFGP analogs were synthesized to further identify the structural motifs that are essential for antifreeze activity, and activity was measured by the Clifton nanoliter osmometer and by examining ice crystal morphology. We also measured CD spectra of the synthetic glycoproteins in aqueous solution to study the relationship between the activity and the conformation of the glycoproteins. These results clearly concluded that the antifreeze activity of AFGP is derived from its intrinsic conformation. The 3-D structure of active syAFGP3 in aqueous solution was determined by NMR using a combination of NOESY and correlation spectroscopy (COSY). The result showed that the peptide backbone folds into a left-handed helix in which three disaccharide moieties are on the same side of the molecules, constructing a hydrophilic face. Ala-CH3 groups and acetyl methyl groups in GalNAc were clustered to form a hydrophobic face. It was surmised that it is likely that antifreeze activity of AFGP strongly depends on the presence of an amphiphatic ordered helix similar to PPII. Further investigations to elucidate the mechanism of action and to examine the potential use of AFGPs in clinical applications are under way.

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3.22.7 Multivalent Enzyme Inhibitor Neuraminidases are found in many pathogens such as viruses, bacteria, and parasites. In some cases, neuraminidases act as a virulence factors.69 Thus, the inhibitor of virus neuraminidase is regarded as a potential agent with antiviral and antibacterial activities. It has been reported that several polymer-based inhibitors of influenza virus are multivalent in neuraminic acid A (NeuAc).8,35,70,71 The synthetic random co-polymers bearing NeuAc via C-glycoside linkage have been shown to inhibit the agglutination of erythrocyte by influenza viruses in vitro.72 For the synthesis of Cglycoside polymer, an enzymatic approach was reported by Linhardt.73 An NeuAc monomer with phenolic aglycon was polymerized with soybean peroxidase to afford a molecular weight of 20000, which is similar to MG2 mucin (MUC7). MG2 contains relatively homogeneous sialylated disaccharides and trisaccharides and binds to a wide variety of microorganisms. The resulting C-glycoside polymer was investigated for the inhibitory effect against neuraminidase from Clostridium perfringens by using 4MU-labeled fluorescent substrate to give a Ki value of 900nM based on the polymer concentration. The observed Ki was over 10-fold lower than the monomeric C-glycoside of NeuAc. Among bacterial neuraminidases, some are known to have one or more carbohydrate-binding domains in addition to a catalytic domain.74,75 The neuraminidases with additional lectin domains were revealed to hydrolyze multivalent substrates with much greater efficiency than the monovalent substrate by Boons et al.76 The multivalent substrate exhibited 100-fold smaller Km than the monovalent derivative. It seems that the catalytic and lectin domains interact simultaneously with the multivalent substrates, leading to the affinity enhancement. Furthermore, the catalytic activity of neuraminidases was effectively inhibited by the multivalent polymer displaying galactose, a ligand for the lectin domain. The Ki of the galactoside polymer against neuraminidase from Vibrio cholerae is 50mM, which is 100-fold smaller than that of monovalent D-galactose. The polymer did not inhibit the neuraminidase from Salmonella typhimurium, which does not have a lectin domain. This new concept of inhibitor that targets lection domains of the enzyme has a great potential to develop a specific inhibitor for bacterial neuraminidase.

3.22.8 Glycopolymer Regulating Cellular Responses Some examples of the use of multivalent glycopolymers to regulate cellular responses will be described below. Many cell surface receptors are known to form dimeric or oligomeric complexes.77 Kiessling et al. demonstrated that the inter-receptor communication within a lattice of receptors on cell surfaces amplify the sensory information in chemotactic bacteria by using multivalent glycopolymer.78 The galactose-bearing polymer was synthesized by ringopening metathesis polymerization (ROMP), which allows the generation of multivalent ligands with distinct valencies. The motion of bacteria in response to the chemotactics was analyzed. It was revealed that the galactose polymer (approximately 25 monomer units) was 100- to 1000-fold more potent a chemoattractant than the monovalent galactose derivative. It seemed that the multivalent ligand mediated the signal amplification by stabilizing the cluster of chemoreceptors on cell surfaces. A unique application of glycopolymer is a carbohydrate–antioxidant hybrid polymer that reduces oxidative damage in spermatozoa and enhances fertility. In this strategy, the cell surface CBP was exploited to deliver antioxidant to the spermatozoa. Among the specific CBPs found on spermatozoa, a galactose-binding protein (GBP) has similarity to the hepatic asialoglycoprotein receptor (AGPR). AGPR allows hepatocytes to internalize Gal-bearing macromolecules through receptor-mediated endocytosis, suggesting that GBP has endocytotic potential. The hybrid polymers containing antioxidant a-tocopherol by enzyme-labile aryl ester linkage, a sperm-targeting galactose as a ligand for GBP, and a solubility-enhancing group were constructed. When the porcine sperm was treated with hybrid polymer, it caused large increase of intracellular level of a-tocopherol and greatly reduced endogenous fatty acid degradation under oxidation stress. It was shown that the hybrid polymer was transferred through GBP into the spermatozoa to release antioxidant, which resulted in enhanced physiological properties, longer half-lives, and fertilization rates. These results demonstrated that the synthetic multivalent carbohydrate ligands could serve as modulators of cellular functions by targeting the cell surface receptors.

3.22.9 Multivalent Glycopolymer Nanoparticles for the Detection of Molecular Recognition It is known that the glycolipids on cell surfaces are assembled with signaling molecules and cholesterol to form dense patches called microdomains, through which various recognition events occur. As a cell-surface model, liposomes composed of phospholipids and glycolipids are widely used. We utilized multivalent polymerized fluorescent

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glycolipid liposomes for the detection of carbohydrate-recognition events (Figure 14).79 Glycolipid mimetics with a diacetylene moiety that can be photopolymerized by UV irradiation was synthesized. Polymerized diacetylene has a long-term conjugated system and exhibits UV absorbance at a visible range and fluorescence emission.80 Carbohydrate-displaying fluorescent nanoparticles were prepared by photopolymerization of the vesicles composed of diacetylene glycolipid and phospholipids. The fluorescence intensity increased with UV irradiation (254nm) and became nearly saturated in 1h, generating spectra with a maximum fluorescence emission at 460nm upon emission at 380nm. The nanoparticle was visualized by scanning electron microscope (SEM), which revealed a particle diameter of around 250nm. The binding of CBPs to the carbohydrate-displaying fluorescent nanoparticle was evaluated by measuring the changes in fluorescence intensity upon addition of a protein solution. The binding of E-selectin to sialyl Lewis X (sLex)-displaying nanoparticle was tested (Figure 15). The intensity of the fluorescence from nanoparticles decreased with an increase in E-selectin concentration, whereas no fluorescence change was induced for the nanoparticle composed of phosphatidylcholine-type lipid alone. For the binding of ConA, a mannopentaosedisplaying nanoparticle was prepared. Decreases in fluorescence intensity were also observed with the addition of ConA to a nanoparticle solution. Next the enzymatic hydrolysis reaction by mannosidase was monitored. Unlike protein binding to the particle, when mannosidase was added to a solution of mannopentaose-displaying nanoparticles, the intensity of fluorescence increased with time. Another experiment (data not shown) revealed that the conformational change in the ene-yne backbone of the nanoparticle upon addition of protein became sensitive

Figure 14 The use of fluorescence to detect protein binding to the carbohydrate-displaying nanoparticle. Reproduced from Nagahori, N.; Nishimura, S.-I. Glyconanotechnology; Yuasa, H., Ed.; Nanotechnology in Carbohydrate Chemistry; Transworld Research Network: Trivandrum, India, 2006; pp 149–166, with permission from Transworld Research Network.

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Figure 15 Florescence detection of the binding of E-selectin to sLex-displaying nanoparticle. Reproduced from Niikura, K.; Osuga, N.; Nagahori, N.; Sadamoto, R.; Shiono, M.; Iwasaki, N.; Monde, K.; Minami, A.; Nishimura, S.-I. Polym. J. 2004, 36, 209–218, with permission form The Society of Polymer Science, Japan.

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when the flexibility of the nanoparticle increased by controlling the degree of polymerization or by introducing flexible lipid dioleoyl L-(a)-phosphatidylcholine (DOPC). Therefore, observed changes in fluorescence are speculated to result from changes in the stress on the diacetylene network in the nanoparticle. The stress was increased with the binding of protein and was decreased by releasing sugars from the particle surface, resulting in respective negative or positive changes in fluorescence. A simple solution-phase sensing system using polymerized glycolipid vesicles does not require special equipment such as surface plasmon resonance (SPR) or MS spectrometry, and thus will be useful in the laboratory for the detection of carbohydrate-recognizing proteins and enzymes.

3.22.10 Two-Dimensional Glycolipid Polymers for the Multivalent Presentation of Proteins As mentioned above, a multivalent glycoliposome prepared with diacetylene-containing glycolipids can be regarded as a model system for molecular recognition on cell surfaces. To understand the mechanism of the formation of a microdomain on cell surfaces and the effect of clustering (assembly) of glycoconjugates, several studies have been performed using the glycolipid monolayers as a model membrane. Artificial lipid membranes, such as monolayers comprised of glycolipids and phospholipids, have suggested that the glycolipid domain with a nanometer scale is formed due to phase separation. Other examples showed that the intensity of the interaction of the glycolipid monolayer with CBPs is strongly dependent on the carbohydrate density in the monolayer, implying the significance of carbohydrate clustering in nature.81 Here, we will describe the preparation of multivalent 2-D glycolipid films and the use of the films as scaffolds to display CBPs in a variety of ordered arrangements.82 Diacetylene-containing glycolipids can be photopolymerized and thus are suitable to prevent undesired lipid movement and to obtain a stable platform. CBPs were assembled on the glycolipid membrane according to the procedure shown in Figure 16. The mixed glycolipid at air–water interface was compressed to form a monolayer and photopolymerized by UV irradiation at 254nm. As polymerization proceeds, the absorbance of the glycolipid films (400–700nm) increased. Then the CBP solution was injected into the subphase to bind to the carbohydrate head of the glycolipid monolayer. The CBPbound monolayer was transferred onto a hydrophobic glass plate. The surface of the CBPs deposited on the glycolipid films was observed by atomic force microscopy (AFM) to show various submicron-sized protein patterns depending on the CBP, glycolipid and lipid structure, and sugar density in the films (Figure 17). Cross-section analysis of the protein array revealed uniform assembly of CBP with a height of 10nm, which corresponds to the diameter of a single CBP molecule. To confirm that the proteins assembled on glycolipid film retained their activity, the binding of glycopolymers bearing lactose residues to peanut lectin (PNA) immobilized on galactolipid film was evaluated by SPR. The binding of the glycopolymer to the PNA array was observed, where Kd was estimated to be 106 M. The interaction between the glycopolymer and the PNA array was inhibited by lactose in the reaction mixture, revealing that the binding was specific and that the immobilized PNA retained its sugar-binding activity. To immobilized proteins on the solid support surface, a random nonspecific covalent approach using conventional chemical coupling reagents or nonspecific noncovalent attachment through physical adsorption including electrostatic, hydrophobic, and van der Waals interactions was widely employed. However, these methods carry the possibility of protein

Surface observation by AFM

(1)

(2)

(3)

Hydrophobic glass plate Figure 16 Protein immobilization on the glycolipid film. (1) Photopolymerization of the glycolipid monolayer; (2) injection of protein for binding to the monolayer; (3) transfer of the monolayer bound with protein to a hydrophobic glass plate. Reproduced from Nagahori, N.; Niikura, K.; Sadamoto, R.; Monde, K.; Nishimura, S.-I. Aust. J. Chem. 2003, 56, 567–576, with permission from CSIRO Publishing.

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30 nm

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Figure 17 AFM images of the proteins displayed on glycolipid LB films. a, L-selectin-IgG on sulfatide-containing LB film; b, MBP on maltotriose-containing LB film; c, an expanded image of (a); d, section analysis of the image (c) at gray line. Cross-section analysis of the protein array showed uniform assembly of protein. Reproduced with permission from Nagahori, N.; Niikura, K.; Sadamoto, R.; Monde, K.; Nishimura, S.-I. Aust. J. Chem. 2003, 56, 567–576 and Nagahori, N.; Nishimura, S.-I. Glyconanotechnology; Yuasa, H., Ed.; Nanotechnology in Carbohydrate Chemistry; Transworld Research Network: Trivandrum, India, 2006; pp 149–166.

denaturation and loss of protein activity and defined orientation.83 Our procedure provides a mild site-specific noncovalent attachment of protein through carbohydrate–protein interaction in which the activity of proteins is maintained and the direction of the active sites of the protein is well controlled. The proteins bound on the glycolipid Langmuir–Blodgett (LB) films assembled to form a protein monolayer with a variety of ordered patterns. This approach may lead to the development of a novel class of biosensors and cell-adhesive substrates that can signal biological messages to the living cells.

3.22.11 Using a Two-Dimensional Glycopolymer as a Sensing Device After CBPs were successfully immobilized on a solid support in their active form, we next tried to utilize the arrayed proteins.84 Real-time monitoring of glycosyltransferase activity is desirable for understanding the mechanism of carbohydrate-related biological functions, since glycosyltransferases are key enzymes for the biosynthesis of glycoconjugates. In addition, a high-throughput assay is also required for screening for novel inhibitors and modulators of glycosyltransferases among combinatorial libraries in the pharmaceutical industry. Thus we applied the protein immobilization process to the preparation of a highly-oriented glycosyltransferase microarray on an SPR biosensor chip to monitor the sugar elongation reactions catalyzed by an enzyme array displayed on the surface of glycolipid LB thin films. In this experiment, galactosyltransferase expressed as a fusion protein with maltose-binding protein (MBP-GalT) was immobilized on glycolipid LB film by the carbohydrate-recognizing function of MBP. Figure 18 details the experiment. A mixed LB membrane of maltotriose-carrying diacetylene lipid 29 and phospholipid 30 was prepared on an SPR sensor chip, and then MBP-GalT was immobilized via maltotriose–MBP specific interaction. Here, it is expected that the catalytic sites of enzymes were displayed with ordered orientation by site-specific immobilization. MPP-GalT immobilized on a gold sensor chip was employed for further evaluation of activity by SPR with the addition of solutions containing glycosyl donor (UDP-Gal) and/or acceptor substrates. Glycopolymer 31 was used as an acceptor substrate because the cluster effect makes it a good substrate for glycosyltransferases.48,49,51,52 It is also expected that the signal intensity of SPR is amplified with a polymer-type substrate. Figure 19 (solid line) shows a typical SPR sensorgram demonstrating the galactose transfer reaction from UDP-Gal to acceptor polymer 31 catalyzed by MBP-GalT on a sensor chip. When polymer acceptor 31 was injected in the presence of 0.5 mM UDP-Gal at the point indicated by A, a large sigmoidal response (2300 RU) was recorded (solid line). The value of 2300 RU seems to be reasonable when it is assumed that a single layer of the polymer covers the sensor surface. On the other hand, no response was

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Figure 18 (a) Detection of the sugar elongation reaction by MBP-GalT immobilized on SPR sensor chip. (b) Chemical structure of photopolymerizable lipids and acceptor substrates. Reproduced from Niikura, K.; Nagahori, N.; Nishimura, S.-I. Kagaku Frontier 13: Nanobioengineering; Kagaku-dojin: Tokyo, 2004; pp 24–36, with permission from Kagaku-dojin.

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Time (s) Figure 19 SPR sensogram upon addition of glycosyl donor and/or acceptor substrates. A, Injection of the substrate mixture. B, Washing the flow cell with buffer solution. Reproduced from Nagahori, N.; Niikura, K.; Sadamoto, R.; Taniguchi, M.; Yamagishi, A.; Monde, K.; Nishimura, S.I. Adv. Synth. Catal. 2003, 345, 729–734, with permission from Wiley-VCH, STM.

detected (dashed line). When monomeric acceptor 32 was injected with UDP-Gal, negligibly low affinity was detected (dashed-dot line). These results correspond with our previous observation that acceptor sugar residues highly branched on the water-soluble polymers become excellent substrates of glycosyltransferase due to a polymeric cluster effect. The large sigmoidal response of SPR found in the presence of both UDP-Gal and polymeric acceptor 31

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suggests that activation of GalT by the predominant binding of UDP-Gal may be an essential step for successful galactose transfer to the acceptor substrate. X-ray crystallographic analysis of the complex of GalT with UDP-Gal also shows a significant conformational change, and this crucial step may be necessary for creating a properly arranged acceptor-binding pocket.85 The simple monitoring method described here will be applied for other glycosyltransferase-MBP fusion proteins.50 The glycosyltransferase microarray chips may become a versatile tool both for investigating the mechanism of enzymatic sugar elongation reactions and for searching for novel inhibitors from synthetic libraries of non-natural sugar nucleotides and related compounds.

3.22.12 Protein Glycomics by Chemoselective Glycoblotting on a Polymer Nanoparticle Protein glycosylation involves a highly dynamic process in which rapid changes in the carbohydrate structure occur in response to cellular signals or cellular stages, resulting in key informational markers of some serious human diseases.58,86 For example, the glycosylation patterns on IgG changes in rheumatoid arthritis87 and specific carbohydrates are used as tumor-associated markers in pancreatic and colon cancers.88 Although there have been substantial advances in our understanding of the effects of glycosylation patterns on some biological systems, we still do not fully understand the specific functional roles of carbohydrates and the relationship between their structures and functions. The major difficulty in carbohydrate sequencing is that the purification of trace amounts of oligosaccharides requires extremely tedious multistep processes, as crude sample mixtures prepared by enzymatic digestion from cells, organisms, serum, etc. usually contain large amounts of impurities such as peptides, lipids, and salts. These technical problems in the sequencing of carbohydrates make it impossible to achieve high-throughput protein glycomics. Thus our attention was focused on the development of an efficient method of oligosaccharide purification and analysis. Once oligosaccharides are released from glycoconjugates, they present an aldehyde or ketone group at the reducing terminal that no biomolecules other than carbohydrates contain.89 This unique chemical characteristic of sugars encouraged us to design a novel polymer nanoparticle for capturing carbohydrates from crude samples on the basis of the chemoselective ligation strategy. We have developed several polymer reagents with reactive and stable oxylamino functional groups that preferentially react with an aldehyde or ketone group at the reducing terminal of carbohydrates.90 A liposome composed of diacetylene-containing lipid derivative 33 and phospholipid derivatives can be readily polymerized by UV irradiation to produce a polymer-based nanoparticle with a diameter of 200–300nm displaying an oxylamino functional group (Figure 20). The feasibility of the polymer as a platform for the ‘trap and release’ of the target oligosaccharides was then evaluated. Figure 21 shows the general protocol for the isolation and analysis of carbohydrates by chemoselective glycoblotting: (1) trapping carbohydrates by mixing nanoparticles with an unpurified proteolytic digest; (2) collection of particles by spin filtration from a crude mixture of peptides, salts, and other impurities; and (3) release and subsequent MALDI-TOF MS analysis of the target carbohydrates. The structural characterization of human IgG N-glycans was carried out using crude IgG samples obtained from human serum (100ml) to test the efficiency of protein glycomics by the present strategy. Then the crude digest was directly subjected to MALDI-TOF MS analysis, and only an extremely complex spectrum was obtained (Figure 22a). On the other hand, the MALDI-TOF MS spectrum of N-glycans of human IgG trapped and released by glycoblotting strategy was remarkably improved (Figure 22b). Sensitive and singly charged precursor on peaks

O O H2N

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Figure 20 Preparation and reaction of polymer nanoparticle. Reproduced from Nagahori, N.; Nishimura, S.-I. Glyconanotechnology; Yuasa, H., Ed.; Nanotechnology in Carbohydrate Chemistry; Transworld Research Network: Trivandrum, India, 2006; pp 149–166, with permission from Transworld Research Network.

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Figure 21 General protocol for chemoselective glycoblotting and MALDI-TOF mass analysis of carbohydrates. Reproduced from Nishimura, S.; Niikura, K.; Kurogochi, M.; Matsushita, T.; Fumoto, M.; Hinou, H.; Kamitani, R.; Nakagawa, H.; Deguchi, K.; Miura, N. Monde, K.; Kondo, H. Angew. Chem., Int. Ed. 2005, 44, 91–96, with permission from Wiley-VCH.

generated in the presence of CHCA was subsequently subjected to LIFT-TOF/TOF analysis for sequencing oligosaccharide structures. Fragmentation patterns produced by analyzing m/z 1486 were identified with the known pyridyl-aminated oligosaccharide. This result indicates that carbohydrate mass fingerprinting (CMF) analysis by data fitting with the mass databases of known oligosaccharide allows precise structural identification of oligosaccharides captured by glycoblotting strategy. The combined use of glycoblotting and high-performance MALDI-TOF/TOF analysis provides a new promising strategy for early diagnosis and tailored treatment of a variety of diseases, as well as high-throughput protein glycomics.

3.22.13 Summary Biomolecular recognition of glycoconjugates in a natural biological system is regulated by the spatial and topological arrangement of the recognition elements at both the molecular and the supramolecular cellular level. Thus, it is

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m/z

Figure 22 MALDI-TOF analysis of human IgG N-glycans. a, MALDI-TOF mass spectrum of crude N-glycans before glycoblotting; b, major ion peaks of N-glycans obtained by glycoblotting. Reproduced from Nishimura, S.; Niikura, K.; Kurogochi, M.; Matsushita, T.; Fumoto, M.; Hinou, H.; Kamitani, R.; Nakagawa, H.; Deguchi, K.; Miura, N.; Monde, K.; Kondo, H. Angew. Chem., Int. Ed. 2005, 44, 91–96, with permission from Wiley-VCH.

indispensable to decipher and reassemble the 3-D topology of carbohydrates to develop the multivalent glycocluster as a biomaterial and a carbohydrate-based drug that controls biological function. Multivalent presentation is a key feature of glycoclusers described in this chapter, which allows these materials to serve as cell surface mimics to understand and manipulate carbohydrate-protein interactions. Various water-soluble glycopolymers with high affinity toward partner CBPs were prepared. The affinity of the glycopolymers with CBPs was found to be density dependent. To control the topology of glycoligands in the polymer network, molecular imprinting was employed. The multivalent water-soluble glycopolymers become good acceptor substrates of glycosyltransferases as well as exhibit high affinity to proteins. Highly efficient enzyme-assisted carbohydrate synthesis was achieved by using the glycopolymers as a molecular platform for the carbohydrate assembly. By combining enzyme-assisted carbohydrate synthesis and solidphase peptide synthesis, the rapid construction of glycopeptide libraries was demonstrated. It was also shown that cyclic and sequential polypeptide backbone served as a scaffold for multivalent presentation of glycoligands with a defined 3-D topology. The supramolecular assemblies of glycolipid mimetics were prepared on a solid support and a nanoparticle as a biomembrane model. These model membranes were applied as devices for detecting molecular recognition and enzymatic activity, in which multivalent carbohydrate presentation plays an important role. Finally, we prepared the polymer nanoparticle that can selectively capture carbohydrate biological samples to achieve highthroughput carbohydrate sequencing.

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Glossary antifreeze glycoprotein (AFGP) Is essential to the survival of many marine teleost fish that reside in polar and subpolar waters where temperatures decline below the colligative freezing points of their body fluids. atomic force microscope (AFM) One of scanning probe microscope. Cantilever (probe) scans the surface of samples to detect the van der Waals force between the tip and sample. hemagglutinin (HA) Found on the surface of influenza virus and is responsible for the binding of virus to the host cells. HA is a trimeric protein and each subunit contains a sialooligosaccharide binding site. Langmuir-Blodgett film (LB film) The film what the monolayer on the surface of aqueous solution is transferred onto the solid plate by vertically dipping the plate passing through the water surface. monolayer A layer with one molecular thickness of amphiphilic compound on air-water interface. surface plasmon resonance (SPR) Surface electromagnetic waves that propagate parallel along a metal/dielectric interface, which is utilized to detect the molecular interaction between immobilized ligand on the sensor chip and injected analyte.

References 1. Graves, B. J.; Crowther, R. L.; Chandran, C.; Rumberger, J. M.; Li, S.; Huang, K. S.; Presky, D. H.; Familletti, P. C.; Wolitzky, B. A.; Burns, D. K. Nature 1994, 367, 532–538. 2. Lee, Y. C.; Townsend, R. R.; Hardy, M. R.; Lonngren, J.; Arnarp, J.; Haraldsson, M.; Lonn, H. J. Biol. Chem. 1983, 258, 199–202. 3. Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321–327. 4. Lee, R. T.; Lee, Y. C. Glycoconj. J. 2000, 17, 543–551. 5. Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102, 555–578. 6. Deng, S. J.; MacKenzie, C. R.; Hirama, T.; Brousseau, R.; Lowary, T. L.; Young, N. M.; Bundle, D. R.; Narang, S. A. Proc. Natl. Acad. Sci. USA 1995, 92, 4992–4996. 7. Ladmiral, V.; Melia, E.; Haddleton, D. M. Eur. Polym. J. 2004, 40, 431–449. 8. Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754–2794. 9. Bovin, N. V. Glycoconj. J. 1998, 15, 431–446. 10. Roy, R. TIGG 1996, 8, 79–99. 11. Andre, S.; Kaltner, H.; Furuike, T.; Nishimura, S.; Gabius, H. J. Bioconj. Chem. 2004, 15, 87–98. 12. Dondoni, A.; Kleban, M.; Hu, X.; Marra, A.; Banks, H. D. J. Org. Chem. 2002, 67, 4722–4733. 13. Connolly, D. T.; Townsend, R. R.; Kawaguchi, K.; Hobish, M. K.; Bell, W. R.; Lee, Y. C. Biochem. J. 1983, 214, 421–431. 14. Schulman, S.; Lee, Y. C.; Vanderberg, J. P. J. Parasitol. 1984, 70, 213–216. 15. Nagahori, N.; Lee, R. T.; Nishimura, S.; Page, D.; Roy, R.; Lee, Y. C. Chem. Bio. Chem. 2002, 3, 836–844. 16. Kitov, P. I.; Sadowska, J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.; Pannu, N. S.; Read, R. J.; Bundle, D. R. Nature 2000, 403, 669–672. 17. Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C. T.; Suzuki, T.; Suzuki, Y.; Monde, K.; Nishimura, S. Angew. Chem., Int. Ed. Engl. 2003, 42, 5186–5189. 18. Tachibana, Y.; Matsubara, N.; Nakajima, F.; Tsuda, T.; Tsuda, S.; Monde, K.; Nishimura, S.-I. Tetrahedron 2002, 58, 10213–10224. 19. Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293–346. 20. de La Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Canada, J.; Fernandez, A.; Penades, S. Angew. Chem., Int. Ed. 2001, 40, 2257–2261. 21. Nagahori, N.; Nishimura, S.-I. Chem. Eur. J. 2006, 12, 6478–6485. 22. Badjic, J. D.; Nelson, A.; Cantrill, S. J.; Turnbull, W. B.; Stoddart, J. F. Acc. Chem. Res. 2005, 38, 723–732. 23. Rojo, J.; Delgado, R. J. Antimicrob. Chemother. 2004, 54, 579–581. 24. Mulder, A.; Huskens, J.; Reinhoudt, D. N. Org. Biomol. Chem. 2004, 2, 3409–3424. 25. Bovin, N. V.; Tuzikov, A. B.; Chinarev, A. A.; Gambaryan, A. S. Glycoconj. J. 2004, 21, 471–478. 26. Schengrund, C. L. Biochem. Pharmacol. 2003, 65, 699–707. 27. Wang, Q.; Dordick, J. S.; Linhardt, R. J. Chem. Mater. 2002, 14, 3232–3244. 28. Flitsch, S. L. Curr. Opin. Chem. Biol. 2000, 4, 619–25. 29. Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71–77. 30. Lee, Y. C. FASEB J. 1992, 6, 3193–3200. 31. Horejsi, V.; Smolek, P.; Kocourek, J. Biochim. Biophys. Acta 1978, 538, 293–298. 32. Roy, R.; Tropper, F. D.; Romanowska, A. Bioconj. Chem. 1992, 3, 256–261. 33. Roy, R.; Park, W. K. C.; Srivastava, O. P.; Foxall, C. Bioorg. Med. Chem. Lett. 1996, 6, 1399–1402. 34. Cao, S. D.; Roy, R. Tetrahedron Lett. 1996, 37, 3421–3424. 35. Choi, S. K.; Mammen, M.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 4103–4111. 36. Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 3789–3900. 37. Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Med. Chem. 1995, 38, 4179–4190. 38. Lees, W. J.; Spaltenstein, A.; Kingery-Wood, J. E.; Whitesides, G. M. J. Med. Chem. 1994, 37, 3419–3433. 39. Nishimura, S.-I.; Matsuoka, K.; Kurita, K. Macromolecules 1990, 23, 4182–4184. 40. Nishimura, S.-I.; Matsuoka, K.; Furuike, T.; Nagami, K.; Ishii, S.; Kurita, K.; Nishimura, K. M. Macromolecules 1991, 24, 4236–4241. 41. Nishimura, S.-I.; Furuike, T.; Matsuoka, K.; Maruyama, K. Macromolecules 1994, 27, 4876–4880. 42. Nishimura, S.-I.; Matsuoka, K.; Furuike, T.; Nishi, N.; Tokura, S.; Nagami, K.; Murayama, S.; Kurita, K. Macromolecules 1994, 27, 157–163. 43. Matsuoka, K.; Nishimura, S.-I. Macromolecules 1995, 28, 2961–2968. 44. Furuike, T.; Aiba, S.; Suzuki, T.; Takahashi, T.; Suzuki, Y.; Yamada, K.; Nishimura, S.-I. J. Chem. Soc., Perkin Trans. 1 2000, 3000–3005. 45. Gambaryan, A. S.; Boravleva, E. Y.; Matrosovich, T. Y.; Matrosovich, M. N.; Klenk, H. D.; Moiseeva, E. V.; Tuzikov, A. B.; Chinarev, A. A.; Pazynina, G. V.; Bovin, N. V. Antiviral Res. 2005, 68, 116–123. 46. Nagahori, N.; Nishimura, S. Biomacromolecules 2001, 2, 22–24.

Glycopolymers

47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92.

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Nishimura, S.; Lee, K. B.; Matsuoka, K.; Lee, Y. C. Biochem. Biophys. Res. Commun. 1994, 199, 249–254. Yamada, K.; Fujita, E.; Nishimura, S.-I. Carbohydr. Res. 1997, 305, 443–461. Yamada, K.; Nishimura, S.-I. Tetrahedron Lett. 1995, 36, 9493–9496. Toda, A.; Yamada, K.; Nishimura, S.-I. Adv. Synth. Catal. 2002, 344, 61–69. Nishimura, S.-I.; Yamada, K. J. Am. Chem. Soc. 1997, 119, 10555–10556. Yamada, K.; Matsumoto, S.; Nishimura, S.-I. Chem. Commun. 1999, 507–508. Fumoto, M.; Hinou, H.; Matsushita, T.; Kurogochi, M.; Ohta, T.; Ito, T.; Yamada, K.; Takimoto, A.; Kondo, H.; Inazu, T.; Nishimura, S. Angew. Chem., Int. Ed. Engl. 2005, 44, 2534–2537. Kurogochi, M.; Nishimura, S. Anal. Chem. 2004, 76, 6097–6101. Hanisch, F. G.; Muller, S. Glycobiology 2000, 10, 439–449. Lloyd, K. O.; Burchell, J.; Kudryashov, V.; Yin, B. W.; Taylor-Papadimitriou, J. J. Biol. Chem. 1996, 271, 33325–33334. Fumoto, M.; Hinou, H.; Ohta, T.; Ito, T.; Yamada, K.; Takimoto, A.; Kondo, H.; Shimizu, H.; Inazu, T.; Nakahara, Y.; Nishimura, S.-I. J. Am. Chem. Soc. 2005, 127, 11804–11818. Dwek, R. A. Chem. Rev. 1996, 96, 683–720. Lowe, J. B. Cell 2001, 104, 809–812. Tsuda, T.; Nishimura, S.-I. Chem. Commun. 1996, 2779. Tachibana, Y.; Fletcher, G. L.; Fujitani, N.; Tsuda, S.; Monde, K.; Nishimura, S. Angew. Chem., Int. Ed. Engl. 2004, 43, 856–862. Fletcher, G. L.; Hew, C. L.; Davies, P. L. Annu. Rev. Physiol. 2001, 63, 359–390. Ewart, K. V.; Lin, Q.; Hew, C. L. Cell. Mol. Life Sci. 1999, 55, 271–283. Harding, M. M.; Anderberg, P. I.; Haymet, A. D. Eur. J. Biochem. 2003, 270, 1381–1392. Yeh, Y.; Feeney, R. E. Chem. Rev. 1996, 96, 601–618. DeVries, A. L.; Komatsu, S. K.; Feeney, R. E. J. Biol. Chem. 1970, 245, 2901–2908. Ikehara, Y.; Kojima, N.; Kurosawa, N.; Kudo, T.; Kono, M.; Nishihara, S.; Issiki, S.; Morozumi, K.; Itzkowitz, S.; Tsuda, T.; Nishimura, S. I.; Tsuji, S.; Narimatsu, H. Glycobiology 1999, 9, 1213–1224. Kono, M.; Tsuda, T.; Ogata, S.; Takashima, S.; Liu, H.; Hamamoto, T.; Itzkowitz, S. H.; Nishimura, S.; Tsuji, S. Biochem. Biophys. Res. Commun. 2000, 272, 94–97. Corfield, T. Glycobiology 1992, 2, 509–521. Reuter, J. D.; Myc, A.; Hayes, M. M.; Gan, Z.; Roy, R.; Qin, D.; Yin, R.; Piehler, L. T.; Esfand, R.; Tomalia, D. A.; Baker, J. R., Jr. Bioconj. Chem. 1999, 10, 271–278. Zanini, D.; Roy, R. J. Am. Chem. Soc. 1997, 119, 2088–2095. Sparks, M. A.; Williams, K. W.; Whitesides, G. M. J. Med. Chem. 1993, 36, 778–783. Wang, Q.; Dordick, J. S.; Linhardt, R. J. Org. Lett. 2003, 5, 1187–1189. Crennell, S.; Garman, E.; Laver, G.; Vimr, E.; Taylor, G. Structure 1994, 2, 535–544. Taylor, G. Curr. Opin. Struct. Biol. 1996, 6, 830–837. Thobhani, S.; Ember, B.; Siriwardena, A.; Boons, G. J. J. Am. Chem. Soc. 2003, 125, 7154–7155. Brewer, C. F.; Miceli, M. C.; Baum, L. G. Curr. Opin. Struct. Biol. 2002, 12, 616–623. Gestwicki, J. E.; Kiessling, L. L. Nature, 2002, 415, 81–84. Niikura, K.; Osuga, N.; Nagahori, N.; Sadamoto, R.; Shiono, M.; Iwasaki, N.; Monde, K.; Minami, A.; Nishimura, S.-I. Polym. J. 2004, 36, 209–218. Charych, D.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113–120. Sato, T.; Serizawa, T.; Okahata, Y. Biochim. Biophys. Acta. 1996, 1285, 14–20. Nagahori, N.; Niikura, K.; Sadamoto, R.; Monde, K.; Nishimura, S.-I. Aust. J. Chem. 2003, 56, 567–576. Tomizaki, K. Y.; Usui, K.; Mihara, H. Chem. Bio. Chem. 2005, 6, 782–799. Nagahori, N.; Niikura, K.; Sadamoto, R.; Taniguchi, M.; Yamagishi, A.; Monde, K.; Nishimura, S. I. Adv. Synth. Catal. 2003, 345, 729–734. Ramakrishnan, B.; Qasba, P. K. J. Mol. Biol. 2001, 310, 205–218. Zachara, N. E.; Hart, G. W. Chem. Rev. 2002, 102, 431–438. Parekh, R. B.; Dwek, R. A.; Sutton, B. J.; Fernandes, D. L.; Leung, A.; Stanworth, D.; Rademacher, T. W.; Mizuochi, T.; Taniguchi, T.; Matsuta, K.; Takeuchi, F.; Nagano, Y.; Miyamoto, T.; Kobata, A. Nature 1985, 316, 452–457. Varki, A. In Essentials of Glycobiology; Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G., Marth, J., Eds.; Cold Spring Harbor Laboratory Press: New York, 1999; pp 537–549. Fischer, E. Ber. Dtsch. Chem. Ges. 1884, 17, 579–584. Nishimura, S.; Niikura, K.; Kurogochi, M.; Matsushita, T.; Fumoto, M.; Hinou, H.; Kamitani, R.; Nakagawa, H.; Deguchi, K.; Miura, N.; Monde, K.; Kondo, H. Angew. Chem., Int. Ed. 2005, 44, 91–96. Nagahori, N.; Nishimura, S.-I. Glyconanotechnology; Yuasa, H., Ed.; Nanotechnology in Carbohydrate Chemistry; Transworld Research Network: Trivandrum, India, 2006; pp 149–166. Niikura, K.; Nagahori, N.; Nishimura, S.-I. Kagaku Frontier 13: Nanobioengineering; Kagaku-dojin: Tokyo, 2004; pp 24–36.

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Biographical Sketch

Noriko Nagahori received Ph.D. in 2002 from Division of Biological Sciences, Graduate School of Science, Hokkaido University, under Professor Shin-Ichiro Nishimura on the thesis ‘‘Template effect on carbohydrate-protein interaction.’’ During doctor course, she studied at Biological Department, Johns Hopkins University (Professor Y. C. Lee), as visiting student. After working as postdoctoral fellow, she joined at Sun Microsystems Laboratory of Computational Molecular Science, Hokkaido University (2002–06). She is now a research associate at Shionogi Laboratory of Biomolecular Chemistry, Hokkaido University. She moved to the present position in April 2006.

Shin-Ichiro Nishimura Ph.D. is Professor of the Graduate School of Advanced Life Science in Hokkaido University and Director of the Drug-Seeds Discovery Research Laboratory in Advanced Industrial Science and Technology (AIST). He received his Ph.D. from Hokkaido University in 1987. He is interested in the fields of life science, bioorganic chemistry, glycotechnology, and drug discovery, especially focusing on clarification of structures and functions of glycoconjugates, signification of post-translational modification of genetic code, discovery of disorder marker molecules by the methods of glycoform-focused reverse genomics and development of basic technology for drug discovery and applications to drug development. From 1982 to the present, 220 academic papers and about 53 reviews/book chapters have been published.

3.23 Neoglycoproteins M. Monsigny, A.-C. Roche, E´. Duverger, and O. Srinivas, CNRS & Universite´ d’Orleáns, Orleáns, France ß 2007 Elsevier Ltd. All rights reserved. 3.23.1 Introduction

478

3.23.2 Historical Aspects

478

3.23.3 Neoglycoproteins

479

3.23.3.1 Serum Albumin

479

3.23.3.2 Neoglycoprotein Synthesis

480

3.23.3.2.1

Conjugation to lysine

482

3.23.3.3 Conjugation to Cysteine

490

3.23.3.4 Conjugation to the Aromatic Amino Acids

490

3.23.4 Avidity

490

3.23.4.1 Evidencing Avidity

491

3.23.4.1.1 3.23.4.1.2

Evidencing avidity with antibodies Evidencing avidity with lectins

491 493

3.23.4.2 Affinity and Avidity Determined by Surface Plasmon Resonance 3.23.4.2.1 3.23.4.2.2 3.23.4.2.3 3.23.4.2.4 3.23.4.2.5

Principle of SPR The classical kinetics analysis used in SPR Determination of avidity in SPR Kinetics analysis according to the Sips distribution in order to take into account the neoglycoprotein heterogeneity SPR applied to study lectin/neoglycoprotein interactions

494 495 496 496 496 497

3.23.5 Use of Neoglycoproteins to Evidence Lectins In Situ

499

3.23.5.1 Evidencing Lectins in Animal Cells

499

3.23.5.1.1 3.23.5.1.2 3.23.5.1.3

Evidencing lectins in animal cells with labeled neoglycoproteins Use of glycosylated markers for diagnostics Visualization of endogenous lectins with neoglycoproteins

3.23.5.2 Use of Neoglycoproteins to Detect Nuclear Lectins and Glycoconjugate Nuclear Import 3.23.5.2.1 3.23.5.2.2 3.23.5.2.3

499 500 500

502

Presence of lectins in the cytosol and the nucleus of animal cells Modulation of the nuclear lectin content Sugar-dependent nuclear import of glycosylated proteins

502 503 503

3.23.5.3 Use of Neoglycoproteins to Decipher the Mechanism of the Acrosome Reaction

505

3.23.5.3.1 3.23.5.3.2 3.23.5.3.3

Binding of neoglycoproteins to spermatozoa Acrosome reaction induced by neoglycoproteins containing Man and GlcNAc Acrosome reaction induced by neoglycoproteins containing Lewis oligosaccharides

505 505 506

3.23.6 Drug Targeting

506

3.23.6.1 Uptake of Neoglycoproteins Assessed by Fluorimetry

506

3.23.6.2 Neoglycoproteins as Drug Carriers

506

3.23.6.2.1 3.23.6.2.2 3.23.6.2.3 3.23.6.2.4 3.23.6.2.5

Targeting antitumoral drugs Targeting antiviral drugs Targeting macrophage immunomodulators Targeting antiparasite drugs Targeting oligonucleotides

507 507 508 508 508

3.23.7 Neoglycoproteins as Sugar-Specific Antigens

509

3.23.7.1 Neoglycoproteins as Immune Response Triggers

509

3.23.7.2 Rational Design of Neoglycoprotein-Based Antibacterial Vaccines

510

477

478

Neoglycoproteins

3.23.7.2.1 3.23.7.2.2

3.23.7.3 3.23.8

Proteins used to prepare antigenic neoglycoproteins Oligosaccharide antigenic epitopes

Neoglycoprotein Vaccines for Cancer Have Neoglycoproteins Any Future?

510 511

512 514

3.23.1 Introduction The presence of both lectins and complex carbohydrates in the biological fluids, on the surface as well as inside cells, is well documented (see Refs.: 1 and 2 for reviews). Specific interactions between lectins and complex carbohydrates (glycoproteins, glycolipids, polysaccharides, or proteoglycans) are involved in several basic phenomena such as phagocytosis, endocytosis, intracellular traffic, signal transduction, cell–cell recognition, inflammation processes, cell matrix adhesion, opsonization, cell growth control, cell regulation and differentiation, acrosome reaction, cellular trafficking, cancer cell metastasis, etc. The physicochemical aspects as well as the biological functions of lectin– glycoconjugate interactions depend, to some extent, on the density of the lectins and on the organization of the sugar moieties of glycoconjugates. The recognition of an individual simple sugar by a lectin is usually in a low-affinity range (about 103 lmol1).3 Conversely, complex oligosaccharides or saccharide clusters, as well as neoglycoproteins, bind lectins in a high-affinity range (up to 108 lmol1). Multivalency is a strategy used by both the binding partners to circumvent the intrinsic low affinity of carbohydrate–protein interactions. On the one hand, the carbohydrate binding proteins (lectins) often exist as multimeric and/or possess multiple carbohydrate recognition domains and on the other hand, carbohydrate moieties are often numerous in glycoconjugates. Multivalency leads to the possibility of establishing multiple separate connections resulting in a strong attractive binding force. Numerous reviews and books dealing with the preparation and the use of neoglycoproteins are available.4–14 The reader of this chapter will find in these materials a considerable numbers of references. Various methods are available for constructing multivalent structures. Some of them frequently include conjugation of carbohydrate ligands with proteins or other polymers:9,10,15 these glycoconjugates are called ‘neoglycoproteins’ (glycosylated proteins) and glycopolymers, respectively. These approaches have been successful, although the products are ambiguous in composition and structure. Alternatively, high-affinity ligands of low molecular weight (complex oligosaccharides from natural or synthetic sources and small oligosaccharide clusters such as glycodendrimers16,17 or glycoclusters18 have been developed and can be used as tools to evidence and to study the functions of endogenous lectins as well as devices to target molecules of interest (see Refs.: 9, 10, 19, and 20 for reviews). Such oligosaccharides or glycoclusters may also be bound to a protein, conferring to the neoglycoprotein a very high apparent affinity.

3.23.2 Historical Aspects Avery and Goebel,21 in 1929, prepared neoglycoproteins referred to gluco-globulin, galacto-globulin, gluco-albumin, and galacto-albumin: horse serum globulin and crystalline egg albumin substituted with glucosides and galactosides, respectively. They used those neoglycoproteins as antigens to induce the production of immune sera. They demonstrated that the sera contained two separate types of antibodies: the anticarbohydrate antibodies present in sera prepared with the neoglycoproteins which were specific of the sugar borne by the neoglycoprotein used as immunogen and the antiprotein antibodies. The antiprotein antibodies exhibit specificity linked to the original protein (globulin or albumin) and the antibodies reactive with the conjugated sugar-proteins are specific for unrelated proteins containing the same carbohydrate (galactosides or glucosides). Their results demonstrated that the carbohydrate moiety and not the protein molecule determine the serological specificity of the conjugated antigen.21–23 Then, Goebel and Avery23 prepared a neoglycoprotein based on the capsular polysaccharide (CS) of type 3 Pneumococcus, a polymer of aldobionic acid.24 This work was initiated on the basis that one of the free hydroxyl groups of the polysaccharide could be replaced by a nitrobenzyl group without incurring a loss in specificity of the polysaccharide. The nitroderivative was then reduced to the amino compound which in turn was coupled through its diazonium derivative to a protein, yielding a neoglycoprotein made with ‘complex’ oligosaccharides. Such a conjugate had a single constituent common to the Pneumococcus cell, namely, the CS and the conjugates should behave as a ‘synthetic antigen’, inducing specific

Neoglycoproteins

479

antibodies upon immunization, which share similar specificity as those produced by immunization with intact bacterial cells. Later, Goebel25 showed that a neoglycoprotein containing the repeating unit of this polysaccharide cellobiuronic acid – but not the neoglycoproteins containing cellobioside, glucoside, or glucuronide – gives rise in rabbits to antibodies which are specific and characteristic of the saccharide constituent of the CS of type 3 Pneumococcus. It was concluded that the antiserum to the synthetic antigen containing cellobiuronic acid conveys passive protection on mice to infection with virulent Pneumococcus types 2, 3, and 8. Several years later, Iyer and Goldstein26 prepared neoglycoproteins in order to study, in a quantitative approach, the interaction between glycoconjugates and concanavalin A, a plant lectin specific for mannosides and glucosides. Similarly, Privat and co-workers27 prepared neoglycoproteins bearing chitin oligomers (GlcNAcb(4GlcNAcb-)n with n¼0–3) and showed that those neoglycoproteins interact with another plant lectin: wheat germ agglutinin, inducing the precipitation of the complex in a concentration-dependent manner, analogous to an immunoprecipitation.

3.23.3 Neoglycoproteins Neoglycoproteins have been prepared from various proteins (bovine and human serum albumin (HSA), ribonuclease, ferritin, diphtheria toxin, streptavidin, etc.) as well as glycoproteins (ovalbumin, horse radish peroxidase, serum globulin, etc.). It is obvious that the most interesting neoglycoproteins are those prepared from sugar-free proteins and serum albumin is the most popular protein selected by many researchers to prepare well-defined neoglycoproteins. Serum albumin is highly soluble in neutral or alkaline medium and its molecular weight is large enough (roughly Mr: 67000) to allow a heavy sugar substitution.

3.23.3.1 Serum Albumin The properties of serum albumin are very nicely presented in reviews by Peters,28 and Carter and Ho.29 Mammalian serum albumin contains about 60 lysines (Table 1). The three-dimensional structure of HSA has been determined by crystallography to a resolution of 0.28nm.30,31 It comprises three homologous domains that assemble to form a heartshaped molecule. Each domain is a product of two subdomains that possess common structural motifs (Figures 1 and 2). The principal regions of ligand binding to HSA are located in hydrophobic cavities in the first subdomains II and III, which exhibit similar chemical properties. The structure explains numerous physical phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin. As shown in Figure 3 from the primary sequence, 60 lysines of bovine serum albumin (BSA) are dispersed throughout the molecule. Serum albumin is also very rich in cysteine, as shown in Figure 2 accounting for the exceptional stability of

Table 1 Amino acid composition of BSA272 Ala Gln Leu Ser

48 21 65 32

Arg Glu Lys Thr

26 58 60 34

Asn Gly Met Trp

14 17 5 3

Asp His Phe Tyr

41 16 30 21

Cys Ile Pro Val

35 15 28 38

14 nm

4 nm

IA

IP −10*

IIA

IIB −8*

IIIA

IIIB 0*

* Net charges Figure 1 Schematic structure of serum albumin, adapted from Peters, 1985:28 the molecule is made of three domains (I, II, III) containing 10, 8, and 0 negative net charges, respectively. Each domain is divided into subdomains A and B.

480

Neoglycoproteins

1 *

#

*

25 #

*

40

*

* * *

68

* *

* *

Domain 1

* * *

*

*

* #

* *

#

*

*

* *

Domain 2

295

* *

317

*

*

*

* *

# #

246 *

*

*

* #

*

402

#

*

*

*

*

*

*

*

*

* *

*

514

* *

* * *

438 492

*

Domain 3

#

362

* #

*

*

560 607

99 115 114 125 147 192 191 200

170 210

*

* *

77 86

127

*

*

# #

107

The cysteines

223 269 268 276 288 302 301 312 339 384 383 392 415 461 460 471 484 500 499 510 537 582 581 590

* Each tenth amino acid. # Domains: 1: 25 → 204; 2: 211 → 396; 3: 403 → 594. Lys: 2, 28, 36, 44, 65, 75, 88, 100, 117, 130, 138, 140, 151, 155, 156, 160, 183, 197, 204, 211, 228, 235, 245, 248, 256, 263, 266, 285, 297, 299, 304, 309, 318, 336, 340, 346, 374, 386, 399, 401, 412, 420, 437, 455, 463, 489, 495, 498, 523, 528, 544, 547, 548, 557, 559, 561, 568, 580, 587, 597. Figure 2 Structure of BSA showing both lysine and disulfide bridges present in the three domains (I, II, III), according to Peters.28

the mature protein. The albumin molecule is not uniformly charged within the primary structure. At neutral pH, Peters28 calculated a net charge of 10, 8, and 0 for domains, I, II, and III for BSA (Figure 1). Interestingly, the lysines are well dispersed on the surface of the protein as shown in Figure 4.

3.23.3.2 Neoglycoprotein Synthesis The coupling of saccharide residues to the surface of proteins has long been used as a straightforward strategy for the creation of high-valence neoglycoproteins; such approaches are still in use.11 During the last two decades, advances in the area of bioconjugate synthesis methods have led to the development of mild methods for the preparation of neoglycoproteins suitable for vaccination purposes.11,20,32–34 The maintenance of three-dimensional conformation or tertiary structures of the protein carriers, especially the bacterial toxins, necessitates the use of mild coupling procedures. In fact, the choice of coupling procedures is often limited by the propensity of carrier protein to undergo changes in the tertiary structure or denaturation under the reaction conditions. The synthesis of neoglycoproteins primarily involves random or defined coupling sites on the surface of the protein carrier and their covalent modification with oligosaccharides at their reducing end or functionalization of the oligosaccharides bearing a spacer arm.

Neoglycoproteins

481

MKWVTFISLL LLFSSAYSRG VFRR DTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA Figure 3 Primary sequence of mature BSA.272 One lysine is in the N-terminal peptide of the preproalbumin and is lost upon processing to the mature albumin, Mr ¼66430.

Figure 4 Four views of lysyl residues (green) in HSA. Credit:284 http://www.ks.uiuc.edu/Research/vmd/.

482

Neoglycoproteins

A variety of chemical conjugation methods have been employed for the attachment of antigenic carbohydrate epitopes by targeting specific amino acid residues present on the carrier protein surface. Although these methods are not site specific and lead to a variable loading of the protein carrier, they have nevertheless been the most effective means to prepare neoglycoproteins so far. Some of the popular approaches are discussed briefly below: for comprehensive reviews on neoglycoproteins and artificial glycoprotein synthesis, see Refs.: 35 and 36 and on structural and synthetic aspects of neoglycoprotein vaccines, see Ref.: 37 and Section 3.23.7.

3.23.3.2.1 Conjugation to lysine As shown in Figure 5, several chemical reactions lead to neoglycoproteins in which lysyl residues are substituted with a glycopolyol or with a carbohydrate moiety through a spacer arm.

3.23.3.2.1.1 Reductive amination The conjugation of the reducing end of the oligosaccharides with the e-amino group of lysine leading to the formation of the Schiff’s base is one of the most frequently employed methods. Since the formation of a Schiff’s base is a reversible equilibrium-driven process, the in situ reduction of the imine formed using sodium cyanoborohydride (NaBH3CN) drives the reaction toward the formation of stable amine adducts.38 The e-amino groups of lysines are useful for direct coupling not only to the reducing terminal of poly- or oligosaccharides but also to aldehyde functionalities generated on short spacer arms linked to the saccharide. The reactive aldehyde functionalities can be easily generated from spacer arms by ozonolysis of unsaturated alkyl spacers or hydrolysis of acetals. Periodate cleavage of saccharide residues to produce reactive aldehydes followed by reductive amination in the presence of cyanoborohydride has also been extensively utilized. Borch and co-workers39 showed that sodium cyanoborohydride (NaBH3CN) reduces a wide variety of organic functional groups with a remarkable selectivity. The reduction of aldehydes and ketones is pH dependent, the reaction proceeds readily at pH 3–4. Reaction of an aldehyde or ketone with ammonia, primary amine, or secondary amine at pH 7 in the presence of BH3CN leads to primary, secondary, or tertiary amines, respectively, via reductive amination of the carbonyl group. Then, Gray38 successfully applied this new method to transform reducing sugars to glycopolyol-amines in a good yield. Knowing that carbonyl groups are not reduced at pH above 5, while the Schiff bases are, Gray and co-workers40 showed that a disaccharide such as cellobiose

HO

O

O

(CH2)n

C

S

H N

O N H

O

O

HO

HO

O

O

OH H N

O

HO

(CH2)n

OH

N H

OH

O

NaBH3CN O

HO

CON3 HO

O

NCS

O NH2

O

NH2

HO

O

O H2 N

O

NH

O

HO

S

O

HO

HO

O

OH

O OEt

OH H N O

O

OMe EtO

O

(CH2)n

NH

O S

HN

Figure 5 Neoglycoprotein synthesis by conjugation of oligosaccharides to lysyl residues.

O

O

N H

N H

(CH2)n

O

O

OH

Neoglycoproteins

483

OH OH HO

O O

HO

HO

O

HO

OH

OH

(a)

HO OH OH HO

OH

O O

HO

HO OH N H (b) HO OH OH HO

OH

O O

HO

HO OH H2 N (c) Figure 6 Preparation of glycosylated serum albumin according to the reductive amination procedure developed by Gray.38 Cellobiose a, (292mmol) reacted at 37 C for about 10 days with 1mmol of BSA at pH 8.0 in the presence of 1590mmol of sodium cyanoborohydride, allowing to reduce the Schiff base b, to a secondary amine c, the neoglycoprotein contained about 26 residues of glucose.

reacted with the amino groups of serum albumin at pH 8.0 leading to a protein substituted with up to 26 sugar moieties according to the reaction shown in Figure 6. The sugar linked to an amino group of the protein is transformed during the reduction step into a linear polyol-amine. For instance, lactose41 has been coupled to the cross-linked dimer of bovine pancreatic ribonuclease A as well as serum albumin; in the last case, up to 20mol of lactose/mol of protein was coupled at the end of a 5-day reaction; while the glycosamination reaction can be carried out at pH 7, the coupling reaction was 2–3 times faster at pH 9, in agreement with Baues and Gray’s data.42 When derivatives of ribonuclease dimer that contained up to eight N-e-1-(1-deoxylactitolyl)-lysine residues per molecule were injected into mice, 69% of this neoglycoprotein was found in the liver after 10min; while with the nonglycosylated enzyme, the liver uptake was only 4%, the majority of the neoglycoprotein being found in the kidneys. Some years later, Lee and co-workers prepared thioglycosides ended with a diacetal derivative (see Figure 7) allowing upon deprotection to o-aldehydo-alkyl 1-thio-glycopyranosides.43 The length of the spacers between the sulfur atom and the amino groups of BSA may be modulated in order to give more freedom to the presentation of sugar moieties.

3.23.3.2.1.2

Use of O- and S-glycosides with a spacer arm

3.23.3.2.1.2.1 Formation of thiourea linkages Phenylisothiocyanate glycosides (see Ref.: 44 for a review) derived from p-amino-aryl glycosides using thiophosgene45 or thiocarbonyldiimidazole46,47 have been extensively used for conjugation of simple sugars or complex oligosaccharides onto lysines (Figure 8). Oligosaccharyl p-nitro-anilide (pNA) pyroglutamyl glycosynthons derived from unprotected oligosaccharides provide an easy approach to synthesize phenylisothiocyanate derivatives of complex oligosaccharides.47

484

Neoglycoproteins

AcO

OAc O NH2

S

AcO AcO (a)

NH

AcO

OAc

O O

O

S AcO

N H

AcO

O

(b) HO

OH

O O

O

S HO

N H

HO

O

(c)

HO

OH

O O S

HO (d)

N H

HO

HO

OH

O

H

O

N

S HO

N HO

(e)

CHO

BSA

H

Figure 7 The tetra-O-acetylgalactosyl-b-pseudothiouronium a, was reacted with chloro-acetylamido-acetaldehyde dimethyl acetal Cl-CH2-CO-NH-CH2-CH(O-CH3)2 in the presence of K2CO3 and NaHSO3 leading to tetra-O-acetylgalactosylb-thio-acetylamidoacetaldehyde dimethyl acetal b, and converted in the presence of NaOCH3 (sodium methoxide) in methanol to galactosyl-b-thio-acetylamidoacetaldehyde dimethyl acetal c, and then in mild acid medium to galactosyl-bthio-acetylamidoacetaldehyde d; finally, d, was allowed to react in the presence of sodium cyanoborohydride (NaCNBH3) with BSA at neutral pH (close to 7), leading to galactosylated BSA, a neoglycoprotein e; as much as 40 thiogalactosides (one of which is shown inside the brackets) were linked to the protein-amino groups (one of which is shown outside the brackets).43,285

The allyl trisaccharide GalNAcb-4[Fuca-3]GlcNAcb-glycoside was transformed in 96% yield into the 3-(2-aminoethylthio)propyl spacer by radical addition of cysteamine hydrochloride under UV irradiation, then activated with thiophosgene and coupled to BSA48 The ligand/protein ratio of the neoglycoprotein, based on protein and amino-sugar analyses, was 6:1mol/mol. 3.23.3.2.1.2.2 Formation of amidine linkages d-Alkylimidate glycosides (2-iminomethoxymethyl thioglycosides) were synthesized49,50 and used to prepare neoglycoproteins (Figure 9). Such neoglycoproteins retain the number of positive charges of the native protein because the linkage is an amidine. 3.23.3.2.1.2.3 Formation of amide linkages Other approaches to prepare neoglycoproteins via coupling to lysines involve condensation of oligosaccharides with spacer arms (carbohydrate or non-carbohydrate-based) such as (1) oligosaccharide containing sugar lactones;51 (2) activated acyl azides derived from oligosaccharyl-acyl hydrazides;52 (3) oligosaccharides that contain an amino spacer with diethyl squarate;53 (4) oligosaccharides with a spacer arm ended with a carboxylic group activated as N-hydroxysuccinimide (NHS)54 or sulfo-NHS esters; (5) a thioglycoside

Neoglycoproteins

HO

485

NO2

OH

OH

O

HO O

HO

O O

OH (a)

OH

HO

NH2

OH

OH

O

HO O

HO

O O

OH (b)

OH

HO

C

N

OH

S

OH O

HO O

HO

O O

OH (c)

OH

HO

H N

H N

OH

OH

O

HO O

HO

O

S

O

OH (d)

OH

Figure 8 Synthesis of a neoglycoprotein d, containing lactosyl-b-phenylthiocarbamyl moieties.5,127,128 A glyco-paranitrophenyl conjugate a, was reduced in the presence of hydrogen and platinium oxide45 or palladium on charcoal127 to a glyco-para-aniline derivative b, then converted into a glyco-phenylisothiocyanate c, by using thiophosgene45 or thiocarbonyl-bisimidazole46 as in Que´tard and co-workers.47 Finally, the last compound c, reacted with BSA at pH 9.0 leading to a neoglycoprotein d, containing up to 54 sugar moieties.

AcO

OAc

AcO

OAc

O AcO (a) AcO

O S

AcO

AcO

Br

(b)

OAc

NH HO

OH

O S

AcO (c)

(d)

HO

S

HO

OH

O

OH NH2

O S

HO OH (e)

NH

O

CN

AcO

NH2

AcO

N

BSA 20

Figure 9 Synthesis of neoglycoproteins according to Lee and co-workers.50 Acetobromogalactose a, was converted in the presence of thiourea CS(NH2)2 into tetra-O-acetylgalactosyl-b-pseudothiouronium b; b, was reacted with chloroacetonitrile in the presence of K2CO3 and NaHSO3 leading to tetra-O-acetylgalactosyl-b-thio-acetonitrile c, and converted in the presence of NaOCH3 (sodium methoxide) in methanol into galactosyl-b-thio-imidate d; the imidate was allowed to react with BSA at pH close to 9, leading to galactosylated BSA, a neoglycoprotein.

486

Neoglycoproteins

corresponding to galactosyl-globoside (SSEA-3), Galb-3GalNAcb-3Gala-4Galb-4Glcb-S-CH2-CH2-COOH activated with N,N,N0 ,N0 -tetramethyl(succinimido)uronium tetrafluoroborate (TSTU) and coupled to BSA to give the corresponding neoglycoprotein (11.6mol of saccharide/mol of BSA);55 (6) the preparation of neoglycoproteins containing Fuca-2Gal and Galb-4GlcNAc glycosides, achieved by transforming 2-bromoethyl glycosides into a methoxycarbonylethylthioethyl derivative which was used to substitute both BSA and keyhole limpet hemocyanin (KLH).56

3.23.3.2.1.3

Use of N-glycosides with a spacer arm

3.23.3.2.1.3.1 Glycosylamines as starting material Glycosylamines (see Refs.: 57 and 58 for reviews) are easily prepared in the presence of an excess of ammonia and can be isolated from such a medium, but they are not stable in neutral or slightly acidic media; conversely, they become stable upon acylation (Figure 10). Glycosylamines made from D-sugars have usually a b-configuration, the a-configuration being less stable than the b-configuration. Upon Nacylation, the N-acylglycosylamides are stable and it is therefore possible to isolate them. N-acylglycosylamides may also be obtained directly by action of ketene on D-glycosylamine. Glycosylamines may undergo Amadori rearrangement: a transformation of glycosylamine into 1-amino-1-deoxy-2-keto derivative was shown to occur with Narylglycosylamines when they were heated for a few hours in ethanol solution with a weak acid as a catalyst. The Amadori rearrangement occurs especially in the presence of both a compound having an activated methylene group and a catalytic amount of a secondary amine or in the presence of glacial acetic acid. The mechanism involves the addition of a proton to the nitrogen atom of the glycosylamine; therefore, when the amine is further substituted by acylation as in N-acetylglycosylamide, the protonation is inhibited and the Amadori rearrangement is limited. Various glycosylamines were obtained, more than a century ago, by Lobry de Bruyn59 in 1895 by dissolving a reducing sugar in warm water and adding anhydrous methanol saturated with ammonia. Within 10 days, the glycosylamine crystallized. A b-glycosylamine can also be obtained by dissolution of the sugar in liquid ammonia followed by evaporation of the solvent.60 More recently, 2-acetamido-2-deoxy-b-D-glucopyranosylamine61 was prepared with a 7010% yield by dissolving N-acetylglucosamine in saturated aqueous ammonium hydrogen carbonate; the solution was kept at 20 C for 45 days or at 30 C for 6 days. The main compound, however, was not the expected glycosylamine but rather a glycosylcarbamate. However, when the crude product was purified by cation exchange chromatography, the expected glycosylamine was eluted with 2M ammonia in a methanol–water mixture.

OH HO HO (a)

OH

O OH

OH

(b)

OH

(c)

HO HO

O

HO HO

NH2 OH

OH O

OH

H N

O

HO HO

CO2

H N

Cl

OH O

(d)

OH HO HO (e)

O

H N

NH3

OH O

Figure 10 Preparation of glycosylamide derivatives. A reducing sugar such as glucose a, may be converted to a glycosylamine derivative b, (mainly as a b-anomer) upon incubation in methanol ammonia mixture,59 liquid ammonia,60 ammonium hydrogen bicarbonate,61 for several days (up to 10 days) at room temperature. With a solution of ammonium hydrogen bicarbonate, byproducts can be obtained such as an N-glycosylcarbamate62 c. A glycosylamine is easily converted back to the initial sugar if it is not acylated. Many activated acids may be used to obtain a stable glycosylamide such as the chloro-acetyl glucosylamide shown d. The last compound may be then converted to a glycyl-glycosylamide e.63

Neoglycoproteins

487

Glycosylamines were also obtained62 by treating reducing sugars with 16M commercial aqueous ammonia in the presence of NH4HCO3. The solution was heated at 42 C for 36h. The yield in the expected glycosylamine was almost quantitative, the presence of glycosylcarbamate being rather low (106 lmol1) while the binding constant of soluble sialylosides is much lower (around 103 lmol1). The binding of lectins on the cell surface is quite strong because cells, which have been incubated with a lectin, may be washed several times in a lectin-free buffer without losing any significant amount of associated lectin. Conversely, the cell-associated lectin was readily released when lectin-coated cells were incubated in the presence of a buffer containing a relevant soluble ligand.99 Wheat germ agglutinin, which is a dimeric lectin (Mr ¼36000) with four binding sites,27 binds with high-affinity glycoproteins bearing a cluster of oligosaccharides ending with Neu5Ac residues100 as in the case of the N-terminal moiety of glycophorin. The increase of affinity based on the spatial density of specific sugars on a ligand has been clearly established by Lee and co-workers101,102 in the case of the Ashwell lectin, a lectin from liver parenchyma cells specific for terminal galactose (or N-acetylgalactosamine) residues at the nonreducing end of an oligosaccharide. The affinity of a single antenna ending with a b-galactopyranosyl residue was found to be around 103 lmol1, while that of the bis-derivative was around 106 lmol1 and these of the tris- and tetrakisderivatives101,102 were around 109 lmol1. Since then the apparent enhancement of the binding constant related to avidity has been largely documented in the field of lectins from plant as well as from animal origin, and has been discussed in detail in several places, including Lee and co-workers.72 The very high affinity, up to 1012 lmol1 of influenza virus to erythrocytes, target cells or sialylated glycoproteins are related to both the density of the viral lectin on the surface of the virus and the density of N-acetylneuraminic acid on the cell surface (or soluble) glycoproteins (see Ref: 103 for instance). Avidity linked to multivalent character of neoglycoproteins can

494

Neoglycoproteins

be easily observed in competitive inhibition experiments. STA, the lectin from Solanum tuberosum, agglutinates rabbit erythrocytes. Its binding is inhibited in the presence of N-acetylglucosamine (GlcNAc). Various glycoconjugates, including di-N-acetylchitobiose-substituted neoglycoprotein, were tested for their capacity to inhibit erythrocytes agglutination.91 As shown in Table 3, BSA substituted with di-N-acetylchitobiosyl residues is a much better inhibitor than di-N-acetylchitobiose itself. Lee and co-workers72 studied the affinity of various galactose terminated BSA to the asialoglycoprotein receptor (hepatic lectin). They compared affinity of neoglycoproteins, differing by sugar density, by measuring their capacity to inhibit the binding of 125I-orosomucoid to the lectin (Figure 17). The affinity of Gal-terminated BSA increases exponentially whatever the form of lectin used. The affinity enhancement, due to increasing number of galactose, can be seen up to 20 residues (rabbit hepatocytes) or 30/35 residues (affinity-purified rabbit hepatic lectin). The avidity of neoglycoproteins can also be observed in in vivo experiments. Beljaars and co-workers104 studied the uptake of HSA modified with mannose-6-phosphate (M6P) by hepatic stellate cells (HSCs) which express the mannose-6-phosphate/insulin-like growth factor II receptor. They synthesized a series of M6P-modified HSA containing 2, 4, 10, or 28 phospho-sugar units and determined total liver uptake. At a low degree of sugar substitution (ratios of 2 to 10) M6P-HSA remains in the plasma (liver accumulation: 9% of total administration). Increasing the M6P/HSA ratio up to 28 caused an increase in liver accumulation (60% of total administration). Furthermore, increase in M6P substitution was associated with an increased accumulation in HSCs.

3.23.4.2 Affinity and Avidity Determined by Surface Plasmon Resonance The evaluation of the avidity exhibited by neoglycoproteins toward various lectins has been carried out by a wide range of assays for the measurement of the binding affinities. The most commonly utilized assays are the determination of

Table 3 Inhibitory effect of GlcNAc and GlcNAc containing glycoconjugates on rabbit erythrocytes agglutination by StA Inhibitors

I50(M)*

Ratio

GlcNAc GlcNAcb-4GlcNAc GlcNAcb-4GlcNAcb-ONP (GlcNAcb-4GlcNAc)n-BSA

700 150 60 0.076

0.2 1 2.5 2000

*

I50, minimal concentration giving 50% inhibition.

10.0 9.5 9.0

−log I50

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 0

5

10

15

20

25

30

35

40

45

Gal/BSA (mol/mol) Figure 17 Inhibition of orosomucoid binding to hepatic lectin by neoglycoproteins containing various galactose moieties: Closed circles, affinity-purified rabbit hepatic lectin; open circles, rabbit hepatocytes. Adapted from72 Lee, Y.C.; Lee, R.T. In Neoglycoconjugates: Preparation and Applications; Lee, Y.C., Lee, R.T., Eds.; Academic Press: San Diego, CA, 1994; pp. 23–50.

Neoglycoproteins

495

inhibition constants by hemagglutination assay (Landsteiner hapten inhibition assay) and the enzyme-linked lectin assay (ELLA). Although, hemagglutination and precipitation inhibition techniques have been used to determine the relative affinity and specificity of lectins for various saccharides using neoglycoproteins, these techniques do not provide thermodynamic or kinetic data about such interactions. In addition, various techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), fluorimetry, and nuclear magnetic resonance have provided important insights into carbohydrate–lectin interactions. In comparison to other techniques, SPR is particularly interesting with regard to neoglycoproteins and glycopolymers, wherein the heterogeneity associated with these derivatives can be incorporated in the elucidation of the kinetic binding constants.

3.23.4.2.1 Principle of SPR The use of SPR allows now to easily access the kinetics data of the binding in addition to the determination of the affinity.105 SPR has been largely used in the field of carbohydrate–lectin interactions (see Ref.: 106 for a review). The physical basis of SPR (see Refs.: 107 and 108 for reviews) is based on the peculiar property of the interaction of polarized light with thin metal films. With a prism near the metal interface, an evanescent wave, present in total reflection, can excite optically the surface plasma wave. The excitation can be seen as a strong decrease in reflection for the transverse magnetic light at a special angle of incidence. The direction of this strong decrease is related to the refractive index of the layer in contact with the film opposite to the prism. This layer contains the immobilized molecule of interest, and the refractive index will change according to the concentration of the compound that will be retained by the immobilized molecule. For instance, a lectin is immobilized on a sensor chip in the layer in contact with the gold film; when a solution of neoglycoprotein passes in that layer, a part of it will be retained by interacting with the lectin. This additional material increases the total amount of organic matter in the layer and as a consequence, the refractive index increases (Figure 18). There is of course no need to label any of the partners in contrast with the interaction monitoring based on spectrophotometry or fluorimetry. Practically, a sensor chip is divided into four flow cells which can be used to individually measure interactions. One of the flow cells can be used as a control. Analyte samples must be prepared and diluted in the running buffer. The refractive index of the layer close to the gold film is monitored in real time allowing the easy determination of both the rate and the equilibrium binding constants of macromolecular interactions.

Analyte flow Association

(a)

Running buffer flow Dissociation phase

(b)

Response (change in refractive index)

Regeneration phase

(c)

(a)

(b)

(c)

Time (s) 1

500

Figure 18 Schematic representation of an optical biosensor experiment: the analyte (a glycoconjugate, for instance) is in solution and passes through the layer containing the receptor (a lectin) immobilized on the biosensor surface. Complex formation is monitored by measuring changes, in the refractive index within the layer, caused by the increase in mass. In a first step, corresponding to the association phase a, the analyte is introduced in the running buffer. Reversible interactions lead to association governed by the association rate constant (kass) and to dissociation governed by the dissociation rate constant (kdiss). In a second step, corresponding to the dissociation phase b, the running buffer alone (analyte-free buffer) is injected, allowing the determination of kdiss. Typically, in order to remove all of the remaining analyte, a regeneration procedure c, is applied before running a new cycle.

496

Neoglycoproteins

In a first step, corresponding to the association phase, which depends on both the association kass and the dissociation kdiss constants and the ligand concentration ‘g’ (concentration of a neoglycoprotein, for instance, when the immobilized receptor is a lectin) according to dR=dt ¼ kass g Rmax Rðkass g þ kdiss Þ

½1

the signal R increases (up to a maximum Rmax) with time (t); the plot of dr/dt versus R gives a curve, of which the slope is ‘(kass gþkdiss)’ and the intercept is ‘kass gRmax’ for dR/dt¼0. In a second step corresponding to the dissociation phase, the buffer alone is injected allowing the determination of the dissociation rate constant kdiss; this constant is used to estimate the association rate constant, kass: kass

L0 þ G Ð LG kdiss

½2

3.23.4.2.2 The classical kinetics analysis used in SPR In the case of a classical bimolecular association, the Langmuir equation109 can be written, (according to Scatchard equation110) as Kðn rÞ ¼ rg 1

or rðn rÞ1 ¼ Kg

½3

where K is the affinity constant between a lectin and a neoglycoprotein expressed as lmol1, n the total number of sites on the sensor chip, r the number of sites occupied by the glycoconjugate, and g the concentration of the glycoconjugate in the analyte solution, expressed as moll1.

3.23.4.2.3 Determination of avidity in SPR The avidity effect is not found when, the lectin being immobilized, a simple soluble oligosaccharide is used as a ligand, but it can be very important when the oligosaccharide is immobilized; the apparent binding constant may increase up to 10000-fold when the sugar density on the chip increased.111 This effect can be explained by considering that a sensor chip, mimicking glycocalyx of a cell surface, is made of a layer which is not stirred, and therefore is separated from the bulk flow or the surrounding medium, respectively. This model is kinetically far more complex than the classical Langmuir two state models. The reaction is limited by the mass transport. The lectin concentration in the unstirred layer is not that of the bulk flow or of the surrounding medium. The lectin may be considered to be entrapped in the layer and therefore did not easily escape spontaneously, except when a small soluble free ligand is added, because in the presence of a highly diffusible ligand at high concentration, the lectin will not be able to interact any more with its immobilized ligands. This avidity effect is about 20 times less important when a monomeric lectin is used instead of a dimeric lectin. Shinohara and co-workers111 proposed a simple model where kass and kdiss are affected by a factor depending on the density of the immobilized ligand D and on a mass transport parameter KT : k0ass ¼ kass =ð1 þ kass D=KT Þ and k0diss ¼ kdiss =ð1 þ kass D=KT Þ

½4

where k0 ass and k0 diss are apparent association and dissociation constants, and kass and kdiss are intrinsic association and dissociation constants, respectively. In addition, the increase in the affinity is mainly related to a modulation of k 0 diss, because, in the ligand layer, a rebinding parameter must be taken into account and then k 00 diss < k 0 diss < kdiss. Therefore, the apparent affinity is A ¼ k0ass =k00diss

½5

Usually k 0 ass is in the 104 to 107 lmol1 s1 range and k00 diss between 101 and 104 s1. Typically, with a k 0 ass ¼104 and k 00 diss ¼103, A is about 107 lmol1. The measurement of rate constants higher than allowed by the conventional technique can also be performed by elution of the analyte with a second analyte of low molecular weight.112 Finally, in some cases, to reach reliable binding parameters, it may be necessary to use more sophisticated methods such as the Sips distribution.

3.23.4.2.4 Kinetics analysis according to the Sips distribution in order to take into account the neoglycoprotein heterogeneity It is well known that when two compounds (A and B) interact, a linear relationship between free ( f ) and bound (b) concentrations of A allows the determination of the equilibrium constant K; this is true when the combination of

Neoglycoproteins

497

A and B is the same for all the combining sites, that is, that there is no heterogeneity (see Ref.: 113 for a more developed presentation). More often, the plot 1/b versus 1/f exhibits a downward curvature attributed to a combining sites heterogeneity.114 The same behavior is relevant in the case of neoglycoproteins and lectins. In this case, the heterogeneity comes from the neoglycoprotein (which contains on average 25 sugars) and from a nonhomogeneous density of immobilized lectins on the chip. The number of sugars present in a neoglycoprotein varies from one molecule to another, because about 25 lysines are glycosylated but more than 25 are not; therefore, the heterogeneity comes from a quantitative aspect but also from a qualitative one: the position of the sugar on the neoglycoprotein. To take into account the heterogeneity of polyclonal antibodies, Nisonoff and Pressman114 found convenient to use the Sips’ distribution function115 that led the data to be fitted to a straight line. The equation was proposed by Sips115 derived from the adsorption of a gas on a solid surface; it may be transformed into an equation applicable to equilibrium in solution; that is the case in SPR experiments where RU (resonance units) was measured at equilibrium. According to eqn [3], logðrðn rÞ1 Þ ¼ a logðKgÞ

½6

or rðn rÞ1 ¼ ðKgÞa As Rmax is related to n, R related to r, it follows: Rmax R1 ¼ nr 1

½7

½Rmax R1 11=a ¼ ðKgÞ1

½8

then

The slope a is called either the heterogeneity coefficient or the Sips coefficient. For a homogeneous group of sites, a is equal to þ1; decreasing values of a correspond to increasing degrees of heterogeneity, showing an avidity effect or reflecting an heterogeneity either related to the ligands or to the receptors. A low value of a corresponds to a high heterogeneity. Because the SPR signal is related to the size of the ligand, it may be advisable to use large ligands such as neoglycoproteins in order to determine the inhibitory strength of a small ligand with a good accuracy. The interactions between small ligands (oligosaccharides or glycoclusters) and their receptors (lectins) can be assessed by using neoglycoproteins: the association constants Ki of the glyco-clusters used as inhibitors74 were calculated from the apparent affinity K of a neoglycoprotein relevant for the immobilized lectin, measured in the presence of various concentrations of the oligosaccharides. In the presence of an inhibitor i, with a Ki, the equation giving K and Ki becomes ðRmax R1 1Þ1=a ¼ ð1 þ iKi ÞðKgÞ1

½9

Experimentally, the number of resonance units R is directly obtained from the sensorgrams and Rmax is deduced from the intercept of the linear transformation: R1 versusð1=gÞa

3.23.4.2.5

½10

SPR applied to study lectin/neoglycoprotein interactions

3.23.4.2.5.1 Differences in affinity of monovalent versus multivalent ligands The combination of solution binding assays and SPR analysis116 provided new insights about the specificity displayed by the mannose/SO 3 -4GalNAc receptor (hepatic endothelial cell lectin involved in the clearance of LH, lutropin hormone). For this purpose, authors used a dimeric mutant, Mut11, which can only engage two terminal sulfated GalNAc (monovalent ligands SO 3 -4GalNAcb-4GlcNAcb-2Mana (S4GGnM) and SO3 -3GalNAcb-4GlcNAcb-2Mana (S3GGnM)) and neoglycoproteins as multivalent ligands (S4GGnM)7-BSA and (S3GGnM)7-BSA. Specificity of the mannose/SO 3 -4GalNAc receptor for monovalent ligands was determined by measuring the inhibition of LH binding to immobilized Mut11. Ki values (25.8 and 16.2mM for S4GGnM and S3GGnM, respectively)

498

Neoglycoproteins

were calculated from the concentration giving 50% inhibition. Ki values for multivalent ligands (0.019 and 0.17mM for (S4GGnM)7-BSA and (S3GGnM)7-BSA, respectively) were determined by solution binding assays. These values result in a 2000-fold increase (95mM for S3GGnM) in the affinity for the multivalent ligand as compared with monovalent S4GGnM, showing that the receptor has a greater specificity for multivalent forms of the same structure. Kinetics for binding both neoglycoproteins were further analyzed. With immobilized Mut1, binding of (S4GGnM)7BSA rapidly reached saturation and little or no dissociation was observed. Compared at the same concentrations, (S3GGnM)7-BSA bound Mut1 to a less extent with no or little dissociation. When dissociation was studied in the presence of 1mM SO 3 -4GalNAc, both neoglycoproteins were rapidly dissociated. Authors concluded that (1) the association rate of (S3GGnM)7-BSA is slower than for (S4GGnM)7-BSA, (2) the individual equilibrium at each site is rapid, and (3) the slow dissociation rate results from multivalent binding. In a second set of experiments, binding of Mut11 to immobilized neoglycoproteins was analyzed by SPR. In both cases, equilibrium was rapidly reached and the affinity constant was 2.5105 lmol1 whatever the neoglycoprotein used. The authors calculated the ratio of Mut11 bound to neoglycoproteins: at saturation, (S3GGnM)7-BSA bound 8 times less Mutl than (S4GGnM)7-BSA (0.09mol/ mol vs. 0.74mol/mol). In this study, by pointing out the utility of neoglycoproteins as multivalent ligands, the authors clearly demonstrated that results with monovalent ligand cannot be used to predict the properties of the multivalent forms. Furthermore, while Mut11 binds with the same affinity to both SO4-neoglycoproteins, there is a strong difference on the maximal binding capacity. Finally, they confirmed and explained all previous results obtained (in vivo, in vitro, and in silico for the Man/SO 3 -4GalNAc receptor: the rapid removal of (S4GGnM)7-BSA from the circulation (12 times faster than for (S3GGnM)7-BSA); inhibition and modeling studies show that the binding site for GalNAc can accommodate the sulfate located at C-3. Based on crystal structure, they hypothesized that S3GGnM exist under two conformations: the favorable form, with the same properties as S4GGnM and the unfavorable form which account for low binding.

3.23.4.2.5.2 Interaction of both multivalent lectins and multivalent ligands Mannan-binding lectin (MBL) is an oligomeric lectin (based on a homotrimeric structural unit) which recognizes neutral sugars (mannose and N-acetylglucosamine) on microbial surface and is involved in innate immunity against infection. Serum purified MBL is a mixture of oligomers with two major forms: MBL-I and MBL-II. Precise chemical structure and biological properties (including carbohydrate binding) of MBL-I and MBL-II were recently investigated.117 MBL-I and MBL-II were demonstrated to consist in trimer and tetramer of the homotrimeric structural unit, respectively. Carbohydrate binding properties of trimeric and tetrameric MBL were studied by SPR with immobilized neoglycoproteins: GlcNAc10-BSA and Man30-BSA; lectins interacted with high affinity with both neoglycoproteins; in contrast, with Gal30-BSA there was no interaction. The apparent binding constants K were in the giga range: 0.5 and 1.8109 lmol1 with Man-BSA for MBL-I and MBL-II, respectively; 0.8 and 1.0109 lmol1 with GlcNAc-BSA for MBL-I and MBL-II, respectively. However, trimeric and tetrameric MBL showed different kinetic rate constants. Whatever the neoglycoprotein used, trimeric showed a faster association (kass) and tetrameric exhibited a slower dissociation (kdiss) and a higher maximal binding capacity. This result clearly shows that lectin oligomeric status has a direct effect on its carbohydrate binding properties: increased binding capacity and higher complex stability of the tetravalent MBL. 3.23.4.2.5.3 Use of neoglycoproteins to assess the binding strength of small ligands to lectins The association constants for a series of lactose clusters and a series of dimannose clusters were deduced from SPR experiments. Frison and co-workers118 chose to immobilize the lectins in order to easily compare the affinity of a series of different glycoconjugates. The analysis of the cluster binding to plant lectins indicated that Rca, a galactosespecific lectin, recognizes lactose clusters, and amongst them, the pentakislactose tetralysine with a high affinity, whereas it did not recognize any dimannose clusters. Similarly, Concanavalin A did not recognize the lactose clusters but did bind the dimannose clusters, the best ligand being the cluster made of Mana-2Man in agreement with published data.119 Glycoclusters with a low molecular mass do not give a significant signal. In addition, the authors favored a procedure based on the inhibition of the binding of a neoglycoprotein on an immobilized lectin. Inhibition constants were derived from experiments using analyte in a 0.25–10mM concentration range using a serial threefold dilution in the running buffer containing 10mgml1 (about 0.125mM) neoglycoprotein Lac-BSA or Man-BSA, for Rca and ConA, respectively; this concentration is the half-saturation concentration which gives the half maximal value, RUmax/2. The Sips heterogeneity coefficient was a¼0.55 for Lac-BSA binding onto immobilized Rca and a¼0.7 for Man-BSA binding onto immobilized ConA.118 Using the same experimental approach, Frison and co-workers

Neoglycoproteins

499

(2003) identified highly specific ligands for dendritic cell receptors: the mannose-receptor and DC-SIGN, the dendritic cell-specific ICAM-3-grabbing nonintegrin. A series of glycoclusters made of di- or oligosaccharides containing either mannose or fucose were tested for their inhibitory effect on the binding of Man-BSA to both immobilized lectins. In both studies, in agreement with the avidity effect, glycoclusters made of four or five lysines appeared to be the best ligands for plant as well as animal lectins.

3.23.5 Use of Neoglycoproteins to Evidence Lectins In Situ 3.23.5.1 Evidencing Lectins in Animal Cells Cytochemical methods leading to the identification, localization, and the quantitative (or semiquantitative) determination of cell glycoconjugates have been developed during the 1970s (see the special issue of Biologie Cellulaire in 1979 and Schre´vel and co-workers);4 more recently, glycosylated cytochemical markers and neoglycoproteins have been used to visualize endogenous lectins. Here, we limit ourselves in examples concerning animals and relevant microorganisms.

3.23.5.1.1 Evidencing lectins in animal cells with labeled neoglycoproteins The ‘asialo-glycoprotein receptor’, ASGP-R, present on the surface of hepatocytes, is the first mammalian lectin known; it was discovered by Ashwell and Morell (see Ref.: 120 for a review) and shown to internalize galactosylterminated neoglycoproteins. By using neoglycoproteins in which glycosides were attached by amidination (GlycAI)n-BSA50 in comparison with synthetic cluster glycosides containing either one, two, or three galactosyl or lactosyl residues per ligand, Connolly and co-workers121,122 have studied the specificity of rabbit hepatocytes. Steady-state and kinetic experiments performed at 37 C revealed the presence of two apparent classes of binding sites totaling 6.3105 sites/cell at 37 C; at this temperature, both classes of sites participated in internalization of bound ligands. The cells were capable of internalizing about 60000 molecules/min per cell. Chicken hepatic lectin was isolated by affinity chromatography on GlcNAc-BSA-substituted Sepharose 4B.123 Many different membrane-bound and soluble lectins have been characterized in various cell types, with variable roles in cell recognition, cell adhesion, or endocytosis (see for reviews Refs.: 3, 19, and 124). Fluorescent neoglycoproteins have been widely used to detect lectins in different cells including lymphocytes125,126 as well as to study the capacity of membrane lectins to internalize their ligands: the glucose receptor on Lewis lung carcinoma cells,127 the galactose and fucose receptors on L1210 lymphoid cells.128 Fluorescent neoglycoproteins bearing a-D-Man, a-Man6P, a-L-Rha, and a-L-Fuc were shown to be internalized by immortalized normal and cystic fibrosis airway epithelial cells.129 Human blood monocytes, freshly isolated, do not contain cell-surface mannose-specific receptors but they bind mannose-6-phosphate and actively take up mannose 6-phosphate-bearing neoglycoproteins (6-P-Man-F-BSA).130 Two different mannose-6-phosphate receptors have been cloned,131,132 the Mr 46000 cation-dependent Man6P receptor and the Mr 215000 cation-independent Man6P receptor (Man6P-R) which is identical with the insulin-like growth factor (IGFII-R). Both Ca2þ-dependent and -independent receptors have been found on most cell types. The Man6P/IGFII-R has both binding sites specific for Man6P and IGF II. This receptor targets newly synthesized lysosomal enzymes from the Golgi to acidic prelysosomal organelles and mediates endocytosis of Man6P-containing ligands and IGF II;133 the uptake of the lysosomal enzyme arylsulfatase A and of a Man6P-neoglycoprotein is stimulated two- to threefold by IGF I and IGF II, and this effect persists for at least 6h. Inhibition studies using chemically synthesized oligomannosides or neoglycoproteins demonstrated that the phosphomonoester Man6P in a terminal nonreducing position is the major binding determinant.134 In the liver, HSCs are the principal cell type responsible for abnormal production of collagen leading to liver fibrosis. The mannose 6-phosphate/insulin-like growth factor II (Man6P/IGF II) receptor is expressed on HSCs and its expression is upregulated upon activation of these cells due to acute or chronic liver injury.135 Beljaars and co-workers104 demonstrated a preferential distribution of Man6P conjugated HSA to HSCs after systemic administration into rats. Recently, Gasanov and co-workers136 identified the Man6P/IGFII-R as a receptor for binding and invasion by Listeria monocytogenes.137 The macrophage Man/Fuc receptor was evidenced by Stahl and co-workers138,139 on cultured primary macrophages and their hybrids; similar lectins were also shown in rabbit alveolar macrophages140 as well as on dendritic cells.141,142 Equilibrium binding analysis performed with 125I,Man43-BSA indicated that the apparent affinity of cell surface receptor for ligand increased, reaching a Ka of 3109 lmol1 under conditions of maximal stimulation of binding.143

500

Neoglycoproteins

Sialoadhesin is a macrophage-restricted adhesion molecule that recognizes Neu5Aca-3Gal structure. A multivalent neoglycoprotein probe carrying this oligosaccharide was prepared allowing the determination of the binding activity of sialoadhesin on native rat macrophages. Macrophages from mesenteric and axillary lymph nodes exhibited 36-fold higher activity than those from the spleen. The Ka values (up to 109 lmol1) of the probe binding to macrophages of the two organs were indistinguishable.144 CD 69 antigen of human lymphocytes is a calcium-dependent carbohydrate-binding protein;145 N-acetylglucosamine and N-acetylgalactosamine are amongst the best inhibitors of this lectin: 2104 lmol1 giving 50% inhibition of binding of CD 69 to GlcNAc-BSA. Neoglycoproteins have been used for detecting carbohydrate-binding proteins in various cell types: in tumor cells,127,146,147 in keratinocytes,148 in brain vessels, and in the developing rat cerebellum.149 Mannose-containing neoglycoproteins were shown to bind ependymal cell cilia and penetrated rapidly the brain tissue. Such a phenomenon was not seen with glucose- or galactose-containing neoglycoprotein molecules. Co-injection of mannosecontaining nonfluorescent neoglycoproteins with the other fluorescent compounds (including fluorescent sugar-free BSA) resulted in the penetration of the fluorescent compounds into the brain tissue. This internalization into brain was attributed to desegregation of junctions between ependymal cells.150 DC-SIGN, DC-specific-ICAM-grabbing nonintegrin, a membrane lectin recently identified on dendritic cells,151 has a single carbohydrate recognition domain with mannose-type specificity. DC-SIGN has functions of cell adhesion receptor mediating both DC migration and T cell activation and also functions as an HIV receptor. Engering and coworkers142 showed that DC-SIGN internalizes antigens and mannosylated BSA; however, as they were not able to inhibit internalization by using specific anti-DC-SIGN antibodies, they suggested that mannosylated BSA was internalized by the mannose receptor also present on DC. By using HeLa cells expressing DC-SIGN upon transfection, we have shown that mannosylated and fucosylated BSA were internalized by such cells75 and that DC-SIGN had a high affinity for oligolysine-based Lewis clusters. MR60, an intracellular D-mannose-specific receptor, from human promyelocytic HL60 cells has been characterized and isolated;152 after permeabilization, promyelocytic, promonocytic cells and monocytes bound fluoresceinylated Man-BSA. Under these conditions, confocal analysis confirmed the intracellular membrane localization of the labeling and the absence of nuclear binding. MR60 was purified by column chromatography, using a mannoside immobilized on a gel, and a cDNA was isolated that matched MR60 peptide sequences.153 MR60 was found to be identical to ERGIC-53 a type 1 integral membrane protein, defined as a marker of the intermediate compartment that recycles between the Golgi apparatus and endoplasmic reticulum; the first direct evidence for a lectin function of ERGIC-53 was further demonstrated: overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein.154

3.23.5.1.2 Use of glycosylated markers for diagnostics Disease progression of tumors is accompanied by structural changes of the glycan chains of cellular glycoconjugates. By introducing neoglycoproteins to histopathological colon cancer analysis, Legendre and co-workers155 addressed the questions whether specific binding sites for main N- and O-glycan components are present and whether they harbor potential for prognostic predictions. The tumor panel included routinely fixed tissue sections from 67 cancer cases and six hepatic metastases as well as 20 normal biopsy specimens as control. Quantitative image analysis determined the labeling index and the mean optical density in each case, separating tumor and peritumoral connective tissue. Specific carbohydrate-dependent binding with inter-individual heterogeneity was observed. The distinct staining profiles were not associated with disease stage or metastasis formation. Strong expression of lactose-binding sites in the peritumoral connective tissue especially in terms of the labeling index was significantly correlated with reduced survival in colorectal cancer patients with a Dukes B type. Therefore, neoglycoproteins bearing lactose appear to have a prognostic value by probing specific lectins with this type of cancer.

3.23.5.1.3 Visualization of endogenous lectins with neoglycoproteins Various glycosylated derivatives of peroxidase and ferritin88 specific for the most common lectins used as tools were prepared and used to visualize glycoconjugates in a two-step procedure: first, a lectin (usually a plant lectin) was added onto the preparation, and after washing, a glycosylated cytochemical marker was added.4,7,156 A differential labeling was observed depending on their capability to diffuse toward the lectins.156 Glycosylated cytochemical markers have also been used to localize endogenous lectins. Mannose-specific lectins were detected at the ultrastructural level in nuclei of lizard granulosa cells in situ by means of mannosylated ferritin.157

Neoglycoproteins

501

Mapping of the bone marrow sinus endothelium with lectins and glycosylated ferritins allowed the identification of differentiated microdomains and their functional significance.158 Similar labeling was made in the rat liver endothelial159 showing segregated distribution of lectin binding labeled with glycosylated ferritins. Biotinylated neoglyoproteins can be used in both fluorescence and electron microscopy in conjunction with fluorescent derivatives of streptavidin and with streptavidin adsorbed on gold beads, respectively. Such approaches have been used to evidence lectins in various tissues as follows: Human breast. The presence of lectins was detected in tissue sections of human breast by employing 13 biotinylated neoglycoproteins. Benign and malignant breast lesions were more stained with mannosylated proteins than normal breast tissue. A mixed pattern of staining localization and intensity was seen for different types of malignancy with this neoglycoprotein. Similarly, receptors for lactose and N-acetylglucosamine could only be detected within the cytoplasm for certain types of malignancy.160 Human carcinomas. The expression of endogenous (b-galactoside)-specific and (N-acetyl-D-galactosamine)specific lectins was localized in oral squamous cell carcinomas.161 Human heart. Spatial differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures.162 Rat cerebellar. Endogenous lectins were studied150 during rat cerebellar development at the optical level on sections of fixed tissue using biotinylated neoglycoproteins, in conjunction with subsequent avidin-peroxidase staining, showing developmental regulation. However, studies with a light microscope could not give information on potential membrane-bound localization of carbohydrate-binding sites. Ultrastructural studies with electron microscope demonstrated that these tools are very efficient in detecting intracellular carbohydrate-binding sites, but failed to detect most of them expressed at the cell surface when using immunocytochemical techniques for known receptors, probably because of the interaction of these carbohydrate-binding sites with endogenous membrane-bound ligands. Plasmodium falciparum. The merozoite surface was specifically labeled with a GlcNAc-BSA in a sugar-dependent manner, as shown by affinity cytochemistry in fluorescence and electron microscopy.163 To ascertain the nature of the sugar receptor, merozoite proteins were blotted and tested by a two-step method using biotinylated GlcNAc-BSA and streptavidin-peroxidase conjugate. Colloidal gold. This was used as an electron dense marker for transmission electron microscopy as well as for scanning electron microscopy; for cytochemical use, macromolecules such as polysaccharides and glycoproteins are adsorbed on gold granules of different sizes.164 Both a one-step method and a two-step method have been developed; in the latter, the lectin and the marker are used sequentially. Dual detection can also be achieved by using gold granules of different sizes: for example, hepatic binding protein specific for galactose and galactose-containing receptors have been simultaneously localized on rat hepatocytes165 by using beads of 5 and 17nm in diameter, respectively: Au5 labeled with asialo-ceruloplasmin and Au17 RCA. Neoglycoproteins adsorbed on colloidal gold particles were used for the ultrastructural localization. Boar spermatozoa. A fucose-binding protein was first detected by means of a specifically developed modified ELLA using fluoresceinylated and glycosylated peroxidase derivatives. Fucose binding was particularly prominent at the apical region of the sperm head. In order to gain more insight into the precise localization of the carbohydrate binding protein, electron microscopy studies were performed using fucosyl peroxidase coupled to colloidal gold. In ultrathin sections as well as in specimens prepared in toto for TEM an intensive binding of fucosyl-peroxidase-colloidal gold was predominantly found at the apical part of the acrosome.166 Guinea pig middle ear mucosa. The incubation of ultrathin sections with gold-labeled p-aminophenyl N-acetylb-glucosaminide-BSA (GlcNAc-BSA-gold beads) led to the binding on mucosal cilia, microvilli, rough endoplasmic reticulum, mitochondria, and nuclei. In contrast, no binding occurred when neoglycoproteins containing mannose, N-acetylgalactosamine, or fucose were used.167 Bovine airway smooth muscle cells. By using Man-BSA-colloidal gold conjugate; a mannose receptor, which mediates mannosyl-rich glycoprotein-induced mitogenesis, was localized by electron microscopy to clathrin-coated pits on the cell surface, and was found to undergo internalization into endosomes.168 Trypanosoma cruzi. GlcNAc- and Gal-binding sites were seen on the surface of about 80% and 5–10% of the trypomastigote forms, respectively.169 Staphylococcus aureus. Neoglycoproteins adsorbed on gold beads allowed to localize sugar-binding sites on Spurr-embedded thin sections. While cellobiosyl-BSA-, lactosyl-BSA-, and melibiosyl-BSA-gold beads did not label the section, GlcNAc-BSA-gold beads led to a heavy labeling of both the cell wall and the cytoplasm. Inhibition of labeling upon treatment with N-acetylglucosaminidase indicated that the labeling was due to N-acetylglucosamine residues.170

502

Neoglycoproteins

3.23.5.2 Use of Neoglycoproteins to Detect Nuclear Lectins and Glycoconjugate Nuclear Import In addition to membrane lectins, animal cells express soluble lectins that are present in both the cytosol and the nucleus.

3.23.5.2.1 Presence of lectins in the cytosol and the nucleus of animal cells Nuclear lectins have been directly or indirectly evidenced by using neoglycoproteins; few of them have been characterized (see Table 4 and Wang and co-workers171 for a review). The first evidence of the presence of lectins associated with the nucleus of mammalian cells was obtained by fluorescence microscopy using fluorescein-labeled neoglycoproteins:172 the nuclei of rat hepatocytes were isolated, fixed in 3.5% formaldehyde and finally incubated in the presence of fluorescent BSA or fluorescent neoglycoproteins; the best labeling was obtained with neoglycoproteins containing either a-N-acetylgalactosaminyl or b-galactosyl residues. However, this labeling, based on nonconfocal microscopy, was interpreted as associated with the nuclear envelope; attempts to label membrane-depleted nuclei in a sugar-dependent manner were not successful: both fluorescent sugar-free BSA and neoglycoproteins labeled the nuclei. Then, Se`ve and co-workers173 succeeded to specifically label membrane-depleted nuclei isolated from baby hamster kidney (BHK) cells and mouse L 1210 leukemia cells with fluorescent neoglycoproteins. The presence of nuclear sugar-binding proteins was evidenced by fluorescence microscopy and by flow cytofluorimetry using a panel of neoglycoproteins. Among the nine neoglycoproteins used, six of them (namely a-glucosylated, b-N-acetylglucosaminylated, a-6-phosphoryl-mannosylated, a-L-rhamnosylated, b-lactosylated, and a-L-fucosylated BSA) strongly labeled nuclei, while a-galactosylated, a-N-acetylgalactosaminylated, and a-mannosylated BSA led to a dull (about one tenth) labeling. These preliminary data were then confirmed174 by flow cytometry in a comparative study using BHK cell nuclei isolated upon cell lysis on the one hand and on permeabilized karyoplasts obtained by enucleation on the other hand. In both cases, the labeling was quite similar, supporting the idea that the binding sites were borne by actual nuclear structures and not by cytoplasmic or membrane-derived contaminants. Neoglycoproteins containing approximately 25 a-glucosyl, b-N-acetylglucosaminyl, b-lactosyl, a-L-fucosyl, or a-L-rhamnosyl moieties gave a strong labeling, all in the same range. However, a neoglycoprotein containing 45 a-L-rhamnosyl moieties gave a fourfold stronger labeling than that obtained with one containing 23 a-L-rhamnosyl moieties, showing that for comparative purposes it is important to use neoglycoproteins with a similar number of sugar units. Indeed, the apparent binding constant depends on the avidity (also called cluster effect, see Section 3.23.4) which increases with the density of sugars linked on a protein.93,94 The intra-nucleolar distribution of sugar-binding sites (the carbohydrate recognition domains of putative lectins) was analyzed in segregated nucleoli of actinomycin D-treated HeLa cells.175 Facy and co-workers176 found, using the human promyelocytic cell line HL60, that the nuclei bound a panel of neoglycoproteins, the highest efficiency being obtained with that containing a-L-rhamnosyl moieties, labeling was twofold less

Table 4 Identified nuclear lectins and associated activity Protein

Carbohydrate specificity

Activity

Galectin-1

b-Galactosides

Galectin-3

b-Galactosides

Pre-mRNA splicing Pre-mRNA splicing

Galectin-7 Galectin-10,-11,-12, -13,-14 Annexin A1 Annexin A2 Annexin A4 Annexin A5 Annexin A11 Pigpen CBP70 Hsc70/Hsp70 Human p32 Avian cdc37

b-Galactosides

DNA replication Glycosaminoglycans (heparin, heparan sulfates, chondroı¨tin sulfates A, B, C, . . .) Heparin, heparan sulfates bisecting N-acetylglucosamine GalNAc, chondroitin sulfate Glc/GlcNAc GlcNAc Hyaluronic acid Glycosaminoglycans (hyaluronan, heparin, heparan sulfates)

Cell division

Neoglycoproteins

503

with that containing a-glucosyl or a-galactosyl residues and ninefold less with those containing either a-fucosyl or b-lactosyl moieties while the labeling with a neoglycoprotein containing a-6-phosphomannosyl residues was dull. These results show that the nuclear lectin content may be cell-type dependent. The presence of nuclear lectins in proliferating 3T3 fibroblasts was further confirmed by Moutsatsos and co-workers177 who were working on a soluble galactose-specific lectin carbohydrate-binding protein-35 (CBP35): an Mr 35000 carbohydrate-binding protein now known as galectin 3 (see Refs.: 178 and 179, for reviews on galectins, a family of lectins recognizing galactosides and N-acetylgalactosaminides).180 Using immuno-fluorescence, with a rabbit antiserum directed against CBP35, they showed that, in proliferating cells, this lectin was predominantly localized in the nucleus. Other nuclear lectins have since been isolated and characterized. Schro¨der and co-workers181 isolated a glucosebinding protein (CBP67: Mr 67000) from rat liver nuclei and suggested a role of this lectin in targeting nuclear ribonuclear particles (RNPs) to nuclear pore complexes. Se`ve and co-workers182 characterized another glucosespecific lectin (CBP70: Mr 70000) from HeLa cells. The same team further showed that CBP70 was a glycosylated nuclear lectin which also interacted with neoglycoproteins bearing N-acetylglucosamine.183 Lauc and co-workers184 identified and purified a stress-associated nuclear lectin (Mr 33000) from rat liver. Lefebvre and co-workers185 have isolated nuclear and cytosolic N-acetylglucosamine specific lectins from adult rat liver by affinity chromatography on immobilized GlcNAc.

3.23.5.2.2 Modulation of the nuclear lectin content Nuclei from exponentially growing cells bound much greater amounts of neoglycoproteins than did nuclei from contact-inhibited cells.174 Proliferating 3T3 mouse fibroblasts contained higher levels of the galactose-specific lectin CBP35 than do quiescent cultures of the same cells;177 when cells in culture reached a quiescent state, the majority of 3T3 cells underwent a general decrease in the overall fluorescence intensity and lost their nuclear staining. Lauc and co-workers186 showed that the exposure of mature rats to stress induced an increase in nuclear CBP35 bound to CBP67 (a glucose-specific lectin) that retained the ability to bind immobilized glucose. In contrast, nuclear extracts from the liver of old rats had a lectin content independent of stress pretreatment. Recently, the expression of a functional CBP70 lectin was shown to be stable during heat shock stress.187 Using isolated and membrane-depleted nuclei, Facy and co-workers176 analyzed the nuclear lectin content of HL60 cells before and during in vitro differentiation into monocytes by PMA treatment and into granulocytes by DMSO treatment. The neoglycoprotein binding was found to be significantly lower in differentiated cells than in undifferentiated cells. Interestingly, the cellular expression of CBP70 was enhanced during cell differentiation but was different in tumoral (HL60-derived) versus healthy (monocyte-derived) cells of the macrophage lineage.188

3.23.5.2.3 Sugar-dependent nuclear import of glycosylated proteins Polet and Molnar189 showed that the N-glycosylated acidic nonhistone proteins were induced to translocate from the cytoplasm to the nucleus in lymphocytes upon mitogenesis induction or by agents such as NaF or eserine; however, they did not document the possibility that the nuclear import could be glyco-dependent. Large proteins (Mr above 40000) do not migrate from the cytosol to the nucleus except when they contain or are substituted with specific nuclear localization signals (NLSs). Accordingly, fluorescein-labeled serum albumin, introduced into the cytosol, did not leave the cytosol. Duverger and co-workers190 showed that neoglycoproteins (BSA substituted with about 25 sugar residues) introduced into cytosol by electroporation migrated into the nucleus under specific conditions related to the nature of the linked sugars, the temperature, and duration of incubation. Serum albumin substituted with a-glucosyl, a-L-fucosyl, or a-mannosyl residues entered the nucleus at 37 C within 30min while sugar-free serum albumin or serum albumin substituted with b-galactosyl or 6-phosphomannosyl residues did not. These results have been confirmed by injecting BSA and neoglycoproteins in the cytosol of living cells (Figure 19). In addition, Duverger and co-workers191 showed that, in digitonin-permeabilized cells, BSA substituted with b-di-N-acetylchitobioside (GlcNAcb-4GlcNAc) was transported from the cytosol to the nucleus in a sugar-dependent manner. At 4 C, all proteins and neoglycoproteins stayed in the cytosol. The glyco-dependent nuclear import of glycosylated proteins from the cytosol could be related to cytosolonuclear lectins shuttling between the cytosol and the nucleus. The glycodependent import and the peptide NLS-dependent import, which have some common properties, have clearly distinct characteristics (Table 5). Duverger and co-workers192 shed light on the mechanism of the glyco-dependent process by using digitoninpermeabilized cells. They used the following fluorescent probes: F-BSA and R-BSA (BSA substituted with a fluorescein or rhodamine derivative, respectively), F-,NLS-BSA and R-,NLS-BSA (F-BSA and R-BSA substituted

504

Neoglycoproteins

F-BSA

Glc-BSA

Galb -4Glc-BSA

NLS-BSA

Figure 19 Nuclear import of Glc-BSA and Galb-4Glc-BSA and NLS-BSA upon cytosolic microinjection. Lissaminerhodamine-labeled glucosylated-BSA (Glc-BSA), lissamine-rhodamine-labeled lactosylated-BSA (Galb-4Glc-BSA) or lissamine-rhodamine-labeled NLS-BSA were injected together with fluorescein-labeled sugar-free BSA (F-BSA) into the cytosol of HeLa cells. After 2h incubation at 37 C, cells were fixed, and the intracellular localization of fluorescent proteins (neoglycoproteins, and NLS-proteins) was analyzed by confocal microscopy, according to Duverger and co-workers.192

Table 5 Properties of both glyco-dependent import and NLS-dependent import Glyco-dependent import (neoglycoprotein: Glc-BSA)

NLS-dependent import (BSA-NLS)

Energy dependence Incubation at 37 C Incubation at 4 C Incubation in the presence of apyrase

þ

þ

Passage through the nuclear pore Incubation in the presence of WGA

Involvment of peptidic NLS Incubation in the presence of large excess NLS-BSA

þ

Involvment of specific factors/proteins NEM treatment Incubation in the presence of nonhydrolizable GTP Incubation in the presence of importin b-specific antibody

þ þ þ

‘þ’ means that nuclear import does occur under experimental conditions; ‘’ means that nuclear import does not occur under experimental conditions.

with about 10 NLS peptides, respectively), and F-,Glc-BSA and R-,Glc-BSA (F-BSA and R-BSA substituted with about 25 a-glucopyranosyl moieties, respectively). The nuclear import of neoglycoproteins was shown to share several traits in common with the peptide NLS-dependent process. Both processes were energy dependent: the nuclear import did neither occur when cells were maintained at 4 C nor when cells were ATP-depleted by treatment with apyrase. Both of them occurred through the nuclear pores: they were inhibited by wheat germ agglutinin, a lectin which binds the nuclear pore glycoproteins and blocks the translocation step of macromolecules through the nuclear pore.193 However, the nuclear import of neoglycoproteins did not use the pathway of proteins bearing the NLS peptide: in digitonin permeabilized cells, the NLS-dependent nuclear import required additional cytosolic factors and was inhibited by treatment of cells with N-ethylmaleimide, while the nuclear import of neoglycoproteins neither required added cytosolic factors nor was sensitive to thiol-alkylation by N-ethylmaleimide. In addition, upon incubation in the presence of a large excess of a protein bearing NLSs, the nuclear import of neoglycoproteins was not inhibited. The specific features of the glyco-dependent nuclear import of glycosylated proteins in intact living cells were further analyzed by Rondanino and co-workers194 by comparing the two nuclear import pathways in a microinjection

Neoglycoproteins

505

approach. The intracellular localization of fluorescent NLS-BSA or Glc-BSA injected into the cytosol was analyzed by confocal microscopy. Glc-BSA migrated less efficiently into the nucleus than did NLS-BSA because Glc-BSA apparently interacted with resident cytosolic components. The import of neoglycoproteins was not affected by microinjection of antibodies specific for the importin/karyopherin-b, a nuclear import factor, whereas the NLS-dependent transport was completely abolished. The nuclear import activity of Glc-BSA was found to be cell cycle dependent in thymidine and hydroxyurea-treated HeLa cells, with greatest efficiency during the G1/S transition and S phases, whereas NLS-BSA was imported with the same efficiency during any stage of the cell cycle but the G2 phase.

3.23.5.3 Use of Neoglycoproteins to Decipher the Mechanism of the Acrosome Reaction Sperm binding to the egg zona pellucida (ZP) is mediated by complementary protein–carbohydrate interaction. Mammalian spermatozoa must undergo an exocytotic event during fertilization, the acrosome reaction (AR). In most species studied, this process is induced by specific glycoproteins of the oocyte extracellular matrix, the zona pellucida and it involves guanine nucleotide-binding regulatory proteins (G-proteins), resulting in an uptake of extracellular calcium by the sperm. The following carbohydrates (N-acetylglucosamine, mannose, fucose, and galactose) have been shown to be involved in human sperm-zona pellucida binding.195

3.23.5.3.1 Binding of neoglycoproteins to spermatozoa Mannose-binding sites on human spermatozoa have been identified on the sperm head in the equatorial, pre- and postequatorial areas.196–198 Zona recognition and binding involve the interactions of sperm surface ‘mannose-specific lectins’ with mannose ligands on the zona pellucida. Sperm surface mannose lectins can be visualized by their ability to bind fluorescein isothiocyanate-conjugated mannosylated bovine serum albumin (Man, Flu-BSA). The Man-SA neoglycoprotein specifically blocked zona binding of swim-up human spermatozoa in a concentrationdependent manner. Unfixed sperm presented fluorescent Man-SA label over the entire acrosomal area or concentrated at the equatorial segment. These sites appear to require free calcium to operate, and their expression changes with capacitation and acrosome reaction.196 It has also been shown that certain cases of male infertility may be related to altered mannose-binding capacity and that the expression of mannose-binding sites depends on capacitation.199,200 The binding of glycodelin-A (a natural inhibitor of human sperm-egg binding) to sperm was suppressed by mannose and fucose neoglycoproteins, while that of glycodelin-F was in addition reduced by acetylglucosamine neoglycoprotein.201

3.23.5.3.2 Acrosome reaction induced by neoglycoproteins containing Man and GlcNAc Several BSA-based ‘neoglycoproteins’ were shown to stimulate the acrosome reaction. The binding of Man,Flu-BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis in capacitated sperm populations from fertile donors.200 N-acetyl-a-glucosamine (a-GlcNAc) and a-mannose (aMan-BSA) may interact with the putative receptor for one protein of zona pellucida: ZP3 in human spermatozoa.195,202–205 This binding results in the exocytosis of the sperm acrosome (AR).206 The GlcNAc-BSA-induced acrosome reaction is inhibited by a small ligand: N-acetylglucosamine (GlcNAc) and by a purified soluble hydrolase, b-N-acetylglucosaminidase. The induction of the AR with Man-BSA was inhibited by mannose, while soluble a-mannosidase was only partially effective. These data suggest that binding sites for GlcNAc and Man seem to be involved in the induction of the AR in human sperm. The induction of AR in human spermatozoa by GlcNAc-BSA could be used to predict their fertilizing ability in vitro.205 The induction of the acrosome reaction by GlcNAc-BSA and Man-BSA has been shown to involve voltagedependent Ca2þ channels and a Gi-like guanine regulatory protein.203,207 The GlcNAc-BSA- or Man-BSA-induced AR was completely inhibited by preincubation of spermatozoa with calcium antagonists, indicating a link between the binding of sugar residues of the neoglycoproteins and channel activation. The pretreatment of spermatozoa with Pertussis toxin (PTx) inhibits GlcNAc-BSA- or Man-BSA-induced AR, whereas cholera toxin has no effect. Therefore, the transduction mechanism for GlcNAc-BSA- and Man-BSA-induced AR involves G-proteins of the inhibitory type (GI).204 The effect of ‘peritoneal fluid’ on the human sperm acrosome reaction (AR) was tested.208 When the AR was induced by GlcNAc-BSA, pre-incubation with peritoneal fluid reduced (60%) the percentage of AR, while peritoneal fluid from either the endometriosis group or infertile patients without endometriosis caused no significant differences. These data indicated that peritoneal fluid possesses a protective factor which prevents premature AR.

506

Neoglycoproteins

3.23.5.3.3 Acrosome reaction induced by neoglycoproteins containing Lewis oligosaccharides Fucosylated glycoconjugates play an important role in fertilization as the recognition signal of the zona pellucida. Using ‘critical’ concentrations of F-, Fuc-BSA neoglycoprotein as molecular probes, population densities of fucosebinding material were found all over the human sperm surface with higher population densities in postacrosomal sheath, neck, and midpiece.209 Lewisx (Lex; Galb-4[Fuca-3]GlcNAc) was shown to be a potent inhibitor of in vitro sperm-ZP binding.210 This glycan is recognized by approximately 70% of the ZP3-binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewisa (Lea; Galb-3[Fuca-4]GlcNAc) is recognized by most of the remaining sites.211 An Lex-containing neoglycoprotein (as well as an Lea-containing neoglycoprotein but to a lower extent) induced the acrosome reaction in a dose- and capacitation-dependent manner.211 In contrast, neoglycoproteins containing b-lactosamine (Galb-4GlcNAc), Lewisb (Fuca-2Galb-3[Fuca-4]GlcNAc), and sialyl-Lex glycans were inactive. Consistent with these results, unconjugated Lex- and Lea-capped glycans were dose-dependent inhibitors, which, at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Lex- and Lea-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional Gi protein. Thus, Lex-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. An Lea-containing neoglycoprotein binds a second ZP3 receptor but is a lesspotent inducer of the acrosome reaction.

3.23.6 Drug Targeting 3.23.6.1 Uptake of Neoglycoproteins Assessed by Fluorimetry The general aim in drug targeting is to increase the efficiency of a given drug by increasing the local drug concentration and/or decreasing its clearance rate. Since Paul Ehrlich’s vision of targeted drugs as ‘magic bullets’ research in this field has expanded, mainly since 1970. Certain membrane lectins induce the internalization of their ligands; therefore, glycoconjugates specifically recognized by these lectins could be used as carriers of antiparasite, antiviral, and antitumor drugs, liver cells, macrophages, and tumor cells being the main target cells. In order to determine the efficiency of glycoconjugates as carriers for targeting to a given cell, we developed a fluorescence-based assay.212 The fluorescence of a fluorescein bound to a neoglycoprotein is partially quenched, but upon proteolytic cleavage the quenching is abolished. The quantum yield is maximal at neutral pH and decrease to a very low value among with medium acidification. During the process of internalization mediated by membrane lectins, ligands are internalized into endocytic vesicles derived from plasma membrane. Although the exact pathway followed by the ligands is not always known, the key step is a rapid acidification of endocytotic vesicles, allowing the dissociation of the ligands from receptors; ligands are usually delivered to lysosomes where proteolysis occurs. Upon cell incubation at 37 C allowing endocytosis of the ligands, the endoplasmic organelles were neutralized by posttreating cells for 30 min at 4 C in the presence of 50mM monensin.128,213 By comparing the fluorescence intensity of cells incubated at 37 C without and with post-treatment with monensin, it is possible to evaluate the extent of uptake and of the degradation.

3.23.6.2 Neoglycoproteins as Drug Carriers Neoglycoproteins have been used successfully to carry and selectively deliver various types of drugs to several types of cells. In order to be released inside a cell, the drug is associated through a linkage which is either: 1. cleavable in acidic medium (in the lumen of endosomes or lysosomes) as in the case of acido-labile heterobifunctional links (maleic acid derivatives); 2. cleavable under reducing conditions as in the case of disulfide bridges; or 3. cleavable by endosomes or lysosome hydrolase as in the case of peptidyl linkers. Trouet and co-workers214 were the first to show the interest of a covalent linkage between a drug (daunorubicin) and proteins that is stable in serum but allows its release by lysosomal hydrolases. In some cases, neoglycoproteins may also bind to the scavenger receptors which are known to recognize polyanionic globular proteins. Indeed, neoglycoproteins made by reaction with O-glycosyl-phenylisothiocyanate on serum albumin have a low isoelectric point. Serum albumin is an acidic protein and by neutralizing some 20 lysine e-amino

Neoglycoproteins

507

groups, the anion/cation ratio increases, and the isoelectric point is still lower. With cells expressing the scavenger receptor, negatively charged serum albumin is quite a good ligand and neoglycoproteins may bind to both the membrane lectin and the scavenger receptor.215

3.23.6.2.1 Targeting antitumoral drugs Since 1980, neoglycoproteins have been shown to be suitable to target toxic drugs, such as Ara-A,216 Gelonin,127 methotrexate,217 daunorubicine218 (see for reviews Refs.: 15, 219, and 220). Gelonin, a single-chain plant lectin which is quite efficient in blocking protein synthesis in cell lysate, has a limited activity on living cells. Gelonin linked through a disulfide bridge to glucosylated serum albumin was 100 times more efficient than free gelonin in killing Lewis lung carcinoma cells which express glucose-specific membrane lectin.127 Knowing that L1210 leukemia cells are sensitive to the cytotoxic effect of methotrexate (MTX) which acts as a metabolic inhibitor within the cells and actively internalized fucosyl serum albumin, MTX neoglycoprotein conjugate was synthesized. The active ester derivative of MTX was added to Fuc-BSA containing 267 sugar units leading to a conjugate with 172 MTX units which was more cytotoxic than free MTX and MTX bound to BSA indicating that MTX bound to neoglycoprotein is more actively internalized than sugar-free serum albumin.128 Hepatocellular carcinoma (HCC) is a major health problem wordwilde. Among the therapeutic approaches, systemic chemotherapy is sought, but the available anticancer drugs have a limited efficacy on HCCs, at the conventional doses. In the treatment of HCCs that maintain the receptor for asialoglycoproteins ASGPs, Fiume and co-workers proposed coupling of drugs to macromolecules that are taken up by this receptor. Twenty years ago, with the aim of improving the chemotherapeutic index of 9-b-D-arabinofuranosyl-adenine 50 monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes.221 They found that this conjugate was quite stable in the mouse bloodstream and inhibited virus hepatitis Ectromelia virus DNA synthesis in mouse liver without affecting cellular DNA synthesis in intestine and bone marrow. The conjugate did not display any recognizable sign of acute toxicity even at doses several fold higher than those pharmacologically active; in addition, when prepared with homologous albumin it was not immunogenic. A clinical study was presented in 1995 by Fiume and co-workers:222 in six patients, who received a 28-day treatment, the conjugate inhibited virus growth without producing any side effects. L-HSA-ara-AMP conjugate must be given by intravenous infusion. New hepatotropic conjugates of ara-AMP have been recently prepared which could be administered by bolus intravenous injection or by intramuscular route. These complexes might assure a better compliance in patients with hepatitis B virus infection for a long-lasting livertargeted antiviral treatment. In view of an adjuvant chemotherapy with Lac-HSA-DOXO after the surgical removal of the tumor, the conjugate of doxorubicin (DOXO) was coupled to lactosaminated human albumin (Lac-HSA) using the (6-maleimidocaproyl) hydrazone derivative of the drug as an acid-sensitive linker.218,223 Lac-HSA-DOXO caused a selective drug accumulation in liver, with low DOXO levels in extra-hepatic tissues. Upon injection of radioactive lactosaminated human albumin (Lac-HSA) in rats with HCCs produced by nitroso-diethylamine, Lac-HSA accumulation in the tumors mainly occurred via the ASGP-R, as indicated by the 20 times lower penetration of non-lactosaminated HSA.224 However, the radioactivity was 2–3 times lower in HCCs than that of surrounding liver, but several times higher than that of extra-hepatic tissues.

3.23.6.2.2 Targeting antiviral drugs The in vitro delivery of the anti-HIV agent 30 -azido-30 -deoxythymidine (AZT) as 50 -monophosphate into human T-lymphocyte MT-4 cells through covalent coupling to neoglycoproteins was investigated by Molema and co-workers.225,226 In vivo application of this drug targeting concept may lead to increased efficacy and/or diminished side effects caused by AZT. Using a phenyl linkage between sugar and HSA, various mannose-, fucose-, galactose-, and glucose-containing neoglycoproteins were synthesized. The intrinsic anti-HIV activity of these neoglycoproteins was tested in vitro in HIV-1-infected MT-4 cells. The derivative having 40mol mannose per mole protein (Man40HSA) shows pronounced anti-HIV-1 activity itself. This effect may be caused by interference of the Man40-HSA with the gp120-CD4-mediated virus/MT-4 cell interaction. After conjugation with the 50 -monophosphate form of the antiviral drug AZT, AZTMP, the mannose- as well as the fucose- and galactose-containing conjugates exhibited a pronounced activity. Conjugates of glucose-HSA and HSA displayed much less activity in spite of the fact that drug loading was considerably higher, compared with the galactose, mannose, and fucose derivatives. In the series of mannoseneoglycoproteins, the AZTMP-Man-HSA conjugate was shown to be more than 30 times as active against HIV-1

508

Neoglycoproteins

compared to HSA-AZTMP and the anti-HIV-1 activity of the AZTMP-Man-HSA conjugate exceeded that of the parent drug by more than four times. The reaction conditions for the synthesis of neoglycoprotein-AZTmonophosphate conjugates were optimized more recently.227 The coupling of the monophosphate derivative of 3-azido-2,3-dideoxythymidine (AZTMP) to glycoproteins by water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) was greatly improved by also using N-hydroxysulfosuccinimide (sulfoNHS) in the conjugation reaction and allowed 2 AZTMP molecules/mol of neoglycoprotein.

3.23.6.2.3 Targeting macrophage immunomodulators Macrophages can be activated by immunomodulators such as lymphokines, LPSs, and muramyl dipeptide (MDP). MDP renders macrophages cytostatic and cytotoxic against tumorigenic target cells. In vitro, the biological effect depends on its internalization by pinocytosis. Since (1) macrophages did not express MDP receptors228 and in vivo, free MDP does not induce any tumoricidal activity and (2) knowing that macrophages express a mannose receptor mediating uptake of mannosylated glycoconjugates; MDP was linked to mannosylated serum albumin.217,229 The covalent attachment of MDP to Man-BSA or BSA (used as a negative) control was performed by carbodiimidemediated formation of amide linkage between the g-glutamyl-carboxyl group of MDP and a primary amino group of the protein carrier; the number of MDP residues bound to Man-BSA was about 10. MDP-Man-BSA activate macrophage both in vitro and in vivo; systemic injection of this conjugate led, in mice, to regression of established spontaneous metastases originating from the primary tumor Lewis lung carcinoma cells.229 Macrophage activation by MDP bound to neoglycoprotein release of cytotoxic factor.230 More recently, Sarkar and Das231 have shown that MDP bound to mannosyl HSA was 50 times more efficient than free MDP in inhibiting the growth of Leishmania donovani inside peritoneal macrophages. Visceral leishmaniasis is a widespread parasitic disease caused by the protozoan Leishmanai donovani, an obligate intracellular parasite in man that resides and multiplies within macrophages. Moreover, in a 60-day murine model of visceral leishmaniasis, 95% of the spleen parasite burden was reduced by MDP-Man-BSA at a dose of 0.5mgkg1 day1 given for 4 days. Free MDP at a similar dose had very little effect.

3.23.6.2.4 Targeting antiparasite drugs In addition to the activation of macrophages infested by Leishmania donovani, several other antiparasite approaches have been described. MTX coupled to Man-BSA (30mol of MTX per mole of neoglycoprotein) was taken up efficiently through the mannosyl receptors present on macrophages.230,232 The drug conjugate eliminated intracellular amastigotes of Leishmania donovani in mouse peritoneal macrophages about 100 times more efficiently than free drug on the basis of 50% inhibitory dose. Inhibitory effect of the conjugate was directly proportional to the density of sugar on the neoglycoprotein carrier. MTX conjugated to BSA or other nonspecific neoglycoproteins such as Gal-BSA and Glc-BSA have leishmanicidal effects comparable to free MTX. In a murine model of experimental visceral leishmaniasis the drug conjugate reduced the spleen parasite burden by more than 85% in a 30-day model, whereas the same concentration of free drug caused little effect. These results indicate that MTX-neoglycoprotein conjugate binds specifically to macrophages, and is internalized and degraded in lysosomes releasing the active drug to act on Leishmania parasites. The anti-leishmanial potency of doxorubicin233 conjugated Man-HSA was tested in experimental visceral leishmaniasis. Conjugated doxorubicin eliminated intracellular amastigotes of Leishmania donovani in peritoneal macrophages almost 12.5 times more efficiently than did the free drug and greatly reduced and possibly eliminated spleen intracellular parasites in four consecutive dosages at 5mgkg1 day1 for 45 days. Free drug at a similar dose had little effect. Antileishmanial effect of the conjugates was competitively inhibited by Man-BSA and mannan and prevented by inhibitors (colchicine and monensin) of receptor-mediated endocytosis.

3.23.6.2.5 Targeting oligonucleotides Oligonucleotides (ODNs) and especially antisense ODNs have been shown to be active in inhibiting the expression of various proteins in several biological systems but the requested concentration to elicit a biological activity is high, around 10mM due to a poor cellular uptake and sequestration in intracellular vesicles. A number of vehicles have been proposed as ODN carriers: they include nanoparticles, micelles, lipoproteins, liposomes, poly-L-lysine, and glycoconjugates. We have shown that ODNs, bound to either a neoglycoprotein or a glycosylated poly-L-lysine neutralized by gluconoylation, are more efficiently taken up by cells expressing a membrane lectin able to bind the glycoconjugate. An ODN substituted on its 30 end with either a fluorescein residue or a

Neoglycoproteins

509

radioactive tag and linked through its 50 end to a glycosylated carrier is internalized up to 40 times more efficiently in cells which express a lectin with the ability to bind the sugars borne by the glycoconjugates.234 As an example, J774E cells (a macrophage cell line) internalize slowly free ODNs and ODNs bound to serum albumin; they internalized quite efficiently ODNs bound to mannosylated serum albumin and still more ODN bound to mannose-6-phosphate containing neoglycoproteins. The ODNs are mainly present in vesicular compartments as shown by confocal microscopy. Furthermore, on the basis of quantitative data obtained by flow cytometry, this type of targeting leads to a clear increase in the quantity of ODNs inside the cells as compared with that present outside the cells. Upon subcellular fractionation, a larger proportion of free ODNs was found in the cytosol and in the vesicular compartment when ODNs were targeted than when they were used free.220 In addition, the biological activity was also significantly increased. For instance, an ODN (specific for the sequence around the codon 12 of oncogene ras) covalently bound to Man6P-BSA was found to be quite active at a concentration for which the same ODN protected at both ends but not coupled to the appropriate neoglycoprotein was almost inactive.235 In all cases, the ODNs were linked to the glycoconjugate through a disulfide bridge: 0 0 Glycoconjugate-NH-CO-ðCH2 Þ6 -SS-ðCH2 Þ6 -O-PO 2 -5 -oligonucleotide-3 -PO2 O-ðCH2 Þ6 -NH-F with F ¼ fluoresceinylthiocarbamyl-or-CO-CH2 -pC6 H4 -OH

Since the coupling step of the ODN derivative to the neoglycoprotein occurs in a low yield, an alternative method has been developed. The ODN was not chemically linked to the neoglycoprotein but it was substituted with biotin: 0 0 Biotinyl-NH-ðCH2 Þ6 -SS-ðCH2 Þ6 -O-PO 2 -5 -oligonucleotide-3 -PO2 -O-ðCH2 Þ6 -NH-F

Independently, streptavidin was glycosylated as in the case of serum albumin. Finally, solutions of biotinylated ODN and of glycosylated streptavidin were mixed, leading to a stable complex which could be isolated by gel filtration chromatography. This complex is efficiently taken up by cells expressing a membrane lectin which binds the sugar borne by the complex.234

3.23.7 Neoglycoproteins as Sugar-Specific Antigens As a functional entity, oligosaccharides present themselves primarily in the form of either a glycoprotein or glycolipid or a proteoglycan. It is now well recognized that oligosaccharide portions present in one of the above conjugate forms and their concomitant interaction with proteins are responsible for a wide range of biological aberrations. Initial recognition events between the host cell surface bound oligosaccharide components with protein receptors are necessary, for example, on infectivity arising from a variety of viral, parasitic, and bacterial pathogens.236–239 Oligosaccharides present at the surface of bacterial cell envelopes CPSs or as LPSs act as bacterial virulence factors and are major targets of the host’s immune response upon infection. As a consequence, the antibodies recognizing the cell surface oligosaccharides of bacteria as well as other microbial pathogens are produced by the host immune system. This has been exploited for the development of purified polysaccharide vaccines. The potential of using polysaccharides as vaccines was first demonstrated in 1931 by Goebel and Avery89 (see Section 3.23.2). In the past few decades, several polysaccharide–protein conjugates have been prepared and their crucial impact in the field of antibacterial vaccination is being increasingly understood.240 A variety of structurally defined CPSs have been applied for the immunoprophylaxis of diseases caused by S. pneumoniae, N. meningitidis, and H. influenzae.241

3.23.7.1 Neoglycoproteins as Immune Response Triggers Cell surface oligosaccharides belong to the class of T-cell-independent antigens, which stimulate B cells to produce antibodies without the involvement of T cells. Upon the binding of the cell surface oligosaccharides of pathogenic organisms with mammalian cell surface-bound immunoglobulins, an immune response is elicited resulting in the formation of antibodies mainly of the IgM type. This event occurs without any role of T-helper cells and consequently no memory B cells are produced. The T-cell-independent antigenic nature of oligosaccharides has been responsible for poor immunological response to polysaccharide vaccines in infants and young children due to the immaturity of their immune system.242 In order to improve the efficacy of the immune response, a popular strategy was to conjugate the oligosaccharide component to a protein, and converting the T-independent antigen to a T-dependent antigen. This strategy is also useful to render smaller oligosaccharide haptens immunogenic by their coupling to protein carriers. Glycoproteins function as T-dependent antigens and are recognized by surface-bound immunoglobulins, leading to proteolysis of the protein part in the B cell to generate peptide fragments that interact with MHC class II molecules.

510

Neoglycoproteins

The recognition of peptide-MHC complex by the T-cell receptor (TCR) initiates the formation of both IgG-type antibody-producing and memory B cells. The use of artificial glycoproteins or neoglycoproteins can be exploited for changing the nature of immune response to the antigenic glycan determinant and inducing a class switch from IgM to IgG antibody. Immunization with neoglycoproteins, in which the cell surface carbohydrate of a microbial pathogen is covalently attached to an appropriate carrier protein, has been shown to exhibit long-lasting immunity to pathogenic microorganisms and has led to the design of various candidates for generating this protective immunity (for review of glycoconjugates on human vaccine, see Ref.: 240). The discovery that polysaccharides could be converted to T-dependent antigens upon conjugation to a protein carrier was a major achievement in the field of antibacterial vaccination, as illustrated by the commercialization of the first glycoconjugate vaccine, targeting Haemophilus influenzae type b.243 Also, carbohydrate-based vaccines against Neisseria meningitidis, Streptococcus pneumoniae, and Salmonella enterica serotype typhi (S. typhi) are already licensed, and many similar products are in various stages of development.

3.23.7.2 Rational Design of Neoglycoprotein-Based Antibacterial Vaccines A clear understanding of the oligosaccharide structure–immunogenicity relationship is necessary for the development of an efficient antibacterial vaccine. In this neoglycoprotein model, the carbohydrate’s antigenic sites are readily accessible to antibodies. The antigenic activity of such neoglycoproteins primarily depends on the density of carbohydrate hapten on the carrier protein. The immune response to neoglycoproteins is dependent on the following factors: (1) the nature of the carrier protein and its origin, that is, inherently immunogenic toxoid protein or nonimmunogenic carrier protein; (2) the sequence as well as the conformation of the oligosaccharide antigenic epitope; (3) the number of covalent linkages between the carbohydrate and carrier protein and also the resulting carbohydrate/protein ratio (carbohydrate loading); and (4) the nature of the spacer arm used for the conjugation of oligosaccharide to the carrier protein. All of these factors contribute to the three-dimensional structure of a neoglycoprotein vaccine and the accessibility of the antigenic oligosaccharide sites to ensure its high level of immunogenicity. Some of the underlying structural principles governing immunogenicity are discussed below.

3.23.7.2.1 Proteins used to prepare antigenic neoglycoproteins The family of neoglycoproteins fit into the category of glycoconjugate vaccines (Table 6) owing to their nontoxic nature and also their ability to present large number of the immunogenic carbohydrate epitopes. A large number of proteins from a variety of sources have been used in the construction of conjugate vaccines, namely, bacterial

Table 6 Neoglycoprotein vaccines for antibacterial immunity Bacterium

Carbohydrate epitope/carrier protein

Status of development

Group B Streptococcus types 1a, 1b, 2, 3, and 4 CPS Haemophilus influenzae type b

CPS derived oligosaccharides – tetanus toxoid CPS (high MW) – tetanus toxoid

Phase II

CPS (synthetic oligosaccharides) – tetanus toxoid Type b CPS (12-mers) – CRM197 Neisseria meningitides Group C

Group A Streptococcus pneumoniae

Group C CPS-derived oligosaccharides – CRM197 De-O-Ac CPS-derived oligosaccharides – tetanus toxoid Group A CPS – CRM197 CPS (extracts from 7 types of S. pneumoniae) – CRM197

ActHib® (Pasteur Me´rieux Se´rums Vaccins S.A.) Omnihib® (Smithkline-Beecham Pharmaceuticals) QuimiHib® (Centro de Ingenierıá Gene´tica y Biotecnologıá (CIGB). Ciudad Habana. Cuba HibTiter® (Wyeth-Ayerst) VaxemHib® (Chiron, Emeryville, CA) Meningitec® (Wyeth Pharmaceuticals) NeisVac C® (Baxter) Phase II Prevenar® (Wyeth Pharmaceuticals)

Neoglycoproteins

511

proteins–toxins and other membrane proteins, non-mammalian proteins such as KLH, horseshoe crab hemocyanin (HCH), and proteins from mammalian sources such as BSA, bovine g-globulin (BGG), HSA, and human immunoglobulin (IgG). The proteins derived from bacteria are attractive on account of their ready availability and immunogenic properties. In comparison to the carrier proteins of nonbacterial origin, the inherent immunogenicity of the bacterial toxins results in its role as protective antigens in human bacterial infections and is more suited for the preparation of neoglycoprotein antibacterial vaccines. In most of the cases, toxoid vaccines are used to protect against infections whose pathology or symptoms are caused by exotoxins, shed by the microorganisms (i.e., diphtheria and tetanus toxoid vaccines). One of the drawbacks in using the bacterial derived toxoids is their inherent toxicity itself, which is reduced by the treatment of the lysine residues with formaldehyde (for detoxification). Despite the blocking of the lysine residues, there are a sufficient number of accessible lysine residues to ensure adequate coupling to polysaccharide components. Most of the human vaccines are derived from tetanus toxoid244 and diphtheria toxoid,245 cross-reacting material of diphtheria toxoid (CRM197),246 and the outer membrane protein complex of Neisseria meningitidis (OMPC).247 in comparison to the protein carriers based on bacterial toxoid proteins, the use of serum albumin-based carriers is useful for (1) carrying preliminary immunological investigations, (2) developing polysaccharide-specific conjugate ELISA assay reagents for measuring antibody responses, and (3) comparison to the conjugates derived from bacterial toxoid counterparts. Using HSA conjugates of synthetic fragments of CPS of Neisseria meningitidis serogroup A, it was possible to know that even a fragment as small as a monosaccharide is sufficient for recognition by polyclonal Meningococcus A antiserum.248 In order to compare the influence of the carrier protein, synthesized fragments of CPS of S. pneumoniae type 3 were coupled to CRM197 (cross-reacting material of diphtheria toxin), KLH, and Tetanus toxoid. Such comparative studies would be helpful in the evaluation of different structural parameters in immunological tests.

3.23.7.2.2 Oligosaccharide antigenic epitopes The search for an efficient immunogenic compound has led to a detailed study of the nature of oligosaccharide segments (i.e., chain length, presented epitope, and conformation), required for inducing carbohydrate-specific immune response. Most of the studies have concentrated on the identification of minimum size required for a decent immunogenic property rather than the identification of the most potent polysaccharide isolated from natural sources. Besides isolated polysaccharides, oligosaccharides derived from the CPS can be used for the preparation of conjugate vaccines. The exact binding domain of an oligosaccharide and its conformation determine the efficiency of binding by the specific antibodies. It has been shown that carbohydrate epitopes ranging from di- to heptasaccharide have been found to be immunogenic.51 The conformation of an oligosaccharide plays an important role in its specific recognition by an antibody and out of the many flexible conformations that a polysaccharide can adopt; antibodies can be raised to recognize distinct conformational states. In addition to the complex nature of oligosaccharide sequences, the number of pathogenic serotypes that are found for each of the bacteria also adds to the complexity in the design of neoglycoprotein vaccines. For instance, S. pneumoniae is divided into different serotypes according to the chemical composition and structure of the CPS and a recent survey gives an update of the known structures of pneumococcal CPS.249 Vaccines consisting of purified CPS antigens, developed against the major serotypes of Gram-negative N. meningitides, have been effective in adults, but were found to be less efficacious amongst infants and young children. Neoglycoproteins incorporating the CPS were subsequently developed and shown to be effective against meningitides in children (see Ref.: 250 for a review on meningococcal vaccines). The polysaccharide fragments from bacterial capsule are usually obtained after hydrolysis and tedious purification of the pure fragments. Depolymerization of CPSs to produce fragments has been extensively employed to derive the carbohydrate moiety. Several groups have, therefore, studied the potency of oligosaccharide–protein conjugates containing the minimum antigenic determinant needed to induce CPS-specific antibodies. Synthetically or chemo-enzymatically derived oligosaccharides are relevant in the context of development of chemically well-defined (homogenous glycoforms) neoglycoprotein vaccines. Furthermore, they can overcome the problems of product heterogeneity and biological contamination associated with the use of native carbohydrate material. It was observed that synthetic tri- and tetrameric fragments of Haemophilus influenzae type b (Figure 20c) could induce anti-CPS antibodies in mice and monkeys.251 A single synthetic repeating unit was found to induce typespecific antibodies in rabbits in the case of a pneumococcal type 23F conjugate (Figure 20b), while the induction of human IgG required a larger epitope.252 Similar observations were found in the case of Streptococcus pneumoniae type

512

Neoglycoproteins

HO

OH O O

OH

O

O HO O

OH CH3 O

HO

OH

O HO

HO

HO

O

P

O

O

OH

O

HO

OH

O HO HO

O

P O O OH

HO

O

O

O OH

H3C HO

HO

OH

HO (a)

H3C O

O

HO

OH

(b) O

HO

O

O

O

(c)

OH

H3C HO

OH HO

OH OH OH O O P OH

O

O O

HO

AcHN HO H3 C HO (d)

O O

O

O HO

O OH

HO

Figure 20 Bacterial antigenic epitopes used for neoglycoprotein vaccines: a, Streptococcus pheumoniae 6B; b, Steptococcus pneumoniae 23 F; c, Haemophilus influenzae type B; and d, Shigella dysenteriae type 1.

6B (Figure 20a) neoglycoproteins for generating protective antibody response in mice, wherein, a single tetrasaccharide fragment constituting the repeating unit of CPS was conjugated to KLH.253 To examine the effect of chain length of oligosaccharide, several pneumococcal oligosaccharide conjugates constituted by fragments of 8, 16, and 27 repeating units for type 3 were tested. Interestingly, no variation in antibody titers was found with increasing chain length,254 suggesting that medium length oligosaccharides (>10 repeating units) can express the same conformational epitope as the polysaccharide and can be utilized for conjugate vaccine preparation. For the development of vaccines to prevent shigellosis, synthetic oligosaccharides (1–4 tetrasaccharide repeating units) of Shigella dysenteriae type 1 (Figure 20d) were conjugated to HSA, with average molar ratios of the saccharides to HSA ranging from 4 to 24.225 It was observed that the protein conjugates of synthetic saccharides elicited higher levels of serum IgG anti-LPS antibodies as compared to the neoglycoproteins derived from the attachment of acidhydrolyzed Shigella LPS saccharide fragments. Also, amongst the synthetic saccharide–KLH conjugates, the conjugate containing the largest carbohydrate, with an intermediate loading of 9, induced the highest IgG levels in mice in comparison, the conjugates with higher loadings showed a lower level of IgG. This result has important implications in the design of neoglycoprotein vaccines, since high saccharide loadings can lead to the inaccessibility of the T-cell epitopes to proteolytic enzymes due to steric shielding of the protein by the carbohydrates. Recently, a synthetic CPS antigen of Haemophilus influenza type b (Hib) was evaluated in clinical trials in Cuba.256 This synthetic conjugate exhibited long-term antibody protective titers similar to the already licensed products prepared from Hib polysaccharide extracted from bacteria.

3.23.7.3 Neoglycoprotein Vaccines for Cancer Abnormal changes in the structure of cell surface oligosaccharides are observed in cancer cells, and have been associated with malignancy.257–260 These changes are a consequence of the aberrant glycosylation due to downregulation or upregulation of glycosyl transferases and resulting in either incomplete glycosylation or incorporation of additional residues in the oligosaccharide sequences present on the cell surface. In comparison to the normal cells, the

Neoglycoproteins

513

mucins on the tumor cells are underglycosylated and bear truncated O-glycosylated structures such as TN, STN, T (Thomsen-Friedenreich) and sialylated T antigens (Figure 21). TN and T antigens are some of the earliest oncogenes discovered and are shielded in healthy tissues but uncovered in approximately 90% of carcinoma cells. Tumor malignancy is also marked by the overexpression of gangliosides (GD3, GD2, GM3, GM2, extended GM2, and fucosyl GMl) which are neuraminic acid-containing glycosphingolipids. Although these glycosphingolipids are also present in normal cells in a low density of distribution, they elicit an immune response due to their clustered presentation in tumor cells. Overexpression of gangliosides has been observed in tumors such as melanomas, sarcomas, neuroblastomas, astrocytomas, and small cell lung cancers. Unlike the antigenic carbohydrate epitopes of bacterial origin, tumor-associated antigens behave as self-antigens and poor immunogens. The problem of immune tolerance and also the possible stimulation of auto-immunity against normal cells also expressing these self-antigens pose difficulties in the development of neoglycoprotein-based antitumor vaccines. In the last decade, neoglycoproteins constituted by the tumor-associated carbohydrate antigens (Figure 21) have been prepared (Table 7) and are being used for stimulating immune response against cancer. One of the first neoglycoproteins investigated systematically for its cancer immunological response was GD3 ganglioside–KLH conjugate.261 Amongst the various protein carriers that can be utilized for the construction of antitumor neoglycoprotein vaccines, KLH has been widely used for the preparation of conjugates of TN, sialyl TN,

TN antigen

GalNAca-O-Ser/Thr

Sialyl TN antigen

Neu5Aca-6GalNAca-O-Ser/Thr

T antigen

Galb-3GalNAca-O-Ser/Thr

Sialyl T antigen

Neu5Aca-3Galb-3GalNAca-O-Ser/Thr

GM2

GalNAcb-4Galb-4Glcb-O-Cer

Fucosyl GM1

Fuca-2Galb -3GalNAcb-4Galb -4Glcb -O-Cer

GD2

GalNAcb-4Galb-4Glcb-O-Cer

Neu5Aca-3

Neu5Aca-3

Neu5Aca-8Neu5Aca-3 Globo H

Fuca-2Galb-3GalNAcb-3Gala-4Galb-4Glcb-O-R

Gb3, Pk, CD 77,

Gala-4Galb-4Glcb-Cer

KH1 epitope Figure 21 Tumor-associated carbohydrate antigens used for development of neoglycoprotein vaccines.

Table 7 Neoglycoproteins used as antitumor vaccines Tumor-associated carbohydrate antigen

Cancer types and occurrence

Protein carrier for the neoglycoprotein vaccines

TN and sialyl TN antigens

Melanomas, colorectal, lung, breast

OSA, BSA, KLH

T and sialyl T antigens GM2

Melanomas Neuroectodermal tumors (melanoma and neuroblastoma) Neuroectodermal tumors (melanoma and neuroblastoma) Adenocarninoma Small cell lung cancer Adenocarninoma

KLH KLH

273–275 262 276, 277

KLH

278,279

KLH KLH KLH

280 281,282 283

GD2 and GD3 Globo H Fucosyl GM1 KH1

References

514

Neoglycoproteins

and T antigens.262 These neoglycoproteins were investigated for clinical trials and the most efficacious response was observed from sialyl TN-KLH, resulting in the production of IgM and IgG antibodies. Synthetic globo H hexasaccharide conjugated to KLH was used as a vaccine for patients with prostate cancer who have relapsed after primary therapies such as radiation or surgery. Globo H is one of several candidate antigens present on prostate cancer cells that can serve as targets for immune recognition and treatment strategies.86 So far, the best results for inducing an antibody response against defined cancer antigens has been observed with KLH and use of the potent saponin adjuvant QS-21. The importance of glycoclustering in the design of neoglycoproteins is exemplified by the superior response of KLH conjugates of clustered TN antigens as compared to monomeric ones. When KLH conjugates of several TN constructs such as monomeric TN, trivalent cluster of TN prepared on a tri-threonine peptide, and TN prepared on a partially or fully glycosylated MUC1263 backbone, were compared by using ELISA assay against TN and FACS reactivity against TN-positive tumor cells.264 The trivalent cluster of TN was found to be more effective than TN, and conjugation to KLH gave the best results as compared to conjugation to BSA or polystyrene beads. However, KLH conjugate of MUC1 peptide partially glycosylated with TN induced the strongest antibody response against TN and tumor cells expressing TN and also antibodies against MUC1. There are several examples of neoglycoproteins loaded with tumor antigenic carbohydrate epitopes (Table 7) and the outcome of immunological studies and clinical trials with these conjugates is useful in the refinement of design of these conjugates to find the most potent tumor vaccines.265,266

3.23.8 Have Neoglycoproteins Any Future? Neoglycoconjugates are not only useful for the basic understanding of protein–carbohydrate interactions but they also have many practical applications. Obviously, some cautions should be mentioned with regard to therapeutic purposes. Due to their antigenic properties they are not suitable when their antigenicity is not the purpose of the therapy. With regard to bovine spongiform encephalopathy and related central nervous diseases, neoglycoproteins made of animal sources must be avoided; however, recombinant proteins did not suffer this restriction. However, because such neoglycoproteins were glycosylated serum bovine or HSA, it was not possible to test such devices in clinics. Similarly, the protein can be replaced by polymers such as polylysine or polyethylenimine; glycosylated polymers have been used to obtain safe glycoconjugates usable as drug carriers267,268 as well as to target plasmids (see Refs.: 13, 129, 220, and 269–271). In conclusion, neoglycoproteins remain excellent tools for several major reasons: 1. they are easily prepared from commercially available material, not only by using monosaccharides derivatives but also by using functionalized complex oligosaccharides of natural origin47,71 or glycoclusters obtained by synthesis;74 2. they are quite flexible: they can be made fluorescent, biotinylated, adsorbed on gold particles; 3. they are highly soluble in usual buffers including physiological serum; 4. they are quite stable: usually they can be freeze-dried and quantitatively solubilized; 5. they are powerful reagents suitable for many cell biology studies; and 6. they are excellent tools for the isolation and characterization of plant and animal lectins, separation of cells, as well as for the targeting of drugs, artificial vaccines, and diagnostic reagents.

Glossary affinity The attractive force exerted in different degrees between molecules that causes them to enter into and remain in chemical combination. Typically, a receptor binds its cognate with an affinity expressed as lmol1, from low affinity as in the case of a monosaccharide interacting with a lectin: about 103 up to very high affinity as in the case of biotin-binding avidin: about 1015 lmol1. avidity A characteristic of antibodies that tends to enhance their rate of combination or firmness of combination with antigens; the avidity is usually related to multivalency and is sometimes called cluster effect. This characteristic is also relevant for the interaction between lectins and (natural or synthetic) glycoconjugates. glycocluster A small molecule made of a skeleton substituted by several (usually 3–6) mono- or oligosaccharides, for instance, an oligolysine substituted by several glycosynthons. glycosynthon A conjugate obtained by substituting a small molecule such as an amino acid with a monosaccharide or an oligosaccharide

Neoglycoproteins

515

multivalency A characteristic of a molecule containing either several ligands such as in a glycocluster, in a neoglycoprotein or a receptor with several binding sites as in IgM with 10 binding sites, in lectins containing 3 to 12 binding sites. neoglycoprotein Any protein that has been substituted in vitro with several mono- or oligosaccharides: typically serum albumin substituted with about 25 carbohydrate moieties SPR (surface plasmon resonance) A method allowing the determination of both kinetic parameters and affinity constants between to interacting molecules. It is based on a very efficient measure of the variation of the refractive index of a layer containing one interacting molecule upon passing a solution containing one of its cognate.

Acknowledgments

This work was partly supported by grants from French foundations: Agence Nationale de la Recherche sur le SIDA (AIDS), Cance´ropole Grand Ouest, Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer, Vaincre la Mucoviscidose, Fondation Recherche Me´dicale. M. M. is Emeritus Professor at the University of Orleáns, A. C. R. is Research Director at Inserm, E. D. is Assistant Professor at the University of Orleáns, O. S. is a post-doctoral fellow supported by Ligue Nationale contre le Cancer.

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H.; Lloyd, K. O.; Livingston, P. O. Cancer Immunol. Immunother. 1995, 41, 185–192. 263. Taylor-Papadimitriou, J.; Burchell, J. M.; Plunkett, T.; Graham, R.; Correa, I.; Miles, D.; Smith, M. J. Mammary Gland Biol. Neoplasia 2002, 7, 209–221. 264. Zhang, S.; Walberg, L. A.; Ogata, S.; Itzkowitz, S. H.; Koganty, R. R.; Reddish, M.; Gandhi, S. S.; Longenecker, B. M.; Lloyd, K. O.; Livingston, P. O. Cancer Res. 1995, 55, 3364–3368. 265. Livingston, P. O. Curr. Opin. Immunol. 1992, 4, 624–629. 266. Croce, M. V.; Segal-Eiras, A. Drugs Today (Bard) 2002, 38, 759–768. 267. Fiume, L.; Bassi, B.; Busi, C.; Mattioli, A.; Spinosa, G.; Faulstich, H. FEBS Lett. 1986, 203, 203–206. 268. Derrien, D.; Midoux, P.; Petit, C.; Ne`gre, E.; Mayer, R.; Monsigny, M.; Roche, A. C. Glycoconj. J. 1989, 6, 241–255. 269. Frese, J.; Wu, C. H.; Wu, G. Y. Adv. Drug Delivery Rev. 1994, 14, 137–152. 270. Roche, A. C.; Fajac, I.; Grosse, S.; Frison, N.; Rondanino, C.; Mayer, R.; Monsigny, M. Cell. Mol. Life Sci. 2003, 60, 288–297. 271. Nishikawa, M. Biol. Pharm. Bull. 2005, 28, 195–200. 272. Hirayama, K.; Akashi, S.; Furuya, M.; Fukuhara, K. I. Biochem. Biophys. Res. Commun. 1990, 173, 639–646. 273. Toyokuni, T.; Hakomori, S.; Singhal, A. K. Bioorg. Med. Chem. 1994, 2, 1119–1132. 274. Holmberg, L. A.; Sandmaier, B. M. Expert Rev. Vaccines 2004, 3, 655–663. 275. Ibrahim, N. K.; Murray, J. L. Clin. Breast Cancer 2003, 3(4), S139–S143. 276. Helling, F.; Zhang, S.; Shang, A.; Adluri, S.; Calves, M.; Koganty, R.; Longenecker, B. M.; Yao, T. J.; Oettgen, H. F.; Livingston, P. O. Cancer Res. 1995, 55, 2783–2788. 277. Chapman, P. B.; Morrissey, D. M.; Panageas, K. S.; Hamilton, W. B.; Zhan, C.; Destro, A. N.; Williams, L.; Israel, R. J.; Livingston, P. O. Clin. Cancer Res. 2000, 6, 874–879. 278. Ragupathi, G.; Livingston, P. O.; Hood, C.; Gathuru, J.; Krown, S. E.; Chapman, P. B.; Wolchok, J. D.; Williams, L. J.; Oldfield, R. C.; Hwu, W. J. Clin. Cancer Res. 2003, 9, 5214–5220. 279. Musselli, C.; Livingston, P. O.; Ragupathi, G. J. Cancer. Res. Clin. Oncol. 2001, 127(2), R20–R26. 280. Gilewski, T.; Ragupathi, G.; Bhuta, S.; Williams, L. J.; Musselli, C.; Zhang, X. F.; Bommann, W. G.; Spassova, M.; Bencsath, K. P.; Panageas, K. S.; Chin, J.; Hudis, C. A.; Norton, L.; Houghton, A. N.; Livingston, P. O.; Danishefsky, S. J. Proc. Natl. Acad. Sci. USA 2001, 98, 3270–3275. 281. Dickler, M. N.; Ragupathi, G.; Liu, N. X.; Musselli, C.; Martino, D. J.; Miller, V. A.; Kris, M. G.; Brezicka, F. T.; Livingston, P. O.; Grant, S. C. Clin. Cancer Res. 1999, 5, 2773–2779. 282. Krug, L. M.; Ragupathi, G.; Hood, C.; Kris, M. G.; Miller, V. A.; Allen, J. R.; Keding, S. J.; Danishefsky, S. J.; Gomez, J.; Tyson, L.; Pizzo, B.; Baez, V.; Livingston, P. O. Clin. Cancer Res. 2004, 10, 6094–6100. 283. Ragupathi, G.; Deshpande, P. P.; Coltart, D. M.; Kim, H. M.; Williams, L. J.; Danishefsky, S.; Livingston, P. O. Int. J. Cancer 2002, 99, 207–212. 284. Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graph. 1996, 14, 27–28 and 33–38. 285. Lee, R. T.; Lee, Y. C. Biochemistry 1980, 19, 156–163.

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Biographical Sketch

Michel Monsigny, Ph.D. (Dr-e`s-Sciences) from the University of Lille, France in 1968, with Jean Montreuil as supervisor, on ‘Synthesis of O-glycosides of serine and threonine and the mechanism of beta-elimination’. Emeritus Professor of Biochemistry at the University of Orleáns, France: Department of Glycobiology, Centre de Biophysique Molećulaire, Centre National de la Recherche Scientifique. He has developed interest in the biochemistry of glycoconjugates and endogenous lectins including their chemical and cellular aspects: synthesis of glycosynthons, glycopeptides, neoglycoproteins and glycosylated polymers/endocytosis and intracellular trafficking of glycoconjugates and endogenous lectins/nuclear and membrane lectins/sugar-dependent nuclear import/sugar-dependent cell–cell recognition, in glycotargeting of drugs/biological response modifiers/anticancer drugs/antiviral drugs/antimalarial drugs, antisense oligonucleotides, antigenic peptides, and more recently, on gene therapy using synthetic carriers: glycosylated cationic polypeptides and glycosylated polyethylenimines, with the aim of targeting tumor cells as well as human airway epithelial cells. He is on the editorial board of several scientific journals: Bioscience Reports, Drug delivery, Plant Science, TIGG. He is author or co-author of over 200 papers and of 15 patents.

Annie-Claude Roche, Ph.D. (Dr-e`s-Sciences) from the University of Orleáns, France, in 1978, with Michel Monsigny as supervisor, on ‘Glycan-protein interactions: isolation and properties of Limulin’. Research Director Inserm in Centre de Biophysique Molećulaire, Centre National de la Recherche Scientifique, Orleáns. She has developed interest: in the biochemistry of glycoconjugates and cellular aspects of endogenous lectins: endocytosis and intracellular trafficking of glycoconjugates and endogenous lectins, sugar-dependent nuclear import, on glycotargeting of drugs (biological response modifiers/anticancer drugs/antiviral drugs/antisense oligonucleotides, on gene therapy using synthetic carriers: glycosylated polymers with the aim of targeting tumor cells, human airway epithelial cells as well as dendritic cells, and more recently on targeting tumor antigenic peptide on dendritic cell in a cellular vaccination purpose. She is author or co-author of over 100 papers and 5 patents.

Neoglycoproteins

Eric Duverger, Ph.D. from University of Orleáns in 1995 with Professor Michel Monsigny as supervisor. During this work, he evidenced a new glycodependent nuclear import pathway which could involve nucleocytosolic lectins. After his Ph.D., he worked with Professor F. Delmotte on plant lectin isolation. Since 1997, he is assistant professor in biochemistry and cell biology at the University of Orleáns and started a new project on lignan properties and synthesis in Chartres. In 2003, he joined back the Vectorology and Intracellular Traffic team, Department of Glycobiology, of which Doctor Annie-Claude Roche is the head, at Centre de Biophysique Molećulaire, CNRS, Orleáns, France) and is presently working on nuclear import of plasmids mediated by karyophilic sugars.

521

Oruganti Srinivas, Ph.D. (2004) from Indian Institute of Science, Bangalore, India, with Professor Narayanaswamy Jayaraman as supervisor, on ‘Multivalent carbohydrate clusters: synthesis and studies of glycoside clusters on photoswitchable and dendritic scaffolds’. Presently, a postdoctoral fellow pursuing the project ‘Glycocluster-tumor antigenic peptide conjugates for cellular vaccination’ (exploring glycotargeting for cellular vaccination) in the laboratory of Doctor Annie-Claude Roche and Professor Michel Monsigny at Centre de Biophysique Molećulaire, CNRS, Orleáns, France. His research interests are related to chemical synthesis of bioconjugates and evaluation of their biological properties, in particular, synthesis of cluster glycosides, glycodendrimers, glycopeptides and glycopolymers/bio-conjugation strategies for neoglycoprotein synthesis/thermodynamic and kinetic analysis of carbohydrate-protein interactions using microcalorimetry and surface plasmon resonance techniques/ molecular modeling/crystal structure determination of small organic molecules and study of packing interactions.

3.24 Siglecs S. Kelm and R. Ravindran, University of Bremen, Bremen, Germany ß 2007 Elsevier Ltd. All rights reserved. 3.24.1 Introduction

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3.24.2 Human Siglecs

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3.24.2.1 Siglec-1 (Sialoadhesin, Sn)

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3.24.2.1.1 3.24.2.1.2 3.24.2.1.3

Structure Specificity Occurrence and biological functions

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3.24.2.2 Siglec-2 (CD22) 3.24.2.2.1 3.24.2.2.2 3.24.2.2.3

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3.24.2.3 Siglec-3 (CD33) 3.24.2.3.1 3.24.2.3.2 3.24.2.3.3

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3.24.2.4 Siglec-4 (Myelin-Associated Glycoprotein, MAG) 3.24.2.4.1 3.24.2.4.2 3.24.2.4.3


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3.24.2.5 Siglec-5 3.24.2.5.1 3.24.2.5.2 3.24.2.5.3

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3.24.2.6 Siglec-6 3.24.2.6.1 3.24.2.6.2 3.24.2.6.3

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3.24.2.7 Siglec-7 3.24.2.7.1 3.24.2.7.2 3.24.2.7.3

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3.24.2.8 Siglec-8 3.24.2.8.1 3.24.2.8.2 3.24.2.8.3

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3.24.2.9 Siglec-9 3.24.2.9.1 3.24.2.9.2 3.24.2.9.3

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3.24.2.10 Siglec-10 3.24.2.10.1 3.24.2.10.2 3.24.2.10.3

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3.24.1 Introduction Siglecs are sialic acid-binding ImmunoGlobulin-like LECtinS. Siglecs are type I transmembrane proteins1 belonging to the immunoglobulin superfamily and have been classified as I-type lectins.2,3 There are 11 known functional human siglecs and one siglec-like molecule. Siglec-1 (sialoadhesin), Siglec-2 (CD22), and Siglec-4 (myelin-associated glycoprotein, abbreviated as MAG) form a separate distantly related evolutionary group. Siglecs 1–4 were the first Siglecs characterized and were first grouped together as the sialoadhesin family.4–7 In contrast, Siglec-3 (CD33) and Siglecs 5–11 share a high degree of sequence similarity in their extracellular and intracellular regions, and are hence collectively referred to as ‘CD33-related Siglecs’.8,9 The genes encoding CD33-related siglecs map very close to each other and are clustered on chromosome 19 in humans. Interestingly, they have been found to be rapidly evolving by different genetic mechanisms.10 Siglecs are characterized by an N-terminal V-set immunoglobulin (Ig)-like domain and 1–16 C2-set Ig-like domains depending on the Siglec on the extracellular side. The V-set domain is responsible for the sialic acid (Sia) binding of these proteins.11–13 It is known that different Siglecs bind to Sia (Figure 1) with different specificities based on the linkage and the underlying glycans.4,13–15 Although ligands for Siglecs were identified using synthetic Sia probes, the in vivo natural binding partners are yet to be characterized. It has been difficult to assess the function of Sia binding by the Siglecs in intact cells because the Sia-binding sites are often masked by endogenous Sia of the cell surface glycoproteins including the Siglecs themselves, which can be unmasked by sialidase treatment or sometimes by cellular activation.16 The relevance for ligand binding and the functions of siglecs will be discussed below for each of the Siglecs. Typical for Siglecs is their differential expression pattern on various blood cells of the circulatory system, with the exception of Siglec-4 which is expressed in the nervous system by oligodendrocytes and Schwann cells.17,18 Most Siglecs contain immuno-tyrosine inhibitory motif (ITIM) and ITIM-like motifs in their cytoplasmic tail region.19 Among the human Siglecs, Siglec-1 and Siglec-4 are notable exceptions.

3.24.2 Human Siglecs 3.24.2.1 Siglec-1 (Sialoadhesin, Sn) Siglec-1 was first identified as a receptor-binding unopsonized sheep erythrocytes.20,21 Later it was purified and characterized as a Sia-recognizing receptor under the name sialoadhesin.22 A few years later, the molecular cloning of the cDNA encoding Siglec-1 demonstrated that sialoadhesin is a typical member of the Ig-superfamily and most closely related to three other proteins of this family with at that time unknown specificities, CD22, CD33, and MAG.23 Whereas the genes encoding these three proteins are located in the same region of the murine and the human genomes, the genes encoding Siglec-1 are not linked to the genes of other Siglecs and map to chromosome 20 in humans and chromosome 2 in mice.24

3.24.2.1.1 Structure Siglec-1 is 185kDa protein with a total of 17 Ig-like domains, 1 V-set domain, and 16 C2-set domains,23 with the N-terminal V-set domain containing the sialic acid-binding domain.25 Like all other Siglecs, sialoadhesin contains COOH OH HO

O OH

AcNH OH

Figure 1

OR

Siglecs

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Siglec-1 (Sialoadhesin)

Figure 2

several potential N-glycosylation sites. Its overall structural features are shown in Figure 2. Unusual is the large number of C2-set domains which is the highest in the Ig superfamily. A pairwise comparison of the C2-set domains led to the conclusion that most likely the large number of C2-set domains has developed from the duplication of sets of two domains.26

3.24.2.1.2

Specificity

In the initial characterization of Siglec-1 as a Sia-recognizing receptor it was shown that Siglec-1 prefers a2-3-linked Sia over a2-6-linked Sia.22 Furthermore, evidence was provided that an additional Sia a2-6-linked to an internal GalNAc enhances binding. The binding specificity of murine Siglec-1 has been investigated in fair detail and it represents one of the most studied Siglecs in this respect. At this point it is worth mentioning that binding characteristics of mouse and human Siglec-1 appear to be identical27 and hence all results with mouse Siglec-1 probably could be extrapolated to human Siglec-1 as well. Binding studies with synthetic oligosaccharides28 and gangliosides29 confirmed that Siglec-1 prefers a2-3-linked Sia but does not differentiate very much between the underlying glycans. However, modifications of Sia-like 9-O-acetylation or hydroxylation of the acetyl group can prevent Siglec-1 binding to sialylated ligands.14 Structural details of ligand binding have been investigated extensively for Siglec-1 compared to other Siglecs using site-directed mutagenesis,25,30 X-ray crystallographic studies,31–34 and nuclear magnetic resonance (NMR) techniques.35,36 As mentioned previously, the Sia-binding site was shown to be located in the N-terminal V-set domain.25 Further mutational studies revealed that the GFCC0 C00 b-sheet centered around a conserved arginine residue in the F-strand to be the region responsible for Sia binding. A conservative mutation of the arginine to lysine completely abolished Sia binding.30 X-ray crystallographic analysis of Siglec-1 complexed with a2-3-sialyllactose revealed that this conserved arginine residue (Arg97 in Siglec-1) is engaged in a salt bridge with the carboxylate group of the bound Sia.31 Furthermore, the crystal structure (Figure 3) showed two conserved tryptophan residues (Trp2 and Trp106) making hydrophobic interactions with the acetyl group at position 5 and the glycerol side chain, respectively. Finally, hydrogen bonds between the hydroxyl groups at C-8 and C-9 of Sia and the NH and C¼O of Leu107 stabilize the interaction as confirmed by inhibition experiments with synthetic Sia derivatives.37

3.24.2.1.3

Occurrence and biological functions

Siglec-1 or sialoadhesin was first discovered in mouse macrophages as a molecule mediating Ca2þ-independent binding and agglutination of sheep erythrocytes.20,21 Later it was shown to be a tissue macrophage-specific molecule which has Sia-specific binding activity.22 Although Siglec-1 is expressed in most resident tissue macrophages, it was not seen in brain microglia.38 Siglec-1 expression is regulated by serum factors. This was concluded from the observation that murine peritoneal macrophages express Siglec-1 only if exposed to mouse serum. Also the lack of Siglec-1 expression on microglia was explained by the blood–brain barrier.38 However, the factors regulating Siglec-1 expression are still unclear. Whereas the cytokine interleukin-4 (IL-4) downregulates Siglec-1 expression in peritoneal murine macrophages,39 in rat macrophages an upregulation has been observed with IL-4, IFN-b, IFN-g, and LPS along with glucocorticoids.40 Its extended structure is thought to allow Siglec-1 to avoid the interactions with sialylated glycoconjugate structures present on the macrophage glycocalyx. This hypothesis has been termed as the ‘rainforest model’.41 Like all other Siglecs, sialoadhesin contains several N-glycans which can serve as binding sites for ‘self-binding’ if appropriately sialylated. In fact, the binding of Siglec-1 has been shown to increase upon its desialylation,42 a phenomenon which is likely to be due to self-binding and is found with many other Siglecs. Also in vivo, the availability of Siglec-1-binding sites appears to be regulated, as it was shown that lymph node rat macrophages express high levels of unmasked Siglec-1 as compared to spleenic macrophages.43

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Figure 3

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Siglec-2 (CD22)

Figure 4

Ultrastructural localization studies revealed that Siglec-1 is highly concentrated at the contact zones between macrophages and myelo-monocytic cells and lymphocytes in murine bone marrow and lymphoid tissues.44 Overlay and in vitro binding assays provided evidence that Siglec-1 can function as a lymphocyte adhesion molecule of tissue macrophages binding to T-cells and B-cells45 and quantitative binding experiments demonstrated that murine Siglec-1 binds preferentially to granulocytes.46 Several studies have been performed to identify the binding partners for Siglec-1. Nevertheless, the situation is still largely unclear. The observation that MUC1, a heavily sialylated mucin isolated from breast cancer cells, can act as a binding partner for Siglec-1 indicates its possible role in cancer.47 Another study suggested that CD43 is a Siadependent binding partner for Siglec-1 on T-cells.48

3.24.2.2 Siglec-2 (CD22) Siglec-2 was originally described as CD22, identified using monoclonal antibodies specifically recognizing B-cells.49 Chromosome mapping studies showed that Siglec-2 mapped to chromosome 19.50 Together with sialoadhesin, CD33, and MAG, CD22 was assigned as a member of the sialoadhesin family within the immunoglobulin superfamily, based on the sequence similarity and the common specificity for sialylated structures.4–7 With respect to its biological function as inhibitory molecule in the process of B-cell activation, Siglec-2 (CD22) is probably the best-characterized Siglec until now.

3.24.2.2.1 Structure One of the earliest studies showed that Siglec-2 is structurally related to MAG and that it mediates adhesion to monocytes and erythrocytes in classical rosetting assays when expressed in COS cells.51 Further analysis of the gene encoding CD22 demonstrated that Siglec-2 is a 140kDa glycoprotein with seven Ig-like domains (Figure 3).50,52 Its overall structural features are shown in Figure 4.

Siglecs

527

Siglec-2 is heavily glycosylated containing 11 potential N-glycosylation sites. Six of these are found in the N-terminal two domains. Whereas desialylation of Siglec-2 enhances ligand binding, the deletion mutation of one N-glycosylation site within the first domain leads to the loss of Sia-binding capacity.53 However, this site is on the face of this domain opposite to the binding site.31,54 Therefore, it is unlikely that this glycan is involved in binding. Since the corresponding Asn is next to the Cys involved in the proposed interdomain disulfide bridge, it is more likely that the loss of binding is due to improper folding of the domain in the mutant protein. It is worth mentioning that this glycosylation site is well conserved in all other Siglecs as well, except for Siglec-1 and Siglec-8.

3.24.2.2.2

Specificity

In the earliest studies trying to identify CD22 ligands 2,6-sialyltransferase and CD45 had been proposed as binding partners on B- and T-cells, respectively. Subsequently, it was shown that these interactions are indeed Sia-dependent55 and Siglec-2 was shown to bind to its ligand in a a2-6-linked Sia-dependent manner.4,56,57 Using epitope mapping with a panel of monoclonal antibodies,58 domain deletion and recombination experiments59,25 identified domains 1 (V-set domain) and 2 (C2-set domain) as the location of the Sia-binding site. The exact Sia binding-site location of Siglec2 was found to be centered around the arginine residue in the F-strand of the GFCC0 C00 b-sheet of the first V-set domain, just like in the case of Siglec-1.54 Interestingly, in contrast to Siglec-1 and Siglec-4, murine Siglec-2 strongly prefers Neu5Gc over Neu5Ac with the same specificity for a2-6-linked Sia.14 Similar to Siglec-1 and Siglec-4, 9-O-acetylation of Sia masks the natural ligands also of Siglec-2.60 As in the case of Siglec-1, human Siglec-2 also binds better upon desialylation and sialylation abrogates adhesion if CD22 contains a2-6-linked Sia.61 Due to the linkage specificity this is not observed if Siglec-2 is expressed in CHOcells that express a2-3-linked Sia almost exclusively. In addition, for murine Siglec-2 this holds only for a2-6-linked Neu5Gc.

3.24.2.2.3


Siglec-2 was discovered as a B-cell-specific molecule.49,62 More recently, it has been shown that Siglec-2 is also expressed in basophils63 and is secreted by neurons.64 It is also worth mentioning that like Siglec-1, Siglec-2 is also downregulated by IL-4.65 The potential of CD22 to mediate the adhesion of monocytes and erythrocytes was identified by conventional cell rosetting assays more than 15 years ago.51 Little later it was also shown that CD22 mediates the binding of B- and T-cells52,66 or neutrophils.67 Although initially characterized as an adhesion molecule, binding to molecules on the same cell surface (cis-binding) has been implicated to be relevant for the biological role of Siglec-2 by many studies, since the first observation that sialylation of Siglec-2 itself can interfere with the detection of its binding activity,61 commonly called masking effect. Obviously, also other sialylated molecules on the same cell surface (cis-binding) can also mask the binding sites of Siglec-2.16 Furthermore, Siglec-2 appears to be constitutively enganged in cis-interactions and is found constitutively unmasked in B-cells of a-2,6-sialyltransferase ST6Gal I knockout mice, showing that the masking effect is due to a2-6linked Sia on cis-ligands.68 The a2-6-linked trans-Sia ligands mediate accumulation of Siglec-2 to the site of cell contact and the cis-masking does not thwart the redistribution of Siglec-2 to these cell contact sites.69 It was believed that CD45, a B-cell surface glycoprotein, and surface IgM, carrying a2-6-linked Sia, are Sia-dependent cis-ligands of Siglec-2 but cross-linking experiments suggested that this is not the case.70 Using a photoaffinity labeling approach, it was elegantly shown that Siglec-2 themselves are their own Sia-dependent ligands on the cell surface, forming homomultimeric complexes via protein–glycan interactions.71 Siglec-2 knockout mice showed that the CD22 molecule is a negative regulator of B-cells and show that upon its knockout the B-cells become hyperreactive,72,73 but some reports proposed that Siglec-2 might be a positive regulator of B-cells as well.74,75 The cytoplasmic tail of Siglec-2 contains three ITIM signaling conveying inhibitory signals. For Siglec-2 these motifs have been shown to function by recruiting signaling molecules, such as SHP-1, PLC-g1, Syk, PI3-kinase, and Lyn.76–80 Further, in vivo functions proposed for Siglec-2 include homing of B-cells.81–85 Also a role for Siglec-2 in the recognition of Sia as ‘self’ has been proposed.86 At this point it is worth reminding that Sia are found only in higher animals and Neu5Gc is not found in humans in contrast to most other Sia-containing animals. Humans have only Neu5Ac as their major sialic acid. If this ‘self-indication’sialylation is modified by pathogenic viruses or bacteria by sialidases, Siglec-2 binding is disturbed and the negative regulation of Siglec-2 is modulated leading to altered threshold levels for B-cell activation in response to the pathogen invasion. Hence, Siglec-2 increases the threshold of B-cell reactivity.85 This also could provide a mechanism for the B-cell avoidance of autoimmunity.86,87

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Siglecs

In accordance with this proposed mechanism it has been shown that interactions of CD22 with sialylated molecules are required for Siglec-2-mediated negative regulation.88,89 From another study90 with knock-in mice expressing Siglec-2 mutants unable to bind Sia, but with intact cytoplasmic tail it was concluded that Siglec-2 functions via two different mechanisms, ligand-dependent and ligand-independent mechanisms. If ligand binding is necessary for the negative regulation of Siglec-2 function, then a loss of Siglec-2 ligands should result in an activated phenotype of the B-cells. However, a recent mouse model with a ST6Gal I knockout showed that Siglec-2 ligand deficiency suppresses the B-cell rather than making it hyperreactive and this suppression was compensated upon additional knocking out of CD22.91,92 This unexpected result might be due to differential mechanisms of Siglec-2 action or due to the presence of another a-2,6-sialyltransferase different from ST6Gal I which makes possible availability of Siglec-2 ligand even in the absence of ST6Gal I. Such an enzyme could be a second a-2,6-sialyltransferase characterized.93,94 However, it is not clear whether this enzyme is expressed on B-cells of ST6Gal I knockout mice. In addition, expression of other Siglecs on B-cells cannot be ruled out which does not require a2-6-linked Sia as ligands. In another study90 it was shown that ligand-binding mutant Siglec-2 expressing B-cells showed neither suppressed phenotype nor were hypersensitive, which is the expected phenotype fitting to the above-mentioned proposed mechanism, but rather showed normal wild-type-like response. However, there is one report which showed that B-cells from mice suffering from systemic lupus erythematosus, which results in an aberrant ligand-binding defective form of Siglec-2 with an intact cytoplasmic tail, does indeed have an activated phenotype.95

3.24.2.3 Siglec-3 (CD33) Siglec-3 has been known as CD33 for over 20 years and has been studied as a myeloid lymphoma marker in the clinical research, long before any other Siglec was known.96 Siglec-3 was mapped to chromosome 19.97 Early work on cDNA isolation and cloning suggested CD33 to be related to MAG (Siglec-4).98 It took another 7 years to identify the Sia-binding activity of Siglec-3 based on its structural similarity to the Siglecs known at that time.5–7

3.24.2.3.1 Structure Siglec-3 has an apparent molecular mass of 67kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) while its predicted mass is 40kDa. This is due to the glycosylation of Siglec-3 with its five potential glycosylation sites. This protein has two Ig-like domains, one V-set domain and one C2-set domain, and is the smallest member of the Siglec family (Figure 5).98 It was shown that Siglec-3 occurs in two alternative splice forms with cell-type-specific expression.99

3.24.2.3.2 Specificity Like Siglec-2, Siglec-3 preferentially binds to a2,6-linked Sia, but also recognizes a2-3-linked Sia.15,100 Siglec-3 always has to be unmasked for its engagement with ligands. Like in the case of Siglec-2, Siglec-3 can also be self-masked and removal of the glycosylation site in the V-set domain increases the binding ability of this lectin by unmasking it.53

3.24.2.3.3 Occurrence and biological functions Apart from being expressed on myeloid lymphoma where it was discovered, Siglec-3 is expressed on umbilical cord blood-derived natural killer cells (NK cells) and normal human activated T-lymphocytes.101,102 A thorough screening of the hematopoietic cells later revealed that Siglec-3 is expressed on monocytes, at lower levels on macrophage subsets and dendritic cell subsets and at low levels on granulocytes.103,104 It was also observed that myeloid precursors and acute and chronic myeloid leukemia cells also express Siglec-3.105–107

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Figure 5

Siglec-3 (CD33)

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Because of its small size Siglec-3 cannot protrude out from the cell surface glycocalyx like Siglec-1. In contrast, it is buried among the cell surface glycoproteins and hence is constitutively masked. Like Siglec-2, Siglec-3 also has ITIM motif and an ITIM-like motif which has been implicated in cell signaling and inhibitory functions through recruitment of SHP-1 and SHP-2 tyrosine protein phosphatases.108,109 Paul et al.110 showed that Siglec-3 is phosphorylated by a Src-family kinase namely Lck. Regarding the biological function of such phosphorylation it was shown that Siglec-3 is involved in inhibition of proliferation of normal and myeloid leukemic cells.105 Moreover, it was also proved that Siglec-3 engagement results in apoptosis of these leukemic cells.111,106 In agreement with the inhibitory role of Siglec-3, it has been described that CD33þ NK cells displayed lower cytotoxic effects than their CD33 counterparts.102 Siglec-3 intracellular signaling is mediated by PI3 kinase and the inhibitory activity of Siglec-3 required Sia recognition.112 It was also observed that Siglec-3 ligation resulted in inhibition of cellular differentiation, such as development of dendritic cells from monocyte or CD34þ precursor cells113 and that cross-linking of Siglec-3 inhibited NKL cell cytotoxicity.99 To further complicate the story, Siglec-3 was shown to be serine/threonine phosphoprotein rather than the classical view of it being a tyrosine phosphoprotein and this phosphorylation also had effects on its ligand binding.114

3.24.2.4 Siglec-4 (Myelin-Associated Glycoprotein, MAG) Although the properties of Siglec-4 as a cell-adhesion molecule have been investigated for many years since its description,115 its specificity for sialylated molecules has remained unnoticed until 1994.4

3.24.2.4.1

Structure

MAG is a heavily glycosylated protein of about 100kDa with 30% of its mass being made up by carbohydrates.115 MAG is expressed in two splice variant forms, L-MAG which is 72kDa and S-MAG which is 67kDa.116 It has eight glycosylation sites, all of which are utilized, and five Ig-like domains (one V-set domain and four C2-set domains) (Figure 6).117

3.24.2.4.2

Specificity

Like sialoadhesin, MAG recognizes Sia in an a2,3 but not in an a2-6 linkage.4,37,118 Like Siglec-1, Siglec-2, and Siglec-3, Siglec-4 also mediates Sia binding through a conserved arginine (R118) in the first V-set domain which is necessary for Sia-binding.119 As in the case of other Siglecs such as sialoadhesin, CD22, and CD33, Sia binding of MAG is mediated mainly by interactions of the carboxyl group, the hydroxyl groups at positions 9, and the N-acyl group.37,28

3.24.2.4.3


Siglec-4 or MAG is the only exception in the Siglec family, in that it is not being expressed by the cells of the hematopoietic system but is rather expressed by oligodendrocytes and Schwann cells of the central and peripheral nervous systems, respectively120–122 and Mu¨ller cells of the retina.123 Siglec-4 contains a tyrosine phosphorylation site in its cytoplasmic tail. However, in contrast to most other Siglecs, this is not in the context of an ITIM. Under normal circumstances, MAG appears to function in the formation and maintenance of intact myelin.17 It came as a surprise when MAG was shown in two independent studies to be one of the major neurite outgrowth-inhibiting molecules in myelin.124,125 In this way Siglec-4 is different because it does not appear to inhibit the signals of the cell in which it is expressed (oligodendrocytes) but rather inhibits a neighboring effector neuronal cell. Along this line, evidence has been provided that also Siglec-4 binding can be regulated by sialylated cis-ligands.126 However, Siglec-4 also binds to sialylated trans-ligands on neurons or the extracellular matrix.127–129 Siglec-4 has also been seen to bind to different gangliosides in a Sia-dependent manner.29,118,130,131

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Figure 6

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Figure 7

This Sia-dependent binding seems to be nonessential for MAG for neuronal inhibition in some studies suggesting that the Sia-binding site is distinct from its neurite outgrowth-inhibition site.119 However, data from other models suggest that inhibition aided by MAG might indeed be Sia dependent.132

3.24.2.5 Siglec-5 Siglec-5 was first identified by molecular cloning of its cDNA from a human activated monocyte cDNA library.133

3.24.2.5.1 Structure It is a 70kDa type I transmembrane protein that exists as 140kDa homodimer on a cell surface like Siglec-3. It has four Ig-like domains with one N-terminal V-set domain and three C2-set domains. The V-set domain contains the conserved arginine necessary for Sia binding. In its cytoplasmic tail, an ITIM and an ITIM-like motif characteristic for most Siglecs can be identified.133,134 Its overall structural features are shown in Figure 7.

3.24.2.5.2 Specificity Siglec-5 bound erythrocytes and soluble glycoconjugates in a Sia-dependent manner and recognized both a2-3-linked and a2-6-linked Sia equally well.133–135 Another study showed that Siglec-5 had a preference for a2-3 over a2-6 linkages.15 It was shown that Siglec-5 can also bind to a2-8-linked Sia and to sialylated Tn epitopes (Neu5Aca26GalNAca).100 Unlike CD33 binding of erythrocytes which strictly require pretreatment of Siglec-3-expressing cells with sialidase to remove cis-Sia ligands to detect its binding activity, Siglec-5 shows binding without sialidase treatment.133 In contrast to Siglec-1, Siglec-2, and Siglec-3, neither desialylation of the molecule nor the mutation of the N-glycosylation site in the first V-set domain resulted in modification of Siglec-5-binding of Sia.136

3.24.2.5.3 Occurrence and biological functions Siglec-5 is expressed very weakly on B-cells, at intermediate levels on monocytes and macrophages, at high levels on neutrophils, and was also found on monocyte-derived and plasmacytoid dendritic cells.103,104,133,137 Furthermore, Siglec-5 expression is also observed on acute myeloid leukemic cells.137 Recent studies provide first clues to Siglec-5 functions. While one study showed that Siglec-5 engagement aided in the priming of neutrophils,138 yet another study demonstrated that Siglec-5 might actually function as an endocytic receptor.104 Siglec-5 behaves as an inhibitory receptor like other Siglecs. It was shown that Siglec-5 inhibits FceR I, an activating Fc-receptor by recruiting SHP-1 and SHP-2 to its ITIM motif. Not only this but the phosphorylation and recruitment of SHP proteins decreased the Sia-binding capacity of Siglec-5, and even more surprisingly the inhibitory function was also seen in the absence of the phosphorylation of the tyrosine in the ITIM motif.139

3.24.2.6 Siglec-6 Siglec-6 was first identified as a CD33 (Siglec-3)-related molecule in the placenta140 and later rediscovered as a leptin-binding protein.141 Due to its amino acid sequence relatedness and Sia-binding capacity, it was categorized as a Siglec.141

3.24.2.6.1 Structure Siglec-6 has three Ig-like domains (one V-set and two C2-set) on the extracellular side and an ITIM and an ITIM-like motif on its cytoplasmic tail (Figure 8). The V-set domain contains all the conserved amino acid residues needed for Sia binding of Siglecs.

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Siglec-6

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Figure 8

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Figure 9

3.24.2.6.2

Specificity

Siglec-6 selectively recognizes sialylated Tn structures.100,141 Usually, Sia-binding of Siglecs requires the carboxylate to interact with the conserved arginine and the glycerol side chain to make contact with a conserved tryptophan. Siglec-6 recognition of Sia seems to be unusual in that Siglec-6 apparently does not require the intact glycerol side chain of Sia for binding.100

3.24.2.6.3


Siglec-6 expression is seen on B-cells albeit to moderate amounts while high expression of Siglec-6 seen on placental trophoblasts is unusual for Siglecs.141 Very recently, Siglec-6 expression was also observed on mast cells.142 Siglec-6 is one of the least studied of the Siglecs and much work needs to be done to understand its possible function.

3.24.2.7 Siglec-7 Siglec-7 like other Siglecs is a type I transmembrane protein and shares structural homology with other Siglecs.143–145 Siglec-7 has been mapped to chromosome 19 in humans, to the region where most Siglec genes are clustered.10,143–145

3.24.2.7.1

Structure

Siglec-7 has three Ig-like domains, one V-set domain which carries the well-conserved arginine necessary for Siabinding, two C2-set domains, and ITIM and ITIM-like motifs in its cytoplasmic tail and has eight potential glycosylation sites.143,144 Siglec-7 has a molecular weight of 75kDa when present on its naturally expressing parent cells but when exogenously expressed on CHO cells or COS cells, it has a slightly lower molecular weight of about 65kDa due to differences in glycosylation.143,144 Its overall structural features are shown in Figure 9. Structural studies of Siglec-7 revealed the involvement of the conserved arginine which makes salt bridge with the carboxylate residue, a conserved tryptophan which makes hydrophobic interactions with the glycerol side chain, a tyrosine which probably makes hydrogen bonding with the N-acetyl carbonyl. This study also suggests that the C–C0 loop could play an important role in different linkage and underlying sugar specificity shown by different Siglecs.146 A detailed structural study demonstrated that a lysine residue in the ligand-binding pocket is as important as the conserved arginine residue which is implicated in Sia binding and a mutation of this lysine to alanine abolishes Sia binding.147

3.24.2.7.2

Specificity

Siglec-7 bound erythrocytes and soluble glycoconjugates in a Sia-dependent manner.143–145 However, while one study reported Siglec-7 bound to both a2-3- and a2-6-linked Sia,143 another study reported that it could bind only Sia in a2-6 linkages.145 This discrepancy may be due to the difference in the assay methods. While the former used cell

532

Siglecs

surface-expressed Siglec-7, the latter used soluble Fc-chimeras. Like most other Siglecs, Siglec-7 requires the glycerol side chains on the Sia for optimum ligand binding.145 A comparison of different glycan structures in an array assay revealed the following linkage preference of Siglec-7 for its Sia ligands: a2-8>branched a2-6>sialylated Tn>a2-6.15 This study also observed that Siglec-7 bound to a2-3-linked Sia negligibly. This Siglec-7 ligand specificity was also confirmed by studies involving gangliosides.148,149 Siglec-7 in not masked that well by endogenously expressed cis-Sia ligands145 and neither desialylation nor the N-glycosylation in the V-set domain appear to regulate ligand binding.136 These studies used exogenously expressed Siglec-7 on COS cells but COS cells have low endogenous levels of a2-6-linked Sia structures which are preferred ligands for Siglec-7. This might explain why the unmasking effect is not observed in these assays.136 Later it was shown that Siglec-7 is, in reality, constitutively masked on NK cell surface where it is naturally expressed.150

3.24.2.7.3 Occurrence and biological functions Siglec-7 is expressed on NK cells, CD8þ T-cell subset, monocytes, macrophages, dendritic cells, and acute myeloid leukemic cells.104,106,107,143,144 Studies to decipher Siglec-7 functions support the predicted role of this Siglec as an inhibitory receptor. Crosslinking cell surface Siglec-7 resulted in reduction of NK cell cytotoxicity and cross-linking with activating receptors resulted in their diminished activity.144 Engagement of Siglec-7 was also seen to inhibit proliferation of normal and leukemic myeloid cells.144,151 As anticipated, Siglec-7 functions in an inhibitory manner upon ligation with transligands and is able to decrease the cytotoxic effects of NK cells.150 Conversely, disengagement of natural in vivo trans-ligands could lead to increase in cytotoxic reactions of NK cells. In this regard, it is seen that human colon cancer cells downregulate expression of disialyl Lea structures which are bound by Siglec-7.152 However, the disialyl Lea structure has not yet been proved to be the natural in vivo ligands of Siglec-7. As Siglec-7 is also expressed on T-cell subsets, its inhibitory function was also tested on these cell types and this was indeed the case.153 Additionally, like other previously characterized Siglecs, the mechanism of Siglec-7 inhibition appears to be also mediated by SHP-1/2 recruitment at the phosphorylated tyrosine of the ITIM motif and this motif is critical for the inhibitory activity.154 Like in the case of Siglec-3 and Siglec-5, for Siglec-7, Sia-binding capacity increases upon decreased phosphorylation of the ITIM motif. Reciprocally, increase in cytoplasmic tail phosphorylation decreases Sia-binding activity.154

3.24.2.8 Siglec-8 Siglec-8 was first discovered from an eosinophil cDNA library.155,156 The Siglec-8 gene has been mapped to chromosome 19 of the human chromosome.156,157

3.24.2.8.1 Structure Siglec-8 has three Ig-like domains in its extracellular side with an N-terminal Sia-binding V-set domain and two C2-set domains (Figure 10).155,156 Unlike other Siglecs, Siglec-8 was first thought not to contain the conserved ITIM motifs in its cytoplasmic tail.155,156 But these were soon proved to be the splice-variant forms and the ITIM containing full length form was characterized.157

3.24.2.8.2 Specificity Sia binding of Siglec-8 was shown with erythrocyte-binding assay initially.155,156 Like Siglec-1, Siglec-8 does not contain an N-glycosylation site in its V-set domain,136 but Siglec-8 is also masked by endogenous Sia structures like other Siglecs.155,156 Initial studies suggested that Siglec-8 prefers a2-3-linked Sia over a2-6-linked Sia.15,156 Later, a detailed screening of different glycans showed that Siglec-8 shows a high affinity toward 60 -O-sulfo-sLex (Neu5Aca2-3 (6-O-sulfo)Galb1-4[Fuca1-3]GlcNAc).158

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Figure 10

Siglec-8

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Siglec-8 expression is very specifically confined to cells involved in the allergy reactions, namely eosinophils, basophils, and mast cells.142,155,156 Siglec-8, like other Siglecs, is an inhibitory receptor by virtue of its function due to the ITIM and ITIM-like motifs in its cytoplasmic tail. It is seen that engagement of Siglec-8 brings about apoptosis in eosinophils159 and that the mechanism of this apoptotic event is dependent on reactive-oxygen species, mitochondrial injury, and caspases.160 Keeping this apoptotic effect of Siglec-8 in mind, the ligand specificity toward 60 -O-sulfo-sLex, and its selective expression on inflammatory cells, it has been postulated that the expression of Siglec-8 may be a way to eliminate these cells that have infiltrated the central nervous system since these 60 -O-sulfo-sLex structures are found largely in brain tissue.158,161 However, the in vivo ligands of Siglec-8 are yet to be characterized.

3.24.2.9 Siglec-9 Siglec-9 was identified in three independent studies: it was isolated from a cDNA library,162 it was discovered during 50 -rapid amplification of cDNA-ends experiment during Siglec-7 cloning,163 and it was identified by computer algorithm (gene prediction program) from the human genome sequence.164 The Siglec-9 gene has been mapped on the ‘Siglec cluster’ on the human chromosome 19.163,164

3.24.2.9.1

Structure

Siglec-9 is a Siglec with three Ig-like domains, one V-set domain which is responsible for Sia binding and two C2-set domains. Like other characterized Siglecs, Siglec-9 also has ITIM and ITIM-like motifs in its cytoplasmic tail.162–164 Its overall structural features are shown in Figure 11.

3.24.2.9.2

Specificity

Siglec-9 binds to erythrocytes in a Sia-dependent manner, recognizing a2-3-linked and a2-6-linked Sia.162,163 Siglec-9 also binds to sialylated Tn-structures similar to Siglec-2, Siglec-3, Siglec-5, Siglec-6, Siglec-7, and Siglec-8, albeit weakly. Only Siglec-6 binds this structure with high affinity.15,100,163

3.24.2.9.3


Siglec-9 has originally been found to be expressed abundantly on monocytes and neutrophils and weakly on NK-cell subsets and T-cell subsets162,163 and later on macrophages, dendritic cells,104 and on acute myeloid leukemic cells.107 In decoding Siglec-9 functions, it has been shown that Siglec-9 inhibits monocyte, NK-cell, and T-cell signaling by engaging SHP-1 and SHP-2 to the ITIM motif.153,154 Later studies have indicated that Siglec-9 could also function as an apoptotic receptor on neutrophils and myeloid leukemic cells.107,165 In addition, Siglec-9 apparently functions as an endocytic receptor on acute myeloid leukemic cells.166 Siglec-10 is also mapped to the ‘Siglec cluster’ on chromosome 19.167–169

3.24.2.10 Siglec-10 3.24.2.10.1

Structure

Siglec-10 is a Siglec that contains one V-set domain and four C2-set domains, a total of five Ig-like domains (Figure 12).167 Several splice variants have been reported for Siglec-10.168–171

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Siglec-9

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Figure 12

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Siglec-11

Figure 13

3.24.2.10.2 Specificity As for all Siglecs, Siglec-10 binding to erythrocyte requires cell surface Sia.167,170 Further investigations of its ligandbinding specificity showed that Siglec-10 can bind to both a2-3-linked and a2-6-linked Sia,15,167,170 while another study provided data suggesting that Siglec-10 prefers a2-6 over a2-3 linkages.168 Siglec-10 is also seen to bind to sialylated Tn-epitope.15

3.24.2.10.3 Occurrence and biological functions Siglec-10 has been detected on eosinophils, neutrophils, monocytes, activated dendritic cells, and macrophages, subsets of NK- and B-cells and on acute myeloid leukemic cells.104,107,167,168,170 Like other Siglecs, Siglec-10 appears to be engaged in binding to cis-ligands on the cell surface. Siglec-10 has three inhibitory motifs in its cytoplasmic tail, two ITIM and one ITIM-like motif which is implicated in its inhibitory functions.167,170,171 Siglec-10 is phosphorylated by tyrosine kinases and the cytoplasmic tail has been shown to recruit SHP-1 and SHP-2 phosphatases after phosphorylation of its ITIMs.168,171 Inhibitory functions of Siglec-10 have not been described yet, although Siglec-10 appears to function as an apoptotic receptor on myeloid leukemic cells.107

3.24.2.11 Siglec-11 Very little is known about Siglec-11 until now, since it has not been studied in detail since its first description.172 The gene encoding Siglec-11 does not map to the Siglec cluster on chromosome 19, but has been found in another location on chromosome 19.

3.24.2.11.1 Structure Siglec-11 is a five-domain Siglec with one V-set and four C2-set Ig-like domains on the extracellular side and with an ITIM and an ITIM-like motif in its cytoplasmic tail.172 Its overall structural features are shown in Figure 13.

3.24.2.11.2 Specificity Unusual binding property of Siglec-11 was observed, with this Siglec showing good binding neither with a2-6-linked nor with a2-3-linked Sia but only with a2-8-linked polymers of Sia.172

3.24.2.11.3 Occurrence and biological functions Siglec-11 is not found on peripheral blood cells like most other Siglecs but it is expressed on tissue macrophages and brain microglia.172 In the same study it was also shown that Siglec-11 could recruit SHP-1 and SHP-2 phosphatases to its ITIM upon its phosphorylation, suggesting an inhibitory role of this Siglec.

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3.24.2.12 Siglec-12 Siglec-12 is not a true Siglec in humans and is called a Siglec-like molecule. This is because it cannot bind Sia as it does not contain the conserved arginine residue. However, it is a functional Siglec in apes in chimpanzees,173 and its gene has been mapped to the ‘Siglec cluster’ on chromosome 19.174,175

3.24.2.12.1

Structure

Siglec-12 contains four Ig-like domains, two V-set and two C2-set domains.173 Two spliced forms of this Siglec have also been reported.174

3.24.2.12.2

Specificity

Restoring the essential arginine in the first V-set domain actually refurbishes Sia-dependent binding of erythrocytes,173 while another study reported Sia binding even without the essential arginine.175

3.24.2.12.3


Siglec-12 is expressed on epithelial cells173 and monocytes/macrophages.175 This study also reported SHP-1 and SHP-2 recruitment to ITIM motif upon its tyrosine phosphorylation,175 implying an inhibitory function of this molecule. How this inhibitory role is carried out in the absence of a ligand-binding function in humans awaits further investigation. It is very interesting to note that this molecule is present as a ligand-binding functional molecule in the great apes. The study of this functional molecule in apes will certainly help to elucidate its role.

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Bochner, B. S.; Alvarez, R. A.; Mehta, P.; Bovin, N. V.; Blixt, O.; White, J. R.; Schnaar, R. L. J. Biol. Chem. 2005, 280, 4307–4312. Nutku, E.; Aizawa, H.; Hudson, S. A.; Bochner, B. S. Blood 2003, 101, 5014–5020. Nutku, E.; Hudson, S. A.; Bochner, B. S. Biochem. Biophys. Res. Commun. 2005, 336, 918–924. Bochner, B. S.; Busse, W. W. J. Allergy Clin. Immunol. 2005, 115, 953–959. Zhang, J. Q.; Nicoll, G.; Jones, C.; Crocker, P. R. J. Biol. Chem. 2000, 275, 22121–22126. Angata, T.; Varki, A. J. Biol. Chem. 2000, 275, 22127–22135. Foussias, G.; Yousef, G. M.; Diamandis, E. P. Genomics 2000, 67, 171–178. von Gunten, S.; Yousefi, S.; Seitz, M.; Jakob, S. M.; Schaffner, T.; Seger, R.; Takala, J.; Villiger, P. M.; Simon, H. U. Blood 2005, 106, 1423–1431. Biedermann, B.; Gil, D.; Bowen, D. T.; Crocker, P. R. Leuk. Res. 2006. Munday, J.; Kerr, S.; Ni, J.; Cornish, A. L.; Zhang, J. Q.; Nicoll, G.; Floyd, H.; Mattei, M. G.; Moore, P.; Liu, D.; Crocker, P. R. Biochem. J. 2001, 355, 489–497. Whitney, G.; Wang, S.; Chang, H.; Cheng, K. Y.; Lu, P.; Zhou, X. D.; Yang, W. P.; McKinnon, M.; Longphre, M. Eur. J. Biochem. 2001, 268, 6083–6096. Yousef, G. M.; Ordon, M. H.; Foussias, G.; Diamandis, E. P. Biochem. Biophys. Res. Commun. 2001, 284, 900–910. Li, N.; Zhang, W.; Wan, T.; Zhang, J.; Chen, T.; Yu, Y.; Wang, J.; Cao, X. J. Biol. Chem. 2001, 276, 28106–28112 (doi: 10.1074/jbc.M100467200). Kitzig, F.; Martinez-Barriocanal, A.; Lopez-Botet, M.; Sayos, J. Biochem. Biophys. Res. Commun. 2002, 296, 355–362. Angata, T.; Kerr, S. C.; Greaves, D. R.; Varki, N. M.; Crocker, P. R.; Varki, A. J. Biol. Chem. 2002, 277, 24466–24474. Angata, T.; Varki, N. M.; Varki, A. J. Biol. Chem. 2001, 276, 40282–40287. Foussias, G.; Taylor, S. M.; Yousef, G. M.; Tropak, M. B.; Ordon, M. H.; Diamandis, E. P. Biochem. Biophys. Res. Commun. 2001, 284, 887–899. Yu, Z.; Lai, C. M.; Maoui, M.; Banville, D.; Shen, S. H. J. Biol. Chem. 2001, 276, 23816–23824.

3.25 Carbohydrate Recognition by Cytokines and its Relevance to their Physiological Activities K. Yamashita and K. Fukushima, Tokyo Institute of Technology, Tokyo, Japan ß 2007 Elsevier Ltd. All rights reserved. 3.25.1 Introduction

539

3.25.2 Lectin-Like Characteristics of Cytokines

539

3.25.2.1 GAG-Binding Cytokines

539

3.25.2.2 High-Mannose-Type Glycan Binding IL-2

542

3.25.2.3 GPI-Anchor Glycan-Binding Cytokines

544

3.25.2.3.1 Attachment of b-N-acetylglucosaminyl phosphate diester in GPI-anchor glycans 3.25.2.3.2 TNF-a, IL-1b, and IL-18 binding to GPI-anchor glycans 3.25.2.3.3 TNF-a and IL-18 distinctly recognize the second and third mannose-6-phosphate diester in the GPI-anchor glycan

3.25.3 Heparan Sulfate Recognition by Both FGF and its Receptor Induces Cell Proliferation

544 546 547

547

3.25.4 Recognition of Both a Specific Peptide Sequence and Man5GlcNAc2 in IL-2 Receptor a Triggers Signal Transduction

550

3.25.5 GPI-Anchor Glycan Recognition of Cytokines in Relation to Their Physiological Activities

553

3.25.5.1 Recognition of GPI-Anchor Glycan by TNF-a Induces Apoptosis of U937 Cells

553

3.25.5.2 Recognition of the Distinct GPI-Anchor Glycan Portion in CD48 by IL-18 and IL-18 Receptor a Induces IFN-g Production in KG-1 Cells 3.25.6 Future Directions

554 558

3.25.1 Introduction Cytokines are mediators of the regulatory network among lymphoid cells, hematopoietic cells, and endothelial cells. They regulate immune response, inflammation, cell proliferation, and differentiation. Many signal transduction mechanisms are evoked after cytokines bind to their respective receptors.1 Most cytokine receptors consist of several specific receptor subunits. These subunits form high-affinity complexes with their respective cytokines, following which various physiological effects are induced.1 Other characteristic cytokine actions include pleiotrophic, redundant, autocrine, and paracrine functions. These complicated cytokine behaviors cannot be explained by the simple interaction between a cytokine and its receptor.1 However, lectin-like characteristics of cytokines (summarized in Table 1) may provide an insight into their multiple functions, although their physiological significance has not generally been elucidated. Nevertheless, there are several cytokines for which functional roles of lectin-like interactions have been determined. In this chapter, we describe two carbohydrate recognition mechanisms that are known to influence the physiological activities of cytokines. One involves a group of cytokines that recognize both the specific N-glycan and the specific peptide sequence within their receptor subunits. This binding triggers high-affinity complex formation and induces downstream intracellular signaling. The other mechanism involves cytokines that form high-affinity complexes with their receptors via distinct glycosaminoglycan (GAG) sites or GPI-anchored glycans sites. It seems that such a tight, oligomeric formation of (cytokine)m/(receptor subunit)n/glycoconjugate amplifies intracellular signaling.

3.25.2 Lectin-Like Characteristics of Cytokines 3.25.2.1 GAG-Binding Cytokines A large number of cytokines have been described as GAG-binding proteins (Table 1). Most GAG-binding cytokines interact with heparin/heparan sulfate (HS), while a few are known to interact with chondroitin sulfate or dermatan

539

540

Carbohydrate Recognition by Cytokines and its Relevance to their Physiological Activities

Table 1 Cytokines that have lectin-like characteristics and their ligands Cytokine

Oligosaccharide ligands

References

IL-1a

Glycans of uromodulin Disialylated biantennary Glycans of uromodulin GM4 Sulfated N-glycan of Tamm–Horsfall glycoprotein GPI-anchor glycan High-mannose type Heparin GD1b Heparan sulfate Chondroitin sulfate 1,7 Intramolecular lactone of N-acetylneuraminic acid Ganglioside GAG Heparin/heparan sulfate HNK-1 epitope Heparin Sialyl-Tn antigen Heparin Heparin/heparan sulfate Heparin/heparan sulfate Heparin GPI-anchor glycan GPI-anchor glycan Heparin Gb3 Ganglioside Ganglioside Heparin/heparan sulfate Heparan sulfate Chondroitin sulfate Heparan sulfate Chondroitin sulfate GM1 Heparin/heparan sulfate Heparan sulfate CD44 Heparin/heparan sulfate Heparin/heparan sulfate Heparan sulfate Dermatan sulfate Heparan sulfate CD44 Heparin/heparan sulfate Heparan sulfate Chondroitin sulfate Heparin/heparan sulfate Heparin/heparan sulfate Chondroitin sulfate Heparan sulfate Chondroitin sulfate Heparin/heparan sulfate Heparin/heparan sulfate Heparin/heparan sulfate Heparin/heparan sulfate Heparin/heparan sulfate Glypican-2 PG-M/vercican Chondroitin sulfate

2 3 2 3 4 5 6 7, 8 9 10,11 12 3 13 14 15 3 16 3 17 18 19 20 21 22 23 24 25 26,27 28–31 32–36 37 32,34,35,38–40 37 41,42 43 32,38,44,45 46 40,47 48 34,35 49 50,51 46 52 50 37 53 54 37 50 37 55 56 57 10,11,58 59,60 61 62 63

IL-1b

IL-2

IL-3 IL-4

IL-5 IL-6 IL-7 IL-8 IL-10 IL-12 IL-18 TNF-a Interferon-a Interferon-b Interferon-g FGF-1 FGF-2

FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF-10 FGF-11 FGF-12 FGF-16 FGF-18 FGF-19 FGF-22 FGF-23 GM-CSF Midkine

Pleiotropin


541

Table 1 (continued) Cytokine

Oligosaccharide ligands

References

Hepatocyte growth factor (HGF)

Heparin/heparan sulfate Sulfoglycolipid Dermatan sulfate Heparin/heparan sulfate GM1/GM2/GM3 Heparin/heparan sulfate GM3 GAG Heparan sulfate Heparin/heparan sulfate Heparan sulfate Heparin/heparan sulfate Heparin/heparan sulfate Heparin/heparan sulfate Heparin/heparan sulfate Sulfatide Heparin/heparan sulfate

50,64,65 66 67 50,68 69,70 50,71,72 73 74 75 76 77 78 79 80 50 81 82–86

Platelet-derived growth factor (PDGF) Vascular endothelial growth factor (VEGF) Nerve growth factor Neuregulin-1 Hepatoma-derived growth factor (HDGF) Platelet factor 4 Follistatin Pigment epithelium-derived factor (PEDF) Connective tissue growth factor (CTGF) Bone morphogenic protein 6 (BMP6) Chemokines

sulfate. The most common method for elucidating GAG-binding abilities involves affinity fractionation of cytokines or GAG on Sepharose columns covalently linked to either GAG or cytokines. The bound proteins are eluted with different concentrations of NaCl, and the concentration required for elutions is generally proportional to the Kd of the interaction. Although this type of assay is restricted to GAG-binding events involving electrostatic interactions, and proteins with low affinity (104–106 M) do not bind under normal conditions, it is an easy and practical way of identifying GAG-binding cytokines. Recently, surface plasmon resonance analysis has been used to reveal the realtime interaction of GAG-binding cytokines and GAG. However, this technique requires specialized equipment – a BIAcore SPR biosensor. NMR and X-ray crystallography provide the most precise insight into these interactions, but these methods require high concentrations of GAG and GAG-binding proteins. From a practical viewpoint, preparation of homogeneous GAG becomes difficult as its size increases. Nevertheless, NMR or X-ray crystallography using complexes of proteins and small disaccharides has provided much important information on these interactions. GAG heterogeneity is one of the biggest obstacles in elucidating the binding specificity of cytokines to GAG. Therefore, early studies focused on fibroblast growth factors FGF-1 and FGF-2, and systematic analyses of GAG structure involved in the interaction with GAG-binding cytokines are still limited. Thus, in this section, historical studies on the binding specificities of FGF-1 and FGF-2, and a systematic analysis of this phenomenon recently reported by Kimata and colleagues, are presented. In 1992, enzymatic or chemical degradation of HS was found to abolish binding of FGF-2 to HS.38,87 Heparinase-, heparitinase treatment, or deaminitive scission at low pH revealed that iduronate-2-sulfates (IdoA2S) were essential for interaction between FGF-2 and HS. A biochemical approach, used to define the minimal structure in HS required for binding of FGF-2, implicated a pentasaccharide sequence with three hexuronic acid units, two N-sulfated glucosamine residues and the reducing terminal IdoA unit being 2-Osulfated.39 Later, a pentasaccharide containing IdoA2S was deduced from crystallographic analyses of a complex between FGF-2 and a fully sulfated heparin hexasaccharide88 and of an FGF-2/FGF-R1/heparin 10-mer complex.89 Furthermore, Ishihara et al. found that both 2-O-sulfate groups and the negative charge of the carboxy group in iduronate residues are required for interaction with FGF-2, utilizing 2-O-desulfated heparin, 6-O-sulfated heparin, carboxy-reduced heparin, and carboxyamidomethylsulfonated heparin.90 At the same time, Ishihara reported that binding of FGF-1 requires 6-O-sulfate groups in addition to 2-O-sulfate groups.91 Moreover, it was found that high affinity for FGF-1 requires an IdoA2S-GlcNS6S-IdoA2S-trisaccharide motif.92 From these early results, it became apparent that the FGF-1-binding region of HS was distinct from the minimal FGF-2-binding region. In addition to these studies on FGF-1 and FGF-2, the HS sequences required for binding of FGF-4,44,45 FGF-8b,51 HGF,65,93,94 and PDGF68 have also been reported. These studies indicated that each HS-binding cytokine may recognize a unique structure. Systematic analysis of the binding specificity of HS-binding cytokines is also limited. However, Kimata’s group recently reported the HS sequences that were specifically bound to various cytokines, including FGF-2, FGF-4,

542


FGF-7, FGF-8, FGF-10, FGF-18, HGF, VEGF, and BMP-6. To do this, they used an octasaccharide library consisting of 2-O-sulfated or 6-O-sulfated octasaccharides synthesized by in vitro reactions with HS 2-O-sulfotransferase or 6-Osulfotransferase.50 On this basis, they proposed the classification of HS-binding growth factors into five groups, as shown in Figure 1. Group 1 has affinity for 2-O-sulfated but not 6-O-sulfated octasaccharides (FGF-2). Group 2 has affinity for 6-O-sulfated but not 2-O-sulfated oligosaccharides (FGF-10). Group 3 has affinity for both 2-O-sulfated and 6-O-sulfated octasaccharides but prefers 2-O-sulfated ones (FGF-18 and HGF). Group 4 proteins require both 2-Osulfate and 6-O-sulfate in octasaccharides for binding (FGF-4, FGF-7). Group 5 requires a longer binding domain than octasaccharide for binding heparin (FGF-8, VEGF, BMP-6). To elucidate the binding specificities of growth factors classified into group 5 or unknown factors, it is important to generate a more divergent oligosaccharide library, by the combined use of various HS modification enzymes and acceptor oligosaccharides of various sizes. As more cytokines have been recognized as HS-binding proteins, there have been more studies of consensus sequences in the HS-binding region. Early studies revealed that XBBXBX and XBBBXXBX motifs, in which B is a basic amino acid, usually Arg or Lys, are common HS-binding sequences for several proteins.95 In fact, X-ray crystallographic analysis suggested that several basic amino acid residues were found to exist opposite a 2-O-sulfate group in HS.88,89,96–98 As shown in Figure 2, in the case of FGF-2, the HS binding region consists of Lys125, Gln134, Lys135, and Ala136,88 whereas that of FGF-1 is composed of Asn18, Lys113, Lys118, and Gln127.99 However, with the characterization of these HS-binding proteins, it was found that binding epitopes are composed of sequentially distant residues that form an optimal binding surface due to their special orientation in the folded state. Kimata’s group indicated that either Gln or Lys residues found in the glycine boxes, which are commonly found in the FGF family and thought to correspond to motifs for HS-binding sites, are conserved in FGF-2,-4,-7,-8, and -18 (Figure 1).50 Further crystallographic analyses may reveal other amino acids that are required for HS-binding sites.

3.25.2.2 High-Mannose-Type Glycan Binding IL-2 IL-2 recognizes high-mannose-type glycans, as shown in Table 1. There is a long history of research on carbohydrate recognition by IL-2. Sherblom et al.100 and Zanetta et al.101 reported that IL-2 recognizes high-mannose-type glycans with five or six mannosyl residues, as determined by the plate method. Later, Fukushima et al.6,102 precisely examined the lectin-like characteristics of IL-2. In a solid-phase [35S]rhIL-2 binding assay, which involved in vitro transcription and translation, ribonuclease B was immobilized in each well by incubation overnight, and the remaining sites were blocked with phosphate-buffered

Groups

Necessary O-sulfate in octasaccharide

Growth Factor

Group 1

2-O-sulfate

FGF-2

118 L K R T G Q Y K L G S K T G P G Q K A I L

Group 2

6-O-sulfate

FGF-10

180 L N G K G A P R R G Q K T R R K N T S A H

Group 3

2-O- or 6-O-sulfate

FGF-18 HGF

153 F T K K G R P R K G P K T R E N Q Q D V H

Group 4

2-O- and 6-O-sulfate

FGF-4

181 L S K N G K T K K G N R V S P T M K V T H

FGF-7

167 L N Q K G I P V R G K K T K K E Q K T A H

FGF-1

111 L K K N G S C K R G P R T H Y G Q K A I H

FGF-8

171 F T R K G R P R K G S K T R Q H Q R E V H

Heparin-binding regions of FGFs Glycine box

Group 5

VEGF BMP-5 Figure 1 Classification of FGFs on the basis of their different heparan-binding affinities. The sequences of the putative heparin-binding regions of FGFs are shown together with the residues involved in 2-O-sulfate-binding (highlighted by a dark background). The square shows the glycine box, which is thought to be the motif responsible for heparin binding.49


01

I5 S2 I3

S6

O4

S4

136 28 102

Asn 18 Lys 118 G1n 127 (a)

543

27

121 135 126

Arg 122 (b)

Figure 2 Heparin binding to FGF-1 (A) and FGF-2 (B). a, Complex consisting of FGF-1 (ribbons) and heparin (ball-andstick). Interactions are shown as a dashed yellow line. b, Complex consisting of FGF-2 and the hexasaccharide fragment. The higher-affinity binding site is colored magenta, whereas the lower-affinity binding site is colored yellow. The side chains and residue numbers of amino acids in FGFs that interact with heparin are indicated. Modified from Middleton, J.; Patterson, A. M.; Gardner, L.; Schmutz, C.; Ashton, B. A. Blood 2002, 100, 3853-3860 and Habuchi, H.; Suzuki, S.; Saito, T.; Tamura, T.; Harada, T.; Yoshida, K.; Kimata, K. Biochem. J. 1992, 285, 805-813.

saline (PBS) containing 3% human serum albumin. [35S]rhIL-2 was added to each well and allowed to stand for 2h at 37 C. The plates were then washed with 0.05% Tween 20 in PBS, and bound [35S]rhIL-2 was measured after being released by 1% SDS. The binding of [35S]rhIL-2 to ribonuclease B-coated plates was concentration dependent. In contrast, [35S]rhIL-2 did not bind to ribonuclease A, which had the same amino acid sequence as ribonuclease B but without any N-glycan. The same results were obtained using EndoH-treated ribonuclease B-coated plates. These results suggest that the binding of [35S]rhIL-2 to immobilized ribonuclease B occurs via Man5GlcNAc2 or Man6GlcNAc2, which are linked to ribonuclease B.103 The inhibitory effects of various high-mannose-type glycans on the binding of [35S]rhIL-2 to ribonuclease B-coated plates are summarized in Table 2. For M5 and M6, the 50% inhibition concentration was estimated to be 0.2mM, whereas for M7, M8, M9, and M3, it was greater than 1mM. Since Man5GlcNAc2Asn, Man5GlcNAc2, and Man5GlcNAc showed the same inhibitory effects, with respect to the structural requirement of the reducing terminal end, neither asparagine nor N,N0 -diacetylchitobiose structure was required, whereas Man5GlcNAc-ol did not show any inhibitory effect. These results indicate that two nonsubstituted a-mannosyl residues linked to the a1!6 side of the trimannosyl core and reducing terminal Manb1!4GlcNAcb1! are required for the inhibitory activity. In order to determine the carbohydrate recognition sites of IL-2, mutant forms of in vitro translated [35S]rhIL-2 with different levels of carbohydrate-binding activities were prepared. It had been reported by Sherblom et al.100 that the N-terminal portion of rhIL2 exhibits a limited degree of sequence homology at Glu15, Leu19, Leu21, Asn26, Gly27, Asn30, Cys58, and Glu67 among human mannose-binding proteins, MBP (H),104 and two rat liver MBPs, MBP (A) and MBP (C).105 Moreover, it is confirmed that Asp20 is the binding site of IL-2 receptor b, Gln126 is that of IL-2 receptor g, and Lys35, Arg38, Phe42, Lys43 are binding sites of IL-2 receptor a.106 On the basis of these data, point mutations were introduced into IL-2 cDNA by PCR, and [35S]rhIL-2 mutants were synthesized in vitro in the presence of [35S] methionine. All of them showed single bands corresponding to 16kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the wild type of [35S]rhIL-2 and eight mutant forms of [35S]rhIL-2 were equally labeled with five [35S]methionine residues per molecule. The carbohydrate-recognition activities of these IL-2 analogs were assayed by a ribonuclease B-coated plate method. Although mutated E67Q showed the same binding activity as the wild type, mutated N26Q showed an improved carbohydrate-binding ability compared to wild-type IL-2, whereas mutated N26D showed diminished carbohydrate-binding activity. The carbohydrate-binding specificities of N26D and N26Q do not seem to be altered in comparison with that of wild-type IL-2, because the binding activities of N26D and N26Q on the ribonuclease B-coated plates were inhibited by M5 andM6, whereas M7, M8, or M9 did not produce any inhibitory effect. The 50% inhibition concentration with M5 or M6 was 0.2mM, whereas those of M7, M8, or M9 were greater than 1mM. Since the side chain of Asn26 is directed toward the outside of the a-helix structure, as determined by X-ray crystallography of IL-2 (as shown in Figure 3),107 even if Asn26 is replaced by Asp or Gln by point mutation, it is thought that the three-dimensional structure of these IL-2 molecules would not be affected. These results imply that Asn26 is involved in the carbohydrate binding of IL-2.

544


Table 2 Inhibition of [35S]rhIL-2 binding to plates coated with ribonuclease B by oligomannosyl derivatives Compounds

Concentration for 50% inhibition

M5:

Mana 1 Mana 1

6 Mana 1 3 6 Manb 1 Mana 1 3

4GlcNAcb 1

4GlcNAc-Asn

0.2mM

Man5 Man5!4GlcNAcb1!4GlcNAc Man5!4GlcNAcb1!4GlcNAc-ol Man5!4GlcNAc Man5!4GlcNAc-ol M6:

Mana 1

6 Mana 1 6 3 Mana 1 Manb 1 Mana 1 2 Mana 1 3

M7:

Mana 1 2

M8:

Mana 1 Mana 1 M9:

M3:

0.2mM 0.2mM 0.2mM >1mM

2 2

4GlcNAcb 1

0.2mM

4GlcNAc-Asn

Mana 1


4GlcNAcb 1

4GlcNAc-Asn

>1mM

4GlcNAcb 1

4GlcNAc-Asn

>1mM

Mana 1


Mana 1 2Mana 1

6 Mana 1 6 3 Mana 1 2Mana 1 Manb 1 3 Mana 1 2Mana 1 2 Mana 1 Mana 1

6 Manb 1 Mana 1 3

4GlcNAcb 1

4GlcNAcb 1

4GlcNAc

4GlcNAc-Asn

>1mM

>1mM

IL-2Ra

IL-2 IL-2Rb

IL-2Rg (a)

(b)

Figure 3 Molecular models of human IL-2 and human IL-2 bound to IL-2 receptor subunits. a, A molecular model of human IL-2 taken from the Brookhaven Protein Database, PDB (1irl), shown as a turquoise ribbon structure. The side chain of Asn26 is shown as a ball and stick structure. Lys35, Arg38, Phe42, and Lys43, which bind to the IL-2R peptide portion, are in red. b, A theoretical model of IL-2 attached to its three receptor subunits, which was also taken from the PDB (1iln). IL-2 is shown in blue; the receptor subunits are in red (a), green (b), and turquoise (g). The Asn26 residue is in white. Reprinted with permission from Faham, S.; Hileman, R. E.; Fromm, J. R.; Linhardt, R. J.; Rees, D. C. Science 1996, 271, 1116–1120. Copyright 1996 AAAS.

3.25.2.3 GPI-Anchor Glycan-Binding Cytokines 3.25.2.3.1 Attachment of b-N-acetylglucosaminyl phosphate diester in GPI-anchor glycans As shown in Table 1, IL-1b, TNF-a, or IL-18 bind to GPI-anchor glycans of GPI-anchored glycoproteins on the cell surface. All GPI-anchor glycans from yeast to mammals have the same core structure, consisting of


545

ethanolamine-PO4-6Mana1!2Mana1!6Mana1!4GlcNa1!6myo-D-inositol-PO4 linked to a lipid moiety.108 Using a hydrogen fluoride treatment method, which cleaves both GlcNAc-phosphodiester and ethanolamine phosphodiester, it was determined that human placental alkaline phosphatase (AP) also has the same core structure.109 However, asialo-AP, CEA, ChE, and T-H glycoprotein bound to a PVL-Sepharose column, which interacts with bGlcNAc residues,110 and it was eluted with 0.3M GlcNAc.111 Since all the N-glycans of these glycoproteins are mature, nonreducing terminal bGlcNAc residues seem to be present in the GPI-anchor glycan of these glycoproteins. Several analytical methods can be used to determine how the bGlcNAc residue is attached as a GPI-anchor glycan side chain. As summarized in Figure 4, after pronase digestion of GPI-anchored glycoproteins, glucosamine residues in GPI-anchor glycans were deaminated by nitrous acid treatment and reduced with NaB3H4. [3H]-anhydromannitol was confirmed by monosaccharide composition analysis. The [3H]-GPI-anchor glycan also bound to a PVL column and was eluted with 0.3M GlcNAc. The PVL-binding ability of the [3H]-GPI-anchor glycan was abolished by digestion with diplococcal b-N-acetylhexosaminidase. The mild acid hydrolysate of [3H]-GPI-anchor glycan also failed to bind the PVL column, indicating that the bGlcNAc residue does not bind to the GPI-anchor glycan via O-glycosidic bonding. Since N-acetylglucosaminyl phosphate diester can be easily hydrolyzed by mild acid treatment,112 a GlcNAcb1!phosphodiester residue (GlcNAc-P) should exist as a GPI-anchor glycan side chain. Chromatofocusing and LC/ESI-MS are useful for further identification of GlcNAcb1!phosphate. For example, purified AP was eluted from a chromatofocusing column at pH 4.6 and pH 4.8 in the ratio of 4:1.111 AP forms a homodimer and contains 4 mol of sialic acid per molecule.113 Sialidase-treated APs were eluted at pH 5.2 and pH 5.0. Although asialo APs were resistant to wheat germ acid phosphatase, after digestion with diplococcal b-N-acetylhexosaminidase, APs were eluted at pH 5.0 and pH 4.8. Subsequently, APs were thoroughly digested by wheat germ acid phosphatase and were eluted at pH 5.4 and pH 5.2, suggesting that they had lost their negatively charged phosphate residues. LC/ESI-MS analysis indicated that the GPI-anchor glycan consisted of two isoforms, one with and one without an ethanolamine phosphate. Since ethanolamine phosphate diester has a single negative charge at pH 5.0, one isoform must contain both a GlcNAc-P residue and ethanolamine phosphate as side chains, while another isoform contains only the GlcNAc-P residue. These results suggest that b-N-acetylglucosaminyl phosphate binds to the GPI-anchor glycans of AP.111 Combined results from LC/ESI-MS, nitrous acid deamination/NaB3H4 reduction, periodate oxidation, chromatofocusing, and several lectin column chromatographies confirmed that the structures of the GPI-anchor glycans derived from AP, CEA, and T-H glycoprotein commonly consisted of GlcNAcb1!phosphate at position C6 of the second mannose residue,21 although the other side-chain portions have different carbohydrate moieties among the respective GPI-anchored glycoproteins.

GlcNAc b1 P GPI-anchored glycoprotein

EtN ± P

Endo-aglucosaminidase?

6 EtN- P -6Mana1→2Mana1→6Mana1→4GlcN

inositol

GPI-anchored glycoprotein

P : PO3−

lipid

GPI glycan HONO/NaB3H4

+Diplococcal b-HexNAcase

Chromatofocusing

P

Pronase

Trypsin

Sialidase Diplococcal b-HexNAcase

PI-PLC

+Wheat germ acid phosphatase LC/ESI-MS

Diplococcal b-HexNAcase

Mild acid hydrolysis

PVL-Sepharose column chromatography (−)

(+)

(−)

Figure 4 Structural analysis of the GPI glycan derived from GPI-anchored glycoproteins. b-N-acetylglucosaminyl phosphate diester is common to GPI-anchored glycans of AP, CEA, and T-H glycoprotein.

546


3.25.2.3.2 TNF-a, IL-1b, and IL-18 binding to GPI-anchor glycans In 1987, Muchmore et al. reported that TNF-a binds to uromodulin corresponding T-H glycoprotein in a lectin-like manner.114 Uromodulin, which has heterogenous N-glycans and a GPI-anchor glycan, is an immunosuppressive glycoprotein that was originally isolated from human urine.115 Since uromodulin also binds to IL-11 and IL-2,100 it had been believed that the glycans on uromodulin may inhibit these cytokine activities. In order to determine the precise carbohydrate-binding specificities of TNF-a, the lectin-like interaction of recombinant human TNF-a (rhTNF-a) with various glycoproteins was investigated on enzyme-linked immunosorbent assay (ELISA) plates. As shown in Table 3, rhTNF-a bound in a dose-dependent manner to AP,111,113 CEA,116,117 and T-H glycoprotein,118,119 which were saturated on the plates. All these molecules carry GPI-anchor glycans. On the other hand, rhTNF-a did not bind to transferrin,120 fetuin,121 orosomucoid,122 ribonuclease B,103 ovalbumin,123,124 and thyroglobulin,125,126 although all of these latter molecules carry various N-linked glycans. These results imply that rhTNF-a recognizes the GPI-anchor glycans of AP, CEA, and T-H glycoprotein. The inhibitory ability of various haptenic sugars derived from GPI-anchor glycans on the carbohydrate binding of TNF-a was subsequently examined by ELISA using AP-coated plates (Table 4).22 Mannose 6-phosphate at 106 M inhibited 50% of TNF-a binding to AP. In contrast, ethanolamine phosphate, inositol phosphate, and N-acetylglucosamine 1-phosphate did not inhibit the interaction between TNF-a and AP, even at concentrations of 1mM. Mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol 6-phosphate also failed to inhibit binding, which indicates that a mannosyl residue substituted with phosphate at the C6 position is required for the sugar-binding ability of TNF-a. Since IL-1b shares amino acid sequence homology with IL-18, it was determined whether IL-1b and IL-18 have the same carbohydrate-binding activities.5,21 First, binding assays were performed using plates coated with various glycoproteins, as shown in Table 3. Since bovine serum albumin (BSA) resulted in relatively high nonspecific binding, human serum albumin (HSA) was used as the blocking reagent. 35S-rhIL-1b and 35S-rhIL-18 were prepared by in vitro translation in the presence of [35S]methionine. The resulting 35S-rhIL-1b and 35S-rhIL-18 proteins were immediately used in the binding assays. Both cytokines bound to AP, T-H glycoprotein, and CEA in a dose-dependent manner. The binding of 35S-rhIL-1b and 35S-rhIL-18 to plates coated with 10mgml1 CEA, AP, or T-H glycoprotein was concentration dependent, similar to TNF-a. These interactions did not change in the presence of 1mM ethylenediaminetetraacetic acid (EDTA), which shows that the binding of both cytokines to AP does not require divalent cations. However, both cytokines failed to bind transferrin, fetuin, orosomucoid, ribonuclease B, ovalbumin, or thyroglobulin, as summarized in Table 3. These results indicate that, similar to TNF-a, IL-1b and IL-18 bind to GPI-anchored glycoproteins. To more precisely determine the carbohydrate-binding specificity of IL-1b and IL-18, the inhibition of 35S-rhIL-1b and 35S-rhIL-18 binding to AP of haptenic sugars derived from the GPI-anchor glycan of

Table 3 Glycan structures of glycoproteins used to survey lectin-like characteristics of TNF-a, IL-1b, and IL-18 Glycoprotein

Structure of glycan (reference)

TNF-a22

IL-1b5

IL-1821

Alkaline phosphatase (AP)

a2!3sialyl biantennary112 GPI anchor110 Bi- to tetraantennary115 GPI anchor116 Bi- to tetraantennary117 High-mannose type117 GPI anchor118 a2!6sialyl biantennary119 Mucin type120 Bi- and triantennary120 Bi- to tetraantennary121 High-mannose type (Man5>6GlcNAc2)102 High-mannose type (Man6,7> Man5GlcNAc2)122 Hybrid type123 Bi- to tetraantennary124 High-mannose type (Man9,8,7GlcNAc2) 125

þ

þ

þ

þ

þ

þ

þ

þ

þ

CEA T-H glycoprotein

Transferrin Fetuin Orosomucoid Ribonuclease B Ovalbumin Thyroglobulin

Plates were coated with each glycoprotein and the level of bound cytokines was measured by ELISA or using radioactivity.

547


Table 4 Ability of haptenic sugars to inhibit the binding of TNF-a, IL-1b, or IL-18 to AP Concentration needed for 50% inhibition Hapten

TNF-a

IL-1b

IL-18

Mannose 6-phosphate Gal2GlcNAc2Man3GlcNAc Ethanolamine phosphate Inositol phosphate GlcNAc 1-phosphate Mannose 6-sulfate Mannose 1-phosphate Glucose 6-phosphate Mannitol 6-phosphate

106 M NDa ND ND ND ND ND ND ND

106 M ND ND ND ND ND ND ND ND

106 M ND ND ND ND ND ND ND ND

a

ND: no inhibitory effect was detected, even when up to 1mM of the haptenic sugar was present. ND, not detected.

AP was examined. The biantennary sugar chain of AP was not inhibitory at concentrations up to 1mM. In contrast, mannose 6-phosphate, which is a constituent of the GPI-anchor glycan of AP, was an effective inhibitor. However, other constituents of the GPI-anchor glycan of AP were not inhibitory, at concentrations up to 1mM. Similarly, mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol 6-phosphate were not inhibitory at concentrations up to 1mM. These results indicate that a second or third mannose 6-phosphate diester in the GPI-anchor glycan is necessary for the carbohydrate binding of IL-1b and IL-18.

3.25.2.3.3

TNF-a and IL-18 distinctly recognize the second and third mannose-6-phosphate diester in the GPI-anchor glycan

A b-N-acetylglucosaminyl phosphate diester residue is attached to the C6 position of the second mannosyl residue in the GPI-anchor glycans of AP, CEA, and T-H glycoprotein, as shown in Figure 4. Thus, it was investigated whether this GlcNAcb1!phosphate!6 mannose residue is involved in the binding of AP to TNF-a.22 Plates were coated with intact AP or mild acid-treated AP and the binding of TNF-a was assessed by ELISA. The mild acid-treated AP retained the ability to bind rhTNF-a. In contrast, mild acid and phosphatase digestion, which specifically releases the GlcNAcb1!phosphate that is attached to the C6 position of the second mannosyl residue of AP, abrogated the binding of AP to TNF-a. These results indicate that TNF-a specifically recognizes the second mannose 6-phosphate diester of GPI-anchored glycans (Figure 5a). In other studies, it was determined which mannose 6-phosphate diester moiety in GPI-anchor glycans is recognized by IL-18.21 The second mannose 6-phosphate diester of AP was removed by mild acid and phosphatase treatment and IL-18-binding was assayed again. Since de-GlcNAc AP and de-GlcNAcb1!phosphate AP maintained their ability to bind 35S-rhIL-18, it seems that IL-18 specifically binds to the third mannose 6-phosphate diester in the GPI-anchor glycan (Figure 5b). Furthermore, since it is known that the first step in the signal transduction cascade induced by IL-18 involves its binding to IL-18Ra, the potential interaction between IL-18Ra and the GPI-anchor glycan was also investigated. Plates were coated with intact AP, de-bGlcNAc-AP, and de-GlcNAcb1!phosphate-sAP, and the binding of an IL-18Ra-Fc chimera was assessed using a mouse antihuman IgG Fc antibody and HRP-conjugated antimouse IgG. While IL-18Ra bound to de-bGlcNAc-AP and did not recognize intact AP or de-GlcNAcb1!phosphate-sAP, IL-18Rb did not bind these AP derivatives. These results suggest that both IL-18 and IL-18Ra can recognize the GPI-anchor glycan and that IL-18 binds to the third mannose 6-phosphate diester while IL-18Ra binds to the second exposed mannose 6-phosphate (Figure 5b).

3.25.3 Heparan Sulfate Recognition by Both FGF and its Receptor Induces Cell Proliferation FGF-1 and FGF-2 are the best-studied examples of how HS interacts with cytokines to modulate their activities. In this section, several studies concerning the requirement for HS in FGF functions, structural information on the FGF/ FGFR/HS complex, and HS specificity required for tissue-specific FGF systems are presented.

548


TNF-a Protein-EtN P

bGlcNAc P

6 6 Mana1→2Mana1→6Mana1→4GlcN-Inositol (a)

P

IL-18

IL-18Ra

Protein-EtN P

P

6 6 Mana 1→2Mana 1→6Mana 1→4GlcN-Inositol (b)

P

Figure 5 Distinct recognition sites in GPI-anchor glycans of TNF-a, IL-18, and IL-18Ra. A, TNF-a; B, IL-18, and IL-18Ra.

Before describing direct roles of HS in mediating signal transduction, it is important to consider the numerous indirect roles for HS, including extracellular stabilization and storage of FGF. In the extracellular matrix, HS binds FGF to protect it from proteases and to create a reservoir of inactive factors. Moreover, HS facilitates the formation of concentration gradients and controls diffusion of the FGF response. Flaumenhaft et al. measured the radius of diffusion of FGF-2 in the presence and absence of HS.127 HS produced effective diffusion of FGF-2, and it is suggested that the FGF-2/HS complex is more effective in stimulating distant cells. As discussed below, HS specificity in respective tissues is also important in mediating these functions. To elucidate direct roles for HS in FGF function, Rapraeger et al. investigated the effects of hepalitinase or sodium chlorate on Swiss 3T3 fibroblast proliferation and MM14-myoblast differentiation.128 Both direct digestion of HS by hepalitinase, and reduced sulfation by sodium chlorate, blocked the mitogenic response of 3T3 cells to FGF-2. The requirement for HS also extended to the FGF-2-mediated repression of MM14 skeletal muscle cell differentiation. The addition of soluble HS recovered FGF-mediated signaling in hepalitinase-treated or sodium chlorate-treated 3T3 cells. Yayon et al. also demonstrated a requirement for HS in high-affinity binding of FGF and FGFR, by using HS-deficient Chinese hamster ovary (CHO) cells.129 HS-deficient CHO cells, which express abundant chondroitin sulfate proteoglycans, do not bind FGF-2, and free HS promotes binding of FGF-2 to its high-affinity receptor. These studies indicate that HS molecules are required for stabilization of the FGF/FGFR complex and for related signal transduction. The binding site for FGF-2 has been identified as a pentasaccharide containing a 2-O-sulfate group (see Section 3.25.2.1). However, a dodecasaccharide sequence is required to promote receptor signaling by FGF-2.33 Rapraeger’s group used chlorate-treated Swiss 3T3 fibroblasts, with impaired synthesis of HS proteoglycan,128 as target cells to assess the ability of exogenous HS-derived oligosaccharides to promote the mitogenic activity of FGF-2. This revealed that a dodecasaccharide, as well as full-size native HS, is an efficient promoter, whereas the corresponding decasaccharides, or smaller oligosaccharides are inactive. Neither selectively 2-O-desulfated nor preferentially 6-O-desulfated heparin is active. Therefore, a dodecasaccharide is the minimal size of HS that can occupy both FGF-2 and FGFR simultaneously. Next, the mechanism by which HS facilitates FGF-mediated signaling was elucidated. Firstly, biochemical analyses revealed that HS promotes the formation of FGF dimers and the arrangement of FGF oligomers along with HS.130–132 Analytical ultracentrifugation of FGF-2 in the presence of HS-derived saccharides shows that HS induces FGF-2 self-association. There are three possibilities for the configuration of FGF-2 molecules bound to HS: (1) the FGF-2 molecules are located in the trans-orientation relative to HS; (2) the FGF-molecules are bound to neighboring sites on HS but are not in contact with each other; and (3) the FGF-2 molecules are bound to neighboring sites (in cis) on HS and are in contact with each other. However, co-crystals of FGF-2 with a heparin-derived oligosaccharide indicate that the latter possibility is the most likely. Secondly, as shown in Figure 6, binding of HS and FGF to FGFR triggers receptor dimerization and both cis- or trans-FGFR autophosphorylation of up to seven


549

Figure 6 A model of FGFR dimerization that involves binding of the extracellular FGFR domain to the heparin-linked FGF dimer.

Figure 7 The FGF1-FGFR2-heparin complex FGFR domains 2 (D2) and 3 (D3) are in cyan and magenta, and FGF1 is in green. The heparin molecule is in the CPK representation.

tyrosines in each cytoplasmic C tail of the assembled FGFR homo- or heterodimers.133 In subsequent molecular structural studies, the nature of the FGFR active complex was addressed and two separate models of FGF ternary complex were proposed. Schlessinger et al. presented the crystal structure of a symmetrical dimer formed by two 1:1:1 FGF2-FGFR1-decasaccharide complexes.89 In contrast, Pellegrini et al.33 described an asymmetrical structure in which two FGFR-2 subunits were bridged by an FGF-1 dimer formed upon a single heparin decasaccharide. It was found that two FGF-FGFR heterocomplexes are thermodynamically stabilized by one molecule of HS.99,134–136 In contrast, further analysis suggested that, under different preparation conditions, both architectures can be formed from the same protein.137 Thus, there appear to be two potential architectures for the 2:2 FGF-FGFR signal complex, both of which can be formed upon a single HS chain in vivo (Figure 7).

550


Twenty-three members of the FGF family have been described along with four FGF receptor tyrosine kinases. The FGFRs share a number of common structural features.138 All have an intracellular split tyrosine kinase domain, a transmembrane sequence, and an extracellular region composed of three immunoglobulin-like domains (D1-D3) (Figure 6). The D2 domain of the FGFRs contains a short stretch of basic amino acids referred to as the K18K loop.130 This region mediates interaction with HS139 and is essential for receptor function. Membrane-associated HS species are far more effective in catalyzing the formation of active FGFR dimers than soluble HS.140 Moreover, different HS and HS fragments of HS sugar moieties show different activities, and can be inhibitory for FGF.141 Knox et al. showed different effects on FGF2 receptor affinity, binding, and signaling by using perlecan isolated from primary endothelial cells, endothelial cell lines, or colon carcinoma cells.142 Berry et al. indicated that different fragments of HS from smooth muscle cells mediate signaling of bound FGF-2 through different FGFRs.143 These studies suggested that HS plays a fundamental role in the tissue-specific binding affinity, specificity, and activity of both FGF and FGFR.

3.25.4 Recognition of Both a Specific Peptide Sequence and Man5GlcNAc2 in IL-2 Receptor a Triggers Signal Transduction IL-21 is a cytokine synthesized by activated T-cells.144 It promotes the proliferation of IL-2-dependent T-cells and functions as an immunomodulator of activated B-cells, macrophages, and natural killer cells.145 IL-2 mediates its physiological functions through interaction with its receptor complex, which consists of three receptor subunits, a, b, and g (IL-2 receptors IL-2Ra,-b, and -g).146 Although none of the receptor subunits has intrinsic tyrosine-kinase activity, intracellular portions of the IL-2Rb and -g subunits do associate with intracellular tyrosine kinases. These observations suggest that a complex consisting of IL-2, IL-2Ra, IL-2Rb, IL-2Rg, and tyrosine kinases might be formed in IL-2-stimulated T-cells. However, when independently expressed, each IL-2 receptor subunit shows only weak binding to IL-2, and the mechanism underlying the formation of a high-affinity IL-2-IL-2Ra,-b,-g complex remains unidentified. However, the observation that IL-2 has carbohydrate recognition ability provided an opportunity to resolve this mechanism. Zanetta et al.101 proposed a cross-linking model in which IL-2 binds to not only the IL-2 receptor via the IL-2 receptor-binding sites, but also to the TCR complex containing glycosylated CD3 of human peripheral lymphocytes. This tentative model was proposed on the basis of analysis of IL-2Rb antibody immunoprecipitates. However, they did not directly show that phosphorylation of Lck kinase co-immunoprecipitated with IL-2Rb subunit occurs, or that a high-mannose-type glycan inhibits IL-2-dependent cell proliferation. Recently, it was found that the lectin activity of IL-2 is indispensable for induction of IL-2-dependent cell proliferation.102 CTLL-2, a mouse T-cell line, proliferates in an IL-2-dependent manner. The extent of CTLL-2 cell proliferation was determined colorimetrically using MTS reagent. After mixtures containing rhIL-2 and highmannose-type glycans at various concentrations were left standing for 2h at 37 C, they were added to the cultured cells. Man5GlcNAc2Asn and Man6GlcNAc2Asn dose-dependently inhibited the proliferative response of these cells to rhIL-2 in vitro, whereas Man7GlcNAc2Asn, Man8GlcNAc2Asn, Man9GlcNAc2Asn, and Man3GlcNAc2 did not show any inhibitory effect (Figure 8). These results suggest that IL-2 lectin activity is required for stimulation of IL-2dependent T-cell proliferation. In the case of IL-2-induced proliferation of CTLL-2 cells, signal transduction occurs via tyrosine kinases including Lck,150 Jak1, Jak3,147,148,149 and Lyn.151 In order to confirm whether the lectin activity of IL-2 modulates the cellular signal transduction mechanism, phosphorylation of Jak1, Jak3, Lck, and Lyn were compared in the presence and absence of Man5GlcNAc2Asn. After culturing the cells in the absence of IL-2 for 6h, CTLL-2 cells in G0 phase were stimulated with IL-2 at 37 C for 30min in the presence or absence of Man5GlcNAc2Asn. Then, the cells were solubilized and proteins in the lysates were immunoprecipitated with antiJak1, anti-Jak3, anti-Lck, or anti-Lyn antibody. Tyrosine-phosphorylated proteins were identified by immunoblotting with an antiphosphotyrosine monoclonal antibody (anti-pTyr). The levels of phosphorylated Jak1, Jak3, Lck, and Lyn increased in IL-2-induced cells as compared to cells in G0 phase. In contrast, phosphorylation of these tyrosine kinases was exclusively reduced in the presence of Man5GlcNAc2Asn, indicating that the carbohydrate recognition of IL-2 modulates signal transduction through Jak1, Jak3, Lck, and Lyn linked to the IL-2Rb and -g. Although it is known that IL-2 induces the formation of an IL-2-IL-2R complex which includes the three receptor subunits a, b, and g (IL-2Ra,-b,-g),146 the independently expressed soluble IL-2Ra,-b, and -g show low-affinity binding to IL-2. That is, the a-subunit binds IL-2 with low affinity (Kd 10nM), the b-subunit binds IL-2 with very low affinity (Kd 100nM), and the g-subunit has no measurable affinity for IL-2.152,153,154 However, as soon as IL-2 forms the high-affinity complex with the IL-2Ra,-b,-g subunits, cellular signaling is triggered. If a lectin-like

Relative proliferation activity (%)


551

M3,M7,or M8

100

M9

50

M6 M5

0 10−9

10−8

10−7 10−6 Concentration [M]

10−5

Figure 8 The effects of high-mannose-type glycans on rhIL-2-dependent proliferation of CTLL-2 cells. The extent of cell proliferation was determined by the MTS colorimetric assay, which consisted of adding to cultures of CTLL-2 cells a mixture of rhIL-2 (5 units/ml) and various high-mannose-type glycans incubated at 37 C for 2h. Results are means of three experiments (P95% of the total protein in the bark. Most plant ricin-B domains preferentially bind to Gal or GalNAc. However, some lectins exhibit a clear preference for sialylated glycans. Moreover, in S. nigra, a type-2 RIP was found that is completely devoid of carbohydrate-binding activity.107 Besides lectins, type-2 RIPs are also enzymes capable of (catalytically) inactivating ribosomes (through the removal of a highly conserved adenine residue in the so-called sarcin/ricin loop of the large ribosomal RNA). Due to this activity, type-2 RIPs are extremely potent cytotoxins, at least at the condition that they succeed in entering the cell. Type-2 RIPs are usually associated, indeed, with toxins. However, apart from ricin, abrin, and a few others, most type-2 RIPs are only moderately or even weakly toxic. The huge difference in toxicity is not linked to differences in the catalytic activity of the A-chain but is primarily determined by the lectin’s ability to penetrate the cell, which itself depends on the sugar-binding specificity of the B-chain.103,108,109

3.26.3.1.8

Other plant lectins

Though the great majority of all characterized plant lectins can be classified in one of the above described families, there are a few exceptions. Some previously described plant lectins, for example, the Euonymus europaeus lectin,110 cannot be classified because no sequence information is available and, in addition, they share no apparent structural similarities with any other lectin. There is also a report of a lectin from Dioscorea batatas that is not related to any other plant lectin but shares aproximately 45% sequence identity with carbonic anhydrase from A. thaliana.111 Since the structure of this lectin has not yet been resolved, it is not clear whether the carbohydrate-binding domain comprises the whole protein or only a subdomain.

3.26.3.2 Evolutionary Relationships between Plant Lectins and Lectins from Other Taxa The occurrence in plants of at least seven distinct lectin families raises the question of their evolutionary origin and possible relationship to structurally related proteins/lectins from other living organisms. To address this issue, a brief overview is given of the documented occurrence (or absence) outside the plant kingdom of the seven lectin domains described in the previous section. Based on the current distribution, the origin of the modern plant lectin domains is discussed. To get a quick overview of the documented occurrence of the lectin domains, the following databases have been explored: (1) NCBI conserved domains (CD)111a and (2) the Pfam Protein Families database.111b In this section, the different plant lectin domains are discussed in the alphabetic order, and whenever appropriate, the corresponding CD and Pfam identifiers are indicated.

3.26.3.2.1

Amaranthin family

Hitherto (putative) proteins with amaranthin domain(s) (pfam07468) were exclusively identified in plants. Though the occurrence in other organisms cannot be excluded on the basis of the available sequence information, it seems likely that the amaranthin domain was only developed by plants.

3.26.3.2.2

Cucurbitaceae phloem lectins

Structural units equivalent to the carbohydrate-binding domain of the Cucurbitaceae phloem lectins (not yet recognized as a pfam or CD) also seem to be confined to plants. Accordingly, one can reasonably assume that this lectin domain was developed in plants rather than acquired by classical vertical inheritance from a prokaryotic ancestor.

578

Plant Lectins

3.26.3.2.3 Heveins Hevein domains (cd00035, ChtBD1, chitin-binding domain; pfam00187, chitin recognition protein) are definitely not confined to plants. Numerous (putative) fungal proteins contain one or more domains that share a high-sequence identity/similarity with plant hevein domains. Moreover, a greater variety of chimerolectins occurs in fungi than in plants. Besides fungi, chimeric proteins with a hevein domain were identified in a diatom (Thalassiosira pseudonana). In Caenorhabditis elegans also, some proteins comprise a small domain that shares a residual sequence similarity with a plant hevein domain. However, since this C. elegans domain is shorter, its relationship to the canonical plant and fungal hevein domains is still unclear. No hevein domains occur in bacterial genomes. The present distribution suggests that the hevein domain developed in an early eukaryote and further evolved through classical vertical inheritance in plants, fungi, and some – but apparently not all – eukaryotic taxa.

3.26.3.2.4 Jacalins Proteins with jacalin domains (pfam01419) are widespread in plants but are absent from prokaryotes and from most – if not all – other eukaryotes. One type of animal protein (the so-called zymogen granule membrane protein 16 found in mouse, rat, and a few other vertebrates) is classified in pfam01419. Since the sequence identity with the plant jacalin domain is low (approximately 30%), and neither the sugar-binding activity nor the structure has been determined, it remains to be demonstrated that the zymogen granule membrane proteins 16 are genuine members of the jacalin family. There is also a recent report of a presumed jacalin-related lectin from a fungus (Grifola frondosa).112 However, this fungal lectin shares only a limited sequence identity (50%) with the ricin-domain mushroom lectins MOA and PSL (see Sections 3.27.2.1.2 and 3.27.2.1.3). This group has also obtained crystals, and a preliminary diffraction to 3.8 A˚ resolution.30 This lectin is likely to prove to be another member of the ricin family, but further refinment of the crystallographic measurement will be necessary to establish its structural relationships.

3.27.2.2 Mushroom Lectin (XCL) Family 3.27.2.2.1

Agaricus bisporus (Agaricaceae)

Agglutination activity in extracts of the meadow mushroom, Agaricus campestris, was first observed by Friedberger and Brossa in 1912.32 Sage and his co-workers33,34 isolated the agglutination activity using ion exchange chromatography, demonstrated its adsorption to type A2 and O erythrocyte ghosts, and determined it to be a heterotetramer of two distinct 16kDa subunits. Subsequently, Presant and Kornfeld35 isolated two very similar agglutinins (isolectins) from the commercial mushroom A. bisporus using the same procedures. Additionally, Eifler and Ziska36 isolated two immunologically cross-reacting (with anti-A. bisporus lectin) lectins from A. edulis (now considered synonymous with A. campestris). Agaricus lectins show greatest specifity for the O-linked T-antigen, Gal(b1-3)GalNAc(a1-O)-Ser/Thr. The free Tdisaccharide is a poor inhibitor of A. bisporus agglutinin (ABA) hemagglutination or glycoprotein binding, and its component monosaccharides are virtually noninhibitory. However, methyl a-N-acetylgalactosaminide at high concentrations is slightly inhibitory, while the N-tosyl a-L-seryl T-disaccharide is a strong inhibitor.37 Interestingly, lactose and Gal(b1-6)Glc exhibit some binding, but N-acetyllactosamine does not. Immunoglobulin IgA-1, which contains an Olinked T-disaccharide in the hinge region of the heavy chain, binds so tightly to ABA that it cannot be eluted from a column of immobilized ABA with 200mM NH4OH. The subclass IgA-2, which lacks the hinge region and contains only N-linked glycans, also binds to the immobilized lectin, but is readily eluted under mildly alkaline conditions.38 Wu et al.39 demonstrated the profound enhancing effect of clustering of epitopes using an enzyme-linked lectinosorbent assay. Clustered T, Tn (GalNAc), and the human blood group precursors type I [Gal(b1-3)GlcNAc] and type II [Gal(b1-4)GlcNAc] glycan epitopes were bound with several orders of magnitude greater affinities than were the monomeric saccharides. A. bisporus lectin has potent antiproliferative affects on human epithelial cancer cells, but is totally nontoxic to normal cells. This is consistent with its specificity for O-linked T-disaccharide, which is expressed on many cancer cell lines but is generally cryptic in normal cells.40 A gene for A. bisporus lectin (ABL) has been cloned and sequenced.41 The sequence was claimed to be unique among T-antigen-binding lectins, although subsequently other Gal/GalNAc-binding mushroom lectins, for example, Xerocomus chr ysenteron lectin (see Section 3.27.2.2.2), were found to exhibit a high degree of homology. Electron densities in a 1.5A˚ X-ray structure42,43 corrected and shortened by 11 residues the amino acid sequence at the Cterminus, and established that no putative O- or N-glycosylation sites were occupied. The protein exhibited a novel structure having a seven-stranded mixed b-sheet and four-stranded antiparallel b-sheet connected by a helix–loop– helix region (Figure 4). Examination of crystals obtained in the presence of a variety of mono- or disaccharides revealed the location of the T-disaccharide binding site, but, surprisingly, revealed a second binding site on each monomer having selectivity for a reducing N-acetylglucosamine, instead of the epimeric N-acetylgalactosamine. This second site could account for the observation noted above by Irazoqui38 that some N-linked structures bind to the lectin, albeit with weaker affinities.

3.27.2.2.2

Xerocomus chrysenteron (Boletaceae)

A potent insecticidal lectin (XCL) was isolated from the Bolete mushroom Xerocomus (Boletus) chrysenteron by affinity chromatography on lactosyl Sepharose.44 This lectin also exhibits high affinity for the T-antigen, as indicated by inhibition of agglutination, and more recently by isothermal titration calorimetry.45 The deduced amino acid sequence had 55% sequence identity (69% similarity) to ABA. Subsequent X-ray crystallography of the lectin to 1.4A˚ resolution,46 whose publication actually preceded that of the ABA structure by a few months, revealed it to have a novel structure that proved to be nearly identical to ABA (Figure 5). Recognizing the highly homologous sequences of several fungal lectins, the authors proposed a new family of lectins, which they designated the XCL family. Several other fungal lectins show significant homology in their primary sequence with XCL and ABA, including sequences that delineate the b-strands and helical regions, so likely belong also to this structural family of lectins (Figure 6). Other basidomycetes (gilled or pored mushrooms) are P. cornucopiae (see Section 3.27.3.3) and Paxillus involutus (Paxillaceae), a toxic mushroom with somewhat intermediate morphology between gilled (Agaricus) and

608

Mushroom Lectins

H

I

J

A

D

C

B

E

F

G

(b)

(a)

N C

C

C

C N

(c)

(d)

Figure 4 Overall structure and folding of ABL. a, Ribbon representation of the ABL monomer. The six-stranded b-sheet is shown in blue, and the four-stranded sheet is shown in red. The two short helices are yellow, and the connections are gray. N-acetylgalactosamine is represented as a ball-and-stick model at the T-antigen binding site (top), and Nacetylglucosamine is represented as a ball-and-stick model at the other site (bottom). b, A topological diagram of the ABL monomer. b-Strands are labeled in the order of their appearance from the N-terminus to the C-terminus using the letters A–J. The 10 strands span the following residues: A, 3–11; B, 19–25; C, 33–37; D, 40–45; E, 51–58; F, 63–72; G, 75–82; H, 113–117; I, 123–130; and J, 135–142. The two helices span residues 90–96 and 101–108. c, Stereodiagram of the ABL tetramer viewed looking down one of the crystallographic dyads. The other two dyads are approximately in the plane of the figure. The two top (or bottom) monomers define the dimer present in the crystallographic asymmetric unit. d, The tetramer viewed looking down the other crystallographic dyad with the T-antigen and N-acetylglucosamine represented as ball-and-stick models. Only two N-acetylglucosamine molecules are visible because the other two are behind the helices represented in the plane of the figure. Reproduced from Carrizo, M. E.; Capaldi, S.; Perduca, M.; Irazoqui, F. J.; Nores, G. A.; Monaco, H. L. J. Biol. Chem. 2005, 280, 10614–10623, with permission from ASBMB.

pore-forming (Xerocomus) mushrooms. Several ascomycetes with lectins of highly homologous sequences, or deduced lectin-like sequences, are the filamentous fungi A. oligospora (see Section 3.27.2.2.3), Gibberella zeae, Podospora anserina (P. pauciseta), and Neurospora crassa (see Refs: 43, 46, and references therein). Homologous structures also occur in cytolysins from species of sea anemones (Figures 4, 5, and 7), except that the cytolysins possess a 31-residue N-terminal extension with structure likely involved in membrane pore formation.46 The toxic action of the Xerocomus lectin is not well defined. Unlike ricin-type toxins, XCL (and ABA, which is also likely to be toxic to insects) is a homodimer and does not possess a ribosome-inhibiting RNase subunit, nor does it possess the membrane-associating region of the cytolysins. Binding and internalization of the lectin in epithelial cells may inhibit uptake of nuclear localization sequence-containing peptides, as has been demonstrated for ABA.47

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(b) Figure 5 Stereo views of the three-dimensional structure of the XCL monomer. a, Ribbon diagram. The four-stranded b-sheet (b2–b5–b6–b7) and the six-stranded b-sheet (b1–b3–b4–b8–b9–b10) are shown in yellow and cyan, respectively. The two short helices flanking the four-stranded b-sheet are shown in orange. b, Stereo a-carbon trace in the same orientation as (a) with balls and numbers marking every 10 residues. Figures were prepared by MOLSCRIPT. Reproduced from Birck C.; Damian, L.; Marty-Detraves, C.; Lougarre, A.; Schulze-Briese, C.; Koehl, P.; Fournier, D.; Paquereau, L.; Samama, J. P. J. Mol. Biol. 2004, 344, 1409–1420, with permission from Elsevier.

3.27.2.2.3

Arthrobotrys oligospora (anamorphic fungi)

A. oligospora is a filamentous ascomycete that is of considerable interest because of its ability to attach to and kill parasitic nematodes, suggesting its application as a biological control of these pests. Rosen et al.48 isolated a lectin (AOL) from the fungal surface, as well as from mycelial extracts, by affinity chromatography on a mucin-Sepharose column. Subsequent studies showed that lectin bound certain sulfated glycoconjugates, a few phospholipids, the Tdisaccharide O-Ser/Thr-linked in glycoproteins, and some galactose-containing N-linked glycans.49 As noted above, the deduced primary sequence of AOL had a high degree of homology with ABA,9 P. cornucopiae lectin,8 and therefore with the other putative members of the XCL family of lectins, although no X-ray structural work has been reported for this lectin.

3.27.2.2.4

Boletopsis leucomelas (Bankeraceae)

The polypore-like mushroom Kurokawa from Japan, B. leucomelas (B. leucomeleana (Persoon) Fayod), contains an agglutinating activity which was isolated on a column of N,N 0 -diacetylchitobiose-Sepharose. Although its N-terminus is blocked, it contains an internal amino acid sequence, MGGSGTSGTIR,50 that conserves five of the six residues in

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Figure 6 Structure-based sequence alignment of XCL with the other members of the XCL family and with the two actinoporins of known structure. Invariant residues between the two families are shown in red background. In each family, invariant and conserved residues are indicated by red type and green type, respectively. The secondary structure elements for XCL, equinatoxin II (EqtII), and sticholysin II (CytII) (same color code as the protein name) are indicated by coils for a-helices and arrows for b-strands. Reproduced from Birck C.; Damian, L.; Marty-Detraves, C.; Lougarre, A.; SchulzeBriese, C.; Koehl, P.; Fournier, D.; Paquereau, L.; Samama, J. P. J. Mol. Biol. 2004, 344, 1409–1420, with permission from Elsevier.

this region (residues 45–57 of ABA) of the six other fungal lectins or putative lectins of this family. The pentultimate residue in this peptide, isoleucine, is leucine in all other sequences, while the threonine (underlined) is conserved in all but the Neurospora crassa-deduced lectin-like sequence, where it is serine (Figure 6). The lectin induces apoptosis in a human monoblastic leukemia cell line, a property not exhibited by ABA, however. Subsequent studies using frontal affinity chromatography showed that the lectin binds to the agalactodiantennary N-linked structure, with or

Mushroom Lectins

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Figure 7 Structural comparison of XCL and StnII. a, Stereo view showing XCL (cyan) and StnII (purple) after superposition of 68 a-carbon atoms from the eight common b-strands in the two structures. The 31-residue N-terminal extension of StnII has been omitted for clarity. In this orientation, the b3–b4 loops and the helical connections appear on opposite sides of the b-sandwich. b, Topology diagram of XCL. a-Helices and b-strands are represented by cylinders and arrows, respectively. The b-strand identifiers and the residue range for each strand are given. Bold parts highlight the dissimilar regions between XCL and StnII. c, Topology diagram of StnII using the same conventions as described above. Reproduced from Birck C.; Damian, L.; Marty-Detraves, C.; Lougarre, A.; Schulze-Briese, C.; Koehl, P.; Fournier, D.; Paquereau, L.; Samama, J. P. J. Mol. Biol. 2004, 344, 1409–1420, with permission from Elsevier.

without a bisecting N-acetylglucosamine. Surprisingly, however, it fails to interact with the N,N0 -diacetylchitobiosyl glycoside, suggesting that it requires non-reducing N-acetylglucosaminide moieties in close proximity.51 Further structural studies will be needed to ascertain if this lectin, from a fairly distant-related mushroom and of different binding specificity, is another member of the XCL lectin family.

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Mushroom Lectins

3.27.2.3 b-Propeller-Fold Lectins 3.27.2.3.1 Aleuria aurantia (Pezizaceae) The L-fucose-binding lectin (AAL) from fruiting bodies of the orange peel fungus, Aleuria aurantia, was first reported by Kochibe and Furukawa.52 Using a combination of ammonium sulfate precipitation, gel filtration, affinity chromatography on desialylated porcine submaxillary mucin peptide coupled to Sepharose 4B with elution by L-fucose, and carboxymethyl-cellulose chromatography, they obtained an electrophoretically homogeneous protein. The lectin was shown to be a homodimer of molecular weight approximately 72kDa, with single polypeptide chains of 31kDa. High levels of hydroxylic and acidic amino acids, glycine, and alanine and the absence of cysteine, methionine, and carbohydrate were reported for the lectin. The lectin agglutinated human A, B, and (O)H erythrocytes, and bound 4 1 52 L-fucose with association constant Ka¼6.110 M , with one binding site per monomer. Although the lectin binds many L-fucosyl-terminated oligosaccharides, it appears to be most specific for the monomeric sugar.52 Cloning and expression of the lectin in E. coli was carried out by Fukumori and colleagues.53,54 The primary sequence of 312 amino acid residues was determined and found to contain six internal repeats of about 50 amino acids.55 The recombinant product, expressed in E. coli, displayed the same immunological and carbohydrate properties as the native lectin and agglutinated human type O erythrocytes.53,54 An immobilized lectin column was shown to bind L-Fuc(a1-6)GlcNAc with highest affinity among the fucosylated oligosaccharides tested. A second group (Amano et al.)55 expressed AAL in Pichia pastoris; the product was active in agglutination assay and exhibited enhanced thermostability over the E. coli-expressed product, which was attributed to its content of carbohydrate. Several reports on the X-ray crystallographic structure of AAL have appeared in recent years.56–58 All are in agreement that the lectin possesses a six-bladed b-propeller fold. Each blade is composed of four b–strands (Figure 8). The first complete study (Wimmerova et al.),57 followed shortly by a second report,58 identified five fucose molecules located in binding pockets between adjacent propeller blades with strong similarities between the sites due to repeats in the amino acid sequences. The fucose-binding sites are exposed on each side of the dimer at distances ranging from 50 to 70A˚ from each other. Aromatic rings from Trp/Tyr stack against the flat nonpolar face of fucose. Since the six b-propeller blades are not identical, there are some differences between the five binding sites. In sites I, III, and V, the fucose is present as the b-anomer whereas it is found in the a-anomeric configuration in site IV and a mixture of the two anomeric forms in site II. From the electron density map it was deduced that the oxygen atoms O3–O5 of fucose are involved in hydrogen bonds with the side chains of the amino acids. The second paper58 confirmed the b-propeller structure but found only three fucose-binding ligands at the five sites. Chemical modification with tetranitromethane and site-directed mutagenesis studies based on the involvement of tyrosine and tryptophan in the binding sites revealed the nonequivalence of the five binding sites.59

Blade-5 Site 5

Site 4

b 5-4

Blade-4

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Blade-6

b 5-1 Site 3 Site 6 Blade-3

Blade-1 N C

Site 1

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Figure 8 Overall structure of the AAL monomer. Blue, cyan, green, yellow, orange, and red ribbons show blades 1–6, respectively. Purple ellipsoids indicate fucose-binding sites 1–5 and the corresponding site 6. Three stick models at sites 1, 2, and 4 show fucose molecules. Reprinted with permission from Fujihashi, M.; Peapus, D. H.; Kamiya, N.; Nagata, Y.; Miki, K. Biochemistry 2003, 42, 11093–11099. Copyright (2003) American Chemical Society.

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Imobilization of AAL to Sepharose 4B allowed isolation of fucosyl glycoproteins from erythrocyte membrane preparations.60 Desialylation improved yields of the fucosylated glycoproteins.

3.27.2.3.2

Melastiza chateri and Aspergillus oryzae

Another related ascomycete bearing fruiting bodies, M. chateri, was found to contain an L-fucose-specific lectin having a high degree of sequence homology to AAL.61 Presumably, it also forms the six-bladed b-propeller fold, although no X-ray structural work has been reported for this lectin. The pathogenic, mycelial ascomycete fungus, A. oryzae (A. flavus v. oryzae), contains a lectin having about 30% sequence identity with AAL, and which possesses six tandem repeats, suggestive of the six-bladed b-propeller structure. Its agglutination activity is also inhibited by L-fucose.62

3.27.2.3.3

Pleurotus ostreatus (Tricholomataceae)

Extracts of the fruiting bodies of the mushroom P. ostreatus (oyster mushroom) contain hemagglutinating activity,63,64 from which a lectin was isolated on immobilized hog gastric mucin. Upon SDS-PAGE, the lectin was revealed to contain two protein bands, at 44 and 41kDa, and it possessed a native molecular weight of 87kDa by HPLC gel filtration. The lectin was later shown to be a homodimer, suggesting that the 41kDa subunit might have arisen as a result of proteolytic cleavage. Amino acid composition was characteristic of lectins in having low amounts of sulfur-containing amino acids and generous amounts of hydroxyl and acidic amino acids. Carbohydrate analysis showed a content of 14% neutral carbohydrate consisting of mannose and N-acetylglucosamine. Among the sugars inhibiting agglutination of erythrocytes, N-acetylgalactosamine was best followed closely by lactose and methyl a- and b-galactosides.64 Subsequently, Wang et al.65 isolated the same lectin using ion exchange matrices and liquid chromatography. The lectin had the same physical–chemical properties as previously reported. Additionally, the authors reported its lability to heat, acid, and alkali. The N-terminal amino acid sequence, as well as sequences of other peptide fragments, was reported and shown to have limited sequence homology with the Aleuria aurentia lectin. Inhibition of hemagglutination by sugars gave somewhat different results from those of Conrad and Rutiger:64 melibiose was the best inhibitor although lactose and galactose were also good inhibitors.65 The lectin was shown to exert potent antitumor activity in mice bearing sarcoma S-180 and hepatoma H-22. The carbohydrate-binding specificity of the P. ostreatus lectin was also studied by surface plasmon resonance.66 It was confirmed that the lectin recognizes b-galactosyl-terminated oligosaccharide chains: lactose followed by Gal(b1-4)[Fuc(a1-3)]Glc and galactose. A preliminary report on the X-ray crystal structure of the oyster mushroom at 4.0A˚ has appeared.67 A lectin that binds N-acetylglucosamine was isolated from fresh sclerotia of the mushroom Pleurotus tuber-regium (Lentinus tuber-regium) by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, followed by absorption and elution by N-acetylglucosamine from an N-acetylglucosamine-agarose column.68 The lectin had a molecular mass of 32kDa and agglutinated trypsintreated rabbit erythrocytes, but not untreated cells. This lectin does not appear to be structurally related to the P. ostreatus lectin, despite the close taxonomic relationship of the two species.

3.27.2.3.4

Ralstonia solanacearum

Finally, although is is outside the general scope of this chapter, the lectin from the plant pathogenic bacterium R. solanacearum should be mentioned (see Chapter 3.28).69 This lectin recognizes L-fucose in a wide variety of structures; like AAL, it agglutinates all H-positive human erythrocytes regardless of their type. The lectin has a subunit molecular mass of about 9kDa, with a tandem repeat that is substantially homologous with the six tandem repeats of AAL. In subsequent work,70 it was found that the lectin possesses two fucose-binding sites per monomer. Furthermore, X-ray crystallographic studies showed that the monomers contain two small four-stranded antiparallel b-sheets, and that they trimerize through a threefold axis to form a six-bladed propeller structure, much like that of AAL. Unlike AAL, however, all six putative binding sites in the trimer are capable of binding ligands.

3.27.2.4 Fungal Immunomodulatory Protein (FIP Fve) of Flammulina velutipes (Tricholomataceae) The edible mushroom Flammulina velutipes, popular in Asian cuisine and commercially available as Enokitake, or Golden Needle mushrooms, contains a lectin that agglutinates human erythrocytes nonspecifically. The lectin was isolated by extraction with 5% acetic acid, followed by precipitation with 95% saturated (NH4)2SO4, and subsequent ion exchange chromatography sequentially on cationic and anionic resins.31 The lectin stimulated proliferation of

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Mushroom Lectins

human peripheral blood lymphocytes, although at a concentration approximately 20-fold higher than required by the powerful mitogen phytohemagglutinin. It protected N,O-bis(trimethylsilyl)acetamide (BSA)-sensitized mice against anaphylaxis, and also against hindpaw edema induced by the antigenic compound 48/80. The agglutination activity was not inhibited by simple monosaccharides, N-acetylamino sugars, or by lactose, but thyroglobulin was found to inhibit hemagglutination,71 suggesting that agglutination involves binding of an extended glycan moiety. Ko et al.31 sequenced the entire protein of 114 amino acids by peptidase digestion and automated amino acid sequencing. The sequence exhibited homology to another immunomodulatory protein, Ling Zhi-8 (LZ-8), isolated from the polypore mushroom G. lucidum.72,73 LZ-8 exhibits hemagglutinating activity toward sheep erythrocytes, but not human cells. As with the Flammulina protein, the agglutinating activity is not inhibited by simple mono- or disaccharides. Homology also occurred with another lectin having immunomodulatory activity, that from the edible paddy straw mushroom Volvariella volvacea (Plutaceae).74 On this basis, FIP was proposed as a new family of proteins. More recently, FIP Fve has been subjected to X-ray crystallographic analysis.71,75 The 1.7A˚ structure revealed that Fve dimer is a dumbbell-shaped molecule in which two monomers exchange N-terminal a-helices, each of which interacts with an adjacent b-strand of the opposite monomer. Seven additional b-strands at the C-terminus form a fibronectin III (FNIII) fold of intermediate structure between the seven-stranded s-type, and the eight-stranded h-type, which was designated as ‘pseudo-h type’. The model also revealed that large areas of the dimer surface have exposed amino acid residues that are common to carbohydrate-binding sites of other lectins, such as aromatic residues and residues capable of H-bonding to sugar hydroxyl groups. This structural feature is consistent with a possible extended binding site(s) capable of reacting with large, complex glycan moieties of cell surfaces or glycoproteins. This structural work further confirms and legitimatizes the proposal of FIPs as a distinct and novel family of lectins.

3.27.2.5 Galectin Family 3.27.2.5.1 Coprinus cinereus (Coprinaceae) Two isolectins having the carbohydrate specificity characteristics of the galectin family of lectins were isolated from the inky cap mushroom C. cinereus.76 Designated Cgl-I and Cgl-II, they were the first galectin-like proteins found outside of the animal kingdom (Chapter 3.24). The source was targeted by screening GenBank for sequences that included a motif characteristic of all known galectins. cDNA sequences encoding the lectins were sequenced. The isolectins exhibited 84% identity of amino acid sequence to each other, and possessed all of the key conserved residues of galectins, although they lacked any cysteine residues found in animal galectins, as well as characteristic Nterminal post-translational modification. rCgl–I and rCgl–II have recently been crystallized; crystals of Cgl–II diffracted to 1.6A˚ resolution,77 although solution of the structures has not yet been reported.

3.27.2.5.2 Agrocybe spp. (Bolbitiaceae) A lectin was purified fron the fruiting bodies of the edible fungus A. cylindracea by ion exchange and gel filtration chromatography. It was homogeneous on SDS-PAGE, had a molecular mass of 30kDa by gel filtration and a subunit mass of 15kDa.5 Cleavage of the lectin by chemical and enzymatic means gave peptides that permitted determination of the lectin’s complete amino acid sequence.6 The calculated molecular mass of the lectin including the blocked N-terminal acetyl group was 17 088 Da. Sequence identity with human galectin-I and the Coprinus lectin I was 19.1% and 36.8%, respectively. A second group7 reported isolation of a heterodimeric lectin from the same source, with subunits of 15.3 and 16.1kDa and a molecular mass of 31.5kDa. This discrepancy is yet to be resolved, but might be due to cultural or strain differences in the source material. Inhibition of hemagglutination indicated that the lectin binds Neu5Ac(a2-3)Gal(b1-4)GlcNAc/Glc with high affinity, does not recognize the Neu5Ac(a2-6)-isomer, binds N-glycolylneuraminic acids with about 100-fold less affinity, and lactose and N-acetyllactosamine weakly.5 The fruiting bodies of the mushroom Agrocybe aegerita contain a lectin which is very similar to the one isolated and characterized from A. cylindracea.78 Currently, however, A. aegerita is considered to be synonymous with A. cylindracea, so that the minor differences reported may reflect strain, culture, or experimental variations. The pure lectin was obtained by a combination of ammonium sulfate fractionation, ion exchange chromatography, and gel filtration. It appears to be a homodimer of molecular mass 32kDa and subunit mass of 15.8kDa. Its amino acid composition was very similar to the A. cylindracea lectin; however, the blocked N-terminus was apparently due to pyroglutamic acid rather than an N-acetyl group. Analysis of the succeeding eight amino acids (QGVNIYNI) showed similarity to residues 6–10 of A. cylindracea. No carbohydrate was present in the lectin. It had an isoelectric point of about 3.9, consistent with its amino acid composition. The complete amino acid sequence was obtained by cloning the gene

Mushroom Lectins

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encoding the lectin. The deduced amino acid sequence contains 158 amino acids. The recombinant lectin was expressed in E. coli and shown to have identical physical–chemical and carbohydrate-binding properties as the native protein. Lactose was shown to bind to the lectin. A second report on the isolation and characterization of A. aegerita lectin by Zhao et al.79 essentially confirmed the paper by Sun et al.78 No simple sugars inhibited agglutination of erythrocytes; among three glycoproteins tested, only hog gastric mucin was an inhibitor at a minimum concentration of 7.32mgml1. The lectin’s ability to agglutinate human and animal erythrocytes was unaffected by acid or alkali treatment. It showed inhibitory activity to infection by tobacco mosaic virus.78 A. aegerita lectin also had activity against several human tumor cell lines, for example, HeLa, SW480, and HL-60,79 and was shown to assert its toxicity via its ability to induce apoptosis.80 Recombinant lectin has been crystallized and preliminary X-ray data at 1.9A˚ and 1.6A˚ for both recombinant lectin and its lactose complex have been published.81

3.27.2.6 Jacalin-Related Lectin 3.27.2.6.1

Grifola frondosa (Meripilaceae)

This important culinary species, which is grown commercially, was recently found to contain a lectin having potent agglutinating activity toward rabbit erythrocytes.82 The lectin was purified from a potent protease activity, a hemolytic activity, and other proteins by heat treatment and ion exchange chromatography. The agglutinating activity was inhibited neither by the limited panel of mono- and disaccharides tested, nor by bovine submaxillary mucin, fetuin, or asialofetuin, but was inhibited by porcine stomach mucin. It contained polypeptides subunits of 24kDa by SDSpolyacrylamide gel electrophoresis, but their association in the native structure was not determined. Full-length cDNA was cloned and sequenced, the full length sequence exhibiting significant homology with members of the jacalin family of plant lectins. This lectin family is named for the jackfruit, Artocarpus integrifolia, the source of firstrecognized member of the family (see Chapter 3.26). Jacalin lectins are found as homo-oligomers, and as oligomers whose monomers are comprised of a small (about 20 residues) and large subunit. The former exhibit specificity to mannose and mannooligosaccharides, while the latter are specific for galactose/N-acetylgalactosamine.83 The heterodimer arises from a post-translational clipping of a single chain, which does not alter the three-fold symmetric b-prism fold characteristic of jacalin-related lectins, but leads to alteration of the active-site topology to accommodate the galactose structure rather than mannose. It appears that the Grifola lectin is a homo-oligomer, at least as initially isolated, although it does not exhibit clear-cut specificity for mannose or any other simple sugar.

3.27.3 Fungal Lectins not Characterized by X-Ray Analysis or Sequence Homology 3.27.3.1 Lyophyllum spp. (Tricholomataceae) From the Japanese culinary mushroom L. shimeji (Kawam.), Hongo, Ng and co-workers isolated a 30kDa monomeric protein having strong hemagglutinating activity (minimum agglutinating concentration of 1mgml1) toward rabbit erythrocytes.84 The agglutining activity was not inhibited by a number of free mono- or disaccharides, aminohexoses, or N-acetylneuraminic acid, nor by the glycoproteins human chorionic gonadotropin and lactoferrin. N-Terminal sequence of approx. 20 residues showed no homology to any other lectin, although some homology was observed to internal sequences from plant MAP-kinases, and animal kinesins. They propose that L. shimeji agglutinin is a novel lectin. Very recently, a group of lectins from the closely related mushroom L. decastes (Fr.;Fr.)Sing has been isolated (Winter and Goldstein, unpublished). Crude extracts exhibit moderately strong agglutination of rabbit erythrocytes, but only very weak activity towards human cells. Purification was accomplished by binding to and elution from a column of melibiose-Sepharose, using a gradient of lactose, whereby at least three peaks were observed. From the latest-eluting fraction, a nearly pure polypeptide of about 10kDa by SDS-polyacrylamide gel electrophoresis was obtained. MALDI-TOF mass spectrometry indicated this protein to have a molecular mass of 10276Da. Approximately 30 residues of the N-terminal sequence have been determined by automated Edman sequencing, which bears no similarity to the L. shimeji lectin sequence described above, to RIP and Lyophyllum antifungal protein (LAP) also described from the same species,85 or to any other fungal or plant lectin. The lectin binds to galactose-containing structures, with apparent highest affinity for the Gal(a1-4)Gal determinant. This specificity is in contrast to MOA, which has almost exclusive specificity for the Gal(a1-3)Gal moiety (see Section 3.27.2.1.2). It precipitates with pigeon ovalbumin, which contains Gal(a1-4)Gal epitopes,86 but only when

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the latter has been desialylated by neuramididase or mild acid treatment. A curious bulk property of the lectin is its propensity to form gels in solutions of greater than about 1 mg ml1 at low temperature, even though the protein contains neither hydroxylysine nor hydroxyproline, nor does it appear to be glycosylated, as is characteristic of the potato or tomato lectins. Other peak fractions eluting earlier in the lactose gradient from melibiose-Sepharose appear to contain the same polypeptide with or without an additional protein, and do not gel in solution. The nature of these apparent isolectins has not been established.

3.27.3.2 Ischnoderma resinosmum (Polyporaceae) A lectin agglutinating human erythrocytes, especially those treated with pronase, was isolated and partially characterized from this shelf-like polypore mushroom,87 which is also known as Polyporus resinosus (Schrader) Fries, but whose currently accepted name is I. benzoinum (Wahlenberg) P. Kunstner. Type B cells were slightly favored over type A or O(H), but not sufficiently so as to consider it a blood type-specific lectin. Best inhibition was exhibited by lactulose (Gal(b1-4)Fru), followed by methyl b-galactoside and lactose. Fucose, galactose, L-arabinose, and a-galactosides were less active in that order, whereas other mono- or oligosaccharides and all aminohexoses were inactive. Its N-terminal amino acid sequence, PANFSIGTYA, resembles a segment in the XCL family lectins beginning at about residue 28 (Figure 5), although the segment is too short to be of great significance. No homology in this short segment with the other polypore lectins having a ricin domain structure, LSL or PSL (see Sections 3.27.2.1.1 and 3.27.2.1.3), is evident. By specific chemical modification and its protection by ligands, as well as by NMR analysis, the binding of ligand was found to involve histidine and tyrosine residues.88 This property is consistent with, but not exclusive to, the XCL family of lectins. No further work with this lectin has been reported in the past 15 years.

3.27.3.3 Pleurotus cornucopiae (Tricholomataceae) The fruiting bodies of the mushroom P. cornucopiae contains three hemagglutinating components – PCL-a,-b,-c – isolated by DEAE- and CM-Toyopearl chromatography. They displayed similar chemical and immunochemical properties and it was concluded that they are isolectins: two homodimers and a heterodimer, consisting of combinations of two subunits of 16 and 15kDa.89 No simple sugars inhibited the agglutination activity of these lectins. However, mucins and their asialo derivatives were potent inhibitors of hemagglutination, and it was suggested that the T-disaccharide [Gal(b1-3)GalNAc] was the structure responsible for the lectins’ binding activity.89 Two genes encoding the lectins were cloned and their products characterized.8 Each of the genes encoded 144 amino acids, with only a difference of five amino acids between them within the coding region. The two lectins were expressed in E. coli; both recombinant lectins showed hemagglutinating activity and were inhibited by porcine submaxillary mucin, similar to the native lectin. The primary structure of one of the lectins was very similar to the lectin of the nematode-trapping fungus from A. oligospora (see Section 3.27.2.2.3). The lectin from mycelial aggregates (PCL-M) becomes active when its 40kDa subunit forms a multimer via disufide bond formation in the presence of Ca2þ. A lectin-deficient strain of P. cornucopiae contains a protein that was isolated, characterized, and crystallized.90 The protein was a homotetramer, each monomer having a mass of 16.5kDa. Comparison of amino acid sequences of this protein with proteins from the lectin-containing strain revealed limited homology, although antiserum against the lectin-containing strain did not cross-react with the lectin-deficient strain. The complete amino acid sequence of the lectin-deficient strain protein was determined. It also contained 144 amino acids, and was blocked at its N-terminus by N-acetylserine.91

3.27.3.4 Sialic Acid-Binding Lectins 3.27.3.4.1 Hericium erinaceus (Hydnaceae) The fruiting bodies of the mushroom H. erinaceus contain a lectin isolated by ammonium sulfate fractionation followed by fractional salt elution from DEAE-Toyopearl and Mono-S columns.92 On SDS-PAGE, in the presence or absence of 2-mercatoethanol, it gave two closely spaced bands of 15 and 16 kDa. By gel filtration, an aggregate mass of 54kDa was estimated, suggesting that it occurs as an A2B2 heterotetramer. Carbohydrate and amino acid analysis revealed a high content of Gly, Glx, Ser, Ala, and 1.5% carbohydrate. Of the sugars tested for their ability to inhibit lectin-mediated hemagglutination, only four monosaccharides and an oligosaccharide were active: Neu5Gc was most active, followed by Neu5Ac, Neu5Ac(a2-3)lactose, and

Mushroom Lectins

617

galacturonic acid. However, asialobovine serum albumin and asialofetuin were much better inhibitors of hemagglutination than their sialylated glycoproteins, placing some doubt on this lectin being specific for sialic acid. Furthermore, sialidase-treated erythrocytes were 50–100 times more readily agglutinated than were the native cells.

3.27.3.4.2

Psathyrella velutina (Coprinaceae)

A lectin (PVL) from the fruiting bodies of the mushroom P. velutina (Lacrymaria lacrymabunda (Bull.) Patoulland) was purified by affinity chromatography on chitin.93 It is a monomeric polypeptide with molecular mass of 40kDa, estimated by gel filtration. High contents of acidic and hydroxylic amino acids and glycine characterize its amino acid distribution. Equilibrium dialysis against N-acetylglucosamine indicated the existence of four sites per polypeptide chain with Ka¼6.4103 M1. Of the monosaccharides tested, the hemagglutination activity of the lectin (human type O erythrocytes) was inhibited only by N-acetylglucosamine, its a- and b-methyl glycosides being essentially indistinguisable. Unlike most other GlcNAc-binding lectins, (b1-4)-linked oligomers of N-acetylglucosamine were less inhibitory than the free sugar and N-acetyllactosamine was a noninhibitor.93 Oligosaccharides containing GlcNAc linked b1-3 to GalNAc or Gal were good inhibitors. Interestingly, L-Fuc linked a1-6, but not a1-3 or a1-4 to GlcNAc (b1-3)Gal, was a very good inhibitor. A mixture of Neu5Ac(a2-3)lactose and Neu5Ac(a2-6)lactose was noninhibitory. Asialofetuin did not inhibit, whereas asialo-agalactofetuin, exposing terminal GlcNAc groups, was a good inhibitor. Immobilized P. velutina mushroom lectin (on Affi-Gel 10 column) was employed to characterize its carbohydratebinding specificity.94,95 It was shown that the lectin interacts with nonreducing terminal N-acetylglucosamine end groups, but with neither N-acetylgalactosamine nor N-acetylneuraminic acid groups. GlcNAc(b1-3)Gal-terminated oligosaccharides bound to the column but the (b1-6)-linked isomer is only retarded. Oligosaccharides with biantennary GlcNAc(b1-2)Man residues bound strongly to the PVL column; however, a bisecting GlcNAc group reduced the affinity of such oligosaccharides to the column.94,95 The claim was made by Ueda et al.96 that PVL recognizes nonreducing terminal N-acetylneuraminic acid residues in glycoproteins and oligosaccharides. In a subsequent study, Ueda et al.97 employed surface plasmon resonance and affinity chromatography to study the binding of PVL to various sialoglycoproteins and their asialo derivatives. It was concluded that PVL has a dual specificity for GlcNAcand (a2-3)-sialyl-terminated glycoproteins, similar to wheat germ agglutinin. It was found that trisialylated glycoproteins were especially recognized by the lectin. Glycoproteins containing (a2-6)-linked neuraminic acid were retarded, but not bound on immobilized PVL. In an important biomedical application of the P. velutina lectin, it was found that the serums of individuals suffering from rheumatoid arthritis express a high percentage of agalacto-N-glycan chains on their immunoglobulin G (IgG) molecules.94,98 This exposes GlcNAc–terminated oligosaccharides which bind to immobilized PVL–Affi–Gel 10 columns, or can be detected using biotinylated PVL in Western blotting on the H-chains of IgG derived from patients with rheumatoid arthritis.94,95,98 Substantial galactose deficiency of IgG molecules has also been demonstrated in individuals with HIV-1 infection.99

3.27.3.4.3

Chlorophyllum molybdites (Gomphaceae)

Extracts of the toxic mushroom from Chlorophyllum molybdites gave a novel N-glycolylneuraminic acid-binding lectin.100 Isolated by affinity chromatography on immobilized bovine submaxillary mucin, the lectin was shown to be a homodimer of subunit molecular mass 16kDa joined by noncovalent interactions. The pI of 3.75 indicated a high content of acidic amino acids (27 mol.% Asx, Glx). There was also one residue each of Cys and Met. A glycoprotein. The lectin was shown to be a glycoprotein having a carbohydrate moiety consisting of GlcNAc: GalNAc:Gal:Man:L-Fuc in the ratio of 1.5:1.9:4.4:4.8:1.0. Although no structural data are yet available, the lectin’s unique specificity suggests that it may be of a novel structure. In hapten inhibition of hemagglutination analysis, N-glycolylneuraminic acid exhibited the strongest specificity among the monosaccharides tested whereas N-acetylneuraminic acid was noninhibitory. GalNAc and Me-a-GalNAc were also inhibitory but only at higher concentrations. In surface plasmon resonance analysis, asialobovine submaxillary mucin, and porcine stomach mucin exhibited the strongest binding affinity. Further analysis indicated the presence of two types of binding sites present on the C. molybdites lectin – one that recognized N-glycolylneuraminic acid and a second which was specific for GalNAc.

3.27.4 Conclusions In this chapter, we have limited coverage to lectins isolated primarily from mushrooms, although a few fungi not forming macroscopic carpophores (fruiting bodies) are included, when they are structurally related to lectins isolated

618

Mushroom Lectins

from mushrooms. Although only a very small fraction of the thousands of extant species of mushrooms have been examined for lectin activities, the number of species in which lectin activity has been detected, purified, and characterized to some degree is too large to be covered in this chapter. Many of the mushroom lectins reported have been only cursorily characterized, sometimes with very questionable conclusions as to binding specificity, so have not been included here. Instead, we have concentrated on those mushroom lectins that have been well characterized and for which a significant amount of structural data are available, allowing their assignments to specific structural families, or to the recognition of their potentially having heretofore unknown structures. It is evident from this approach that the ricin-like domain lectins, identified and named from the toxin, and the toxic lectin from the castor bean, Ricinis communis, is prevalent in the fungal kingdom as well as in vascular plants. This suggests that the b-barrel motif characterizing this family is of ancient origin, or else under some unrecognized selection pressure for convergent evolution. Otherwise, apart from some preliminary indication of galectin-like structures (see Section 3.27.2.5) and one possible member of the jacalin-related family (see Section 3.27.2.6), the fungal lectin structures (XCL, b-propeller fold, and FIP families) are distinct from the prevalent plant lectin families (monocot mannose binding, legume lecin). Despite these different structures, all lectins, both fungal and plant, are characterized by a high content of b-sheet structure, and by a similar spectrum of carbohydrate recognition. As noted above, certain mushroom lectins are already finding specific biomedical application, as has been the providence of plant lectins and some animal lectins for many years. The large number of mushroom and other fungal lectins not yet well characterized, and the vast number of species not yet examined, suggests that this kingdom of biotica will be a rich source for glycobiological material and study for many years.

Glossary mushroom Macroscopic fruiting body of Ascomycota or Basidomycota fungi. Most lectins discussed are extracted from the fruiting body, although in the few cases that have been investigated, the non-fruiting mycelium may also contain the same or similar lectin. RIP Ribosome inactivating protein: hetero-oligomeric proteins containing a carbohydrate-binding (lectin-like) subunit and a ribosome-inhibiting subunit (RNAse) whose entry into the cell is facilitated by the lectin subunit binding. MAP kinase Mitogen-activated protein kinase: serine/threonine specific protein kinase that responds to extracellular stimuli (mitogens) to regulate various cellular activities. SDS-PAGE Polyacrylamide gel electrophoresis conducted in the presence of the anionic denaturant sodium dodecyl sulfate.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86.

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Mushroom Lectins

Kawagishi, H.; Mizuno, T. FEBS Lett. 1988, 227, 99–102. Kawagishi, H.; Mori, H. Biochim. Biophys. Acta 1991, 1076, 179–186. Yoshida, M.; Kato, S.-I.; Oguri, S.; Nagata, Y. Biosci. Biotech. Biochem. 1994, 58, 498–501. Oguri, S.; Yoshida, M.; Nagata, Y. Biosci. Biotech. Biochem. 1994, 58, 502–506. Oguri, S.; Nagata, Y. Biosci. Biotech. Biochem. 1994, 58, 507–511. Kawagishi, H.; Mori, H.; Uno, A.; Kimura, A.; Chiba, S. FEBS Lett. 1994, 340, 56–58. Kochibe, N.; Matta, K. L. J. Biol. Chem. 1989, 264, 173–177. Endo, T.; Ohbayashi, H.; Kanazawa, K.; Kochibe, N.; Kobata, A. J. Biol. Chem. 1992, 267, 707–713. Kobata, K.; Koshibe, Endo, T. Meth. Enzymol 1994, 247, 228–237. Ueda, H.; Kojima, K.; Saitoh, T.; Ogawa, H. FEBS Lett. 1999, 448, 75–80. Ueda, H.; Matsumoto, H.; Takahashi, N.; Ogawa, H. J. Biol. Chem. 2002, 277, 24916–24925. Tsuchiya, N.; Endo, T.; Matsuta, K.; Yoshinoya, S.; Takeuchi, F.; Nagano, Y.; Shiota, M.; Furukawa, K.; Kochibe, N.; Ito, K.; et al. J. Immunol. 1993, 151, 1137–1146. 99. Moore, J. S.; Wu, X.; Kulhavy, R.; Tomana, M.; Novak, J.; Moldoveanu, Z.; Brown, R.; Goepfert, P. A.; Mestecky, J. AIDS 2005, 19, 381–389. 100. Kobayashi, Y.; Kobayashi, K.; Umehara, K.; Dohra, H.; Murata, T.; Usui, T.; Kawagishi, H. J. Biol. Chem. 2004, 279, 53048–53055. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98.

Mushroom Lectins

621

Biographical Sketch

Irwin J. Goldstein – Born 8 September 1929. B.S. Chemistry, Syracuse University, 1951; Ph.D. in Biochemistry, University of Minnesota, 1956; Research Fellow and Lecturer, Department of Biochemistry, University of Minnesota; Guggenheim Memorial Fellowship, Lister Institute of Preventive Medicine in London with Dr. W. J. Whelan, 1959–60; NIH Special Fellow, University of Stockholm, Sweden, 1960–61; Asst. Prof. Biochemistry, State University of New York at Buffalo, 1961–65; Assoc. Prof. Biological Chemistry, University of Michigan, Medical School, 1965–72; Prof. Biological Chemistry, University of Michigan Medical School, 1972–99; Professor Emeritus, University of Michigan, 2000–present; Associate Dean for Research and Graduate Studies, University of Michigan Medical School, 1986–98; Prof. and Interim Chair, Department of Biological Chemistry, University of Michigan Medical School, 2001–03; American Heart Association Established Investigator, 1963–69; Distinguished Faculty Achievement Award, University of Michigan, 1985; University of Michigan Medical School Distinguished Faculty Lectureship in Biomedical Research; Claude C. Hudson Award in Carbohydrate Chemistry, an international American Chemical Society award, 1993; Outstanding Achievement Award of the University of Minnesota, 1996. Research includes synthesis, biochemistry, and immunochemistry of oligosaccharides; protein–carbohydrate interaction; isolation, characterization, and biomedical application of plant lectins.

Harry C. Winter – Born 26 February 1941. B.S. Agricultural & Biological Chemistry, Penn State University, 1962; Ph.D., Biochemistry, University of Wisconsin-Madison, 1967; NSF Postdoctoral Fellow, University of California-Berkeley, Asst. Professor Biological Chemistry, Penn State University 1968–75; Lecturer and Research Scientist, University of Michigan, 1975–present. Previous research areas – biological nitrogen fixation, plant and bacterial amino acid enzymology and metabolism. Present research, since 1992, protein–carbohydrate interaction; isolation, characterization, and biomedical application of plant lectins.

3.28 Microbial Lectins N. Sharon, The Weizmann Institute of Science, Rehovot, Israel I. Ofek, Sackler School of Medicine, Tel Aviv, Israel ß 2007 Elsevier Ltd. All rights reserved. 3.28.1 Introduction

623

3.28.2 Viral Lectins

624

3.28.2.1 Influenza Viruses

624

3.28.2.1.1 3.28.2.1.2

Influenza A virus hemagglutinin Influenza C HEF

625 631

3.28.2.2 Rhesus Rotavirus

631

3.28.2.3 Polyoma Virus

632

3.28.2.4 Foot-and-Mouth Disease Virus

634

3.28.3 Bacterial Lectins

636

3.28.3.1 Surface-Bound Lectins

636

3.28.3.1.1 3.28.3.1.2 3.28.3.1.3

Type 1 fimbriae P fimbriae F17 fimbriae

637 642 642

3.28.3.2 Soluble Bacterial Lectins 3.28.3.2.1 3.28.3.2.2 3.28.3.2.3 3.28.3.2.4

644

Pseudomonas aeruginosa lectins Ralstonia solanacearum lectins Cyanovirin-N Microsystis viridis lectin

644 650 653 654

3.28.1 Introduction Numerous microorganisms produce lectins of different kinds that are specific for animal cell surface carbohydrates.1,2 In viruses, the lectins occur as membrane constituents, in bacteria they are generally present on the cell surface, often as submicroscopic multisubunit surface appendages known as fimbriae or pili, and occasionally in intracellular soluble form, while in protozoa they are constituents of the cell envelope. Most of these lectins serve to tether the organisms to a substratum (e.g., soil bacteria to clays or marine bacteria to corals) in order to better access nutrients, escape deleterious agents, and withstand cleansing. In animals, they serve for the attachment (or adhesion) of the microorganisms to host tissues or cells, a prerequisite for infection, and are therefore among the virulence factors of the microorganisms. Detailed knowledge of the specificity and combining sites of these lectins is of importance not only for the understanding of a basic mechanism conserved throughout evolution, but also for the development of powerful inhibitors of the lectins for blocking microbial adhesion to tissues. This is the aim of antiadhesion therapy of microbial diseases.3,4 Most of the microbial lectins have only been poorly characterized. Their existence and specificity have been inferred primarily from inhibition experiments, in which the effect of different carbohydrates is examined on adhesion of the organisms to animal cells or on the agglutination of erythrocytes or other kinds of cell, such as yeasts.1,2 Specificity for the carbohydrate of glycolipids or glycoproteins is usually assessed by binding of the bacterial lectins to these glycoconjugates separated on thin layer chromatograms or on blots of gel electrophoresis, respectively. Recently, the application of glycan microarrays for specificity studies of viral and bacterial lectins has been described.5,6 In a few cases, the specificity and association constants of carbohydrates to the isolated lectins have been measured by physicochemical techniques, for example, by surface plasmon resonance.64 Binding of carbohydrates to lectins, including those of microbial sources, is generally characterized by a low affinity for monovalent ligands, a drawback balanced by multivalency, which provides high avidity for complex glycans or cell surfaces. In general, millimolar affinity is observed for lectin binding to monosaccharides, although cases of lectins

623

624

Microbial Lectins

with micromolar affinities are known.7 When oligosaccharides act as ligands, corresponding to an extended binding site on the lectins, increased affinity up to the micromolar range can be observed. These interactions are typified by a favorable enthalpy term, due to the high number of hydrogen bonds that is offset by an unfavorable entropy contribution, attributed either to solvent rearrangement or loss of ligand conformational flexibility. An increase of several orders of magnitude in the affinity of carbohydrates to lectins can be achieved by suitable chemical derivatization, for example, by attachment to hydrophobic aglycones. Similar increase is obtained by their attachment to polymeric carriers, to form multivalent ligands such as dendrimers (see Chapter 3.36). For a small number of the microbial lectins, detailed information is available on their structure as well as that of their combining sites and their mode of interaction with ligands. It is based primarily on high-resolution X-ray crystallography of the lectins and their complexes with ligands (see Chapters 2.06 and 2.07). Other inputs include binding experiments with different sugars and their derivatives using state-of-the-art techniques, site-directed mutagenesis of the proteins, and, to a limited extent, also NMR experiments and molecular modeling. Such studies have shown that like the lectins themselves, the sites are diverse, even when their specificity is the same (although within a given protein family the sites may be similar).8 In general, the sites appear to be preformed, since conformational changes occur rarely upon ligand binding. The amino acids that form the combining sites are not necessarily contiguous, and are brought together in space by the folding of the polypeptide chain. Changes in the spatial position of the amino acid residues may occur upon binding of the carbohydrate, bringing them to an orientation which improves stereocomplementarity with the ligand. The fit between the binding site on the protein and the carbohydrate is also affected by the shape of the ligand. Oligosaccharides are flexible molecules with considerable freedom of rotation around the glycosidic bonds connecting the individual monosaccharide constituents. As a result, they may assume different shapes, only one of which may fit the combining sites. The types of bonds involved in the formation of lectin–carbohydrate complexes are in principle not different from those involved in the formation of complexes of proteins with other ligands, such as peptides, oligonucleotides, or various small molecules (see Chapter 3.21). Binding between proteins and carbohydrates is stabilized primarily by a network of hydrogen bonds and hydrophobic interactions; in rare cases, electrostatic interactions (or ion pairing) and coordination with metal ions also play a role.9–11 Bonding is sometimes mediated by one or more water molecules. Although in a single protein a limited set of amino acid residues contribute to the interactions with the ligand, in general most of the side chains of the 20 amino acids can participate in ligand binding.

3.28.2 Viral Lectins Viruses contain sugar-specific surface proteins or glycoproteins that act as hemagglutinins and are therefore classified as lectins (Table 1). These lectins are structurally diverse and no common features can be discerned. Detailed information based largely on X-ray crystallography is available on the interaction with carbohydrates of influenza virus hemagglutinins. Complexes with ligands of the hemagglutinins of rotavirus, polyoma virus, and foot-and-mouth virus have also been well studied by the same technique. No other viral lectins have however been similarly characterized.

3.28.2.1 Influenza Viruses Influenza A and B virions are studded with two different types of spikes, the hemagglutinin (HA), specific for N-acetylneuraminic acid that mediates receptor binding and pH-dependent membrane fusion and the neuraminidase (correct name, ‘sialidase’), originally known as the viral receptor-destroying enzyme (RDE). In contrast, influenza C virus possesses only one type of spike, specific for 9-O-acetyl-N-acetylneuraminic acid, which combines all three functions, and is commonly referred to as the hemagglutinin-esterase-fusion protein (HEF). Influenza virus A is attracting much interest because an understanding of the structural determinants and molecular mechanisms involved in its binding to human cell receptors is central to the identification of viruses that pose a pandemic threat (see Chapter 4.25). The receptors recognized by influenza virus are sialic acids of cell surface glycoproteins and glycolipids. Sialic acids are usually found in either a2-3 or a2-6 linkages to galactose, the predominant penultimate sugar of N-linked carbohydrate side chains. The binding preference of a given HA for one or the other of these linkages correlates with the species specificity of the virus.22,23 Thus, the HAs of all 15 antigenic subtypes found in avian influenza A and B viruses bind preferentially to sialic acid in a2-3 linkage, and it is this form of the sialosaccharide that predominates in avian enteric tracts where these viruses replicate. Swine influenza viruses bind sialic acid in a2-6 and sometimes also

Microbial Lectins

625

Table 1 Viral lectins Virus Flaviviruses Dengue virus Herpes viruses Herpes simplex Myxoviruses Orthomyxo Influenza A & B, human strains Porcine strains Avian strains Influenza C Paramyxo Newcastle disease Sendai Rotavirus Nidoviruses Coronavirus, bovine Totovirus, bovine Papoviruses Polyoma Picornaviruses Foot-and–mouth disease Retroviruses HIV

Specificity

References

Heparan sulfate

12

Heparan sulfate

13

Neu5Aca6Galb4GlcNAc Neu5Aca3/6Galb4GlcNAc Neu5Aca3Galb4GlcNAc Neu5,9Ac2a3Galb4GlcNAc

See text ’’ ’’ ’’ 14,15

Neu5Aca3Galb4GlcNAc Neu5Aca8Neu5Ac Neu5Aca

See text 16,17

Neu5,9Ac2 Neu5,7,9Ac3 Neu5Aca3Galb4GlcNAc Neu5Aca3Galb3(Neu5Aca6)0,1GalNAc

See text

Heparan sulfate

See text

ManOS, heparan sulfate

18–21

a2-3 linkages, and sialic acid in both linkages is detected in porcine tracheae. Human viruses of the H1, H2, and H3 subtypes that are known to have caused pandemics in 1918, 1957, and 1968, respectively, recognize 2-6-linked sialic acid, the major form found on cells of the human respiratory tract, and are now the focus of intense attention.

3.28.2.1.1

Influenza A virus hemagglutinin

This is the most thoroughly investigated viral lectin.23,24 Its subunit is composed of two polypeptides, HA1 and HA2, m.w. 36 and 26kDa, respectively, covalently linked by a single disulfide bond22 (Figure 1). The hemagglutinin is a glycoprotein, with six N-linked oligosaccharides attached to HA1 and one to HA2. One of these glycans is an oligomannoside, while the others are complex bi- or triantennary structures, several of which contain sulfated galactose. X-ray crystallography showed that the hemagglutinin subunit consists of a hydrophilic, C-terminal domain, a hydrophobic membrane spanning region of 24–28 residues, an elongated triple-helical coiled stem, and a globular domain projecting 135A˚ from the membrane. The globular domain consists of HA1 only, and contains the carbohydrate-binding site of the lectin. The subunits associate noncovalently to form trimers. The affinity of the hemagglutinin of the influenza virus for its receptor is downregulated by the two N-linked oligosaccharides attached to Asn123 and Asn129 in the vicinity of the combining site.25 Thus, a mutant hemagglutinin devoid of these oligosaccharides bound to human erythrocytes much more strongly than the native lectin. To date, crystal structures of several human, avian, and swine influenza hemagglutinins, mostly in complexes with the trisaccharide Neu5Aca6Galb4Glc (Sia2-6Lac) or Neu5Aca3Galb4Glc (Sia2-3Lac) and the pentasaccharide Neu5Aca6Galb4GlcNAcb3GalbGlc (LSTc) or Neu5Aca3Galb4GlcNAcb3Galb4Glc (LSTa), analogs of the Neu5Aca2-6- and Neu5Aca2-3-human and avian receptors, respectively, have been determined. A complete description of all the structures obtained is beyond the scope of this chapter. Here, only selective examples will be given of the structures of HAs and how they interact with the corresponding ligands. Early sequence studies have shown that for the HAs of the H2 and H3 human viruses, a minimum of two changes in receptor-binding site amino acids, Gln226 to Leu226 and Gly228 to Ser228, correlate with the shift from avian to human receptor binding.26 By contrast, HAs of human H1 viruses acquire the ability to bind to human receptors while retaining Gln226 and Gly228. An understanding of how these changes occur, and a better insight into the combining sites of the HAs, have been provided by the recent study of John Skehel and co-workers of the structures of HAs from the 1918 pandemic virus (1918-human) with the use of HA expressed from the DNA recovered from tissues infected with virus in 1918 and from the prototype human (1934-human) and swine (1930-swine) H1 influenza viruses.27

626

Microbial Lectins

Figure 1 Influenza virus hemagglutinin trimer (a, side view, and b, top view) and monomer (c). In the monomer, the broad ribbon at the top represents the HA1 subunit and the narrow one the HA2 subunit. Courtesy J. J. Skehel, National Institute for Medical Research, Mill Hill, London.

Overall structure and receptor binding subdomain. The overall trimeric structures of the HAs of the three H1 viruses (1918-human, 1934-human, and 1930-swine) HAs are similar (Figure 2), but they show notable differences to HAs of other subtypes with respect to the arrangements of the receptor-binding subdomain among others, both within the HA trimer and also within individual monomers. The receptor-binding sites are located as in other influenza A virus HAs at the membrane-distal tip of each subunit of the HA trimer. Three secondary structure elements, the 190 helix (residues 190–198), the 130 loop (residues 135–138), and the 220 loop (residues 221–228), form the sides of each site, with the base made up of the conserved residues Tyr98, Trp153, His183, and Tyr195. The conformations adopted by the 130 and 220 loops of the three H1 HAs are similar, but they are significantly different from those of the equivalent loops in the HAs of other influenza subtypes (Figures 2b, 3, and 4). As observed with other HAs, the terminal sialic acids of the human and avian receptors interact with binding site residues through a series of conserved hydrogen bonds to the carboxyl and amide of the sialic acid (Figure 3). The 1934-human HA/human receptor complex. The electron density maps reveal well-ordered features for the Sia-1, Gal-2, and GlcNAc-3 of the sialopentasaccharide (the numbers in bold refer to the positions of the monosaccharides in the ligand) in this complex (Figure 3a). Gal-2 forms five hydrogen bonds that have not been previously observed in other HA/receptor complexes. Four bonds are possible between the 2- and 3-hydroxyls of Gal-2 and the side chains of Lys222 and Asp225, and a fifth between the 4-hydroxyl of Gal-2 and the main-chain amide of 227 that is mediated by a water molecule. The 1934-human HA/avian receptor complex. Again, only the Sia-1, Gal-2, and GlcNAc-3 moieties of the sialopentasaccharide are ordered in this complex (Figure 3b). This observation is consistent with the results of hemagglutination assays showing dual binding specificity for this HA. The side-chain carbonyl of Gln226 forms a hydrogen bond with the 4-hydroxyl of Gal-2, as observed in other HA/avian receptor complexes. In addition, there is a previously unknown water-mediated interaction between the 4-hydroxyl of Gal-2, the main-chain carbonyl of residue 225, and the side chain of Lys222 (Figure 3b).

Microbial Lectins

627

Figure 2 Structures of 1918-human, 1934-human, and 1930-swine HAs. a, Ribbon diagram of the trimer of 1918-human HA. Monomers 2 and 3 are in silver and gold, respectively, and the featured monomer is colored according to its individual subdomains: receptor binding (RB) in blue, vestigial esterase (E) in yellow, and fusion subdomains (F0 and F) in magenta and red, respectively. b, An expanded view of the superposed polypeptide backbone of the receptor-binding site of all three H1 HAs. The position of the three secondary structure units making up the site, the 190 helix, and the 130 and 220 loops, are indicated. Also shown are the side chains of some residues important for receptor binding. Certain Ca positions are indicated by black spheres for residues discussed in the text. Overall, the three H1 structures are very similar. c, An expanded view of a region of the F subdomain indicating differences between H1 and H5 subtype HAs in the position of the loop connecting helix A to helix B. Interactions between the C-terminal region of the loop and the RB and E subdomains (110 helix) influence the dispositions of the subdomains relative to the central coiled coil formed in the trimer by the B helices. Reproduced from Gamblin, S. J.; Haire, L. F.; Russell, R. J.; Stevens, D. J.; Xiao, B.; Ha, Y.; Vasisht, N.; Steinhauer, D. A.; Daniels, R. S.; Elliot, A.; Wiley, D. S.; Skehel, J. J. Science 2004, 303, 1838–1842.

The 1930-swine HA/human receptor complex. The sialic acid of the receptor is located similarly in the 1930-swine and 1934-human HA receptor binding sites, but in the 1930 swine complex all five saccharides of the receptor analog are detected (Figure 3c). Lys222 again forms hydrogen bonds with the 2- and 3-hydroxyls of Gal-2, although in the present case this residue sits higher in the binding site. Asp190 hydrogen bonds to the amino nitrogen of GlcNAc-3, Ser193 hydrogen bonds to the 2-hydroxyl of Gal-4, and there is a water-mediated interaction between Thr189 and GlcNAc-5. The last three interactions have not been observed before in HA receptor complexes. In addition, the sialopentasaccharide exits the binding site in an orientation not previously seen, crossing the 190 helix near its N-terminus, about parallel to the threefold symmetry axis of the HA trimer. The 1930-swine HA/avian receptor complex. The electron density for the avian receptor analog bound to the 1930swine HA is weak and mainly represents the sialic acid moiety (Figure 3d). A similar situation was observed for an

628

Microbial Lectins

1934-human/avian receptor complex

Asn-193

1934-human/human receptor complex

Asn-193

190-Helix

190-Helix

GlcNAc-3

Glu-190

GlcNAc-3

Glu-190 Gal-2

Gal-2 4

2 3

Lys-222

Lys-222

4

Sialic acid

227 Gln-226 220-Loop

130-Loop

(a)

Gln-226

Asp-225

Thr-136

137

135

225

135 Asp-225

Sialic acid

227

220-Loop

130-Loop Thr-136

137

(b)

GlcNAc-5

1930-swine/human receptor complex

Gal-4

Ser-193

Ser-193

1930-swine/avian receptor complex

GlcNAc-3 Asp-190

190-Helix

190-Helix Asp-190

N Gal-2 2

Lys-222

3

4

Lys-222 227 Gly-225

(c)

Gly-225

135 Gln-226

220-Loop

Sialic acid

Sialic acid

137

135

Gln-226

130-Loop Thr-136

220-Loop

130-Loop 137

Thr-136

(d)

Figure 3 Interactions of 1934-human HA (top) and 1930-swine HA (bottom) with human receptor and with avian receptor analogs. The view of the receptor binding site is about the same as in Figure 2b. The three secondary structure components of the binding site are labeled in this backbone representation together with some of the most relevant side chains. The broken lines indicate potential hydrogen bond interactions between the protein and the receptors; residues making interactions via main-chain carbonyl groups are shown as red spheres, whereas those interacting via main-chain nitrogens are shown as blue spheres. In all four panels, the sialosaccharides are colored yellow for carbon atoms, blue for nitrogen, and red for oxygen. Water molecules are indicated by green spheres. a, 1934-human HA in complex with human receptor; b, 1934-human HA in complex with avian receptor; in both cases, the HA is colored in green for backbone and carbon atoms. c, 1930-Swine HA in complex with human receptor; d, 1930-swine HA in complex with avian receptor; in these cases, the backbone and carbon atoms of the HA are colored in blue. The small black arrows in (a), (b), and (c) indicate that for the two human receptor complexes the Sia-1–Gal-2 linkage adopts a cis-conformation about the glycosidic bond, whereas for the avian complex it adopts a trans-conformation. The large black arrow in (c) indicates the direction of an axis parallel to the trimer threefold axis. Reproduced from Gamblin, S. J.; Haire, L. F.; Russell, R. J.; Stevens, D. J.; Xiao, B.; Ha, Y.; Vasisht, N.; Steinhauer, D. A.; Daniels, R. S.; Elliot, A.; Wiley, D. S.; Skehel, J. J. Science 2004, 303, 1838–1842.

H5 avian HA in complex with a human receptor analog, where only a subset of the atoms for the sialic acid could be located. These observations probably reflect the low affinity of the HAs for their respective ligands, consistent with the preference of the 1930-swine virus for human receptor in hemagglutination assays. Human receptor complexes. Complexes of the human receptor analog bound to 1934-human HA (green) and 1930swine HA (blue) and to human H3 HA (red) are superimposed in Figure 4a. Perhaps the most important feature of this comparison is the difference in structure adopted by the 130 and 220 loops of the receptor-binding site between

Microbial Lectins

629

Avian receptor complexes 1930-swine 1934-human Avain H3

Human receptor complexes 1930-swine 1934-human Human H3

190-Helix

190-Helix

1A

2A

Leu-226 220-Loop

1A

1A Gln-226

(a)

220-Loop

Gln-226

130-Loop

130-Loop

(b)

Figure 4 Differences in the orientations of bound receptors in the receptor-binding sites of three different HAs. The receptor-binding sites of 1934-human HA (green), 1930-swine HA (blue), and human H3 HA (red) are overlaid. The view matrix is about the same as in Figure 3. The sialopentasaccharides are colored according to the HAs to which they are bound. The side chains of Gln226 (H1 HAs) and Leu226 (H3 HA) are shown. a, Human receptor complexes. b, Avian receptor complexes. Reproduced Gamblin, S. J.; Haire, L. F.; Russell, R. J.; Stevens, D. J.; Xiao, B.; Ha, Y.; Vasisht, N.; Steinhauer, D. A.; Daniels, R. S.; Elliot, A.; Wiley, D. S.; Skehel, J. J. Science 2004, 303, 1838–1842.

the H1 and H3 HAs. One consequence of the change in the 130-loop structure is that the sialic acid of the receptor is tilted about 10 into the receptor-binding sites of the H1 HAs. This effect, together with different orientations about the glycosidic bond, contributes to Gal-2 being located almost 2A˚ lower in the H1 HAs than in the human H3 HAs. Gal-2 is able to adopt this position because structural differences in the 220 loop locate Gln226 lower in the binding site than the equivalent Leu226 of human H3 HA. Consequently, in the H1 HAs, Gal-2 is located closer to the 220 loop and is able to form hydrogen bonds with Lys222. In the case of the 1934-human HA, Gal-2 also interacts with Asp225. Thus, a combination of factors relating to the structure of the 130 and 220 loops enable the H1 HAs to make favorable hydrogen bond interactions with Gal-2 of the human receptor. Gln226 plays an essentially passive role in this process, in marked contrast to the role played by Leu226 in the binding of human H3 HA to human receptor. In that case, Gal-2 makes hydrophobic contacts with Leu226, and the higher position and the nature of this side chain are important for human receptor binding. Avian receptor complexes. Complexes of avian receptor analogs with 1934-human HA (green), 1930-swine HA (blue), and an avian H3 HA (red) are overlaid in Figure 4b. Again, the differences in the structure of the 130 loop between the H1 and H3 HAs result in the sialic acid of the avian receptor being located lower in the receptor binding site of the H1 HAs. Comparison of the 1934-human and avian H3 complexes also reveals that Gal-2 of the avian receptor is located about 1A˚ lower in the binding site of the H1 complex, as is Gln226. In both complexes, the 4-hydroxyl of Gal-2 hydrogen bonds with the side-chain carbonyl of Gln226 (Figure 4b), and the coordinated differences in position of the bound receptor and Gln226 enable this interaction to be conserved. It seems therefore that the 1934-human HA is able to bind the avian receptor in a manner reminiscent of avian HAs, with Gln226 playing a key role. The above data for the different complexes show the overall structures of 1930-swine and 1934-human HA receptorbinding sites are very similar (Figure 2b), and that both contain a glutamine residue at position 226. It has been suggested that the 1930-swine HA binds less effectively to avian receptors than the 1934-human HA because the position adopted by Gln226 in 1934-human HA is about 1A˚ higher in its complex with the avian receptor than it is either in the human receptor complex or in the uncomplexed form (Figure 5). By contrast, the position of Gln226 in 1930-swine HA is about the same as uncomplexed and in the human and avian receptor complexes. The apparent

630

Microbial Lectins

1934-human uncomplexed and 1934-human/avian receptor complx

190-Helix

Gal-2

Glu-190 4

Lys-222

1A Asp-225

Sialic acid

Gln-226 220-Loop

Thr-136

130-Loop

Figure 5 Superimposition of the binding site of 1934-human HA in its uncomplexed state and complexed with avian receptor analog. The HA is shown in green in both cases, and the avian receptor is colored as in Figure 3. Two water molecules, shown as green spheres, link Glu190 to Gln226 in the avian receptor complex. This hydrogen-bonded network is not formed in the uncomplexed structure or in the human receptor complex not shown. Reproduced from Gamblin, S. J.; Haire, L. F.; Russell, R. J.; Stevens, D. J.; Xiao, B.; Ha, Y.; Vasisht, N.; Steinhauer, D. A.; Daniels, R. S.; Elliot, A.; Wiley, D. S.; Skehel, J. J. Science 2004, 303, 1838–1842.

inability of Gln226 to adopt a higher position in the receptor-binding site seems to explain the failure of 1930-swine HA to interact as effectively with the avian receptor. This explanation is supported by the structural observation that in the 1934-human HA complex with avian receptor Glu190 interacts through two water molecules with Gln226 (Figure 5). This network of hydrogen bonds may be necessary to position Gln226 in the binding site for its interaction with Gal-2. Glu190 is conserved in avian H1 HAs, all of which specifically bind a2-3-linked receptors. By contrast, residue 190 of 1930-swine HA is an aspartic acid, which does not interact with either the 9-hydroxyl of Sia-1 or Gln226 and is thus unable to facilitate binding to avian receptor. Irrespective of the single amino acid difference of Asp or Gly at residue 225 between the sequences of 1918-human HAs, by recognizing human receptors, all would fulfill the first requirement of an epidemic virus: the ability to spread in the human population. The importance of this requirement was emphasized in the 1997 outbreak of H5 ‘chicken’ influenza in Hong Kong, when the virus was extremely virulent but did not acquire the ability to bind 2-6-linked sialosides and was therefore unable to spread. With the ability to ensure the efficiency of the initial stages of virus infection, coupled with novel antigenicity, the human-1918 HA may have been the prime determinant of extensive mortality in the 1918 pandemic. Previous crystallographic analysis of the trisaccharide human receptor analog, Sia2-6Lac, bound to human H3 HA, revealed an extended conformation24 rather than the folded conformation of the pentasaccharide human receptor analog, LSTa. This conformation was presumed not to be representative of natural receptors because of the presence of glucose rather than N-acetylglucosamine at position 3 and the lack of glycosylation beyond this position. The mode of Sia2-6Lac binding, however, further demonstrates the plasticity of the HA receptor binding site in accommodating oligosaccharides in different conformations. By contrast, Sia2-6Lac binds in a manner very similar to that of the pentasaccharide, LSTa, again suggesting that there is no role in avian receptor binding for the saccharides beyond GlcNAc-3. It is therefore possible that some avian HAs are capable of binding sialic acid in a2-6 linkage to galactose, but are not capable of infecting humans because the saccharides beyond GlcNAc-3 sterically clash with the HA. For an avian HA to change its binding specificity from avian to human receptors may therefore involve two sorts of changes, the first, to allow binding of the a2-6-linkage, either via mutation of Gln226 to Leu226 as in H2 and H3 HAs or via a specific positioning of Gln226 as in H1, and the second, to accommodate the additional saccharides linked to Gal-2 in the natural receptor side chain.

Microbial Lectins

631

Receptor

Enzyme

CH3

O

O

HO HN O C CH3

(a)

COO−

OH C O

OH

OH

(b)

Figure 6 HEF glycoprotein structure. a, The structure of the HEF trimer. HEF1 (blue), HEF2 (red), receptor analog and enzyme inhibitor ligands, (yellow); N-linked carbohydrate ball and stick (purple). HEF1 is linked to HEF2 by a disulfide bond from Cys6 of HEF1 to Cys137 of HEF2. b, Monomer surface of HEF showing 9-O-acetylsialoside receptor-binding site (top) and 9-O-acetylesterase site (bottom). Inset, the esterase removes the acetyl group of 9-O-acetylsialic acid (see arrow). Reprinted by permission from Macmillan Publishers Ltd: Nature (Rosenthal, P. B.; Zhang, X.; Formanowski, F.; Fitz, W.; Wong, C. H.; Meier-Ewert, H.; Skehel, J. J.; Wiley, D. C. Nature 1998, 396, 92–96.), Copyright (1998).

3.28.2.1.2

Influenza C HEF

Like its homologous influenza A and B virus hemagglutinins, to which it bears limited primary sequence identity, HEF is a homotrimer of an N-glycosylated type I membrane glycoprotein. It consists of a 65kDa N-terminal subunit HEF1 and a 30kDa C-terminal subunit HEF2 that is membrane anchored. Despite an overall primary sequence identity of only 11%, the similarity between the three-dimensional (3-D) structure of HEF and the hemagglutinin is astounding (Figure 6).16,28,29 The main differences are within the N-terminal subunits, which form the globular part of the spikes and which, in the case of HEF, contain the receptor-binding R and RDE esterase domains. The C-terminal HEF2 subunit, together with segments of HEF1, comprises the fusion domain F. In the trimer, HEF2 subunits constitute the elongated membrane-anchored stalk, at the core of which long central helices form a triple-stranded interphase. Each HEF1 subunit contains two sites that can accommodate Neu5,9Ac2. The actual receptor-binding sites are cavities at the tip of the globular head domain with Tyr127, Thr170, and Gly172 involved in ligand binding. Interaction with the 9-O-acetyl group, the most critical determinant for receptor recognition, is mediated among others by the hydroxyl group of Tyr224 and the guanido group of Arg236, which contact the acetyl carbonyl oxygen. Moreover, Phe225, Phe293, and Pro271 form a nonpolar pocket into which the acetyl methyl group can be fitted (Figure 7). The importance of the latter site for receptor recognition is underlined by the observations that mutations of residues adjacent to Pro271 (Asp269Asn, Thr270Leu or Ile, and Thr272Ile) are associated with increased binding efficiency to Neu5,9Ac2 receptors.

3.28.2.2 Rhesus Rotavirus The rotavirus hemagglutinin binds the alpha anomer of N-acetylneuraminic acid with an association constant (Ka) of 8105 M–1. It requires no additional carbohydrate moiety for binding to the sialic acid, does not distinguish between 30 and 60 sialyllactose, and has approximately 10-fold lower affinity for N-glycolylneuraminic acid than for N-acetylneuraminic acid.30 The rotavirus outer capsid is made of the coat glycoprotein VP7 and the spike protein VP4. Trypsin cleaves VP4 into an N-terminal fragment, VP8*, consisting of amino acids 46–231, that contains the viral hemagglutinin

632

Microbial Lectins

Arg292 Arg236

Thr186

Phe293

Tyr227 Leu184

Pro271 Phe225

O Thr170 Tyr127 NH Gly172 Figure 7 HEF receptor binding. Ligand bound to the receptor-binding site. Potential hydrogen bonds are indicated in green or red for those conserved in HA ligand binding. Four polar contacts are formed with the ligand identically in HEF and HA: two from the hydroxyl group of HEF1 Tyr127 (Y98 in HA1) to the 8-hydroxyl and 9-amide of the ligands and two from main-chain atoms: the carbonyl oxygen of HEF1 residue 170 (135 in HA1) to the 5-amide of the ligand and the amide of HEF1 172 (137 in HA1) to the carboxylate of the ligand. The acetyl methyl group binds in a nonpolar pocket unique to HEF, formed by Phe225 and 293, and Pro271; the acetyl carbonyl oxygen contacts the hydroxyl group of Tyr224 and the guanidino group of Arg236. Reprinted by permission from Macmillan Publishers Ltd: Nature (Rosenthal, P. B.; Zhang, X.; Formanowski, F.; Fitz, W.; Wong, C. H.; Meier-Ewert, H.; Skehel, J. J.; Wiley, D. C. Nature 1998, 396, 92–96.), Copyright (1998).

(residues 93–208) and a C-terminal fragment, VP5*, the function of which is to permeabilize the membrane to which the virus binds. No crystals of free VP8* could be obtained, but it crystallized in complex with methyl a-linked N-acetylneuraminic acid. NMR analyses of VP8* in solution, and X-ray studies of its complex with the above ligand, revealed the same basic protein structure.31 It is a single, compactly folded globular domain with two cysteines (C203 and C216) and two prolines (P68 and P182) in the cis-configuration. The crystals contain a mixture of molecules with the peptide bond G156–P157 in either the cis- or trans-configuration. The tight fold of the b-sandwich, the cross-bracing of the b-sheets by the b-ribbon and the C-terminal a-helix, the short loops between the strands and the dense hydrophobic cores between the major structural elements all suggest a compact, rigid structure, that accounts for the protease resistance and stability of the VP8* core (Figure 8). The b-sandwich of the rotavirus sialic acid binding domain has the same fold as the S-carbohydrate recognition domain (S-CRD) of the galectins, despite the absence of a significant sequence similarity between the two (9% identity with human galectin-3 in structurally equivalent residues). The sialic acid-binding site of the rhesus rotavirus hemagglutinin lies above the cleft between the two b-sheets and appears to be an open-ended, shallow groove. Of the four amino acids (Arg101, Tyr155, Tyr188, and Ser190) seen to be involved in sialic acid binding, the last three were previously identified as likely ligand-binding residues by mutagenesis studies. In addition to the seven hydrogen bonds (Figure 9), the sialic acid makes several van der Waals contacts with the side chains of six amino acids, three of which are tyrosines.

3.28.2.3 Polyoma Virus This virus is a nonenveloped, icosahedrically symmetrical particle, with a circular, double-stranded DNA genome.32 Its carbohydrate binding site is located in viral protein 1 (VP1, m.w. c. 42kDa), the major constituent of the outer shell (capsid) of the virion. Each virion contains 360 copies of VP1, arranged in pentamers. VP1 has two antiparallel b-sheets with a topology that resembles the jellyroll fold; some loops that connect the b-strands are extensive and contain additional secondary structural elements (Figure 10). The most striking feature of the capsid is the way the individual pentamers are tied together by the C-terminal arms of the monomers; the last 63 residues emerge from each monomer and protrude into a subunit of another pentamer, where they form a b-strand that augments a sheet in the target subunit. Two types of strain of murine polyoma virus are known that differ in their tumorogenicity in mice and in their specificity for sialic acid oligosaccharides. The critical difference in the structure of the viral protein (VP1) that contains the carbohydrate-binding site of these strains is in residue 91, which is glycine in the poorly tumorogenic strains and glutamic acid in the highly tumorogenic ones.33 Crystallographic studies at low resolution of the intact poorly tumorogenic virus particle in complex with Sia2-3Lac34 and with a branched hexasaccharide containing both a2-3- and a2-6-linked N-acetylneuraminic acid35,36 have located the combining site in a shallow groove and have

Microbial Lectins

633

bK

VP8* core

bF bC bL

bD

bD

bG

aA bI bJ bB

bH

bK

bM aB

NH2

COOH

Figure 8 Ribbon diagram of the sialic acid-binding domain of rotavirus. The central structural feature of VP8* is an 11stranded antiparallel b-sandwich, formed from a five-stranded and a six-stranded b-sheet, with an interrupted top strand. The two b-sheets are joined by five short intersheet loops, as well as by a brief stretch of a parallel b-structure between strand bH0 of the six-stranded sheet and strand bJ of the five-stranded one. The cleft between the sheets is filled by a dense core of hydrophobic side chains, contributed by all strands of the sheets, except for bH. The domain contains three other structural elements, namely a short a-helix (aA) as part of the intersheet loop, a longer a-helix (aB) at the C-terminus, and an extended b-ribbon made up of strands bE and bF. Sialic acid is shown as a stick and ball model. Reproduced with permission from Dormitzer, P. R.; Sun, Z. Y.; Wagner, G.; Harrison, S. C. Embo J. 2002, 21, 885–897.

bH

Y155

H2O bG

O7

O9 bD

O8 O2 R101

O1B

O4 H2O O1A

bK S190

Y188

Figure 9 Binding of MeaNeu5Ac by rhesus rotavirus. The hydrogen bonds anchoring the ligand to the protein are: two bonds from the side-chain guanidinium of Arg101 to the ligand glycerol chain (atoms O8 and O9); one from the side-chain hydroxyl of Tyr155 to the glycerol side chain (O9) via a water bridge; one from the side-chain hydroxyl of Ser190 to the carboxylate (atom O1A); one from the main-chain amide of Ser190 to the carboxylate (atom O1B); one from the main-chain carbonyl of Tyr188 to the acetamide nitrogen of the ligand; and one from the main-chain amide to 4-OH via a water bridge. The ligand is shown as a ball-and-stick model; hydrogen bonds are indicated by dotted lines. From Dormitzer, P. R.; Sun, Z. Y.; Wagner, G.; Harrison, S. C. Embo J. 2002, 21, 885–897.

634

Microbial Lectins

Figure 10 Structure of the recombinant VP1 pentamer of polyoma virus. Ribbon drawing of the VP1 pentamer complexed with the disialylated oligosaccharide Neu5Aca3Galb3(Neu5Aca6)GlcNAc. One monomer is shown in red, the others in gray. The receptor fragments are shown as ball-and-stick models. The rearranged N-terminal segments are shown in yellow (red monomer) and orange. Reproduced from Stehle, T.; Harrison, S. C. Embo J. 1997, 16, 5139–5148.

shown that the sialic acid as well as the galactose form contacts with the protein (Figure 11). The inability of the highly tumorogenic strains to bind the branched ligand is due to electrostatic repulsion between the side chain of Glu91 and the carboxylate of the a2-6-linked N-acetylneuraminic acid. These results have been confirmed and extended in a high-resolution crystallographic study of a pentamer of VP1 from the poorly tumorogenic virus in complex with the above pentasaccharide.32

3.28.2.4 Foot-and-Mouth Disease Virus This virus has the basic picornavirus structure (Figure 12). Its icosahedral capsid comprises 60 copies each of four virus-encoded proteins, VP1–VP4. X-ray crystallography of the complex of the virus with its ligand, heparin, revealed that the combining site is located in a shallow depression on its surface, consists of contributions from the three major capsid proteins, VP1, VP2 and VP3 and is located at the junction of these proteins. VP1, VP2, and VP3, virtually identical for the type A and O viruses.37,38 There were virtually no changes in the protein to accommodate the sugar. In the complex with a sulfated heparin trisaccharide HSO36GlcNH(HSO3)a[(HSO3)Idoa][(HSO3)2GlcN(HSO3)], the key binding residue was Arg56 of VP3, which interacts ionically with two of the sulfates of the trisaccharide. The importance of this residue is supported by the finding that field isolates of the virus, where the arginine is replaced by histidine, do not bind heparin. Additionally, nonionic interactions observed in the virus–ligand complex include bonds between His195 of VP1 and the iduronic acid, and a stacking interaction between the imidazole ring of this amino acid and the hydrophobic face of the glucosamine that follows the iduronic acid.38 In strain O1BFS, the most tightly bound heparin residues were GlcN2, Ido3, and GlcN4, and, for A1061, these were the only ones for which there was clear electron density. The ligands were the basic and polar residues Arg56 and Asn88 of VP3, Thr134 and Arg135 of VP2, and Lys193 of VP1, and bridging water molecules were important (Figure 13b). As for virus strain O1BFS, there was no direct involvement of the VP1 C-terminal residues 200RHKQI205 in binding; however, the neighboring residues His195 (O1BFS) (Figure 4b) and Lys-193 (A1061) of VP1 (Figure 13a) did contact the heparan substrate (HS), and the C-terminus may stabilize these residues in a suitable position for HS binding.

Microbial Lectins

635

Neu5Ac-1 Glu87 Asn293 ND

Thr291 OG CG

O8

OE1

Neu5Ac-2

Val296 CG1

GlcNAc

O10 O1A

Asp85# NS

W

NH2 Arg289

O1A

Gal

W

OH Tyr72

W

O6

W

NE His298

O4

O

O8

O4

O1B

Thr83 CG

O1B

O9

W

NH2 NE Arg77 NH1

ND Asn93

N O Gly78

O Leu81

CG

N Ser89 OG

ND Asn80 OD

CA Gly91 N

Thr94 NZ

OE1

Lys186

Gln59 Figure 11 Schematic view of the combining site of polyoma virus with the bound tetrasaccharide Neu5Aca3Galb3 (Neu5Aca6)GlcNAc. Hydrogen bonds are represented by thin broken lines, and hydrophobic contacts are shown as thick gray broken lines. Asp85, located at the tip of the BC2-loop of the clockwise VP1 neighbor, approaches the N-acetyl group of Neu5Ac-1. The small circles labeled W0 represent water molecules Reproduced from Stehle, T.; Harrison, S. C. Embo J. 1997, 16, 5139–5148.

Figure 12 Model of foot-and-mouth disease virus in complex with the heparin trisaccharide HSO36GlcNH(HSO3)a(HSO3) Idoa][(HSO3)2GlcN(HSO3)]. The structure shown is a pentamer; 12 such structures make up the icosahedral viral capsid. The trisacharide is shown as a CPK model. Reproduced from Mulloy, B.; Linhardt, R. J. Curr. Opin. Struct. Biol. 2001, 11, 623–628.

636

Microbial Lectins

Lys 1931 His 1951 Arg 1352 Asn 883

Asn 883 Arg 563

Arg 563

Arg 1352

Gly 593

Thr 1342

(a)

Idu 1 Gcn 2

Gcn 2 Idu 5

Thr 1342

Gly 503 Idu 3

Arg 563

Arg 563 Idu 3 Gcn 4 Lys 1342

Gcn 4

His 1951

Lys 193 Asn 883 Arg 1352

(b)

Asn 883

Arg 1352

Figure 13 Heparin-binding site of different strains (A1061 and O1BFS) of foot-and-mouth virus. a, Ligand-binding site in A1061 (left) and O1BFS (right). Viral proteins are depicted as ribbons with standard color coding. The side chains of ligand residues (labeled) are drawn as balls and sticks in the same color as the corresponding protein. They are labeled with the residue number and the chain ID as the least significant digit,for example, 563 is residue 56 of VP3. The sugar residues are depicted as purple balls and sticks, with sulfur atoms in yellow. b, Sugar–protein interactions. A1061 (left) and O1BFS (right) are shown each with the pentasulfated trisaccharide GlcN-IdoA-GlcN (its constituents marked as Gcn 2, Idu 3, and Gcn 4, respectively). Only protein side chains that interact directly are shown and these are rearranged to clarify the hydrogenbonding pattern. Ligand bonds are drawn in purple and nonligand bonds in brown. Hydrogen bonds are depicted by olivegreen, dashed lines. Nonligand residues involved in hydrophobic contacts are shown as red, fringed semicircles. Water molecules are colored red. Reproduced from Fry, E. E.; Newman, J. W.; Curry, S.; Najjam, S.; Jackson, T.; Blakemore, W.; Lea, S. M.; Miller, L.; Burman, A.; King, A. M.; et al. J. Gen. Virol. 2005, 86, 1909–1920.

Two ligating residues were conserved between the type A and O complexes: Arg-56 of VP3 and Arg-135 of VP2 (Figures 13a and 13b). The former switches from a histidine to an arginine in type O viruses on adaptation to tissue culture and is a key ligand in both complexes. In the O1BFS complex, it acts as a bidentate ligand, stabilizing sulfate groups from rings 2 and 4, whereas in the A1061 complex, it only interacts with ring 4. Arg-135 makes a hydrophobic interaction in both complexes and appears to polarize Asn-88 of VP3, increasing its affinity for HSO3-6-NH-SO3.

3.28.3 Bacterial Lectins 3.28.3.1 Surface-Bound Lectins A variety of bacterial species and genera express surface lectins, frequently of more than one type and with distinct specificities2,3,39 (Table 2). A few bacteria are known to produce intracellular and soluble lectins. The diverse

Microbial Lectins

637

Table 2 Bacterial surface lectins Organism

Carbohydrate

C. jejuni E. coli Type 1 P S CFA/1 F1C F17 K1 K99 H. influenzae H. pylori

Fuca2Galb4GlcNAc Mana3Mana6Man Gala4Gal Neu5Aca3Galb3GalNAc Neu5Aca8 GalNAcb4Galb GlcNAc GlcNAcb4GlcNAc Neu5Aca3Galb4Glc Neu5Aca30,1Galb4GlcNAcb3Galb4GlcNAc Neu5Aca3Galb4GlcNAc Fuca2Galb3(Fuca4)Gal Man Galb4GlcNAc Neu5Aca30,1Galb4GlcNAcb3Galb4GlcNAc Galb3Glc(NAc)b3Galb4Glc Man Neu5Aca30,1Galb4GlcNAcb3Galb4GlcNAc Gala4Galb4Glc

K. pneumoniae N. gonorrhoea N. meningitidis P. aeruginosa S. typhimurium S. pneumoniae S. suis Modified from Sharon, N. Biochim. Biophys. Acta 2006, 1760, 527–537.

specificities of most of the surface lectins are among the factors determining the organ and animal tropism of the bacteria. It is not known whether individual cells co-express multiple lectins or if each lectin is confined to a distinct cell subpopulation of the species. We know, however, that the expression of the surface lectins is commonly regulated by a mechanism known as phase variation, that controls the back-and-forth conversion of the lectin-expressing cells in a bacterial population to nonexpressing ones.40 In Escherichia coli, Klebsiella pneumoniae, and Salmonella spp., the lectins often are in the form of submicroscopic hair-like appendages, named fimbriae or pili, that protrude from the surface of the cells. During the fimbriated phase, a typical Gram-negative bacterium carries 200–500 peritrichously arranged fimbriae. The most prevalent and best characterized bacterial surface lectins with respect to structure, biosynthesis, and function are those expressed by E. coli, namely the mannose-specific type 1 fimbriae, the galabiose-specific P fimbriae, and the N-acetylglucosamine-specific F17 fimbriae. They all consist of an assembly of different classes of subunit and belong to the family of flexible ‘Cup’ fimbriae, referred to as such because they are assembled by the chaperone/usher pathway.41 The carbohydrate-binding subunit is typically located at the end of the assembled structure.

3.28.3.1.1

Type 1 fimbriae

Type 1 fimbriae (Figures 14 and 15) are expressed by a large number of E. coli strains, and are found in more than 95% of E. coli isolates from intestinal and extraintestinal infections such as urinary ones.1,3,39,42 They are also produced by other enterobacterial species, among them of K. pneumoniae, Salmonella typhimurium, and Salmonella enteritidis. The affinity of different phenotypes of E. coli type 1 or of the isolated fimbriae to mannose or MeaMan may differ within a factor of 15 and they can be functionally subdivided into either low-mannose-binding (M1L) or high-mannosebinding (M1H) phenotypes.43 E. coli exhibiting these two basic phenotypes have been found to predominate in different niches. Most isolates from the large intestine of healthy humans (c. 80%) express a distinct M1L phenotype, whereas most isolates from urinary tract infections (>70%) express M1H variants. These naturally occurring variations dramatically change the tissue tropism of E. coli and can be a major factor in shifting the bacterial adaptation from commensal to pathologic habitats, a phenomenon known as pathoadaptation.44 Typically, type 1 fimbriae of E. coli have a considerably higher affinity (up to 40 times as compared to mannose) for oligosaccharides such as Mana3Manb4GlcNAc or Mana6(Mana3)Mana6(Mana3)Man that are constituents of cell surface glycoproteins.45 In the urinary tract, the fimbriae mediate binding of the bacteria to the oligomannosides Man9GlcNAc2 to Man6GlcNAc2 of uroplakin Ia, a major glycoprotein of urothelial apical plaques.46,47 Anchorage of E. coli to the urothelial surface via type 1 fimbriae–uroplakin I interactions may play a role in their colonization of the bladder and eventual ascent through the ureters, against urine flow, to invade the kidneys. Structurally, the fimbriae are 1–2mm long and 7nm thick fibers, that are rod like, made up largely of repeating immunoglobulin-like FimA subunits (m.w. 17kDa) arranged helically in a structure referred to as ‘shaft’ (Figure 16). 48,49

638

Microbial Lectins

Figure 14 Type 1 fimbriated E. coli. Magnification 20000. Courtesy Dr. David Hasty.

Figure 15 Electron micrograph of isolated type 1 fimbriae. Reproduced from Eshdat, Y.; Silverblatt, F. J.; Sharon, N. J. Bacteriol. 1981, 148, 308, with permission from American Society for Microbiology.

The shaft is joined to a short 3nm thick distal tip fibrillum that consists of two adapter proteins, FimF and FimG, and a third of a different kind, FimH (m.w. 29–31kDa). The latter is the only subunit that possesses a carbohydrate-binding site and is thus responsible for the sugar-binding activity of the fimbriae.50 FimH is also present in small numbers at intervals along the fimbrial filament, but only the subunit at the tip appears to be able to mediate mannose-specific adhesive interactions, whereas the subunits at the other positions are inaccessible to the carbohydrate ligand.51 Minor sequence variations in FimH alleles from different clinical isolates have been shown to correlate with altered carbohydrate-binding profiles of the fimbriae.44 The FimH subunits of E. coli and K. pneumoniae are 88% homologous. Still, the two organisms differ in their fine specificity, for example, in their relative affinity for Mana3Manb4GlcNAc and p-nitrophenyl a-mannoside.52–54 Other aromatic a-mannosides are also high-affinity ligands (up to 1000 times stronger than MeaMan) for E. coli type 1, suggesting the presence of a hydrophobic binding region next to the monosaccharide-combining site of FimH. With

Microbial Lectins

639

Figure 16 Structure of type 1 fimbriae. Top, schematic representation, depicting the distal end of the shaft as well as the tip of a fimbrium (or pilus) and a typical attachment site on host cells; A, F, G, and H denote the fimbrial subunits. Bottom, electron micrograph, in which arrowhead indicates the fimbrial tip containing the FimH subunit. Reproduced from Schilling, J. D.; Mulvey, M. A.; Hultgren, S. J. J. Infect. Dis. 2001, 183(1), S36–S40.

several Salmonella species examined, aromatic a-mannosides, as well as the trisaccharide Mana2Manb4GlcNAc, were weaker inhibitors than MeaMan. The combining site of Salmonella species appears thus to be smaller than that of E. coli and K. pneumoniae, and to be devoid of an adjoining hydrophobic region. Different combining sites were found in other mannose-specific bacterial lectins. Therefore, although classified together on the basis of their monosaccharide (primary) specificity, these lectins differ in their fine specificity. Swapping experiments with genetically engineered hybrid fimbriae, in which the FimH of one species (e.g., E. coli) was presented on the shaft of the other species (K. pneumoniae), have shown that the shaft plays a role in modulating the specificity of fimbriae, probably by imposing conformational constraints on the carbohydrate-binding subunit.55 The first 3-D structure of a fimbrial carbohydrate-binding subunit to be solved was that of FimH in complex with the FimC chaperone (see below) to which the mannose analog cyclohexylbutanoyl-N-hydroxyethyl-D-glucamide was bound.56 FimH is seen folded into two all-b class domains connected by a short extended linker (Figure 17). One of these, located in the N-terminal half of the subunit (residues 1–156), is the lectin domain, with the mannose-binding site at its tip. The C-terminal half (residues 160–279), known as the pilin domain, serves to anchor the subunit to the fimbriae. It binds in the cleft of FimC, although there is only limited contact between FimH and the C-terminal domain of the chaperone. The carbohydrate recognition domain of FimH is an 11-stranded elongated b-barrel with a jellyroll-like topology, while the pilin domain has an immunoglobulin fold that lacks the seventh (C-terminal) b-strand present in the canonical immunoglobulin fold. In the FimC–FimH complex, the missing b-strand is donated by the seventh strand of the N-terminal domain of the chaperone to complete the immunoglobulin-like fold of FimH. This kind of ‘donor strand complementation’ is thought to initiate folding of FimH directly on the chaperone, thus accounting for the function of the latter in biogenesis of the fimbriae.57 The 3-D structure of the FimC–FimH in complex with bound mannose has also been elucidated by X-ray crystallography.58 Although mannose exists in solution as a mixture of a- and b-anomers, only the former was found in the crystal (Figure 18). It is buried at a deep and negatively charged site at the edge of FimH, opposite to the region through which the latter combines with the chaperone. All the mannose hydroxyls, except the anomeric one, interacted extensively with combining site residues, almost all of which are situated at the ends of b-strands or in the loops extending from them. Part of the hydrogen-bonding network is identical to that found in mannose complexes of other lectins. Thus, Asp54 makes cooperative hydrogen bonds with the 4- and 6-OH and Asn140 with the 3- and 4-OH of the ligand, similar to those made by the equivalent residues with the same sugar in the combining sites of legume lectins with the same primary specificity.59 In addition, the N-terminal amine of the FimH polypeptide is H-bonded to the 2-OH, 6-OH, and the ring oxygen of the mannose.58 This is one of the rare cases in which an N-terminal amine of a protein participates in ligand binding. Phe142 of FimH interacts hydrophobically

640

Microbial Lectins

Figure 17 Crystal structure of FimH, the carbohydrate-binding subunit of type 1 fimbriae, in complex with the FimC chaperone. The asterisk indicates the mannose-binding pocket of FimH. Also shown is the insertion of the G1 strand of the FimC chaperone into the hydrophobic groove formed between the A and F strands of the FimH pilin domain, an interaction known as donor-strand complementation. Reproduced from Schilling, J. D.; Mulvey, M. A.; Hultgren, S. J. J. Infect. Dis. 2001, 183(1), S36–S40.

Wat1 2

Phe1

Phe142 3

1

Asp47

5 6

Asp140

Gln133

Asn135

4

Asn46

Asp54

Figure 18 The a-anomer of mannose in the combining site of FimH. The eight marked residues combine with the mannose by hydrogen bonds and hydrophobic interactions. Contact residues are shown as ball-and-stick model. Wat stands for water. From Hung, C. S.; Bouckaert, J.; Hung, D.; Pinkner, J.; Widberg, C.; DeFusco, A.; Auguste, C. G.; Strouse, R.; Langermann, S.; Waksman, G.; et al. Mol. Microbiol. 2002, 44, 903–915.59

with the C2–C3 bond of the mannose. The same residue, together with Ile13, Tyr 48, and Ile52, form part of a hydrophobic ridge that surrounds the site and which may help to direct the ligand into it. Site-directed mutagenesis showed that combining site residues Asp54, Gln133, Asn135, and Asp140 are essential for carbohydrate binding by FimH, since their replacement by alanine, asparagine, or aspartic acid resulted in complete loss of this activity. Examination of the binding site region of FimH has provided confirmation of the suggestion, made some two decades ago, that the combining site of this lectin is extended, and fits best mannose-containing trisaccharides such as Mana3Manb4GlcNAc or Mana6(Mana3)Man.45 X-ray crystallography and modeling studies of the complexes of FimH with hydrophobic mannosides60 have furnished a molecular explanation for the high affinity of type 1 fimbriae to such compounds (Figure 19). It is likely that the hydrophobic character of the ridge of the mannose-binding site of FimH distinguishes E. coli type 1 fimbriae from those of Salmonella species that neither exhibit an increased affinity for mannose with hydrophobic substituents nor for the oligomannosides mentioned earlier.

Microbial Lectins

641

Thr 51

Thr 51 Tyr 48

Tyr 48

Tyr 137

Tyr 137

Asp 47

Asp 47

Asn 135

Asn 135 Phe 1 Asp 54

Phe 1 Asp 54

Asp 140

Asp 140

Gln 133

Gln 133

(a) Tyr 48

Tyr 48

Tyr 137

Tyr 137

Asn 46

Asn 46 Asp 47

Asp 47 Asn 135 Phe 1 Asp 54

Asn 135 Phe 1

Asp 140 Gln 133

Asp 54 Asp 140 Gln 133

(b) Figure 19 Stereodiagrams of MeUmbaMan (a) and pNPheaMan (b) docked in the FimH-binding site. The crystallographically determined position of the a-anomer of mannose is shown in orange for comparison. From Bouckaert, J.; Berglund, J.; Schembri, M.; De Genst, E.; Cools, L.; Wuhrer, M.; Hung, C. S.; Pinkner, J.; Slattegard, R.; Zavialov, A.; et al. Mol. Microbiol. 2005, 55, 441–455.

Nearly all mutations in the combining site of FimH abolished or decreased its binding not only to mannose, but also to urinary epithelial cells, indicating that the site may be highly conserved.44,61 Support for this conclusion comes from the finding that there are very few variations in the sequences of the mannose-binding site of over 200 uropathogenic strains of E. coli examined, in contrast to enterohemorrhagic strains of E. coli, in which there are sequence variations at this site.61 On the other hand, replacement of residues 185–279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial subunit FocH has led to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct mannose-specific (i.e., monovalent, M1H) binding capability. Bacteria expressing the monovalent hybrid FimH were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeasts or bind human buccal cells, functions readily accomplished by the E. coli expressing mannotriose-specific FimH variants. Based on the relative potency of

642

Microbial Lectins

Figure 20 Structure of P fimbriae (pili). On the left, a schematic diagram of a single P fimbrium, showing the location of each subunit within the structure; on the right, electronmicrograph showing the two subassemblies of a P fimbrium. Reproduced from Dodson, K. W.; Pinkner, J. S.; Rose, T.; Magnusson, G.; Hultgren, S. J.; Waksman, G. Cell 2001, 105, 733–743.

inhibitory compounds of different structures, it was concluded that the receptor-binding site within the monovalent FimH–FocH subunit has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. Another class of high-affinity ligand for type 1 fimbriated E. coli are mannose-derived neoglycoproteins and dendrimers (see Chapter 3.23).61a The former are proteins to which varying numbers of a-mannose residues are covalently attached, while the latter are multifunctional spherical branched polymers of well-defined molecular size that carry large numbers of such residues on their surface.

3.28.3.1.2 P fimbriae In contrast to type 1 E. coli, that recognize structures present only in glycoproteins, P-fimbriated E. coli are specific for galabiose (Gala4Gal), a structure found in glycoproteins (see Chapter 3.14) as well as in membrane glycosphingolipids of the globo-series.62–64 These bacteria bind the disaccharide when it is present either at the nonreducing position or at an internal one of such glycolipids. They adhere mainly to the upper part of the kidney, where galabiose is more abundant. P fimbriae are similar to type 1 fimbriae in that they too are composite structures consisting of a long, rigid rod and a short, flexible, open helical part (Figure 20). The rigid section is about 7nm in diameter and is composed mainly of about 1200–2400 copies of the PapA subunit (16–22kDa) arranged in a tightly packed right-handed helix. The short flexible tip, consisting of PapE monomers, is 2 nm in diameter and is joined to the rigid rod by PapK adapter subunit. The carbohydrate-binding subunit, PapG, is located exclusively at the N-terminal domain of the flexible tip and appears to be the sole determinant of binding specificity. It has mostly a b-sheet structure that can be subdivided into two regions (Figure 21). One is in the form of a b-barrel similar to the corresponding region of FimH; the other, with a structure that has not been encountered elsewhere, contains the carbohydrate-binding site. Like in type 1 fimbriae, donor strand complementation between PapG and PapK has been observed in the crystal structure of the chaperonesubunit complex of the P fimbriae. As with type 1 fimbria, allelic variations in PapG are found with distinct receptor specificity. The combining site of PapG is extended and accommodates galabiose, the primary specificity determinant, binding the disaccharide by an array of hydrogen bonds, some mediated by water molecules, and hydrophobic interactions, with space for additional monosaccharide residues at its nonreducing end (Figure 22).

3.28.3.1.3 F17 fimbriae These fimbriae, produced by enterotoxigenic E. coli, are 3nm wide, flexible and wire-like organelles, built up of the major pilin subunit F17-A and exposing the F17-G subunit at their tip.65 They mediate binding of the bacteria to N-acetylglucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants, leading to diarrhea or septicemia. Binding of F17 E. coli to the microvilli is inhibited by N-acetylglucosamine as well as its

Microbial Lectins

f

2

N

f

l

N l

e m

a

C h

p

e

a

m

g k B

b

D

C

O

d

O

643

d

B

A

n

c

b

i

o

p h

D

C

o g k

A

n

i

1 C

C

Figure 21 Ribbon presentation of the N-terminal region of PapG. The upper part, made up of seven b-strands, forms a b-barrel. The lower part is composed of a six-stranded central antiparallel b-sheet, flanked on one side by two doublestranded b-sheets and, on the other side, by an a-helix and a large loop connecting this helix to the central b-sheet. The upper part contains the carbohydrate-binding site, shown as a stick model of the tetrasaccharide ligand GbO4 (GalNAcb3Gala4Galb4Glc), starting with A at the nonreducing end. N- and C-mark the respective ends of the polypeptide. Regions 1 and 2 of the structure are indicated by the vertical black arrows and labeled 1 and 2, respectively. Reproduced from Dodson, K. W.; Pinkner, J. S.; Rose, T.; Magnusson, G.; Hultgren, S. J.; Waksman, G. Cell 2001, 105, 733–743.

R170 NH2 R170 NH1 R170 NE

OH 02 W107 NEI

C2 O 05 D C3 C5

Y60 O G104 O Y105 N K106 O K106 N

CH3 C8

HO 06

HO 02 O 03

C3 A C2 OH 03

C4

O C5 05 C6

OH 04 Q92 N Y175 OH

C4 C6 O

05 O C

C6

HO 07 N2 C1 C7 N

OH 06

03 HO

E59 OEI

I61 L102 K172

OR 01 C1

OH C6 04

06 OH

C1 C2

C5

O 04

C3 B C2 C5 C4

04

C4 C1 OH 03 O05

OH 06

K172 Nζ

W107 OH 07

C3

G104 O G104 N K103 Nζ E91 OE2 E91 OE1

E91 OEl

Figure 22 Schematic representation of interactions between PapG and its ligand. Polar interactions, either direct or water mediated, are shown by arrows. Brackets and arrows shown as dashed lines indicate contacts with aromatic/hydrophobic platforms. Reproduced from Dodson, K. W.; Pinkner, J. S.; Rose, T.; Magnusson, G.; Hultgren, S. J.; Waksman, G. Cell 2001, 105, 733–743.

644

Microbial Lectins

Figure 23 Localization of the sugar-binding sites of F17a-G (a), FimH (b), and PapGII (c). The proteins (represented in gray) were superimposed, based on the structural core of the immunoglobulin fold, which was identified in the three carbohydratebinding subunits. Representative carbohydrate ligands are shown in black. The C-termini of the lectin domains, which precede the linker to the pilin domain, coincide approximately. Structurally equivalent strands are labeled with their names as defined for F17-G. The two parts of the PapGII domain are indicated: part 1 has the immunoglobulin-like core, whereas part 2 holds the sugar-binding site. From Buts, L.; Bouckaert, J.; De Genst, E.; Loris, R.; Oscarson, S.; Lahmann, M.; Messens, J.; Brosens, E.; Wyns, L.; De Greve, H. Mol. Microbiol. 2003, 49, 705–715.

b1-4-linked oligomers. The appearance and molecular organization of F17 fimbriae are similar to those of type 1 and P fimbriae, being composed of a flexible tip fibrillum with an open helical structure connected to the end of a tightly wound helical rod made up of their major fimbrial subunit. The high-resolution crystal structure of the lectin domain of F17-G, in complex with N-acetylglucosamine, revealed that the monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered 65 (for a study of the lectin domain of a closely related strain, see Ref.: 66). Despite lack of any sequence identity, it was unexpectedly found that the lectin domain of F17-G is similar to that FimH and PapG, and that all three share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae (Figure 23). Analogous to the other fimbrial subunits, F17-G is a two-domain protein in which a C-terminal pilin domain is linked with an N-terminal carbohydrate-specific lectin domain. The binding site of the lectin domain of F17a-G, one of the several cloned variants of F17-G examined in complex with the N-acetylglucosamine, is formed by the carbonyl group of Ala43, the side chains of Asp88, Thr89, Trp109, Ser117, Thr118, Gln119, and the nitrogen of Gly120 (Figure 24). Interactions between the carbohydrate and the protein include 11 possible hydrogen bonds, of which four are mediated by water molecules, as well as the hydrophobic stacking of the Trp109 side chain against the C5 and C6 atoms of the sugar. The N-acetyl group of N-acetylglucosamine contributes significantly to the affinity of the ligand to F17a G, due to a good complementarity of van der Waals surfaces between this group and the side chains of Thr118 and Asn44, as well as the carbonyl group of Ala43.

3.28.3.2 Soluble Bacterial Lectins Only small number of bacterial lectins that are soluble are known (Table 3). The 3-D structures of all these in complexes with ligands have been investigated by X-ray crystallography and in some cases also by NMR.

3.28.3.2.1 Pseudomonas aeruginosa lectins Two soluble lectins, originally isolated from Pseudomonas aeruginosa by Nechama Gilboa-Garber in the 1970s,67,68 have been well characterized.7 The galactose-specific PA-IL is a tetramer of four identical subunits, each consisting of

Microbial Lectins

39

Trp109

Ser117

39

Trp109

Ser117 3.07

3.07 3.02

32

Gln119

2.63

3.08 2.72 2.92 3.46 3.41 44

2.49 46 3.17 2.86 2.62

3.02

32

3.08

645

Thr118 2.90 Gln119

2.68 Ala43(0)

46 2.49 3.17

3.46 3.4144

Thr118 2.90

2.86

2.63 2.62

Gly120(N)

2.72 2.92

2.68 Ala43(0) Gly120(N)

3.31

3.31 Asp88

Asp88 27

27 Thr89

Thr89

Figure 24 Stereo view of N-acetylglucosamine in the binding site of F17a-G. Hydrogen bonds are indicated by green dotted lines with heavy atom distances in A˚. Water molecules are represented as small spheres. From Buts, L.; Bouckaert, J.; De Genst, E.; Loris, R.; Oscarson, S.; Lahmann, M.; Messens, J.; Brosens, E.; Wyns, L.; De Greve, H. Mol. Microbiol. 2003, 49, 705–715.

Table 3 Soluble bacterial lectinsa Source

Name

Pseudomonas aeruginosa

PA-IL PA-IIL RSL RS-IIL Cyanovirin N MVL

Ralstonia solanaceareum Nostoc ellipsosporum Microcystis viridis a

Specificity Galactose L-Fucose>mannose L-Fucose Mannose> L-fucose M(9), M(8)

For references, see text.

121 amino acids (m.w. 12.7kDa). The subunit of the fucose- and mannose-specific PA-IIL, also a tetramer, is similar in size to that of PA-IL, but its primary structure is different.69 The activities of both proteins are dependent upon the presence of divalent cations. The Pseudomonas-soluble lectins are found mainly in the bacterial cytoplasm. They are released by a subpopulation of the bacterial cells and serve to tether the bacteria to a substratum by binding to ill-defined structures on the bacterial surface and to a receptor on animal cells that contain the corresponding sugar. PA-IL displays medium-range affinity for galactose, with a Ka of 3.4104 M–1.70,71 Among monosaccharides, the specificity is strictly for galactose, with the exception of N-acetylgalactosamine, albeit with a lower affinity than that for galactose. The presence of a hydrophobic group on the anomeric position of the monosaccharide, either in a- or b-configuration, enhances the affinity, as observed with other lectins, such as E. coli type 1 fimbriae, with the tightest binding obtained for phenyl b-thiogalactoside. As for disaccharides, only those containing a terminal a-galactose residue are recognized, such as Gala3Gal, Gala4Gal, and Gala6Glc, to which the lectin displays the highest affinity; PA-IL binds also to a large number of glycoconjugates.72,73 The latter include compounds containing terminal unsubstituted Gala4Gal disaccharide, that is, the human P1 and Pk blood group antigens, present on either red blood cell glycosphingolipids or on pigeon egg white glycoproteins, as well as Gala3Gal, the human B blood group antigen, and the xenoantigen present on pig tissues.

646

Microbial Lectins

PA-IIL is characterized by an unusually high affinity for L-fucose, with a Ka of 1.6106 M–1 and binds the Lea trisaccharide [Galb3(Fuca4)GlcNAc] more strongly, with a Ka of 4.7106 M1.7,74 Thermodynamic studies indicate that these interactions are dominated by enthalpy. The entropy contribution is slightly favorable when binding to fucose and to the highest-affinity ligand Lea. The high-resolution X-ray structures of two complexes of PA-IIL with Lea and LNnFP-V (lacto-N-neofucopentaose-5, Galb4GlcNAcb4(Fuca3)Galb4Glc), allowed the precise determination of the conformation of a trisaccharide and a pentasaccharide (see later, Figure 28). The different types of interaction between the oligosaccharides and the protein involve not only hydrogen bonding, but also calcium- and water-bridged contacts, allowing a rationalization of the thermodynamic data. Three crystal structures of PA-IL have been reported thus far: the native lectin that contains one calcium ion per monomer, the calcium-free lectin, and the lectin with bound monosaccharide that contains both calcium and galactose (Figure 25).75 In all cases, the quaternary structure is a tetramer, each of which monomers adopts a small jellyroll b-sandwich fold, consisting of two curved sheets, each one constructed from four antiparallel b strands. The most striking feature of the PA-IIL structure is the involvement of the C-terminal carboxyl group (Gly114) of each monomer in the carbohydrate-binding site of the other monomer. Dimerization of the PA-II subunit is the result of a head-to-tail association of two monomers that make contact through the curved five-stranded b sheet. It largely involves hydrophobic contacts in the strands outside the Greek key motif of the protein and several hydrogen bonds at the extremity of these strands.7,76 The buried surface is 1500A˚2 per monomer. The dimers form a tetramer mainly by the antiparallel association of b-strands comprised of amino acids 79–85 (strand 6) from each dimer with their counterparts in the other dimer. The surface buried by the association of the dimers into tetramers is 500A˚2 per monomer. Both lectins are unusual in binding monosaccharides via direct interaction with metal ions.7 The sugar-binding sites of PA-IL and PA-IIL contain one and two calcium ions, respectively. In the PA-IL-galactose complex, one sugar residue is located in each monomer, close to the calcium atom. Oxygen atoms O3 and O4 of galactose are directly involved in the coordination sphere of the calcium ion (Figure 26). The lectin–sugar interaction also involves six direct hydrogen bonds between the sugar hydroxyl groups and the protein and an additional one bridged by a water molecule. Some limited hydrophobic contacts are established between the apolar face of the galactose residue and certain hydrophobic amino acids. In the PA-IIL-fucose complex, the fucose residue is locked onto a pair of calcium ions. Such a protein–carbohydrate binding mode involving two calcium ions has never been observed before (Figure 27). Three hydroxyl groups of fucose participate in the coordination of those calcium ions: O2 to the first calcium, O4 to the second calcium, and O3 to both of them. In addition to these electrostatic interactions, the three hydroxyl groups of the monosaccharide participate in hydrogen bonds with acidic groups of the calcium-binding site. The ring oxygen of fucose is involved in one hydrogen bond with the main chain, resulting altogether in a network of seven direct hydrogen bonds between the

Figure 25 Crystal structures PA-IL and PA-IIL. a, Tetramers with stick representation of monosaccharides and spacefilling representation of calcium ions. b, Monomers with the two b-sheets represented by different colors. From Imberty, A.; Wimmerova, M.; Mitchell, E. P.; Gilboa-Garber, N. Microbes Infect. 2004, 6, 221–228.

Microbial Lectins

647

(a)

(b)

S23 T45 W2 05 N21

D96 01

04 D104

02

W1

G114∗ CA2

03 CA1 T98

E99

N103

E95

D101

(c) Figure 26 Structure of the PA-IIL-fucose complex. a, Ribbon representation of the tetramer consisting of the asymmetric unit with stick representation of sugar and sulfate and CPK representation of the calcium ions. b, Electron density map around the monosaccharide residue, water molecules, and calcium ions, omitting the hydrogen atoms riding the fucose carbons. c, Interactions of PA-IIL with calcium ions and fucose with modeled hydrogen atoms. Coordination contacts are indicated by orange solid lines and hydrogen bonds by blue dashed lines. The asterisk symbol indicates the terminal glycine from the other monomer. Reproduced from Proteins Mitchell, E. P.; Sabin, C.; Snajdrova, L.; Pokorna, M.; Perret, S.; Gautier, C.; Hofr, C.; Gilboa-Garber, N.; Koca, J.; Wimmerova, M.; Imberty, A.; Copyright © (2005, Wiley); Reprinted with permission of Wiley-Liss, Inc., a subsidary of John Wiley & Sons, Inc.

648

Microbial Lectins

H50

P51

02 Q53

Y36

O6

N107 O3

O4

N108

D100

V101

T104

PA-IL/galactose

G114∗ N21

S23

O4

N103 O3 D104 O2

D101 D99

D96

E95

T98

PA-IIL/fucose

G114∗

N21

S23

O2

N103 O3 D101

D104

O6

O4

D99 D96

E95

(a)

T98

PA-IIL/mannose

(b)

Figure 27 Binding sites in the crystal structures of the PA-IL-galactose, PA-IIL-fucose, and PA-IIL-mannose complexes. a, Stick representation of the amino acids involved in binding. Ca2þ coordination bonds are shown as solid orange lines; hydrogen bonds as dashed green lines. Color coding: red, oxygen; blue, nitrogen; black, carbon; pink, Ca2þ. b, Electrostatic surface representation (color coding from violet for negative to orange for positive) of the protein-binding site, with Ca2þ as large pink spheres and monosaccharides as stick models From Imberty, A.; Wimmerova, M.; Mitchell, E. P.; Gilboa-Garber, N. Microbes Infect. 2004, 6, 221–228.

sugar and the protein. A water molecule mediates an additional hydrogen bond. The methyl group at position 6 of fucose is located in a shallow pocket with hydrophobic character. Analysis of the mode of binding of Lea by PA-IIL revealed a large number of contacts between the sugar and the protein (Figure 28). All monosaccharide units of the trisaccharide interact with the protein surface either directly (hydrogen bonding and coordination of calcium) or indirectly through bridging water molecules. In addition to the nine direct hydrogen bonds between the fucose of Lea and the protein described, the GlcNAc residue establishes a hydrogen bond between its O6 atom and the main-chain carbonyl group of Asp96. Two water molecules play an important bridging role in the interaction: one bridges O1 and O2 of fucose to the amide nitrogen of Thr98, whereas the other bridges the Gal residue (O2 and O1) to the side chain of Ser23. Hydrophobic contacts are very limited and

649

Microbial Lectins

07 Gal 02

06

GlcNAc 02

GlcNAc Gal

01 Gal

06

Ser23

Thr98

Asn21 CA2 Gly114*

Asp96

02 Thr98

Asp99

CA1

Asp104 Asp101

Ser23 06 Fuc

Fuc Asp99

Glc

CA1 Asn21 CA2 Gly114* Asp104 Asp101

Glu95

Asn103

Asp96

Glu95

Asn103

(a)

(b)

(c)

(d)

Figure 28 Complexes of PA-IIL with oligosaccharides. a, Interactions of PA-IIL with calcium ions and Lea. Coordination contacts are indicated by orange solid lines and hydrogen bonds by green dashed lines. b, Same representation for the interaction of PA-IIL with calcium ions and LNnFP-V. c, Interaction between two pentasaccharides in the PA-IIL–LNnFP-V complex. Hydrogen bonds are represented by dotted lines. d, Superimposition of the binding sites of the PA-IIL–Lea and PA-IIL–LNnFP-V complexes. From Perret, S.; Sabin, C.; Dumon, C.; Pokorna, M.; Gautier, C.; Galanina, O.; Ilia, S.; Bovin, N.; Nicaise, M.; Desmadril, M.; et al. Biochem. J. 2005, 389, 325–332.

only involve the methyl group of fucose with that of Thr145. The conformation adopted by Lea in the binding site is similar to that predicted by those earlier studies, confirming the rigidity of the trisaccharide. Among the four independent molecules of the PA-IIL–LNnFP-V complex, clear electron density can be observed for two complete pentasaccharides (Figure 28), whereas only a tetrasaccharide and a trisaccharide can be located for the two remaining sites. The fact that two of the oligosaccharides can be completely visualized is due the rigidity of their linear part resulting from fortuitous crystal packing that generates extensive contacts between two adjacent sugars centered on a pseudo twofold axis of symmetry (Figure 28c). It is this interaction that allows the very highresolution structure of milk pentasaccharide to be described here for the first time. The protein-binding site, together with the calcium ions and fucose moiety, are identical with those described previously. Additional interactions with the protein surface are established by the glucose and galactose residues of the Lex glucose analog moiety (Figure 28b). The glucose establishes a direct hydrogen bond between its hydroxyl group at position 2 and the side chain of Asp-96. In addition, five interactions are mediated by a water molecule, involving O1 of fucose, O1 and O6 of glucose, and O1, O2, and O6 of galactose. Hydrophobic interactions are observed for the methyl group of fucose, but also for C6 of galactose, which interacts with the carbon of a glycerol molecule located at the protein surface. The other part of the pentasaccharide, that is, the Glcb4GlcNAc linked to position 3 of galactose, does not interact with the protein surface, but instead is involved in extensive interactions with the neighboring carbohydrate in the crystal lattice (Figure 28c). PA-IL and PA-IIL are the first calcium-dependent lectins of bacterial origin to be structurally characterized. They do not seem to be related evolutionarily, even though they both adopt a jellyroll fold and they both use calcium for carbohydrate binding. In PA-IL, calcium, together with a unique network of hydrogen bonds, generates a binding site

650

Microbial Lectins

endowed with a selective specificity for galactose. In contrast, in PA-IIL, the two calcium ions are associated with a very high affinity (micromolar range) and a wide specificity for binding different monosaccharides. The occurrence of metal ions in lectins is well known. Carbohydrate binding by legume lectins depends on the presence of manganese and calcium ions, whose main role is to maintain the architecture of the binding site and not to make contacts with the sugar ligand. Direct calcium–sugar interactions have been observed in only two families of animal lectins: pentraxins and C-type lectins. The first family includes serum amyloid P component (SAP) and C-reactive protein (CRP), which contain two close calcium ions involved in binding of diverse ligands. The lectin activity of SAP differs from that of PA-IIL, since it is only directed to sulfated or phosphorylated carbohydrates. Despite the lack of sequence or fold similarity, the galactose-binding mode of PA-IL shows a striking resemblance to those of two C-type animal lectins complexed with galactose, that is, the QPDWG mutant of rat mannose-binding protein A and the tunicate galactosespecific C-type lectin, TC14, from Polyandrocarpa misakiens.7,78 Nevertheless, in PA-IL, the occurrence of an additional strong interaction with the galactose O6 group can explain the 10-fold higher galactose affinity compared to that of the C-type animal lectins.

3.28.3.2.2 Ralstonia solanacearum lectins Two soluble bacterial lectins have been recently isolated from the plant pathogen Ralstonia solanacearum, each with different affinity to fucose. One of these is RSL (subunit M(r) 9.9 kDa), related to fungal lectins,79 and the other is RS-IIL (subunit M(r) 11.6kDa), a tetrameric lectin, with high sequence similarity to PA-IIL.80 RSL consists of 90 amino acids with a tandem repeat in its amino acid sequence. It binds fucose with Ka of 5105 M–1. Among the monosaccharides tested, only L-galactose and D-arabinose, differing from fucose just by the substituent at C6, displayed some binding, albeit with affinity 4–5 times lower than that of fucose. RSL shows a preference for binding to Fuca2Gal and Fuca6Gal epitopes. It possesses two binding sites per monomer and an unusually high affinity for Fuca2Gal-containing oligosaccharides (Ka of 4106 M–1 for 2-fucosyllactose). The lectin has been crystallized with MeaFuc and with the highest-affinity ligand 2-fucosyllactose. The X-ray crystal structure of the RSL-MeaFuc complex revealed that each monomer consists of two small four-stranded antiparallel sheets (Figure 29). Trimerization through a threefold or pseudo-threefold axis generates a six-bladed b-propeller architecture, very similar to that previously described for the fungal lectin of Aleuria aurantia. Each monomer presents two fucose-binding sites, resulting in six symmetrically arranged sugar-binding sites for the b-propeller. The hydrogen-bonding network between fucose and protein consists of six bonds. The fucose hydroxyl O2 is bridged to the main chain by the amide group of Ala40a in site 1 (Ala85 in site 2). Hydroxyl O3 receives hydrogen from NE1 of Trp81 (Trp36 of neighboring monomer in site 2) and donates to the carboxyl group of Glu28 (Glu73 in site 2). The other acidic oxygen of this carboxyl receives a hydrogen bond from O4 of fucose that is also hydrogen-bonded to NE of Arg17 (Arg62 in site 2). Finally, the ring oxygen O5 receives a hydrogen bond from the terminal NH2 of the same arginine. In the high-resolution structure of RSL complexed with MeaFuc, water molecules are observed bound to the most accessible oxygens of fucose, O1, and O2, but they are not present in all sites and do not bridge to the protein. Hydrophobic contacts play an important role in the binding of fucose; Trp76 (Trp31 of the neighboring monomer in site 2) stacks to the fucose hydrophobic face (C3, C5, and C6) with distances smaller than 4A˚. On the other face of fucose, the sulfur atom of Cys30 (Cys75 in site 2) establishes a hydrophobic contact with the methine carbon C2. The methyl group at C6 is inserted in a hydrophobic pocket made by isoleucine residues 59 and 61 (monomer Pro14 and Ile16 in site 2) and tryptophan residues 10 and 81 (Trp53 of the same monomer and Trp16 of the neighboring one in site 2). The same fucose-binding mode is observed in all of the complexes studied, with a very limited variation in the fucose orientation. The only difference between site 1 and site 2 is the Ile59/Pro14 substitution in the hydrophobic pocket. In all binding sites, the hydrogen bond network is fully conserved. In the RSL-FucLac complex (intramonomer (site 1) and intermonomer (site 2)), the trisaccharide adopts very similar conformations and establishes the same contacts with the protein. The interactions involving the fucose residue have been described above. The galactose does not interact with the protein surface but makes an interresidue Gal.O2-Fuc.O3 hydrogen bond with the fucose residue. The glucose residue is folded back on the protein surface with its hydrophobic face, made by the CH of C1, C3, and C5, establishing van der Waals interactions with the plane surface created by a side chain of Asp32 that creates a salt bridge with Arg17 (Asp77 and Arg62 in site 2). In site 2, there is an additional stabilization by O6 interacting with the side chain of Trp53 through a bridging water molecule. RS-IIL recognizes fucose but displays much higher affinity for mannose and fructose, which is opposite to the preference of PA-IIL. The binding loop of the cations is fully conserved in RS-IIL as in PA-IIL, whereas the preference for mannose versus fucose can be attributed to the change of a three-amino-acid sequence in the ‘specificity loop’. The RS-IIL crystal contains one monomer of 113 amino acids per asymmetric unit, together with two Ca2þ ions

Microbial Lectins

651

Site 2 C-term Site 1

N-term N-term C-term

(a)

(b)

W10

W53

I59

I61

P14

R17

I16 C30

R62

C75

W76 W31

E28 W81

A40

W36

E73 A85

(d)

(c)

(e) Figure 29 RSL trimer complexed with fucose. a, Ribbon diagram of the complex with fucose (shown as sticks). b, Same representation for Aleuria aurantia agglutinin (AAL) monomer complexed with fucose. c, Fucose in binding site 1 with hydrogen bonds represented by dashed lines. d, Same representation for binding site 2. e, Final electron density map around the fucose molecule. From Kostlanova, N.; Mitchell, E. P.; Lortat-Jacob, H.; Oscarson, S.; Lahmann, M.; GilboaGarber, N.; Chambat, G.; Wimmerova, M.; Imberty, A. J. Biol. Chem. 2005, 280, 27839–27849.

and one MeaMan molecule (Figure 30). The overall topology of RS-IIL is that of a nine-stranded antiparallel b-sandwich consisting of two sheets of four and five strands respectively. In the tetrameric quaternary structure, one interface between two monomers is created by strong complementary packing of the one hydrophobic curved b-sheet to its counterpart, and the other interface is created via sheet extension (Figure 30). Both monomeric and tetrameric structures are very similar to those described in detail for several crystal structures of PA-IIL.77,78 The binding site is occupied by two Ca2þ ions and a mannose residue. The two ions are located close together, 3.75A˚, and

652

Microbial Lectins

(a)

(b)

A23

G113∗ N21

(c)

A23 S23 A22 S22

O2

G24

N24

O6 N102 O3 D103

O4 D96 D95

D100

D95

D98

E94

T97

(d)

(e)

Ser23

Als23 a

Asn24

(f) Figure 30 a, Crystal structure of the RSL-II-MeaMan complex with stick representation of the calcium ions. b, Representation of the molecular tetramer. c, Final electron density map around the monosaccharide residue. d, Calcium and MeaMan binding site with hydrogen bonds represented as dashed green lines and coordination contacts as orange solid lines. Calcium ions and water are represented by violet spheres respectively. e, Superimposition of MeaMan in RS-L binding site (grey and black lines and labels) and aMan in PA-IIL binding site (green lines and labels) from structure 1OVS. f, Electrostatic surface representation (color coding from violet for negative to orange for positive) of the protein binding site with Ca2þ as large pink spheres and monosaccharides as stick models with RS-IIL on the left and PA-II-MeaMan (1OVS) on the right. From Sudakevitz, D.; Kostlanova, N.; Blatman-Jan, G.; Mitchell, E. P.; Lerrer, B.; Wimmerova, M.; Katcoff, D. J.; Imberty, A.; Gilboa-Garber, N. Mol. Microbiol. 2004, 52, 691–700.

both display a classical seven-ligand coordination, mainly involving one loop, the conserved ‘calcium-binding loop’, via the side chains of Glu94, Asp98, Asp100, Asn102, and Asp103. A second loop is involved through the side chain of Asn21. In addition, the acidic group of the C-terminus Gly113 of the other monomer, which is involved in the head-totail interaction, also participates in the coordination of the Ca2þ ions. All amino acids of the calcium-binding site are strictly conserved between PA-IIL and RS-IIL, and the Ca2þ ions are bound in a similar manner. In the RSLII-MeaMan complex (Figure 30), O2, O3, and O4 participate directly in the coordination of the Ca2þ ions, with a special position for O3, which participates in the coordination sphere of both. The O3, O4, and O6

Microbial Lectins

653

hydroxyl groups of mannose are involved in hydrogen bonds with acidic amino acids of the calcium-binding loop extending from Glu94 to Asp103. The O2 hydroxyl group establishes hydrogen bonds with the backbone oxygen of Asn21 as well as with the C-terminus from the adjacent monomer. The ring oxygen O5 receives a hydrogen bond from the backbone nitrogen of Ala23. One water molecule makes an additional interaction by bridging between O4 and the backbone nitrogen of Thr97. Hydrophobic contacts appear to be very limited: only the methyl group of the MeaMan ligand is in relatively close position to Ala23. The comparison with the binding mode of mannose in PA-IIL shows similar contacts except in the region of O6.

3.28.3.2.3

Cyanovirin-N

Cyanovirin-N (CVN) is an 11kDa lectin from the cyanobacterium (blue-green alga) Nostoc ellipsosporum with potent virucidal activity, that possesses the ability to inactivate all strains of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) as well as other viruses such as those of influenza and Ebola.81,82 In addition, it binds very strongly the mammalian oligomannosides Man(9) and Man(8) (Figure 31), its minimal target being Mana2Mana, the termini of the branches of Man(8) and Man(9). The lectin consists of 101 amino acid residues organized in two domains, with a high degree of internal sequence and structural similarity. The solution structure solved by NMR revealed that CVN is an elongated, largely b-sheet protein that displays internal twofold pseudo-symmetry83,84 (Figure 32). The two sequence repeats (residues 1–50 and 51–101) share 32% sequence identity and are superimposable; HO

D3

HO O

HO HO

HO

D2

HO

HO

HO HO

O

HO HO D1

O

B

O

OO HO

HO OH

O

HO

O

O

4⬘

O

O

HO O

HO

HO OH HO OH C

HO

HO

O

O

HO

Man9GlcNAc2

O

HO OH A

O

HO

HO O

O HO

3

O

O 2

O HO NHAc

1

OH NHAc

HO OH 4

Figure 31 Structure of the N-linked undecasaccharide Man9GlcNAc2 (Man(9)) where the outer mannose residues are labeled as D1, D2, and D3. In the corresponding decasaccharide (Man(8)), the D2 arm is missing. From Bewley, C. A.; Kiyonaka, S.; Hamachi, I. J. Mol. Biol. 2002, 322, 881–889.

Site 2

* 50 30

20

60

40 C

10

90

80 70

Site 1

*

N

Figure 32 Ribbon drawing of the solution structure of a 1:2 CVN:Mana2Mana complex (PDB coordinates 1IIY) The opposing carbohydrate-binding sites are colored yellow and blue, and labeled as site 1 and site 2, respectively. The disaccharide Mana2Mana is shown in gray bonds with oxygen atoms colored red, and the reducing hydroxyl group is indicated by an asterisk. From Bewley, C. A.; Kiyonaka, S.; Hamachi, I. J. Mol. Biol. 2002, 322, 881–889.

654

Microbial Lectins

they do not form, however, separate domains, since the overall fold is dependent on numerous contacts between them. Rather, two symmetrically related domains are formed by strand exchange between the two repeats. Among the properties of CVN is its ability to block HIV envelope-mediated fusion. The affinity of oligosaccharide ligands to CVN was measured by their ability to inhibit the fusion-blocking activity of CVN in an HIV fusion assay. By this assay, it was found that CVN binds to gp120 with an equilibrium association constant Ka of 2.4107 M–1 and an apparent stoichiometry of 2 equiv. of CVN per gp120; Man(8) acts as a divalent ligand (2 CVN:1 saccharide) with a Ka of 5.4107 M–1, and Man(9) functions as a trivalent ligand (3 CVN:1 saccharide) with a Ka of 1.3108 M–1. Isothermal titration calorimetry experiments of CVN binding to Man(9) confirmed the nanomolar affinity of Ka of 1.5108 M–1, and the fitted data indicated a stoichiometry equal to approximately 1:1 CVN:Man(9). The 1:1 stoichiometry at micromolar concentrations suggested that CVN has not only a high-affinity binding site relevant to the studies at nanomolar concentrations, but a lower-affinity site as well that facilitates cross-linking of CVN-oligomannose at micromolar concentrations or higher. The high-affinity site has a Ka of 7.2106 M–1 and the low-affinity site a Ka of 6.8105 M–1, as determined by isothermal titration calorimetry. The specificity studies indicated that the minimum structure required for high-affinity binding comprises the Mana2Mana that represents the terminal disaccharide of all three arms of Man(9). High-resolution NMR experiments demonstrate that CVN binds the above disaccharide via two distinct binding sites of differing affinities located on opposite ends of the protein.83 Further NMR analysis of the solution structure of a complex of CVN-Mana2Mana at a molar ratio 1:2 revealed that the lectin recognizes the stacked conformation of the disaccharide through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semicircular cleft on the other (site 1)85 (Figure 33). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side chains comprising the binding pocket. The approximately 40A˚ spacing of the two binding sites provides a simple model for CVN:gp120 binding. The CVN: Mana2Mana complex provides the first high-resolution structure of a mannose-specific protein–carbohydrate complex with nanomolar affinity and presents a new carbohydrate-binding motif, as well as a new class of carbohydrate-binding protein, that facilitates divalent binding via a monomeric protein. Site 1 of CVN is able to discriminate between the three related trisaccharides Mea(Mana2Mana2Man), Mea(Mana2Mana3Man), and Mea(Mana2Mana6Man) with remarkable selectivity, and binds these trisaccharides with Ka values ranging from 8.1103 M1 to 6.6106 M1. Site 2 is less selective in that it binds all three trisaccharides with similar Ka values ranging from 1.7 to 3.7105 M1, but overall binds these trimannosides with higher affinities than site 1.

3.28.3.2.4 Microsystis viridis lectin Recently, another cyanobacterial protein known as MVL, originally isolated from a laboratory culture of Microcystis viridis NIES-102.86 MVL has also been shown to inhibit HIV-1 fusion at nanomolar concentrations.87 Like cyanovirin, MVL is unusual in that it binds oligosaccharides with very high affinity in the absence of multivalent interactions. Asn53

Glu56

C

C

O

Ser52 NH2

O

O

HO

C

NH2

HO

O

Asp44

O

O

C

HO

6⬘ Asn37

HO

HO

3⬘

Glu41

O C − O

Thr57-OH

H H 2N + C N H2 N

1⬘

M2

O

Gln50

C O

Arg76 O C Thr75

HO 2

NH2

O

1

4 O 5 M1

H2N Lys74 C O

OH 6

OH NH2

O

OH H2N

O C

C Asn42

Gln78

Figure 33 Schematic showing the potential hydrogen-bonding network between mannose hydroxyls, interfacial side chains, and proximal polar residues (for example, Asn37 and Gln50) of the high-affinity domain of CVN. Solid arrows indicate distances between donor and acceptor atoms that allow for direct hydrogen bonds, and dashed arrows denote distances that would accommodate water-mediated hydrogen bonds. From Bewley, C. A. Structure 2001, 9, 931–940.

Microbial Lectins

655

However, it is is distinct among oligomannose-binding proteins in that it exhibits fine specificity for the Mana6Manb4GlcNAcb4GlcNAc tetrasaccharide core found in N-linked oligomannosides. MVL in solution is a monodisperse homodimer with four independent carbohydrate-binding sites. Each subunit comprises two homologous domains, each consisting of 54 residues with 50% sequence identity between them, connected by a five-residue linker. MVL exhibits no significant sequence similarity to any other known protein family. X-ray crystallography of MVL free and bound to the pentasaccharide Man3GlcNAc2 confirmed that the protein is a homodimer stabilized by an extensive intermolecular interface between monomers87,88 (Figure 34). Each

1 a1

113

b2

N

N b3 b1

113

N-domain

1 b4

1

b5 a2

(a)

C

C

C-domain

b6

113

(b)

1 N

C

(c)

N

113

113

C

(d)

Figure 34 Overview of the crystal structures of MVL free and bound to Man3GlcNAc2. a, Ribbon diagram of a single monomer of MVL from the crystal structure of the free protein. In the N-domain, strands 1, 2, and 3 and helix 1 comprise residues 2–10, 32–41, 45–53, and 14–29, respectively; in the C-domain, strands 4, 5, and 6 and helix 2 comprise residues 61–69, 91–100, 104–112, and 73–88, respectively. b, Ribbon diagram of the free MVL dimer with one subunit in blue and the other in red, together with water molecules (green spheres) and associated side chains that participate in water-bridged hydrogen bonds between the two monomers. c, Ribbon diagram of the MVL-Man3GlcNAc2 complex with the bound carbohydrate depicted as cylinders and spheres (carbon, yellow; nitrogen, blue; and oxygen, red). d, Combined surface, tube, stick, and space-filling representation of the MVL dimer illustrating the water-filled channel formed between monomers in the dimer. A surface diagram of one monomer (red) and tube diagram of the second monomer (blue) are shown with space-filling rendering of water (gray) and ethylene glycol (green) molecules that fill the channel between the monomers. A surface diagram of carbohydrate bound to the surface model of one monomer of MVL and a stick diagram of carbohydrate bound to the tube model of the second monomer are shown in yellow. From Williams, D. C., Jr.; Lee, J. Y.; Cai, M.; Bewley, C. A.; Clore, G. M. J. Biol. Chem. 2005, 280, 29269–29276.

656

Microbial Lectins

Asn15(74) C O

Gly35(94)

H

OH

H 2N

HO O 5 4⬘

HO HO

H O Thr39(98)

C Gln36(95)

Thr38

OH

O HO HO 6

NH2

HO O

O

6 HO O 4 3

HO 6 O HO

4

Asn15(74)

H N

OH

O 2 NH O

Trp37(96) HOOH

N H

HO

O 3 HO

1 O

CH3

1 OH NH

O C

Leu12(71)

Leu12(71) Trp13(72) Pro11(70) Thr39(98) CH3

H N Ser43 (Gly102)

O

Figure 35 Schematic diagram of hydrogen-bonding interactions between MVL and Man3GlcNAc2. The carbohydrate structure is shown in red and the contacting protein residues in black. Dashed lines indicate hydrogen bonds, and the dashed arcs represent hydrophobic contacts. From Williams, D. C., Jr.; Lee, J. Y.; Cai, M.; Bewley, C. A.; Clore, G. M. J. Biol. Chem. 2005, 280, 29269–29276.

monomer contains two structurally homologous domains with high sequence similarity connected by a short fiveamino-acid residue linker. Intriguingly, a water-filled channel is observed between the two monomers. Man3GlcNAc2 binds to a preformed cleft at the distal end of each domain such that a total of four independent carbohydrate molecules associate with each homodimer. The binding cleft provides shape complementarity, including the presence of a deep hydrophobic hole that accommodates the N-acetyl methyl at the reducing end of the carbohydrate, and specificity arises from seven to eight intermolecular hydrogen bonds, one water-mediated bond, and a number of hydrophobic interactions (Figure 35).

References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Ofek, I.; Hasty, D. L.; Doyle, R. J. Bacterial Adhesion to Animal Cells and Tissues. ASM Press: Washington, DC, 2003; 416pp. Sharon, N.; Lis, H. Lectins, Kluwer/Springer: Dordrecht, The Netherlands, 2003; 454pp. Ofek, I.; Hasty, D. L.; Sharon, N. FEMS Immunol. Med. Microbiol. 2003, 38, 181–191. Sharon, N. Biochim. Biophys. Acta. 2006, 1760, 527–537. Blixt, O.; Head, S.; Mondala, T.; Scanlan, C.; Huflejt, M. E.; Alvarez, R.; Bryan, M. C.; Fazio, F.; Calarese, D.; Stevens, J.; Razi, N.; Stevens, D. J.; Skehel, J. J.; van Die, I.; Burton, D. R.; Wilson, I. A.; Cummings, R.; Bovin, N.; Wong, C. H.; Paulson, J. C. Proc. Natl. Acad. Sci. USA 2004, 101, 17033–17038. Ratner, D. M.; Adams, E. W.; Su, J.; O’Keefe, B. R.; Mrksich, M.; Seeberger, P. H. Chembiochem 2004, 5, 379–382. Imberty, A.; Wimmerova, M.; Mitchell, E. P.; Gilboa-Garber, N. Microbes. Infect. 2004, 6, 221–228. Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321–327. Lemieux, R. U. Acc. Chem. Res. 1996, 29, 373–380. Sharon, N. Biochemist 2006, 28, 13–17. Chen, Y.; Maguire, T.; Hileman, R. E.; Fromm, J. R.; Esko, J. D.; Linhardt, R. J.; Marks, R. M. Nat. Med. 1997, 3, 866–871. Spillmann, D. Biochimie 2001, 83, 811–817. Lamb, R. A.; Kolakofsky, D. Paramyxoviridae: The viruses and their replication. In Fields Virology, 3rd ed.; Fields, B. N., Knippe, D.M, Howley, D. R., Eds.; Lippincott-Raven: Philadelphia, PA, 1996; pp 1177–1204. Villar, E.; Barroso, I. M. Glycoconj. J. 2006, 23, 5–17. de Groot, R. J. Glycoconj. J. 2006, 23, 59–72. Schwegmann-Weisels, C; Herrler, G. Glycoconj. J. 2006, 23, 51–58. Vives, R. R.; Imberty, A.; Sattentau, Q. J.; Lortat-Jacob, H. J. Biol. Chem. 2005, 280, 21353–21357. Haidar, M.; Gluckman, J. C.; Gattegno, L. Glycobiology 1992, 2, 429–435. Mbemba, E.; Gluckman, J. C.; Gattegno, L. Glycobiology 1994, 4, 13–21. Rider, C. C. Glycoconj. J. 1997, 14, 639–642. Wiley, D. C.; Skehel, J. J. Annu. Rev. Biochem. 1987, 56, 365–394. Skehel, J. J.; Wiley, D. C. Annu. Rev. Biochem. 2000, 69, 531–569. Russell, R. J.; Stevens, D. J.; Haire, L. F.; Gamblin, S. J.; Skehel, J. J. Glycoconj. J. 2006, 23, 85–92.

Microbial Lectins

657

25. Ohuchi, M.; Ohuchi, R.; Feldmann, A.; Klenk, H. D. J. Virol. 1997, 71, 8377–8384. 26. Connor, R. J.; Kawaoka, Y.; Webster, R. G.; Paulson, J. C. Virology 1994, 205, 17–23. 27. Gamblin, S. J.; Haire, L. F.; Russell, R. J.; Stevens, D. J.; Xiao, B.; Ha, Y.; Vasisht, N.; Steinhauer, D. A.; Daniels, R. S.; Elliot, A.; Wiley, D. S.; Skehel, J. J. Science 2004, 303, 1838–1842. 28. Rosenthal, P. B.; Zhang, X.; Formanowski, F.; Fitz, W.; Wong, C. H.; Meier-Ewert, H.; Skehel, J. J.; Wiley, D. C. Nature 1998, 396, 92–96. 29. Dormitzer, P. R.; Sun, Z. Y.; Blixt, O.; Paulson, J. C.; Wagner, G.; Harrison, S. C. J. Virol. 2002, 76, 10512–10517. 30. Dormitzer, P. R.; Sun, Z. Y.; Wagner, G.; Harrison, S. C. EMBO. J. 2002, 21, 885–897. 31. Stehle, T.; Harrison, S. C. EMBO. J. 1997, 16, 5139–5148. 32. Freund, R.; Garcea, R. L.; Sahli, R.; Benjamin, T. L. J. Virol. 1991, 65, 350–355. 33. Stehle, T.; Yan, Y.; Benjamin, T. L.; Harrison, S. C. Nature 1994, 369, 160–163. 34. Stehle, T.; Harrison, S. C. Structure 1996, 4, 183–194. 35. Stehle, T.; Gamblin, S. J.; Yan, Y.; Harrison, S. C. Structure 1996, 4, 165–182. 36. Fry, E. E.; Lea, S. M.; Jackson, T.; Newman, J. W.; Ellard, F. M.; Blakemore, W. E.; Abu-Ghazaleh, R.; Samuel, A.; King, A. M.; Stuart, D. I. EMBO. J. 1999, 18, 543–554. 37. Mulloy, B.; Linhardt, R. J. Curr. Opin. Struct. Biol. 2001, 11, 623–628. 38. Fry, E. E.; Newman, J. W.; Curry, S.; Najjam, S.; Jackson, T.; Blakemore, W.; Lea, S. M.; Miller, L.; Burman, A.; King, A. M.; Stuart, D. I. J. Gen. Virol. 2005, 86, 1909–1920. 39. Nataro, J. P.; Cohen, P. S.; Mobley, H. L. T.; Weiser, J. N., Eds.; In Colonization of Mucosal Surfaces; ASM Press: Washington, DC, 2005; 456pp. 40. Gally, D. L.; Bogan, J. A.; Eisenstein, B. I.; Blomfield, I. C. J. Bacteriol. 1993, 175, 6186–6193. 41. Remaut, H.; Waksman, G. Curr. Opin. Struct. Biol. 2004, 14, 161–170. 42. Soto, G. E.; Hultgren, S. J. J. Bacteriol. 1999, 181, 1059–1071. 43. Sokurenko, E. V.; Chesnokova, V.; Dykhuizen, D. E.; Ofek, I.; Wu, X. R.; Krogfelt, K. A.; Struve, C.; Schembri, M. A.; Hasty, D. L. Proc. Natl. Acad. Sci. USA 1998, 95, 8922–8926. 44. Sokurenko, E. V.; Feldgarden, M.; Trintchina, E.; Weissman, S. J.; Avagyan, S.; Chattopadhyay, S.; Johnson, J. R.; Dykhuizen, D. E. Mol. Biol. Evol. 2004, 21, 1373–1383. 45. Sharon, N. FEBS Lett. 1987, 217, 145–157. 46. Zhou, G.; Mo, W. J.; Sebbel, P.; Min, G.; Neubert, T. A.; Glockshuber, R.; Wu, X. R.; Sun, T. T.; Kong, X. P. J. Cell. Sci. 2001, 114, 4095–4103. 47. Xie, B.; Zhou, G.; Chan, S. Y.; Shapiro, E.; Kong, X. P.; Wu, X. R.; Sun, T. T.; Costello, C. E. J. Biol. Chem. 2006, 281, 14644–14653. 48. Madison, B.; Ofek, I.; Clegg, S.; Abraham, S. N. Infect. Immun. 1994, 62, 843–848. 49. Ofek, I.; Hasty, D. L.; Abraham, S. N.; Sharon, N. Adv. Exp. Med. Biol. 2000, 485, 183–192. 50. Schembri, M. A.; Kjaergaard, K.; Sokurenko, E. V.; Klemm, P. J. Infect. Dis. 2001, 183(Suppl 1), S28–S31. 51. Abraham, S. N.; Hasty, D. L.; Simpson, W. A.; Beachey, E. H. J. Exp. Med. 1983, 158, 1114–1128. 52. Schilling, J. D.; Mulvey, M. A.; Hultgren, S. J. J. Infect. Dis. 2001, 183(1), S36–S40. 53. Firon, N.; Ofek, I.; Sharon, N. Carbohydr. Res. 1983, 120, 235–249. 54. Firon, N.; Ashbenazi, S.; Miralman, D.; Ofek, I.; Sharon, N. Infect. Immun. 1987, 55, 472–476. 55. Duncan, M. J.; Mann, E. L.; Cohen, M. S.; Ofek, I.; Sharon, N.; Abraham, S. N. J. Biol. Chem. 2005, 280, 37707–37716. 56. Choudhury, D.; Thompson, A.; Stojanoff, V.; Langermann, S.; Pinkner, J.; Hultgren, S. J.; Knight, S. D. Science 1999, 285, 1061–1066. 57. Normark, S. Proc. Natl. Acad. Sci. USA 2000, 97, 7670–7672. 58. Hung, C. S.; Bouckaert, J.; Hung, D.; Pinkner, J.; Widberg, C.; DeFusco, A.; Auguste, C. G.; Strouse, R.; Langermann, S.; Waksman, G.; Hultgren, S. J. Mol. Microbiol. 2002, 44, 903–915. 59. Sharon, N.; Lis, H. Adv. Exp. Med. Biol. 2001, 491, 1–16. 60. Bouckaert, J.; Berglund, J.; Schembri, M.; De Genst, E.; Cools, L.; Wuhrer, M.; Hung, C. S.; Pinkner, J.; Slattegard, R.; Zavialov, A.; Choudhury, D.; Langermann, S.; Hultgren, S. J.; Wyns, L.; Klemm, P.; Oscarson, S.; Knight, S. D.; De Greve, H. Mol. Microbiol. 2005, 55, 441–455. 61. Sokurenko, E. V.; Schembri, M. A.; Trintchina, E.; Kjaergaard, K.; Hasty, D. L.; Klemm, P. Mol. Microbiol. 2001, 41, 675–686. 61a. Pieters, R. J. Trends Glycosci. Glycotech. 2004, 16, 234–254. 62. Stromberg, N.; Nyholm, P. G.; Pascher, I.; Normark, S. PNAS 1991, 88, 9340–9344. 63. Larsson, A.; Ohlsson, J.; Dodson, K. W.; Hultgren, S. J.; Nilsson, U.; Kihlberg, J. Bioorg. Med. Chem. 2003, 11, 2255–2261. 64. Dodson, K. W.; Pinkner, J. S.; Rose, T.; Magnusson, G.; Hultgren, S. J.; Waksman, G. Cell 2001, 105, 733–743. 65. Buts, L.; Bouckaert, J.; De Genst, E.; Loris, R.; Oscarson, S.; Lahmann, M.; Messens, J.; Brosens, E.; Wyns, L.; De Greve, H. Mol. Microbiol. 2003, 49, 705–715. 66. Merckel, M. C.; Tanskanen, J.; Edelman, S.; Westerlund-Wikstrom, B.; Korhonen, T. K.; Goldman, A. J. Mol. Biol. 2003, 331, 897–905. 67. Gilboa-Garber, N.; Mizrahi, L.; Garber, N. Can. J. Biochem. 1977, 55, 975–981. 68. Gilboa-Garber, N. Biochim. Biophys. Acta 1972, 273, 165–173. 69. Gilboa-Garber, N.; Katcoff, D. J.; Garber, N. C. FEMS Immunol. Med. Microbiol. 2000, 29, 53–57. 70. Garber, N.; Guempel, U.; Belz, A.; Gilboa-Garber, N.; Doyle, R. J. Biochim. Biophys. Acta 1992, 1116, 331–333. 71. Stoitsova, S. R.; Boteva, R. N.; Doyle, R. J. Biochim. Biophys. Acta 2003, 1619, 213–219. 72. Chen, C. P.; Song, S. C.; Gilboa-Garber, N.; Chang, K. S.; Wu, A. M. Glycobiology 1998, 8, 7–16. 73. Lanne, B.; Ciopraga, J.; Bergstrom, J.; Motas, C.; Karlsson, K. A. Glycoconj. J. 1994, 11, 292–298. 74. Perret, S.; Sabin, C.; Dumon, C.; Pokorna, M.; Gautier, C.; Galanina, O.; Ilia, S.; Bovin, N.; Nicaise, M.; Desmadril, M.; Gilboa-Garber, N.; Wimmerova, M.; Mitchell, E. P.; Imberty, A. Biochem. J. 2005, 389, 325–332. 75. Cioci, G.; Mitchell, E. P.; Gautier, C.; Wimmerova, M.; Sudakevitz, D.; Perez, S.; Gilboa-Garber, N.; Imberty, A. FEBS Lett. 2003, 555, 297–301. 76. Loris, R.; Tielker, D.; Jaeger, K. E.; Wyns, L. J. Mol. Biol. 2003, 331, 861–870. 77. Mitchell, E.; Houles, C.; Sudakevitz, D.; Wimmerova, M.; Gautier, C.; Perez, S.; Wu, A. M.; Gilboa-Garber, N.; Imberty, A. Nat. Struct. Biol. 2002, 9, 918–921. 78. Poget, S. F.; Legge, G. B.; Proctor, M. R.; Butler, P. J.; Bycroft, M.; Williams, R. L. J. Mol. Biol. 1999, 290, 867–879. 79. Kostlanova, N.; Mitchell, E. P.; Lortat-Jacob, H.; Oscarson, S.; Lahmann, M.; Gilboa-Garber, N.; Chambat, G.; Wimmerova, M.; Imberty, A. J. Biol. Chem. 2005, 280, 27839–27849. 80. Sudakevitz, D.; Kostlanova, N.; Blatman-Jan, G.; Mitchell, E. P.; Lerrer, B.; Wimmerova, M.; Katcoff, D. J.; Imberty, A.; Gilboa-Garber, N. Mol. Microbiol. 2004, 52, 691–700. 81. Dey, B.; Lerner, D. L.; Lusso, P.; Boyd, M. R.; Elder, J. H.; Berger, E. A. J. Virol. 2000, 74, 4562–4569.

658 82. 83. 84. 85. 86. 87. 88.

Microbial Lectins

Barrientos, L. G.; O’Keefe, B. R.; Bray, M.; Sanchez, A.; Gronenborn, A. M.; Boyd, M. R. Antiviral. Res. 2003, 58, 47–56. Bewley, C. A.; Gustafson, K. R.; Boyd, M. R.; Covell, D. G.; Bax, A.; Clore, G. M.; Gronenborn, A. M. Nat. Struct. Biol. 1998, 5, 571–578. Bewley, C. A.; Kiyonaka, S.; Hamachi, I. J. Mol. Biol. 2002, 322, 881–889. Bewley, C. A. Structure 2001, 9, 931–940. Yamaguchi, M.; Ogawa, T.; Muramoto, K.; Kamio, Y.; Jimbo, M.; Kamiya, H. Biochem. Biophys. Res. Commun. 1999, 265, 703–708. Bewley, C. A.; Cai, M.; Ray, S.; Ghirlando, R.; Yamaguchi, M.; Muramoto, K. J. Mol. Biol. 2004, 339, 901–914. Williams, D. C., Jr.; Lee, J. Y.; Cai, M.; Bewley, C. A.; Clore, G. M. J. Biol. Chem. 2005, 280, 29269–29276.

Microbial Lectins

659

Biographical Sketch

Nathan Sharon is Emeritus Professor at the Weizmann Institute of Science, which he joined in 1954, after receiving his Ph.D. Degree from the Hebrew University, Jerusalem. Between 1956 and 1959 he was postdoctoral fellow at the laboratories of Fritz Lipmann and Roger W. Jeanloz, both at the Massachusetts General Hospital, and of Dan Koshland at Brookhaven National Laboratory, and in subsequent years was, among others, visiting professor at University of California, Berkeley, and Harvard University, and Fogarty Scholar at the NIH. He has made seminal contributions to glycobiolology in his studies amino sugars and glycoproteins, of lectin-carbohydrate interactions and their role in cell recognition, and the application of the knowledge accrued to medical problems such as the treatment of microbial infections (together with I. Ofek) and bone marrow transplantation. In addition, he has been a tireless and highly effective advocate for glycobiology worldwide. Sharon’s numerous publications include several widely cited reviews, as well as three books, ‘Complex Carbohydrates’ (1975), and ‘Lectins’, with H. Lis (1989 and 2006), which also appeared in Japanese. He is the recipient of many awards and honors, which include the Israel Prize in biochemical research, membership of the Israel Academy of Sciences and of the European Molecular Biology Organization, Honorary Doctorate from the University of Paris, and Honorary Membership of the American Society of Biochemistry and Molecular Biology and of the American Society of Microbiology.

Itzhak Ofek is Professor of Human Microbiology at Tel Aviv University, which he joined in 1979. After receiving his Ph.D. from the Hebrew University, Jerusalem, in 1972 he spent three years as postdoctoral fellow at the laboratories of Gene Stollerman and the late Edwin Beachey, both at the University of Tennessee, Department of Medicine Infectious Diseases Division, Memphis, TN. During 1975 and 1979 he was faculty member of The Departments of Microbiology at College of Medicine in Memphis, TN and Hadassah Medical School, in Jerusalem, Israel. He was visiting professor at the Department of Pathology, Washington University, St Louis with Erika Crouch and at the Research Center for Infectious Diseases, Wurzburg, Germany with Jorg Hacker. He has made extensive research of bacterial adhesion with special emphasis on the role of lectin–carbohydrate interactions (with N. Sharon) and on innate immunity against bacterial infections (with Yona Keissari, Hanny Sahly, and Erika Crouch). Ofek’s publications include more than 150 original articles, several of which are highly cited, as well as many reviews and two books (coauthored with the late Ron Doyle and with David Hasty), one of which appeared in Italian as well. He also co-edited three books, two of which are on microbial adhesion and one on innate immunity. Ofek together with Sharon have been the main advocates for anti-adhesion therapy of infectious diseases with agents such as carbohydrates or natural food products.

3.29 Carbohydrate–Protein Interactions: Carbohydrate-Binding Modules A. B. Boraston, A. Lammerts van Bueren, E. Ficko-Blean, and D. W. Abbott, University of Victoria, Victoria, BC, Canada ß 2007 Elsevier Ltd. All rights reserved. 3.29.1 Introduction

662

3.29.2 Paradigms of CBM Classification

663

3.29.2.1 Sequence-Based Families

664

3.29.2.2 Fold-Families

664

3.29.2.3 CBM Types

665

3.29.2.3.1 3.29.2.3.2 3.29.2.3.3

Type A surface-binding CBMs Type B glycan chain-binding CBMs Type C CBMs

665 665 666

3.29.3 CBM Functions

667

3.29.3.1 Polysaccharide Degradation

667

3.29.3.1.1 3.29.3.1.2 3.29.3.1.3 3.29.3.1.4

The proximity effect The targeting effect The disruptive effect CBM tandems and multivalency

667 668 669 669

3.29.4 Interactions with Polysaccharides

669

3.29.4.1 Cellulose: The Major Structural Plant Cell Wall Polysaccharide

669

3.29.4.1.1 3.29.4.1.2 3.29.4.1.3

Cellulose structure Molecular determinants of cellulose recognition by CBMs The thermodynamics of cellulose recognition

671 671 673

3.29.4.2 Chitin

673

3.29.4.2.1 3.29.4.2.2 3.29.4.2.3

673 673 675

Chitin structure Molecular determinants of chitin binding Chitin-binding CBMs that use a unique mechanism of ligand recognition

3.29.4.3 Xylan: Threefold Helical Plant Cell Wall Polysaccharide 3.29.4.3.1 3.29.4.3.2 3.29.4.3.3

3.29.4.4

b-Glucans

676 676 676 678

678

b-Glucan has a unique coiled structure Molecular determinants of b-glucan binding

678 679

a-Glucans: The Premier Storage Polysaccharides

680

3.29.4.4.1 3.29.4.4.2

3.29.4.5

Xylan structure Molecular determinants of xylan recognition The outliers: unique xylan-binding CBMs

3.29.4.5.1 3.29.4.5.2 3.29.4.5.3

a-Glucan structures and sources Molecular determinants of a-glucan recognition a-Glucans and multivalency: a role in starch disruption

3.29.4.6 Mannan and CBMs 3.29.4.6.1 3.29.4.6.2 3.29.4.6.3 3.29.4.6.4 3.29.4.6.5

680 680 682

682

Mannan structure Molecular determinants of mannan binding CBM27: detailed analysis of mannan oligomer-specific recognition A special case: the cooperative and synergistic effects of tandem CBM29s A dynamic event: CBM35 undergoes conformational changes upon binding

3.29.4.7 CBMs with Unique Polysaccharide Binding Specificities

661

682 682 682 684 684

684

662

Carbohydrate–Protein Interactions: Carbohydrate-Binding Modules

3.29.5

Interactions with Complex Glycans

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3.29.5.1

Oligosaccharide Synthesis

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3.29.5.2

Toxin Delivery

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3.29.5.3

Host–Microbe Interactions

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3.29.5.4

Implications for the Recognition of Complex Glycans by CBMs

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3.29.6 3.29.6.1

CBMs in Biotechnological Applications Exploiting the Native Properties of CBMs

3.29.6.1.1 3.29.6.1.2 3.29.6.1.3 3.29.6.1.4

3.29.6.2

CBMs and fiber modification Modified CBMs aid in plant growth CBMs as diagnostic tools CBMs as molecular scaffolds

CBM Fusion Proteins

3.29.6.2.1 3.29.6.2.2 3.29.6.2.3

CBMs as affinity tags CBMs as immobilization tools CBMs used to enhance enzyme efficiency

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3.29.1 Introduction Carbohydrate-binding modules (CBMs) are loosely defined as contiguous amino acid sequences that adopt an independent fold, have carbohydrate-binding activity but no catalytic activity, and are found within the primary structures of carbohydrate-active enzymes (e.g., glycoside hydrolases, glycosyltransferases, polysaccharide lyases, and carbohydrate esterases). Of course, there are exceptions: some of the first CBMs discovered were found in the noncatalytic scaffold in proteins of clostridial cellulosomes.1,2 However, given that these proteins were involved in the attachment of polysaccharolytic enzymes to cellulose they do fall under this loose definition. The boundaries of this definition have blurred more recently as many more ‘CBM-like’ modules that are not appended to carbohydrateactive enzymes have been discovered. For example, the Arabidopsis thaliana genome sequencing project has identified putative independent xylan-binding modules in this genome; wheat germ agglutinin (WGA) is considered a lectin but WGA-like modules are now frequently found appended to carbohydrate-active enzymes, usually enzymes active on chitin. Furthermore, modules falling into the CBM classifications (see Section 3.29.2) are occasionally found appended to modules known to lack activity on carbohydrates, such as the proteolytic clotting factor C a-subunit of Tachypleus tridentatus.3 Thus, in application this CBM definition is somewhat liberal but does, for the most part, separate these unique modules from other carbohydrate-binding proteins, such as lectins, antibodies, and transport proteins, which are typically not found associated with catalytic domains. The field of CBM research is rather new as its beginning is in c. 1986 when van Tilbeurgh et al. observed that limited protease treatment of a Trichoderma reesei (Hypocrea jecorina) cellulase resulted in the separation of a polypeptide retaining the cellulolytic activity and a small polypeptide have cellulose-binding activity.4 Over the following 7 years two more separate types of cellulase-associated domains with cellulose-binding activity were described.2,5 These domains were collectively referred to as cellulose-binding domains (CBDs). By 1996, an amino acid sequence-based classification had emerged that included 13 different families 6 and the three-dimensional (3-D) structures of four CBDs representing four of these families were published.7–10 However, by 1999 it became clear that numerous so-called ‘CBDs’ were being discovered that specifically recognized carbohydrates other than cellulose. The more inclusive term carbohydrate-binding module (CBM) was proposed to more accurately describe this class of carbohydrate-binding protein11,12 and this terminology now appears to be generally accepted. There are now over 40 sequence-based CBM families with 3-D structures deposited for representatives of over 30 families. The recent explosion in CBM research is being driven by a number of factors. First, CBMs are found appended to a large number of carbohydrate-active enzymes and these enzymes are frequently observed to have vastly reduced capacities to perform their functions when their CBMs are genetically or proteolytically removed. Thus, understanding CBM activity is a key aspect to understanding the overall biochemistry and biology of this large class of enzymes. Second, and more generally, CBMs make up an interesting group of model systems for the study of protein–carbohydrate interactions. Lectins, and to some degree antibodies and carbohydrate-transport proteins, have been considered the benchmark in this field for many years. However, the ability to obtain large quantities of CBMs and the ease with which they can be genetically manipulated makes them ideal model systems. Furthermore, because many have unique properties, such as specificity for crystalline ligands or highly polymerized ligands, these systems are yielding new


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information. Lastly, CBMs have use in numerous biotechnological applications from engineering the modular structures of industrially relevant glycoside hydrolases to research tools. Overall, CBM research is found in diverse fields from industrial settings to biochemistry laboratories. The purpose of this chapter is to provide a foundation for understanding the current state of this field of research. This will touch upon methods of classification, biological functions, molecular determinants of specific CBM–carbohydrate interactions, and the biotechnological uses of CBMs.

3.29.2 Paradigms of CBM Classification While questions surrounding the functions and structures of CBMs drive CBM research, the paradigms of CBM classification provide context for the findings and a platform for formulating new questions. Three classification schemes that employ structural and/or functional features are now in general use: an amino acid sequence-based classification, a tertiary structure classification, and a structure–function classification. Table 1 summarizes the three classifications. Table 1 The CBM families, their structures and properties Family CBM1 CBM2 CBM3 CBM4 CBM5 CBM6 CBM7 CBM8 CBM9 CBM10 CBM11 CBM12 CBM13 CBM14 CBM15 CBM16 CBM17 CBM18 CBM19 CBM20 CBM21 CBM22 CBM23 CBM24 CBM25 CBM26 CBM27 CBM28 CBM29 CBM30 CBM31 CBM32 CBM33 CBM34 CBM35 CBM36 CBM37 CBM38 CBM39 CBM40 CBM41 CBM42 CBM43 a

Specificity Cellulose Cellulose/xylan Cellulose Cellulose/xylan/b1-3-glucan/mixed b-glucan Chitin/cellulose Cellulose/xylan/b1-3-glucan/mixed b-glucan deleted Cellulose Cellulose/xylan Cellulose b1-3-Glucan/mixed b-glucan Chitin/cellulose Complex glycans/xylan Chitin Xylan Chitin Cellulose Chitin Chitin Starch Starch Xylan/mixed b-glucan Mannan a1-3-Glucan Starch Starch Mannan Cellulose Cellulose/mannan Cellulose b1-3-Xylan Complex glycans Chitin Starch Xylan/mannan/galactan Xylan Xylan/chitin/cellulose Inulin b1-3-Glucan Sialic acid Starch Arabinose b1-3-Glucan

N/A, not available

Ka range (M1) 5

10 103–106 106 104–105 104–105 104–105 deleted 104 104–106 105–106 103–105 106 103–104 N/Aa 104–105 N/Aa 105–106 104–105 N/Aa 104–105 N/Aa 104–106 105–106 107 104 104 105–106 105–106 104–105 104 105 103 106 N/Aa 104 104 N/Aa 105 N/Aa 104 104–106 N/Aa N/Aa

Structure/fold

Type

Cysteine knot b-Sandwich b-Sandwich b-Sandwich Unique b-Sandwich deleted N/Aa b-Sandwich O/B b-Sandwich Unique b-Trefoil Unique/hevein-like b-Sandwich N/Aa b-Sandwich Hevein N/Aa b-Sandwich N/Aa b-Sandwich N/Aa N/Aa b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich b-Sandwich N/Aa N/Aa N/Aa b-Sandwich N/Aa b-Trefoil N/Aa

A A and B(?) A B A B deleted ? C A B ? C C B ? B C ? B(?) ? B ? ? B(?) B(?) B B B B ? C ? B(?) B B ? ? ? C ? C ?

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3.29.2.1 Sequence-Based Families The premier classification of CBMs is based on amino acid similarity. There are currently 43 sequence-based CBM families the details of which are publicly available through the continuously updated CAZy database.12a Initial ‘seeding’ of a CBM family relies on the demonstration of carbohydrate-binding activity by an isolated module or providing reliable evidence that a module within the context of the entire enzyme has carbohydrate-binding activity. Additional members are then included in the family on the basis of sequence identity and, thus, considered putative CBMs. Unlike the related glycoside hydrolase and glycosyltransferase classifications,13 the CBM classification is not predictive of functional elements. In general, the sequence identity that defines a CBM family reflects the conservation of fold and topology within the family. Different features, such as loops and side chains, can be grafted onto the fold to create different carbohydrate-binding functionalities. For instance, family 6 comprises CBMs that bind cellulose, xylan, b1-3-glucans, and mixed b-glucans. The different specificities are achieved through altered architectures of the same binding site or binding sites located on entirely different faces of the protein. Within family 4, the addition or deletion of loops contour the binding sites to accommodate the structurally different sugars, cellulose or b1-3-glucan, while the ‘core’ of the proteins still have high sequence identity. The utility of this classification, therefore, is not in a predictive capacity but in a capacity to reveal differences in the context of similar structures. This has made the sequence-based classification the workhorse of CBM research and has resulted in the identification of numerous CBMs with previously unknown carbohydrate-binding specificities.

3.29.2.2 Fold-Families CBMs have been classified into seven ‘fold-families’ based on their tertiary structures (Figure 1; Table 1).14 The observation that CBMs can adopt any of the seven different folds is testament to their diversity. However, by far the most common fold for CBMs is the b-sandwich (Figure 1), which the vast majority of the CBM families display. In the context of lectins, this fold is shared with plant legume lectins, animal galectins, pentraxins, spermadhesins, calnexin, and ERGIC-53, though no CBM has significant amino acid sequence similarity with these other proteins.

(a)

(b)

(d)

(e)

(c)

(f)

(g)

Figure 1 CBM fold families shown as ‘cartoon’ representations. a, the b-sandwich represented by CBM22 from Clostridium thermocellum (PDB code 1DY0); b, the b-trefoil represented by CBM13 from Streptomyces lividans (PDB code 1MC9); c, the oligonucleotide–carbohydrate binding fold (OB) represented by CBM10 from Cellvibrio japonicus (PDB code 1QLD); d, a unique fold represented by CBM5 from Erwinia chrysanthemi (PDB code 1AIW); e, the knottin fold represented by CBM1 from Hypocrea jecorina (PDB code 1CBH); f, the hevein fold represented by CBM18 from Urtica dioca (PDB code 1EN2); g, the hevein-like fold represented by CBM14 (tachychitin) from Tachypleus tridentatus (PDB code 1DQC).


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In total, CBMs with this fold recognize the major biological polysaccharides: crystalline cellulose, noncrystalline cellulose, chitin, b1-3-glucans and b1-3,1-4-mixed linkage glucans, xylan, mannan, and starch. Other families of b-sandwich CBMs are beginning to emerge with more complex glycan-binding specificities. Thus, the b-sandwich is an extremely versatile scaffold to support the machinery for recognizing diversely structured carbohydrates. The b-trefoil fold-family is second largest though it only encompasses two sequence-based families, 13 and 42 (Figure 1). This fold was made infamous by the Ricinus communis toxin carbohydrate-binding B-chain, which is classified into CBM family 13. This fold possesses an internal threefold symmetry that can provide a scaffold for three functional carbohydrate-binding sites, as in the Streptomyces sp. xylan-binding modules.15–17 This fold family appears well suited to recognize a diverse array of polysaccharides and complex glycans. The remaining five fold-families have somewhat simpler folds and appear specialized for the recognition of cellulose and/or chitin (Figure 1).14

3.29.2.3 CBM Types CBM families can be grouped into families on the basis of amino acid identity; however, as discussed, such groupings are predictive of fold but not functional elements. Enough diversity exists within a given family such that functional elements, either specific amino acids or binding site topographies, are not conserved. An additional classification of CBMs has emerged and is gaining wider acceptability for general use. This classification is much broader and is based on both structural and functional similarities. Thus, three CBM ‘types’ have been proposed in which these protein modules have been grouped into ‘surface-binding’ CBMs (type A), ‘glycan chain’-binding CBMs (type B), and ‘small sugar binding’ CBMs (type C). The strength of this classification is that it relates how binding site architecture leads to general functional properties.

3.29.2.3.1

Type A surface-binding CBMs

This class of CBM is arguably the most unique as its properties differ significantly from other types of carbohydratebinding proteins. It includes members of CBM families 1, 2, 3, 5, and 10 that bind to insoluble, highly crystalline cellulose and/or chitin. By definition this type of CBM shows little or no affinity for soluble carbohydrates. Aromatic amino acid residues are a nearly universal feature of carbohydrate-binding sites; thus, their prevalence in type A CBMs is consistent with the majority of carbohydrate-binding proteins. However, the flat or platform-like binding sites are indigenous to this unique kind of carbohydrate-binding protein (Figure 2). The planar architecture of the binding sites is thought to be complementary to the flat surfaces presented by cellulose or chitin crystals.9,18 In support of this, site-directed mutagenesis studies consistently highlight the importance of the aromatic side chains in binding (see, e.g., Refs. 18 and 19). There is currently no compelling evidence to support the importance of hydrogen-bonding interactions. This type of CBM is best understood in the context of crystalline cellulose binding (see Section 3.29.4.1.2.1 for additional details).

3.29.2.3.2

Type B glycan chain-binding CBMs

In direct contrast to the type A CBMs, the type B CBMs, which currently includes examples from families 2, 4, 6, 11, 15, 17, 22, 27, 28, 29, 35, and 36, have clearly evolved binding-site topographies that are equipped to interact with individual glycan chains rather than crystalline surfaces (Figure 2). Though somewhat ambiguous, members of families 20, 25, 26, and 34 may be included in this classification. The degree of polymerization of the carbohydrate ligand is a key aspect in the binding proficiency of this class of CBMs as biochemical studies frequently demonstrate increased affinities up to hexasaccharides and negligible interactions with oligosaccharides with a degree of polymerization (DP) of 3 or less. Thus, these CBMs are considered to be ‘chain binders’. The structural basis of this ligand requirement has been revealed by nuclear magnetic resonance (NMR) and X-ray crystal structures which show that these carbohydrate binding sites are extended (>15A˚) and are often described as grooves or clefts with depths that vary from quite shallow to being able to accommodate the entire width of a pyranose ring. As with type A CBMs, aromatic residues play a pivotal role in ligand binding, and the orientation of these amino acids are key determinants of specificity.14,20 In contrast to the type A CBMs, direct hydrogen bonds also play an important role in defining the affinity and ligand specificity of type B glycan chain binders.21–23 An analysis of the hydrogen bonding patterns of type B CBMs reveals a hydrogen bonding density of 2.11 hydrogen bonds per 100A˚2 of polar surface area buried upon complex formation.14 The analogous value for lectins is 3.4524 highlighting a significant difference at the molecular level in how type B CBMs recognize polysaccharides in comparison to lectins.

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Type A

Type B

Type C

Figure 2 Structures of type A, B, and C CBMs. Type A is represented by CBM2 from the Cellulomonas fimi xylanase 10A (PDB code 1EXG); type B is represented by CBM27 from the Thermotoga maritima mannanase 5 (PDB code 1OH4); type C is represented by CBM9 from the Thermotoga maritima xylanase 10A (PDB code 1I82). Solvent-accessible surfaces are shown in transparent gray; carbohydrate ligands are shown in ‘licorice’ representations; the secondary structure representations are shown as ‘cartoons’.

3.29.2.3.2.1 Subsite analysis of type B CBMs Type B CBM-binding sites comprise several subsites, each able to accommodate an individual sugar unit of the polymeric ligand. Subsites are usually identified by the analysis of X-ray crystal structures of CBM–ligand complexes; however, subsites can be tentatively identified on the basis of biochemical analyses.25 Similar to the IUPAC subsite nomenclature adopted for glycoside hydrolases, CBM-binding subsites are numbered sequentially, starting at 1, from the reducing-end sugar. Unfortunately, because this system is not centered on catalytic machinery as a reference point, as in the glycoside hydrolase system, it can lead to ambiguity if the subsite analysis is limited to only a portion of the subsites. The most thorough subsite analysis of a CBM was done by Lammerts van Bueren and Boraston, who used a combined structural and thermodynamic approach to analyze the occupation of binding subsites by b1-4-xylooligosaccharides in a family 6 xylan-binding CBM from Clostridium stercorarium.26 The results indicated four subsites with the presence of ‘primary’ subsites that provided the bulk of the binding energy. Additional subsites were occupied in a defined order as the ligand length increased. While each CBM may differ in the order in which the subsites are occupied, this study supplies a working model of how sequential subsite occupation builds up the free energy of binding to polymeric ligands.

3.29.2.3.3 Type C CBMs This class of CBM has the lectin-like property of binding optimally to mono-, di-, or trisaccharides, and thus, lacks the extended binding-site grooves of type B CBMs (Figure 2). The type C CBMs include examples from families 9, 13, 14, 18, 32, 40, and 42. Members of families 13 (e.g., Ricin toxin B-chain), 14 (e.g., tachycitin), and 18 (e.g., WGA) were first discovered as lectins with small-sugar binding activity and have only subsequently been included as CBMs due to the discovery of homologs in a number of glycoside hydrolases. The only characterized member of family 9 is the C-terminal CBM from Thermotoga maritima xylanase 10A.27–29 In general, this family of CBM is found exclusively in xylanases and this particular CBM from Th. maritima has the remarkable property of recognizing the reducing-end sugars of xylans and cellulose. Family 32 is a relatively new CBM family whose only currently characterized member is the C-terminal module from the Micromonospora viridifaciens sialidase.30,31 This CBM has a very similar fold to the


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fucose-specific lectin from Anguilla anguilla and appears to bind galactose.30 Overall, identification and characterization of type C CBMs is lagging behind type A and B CBMs, likely due to their limited presence in plant cell wallactive glycoside hydrolases. Rather, the type C CBMs, particularly CBMs from families 13 and 32, appear more prevalent in bacterial toxins or enzymes (glycoside hydrolases and glycosyltransferases) that are active on eukaryotic cell surface glycans or extracellular matrix glycans (see Section 3.29.5). It should be made clear, however, that the distinction between type B and type C CBMs can be slight. For example, the type B CBM6 module of the Cl. stercorarium xylanase has a very similar fold to the type C lectinlike CBM32 family,30 but apparently binds longer oligosaccharide ligands. Furthermore, the Cellvibrio mixtus Cel5A CBM6 contains two discrete binding sites that display characteristics of type B and type C modules, respectively.32,33 However, an analysis of the hydrogen-bonding patterns in type C CBMs gives a hydrogen-bonding density that agrees very well with lectin patterns (3.7 hydrogen bonds per 100 A˚2 of polar surface area buried upon complex formation vs. 3.45); thus, based on this criterion, it is apparent that the hydrogen-bonding network between protein and ligand is more extensive in type C CBMs than type B modules.

3.29.3 CBM Functions CBMs are found to play several roles in nature. The primary and originally discovered role is that as an accessory module in polysaccharide degradation, which is discussed below. CBMs are also found to function in the anabolic process of glycan synthesis as well as in protein toxin delivery and host colonization by bacterial pathogens. However, the latter roles are discussed in Section 3.29.5 in the context of complex glycan recognition.

3.29.3.1 Polysaccharide Degradation The presence of CBMs in glycoside hydrolases can be a key factor in their ability to efficiently breakdown insoluble polysaccharides. There are three general effects that CBMs have during polysaccharide degradation: a proximity effect, a targeting effect, and a disruptive effect. Each of these will be discussed along with how the frequent occurrence of tandem CBMs (CBMs found in multiples within glycoside hydrolases) exert their influence.

3.29.3.1.1

The proximity effect

CBMs are noncatalytic proteins that in the most basic interpretation serve one single function: to bind carbohydrate. In the context of an entire glycoside hydrolase having both a catalytic module and a CBM means a tighter association of the enzyme with the substrate, consequently keeping all of the components in proximity (Figure 3). This was first hinted at by van Tilbeurgh et al. who papain treated cellobiohydrolase I (CBH I) from H. jecorina to separate the family 1 CBM from the catalytic module.4 They observed that the activity on insoluble cellulose (Avicel) was abolished and adsorption of the proteolyzed CBH I to Avicel was correspondingly decreased relative to the untreated enzyme.

(a)

(b)

Figure 3 A schematic representation of the proximity effect. In both panels, the cellulose is shown as a dash-filled box and the catalytic enzyme modules as gray circles. In panel b, the enzyme is shown as a catalytic module (gray circle) with an appended CBM (black circle). Without a CBM, the enzymes do not interact tightly with the cellulose and are mainly in solution (a). With a CBM, the enzymes adhere tightly to the cellulose and, thus, are mainly in a bound state in close proximity to the cellulose (b).

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A similar effect was observed for a Cellulomonas fimi cellulase having a family 2 CBM.5,34,35 A more recent study by Bolam et al. is generally attributed to have stated the effect of the CBM as a ‘proximity effect’ where substrate and enzyme are kept in prolonged contact.36 Subsequently, the reduction of activity on insoluble substrate upon genetic or proteolytic removal of the CBM has been shown in numerous systems including noncellulolytic enzymes such as amylases, xylanases, and chitinases (see, e.g., Refs. 37–39). As one might expect, CBMs rarely have an influence on the activity of glycoside hydrolases on soluble substrates. This appears to be due to the possibility that in such reactions, which are typically well dispersed, the catalytic modules make frequent collisions with the polysaccharide. In contrast, with aggregated polysaccharides the reactions may be ‘collision limited’ and thus aided when the enzyme is concentrated to the aggregate by the action of the CBM. This proximity effect is not limited to free enzymes. Cellulosomes are large hydrolytic enzyme complexes produced by some anaerobic bacterial and fungal species 40–44 that degrade cellulose and hemicellulose.45,46 The glycoside hydrolases are not free in solution but bound to a scaffoldin molecule through protein–protein interactions.47 Such large enzyme complexes have been described in Cl. thermocellum where there is a family 3 CBM found within the modular scaffoldin subunit.48 The family 3 CBM binds crystalline cellulose9,49 and mediates the interaction between the scaffoldin molecule, any adhered enzymes, and cellulose. Thus, the CBM targets the cellulosome to the insoluble cellulose substrate. The ability to combine more than one type of glycoside hydrolase module in tandem on the cellulosome substructure may allow a synergistic attack on the cellulose or hemicellulose of plant cell walls.48,50 For example, the presence of endo- and exoglycoside hydrolases would efficiently degrade a polysaccharide in a concerted manner.51 Many cellulosomes have varying hydrolytic components, including xylanases, mannanases, lichenases, pectate lyases, chitinases, and cellulases. These glycoside hydrolases may also contain CBMs for recognition of their specific substrates.

3.29.3.1.2 The targeting effect Discussion of the ‘proximity effect’ resulting from the ability of CBMs to bind carbohydrates implies a rather inelegant and nonspecific method of attaching an enzyme to an insoluble substrate. However, this does not appear to be strictly the case with CBMs. These modules are carbohydrate specific and more often than not the specificity of the CBM matches the specificity of the catalytic module. But beyond the somewhat crude partitioning of enzyme to its preferred type of polysaccharide, it is now becoming clear that CBMs have fine specificity for polysaccharide substructures (Figure 4). This is best understood with the cellulose-specific CBMs which display two levels of specificity. First, the cellulose-binding type A CBMs (e.g., family 2 and 3 CBMs) are highly specific for crystalline cellulose. In contrast, the cellulose-binding type B CBMs (e.g., family 4 CBMs) display no affinity for crystalline cellulose but do bind to soluble derivatized cellulose, cellooligosaccharides, and noncrystalline preparations of cellulose. Thus, CBMs are able to discriminate between crystalline and noncrystalline cellulose. In one instance, replacing the family 2 CBM with a family 4 CBM in a crystalline cellulose-specific enzyme resulted in a preference of the chimeric enzyme for noncrystalline cellulose demonstrating the influence that this specificity can have on enzyme function.52 CBMs specific for either crystalline or noncrystalline cellulose are further able to discriminate between as yet unknown substructures in these kinds of cellulose. Using chimeric enzymes, Carrard et al. showed that a catalytic module with crystalline cellulose-specific family 1, 2, or 3 CBMs preferentially hydrolyzed different regions of crystalline cellulose.53 Likewise, direct competition binding studies using noncrystalline cellulose-specific family 4, 17 and 28 CBMs demonstrated that these CBMs preferentially bind different substructures of noncrystalline

Figure 4 A schematic representation outlining the targeting of CBMs to polysaccharide structures. Xylan is represented by wavy dashed lines. Crystalline cellulose is shown in two possible different forms as straight solid lines and straight dotted lines. Noncrystalline cellulose is shown as ‘curvy’ solid lines. A chain-end binding CBM is shown as yellow circles. A xylanbinding CBM is shown as green circles. Red and blue circles represent cellulose-binding CBMs that bind to different forms of crystalline cellulose. Likewise, the purple and orange circles represent cellulose-binding CBMs that bind to different forms of noncrystalline cellulose. This figure is schematic only and not intended to accurately represent the structures of plant cell wall polysaccharide composites.


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cellulose.54,55 This was suggested to influence how efficiently noncrystalline cellulose was hydrolyzed by an enzyme containing both family 17 and 28 CBMs.54 Clearly, CBMs have developed the capacity for very specific interaction with their ligands. The influence of this targeting effect is not well studied, but results to date are intriguing and suggest that this function may give enzymes much narrower window of activity than previously thought, thus giving rise to the concept of ‘enzyme niches’. Curiously, this degree of specificity has not yet been investigated in non-cellulose-binding CBMs. Indeed, most plant cell wall polysaccharides are highly decorated evoking ideas of other highly specialized CBMs such as those that incorporate both the polysaccharide backbone components and side-chain decorations as recognition determinants.

3.29.3.1.3

The disruptive effect

Some type A CBMs appear to have the ability to disrupt the ordered structure of cellulose fibers allowing enzymatic modules to hydrolyze previously inaccessible fibers.56–59 Din et al. proposed that there is ‘intramolecular synergism’ between the CBM and the catalytic module.56 The CBM may bind and infiltrate the crystalline cellulose, disrupting and causing release of any particulate, noncovalently-attached cellulose. Together, the CBM and catalytic modules act synergistically, through disruption of the cellulose fibers causing release of particulate carbohydrate, and exposing areas of noncrystalline cellulose for hydrolysis.56 The molecular mechanism of this phenomenon remains unknown.

3.29.3.1.4

CBM tandems and multivalency

Glycoside hydrolases often contain multiple CBMs. These may or may not be from the same family and though frequently adjacent, are not necessarily so. Studies of CBMs in tandem often,60–62 but not always,63 show increased association constants relative to the individual CBMs. This appears to occur through an avidity effect whereby attached CBMs simultaneously interact with sugar-binding sites that are proximal in 3-D space (Figure 5). The frequency of tandem CBMs in enzymes has been observed to correlate with the growth temperatures of the source organisms suggesting that tandem CBMs and the conferred avidity effect is a way of compensating for the loss of affinity incurred at higher temperatures.62 In the case of the CBM17 and CBM28 story, the natural placement of these two CBMs with differing noncrystalline cellulose specificity in tandem creates both greater affinity and specificity for a subclass of binding sites within noncrystalline cellulose.54

3.29.4 Interactions with Polysaccharides 3.29.4.1 Cellulose: The Major Structural Plant Cell Wall Polysaccharide Cellulose is the most abundant organic biomass on the planet. Comprising linear chains of b1-4-linked glucose monomers (Figure 6), it is the most common structural polysaccharide in plants. The noncovalent interactions between cellulose chains promote their aggregation and ultimately the resulting structural organization provides the necessary strength to maintain the integrity of the plant cell wall. A

B

Ka,B

Ka,A

A

B A

B

Ku,A

Ku,B A

B

Figure 5 Schematic representation of the proposed two-step binding of a CBM tandem to a polysaccharide. The filled ellipses represent the CBMs. Equilibrium constants according to the individual steps are labeled. Ka is used to denote a standard association constant. Ku indicates an unimolecular interaction.

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OH OH

HO O

O

O O HO

O HO

OH

OH

OH

OH

O

O

O

O

O OH HO

OH

O OH HO

O HO

OH

OH O HO

OH

OH O HO

O HO

O OH HO

OH

OH

O HO

CH3 HO O

O x

CH3 OH

NH O O HO OH

O

n O OH HO

O

nO

O

OH O

OH O

O

O OH HO

n OH

OH

(c)

OH O

OH O

O

O

O HO

O

OH

OH

OH

OH

HO O

O

O HO O

O

OH

O

OH

O

O HO

O

NH

OH

NH

NH O

CH3

(a)

O n

CH3

(d)

HO

OH

HO O OH

O O HO

O

OH

HO O

OH

O OH

HO O HO

O OH

O n

(e)

OH O HOO HO OH O

HO HO

OH O

OH O HO O OH

OH O HO OH O

OH

OH

O HO

OH HO HO O HO

(b)

O O HO OH

OH O

HO HO O HO

O O HO OH

OH O n

O

(f)

Figure 6 The structures of the major polysaccharides involved in energy storage and plant cell wall structure. Sugars coordinates were extracted from X-ray crystallography and NMR structures for CBMs in complex with ligands and compared to stereochemically depicted two-dimensional structures. a, amylose (a1-4-linked glucose): B. halodurans CBM25 (PDB code 2C3X). The stereochemical representation of amylopectin is also displayed (a1-4-linked glucose with a1-6-glucose branches). b, laminarin (b1-3-linked glucose): B. halodurans CBM6 (PDB code 1W9W). c, cellulose (b1-4-linked glucose): Cl. cellulovorans CBM17 (PDB code 1J84). d, chitin (b1-4-linked N-acetylglucosamine): Hevea brasiliensis hevein (PDB code 1T0W). e, xylan (b1-4-linked xylose) Cl. stercorarium CBM6 (PDB code 1UY4). f, mannan (b1-4-linked mannose): Thermotoga maritima CBM27 (PDB code 1OF3).


3.29.4.1.1

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Cellulose structure

Cellulose is typically thought of as existing in two forms: crystalline and noncrystalline. As cellulose chains increase in length beyond six glucose units, they develop a strong propensity to self-associate. Extensive inter- and intramolecular hydrogen bonding networks and van der Waals interactions result in arrays of aligned chains forming the ordered structure associated with crystalline cellulose. In nature there is a distinct hierarchy of crystalline cellulose organization: cellulose chains associate to form fibrils and fibrils associate to form microfibres. The strong intermolecular interactions in crystalline cellulose render the structure completely insoluble in water and provide it with substantial resistance to attack by most enzymes. In contrast, noncrystalline cellulose, or amorphous cellulose, lacks the same degree of higher order. The cellulose chains in noncrystalline cellulose are thought to have limited intermolecular interactions. As a result, the cellulose chains are more solvent exposed and this form of cellulose is more susceptible to enzymatic attack.

3.29.4.1.2

Molecular determinants of cellulose recognition by CBMs

There are 15 CBM families containing CBMs with specificity for cellulose (Table 1) from over 500 enzymes. Competition binding studies using fluorophore-labeled CBMs have clearly demonstrated that CBMs are specific for crystalline cellulose or noncrystalline cellulose, but not for both.55 Perhaps not surprisingly, the CBMs that are specific for cellulose are type A and those specific for noncrystalline cellulose are type B CBMs.

3.29.4.1.2.1 Type A cellulose-binding CBMs The type A cellulose-binding CBMs include members of families 1, 2, 3, 5, and 10. Structurally, only families 2 and 3 are related but all share similar binding-site architectures. The binding sites of these CBMs comprise planar amino acid side chains, usually aromatic amino acids, which have an obvious linear and co-planar arrangement (Figure 7). Furthermore, the aromatic amino acid side chains are spaced on intervals of roughly 5 or 10A˚ such that they overlap very well with the pyranose rings of the cellulose chains, thus optimizing the potential for CH–p interactions. This architecture has been assumed to be complementary to the flat surfaces presented by crystalline cellulose. However, there has been substantial controversy around identifying the location of the type A CBM binding site on crystalline cellulose. Tormo et al. first proposed that the binding site comprises the hydrophobic 110 face of crystalline cellulose where the flat a- and b-faces of the glucose rings are solvent exposed.9 However, on the basis of experimentally determined binding capacities McLean et al. argued that in perfect cellulose crystals, the surface area presented by this crystal face is too small to account for the binding capacity of CBMs.18 Thus, it was proposed that the binding faces of the cellulose crystal are more likely to be the hydrophilic 110 and 010 faces, where the edges of the cellulose chains and its hydroxyl groups are solvent exposed. Lehtio et al. weighed in on the debate with an elegant transmission electron microscopy study.64 Using this method they probed the location of the CBM-binding site on crystalline

(a)

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Figure 7 The interaction of type A CBMs with cellulose. Side and bottom views of the CBMs are shown with cellopentaose molecules included for reference. Amino acid residues important in cellulose binding are shown in ‘licorice’ representation. The panels are as follows: a, CBM1 from H. jecorina (PDB code 1CBH); b, CBM2 from Cellulomonas fimi xylanase 10A (PDB code 1EXG); c, CBM3 from Cl. thermocellum CipA (PDB code 1NBC); d, CBM5 from E. crysanthemi (PDB code 1AIW); e, CBM10 from Cellvibrio japonicus (PDB code 1QLD).

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Valonia cellulose and found that both CBM1 and CBM3 modules bound to the hydrophobic 110 face, the original face proposed by Tormo et al. They argued that cellulose crystals are often severely disrupted and chains on the small hydrophobic 110 face are often peeled off to reveal a larger surface area than would be calculated for a perfect crystal. This would account for the previous discrepancies concerning the likely binding site and its capacity to bind CBM. The debate is somewhat complicated by two observations. First, as discussed in Section 3.29.3.1.2, Carrard et al. suggested that various type A cellulose-binding CBMs have different binding sites on crystalline cellulose.53 Second, Creagh et al. used both isothermal titration calorimetry and fluorescence microscopy to detect at least two classes of binding sites on bacterial microcrystalline cellulose (BMCC).65 In light of all the evidence, it appears unlikely that binding of these CBMs is limited to any one face of cellulose crystals. Furthermore, it also seems unlikely that all of the type A cellulose-binding CBMs bind to the same face(s). A unique feature of many of the type A cellulose-binding CBMs is the apparent irreversibility of their adsorption to crystalline cellulose. This property is exploited in the biotechnological uses of CBMs to create cellulose-immobilized fusion proteins (see Section 3.29.6.1.2) but creates a conundrum with respect to the biological function of CBMs: if the CBM containing enzyme is fixed in one position on the substrate how does it effectively and continually perform its function and how is inactivated enzyme replaced on the substrate surface? This was partially resolved by Jervis et al. who used fluorescence-recovery-after-photobleaching studies to show that a type A family 2 CBM was able to diffuse across the surface of crystalline Valonia cellulose providing a mechanism for enzyme mobility. 66 McLean et al. showed that the same type A family 2 CBM in solution was able to exchange with itself when bound to crystalline cellulose suggesting that although the overall binding reaction appears macroscopically irreversible, it is indeed microscopically reversible.55 This not only suggested a mechanism for the removal of inactivated enzyme from the cellulose surface by exchange but also highlighted the complex mechanism of cellulose adsorption that type A CBMs might utilize.

3.29.4.1.2.2 Type B cellulose-binding CBMs Known cellulose-binding type B CBMs are found in families 4, 17, 28, and 30. All of these families adopt a b-sandwich fold and have binding grooves that are open at both ends to allow for internal binding on cellulose chains. Furthermore, the binding sites are all present on the same b-sheet face of the CBMs; however, the amino acid side chains that make specific protein–carbohydrate interactions are not conserved at the primary or tertiary structure levels. Structural studies by X-ray crystallography show a number of CH–p interactions between the pyranose rings of the bound sugar and aromatic amino acid side chains (Figure 8). This is complemented by a number of hydrogen-bonding interactions (Figure 8). The diversity of type A cellulose-binding CBMs is reflected in the apparent heterogeneity of binding sites on crystalline cellulose. A similar story has developed with the type B cellulose-binding CBMs. Using competition binding experiments with fluorophore-labeled versions of CcCBM17 from Cl. cellulovorans and Cf CBM4–1 from Cellulomonas fimi, McLean et al. demonstrated that these two CBMs only partially competed for binding sites on noncrystalline cellulose.55 Boraston et al. used similar experiments to compare the binding of CcCBM17 and BspCBM28 from Bacillus sp. 1139.54 These experiments showed that CcCBM17 and BspCBM28 did not compete at all for binding sites on noncrystalline cellulose. Furthermore, quantitative binding experiments using both

Figure 8 A detailed view of the interactions in the binding site of the family 4 type B cellulose-binding CBM from Cellulomonas fimi cellulase 9A (PDB code 1GU3). The bound cellopentaose molecule is shown as blue in a ‘licorice’ representation. Amino acid side chains involved in binding the sugar are shown as gray in a ‘licorice’ representation. Potential hydrogen bonds are shown as dashed lines.


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depletion isotherms and isothermal titration calorimetry suggested two classes of binding sites on noncrystalline cellulose for each of CcCBM17 and BspCBM28.54,67 Taken together these results suggest at least three structurally different kinds of binding sites, probably more, in noncrystalline cellulose that type B CBMs can recognize. Overall it is clear that natural cellulose, an amalgam of crystalline cellulose and noncrystalline cellulose, is a highly heterogeneous molecule presenting many morphologically different binding sites that CBMs must contend with.

3.29.4.1.3

The thermodynamics of cellulose recognition

The interactions of proteins with soluble carbohydrates have an almost universally observed thermodynamic signature: favorable changes in enthalpy (DH), partially offsetting unfavorable changes in entropy (DS), and small negative changes in heat capacity (DCp). Seminal work by Creagh et al. asked the question of whether this signature applied to the interaction of a CBM with crystalline cellulose. Using isothermal titration calorimetry and the type A family 2 CBM from Cellulomonas fimi xylanase 10A as a model protein they probed the thermodynamics of the interaction with BMCC.65 The interaction was found to have a favorable DS with a small but favorable DH, demonstrating that the thermodynamic forces that drive the binding of CBMs to crystalline ligands are relatively unique among carbohydrate-binding proteins.68 It was argued that the water molecules expelled from the protein and cellulose surfaces when CBMs bind to their target carbohydrates increases the entropy of the system. In the case of soluble saccharides, this is postulated to be more than offset by the conformational restriction of the bound ligand leading to a net reduction in entropy; however, because crystalline cellulose has rigid structure, such a phenomenon would not occur giving the overall favorable DS. This results in the obvious question of what drives the interaction of type B CBMs with noncrystalline cellulose. Would the aggregated structure of this type of cellulose also ultimately result in favorable DS due to lack of conformational restriction upon binding? Contrary to expectation, Boraston used Cc CBM17 and BspCBM28 as model proteins to show that the interaction of these type B CBMs with noncrystalline cellulose was thermodynamically very comparable to the interactions of these CBMs with soluble saccharides: enthalpically favorable and entropically unfavorable.67 Thus, the thermodynamic signatures of binding to crystalline versus noncrystalline cellulose are quite different, highlighting different mechanisms of type A and type B CBM interactions with cellulose.

3.29.4.2 Chitin 3.29.4.2.1

Chitin structure

Chitin is the second most abundant organic polymer in nature. It consists of repeating subunits of N-acetylglucosamine (GlcNAc) linked through b1-4 glycosidic bonds (Figure 6). Chitin is primarily found in fungal cell walls and the exoskeletons of arthropods, and is notably absent from plants and vertebrates. The tensile strength of this insoluble polysaccharide results from intermolecular hydrogen bonding between chains of GlcNAc orientated in either a parallel or antiparallel fashion. Chitinases, enzymes invovled in chitin degradation, are found in diverse organisms such as viruses, bacteria, plants, and humans. The structure–function relationship of CBMs in this process will be described below.

3.29.4.2.2

Molecular determinants of chitin binding

Chitin-specific CBMs are found in many different families and in some cases utilize a distinct mechanism of chitin recognition. Type A CBMs that recognize the crystalline form of chitin fall into families 1, 2, 5, and 37; whereas lectinlike type C chitin-binding CBMs are found in families 14 and 18 (Table 1). Interestingly, there is a third major of group of chitin-binding CBMs, in which the mode of binding remains to be determined; these include families 12 and 33.

3.29.4.2.2.1 Type A chitin-binding CBMs Type A CBMs are generally involved in glycoside hydrolase targeting and polysaccharide remodeling, which is important for nutrient generation. The best-defined family of insoluble chitin-directed CBMs is family 5, which was previously known as CBD V under the old nomenclature and is distantly related to the family 12 CBMs (see Section 3.29.4.2.3). The recent crystal structure of CBM5 from Serratia marcescens (SmCBM5) 69 has made an important contribution towards elucidating how this family interacts with chitin. SmCBM5 is a C-terminal chitinbinding module of an exochitinase belonging to the GH18 family of glycoside hydrolases.70,71 The 3-D structure of the CBM consists of three antiparallel b-strands connected with elongated loops. Of interest, there are two solventexposed aromatic residues that have structural identity to the Erwinia chrysanthemi endoglucanase Cel5 CBM

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binding site.72 Using molecular modeling and structure alignments, the authors were able to predict a 55A˚ pathway of evenly spaced aromatic residues that extends from the conserved aromatic pair into the catalytic active site.69 This fascinating observation provides structural evidence for coordinated substrate binding and orientation by SmCBM5, and concomitant exolytic hydrolysis by the appended catalytic domain SmGH18. There are several other families of type A CBMs that recognize chitin, including 1, 2, and 37. These families interact with a variety of other heterogeneous structural polysaccharides such as cellulose and xylan. An example of a chitinspecific CBM1 is from the mycoparasitic soil fungus Tr. virens chitinase.73 This enzyme, entitled Tv-Cht1, displays a modular structure with an N-terminal catalytic domain and a C-terminal chitin-binding CBM. When aligned with a biochemically characterized cellulose-binding CBM from this family (CBM1 of CBH I from H. jecorina), there are four strictly conserved aromatic residues implicated in ligand binding, and four cysteine residues involved in disulfide bond formation.74 Interestingly, using protein engineering Limon and colleagues 75 demonstrated that the enzymatic efficiency of chitinase Chit33 and Chit42 from Tr. harzianum can be increased by fusion of the cellobiohydrolase II CBM from H. jecorina. The other type A families (2 and 37) are poorly understood. The classification of chitin-specific CBM2s is based upon sequence comparisons and requires biochemical characterization. Recently, recombinant forms of family 37 CBMs from the ruminal bacterium Ruminocoocus albus were determined to interact broadly with different insoluble polysaccharides, including xylan, cellulose and chitin, which is likely important for cell surface adhesion onto heterogeneous substrates.76

3.29.4.2.2.2 Type C chitin-binding CBMs The function of type C chitin-binding CBMs appears to be important for host defense as opposed to nutrient metabolism. These modules contain small ‘hevein-like’ domains that utilize an extensive hydrogen-bonding network for chitin, chitooligosaccharide, and N-acetylglucosamine recognition. The fact that chitin is a component of many pathogenic microbes, including some bacteria and fungi, makes it a functional target for control of infectious diseases. The chitin/N-acetylglucosamine-specific family 14 and 18 CBMs have both been classified as type C CBMs. Interestingly, despite the fact that these families have no significant sequence homology,77 they display remarkable structural similarity (Figure 9). Family 14 chitin-binding CBMs are found throughout the animal kingdom, being identified in the genome analysis of many model organisms, including Drosophila,78 and Caenorhbditis elegans.79 It is a large family of chitin-binding CBMs, which have been documented to be active in nutrient digestion, host defense, and parasitism. Two different human chitinases involved in innate immunity that contain family 14 CBMs have been described: chitotriosidase, which is present in polymorphonuclear neutrophils and macrophages;80,81 and acidic mammalian chitinase found in the gastrointestinal tract and lungs.82 Family 18 is the largest family of CBMs with documented chitin-binding activity. CBM18s can be found appended to chitinases or as independent chitin-binding proteins that exist as single modules or tandem repeats. The majority of CBM18s are restricted to green plants with a few cases being identified in fungi. There are two very well-studied antimicrobial proteins from this family: hevein83 and WGA, which consists of homodimer containing two hevein-like folds.84 These proteins display structural and functional conservation to carbohydrate-binding homologs in a variety of organisms.85 The hevein domain is characterized by 3–5 intramolecular disulfide bridges, which stabilizes the chitinbinding motif. The interaction between hevein and chitin derivatives has been analyzed with a variety of biophysical techniques, including X-ray crystallography,83,86 NMR spectroscopy,87–90 calorimetry,91–94 and sedimentation analysis (Asensio, 2000 #279).90,95 The affinity of hevein for chitin oligosaccharides was determined to be in the millimolar range and enthalpically driven.90,92 A series of conserved hydrophobic and hydroxylated amino acid residues have emerged as key stabilizing factors in the CBM18-chitin complexes for hevein and HEV32,88,89,92,93 WGA,96–98 pseudohevein,90 Amaranthus caudatus peptides (Ac-AMP),99 and Urtica dioica agglutinin isolectin.100 Chitin specificity and the conservation of the hevein fold may be explained by CH–p interactions between the acetoamide methyl group at C2 of GlcNAc and the aromatic residues Trp21 and Tyr30 of CBM18. 88,89,92 Hydrogen bonds between Ser19 and Tyr30 and the nonreducing sugar C2-acetoamido carbonyl group and the hydroxyl group of C3, respectively, further stabilize the complex.88,89,92 Interestingly, the binding properties of CBM18 family members are dynamic and not restricted to the nonreducing end of the polymer.92 Indeed, the chitin polymer has been observed to move along the flat binding surface of hevein and occupy different binding subpockets. In 2000, the 3-D structure of tachychitin, a chitin-binding antimicrobial noncatalytic protein found in the horseshoe crab Tachypeus tridentatus,101,102 was determined by NMR.103 This protein is stabilized by five intramolecular disulfide bonds.101 The tertiary structure of tachycitin consists of a three-stranded b-sheet in the N-terminus followed by a two-stranded b-sheet and an a-helical turn in the C-terminus. The N- and C-terminal b-sheets interact


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W21 W23 Y49 S19

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Tachychitin Hevein

CPKG LHYNA YLKV CDWPSK-AG CPNN LCCSQ W-GW CGSTDEYCS

(Residues: 40−60) (Residues: 12−32)

Figure 9 Superimposition of family 14 tachychitin from Tachypleus tridentatus (PDB code 1DQC) and family 18 hevein from Hevea brasiliens (PDB code 1HEV). a, Proteins are rendered in cartoon format with hevein represented in green and tachychitin in light blue. The structural similarity is found in the chitin-binding domain of tachychitin, which is highlighted as a truncated structure in b. Amino acids involved in chitin binding are shown in stick format: Asn47, Trp49, and Trp23 (hevein) and Ser19, Tyr21, and Val52 (tachychitin). The disulfide bond (C–C) is a well-conserved structure within the hevein-fold family. c, Primary structure alignment of tachychitin and hevein. Although there is strong three-dimensional similarity between the two hevein-fold domains, there is little amino acid conservation. The disulfide bridge and amino acids involved in chitin binding are indicated.

as a distorted b-sandwich connected by a bending loop. Although there is distinctive primary sequence divergence, the chitin-binding domain of this protein 101 displays remarkable structural similarity to hevein. This observation is in support of a convergent evolutionary origin for these modules.77,103

3.29.4.2.3

Chitin-binding CBMs that use a unique mechanism of ligand recognition

Families 12 and 33 contain chitin-directed CBMs each of which has a unique mechanism of recognition. Biophysical studies of a CBM12 from B. circulans WL-12 (BcCBM12) with chitin derivatives has been reported by several different groups. Over 10 years ago, the C-terminus of the protein was observed to be essential for binding to colloidal chitin.37 More recently, purified recombinant BcCBM12104 was found to associate with insoluble chitin over a wide range of pHs (optimized near the pI of the protein) and the interaction was strengthened in the presence of salt. These data are in agreement with a type A mechanism and suggest that BcCBM12–chitin complex formation is mainly driven by hydrophobic interactions. However, following the determination of the solution structure in 2000,105 the binding mechanism has proved to be unique. Interestingly, although BcCBM12 displays 3-D homology to the CBM5 of the E. chrysanthemi endoglucanase Cel5, which binds cellulose,72 it lacks the characteristic linearly arranged aromatic triad on its surface for planar polysaccharide recognition (Figure 10).105 Alternatively, by analyzing for amino acid conservation, solvent accessibility, and hydrophobic character, the authors suggested that Thr682 and Pro689 may be involved in chitin adsorption. Also, two different groups have recently reported that the nonconservative substitution of a unique tryptophan residue (W687) within the binding site severely compromised its binding potential (Figure 10).106,107 This structural evidence suggests that chitin-binding CBM12 molecules interact with chitin with a substantially different mechanism than other CBM families. Family 33 CBMs are found in a diverse group of bacteria, including many pathogenic microorganisms such as Bacillus anthracis, Listeria monocytogenes, Vibrio parahaemolyticus, and Yesinia pestis. The majority of CBM33s have been identified by genome analysis; however, the chitin-binding protein from Serratia marcescens 2170 (CBP21) has been purified,108 cloned, and characterized,109 and its crystal structure has been determined.110 Very surprisingly, aromatic

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Figure 10 Family 12 CBMs have a unique mechanism of chitin binding. Structures of the CBM5 chitin-binding module from E. chrysanthemi endonuclease Z (PDB code 1AIW) (a) and CBM12 from B. circulans WL-12 chitinase A1 (PDB code 1ED7) (b) are displayed. Although both CBMs contain a structurally similar three-stranded anti-parallel b-sheet, there are critical differences in side-chain architecture important for carbohydrate interaction. Importantly, the traditional aromatic platform of type A-binding CBMs found in CBM5 is notably absent within CBM12. Also, polar residues T682, H681, P689, and P693; and an indispensable tryptophan (W687) are implicated in chitin recognition.

residues previously believed to be involved in chitin adherence are buried within the hydrophobic core of the protein. Using mutagenesis and depletion assays, the authors demonstrated that a series of surface-exposed residues capable of polar interactions (Tyr54, Glu5, Glu60, His114, Asp182, and Asn185) were important for chitin binding. This suggests that CBP21 utilizes a type C lectin-like mechanism to adhere to crystalline chitin. Interestingly, CBP21 is expressed as a noncatalytic independent protein that is not directly involved in the hydrolysis of chitin;109 rather, it is believed to indirectly assist in chitin degradation by substrate disruption leading to greater enzyme accessibility.111

3.29.4.3 Xylan: Threefold Helical Plant Cell Wall Polysaccharide 3.29.4.3.1 Xylan structure Along with cellulose in the plant cell wall are other accessory polysaccharides collectively called hemicellulose. The most abundant component of hemicellulose is xylan. The backbone of plant xylan comprises b1-4-linked xylose, a 5-carbon sugar that is structurally similar to glucose except that the C6 hydroxymethyl group is replaced by hydrogen. This polymer adopts an extended linear conformation with each monosaccharide unit rotated about the glycosidic bond by 120 relative to one another resulting in a threefold helix (Figure 6). Xylan is commonly decorated with arabinofuranose or glucuronic acid side chains, with the degree and type of decoration depending on the source of the xylan.

3.29.4.3.2 Molecular determinants of xylan recognition CBMs specific for b1-4-linked xylose polymers are found in a number of CBM families (Table 1). Because plant cell wall xylan does not have a crystalline form, CBMs that bind this polysaccharide are almost exclusively type B. The only possible exception is the xylan-binding subclass of family 2 CBMs (often referred to as family CBM2b). The clear type B xylan-binding CBMs have deep binding clefts that accommodate a single xylan chain (Figure 11a). The architecture of these binding sites complements the helical structure of the xylan chain. As with the type B cellulose-binding CBMs, the major interactions are stacking interactions between the aromatic amino acid side chains (Figure 11a) and the pyranose rings as well as hydrogen bonding between the sugar hydroxyls and polar amino acid side chains. Interestingly, the family 2 xylan-binding CBMs share many features with the type A cellulosebinding family 2 CBMs. First, the binding site of the family 2 xylan-binding CBMs retain the shallow nature of the type A CBMs; however, only two of the three aromatic amino acid residues are conserved and one of these is tilted 90 to break the planar nature of the binding site (Figure 11b).20 The effect is to create an aromatic platform that rather than recognizing the flat surface of a crystal accommodates the helical structure of xylan. Despite shared features with type A CBMs, because of their preference for single soluble xylan chains, the family 2 xylan-binding CBMs are generally considered type B CBMs.


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Figure 11 The binding sites of xylan-binding CBMs. Panel a reveals the 3-fold helix of the xylopentaose molecule bound to the CBM15 from Cellvibrio japonicus (PDB code 1GNY). The bound xylopentaose molecule is shown as blue in a ‘licorice’ representation while amino acid side chains involved in binding the sugar are shown as gray in a ‘licorice’ representation. Panel b shows the shallow binding site of the xylan-binding CBM2-1 from Cellulomonas fimi xylanase 11A (PDB code 2XBD). The solvent surface is shown in transparent gray, the backbone in ‘cartoon’ representation, and the tryptophan side chains oriented at right angles in gray in a ‘licorice’.

Figure 12 The locations of binding sites in xylan-binding CBMs having a b-sandwich fold. The overlapped Ca-traces of CBM6-1 from Clostridium stercorarium (green; PDB code 1UY4) and CBM15 from Cellvibrio japonicus (blue; PDB code 1GNY).

The xylan-binding CBMs are quite diverse and stand out from other groups of CBMs in two respects: the varying location of their binding sites and the calcium dependence of binding in some examples. Excluding the family 13 xylan-binding CBMs, all of the xylan-binding CBMs adopt a b-sandwich fold but the binding sites are found built into different regions of the modules. Like the type B cellulose-binding CBMs, some xylan-binding CBMs have their binding sites built into the concave b-sheet such that the binding groove runs perpendicular to the direction of the b-strands making up the b-sheet (Figure 12). This applies to CBMs in families 4,112 15,113 and 22.114 In contrast, the X-ray crystal structures of family 630,115 and 36 116 CBMs revealed their binding sites to be located at one edge of the b-sandwich where the loops connecting the b-strands make up a large portion of the binding site (Figure 12). The family 36 xylan-binding CBM from Paenibacillus polymyxa xylanase 43 is currently the only CBM for which the structural basis of its dependence upon calcium for binding is known.116 In the case of this CBM, the binding platform comprises mainly the side chains of two tyrosine residues. A single calcium atom sits adjacent to these residues and

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Figure 13 The role of calcium in xylan recognition by the family 36 CBM, PpCBM36, from Paenibacillus polymyxa xylanase 43. The calcium atom bound in the binding site of PpCBM36 is shown as a blue sphere. The amino acid residues involved in coordinating the calcium and binding the sugar are shown in gray ‘licorice’, and the bound xylooligosaccharide is shown in blue ‘licorice’.

contributes two coordination bonds to the bound xylan molecule (Figure 13). The apo form of this CBM is unable to bind carbohydrates showing the importance of this calcium-mediated interaction. A family 35 xylan-binding module from Cellvibrio japonicus has also been shown to be calcium dependent.117 However, it is currently unknown if this is due to indirect effects, such as the metal ion stabilizing the conformation of important binding residues, or if it reflects a direct interaction as in the family 36 CBM.

3.29.4.3.3 The outliers: unique xylan-binding CBMs Xylan-binding CBMs are also found in families 9 and 13. These CBMs differ in that they show similarities to lectins and are thus classified as type C CBMs. The only characterized member of family 9 is the C-terminal module from the Th. maritima xylanase 10A.27–29 This CBM, called TmCBM9-2, is quite promiscuous showing the ability to bind xylan, cellulose, other b-glucans, and even starch.28 However, considering that its parent enzyme is a xylanase, the biological ligand of this CBM is likely xylan and is better discussed under the umbrella of xylan recognition. The unique aspect of this CBM is its strict requirement for binding to the two monosaccharide units at the reducing ends of polysaccharides. These two sugar units must be linked by a 1-4 glycosidic bond and have equatorial hydroxyl groups at C1, C2, and C3. TmCBM9-2 appears to tolerate a or b glycosidic linkages, the presence or absence of the C6-hydroxymethyl group, and axial hydroxyls at C4 giving it a degree of plasticity with respect to potential ligands.29 One biotechnological advantage of this plasticity is the ability to use inexpensive disaccharides such as maltose or lactose as competitive eluants to remove TmCBM9-2 from insoluble matrices making this CBM an excellent affinity tag for purifying proteins to which it is fused.118 Two xylan-binding CBM13s from Streptomyces sp. have been well characterized structurally and biochemically.15–17,119 These CBMs have the b-trefoil fold most commonly associated with the ricin-toxin B-chain (RTB). Unlike RTB, all of the a, b, and g subdomains of the streptomycetes CBM13s have functional carbohydrate-binding sites giving these modules their trivalency and ability to interact reasonably tightly with xylan. These CBMs are classified as type C CBMs on the basis of their binding-site architectures. Though their binding sites appear to have evolved to recognize internal binding sites on xylan, they only accommodate three monosaccharide units in a shallow depression. Furthermore, the family 13 CBM from Streptomyces lividans xylanase 10A has retained the lectin-like ability to bind a selection of small sugars including galactose and lactose,120 much like its RTB relative.

3.29.4.4 b-Glucans 3.29.4.4.1 b-Glucan has a unique coiled structure b-Glucans are naturally occurring polysaccharides found in the cell walls of plants, grasses, cereals, and grains. The b-glucans discussed here are of two general types: those having backbones of b1-3-linked glucose and those with a backbone comprising both b1-3- and b1-4-linked glucose (mixed linkage b-glucans). b1-3-Glucans are found in algae (e.g., laminarin from the brown alga Laminaria saccharina), yeast (e.g., curdlan, zymosan, pachyman, and scleroglucan), and plants (e.g., callose). The b1-3-linked glucose backbone gives these sugars the propensity to form helical structures while differing degrees of b1-6-glucose substitutions on their backbones give them variable solubility in


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water (Figure 6). For instance, pachyman from Poria cocos is essentially unsubstituted and is insoluble in water. In contrast, laminarin has approximately one substitution per 10 backbone sugars, which does not interfere with the ability of this polysaccharide to form a triple helix but does confer considerable solubility in water. Mixed linkage b-glucans are most commonly associated with grasses, cereals, and grains. As a common example, barley b-glucan comprises, on average, b1-4-glucotriose or b1-4-glucotetraose units linked b1-3. The frequency of b1-3-linkages can vary depending upon the source of the glucan. The alternating pattern of b1-4 and b1-3 linkages is thought to give these polysaccharides extended ribbon-like conformations.

3.29.4.4.2

Molecular determinants of b-glucan binding

CBMs that are specific for noncellulosic b-glucans are found in families 4, 6, 11, 13, 22, 39, and 43 (Table 1). The bestunderstood examples are those from families 4, 6, and 11, which are all type B CBMs. TmCBM4-2 from the Th. maritima Lam16 and BhCBM6 from the B. halodurans Lam81 are both specific for b1-3-glucans. Each have binding sites tailored to accommodate the helical conformation of b1-3-glucans but their architectures are uniquely different. TmCBM4-2 has a deep groove and the bottom of the ‘U-shape’ formed by the coil of the b1-3-glucan sits in the bottom of this groove (Figure 14).121 An array of aromatic amino acid side chains line the sides of the groove and provide the CH–p stacking interactions common to protein–carbohydrate interactions. Polar amino acid side chains make a number of hydrogen bonds with the sugar hydroxyl groups that extend away from the outer edge of the ‘U’. In contrast, BhCBM6 binds the nonreducing end of a b1-3-glucan chain in a small, blocked-off groove (Figure 14).122 The sugar curls around the module making contacts with a protein surface specifically contoured by appropriately structured loops. In this case, hydrogen-bonding interactions are made with the sugar hydroxyls on the inside of ‘U’ formed by the sugar. Though these CBMs recognize the same ligand they have evolved uniquely different methods of providing this specific interaction. Two CBMs are known to prefer mixed linkage b-glucans, the family 11 CBM, CtCBM11, from the mixed function lichenase/cellulase from Cl. thermocellum; and the family 6 CBM, CmCBM6, from the Cellvibrio mixtus endoglucanase 5A. CtCBM11 binds with a roughly two- to threefold preference for mixed b1-3,1-4-glucans versus b1-4-glucans but does not bind to b1-3-glucans.123 CmCBM6 has two binding clefts, A and B, which are both utilized in b-glucan binding (Figure 15a).32,33 Both cleft A and B in CmCBM6 can accommodate laminarin and cellooligosaccharides, whereas only cleft B binds to glucans with mixed b1-3,1-4-linkages such as lichenan and barley b-glucan.32 Analysis of a CmCBM6-b1-3,1-4-glucooligosaccharide complex by X-ray crystallography suggested that subsites 2 and 3 of cleft B specifically accommodated glucose residues linked b1-4 while subsite 4 required the glucose bound in this site to be b1-3-linked to the glucose in subsite 3 (Figure 15b).33 Subsite 1 displayed no preference for linkage.

(a)

(b)

Figure 14 Recognition of b1-3-glucans by two different CBMs. Panel a shows the C-terminal family 4 CBM from Thermotoga maritima Lam16 (PDB code 1GUI) while panel b shows the C-terminal family 6 CBM from B. halodurans Lam81 (PDB code 1W9W). The solvent accessible surfaces are shown with surface contributed by relevant aromatic amino acid side chains involved in binding, shown in purple. Bound b1-3-glucohexaose (laminariohexaose) molecules are shown in blue ‘licorice’ representations.

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1

2

3

4

R NR

3 OH

(a)

4 OH

(b)

Figure 15 b-Glucan recognition by the family 6 CBM, CmCBM6, from Cellvibrio mixtus. CmCBM6 has two binding sites located on different faces of the protein (a). Schematic showing the binding subsites in binding site B of CmCBM6 (b). The subsite numbering is indicated above. The reducing and nonreducing ends of the sugar are indicated by R and NR, respectively. Relevant hydroxyl groups that determine subsite specificity are indicated.

3.29.4.5 a-Glucans: The Premier Storage Polysaccharides 3.29.4.5.1 a-Glucan structures and sources Collectively, a-glucans are polysaccharides comprising polymerized glucose joined by a-glycosidic linkages. The most commonly studied a-glucans with respect to CBM research are starch, glycogen, and pullulan. Starch and glycogen are closely related polysaccharides that function as the primary storage carbohydrates in plants and animals, respectively. Starch is a composite of amylose and amylopectin, the former being a nearly pure form of a1-4-linked glucose and the latter comprising an a1-4-linked glucose backbone punctuated by a1-6 branch points occurring approximately every 24–30 glucose residues (Figure 6). Glycogen is essentially the mammalian form of amylopectin but has a1-6 branch points occurring approximately every 8–12 glucose residues along the backbone. Both starch and glycogen are typically found as granules that have a relatively well-organized superstructure. a1-4Linked glucose chains have a propensity to form double helices and adjacent helices can pack to form crystalline arrays giving rise to a dense polysaccharide granule. However, the a1-6 branch points disrupt this regular packing resulting in regions of the polysaccharide granule with less crystalline or more ‘amorphous’ properties. Like the cellulose-binding CBMs which must contend with structural heterogeneity, starch-binding CBMs also have to contend with multiple possible presentations of their ligands. Pullulan is a polysaccharide found as a cell wall component of the fungus Aureobasidium pullulans that can be described as a polymer a1-4-linked glucotriose joined by a1-6-linkages. Though this polymer is not a polysaccharide of major biological relevance, it is very important in industry and often serves as a model polysaccharide in a-glucanase research. Furthermore, enzymes that are apparently selective for this polysaccharide contain CBMs.

3.29.4.5.2 Molecular determinants of a-glucan recognition CBMs with specificity for starch, glycogen, and/or pullulan currently fall into the sequence-based families 20, 21, 25, 26, 34, and 41 (Table 1). However, given the number of enzymes with proven or putative a-glucanase activity and their content of modules with unknown function, it is highly likely that the number of families containing a-glucanbinding CBMs will continue to expand. Characterized members of all of these families share the ability to binding soluble a-glucooligosaccharides as well as the granular forms. However, association constants vary over three orders of magnitude between 103 and 106 M–1 depending on the solubility of the ligand or, if the ligand is an oligosaccharide, its length.124–127 3-D structures determined by NMR or X-ray crystallography are available for several members of family 20 (see, e.g., Ref. 128), and one or two members of families 25,127 26,127 and 34.129,130 The unifying feature of their structures is the presence of a twisted b-sandwich fold with an Ig-like topology (Figure 16).127 CBM–ligand complexes determined by X-ray crystallography ubiquitously reveal solvent-exposed aromatic amino acid side chains that form


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Figure 16 The overall secondary structure of the family 25 CBM from B. halodurans (PDB code 2C3V) as representative of the fold and topology of starch-specific CBMs.

Figure 17 The binding sites of starch-binding CBMs have a conserved curved architecture. Shown are the binding sites of CBM25 from B. halodurans (blue; PDB code 2C3V), CBM26 from B. halodurans (green; PDB code 2C3G), the family 34 CBM from T. vulgaris TVAI (magenta; PDB code 1UH2), and the family 20 CBM from B. circulans (strain 251) cylcodextrin glucanotransferase (yellow; PDB code 1CDG).

the carbohydrate-binding platforms (Figure 17). These protein–ligand complexes have been particularly informative in rationalizing how CBMs recognize granular starch or glycogen. As discussed above, a1-4-linked glucose chains selfassociate into double helices. In these helices, the a-faces of the glucopyranose rings face away from the core of the helix. Thus, in densely packed polysaccharide granules, the solvent-exposed surfaces comprising the a-faces of the glucose residues make the most likely recognition determinant. Indeed, the structures of CBMs in complex with amylose fragments, maltooligosaccharides, clearly show that these proteins interact with the convex a-face surface formed by the looping maltooligosaccharides (Figure 17).127 The dependence of these CBMs on recognizing a surface having a particular conformation is reminiscent of type A CBMs. However, the ability of this class of CBM to bind soluble sugars distinguishes them from type A CBMs. From a nonstructural perspective, the preference of most a-glucan-binding CBMs for soluble sugars containing three or more monosaccharide units suggests their classification as type B CBMs. Examination of their structures, however, reveals a maximum of three of subsites within their carbohydrate-binding sites and hydrogen bonding densities between the protein and ligand of roughly 2.8 hydrogen bonds per 100A˚2 of polar surface area buried upon complexation. This suggests properties similar to the type C CBMs. Thus, as a class, the a-glucan-binding CBMs are somewhat enigmatic with respect to the type A, B, and C classification, as they appear to have qualities particular to each type.127

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3.29.4.5.3 a-Glucans and multivalency: a role in starch disruption The family 20 starch-binding CBM from Aspergillus niger glucoamylase (GlaA) has been demonstrated to possess two functional maltooligosaccharide binding sites. Each site has relatively weak affinity for soluble ligands but appear to function cooperatively in binding granular starch.124 More remarkably, some nice work has established that these two binding sites work in concert to unravel the double helix of amylose, thus exposing the sugar chain for hydrolysis by the catalytic module.131,132 This function has only been demonstrated for this particular CBM and indeed it appears that not all family 20 CBMs possess two functional binding sites. Nevertheless, X-ray crystallography studies have also suggested two binding sites in family 25127 and 34 CBMs,130 though in the family 25 example the secondary binding site was suggested as non-binding with appreciable affinity to soluble sugars.127

3.29.4.6 Mannan and CBMs 3.29.4.6.1 Mannan structure Mannan is an insoluble homopolymer of b1-4-linked mannose residues found in the cell walls of plants (Figure 6). Heteropolymer derivatives of mannan are also found in nature, where the repeating sugar unit alternates between glucose and mannose to form glucomannan. Galactose is also commonly found linked to mannan and glucomannan polysaccharide backbones through a1-6 linkages to form galactomannan and galactoglucomannan, respectively. Galactomannan is mainly a structural polysaccharide and galactoglucomannan is commonly utilized for energy storage.

3.29.4.6.2 Molecular determinants of mannan binding Degradation of mannan results from the activity of bacterial and fungal enzymes found in glycoside hydrolase families 5 and 26 (CAZy). The following section will focus on the activity of selective type A (1, 2a, and 10) and type B CBM (23, 27, 29, and 35) families (Table 1).

3.29.4.6.2.1 Type A mannan-binding CBMs Most type A mannan-binding CBMs have been identified by sequence analysis. However, there are some examples of the biochemical characterization of purified modules. A family 1 CBM from an Oripinomyces sp. mannanase was shown to bind insoluble lignocellulosic substrates by depletion assays and affinity electrophoresis.133 In Cellvibrio japonicus, a cluster of GH5 mannanases (Man5A, Man5B, and Man5C) contain variable combinations of CBMs from families 2a, 5, and 10; interact with crystalline forms of mannan; and in some cases do not show selectivity between these substrates and insoluble cellulose.134 This observation may explain in part why there is an apparent lack of type A mannanbinding CBMs, as the stereochemical difference between glucose and mannose may not be selectively accessible in the crystalline forms of cellulose and mannan. 3.29.4.6.2.2 Type B mannan-binding CBMs The majority of mannan-specific CBMs fall into the type B class. This is not surprising as mannan is rarely found in nature in a homogeneous crystalline form. Commonly, mannan is substituted with a-1-6 linked decorations, which disrupt ordered packing and present many potential binding surfaces for type B CBMs. The first documented mannan-binding domain was discovered in the soil bacterium Cellulomonans fimi. 135 This module was classified within family 23 and does not have sequence similarity to any other protein in the database. The best-characterized mannan-binding CBMs fall into families 27, 29, and 35. Examples from these families will be discussed below.

3.29.4.6.3 CBM27: detailed analysis of mannan oligomer-specific recognition Family 27 is a small family of mannan-binding modules found in thermophilic bacteria.136,137 The binding energetics and crystallographic structures in complex with diverse ligands for two CBM27s have been recently published.138,139 CBM27 from the Th. maritima (TmCBM27) mannanse Man5 binds to oligomannans (d.p.¼M3–M5), carob galatomannan, and konjac glucomannan with association constants of 105–106 M–1, and substituted mannan derivatives with one- to twofold orders of magnitude less.138 Its counterpart from Caldicellulosiruptor, CsCBM27–1, displays even higher affinity for mannose oligomers, with an increasing relationship between polymerization length from mannotriose (Ka ¼2.0104 M–1) to mannohexaose (Ka ¼1.0107 M–1).139 In both cases, the interaction is enthapically driven with minor entropic penalties. In the case of mannohexaose, a unique binding model was observed for TmCBM27.138 In an entropically favorable event, a second CBM bound the same ligand with 100-fold less affinity to form a CBM1-ligand-CBM2 dimer. It was postulated that the entropic penalties for conformational restriction of the


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ligand were paid by the binding of CBM1, and the loss of ordered waters created a favorable situation for binding of CBM2. The biological relevance of this event remains to be determined; however, it is tempting to speculate that TmCBM27 may cluster the catalytic modules of Man5 on mannan substrates. The structure of CBM27 in both Th. maritima and Caldicellulosiruptor consists of a jelly-roll fold with a coordinated Ca2þ ion important for modular stability. Although there is only 22% sequence identity, they have a highly similar structural arrangement as 81 of 170 amino acids align with a main-chain rms deviation of 1.42A˚. Interestingly, the structure of TmCBM27 in complex with heterogeneous ligands provided some insight into the mechanism of ligand recognition.138 It is apparent that mannose selectivity provided by the preferential binding of axial 20 -hydroxls at subsites 2–4 (Figure 18). TmCBM27 can accomodate a1-6-linked substitutions within the binding cleft at subsites 1, 2, and 5 because the C6-OH groups at these positions are directed towards the solvent. This observation provides a molecular and biochemical explanation for how TmCBM27 can recognize substituted mannan and target glycoside hydrolases to these heterogeneous polysaccharides within the cell wall. N

NH2

Arg64

NH2 5 4 OH HO O HO O O O HO OH HO

HO HO HO

O

NH2

6

HO HO

O

O O HO OH

OH

Trp24 O

O

H2N N H

6

HO

HO

O O HO OH

1

OH O

HO O

OH

OH

OH

O OH O

NH3

NH2

O

Ala118

O Lys74

Glu116

O O HO OH

4 OH O HO O

Trp24

HO

3

O NH2

H2N HN

Glu78

1

2 HO

O O HO OH

NH2

O

O

(c)

2 OH

NH2 Glu78

5 OH O HO O

N H

Arg95

HN Arg112

(b)

HO HO HO

HO O

N H

Asp59

OH

OH

O HO O OH

OH

NH

O

3

4

OH O

H2 N

O

O

Lys106

5

OH

HO

O Asp110

Glu62

1

O O HO OH

HO

O (a)

2

3 OH O HO O

OH O

HO NH3

OH

O OH O Ala118

O Glu116

HO O

Lys74

Arg112

Figure 18 Schematics of the hydrogen-bonding patterns of mannan-binding CBMs reveal the use of C2-OH groups as specificity determinants. Panel a shows the hydrogen bonding pattern between mannopentaose and the family 27 CBM from Thermotoga maritima Man5. Panels b and c show the hydrogen-bonding patterns between cellohexaose and mannopentaose, respectively, and the family 29 CBM from Piromyces equi.

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3.29.4.6.4 A special case: the cooperative and synergistic effects of tandem CBM29s Although CBM29s have only been found in the fungus Piromyces equi, it is a fascinating example of the coordinated process of plant cell wall degradation. Clostridial cellulosomes contain type A CBMs from family 3 (see above); however, in this case, the hydrolytic machinery is directed to mannan and mannan-containing derivatives by noncatalytic protein-1 (NCP1).140 This protein is composed of three N-terminal dockerin modules and two closely related C-terminal CBM29s. It has been suggested that this system provides an alternative mechanism for polysaccharide degradation, as promiscuous manno-derivative binding may drive the recruitment of cellulases and hemicellulases through dockerin–cohesin interactions.140 Affinity gel electrophoresis demonstrated that CBM29-1 and CBM29-2 both bind primarily glucomannan; and with less affinity b-mannan, b-glucan, and cellulose.140 Interestingly, when analyzed by ITC, CBM291-2 displayed a 4–45 fold increase in affinity for the same ligands, which is well above a simple additive effect. This observation suggests that with these tandem CBMs there is a synergistic effect in polysaccharide recognition. The structure of CBM29-2 in complex with cello- and mannohexose was recently determined and provides a molecular explanation for the binding of diverse ligands.141 Subsites 5, 4, and 3 favor binding mannose over glucose due to stereospecific hydrogen bond contacts with the C2 of mannose (Figure 18). Additionally sugars at positions 5, 3, and 1 have C6-hydroxymethyl groups oriented toward the surface of and in hydrogen-bond contact with CBM29–2. In agreement with what was observed for family 27 CBMs, the C6–O6 groups of mannosyl residues 6, 4, and 2 are directed towards the solvent which suggests that this binding architecture would accommodate a heterogeneously substituted mannan derivative, such as galactomannan.141 This ‘flexible’ ligand recognition may specialize the targeting of fungal CBMs towards diverse polysaccharide substrates within the plant cell wall.

3.29.4.6.5 A dynamic event: CBM35 undergoes conformational changes upon binding CBM35s have been found appended to xylanases, cellulases, and mannans (CAZy); however, this family shows weak homology to the type B CBM6s.142 Recently, the solution structure for CBM35 (formerly X4) from Cellvibrio japonicus CjCBM35 was recently published, which provided some provocative insights into the binding mechanism of this family.117 This module is appended to an N-terminal CBM10 and CBM5, and the C-terminal mannase Man5C catalytic module.142 In contrast to the majority of research accumulated to date, which has indicated for the most part that CBMs are structurally rigid, CjCBM35 undergoes noticeable changes in conformation upon ligand binding.117 The main rearrangements occur in the loop containing amino acids 80–84, which widens the binding groove to accommodate oligomannans. Binding is driven by CH–p interactions between aromatic side chains (Tyr60, Trp109, and Tyr111) and mannosyl faces, and hydrogen-bonds between Lys63 and the axial C2-hydroxyl of mannose as well as several others. The authors also used sequence comparisons to successfully identify several other members in this family. Biophysical analysis of CBM–polysaccharide interactions will clarify whether conformational rearrangement upon binding is a conserved phenomenon for this family as calcium dependence has been reported for a CBM35 from a arabinofuranosidase.117

3.29.4.7 CBMs with Unique Polysaccharide Binding Specificities The CBM binding specificities covered to this point represent the major classes of polysaccharides recognized by CBMs. However, there are a number of less-common polyaccharides or components of polysaccharides that are recognized by some CBMs. These are b1-3-galactan, mutan, inulin, b1-3-xylan and arabinose (see Table 1). Phanerochaete chrysosporium produces a b1-3-galactan-specific family 43 glycoside hydrolase.143 Appended to this enzyme is a CBM which, though published as a family 6 CBM, is formally classified as a CBM35 (this highlights the similarities between family 6 and 35). This CBM has been shown to be b1-3-galactan specific. Similarily, the fungus Penicillium purpurogenum produces an a1-3-glucan (mutan)-specific family 71 glycoside hydrolase having a family 24 CBM specific for this unique glucan.144 A family 38 b1-2-linked polyfructose (inulin)-binding module was found at the C-terminus of a Bacillus cycloinulinooligosaccharide fructanotransferase.145 The structural basis of the interactions of these CBMs with these unique polysaccharides remains unknown. b1-3-Xylan is a polymer of xylose found primarily in the cell walls of red and green marine algae. Marine bacteria, specifically of the genera Vibrio, Alcaligenes, and Pseudomonas, produce xylanases specific for this insoluble polysaccharide. Perhaps not surprisingly, these enzymes contain family 31 CBMs that potentiate their activity on this sugar.146 The recent X-ray crystal structure of an Alcaligenes CBM31 revealed a b-sandwich Ig-like fold similar to that of the starch specific CBMs.147 Though this structure is known, the molecular basis for the interaction remains to be determined.


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Aspergillus kawachii produces a family 54 a-L-arabinofuranosidase with an intriguing C-terminal family 42 CBM.148 This CBM was identified in the X-ray crystal structure of this enzyme and identified as a CBM by its ability to bind arabinose in the crystal. The fold of this module is a b-trefoil, like the family 13 CBMs, but it shows no sequence identity with this family, thus its classification into family 42. This enzyme is responsible for removing the arabinofuranose decorations from arabino-xylan.148 Given the apparent specificity of this CBM, we are left with the tantalizing suggestion that this CBM42 is responsible for recognizing the arabinose side chains, thereby localizing the enzyme to regions rich in decorations. In most cases, CBMs have developed a tolerance for side-chain decorations while recognizing the polysaccharide backbone. It appears that CBM42 is truly unique in its specificity for the decorations.

3.29.5 Interactions with Complex Glycans A number of CBMs are known to bind to complex glycans such as those found on eukaryotic glycoproteins or glycolipids. These CBMs fall into three families, 13, 32, and 40, and are involved in performing three general functions: oligosaccharide synthesis, protein toxin delivery, and host–microbe interactions.

3.29.5.1 Oligosaccharide Synthesis The function of CBMs is not limited to roles in catabolism. A relatively recent and intriguing study revealed the role of CBMs appended to glycosyltransferases in anabolic processes. O-Glycosylation of mucin is stepwise149 and first begins through the transfer of a GalNAc activated with UDP to a serine or threonine residue of the core mucin protein. This reaction is catalyzed by UDP-GalNAc:polypeptide a-N-acetylgalactosaminyltransferases (GalNAc transferases) from family 27 of the retaining glycosyltransferases, a large family of homologous enzymes.150,151 Many family 27 glycosyltransferases have C-terminal family 13 CBMs, CBMs related to RTB. Different members of the GalNAc transferases have different kinetic properties and glycosylate different sites on the core protein.152–155 There is also differential expression of some isoforms in human tissue.154–156 Individual GalNAc transferases have independent activities that may complement each other based on expression patterns. For example, human GalNAc-T7 recognized incompletely glycosylated tandem repeats of human MUC2 and rat submaxillary gland mucin as acceptor substrates but did not show activity on nonglycosylated substrates.157 Functional studies on the CBM13 domain of GalNAc-T4 demonstrated that mutation of specific residues in the CBM13 module blocked O-glycan attachment to two specific Muc1 tandem repeats on a partially glycosylated peptide acceptor. Glycosylation of nonglycosylated peptides was not affected.158 It appears that O-glycosylation initiation is regulated by a multiple GalNAc transferases and recognition of the partially glycosylated polypeptide substrates is mediated by the family 13 CBM.158 The crystal structure of the murine ppGalNAc-T glycosyltransferase revealed its bimodular structure comprising the family 13 CBM and the family 27 glycosyltransferase module (Figure 19).159 Thus far, the family 27 GalNAc transferases are the sole glycosyltransferases shown to contain CBMs.160

Figure 19 A ‘cartoon’ representing the structure of the family 27 glycosyltransferase from Mus musculus (PDB code 1XHB). The catalytic module is shown in blue. The family 13 CBM is shown in green looking down the pseudo-3-fold axis of the b-trefoil fold.

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3.29.5.2 Toxin Delivery CBMs play roles in the delivery of three general types of toxins: the ribosome-inactivating protein (RIP) toxins, poreforming toxins, and botulinum neurotoxins. These toxins are not carbohydrate-active enzymes and by strict definition the modules responsible for carbohydrate binding in these proteins would not be classified as CBMs. However, in these cases discussed these modules show sequence identity with family 13 CBMs. Thus, by the liberal definition of CBM (see above in Section 3.29.1) these modules are included as CBMs. The protein toxin ricin is a potent RIP derived from castor bean seeds, R. communis. The ricin toxin itself consists of two individual polypeptides: the toxic ricin A chain linked covalently through a disulfide bond to the carbohydratebinding ricin B chain, whose two domains are classified as family 13 CBMs. The B subunit targets ricin to the surface of the cell through recognition of galactose and N-acetylgalactosamine161,162 whereupon ricin is endocytosed, traveling through the internal membrane system to reach the ER. In the ER the disulfide bond linking the two polypeptides is reduced and the ricin A chain is translocated across the membrane into the cytosol. The ricin A chain interacts specifically with the large ribosomal subunit, and through its N-glycosidase activity hydrolyses an N-glycosidic bond between an adenine residue and a ribose in the 23S RNA. This inactivates the ribosome through depurination and destruction of protein synthesis163–165 A single Ricin A chain is able to inactivate many ribosomes contributing to its cytotoxicity. Other plants, such as barley, produce only the A chain which has ribosome-inactivating function; however, due to the absence of the B chain it is not cytotoxic.166 The B chain, that is, family 13 CBMs, are essential for cytotoxicity as without them ricin would not bind the cell and would not be internalized. Numerous RIPs, including those from Sambucus nigra, Viscum album, and Abrus precatorius, contain family 13 CBMs in their A/B toxin structure. The pore-forming, hemagglutinating, hemolytic CEL-III protein produced by the sea cucumber Cucumaria echinata contains two N-terminal family 13 CBMs.167–170 The C-terminal domain consists of a b-sandwich with two alpha helices that appear to potentiate the oligomerization function of CEL-III.171,172 Five of the six a, b, and g subdomains of the family 13 CBMs bind Ca2þ although it has not been established whether Ca2þ plays a role in carbohydrate binding. CEL-III binds cells through protein–carbohydrate interactions upon which it undergoes oligomerization to form pores within the cell membranes. The N-terminal family 13 CBMs are thought to provide the galactose/GalNAcbinding function of this protein172 and it has been proposed that it is the recognition of carbohydrate that promotes exposure of hydrophobic surface area,173 forcing oligomerization and pore formation in the cell membrane. Cl. botulinum is an organism that secretes one of the most deadly neurotoxins known to man, the botulinum neurotoxin (BoNT) of which there are currently seven known serotypes (A–G).174 It is estimated that the human lethal dose is 0.1 ng kg–1.175 Exposure to toxic forms prevents acetylcholine release at the neuromuscular junction resulting in flaccid muscle paralysis.176–178 This toxin is found associated with nontoxic proteins and the complexes are referred to as progenitor toxins. The associated proteins act to protect BoNT in the acidic environment in the stomach and from proteolytic attack.179–182 Nontoxic hemagglutinating proteins (HAs), which are classified into the family 13 CBMs, are often associated with BoNT.183,184 The HA-positive progenitor toxins have demonstrated interactions with complex glycans found in the intestinal microvilli. HA of serotype C binds glycolipids and glycoproteins having sialic acid residues.185,186 HA of serotype A binds glycolipids and glycoproteins containing Galb1-4GlcNAc (N-acetyllactosamine).187,188 The interactions with the oligosaccharides are likely involved in internalization of BoNT into the body in the intestines.189 The unifying feature of CBMs found in toxins is their function in delivering the toxin component of the protein to cell surfaces via a carbohydrate receptor–CBM interaction. This allows toxin internalization or insertion into the membrane, steps which are critical to the toxic activity of these proteins.

3.29.5.3 Host–Microbe Interactions Virulent strains of Vibrio cholera produce a sialidase responsible for cleaving terminal sialic acid moieties from gangliosides in order to expose GM1, the cholera toxin receptor.190 Structurally the Vibrio cholera sialidase is composed of a central six bladed b-propellor catalytic domain flanked by b-sandwich lectin-like ‘wing’ domains. Recent discovery that the N-terminal ‘wing’ domain binds sialic acid resulted in the classification of these domains as family 40 CBMs191 (Figure 20). CBM40–sialic acid interactions occur on the curved b-sheet face of the CBM through 11 direct and water-mediated interactions. A unique feature of this interaction is that there are no aromatic amino acid side chains involved in the interaction as is common to protein–carbohydrate interactions. The role of the CBM is likely to concentrate the enzyme onto sialic acid-rich surfaces in the intestine where the action of the enzyme then unmasks receptors for toxin binding.


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Figure 20 A ‘cartoon’ representing the structure of the family 33 glycoside hydrolase from Vibrio cholerae (PDB code 1W0P). The catalytic module is shown in blue. The family 40 CBMs are shown in green with a bound sialic acid molecule shown in blue ‘licorice’ representation.

The family 32 CBMs are possibly involved in a number of host–microbe interactions. For instance, members of this family have been identified by bioinformatic methods in the m-toxin, a sialidase, and other putative glycoside hydrolase toxins secreted by Cl. perfringens, a bacterium known to cause food poisoning and gas gangrene. The m-toxin is thought to be a hyaluronidase involved in tissue destruction and bacterial spread. Other glycoside hydrolases may be involved in sugar hydrolysis to aid in host colonization and/or bacterial nutrition. The role of the family 32 CBMs is likely to promote adherence of the enzymes to sugar-bearing tissues. Other commensal gut organisms, such as Bacteroides thetaiotaomicron, harbor a different battery of enzymes containing CBM32s. These enzymes likely play a role in breaking down the complex carbohydrates in the human diet, suggesting a different role for CBM32-containing enzymes in host–microbe interactions.

3.29.5.4 Implications for the Recognition of Complex Glycans by CBMs The functions of CBMs in protein glycosylation, toxin binding, and host–microbe interactions, through binding complex glycans are being uncovered only now. However, the results suggest that CBMs involved in these processes may eventually turn out to be targets for carbohydrate-based therapeutics. Antiadhesive drugs targeting the carbohydrate-binding functions of the ‘shiga-like’ toxins are already being explored, suggesting this approach may be viable. Antiadhesive carbohydrate-based therapeutics in combination with inhibitors of catalytic activity may also have potential in combating the action of glycoside hydrolases in infections caused by bacteria. This is one of the exciting new frontiers in CBM research.

3.29.6 CBMs in Biotechnological Applications Interest in CBMs as a biotechnological tool developed early on in CBM research due to the affordability of cellulose as an adsorbent. Furthermore, the use of CBMs is advantageous as they typically do not exert any modifications on substrates or on the proteins to which they may be fused. Applications for their use may be dependent on their affinity for the target carbohydrate; some CBMs have considerably high affinity (TmCBM9–2 106 M–1) 28 while others have very low affinities (MvCBM32 103 M–1).30 CBMs are easily manipulated to generate tandem repeats to create a macromolecule with dual specificity192 or even to suit recombinant production in other hosts.193 Biotechnological applications have exploited the native properties of CBMs in the absence of a catalytic partner as well as the properties of chimeric proteins created by fusing CBMs to partners with desired functions.

3.29.6.1 Exploiting the Native Properties of CBMs 3.29.6.1.1

CBMs and fiber modification

CBMs have been demonstrated to strengthen bound cellulose fibers, creating a more tensile and robust paper base.194 Cellulose-binding CBM-based additives are also used to enhance paper strength and resistance.195 Two CBMs, the cellulose-binding CcCBM17 from Cl. cellulovorans and starch binding AnCBM20 from Aspergillis niger, were

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fused by an elastin-like linker. The resultant cellulose/starch cross bridging protein (CSCP) was initially bound to microcrystalline cellulose and subsequently mixed with solublized cornstarch and made into paper sheets. The mechanical strength and durability of the various starch-containing paper samples significantly increased the tensile strength of paper using CSCP.193 The success of CSCP can be attributed to the CBM’s affinity for linear polymers through their type B binding sites. This application has become more desirable due to environmental and forest management pressures to use renewable resources in paper manufacturing. Scouring is the treatment through which textiles, such as wool, undergo to remove any dirt, oils, waxes, or other contaminants. Pressure is on the textile industry to develop alternative and environmentally safe methods for scouring. Degani et al. developed a recombinant CBM fused with a b-glucuronidase reporter (CBD-GUS) to test for scouring efficiency.196 The chimeric enzyme was used as an effective tool for measuring scouring and wettability of cotton fabrics. CBD-GUS binding increased with hydrophilicity of the fabric due to the mounting harshness of the scouring treatment. This has led to the development of ‘bioscouring’ using extracellular degradative enzymes, such as pectinases and a cutinase from the bacterium Pseudomonas mandocino,197 fused to CBMs to target the enzymes to the fiber for increased scouring efficiency.

3.29.6.1.2 Modified CBMs aid in plant growth Extension of the fiber matrix in the plant cell wall during growth and differentiation is a necessary part of plant development. This is achieved with the use of various proteins to loosen the fibers, making them accessible for addition of new cell wall polymer.198 Swollenin from H. jecorina is involved in separating cellulose fibrils during cell wall expansion and cell growth and thus may be used in enhancing substrate accessibility. CBMs for these enzymes may therefore be manipulated for industrial processes to open up or swell targeted plant polymers.199 The exogenous addition of cellulose-binding CcCBM3a from Cl. cellulovorans CipA to Arabidopsis seeds through soaking increased the rate of synthesis of cellulose, causing elongated roots in seedlings. This enhanced plant growth resulted in splayed cellulose ribbons but did not affect the hypocotyls.200

3.29.6.1.3 CBMs as diagnostic tools CBMs can be utilized for detecting polysaccharide content in plants and microorganisms. Immunofluorescently labeled cellulose-binding TrCBM1 and 2 from H. jecorina cellobiohydrolase I and II (Cel6A) were used to target the protozoan cyst Acanthamoeba which contains cellulose in the cyst wall.201 Acanthamoeba act as carriers of bacterial pathogens such as Legionella pneumophila and thus diagnosing the presence of Acanthamoeba may aid in preventing the spread of pathogens. The CBMs were able to recognize Acanthamoeba cyst wall cellulose and did not bind the chitincontaining cyst walls of other protozoans such as Giardia intestinalis, thus distinguishing between two types of cysts. Other CBMs used as diagnostic tools include SoCBM33 from Streptomyces olivaceoviridis which was used to detect a-chitin present between budding mother and daughter cells of Saccharomyces cerevisiae.202 McCartney et al. utilized various plant cell wall-binding CBMs (Pyromyces equi mannanase PeCBM29, Cl. thermocellum xylanase CtCBM6, and a Cellvibrio japonicus cellulase CjCBM2a) for determining polysaccharide content in developing maize coleoptiles.203 Immunofluorescent anti-His6 antibodies bound to His6-tagged CBMs revealed CBMs localized to their corresponding plant cell wall ligands, such as xyloglucan and cellulose, thus revealing polysaccharide location and composition in maize leaves and roots.

3.29.6.1.4 CBMs as molecular scaffolds A molecular scaffold serves as a model for developing random mutation libraries of a protein active site and testing each member in hopes of the mutation changing the overall function of the protein. Modification of CBMs may make it possible to engineer binders for enhancement of drug design systems or for environmental purposes. Thermostable xylan-specific RmCBM4–2 from xylanase 10A of Rhodothermus marinus was successfully utilized as a molecular scaffold.204,205 A library of 1.6106 clones yielded selected variants with mutationally developed specificities from birchwood xylan to ivory nut mannan, and human IgG4. The different specificities were correlated with amino acid differences in the CBM-binding site. H. jecorina Cel7A TrCBM1 modification yielded a mutant that was able to bind alkaline phosphatase through protein–protein interactions.206 Metal-binding sites have also been engineered from CBM1 of Tr. reseei Cel7A with combinatorial variants that are surface displayed on Staphylococcus carnosus cells.207 Eight TrCBM1 mutational variants were evaluated for nickel-ion binding on Ni2þ-magnetic agarose bead. Two variants showed a significantly enhanced nickel-binding capacity over the parental strain. Such tailor-made bacterial strains could one day be engineered as bioadsorbants for water filtration to capture heavy metals, such as mercury.


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3.29.6.2 CBM Fusion Proteins 3.29.6.2.1

CBMs as affinity tags

For purification purposes in the pharmaceutical and biotechnological industries, which require highly homogenous protein preparations, it can be beneficial to have an affinity tag for effective, single-step protein purification. The use of CBMs as affinity tags has been successfully applied for many purification procedures.106,118,212–214 The most commonly used affinity matrix for this purpose is cellulose as it is easily available, inexpensive, chemically inert, and nontoxic. CBMs are practical affinity tags as they have strong specific binding capabilities while remaining catalytically inactive; due to their small size, 30–200 amino acids, CBMs generally do not sterically interfere with the biological activity of the target protein. They are often stable over long periods of time and over a wide pH range, and CBMs that are thermostable up to 90 C are available.208,209 Elution of fused proteins in a polysaccharide column matrix is dependent on the affinity of the CBM for the matrix. Weakly bound CBMs can be eluted with monosaccharide ligand. More strongly bound CBMs may require denaturation for elution or, in turn, the immobilization on the matrix may itself be the purification step.210 Removal of the CBM affinity tag after purification may occur via an engineered protease cleavage site such as thrombin, enterokinase, and factor Xa as well as a site for chemical cleavage,211 with easy removal of the contaminating CBM with a final pass of the cleaved protein preparation over the polysaccharide column. Naturally occurring CBMs within glycoside hydrolase enzymes can be exploited for affinity purification. The xylanase SoXyn10A from Streptomyces olivaceoviridis has a C-terminal type C family 13 CBM.215 In a simple one-step purification procedure on lactosyl-sepharose column, highly pure recombinant SoXyn10A was obtained.216 CBMs as affinity tags can increase the yield of the fused protein,217,218 increase or decrease solubility,200 and even decrease aggregation upon refolding after denaturation.219 CBMs may protect against proteolytic degradation and provide protection again thermal denaturation.220 Packaging into inclusion bodies occasionally occurs upon expression of CBM fusions.218 This may be advantageous as inclusion bodies are easily purified and in this insoluble state are protected from proteolysis.221

3.29.6.2.2

CBMs as immobilization tools

Immobilization tools are used to immobilize a given substrate on to a solid matrix. This process may be used for targeting an enzyme to substrate, for immobilizing bacteria,222 for cross-linking purposes,193 and even to bind vaccine to adjuvant.223 CBMs can be used as immobilization tools by fusing them to target proteins. CBMs can be fused to the target proteins in tandems of two or more producing a cooperative effect.60,224 An example of an immobilized chimera involves the fusion of cellulose-specific CfCBM2a from Xyn10A of Cellulomonas fimi to the N-terminus of the light chain of the protease Factor Xa containing a self-activation site.217 The fusion resulted in a stable, self-activating, cellulose-binding construct that could be immobilized on cellulose and retained similar activity as wild-type Factor Xa. Immobilized CBMs have potential environmental applications such as detoxification of wastewater.218 The cellulose-binding CcCBM3 from Cl. cellulovorans CipA scaffoldin was fused to horseradish peroxidase (HRP). Once immobilized to cellulose, CcCBM3-HRP was stable and showed increased longevity in the presence of harmful peroxide oxidants, while the cellulose scaffold adsorbed products of the oxidation reaction. When HRP oxidation of 4-bromophenol, a model toxic phenol pollutant, was monitored, the immobilized CcCBM3-HRP oxidized more 4-bromophenol than unbound CcCBM3-HRP. CBM fusions show potential for use in the economical treatment of wastewater toxic phenols as peroxidase stability in the presence of peroxide is a concern that forces constant availability of new and active enzyme.218

3.29.6.2.3

CBMs used to enhance enzyme efficiency

Recombinant fusions of glycoside hydrolases to a CBM for targeting to substrate may result in increased catalytic efficiency.225 In industry, CBMs have even been appended to catalytic modules other than glycoside hydrolases, such as lipases, in order to direct the enzyme to the desired target site: for example, directing lipases to cotton fibers in laundry detergents.226,227 By appending a CBM to an enzyme, a reduced amount of enzyme is required which is more economically viable in terms of enzyme production for industry. Xylanases are of importance for the pulp and paper industry for the enzymatic degradation of hemicellulose.228 Kittur et al. fused the xylan-binding type B CBM2b STX-II of Streptococcus thermoviolaceus 225,229 to the catalytic C-terminus of XynB from Th. maritima. Both of these modules are from thermostable organisms and the

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CBM2b–XynB fusion protein was heat stable up to 90 C and was stable at 70 C over a pH range of 5–11. The fusion significantly increased the activity of XynB toward soluble birchwood xylan relative to wild type. The thermostability of the chimeric xylanase makes it optimal for use in the pulp and paper industry where high temperatures are often employed.

Glossary amylopectin A polymer of a1-4-linked glucose with a1-6-linked branches found as a component of starch. amylose A polymer of a1-4-linked glucose found as a component of starch. carbohydrate-binding module (CBM) Found appended to an enzyme as a distinct entity with its own fold and function; facilitates targeting of the enzymatic module on to its substrate. cellulose A polymer of b1-4-linked glucose found predominantly as a structural polysaccharide in cell wall of plants; usually insoluble and often crystalline. cellulosome A large complex of noncovalently linked carbohydrate-active enzymes, usually targeting cellulose. chitin A polymer of b1-4-linked N-acetylglucosamine found predominantly as a structural polysaccharide in insect exoskeletons; usually insoluble and often crystalline. glycogen Mammalian amylopectin but with a higher degree of branching; used for energy storage. glycoside hydrolase Enzyme that degrades carbohydrates by addition of water to glycosidic bond within a polysaccharide chain or between a carbohydrate and noncarbohydrate moiety, for example, cellulase, xylanase, and hyaluronidase. glycosyltranferase Enzyme that adds a sugar via UDP-sugar substrate on to a carbohydrate chain, releasing water. DH, DS, DCp, and DG Changes in enthalpy, entropy, heat capacity, and free energy, respectively. mannan A polymer of b1-4-linked mannose. starch A composite of amylose and amylopectin. xylan A polymer of xylose, usually b1-4-linked as found in plant cell walls but sometimes b1-3-linked as in algal xylan. Often decorated with arabinose or glucuronic acid side chains.

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Biographical Sketch

Alisdair B. Boraston obtained his Ph.D. in 2000 at the University of British Columbia (UBC), Vancouver, BC, Canada. Following this he did post-doctoral fellowships at UBC and then the University of York, United Kingdom, with Professor Gideon Davies. In 2003 he assumed his current position as an assistant professor in the Department of Biochemistry and Microbiology at the University of Victoria, Victoria, BC, Canada. He presently holds a Tier II Canada Research Chair in Molecular Interactions. His research focuses onthe molecular determinants of protein–carbohydrate interactions with a focus on carbohydrate-binding modules found in carbohydrate-active enzymes.

Alicia Lammerts van Bueren is a Ph.D. candidate at the University of Victoria, Victoria, BC, Canada, in the Department of Biochemistry and Microbiology. She did her Bachelor of Science degree in biochemistry at the University of Victoria. Alicia currently studies the carbohydrate interactions of streptococcal species with host glycans and thermodynamic properties of CBM interactions with plant cell wall polysaccharides.

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Wade Abbott graduated with his Ph.D. in Biochemistry from the University of Victoria in 2005. His research focused on how histone H2A variants exert function on chromatin structure through alteration of nucleosome stability and chromatin fiber folding. His thesis received notable acclaim and was awarded the 2006 University of Victoria Gold Medal and nominated for the NSERC Silver Medal. Currently, he is working as a post-doctoral research fellow in the Boraston lab investigating the structural biology and thermodynamics of pectin degradation in Yersinia enterocolitica.

Elizabeth Ficko-Blean is a Ph.D. candidate at the University of Victoria, Victoria, BC, Canada, in the Department of Biochemistry and Microbiology. She did her Bachelor of Science Honours degree in biochemistry at the University of British Columbia, Vancouver, BC, Canada. She is currently researching the glycoside hydrolase exotoxins of pathogenic Clostridia, particularly the molecular aspects of their protein–carbohydrate interactions.

3.30 Lipid Membrane Domains in Glycobiology A. Prinetti, L. Mauri, V. Chigorno, and S. Sonnino, University of Milan, Milan, Italy ß 2007 Elsevier Ltd. All rights reserved. 3.30.1 Membrane Lipids

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3.30.2 Structural and Chemicophysical Properties of Glycosphingolipids Leading to Lipid Segregation

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3.30.2.1 The Hydrophilic Head Group of Gangliosides

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3.30.2.2 The Lipid–Water Interface of Gangliosides

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3.30.2.3 The Hydrophobic Chains of Gangliosides

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3.30.3 Segregation of Membrane Lipids

704

3.30.3.1 Lipid Membrane Domains in Artificial Membranes

705

3.30.3.2 Detergent Insolubility and Lipid Membrane Domains

706

3.30.3.3 Preparation of Cellular Fractions Enriched in Lipid Membrane Domains

708

3.30.3.4 Preparation of Lipid Membrane Domains from Tissues

709

3.30.3.5 Isolation of Heterogeneous Membrane Subdomains

710

3.30.3.6 Lipid Membrane Domains in Natural Membranes

711

3.30.3.7 Lipid Membrane Domains, Caveolae and Caveolins, Noncaveolar Domains

711

3.30.3.8 Lipid Membrane Domains from Intracellular Membranes

713

3.30.3.9 Artificial Manipulation of Lipid Membrane Domain Composition

714

3.30.3.9.1 3.30.3.9.2

Drugs affecting cholesterol availability Drugs affecting sphingolipid metabolism

714 716

3.30.4 Roles of Glycosphingolipids in Lipid Membrane Domain Functions

717

3.30.4.1 Glycosphingolipids and Lipid Membrane Domains in the Nervous System

717

3.30.4.2 Lipid Membrane Domains in Oncogenic Transformation: GM3 and the Glycosynapse

720

3.30.5 Perspectives, Critiques and Conclusions

722

3.30.1 Membrane Lipids Glycerophospholipids, sphingolipids, and cholesterol are the lipid components of cell membranes. Among these, sphingolipids are minor components. They mainly reside in the external layer of the plasma membrane,3 with the hydrophilic head group protruding toward the extracellular environment. Glycosphingolipids are components of all animal cell membranes and, among these, gangliosides, that contain sialic acid residues, are particularly abundant in the plasma membranes of neurons. In 1935, E. Klenk extracted from the brain of a Niemann–Pick disease patient something new, that he named ‘substance X’.4 In the following years, he understood5 that ‘substance X’ was a mixture of different compounds that he named ‘gangliosides’. Gangliosides attracted immediately the interest of many investigators, but in spite of this, progresses in elucidating their structures were slow (see Chapter 1.03). In 1947, the structure of sphingosine was elucidated,6 and in 1955 that of sialic acid.7 Finally, in 1963, the first ganglioside structure was described.8 Then, studies were mainly devoted to understand ganglioside biological functions. This research is still far to be considered exhausted, but today there is a general agreement to consider gangliosides as functional molecules involved in modulation of enzyme properties, of cell signaling, cell adhesion, and protein sorting (see Chapter 4.15).9–28 The lipid moiety of glycosphingolipids, as well as that of all sphingolipids, is named ceramide29 and is constituted by a long-chain amino alcohol, 2-amino-1,3-dihydroxy-octadec-4-ene, whose trivial name is sphingosine, linked to a fatty acid by an amide bond. Of the four possible configurations of sphingosine, only the 2S,3R is present in nature.6,30 ‘Sphingosine’ identifies also structures with shorter and longer alkyl chain and structures with and without unsaturation (whose names should be sphingenine and sphinganine, respectively).

697

698

Lipid Membrane Domains in Glycobiology

The hydrophilic head group of sphingolipids is phosphocholine in the case of sphingomyelin (SM, the only known phosphosphingolipid in mammalian cells), or an oligosaccharide chain in the case of glycosphingolipids. The oligosaccharide chain of gangliosides is very variable due to the sugar structure, content, sequence, and connections. This, together with the heterogeneity of the lipid moiety, makes gangliosides a very large group of compounds. Table 1 shows several ganglioside structures together with the trivial and correct abbreviation.412,413 Sialic acid is the sugar that differentiates gangliosides from neutral glycosphingolipids and sulfatides. Sialic acid31 is the name that identifies all the derivatives of 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, or neuraminic acid. Three main sialic acids are known: the 5-N-acetyl-, the 5-N-acetyl-9-O-acetyl-, and the 5-N-glycolylderivative. Healthy humans have only the first two.32–35 Gangliosides containing polysialyl chains where the sialic acids are linked together with ketosidic and ester linkage (ganglioside lactones) have been found in human brains.36

3.30.2 Structural and Chemicophysical Properties of Glycosphingolipids Leading to Lipid Segregation On the basis of the lipid molar composition of the detergent-resistant membrane (DRM) fraction putatively representing isolated lipid membrane domains (discussed in Section 3.30.4.2), lipid membrane domains are membrane districts highly enriched in SM, glycosphingolipids, cholesterol, and dipalmitoylphosphatidylcholine that should cover roughly 10–20% of the cell surface.37 On the other hand, lipid membrane domains contain less than 3% of the total cell proteins. Thus, it can be easily predicted that the chemical and physicochemical properties of membrane lipids are the driving forces governing the existence and organization of lipid membrane domains. In the following paragraphs, we discuss these properties, taking in account the geometrical properties of hydrophilic head groups of membrane lipids, the transition temperature of membrane lipids, the hydrogen bond network at the lipid–water interface, the side-byside oligosaccharide interactions and the carbohydrate–water interactions, and the properties of the hydrophobic tails, as driving forces to segregation phenomena.

3.30.2.1 The Hydrophilic Head Group of Gangliosides Gangliosides are a very heterogeneous family of compounds with different content of sugar residues (see Table 1). With the progressive increasing of the head group complexity, the molecules inside the membrane require a progressively larger interfacial area to host the hydrophilic head group. Clustering of some components in a membrane system is favored when the components show large differences in the geometrical characteristics of their head groups, clustering being a spontaneous process due to minimization of the interfacial free energy. This is the case of gangliosides inserted in a glycerophospholipid surface. The larger is the interfacial area required by ganglioside oligosaccharide structure, the more positive is the membrane curvature and the more pronounced is the segregation. Figure 1 shows the volume occupied by the pentasaccharide chain of ganglioside GM1,38 in comparison with that occupied by phosphocholine, the largest group of phospholipids. The volumes were determined combining all the minimum energy of possible conformers. Ganglioside GM3 and gangliosides of the ganglio-series have been deeply studied for their geometrical (Table 2) and dynamic (Table 3) properties. The disaccharide -b-Gal-(1!4)-b-Glc-, the lactose, is linked to ceramide in all these gangliosides. Many conformers in a very reduced range of minimum energy have been determined on the basis of molecular calculations and a few NMR data.38,39 In addition to this, 13C T1, T1r and 13C(1H) NOE measurements40 suggest that the spatial arrangement of the two linkages can be described by fluctuations in a large energy minimum rather than by the sampling of different conformers with short lifetimes. A character of low motional freedom is expected for the Glc residue,41,42 due to some restrictions of motion imposed by the surrounding membrane surface.43 The addition of sialic acid to lactose gives the oligosaccharide structure of ganglioside GM3. In GM3, the sialic acid is mobile, the ketosidic linkage existing in two main conformations (Table 3). Moving to the more complex gangliosides of the ganglioseries 3, 4, and 5, we found the trisaccharide sequence -b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-. This trisaccharide as a consequence of several inter-residual interactions belongs like a very rigid block.38,40,44–46 The side chain of sialic acid, which is in a rigid conformation,38,47–49 strongly interacts with the N-acetylgalactosamine, giving a strong association between the Neu5Ac and GalNAc units, this association being stabilized by a hydrogen bond between the GalNAc amide proton and the Neu5Ac carboxyl group.50 The association between Neu5Ac and GalNAc is very important in defining the rigid conformation of the -b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-trisaccharide. The shift of GalNAc from the position 4 to position 6 of Gal, as in the synthetic compound 60 -GM2, determines a dramatic modification of the

Table 1 Ganglioside structures Abbreviation(413)

Structure of the ganglioside oligosaccharide

Galacto Lacto Lacto Lacto Ganglio-3 Ganglio-3 Ganglio-4 Ganglio-4 Ganglio-4 Ganglio-5 Neolacto-4 Ganglio-4 Ganglio-4 Ganglio-5

GM4 GM3 GD3 O-Acetyl-GD3 GM2 GD2 GM1 GM1b Fuc-GM1 GalNAc-GM1 30 -LM1 GD1a GD1a GalNAc-GD1a

Neu5AcGalCer II3Neu5AcLacCer II3(Neu5Ac)2LacCer II3[Neu5,9Ac2-(2-8)-Neu5Ac]LacCer II3Neu5AcGg3Cer II3(Neu5Ac)2Gg3Cer II3Neu5AcGg4Cer IV3Neu5AcGg4Cer IV2aFucII3Neu5AcGg4Cer II3Neu5AcGg5Cer IV3nLc4Cer IV3Neu5AcII3Neu5AcGg4Cer IV3Neu5AcIII6Neu5AcGg4Cer IV3Neu5AcII3Neu5AcGg5Cer

Ganglio-4 Ganglio-4 Ganglio-4

GD1b GD1b-lactone Fuc-GD1b

II3(Neu5Ac)2Gg4Cer II3[Neu5Ac-(2-8,1-9)-Neu5Ac]Gg4Cer IV2aFucII3Neu5Ac2Gg4Cer

Ganglio-4

GT1a

IV3(Neu5Ac)2II3Neu5AcGg4Cer

Ganglio-4

GT1b

IV3Neu5AcII3(Neu5Ac)2Gg4Cer

Ganglio-4

O-Acetyl-GT1b

IV3Neu5AcII3[Neu5,9Ac2-(2-8)-Neu5Ac]Gg4Cer

Ganglio-4

GT1c

II3(Neu5Ac)3Gg4Cer

Ganglio-4

Chol-1a-a

IV3Neu5AcIII6Neu5AcII3Neu5AcGg4Cer

Ganglio-4

Chol-1b

III6Neu5AcII3(Neu5Ac)2Gg4Cer

Ganglio-4

GT1a

IV3Neu5AcIII6(Neu5Ac)2Gg4Cer

Ganglio-4

GQ1b

IV3(Neu5Ac)2II3(Neu5Ac)2Gg4Cer

Ganglio-4

GQ1c

IV3Neu5AcII3(Neu5Ac)3Gg4Cer

Ganglio-4

GQ1a

IV3(Neu5Ac)2III6(Neu5Ac)2Gg4Cer

Ganglio-4

Chol-1a-b

IV3Neu5AcIII6Neu5AcII3(Neu5Ac)2Gg4Cer

Ganglio-4

GP1c

IV3(Neu5Ac)2II3(Neu5Ac)3Gg4Cer

a-Neu5Ac-(2!3)-b-Gala-Neu5Ac-(2!3)-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)-b-Gal-(1!4)-b-Glca-Neu5,9Ac2-(2!8)-a-Neu5Ac-(2!3)-b-Gal-(1!4)-b-Glcb-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glcb-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glcb-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-b-Gal-(1!4)-b-Glca-Fuc-(1!2)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glcb-GalNAc-(1!4)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!4)-b-GlcNAc-(1!3)-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-[a-Neu5Ac-(2!6)]-b-GalNAc-(1!4)-b-Gal-(1!4)-b-Glcb-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-bGal-(1!4)-b-Glcb-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glcb-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8,1!9)-a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Fuc-(1!2)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-Gal(1!4)-b-Glca-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-Gal(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-Gal(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5,9Ac2-(2!8)-a-Neu5Ac-(2!3)]-bGal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!8)-aNeu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-[a-Neu5Ac-(2!6)]-b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)-b-Gal(1!4)-b-Glcb-Gal-(1!3)-[a-Neu5Ac-(2!6)]-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-Gal(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!6)]-b-GalNAc-(1!4)-b-Gal(1!4)-b-Glca-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!8)a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)-b-Gal-(1!3)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!6)]b-GalNAc-(1!4)-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!3)-b-Gal-(1!3)-[a-Neu5Ac-(2!6)]-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glca-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-Gal-(1!4)-b-Glc-

699

Abbreviation(412)


Series

700


Figure 1 Van der Waals sphere volumes calculated for choline and GM1 oligosaccharide conformers.

Table 2 Hydrodynamic radius Rh (A˚), axial ratio Ra/Rb, molecular mass M (kDa) of the aggregates, and surface area a0 (A˚2) of the monomer in aggregate.85 Natural compounds have heterogeneous ceramide moiety. Purified species with defined ceramide structure are indicated Rh (A˚) GM4, from bovine brain GM3, from bovine brain GM2, from bovine brain GM1, from bovine brain GM1(d18:1,18:0), previously warmed at 60 C GM1(d18:1,18:0), previously warmed at 40 C GM1(d18:1,18:0), previously warmed at 25 C Fuc-GM1, from pig brain GD1a, from bovine brain GalNAc-GD1a, from bovine brain GD1b, from bovine brain GD1b-lactone, synthesis from bovine brain GD1b GT1b, from bovine brain

Vesicle Vesicle Micelle Micelle Micelle Micelle Micelle Micelle Micelle Micelle Micelle Micelle Micelle

300 250 66.0 58.7 52.8 56.2 58.7 61.0 58.0 60.0 52.0 57.0 53.2

a0 (A˚2)

Ra/Rb

M (kDa)

3.1 2.3

18,270 16,700 740 470

80 80 92.0 95.4

394 418 509 311 424 378

97.8 98.1 97.0 100.8 97.6 100.8

2.1 2.0 1.8 2.1 1.8

trisaccharide dynamics. In the trisaccharide -b-GalNAc-(1!6)-[a-Neu5Ac-(2!3)]-b-Gal-, the GalNAc-(1!6)-b-Galglycosidic bond is flexible, sampling three main conformations. On the other hand, the Neu5Ac residue modifies its spatial disposition due to the loss of the interactions with GalNAc that are present in the rigid trisaccharide GalNAc(1!4)-[a-Neu5Ac-(2!3)]-b-Gal-(1!4)-trisaccharide of GM2 and other gangliosides. From the ganglio-series 3 to the ganglio-series 4, we have the addition of galactose to hexosamine. The disaccharide b-Gal-(1!3)-b-GalNAc- is mobile, allowing the existence of two main conformations of the glycosidic linkage (Table 3). Thus GM2, carrying only rigid linkages in the outer portion of the oligosaccharide chain, is present only in one preferred conformation, while GM1 carrying the external mobile linkage b-Gal-(1!3)-b-GalNAc is in two and GD1a carrying the external a-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAc in four (Table 3). According to this, the ganglioside oligosaccharide moieties are represented by a number of conformers which participate to determine the solid angle occupied by the different ganglioside monomers within the surface. It follows that the volume requested to host the GM1 and GD1a oligosaccharide becomes different, in this case much higher, than that expected to host the chain of GM2 plus one or two additional sugar units. About this consideration, it is interesting to analyze ganglioside GalNAc-GD1a. In this ganglioside, the addition of a GalNAc to GD1a gives a second b-GalNAc-(1!4)-[a-Neu5Ac(2!3)]-b-Gal-rigid trisaccharide directly bound to the first one. Thus in GalNAc-GD1a we have two rigid blocks joined together with a mobile linkage, thus reducing the number of conformers from four to two. The four GD1a conformers fill, all together, c. 1.760nm3, calculated as van der Waals sphere volume, versus c. 1.420nm3 occupied by the two GalNAc-GD1a conformers. Thus, the surface are of GalNAc-GD1a is slightly lower than GD1a (Figure 2).46


701

Table 3 Inter-residue proton NOE interactions for ganglioside saccharide sequences, and glycosidic torsional angle pairs (f,c) (accuracy is 15 ) NOE interactions a-Neu5Ac-(2!3)-b-Gal-(1!3)-b-GalNAcNeu5Ac-3ax : Gal-3 Gal-1 : GalNAc-3 Neu5Ac-OH8 : Gal-3 Gal-1 : GalNAc-4 Neu5Ac-8 : Gal-3 Gal-1 : GalNAc-2 Neu5Ac-3eq : Gal-OH2 Gal-1 : GalNAc-NH f a-Neu5Ac-(2!3)-b-Gal76 159 b-Gal-(1!3)-b-GalNAc þ36 26 b-GalNAc-(1!4)-[a-Neu5Ac-(2!3)]-b-GalGalNAc-1 : Gal-4 Neu5Ac-3ax : Gal-3 GalNAc-NH : Gal-2 Neu5Ac-3ax : Gal-OH2 Neu5Ac-3eq : Gal-OH2 GalNAc-CH3 : Gal-OH2 GalNAc-CH3 : Gal-2 Neu5Ac-OH7 : Gal-4 GalNAc-OH6 : Gal-OH6 Neu5Ac-OH8 : Gal-4

a-Neu5Ac-(2!3)-b-Galb-GalNAc-(1!4)-b-Gal-

f 162 þ31

Neu5Ac-8 : GalNAc-CH3

c þ2 18 þ8 21 Neu5Ac-8 : GalNAc-1 Neu5Ac-OH8 : GalNAc-1 Neu5Ac-OH8 : GalNAc-5 Neu5Ac-OH8 : GalNAc-NH Neu5Ac-OH9 : GalNAc-OH6 Neu5Ac-OH9 : GalNAc-5 Neu5Ac-9R : GalNAc-OH6 c 28 þ18

b-Gal-(1!3)-b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-GalGalNAc-1 : Gal-4 Neu5Ac-3ax : Gal-3 Neu5Ac-3eq : GalNAc-1 Neu5Ac-8 : Gal-4 Neu5Ac-3eq : GalNAc-5 Neu5Ac-NH : Gal-OH6 b-Gal-(1!3)-b-GalNAcb-GalNAc-(1!4)-b-Gala-Neu5Ac-(2!3)-b-Gala-Neu5Ac-(2!8)-a-Neu5Ac-

þ53 30 þ31 þ175 þ89

Neu5Ac-8 : GalNAc-1 Neu5Ac-8 : GalNAc-5 þ10 25 þ18 þ5 þ10

f 5!55

c 50!0

Neu5Ac-3ax : Neu5Ac-6 Neu5Ac-6 : Neu5Ac-9a Neu5Ac-8 : Neu5Ac-9a Neu5Ac-3eq : Neu5Ac-8

b-Gal-(1!4)-b-GlcGal-1 : Glc-4 Gal-1 : Glc-6 Gal-1 : Glc-OH3 Gal-OH2 : Glc-6 Gal-OH2 : Glc-60 Gal-OH2 : Glc-OH6 Gal-1 : Glc-OH6 Gal-6 : Glc-OH3

A further group of gangliosides to be considered is that of structures containing a disialosylchain linked to the inner galactose. This is the case of GD1b and GT1b, while more complex polysialylated gangliosides have not been studied up to date. In the tetrasaccharide -b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)-a-Neu5Ac-(2!3)]-b-Gal-, the inter-residual contacts between GalNAc and Neu5Ac linked to Gal are not existing, while interactions occur between GalNAc and the external Neu5Ac. According to these constraints, the tetrasaccharide chain -b-GalNAc-(1!4)-[a-Neu5Ac-(2!8)a-Neu5Ac-(2!3)]-b-Gal-is arranged in a 3A˚ wide circle with a hole about the center.51 This circle seems to be large enough to accept cations. Such a conformation confers a bulkier character to the ganglioside portion closer to the hydrophobic–hydrophilic interface and explains the larger value of surface area of GD1b in comparison to that of its isomer GD1a. It is interesting to note that the solid angle required by the disialosyl chain is wide enough to host an additional sialic acid unit in an external position. In fact, although GT1b carries one more sugar linked to the external galactose unit, it requires surface area very similar to that of GD1b.

702


Figure 2 van der Waals sphere volumes calculated for GD1a and GalNAc-GD1a oligosaccharide conformers.

GD1b, in part, has been found to exist in lactonic form, GD1b-lactone, in human neurons. The conversion process between GD1b and GD1b-lactone has been proposed as a process capable to modulate the activities of membrane proteins. When the external sialic acid carboxyl group esterifies the inner sialic acid residue, the interactions between GalNAc and the external Neu5Ac no longer take place, while the rigid conformation of the trisaccharide-b-GalNAc(1!4)-[a-Neu5Ac-(2!3)]-b-Gal- observed in GM2, GM1, GD1a, and GalNAc-GD1a is restored. This forces a better lining up of the disialosyl chain with the neutral oligosaccharide chain, reducing the angle between the neutral chain and the inner sialic acid axis.51 Thus the geometry of GD1b-lactone is closer to GD1a than to GD1b (Table 2). In Table 4, we propose a general scheme that simplifies the dynamic properties of gangliostructures. The number of carbohydrate rings and the dynamics of glycosidic linkages are the two main parameters determining the head group large size of gangliosides, but a further factor must be considered. This is the hydrating water that interacts with the oligosaccharide chain. Good information is available on the head-to-head interactions;52 nevertheless, data proving direct side-by-side oligosaccharide interactions are not available. Also using as experimental model, a micellar aggregate of gangliosides,44 where at the surface single monomers are very close to each other, inter-monomer carbohydrate–carbohydrate interactions or changes in the oligosaccharide conformation could not be identified by NMR experiments. This is due to the large amount of water present in the hydrophilic layer.53 In fact, it should be noted that the actual environment of each monomer at the membrane hydrophilic layer includes a solvent.53 Water is a natural component of the sugar shell being attracted by the hydrophilic character of sugars and by the necessity to avoid repulsion between the negative-charged oligosaccharide.53 Calculations performed on GM2 micelles54 indicated a difference of about 5 A˚ between the dry and hydrated micellar radius. Experimental data show that GM2 micelle is an oblate aggregate, but calculations can be performed only on an equivalent spherical micelle having the same mass. This does not allow to know the number of water molecules but suggests that several molecules of water are interacting with the oligosaccharide chains, this being in agreement with calorimetric studies suggesting that each chain is surrounded by 40–70 water molecules.55 A strong interaction between water and GM1 sugars, sialic acid, and the inner galactose was observed by NMR.44 Water bridges between saccharides have been observed in hyaluronan where they were strong enough to determine and stabilize the tridimensional structure of the molecule.56 Of course, these results and considerations would exclude any direct inter-monomer side-by-side carbohydrate interactions at the level of cell membrane, but are in favor of a specific role of water in organizing a net of hydrogen bonds able to stabilize the glycosphingolipid clustering. Finally, we recall that the geometry of the monomer of gangliosides determined by the size of the head groups and their structural differences has also an important effect in modulating the transition temperature of the lipid moiety, thus modulating the fluidity of the membrane lipid core. The bigger the head group, the lower the transition temperature (Table 5). This is a further opportunity to modulate the segregation process as a function of the head group structure.


703

Table 4 Dynamics of the ganglioside glycosidic linkages as a function of the primary and secondary structure of gangliosides

a-Neu5Ac-(2----3)-b-Gal-(1•••4)-b-Glc-(1••• b -GalNAc-(1—4)-b -Gal-(1•••4)-b -Glc-(1••• / a-Neu5Ac-(2—3) b -Gal-(1---3)-b -GalNAc-(1—4)-b -Gal-(1•••4)-b -Glc-(1••• / a-Neu5Ac-(2—3) a-Neu5Ac-(2----3)-b -Gal-(1----3)-b -GalNAc-(1—4)-b -Gal-(1•••4)-b -Glc-(1••• / a-Neu5Ac-(2—3)

b -GalNAc-(1— 4)-b -Gal-(1----3)-b -GalNAc-(1—4)-b -Gal-(1•••4)-b -Glc-(1••• / / a-Neu5Ac-(2—3) a-Neu5Ac-(2—3) b -Gal-(1----3)-b -GalNAc-(1—4)-b -Gal-(1•••4)-b -Glc-(1••• \ a-Neu5Ac-(2—3)-a-Neu5Ac-(2—3) Rigid linkage –––; dynamic linkage ----; flexible linkage .

Table 5 Transition temperature Tt ( C) of sphingolipids aggregates. Natural compounds have heterogeneous ceramide moiety Tt ( C ) reference 64 GlcCer, from bovine spleen LacCer, from bovine adrenal medulla Gg3Cer, by hydrolysis of bovine brain GM2 Gg4Cer, by hydrolysis of bovine brain GM1 GM3, from bovine adrenal medulla GM2, from bovine brain GM1, from bovine brain GM1(d18:1,18:0), previously warmed at 60 C GM1(d18:1,18:0), previously warmed at 40 C GM1(d18:1,18:0), previously warmed at 25 C GM1(d20:1,18:0), previously warmed at 60 C FucGM1, from pig brain FucGM1(d18:1,18:0), previously warmed at 60 C FucGM1(d20:1,18:0), previously warmed at 60 C GD1a, from bovine brain GD1a(d18:1,18:0), previously warmed at 60 C GD1a(d20:1,18:0), previously warmed at 60 C GT1b, from bovine brain SM, from several sources

83.7 74.4 60.8 54.0 35.3 29.3 19.3

15.2

414,415

416

19.7 11.7 15.0 17.6 23.2 13.2 10.0 18.3 16.0 10.1 19.2

7.3 35.75.8

Cellular membranes represent a polymorph system where several lipid organizations can occur and where positive and negative surface curvatures are in sequence. The availability of ganglioside structures with large hydrophilic head group showing small differences is a good opportunity to stabilize lipid membrane domains and subdomains57 with positive curvature.

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3.30.2.2 The Lipid–Water Interface of Gangliosides The head group sizes and the high transition temperature of the hydrophobic chains of gangliosides favor the segregation process, and the oligosacharide water environment through intermolecular water bridges stabilizes it. In addition to this, other relevant events occur at the membrane surface and participate to stabilize the lipid membrane domains. In fact, lipid membrane domains exist in cells that contain SM but have low amount or are lacking glycosphingolipids,58 and in subdomains with SM but a very low content of neutral glycosphingolipids and no content of gangliosides.59,60 Thus, something related to ceramide, the moiety belonging to all sphingolipids, and therefore also to gangliosides, is strictly related to lipid membrane domain stabilization, and there is a general consent that this is related to the less hydrophobic portion of ceramide, belonging to the water/lipid interface. At the water/lipid interface, we have the amide group of ceramide as a rigid system comprising six atoms in a planar conformation, together with the hydroxyl group at position 3. The availability of an amide nitrogen, of a carbonyl oxygen, and of a hydroxyl group enables sphingolipids to form hydrogen bonds, acting as hydrogen bond donors and acceptors at the same time. Therefore, this feature allows sphingolipids to form a stable net of interactions, this becoming very important in the case of gangliosides where head groups per se promote the clustering process. Concerning the other membrane complex lipids, glycerophospholipids do not have this property because they can act only as acceptors of hydrogen bonds and cholesterol has very limited capacity to form hydrogen bonds. The van der Waals forces between hydrocarbon chains have been estimated to about 2–3kcal per hydrocarbon chain. The formation of hydrogen bonds at the water/lipid interface contributes with 3–10kcal to the lipid–lipid interaction. Thus, the orientation of the hydrogen bond donor and acceptor groups of sphingolipids optimal to form lateral interactions and the considerable increase of stability in the lipid association are very good candidates to promote the formation of a membrane rigid zone where a network of hydrogen bond-connected lipids are segregated together with cholesterol.

3.30.2.3 The Hydrophobic Chains of Gangliosides The group has a perpendicular orientation toward the axes of the two hydrocarbon chains, whose parallel orientation is stabilized by the D4,5 instauration of sphingosine.61 Thus, the ceramide moiety can be considered a rigid structure and addition of glycosphingolipids to cells was shown to reduce the original membrane fluidity.62 Membrane complex lipids are highly heterogeneous in their lipid moieties. Many of them contain unsaturated alkyl chains. Thus, it is an essential requirement to have fluid membranes so that protein conformational changes and lipid organization changes are allowed. But complex lipids with saturated chains are also membrane components. In the membrane, the components that contain rigid saturated alkyl chains with high transition temperatures are excluded from those that contain unsaturated chains with low transition temperature. Phosphatidylcholine (PC) is the major membrane glycerophospholipid. It comprises several molecular species, differing in the lipid moiety. Within these, dipalmitoylphosphatidylcholine is the main species in DRMs.37,63 Palmitic and stearic acid are the main fatty acids of gangliosides. Thus, if we recall that over 60% of total membrane gangliosides are inside the lipid membrane domain fractions, it follows that lipid membrane domains are highly enriched with unsaturated chains.63,64 In this rigid environment, cholesterol, which alone has a melting point of 148.5 C, would find a correct position.

3.30.3 Segregation of Membrane Lipids The interest for lipid membrane domains, that is, for zones of the membrane with a peculiar lipid composition, different from that of the majority of bilayer, became very strong during the last 15 years, when many proteins deputed to cell signaling were found to be preferentially associated with an environment of lipids highly enriched in sphingolipids and cholesterol. A possible role of lipid membrane domains in the transport of glycosylphosphatidylinositol (GPI)-anchored proteins from the Golgi apparatus to the apical plasma membrane of polarized cells was also suggested, hence the term ‘lipid rafts’ was used to define these domains.65 Biochemical studies on the composition, organization and biological role of lipid membrane domains were carried out mainly starting from 1992, when a method capable to separate them from total cell membranes became available.66 However, a massive biophysical work on artificial membranes was carried out from the 1970s to understand the basis of the organization of amphiphilic compounds within biological membranes, leading to establish the existence of segregation phenomena.


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Today, different terms are used throughout the literature to define lipid membrane domains. Every one of them implies a form of segregation of certain components within the cell membrane. Some define specific domains, as in the case of ‘caveolae’, membrane invaginations containing the protein caveolin67 or ‘lipid rafts’, membrane domains that sort and/or transport proteins inside cells. Others define membrane portions on the basis of chemicophysical or compositional features, such as detergent-insoluble material (DIM),68 detergent-insoluble substrate attachment matrix (DISAM),68 detergent-insoluble glycolipid-enriched (DIG) material,69 detergent resistant membranes (DRMs),70 sphingolipid-enriched membrane fraction (SEMF),71 and glycosignaling domains (GSDs).72 To these authors, DRM is a very reasonable definition, based on the operational way commonly used to obtain biochemical preparation enriched in lipid membrane domains, that is based on their relative insolubility in nonionic detergents under specific experimental conditions.

3.30.3.1 Lipid Membrane Domains in Artificial Membranes The lines of earliest evidence supporting the existence of lipid membrane domains, conceived as areas in the membrane different in lipid composition from other areas in the membrane, were obtained studying artificial membrane models represented by phospholipid bilayers, containing glycosphingolipids, SM, ceramide or/and cholesterol, sphingolipid micelles, and lipid monolayers on an air/water interface or on solid support. Membrane lipids, not only sphingolipids,73–75 exist in multiple phases, and this was probably the first evidence leading to the concept of lipid membrane domains. In the case of glycosphingolipids, their unique properties like the geometry of the monomer inserted into the membrane, the capability of the amide linkage of ceramide to form a network of hydrogen bonds at the water–lipid interface of cell plasma membrane, the D4 double bond of sphingosine near the water–lipid interface, the capability of the oligosaccharide chain to interact with water and the specific content of saturated alkyl chains, suggest a strong tendency to form segregated compositional domains in phospholipid bilayers. Starting from the early 1980s, this was clearly shown for a number of neutral glycosphingolipids (reviewed in Ref. 76).41,73,77–84 Much more controversial appeared the situation for gangliosides that are unique among glycosphingolipids for their aggregative properties in aqueous solutions (reviewed in Ref. 85). By means of spin-label probes,62,86 gangliosides were shown, even at low concentrations, to reduce fluidity and hydrocarbon chain mobility in PC bilayers, due to lateral cooperative interactions between the ganglioside molecules, that is, to the formation of ganglioside clusters. This suggested a possible biological relevance of lipid membrane domains.62,87 The membrane fluidity that resulted further decreased by addition of Ca2þ. This suggested the involvement of ganglioside head groups in the process of ganglioside phase separation.62 Nevertheless, other studies reported different results, at least for small amounts of gangliosides dispersed in PC bilayers.88–92 Electron microscopy (EM) identification of GM1 ganglioside after surface labeling with cholera toxin showed that the lipid was randomly distributed in phospholipid bilayers. Instead, with the same technique, asialo-GM1 was found segregated in microdomains.84,93–95 When similar studies were performed using multilamellar liposomes of phospholipid mixtures, which exhibit laterally separated fluid- and gel-phase regions, ganglioside GM1 and its neutral derivative asialo-GM1 were found preferentially into gel-phase regions.73 The possible role of glycosphingolipid lipid moiety in determining lipid segregation was studied using synthetic compounds.96 Altogether, results showed97,98 that (1) ganglioside phase separation occurs in one- or two-component PC bilayers, the latter characterized by the presence of two distinct PC phases;99–106 (2) the extent of ganglioside lateral phase separation depends upon the length and unsaturation differences between the ganglioside long-chain base and PC acyl chains;99–101 (3) a decrease in the acyl chain length, or an increase in its unsaturation, of ganglioside GM1 increased the ganglioside in the liquid phase of the bilayer;103 (4) for a given lipid moiety composition, the extent of ganglioside phase separation is dependent upon the number of sugars in the oligosaccharide head group;100,101 and (5) the addition of Ca2þ promotes phase separation,62 by a passive ganglioside exclusion from PC-rich regions of the bilayer, that are perturbed by Ca2þ.99,101 When ternary SM/GM1/cholesterol vesicles were analyzed by differential scanning calorimetry (DSC), the formation of separate GM1-enriched and cholesterol-enriched domains was shown.106 Gangliosides in diluted aqueous solution generally form micelles of large molecular mass.85 This feature allowed to obtain further information about the lateral segregation of gangliosides using mixed micelle systems, that can be conveniently studied by laser light scattering. In mixed micelles of the two gangliosides GM2 and GT1b, with similar hydrophobic moiety composition, monomers are not randomly distributed in the ellipsoidal micelle.54 The segregation of one ganglioside with respect to the other in this artificial system is a spontaneous process explained on the basis of the different geometrical properties of ganglioside head groups. A similar segregation as well due to the geometrical differences between these two gangliosides was shown in mixed micelles of GD1b and GD1b-lactone.107

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3.30.3.2 Detergent Insolubility and Lipid Membrane Domains Originally, the existence of a membrane fraction characterized by a peculiar lipid composition leading to a liquidordered or highly organized phase was operationally defined on the basis of the insolubility in aqueous nonionic detergents. Most components of the cell membrane (including glycerophospholipids, the bulk membrane lipid components, and intrinsic membrane glycoproteins) are solubilized by detergents and chaeotropic agents.108 In contrast, many cellular components are insoluble in nonionic (Triton X-100) or zwitterionic detergents (Empigin BB) under certain experimental conditions (where temperature, detergent concentration, and detergent-to-cell ratio seem the most critical parameters). This detergent insoluble material (DIM) was originally shown to be enriched with pericellular matrix proteins (such as fibronectin, tenascin, Gp140), cell attachment site components (including cytoskeletal elements),109 and glycosphingolipids, including GM1.68,110,111 It appeared clear quite soon that these detergent-insoluble fractions were even more complex. In particular, they contain several membrane components other than gangliosides, including other sphingolipids (glycosphingolipids and SM),112–114 cholesterol,113 lipid-anchored proteins (containing GPI or fatty acid modifications),66,114–119 and other hydrophobic plasma membrane proteins, such as caveolin.120 Thus, the concept emerged that DRM is at least in part represented by DRMs, such as those belonging to the apical compartment of polarized epithelial cells (Madin–Darby canine kidney (MDCK)) or to the caveolar membrane system. After detergent treatment, the detergent insoluble membrane domain can be separated from the rest of the cell thanks to its relative light density (buoyancy),66 using continuous or discontinuous sucrose density gradients. The low density of the DIM is likely due to its richness in lipids, that is, to the high lipid-to-protein ratio in this fraction. Lowdensity, detergent-insoluble fractions were isolated from a wide variety of cultured cells, including almost all mammalian cell types investigated so far (normal and tumor epithelial cells,60,66,120,121 lymphocytes122 and lymphoid tumor cells,123 neutrophils,124 platelets,125 erythrocytes,126 fibroblasts,60,127 neurons,71,128–130 and neuroblastoma cells)131–133 as well as yeast (Saccharomyces cerevisiae)134 and the ciliate protozoan Tetrahymena mimbres.135 Similar fractions have been prepared as well from tissues136–142 (discussed in Section 3.30.3.1). Doubtless, the availability of a method to purify a lipid membrane domain-containing fraction dramatically improved the biochemical characterization of these structures, providing basic pieces of information that critically contributed in orienting the studies of many research groups about the structure and function of lipid membrane domains. Compositional information of lipid membrane domains has been largely obtained by mean of the procedure published by Brown and Rose,66 based on the insolubility in aqueous nonionic detergents of those cell lipids (including cholesterol, sphingolipids, and saturated PC) that in membrane models tend to segregate into a liquidordered phase. In Tables 6 and 7, the detailed lipid composition of a detergent-insoluble membrane fraction prepared from differentiated rat cerebellar neurons using the Brown and Rose method is reported. However, several criticisms were raised about the significance of the biochemical data obtained analyzing such a fraction (for excellent reviews on this topic, with very different points of view, see Refs. 143–146). It was argued that detergent insolubility of some cellular components might be due to an artificial rearrangement induced by the detergent itself. This major concern about the possible artifact nature of a fraction prepared by the mean of detergent stimulated comparative studies performed using a wide range of different detergents.139,140,147,148 These studies showed that a detergent-resistant fraction enriched in cholesterol and sphingolipids, as well as in certain proteins usually regarded as lipid membrane domain markers (in particular, GPI-anchored proteins and acylated proteins), can be prepared using detergents with different stringency. However, the association of other proteins (especially integral membrane proteins) with the detergent-resistant lipid membrane domain fraction is strongly affected by the type of detergent used and the detergent/protein ratio. Moreover, the DRM obtained in the presence of Triton X-100, CHAPS, Brij 96, and Triton X-102 float at different densities, suggesting that different lipid or a different lipid/protein ratio characterize domains insoluble in different detergents. This indicates that either different lipid membrane domains exist, that can be separated by differential solubilization (discussed in Section 3.30.3.2), or different degrees of lateral order exist within the same lipid membrane domain. In the latter case, the use of experimental protocols involving different detergents might be a useful tool to dissect the fine structure of lipid membrane domains. However, in some cases, it cannot be excluded that the detergent insolubility of a protein is determined by factors other than its association with a lipid membrane domain. Thus, detergent insolubility per se is not a sufficient criterion to establish the association of a protein with a lipid membrane domain. The analysis of the complex environment of the protein, and especially of its lipid composition, is essential to clarify the protein association with a lipid-rich, highly organized membrane microdomain. Unfortunately, such studies are almost completely absent. To overcome some of the problems related to the use of detergents, many authors struggled for the development of ‘detergent-free’ methods for the separation of low-density membrane fractions corresponding to lipid membrane domains.


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Table 6 Protein and lipid composition in rat cerebellar granule cells differentiated in culture, and in the detergent-resistant membrane (DRM) prepared by cell lysis with Triton X-100 followed by ultracentrifugation on sucrose gradient 37,71 Cell homogenate

DRM

nmol/106cells Proteins Cholesterol Sphingolipids Ceramide Sphingomyelin Gangliosides GM3 GM1 GD3 GD1a GD1b O-Ac-GT1b GT1b O-Ac-GQ1b GQ1b Glycerophospholipids PE PPE PC PPC PS PI PPI PIP PIP2

1.25 4.80 2.01 0.22 1.00 0.79

0.02 1.91 1.25 0.11 0.67 0.47 ND 0.04 0.02 0.11 0.05 0.06 0.17 0.02 0.67 3.95 0.35 0.05 3.41 0.01 0.18 0.05 ND 0.01 0.01

ND 0.06 0.04 0.21 0.09 0.08 0.26 0.01 0.02 32.84 6.64 4.13 16.41 0.35 2.59 1.22 ND 0.15 0.24

ND, not detected.

Table 7 Protein and lipid distribution (%) in rat cerebellar granule cells differentiated in culture, and in DRM fraction prepared by cell lysis with Triton X-100 followed by ultracentrifugation on sucrose gradient 37,71 Cell homogenate

DRM (%)

Proteins Glycerophospholipids Cholesterol Sphingomyelin Ceramide Gangliosides

3.05 80.29 11.73 2.44 0.53 1.93

0.28 55.39 26.78 9.39 1.54 6.59

The rationale for the setup of these methods was the assumption that resistance to detergent solubilization might be only one peculiar aspect of a more general feature, that is, the resistance to different treatments that would be able to disrupt the structure of less ordered membrane areas, but not that of highly organized, ‘rigid’ and thermodynamically favored lipid membrane domains. Indeed, several different techniques are available for the separation of a lipid membrane domain-rich fraction from the rest of the cells without the use of detergents. The disruption of cells in the presence of high pH, hypertonic sodium carbonate149 or by the means of mechanical treatments (sonication under carefully controlled conditions),150 leaves behind membrane fragments that can be separated by density gradient centrifugation. When comparatively analyzed, low-density membrane fractions obtained after cell lysis under the dramatically different experimental conditions described above are very similar but not identical.59,71,120,129,133,151–160 Independently from the method used to disrupt the bulk cell membrane, low-density membrane fractions always contain a highly resistant supramolecular structure possibly corresponding to the native core of lipid membrane domains. These results

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seem to indicate that the low-density membrane fraction composition corresponds to that of physiological lipid membrane domains, and that it is not determined by a random rearrangement of cell components induced by the experimental conditions used. Independently from the method used for its preparation, the low-density membrane fraction always contains membranous material in the form of vesicles with different size (ranging from 30 to 400 nm) and different electron density, mixed with planar or curved heterogeneous membrane fragments without vesicular structure in different ratios.120,125,136,137,139,150–152,154,158 However, low-density membrane fractions prepared using different experimental tools, as mentioned above, are not identical. The differences observed by some authors might be simply due to contingent situations. As mentioned above, standardization of the experimental procedures is sometimes difficult, and the overall composition of DRM fractions or the association of specific molecules with it seems to be affected by even tiny modifications of several conditions, including agents used for membrane disruption (detergents vs non-detergents,137,150,154 different detergents or different detergent concentrations),120,139,142,147 mechanical procedures used to obtain or aid membrane solubilization (sonication, homogeneization),154 temperature,66,147,160,161 pH), and ratio between detergent and biological material.142 For example, using Triton X-100, for a given type of biological material (cell or tissue), there is a certain threshold value for the detergent-to-sample ratio, above which it is impossible to prepare a low-density DRM fraction. The amount of lipid and protein material associated with the low-density DRM fraction remains constant for a wide range of detergent-to-sample ratios, but it suddenly drops to barely detectable quantities above the threshold value (A. Prinetti and S. Sonnino, unpublished observation). Not surprisingly, temperature seems one of the most sensitive parameters, and performing the preparation in the cold (ice immersion or þ4 C) is particularly critical.66 Actually, probably one of the best methods to disrupt the organization of DRM domain is to incubate it at room temperature or at þ37 C.161 This aspect has often been regarded as a strong limit for the physiological significance of detergent-insoluble fractions prepared in the cold (an experimental condition that can hardly be extrapolated to those of living cells). However, more recently, it has been shown that low-density, detergent-insoluble fractions can be prepared at 20 or 37 C.142,147,160,162 Whether DRM prepared under different conditions do represent the same entity or not still remains to be answered. Less dramatically, a given combination of experimental conditions used for the preparation of DRM might be stringent enough to allow the solubilization of some components of these fractions, or not stringent enough to remove all components not resident in this fraction. Moreover, as mentioned above, detergent-insoluble material is in part also represented by molecules not belonging to DRM, and DRM components might interact with detergent-soluble molecules (e.g., cytoskeletal elements) strongly enough to be driven off DRM during the preparation. All these considerations should be carefully kept in mind when comparing the features of low-density DRM fractions obtained by different methods or authors, and it is worth mentioning that only seldom were these methodological aspects systematically and comparatively analyzed. Indeed, the separation conditions described above are considered more or less equivalent, and insolubility in Triton X-100 is still accepted by many authors as the working definition of lipid membrane domains. However, several studies indicate that at least in some cases the differences observed in the composition of lowdensity DRM fractions isolated by different methods might reflect the existence of different levels of order within lipid membrane domains and/or of biochemically distinct lipid membrane domains within the plasma membrane of the same cell. This is particularly clear if the results obtained by the use of different detergents are compared.66,147,163–166 Differential solubilization by nonionic detergents (Triton X-100 vs Brij 96) was used to infer that two functionally unrelated neuronal GPI-anchored proteins, Thy-1 and PrP, belong to structurally different lipid membrane domains characterized by a different degree of order.139 The use of nonionic (Triton X-100, Brij 96, Triton X-102) or zwitterionic (CHAPS) detergents allowed one to separate biochemically distinct detergent-specific domains from myelin membrane.147 Thus, differential detergent solubilization might prove to be a powerful tool to study different lipid membrane domain subpopulations (see Section 3.30.3.2). On the other hand, the above-reported notion introduces a further methodological concern in the use of detergent solubilization for the preparation of lipid membrane domainrich fractions. In fact, low-density DRM may contain membrane fragments derived by the coalescence of distinct lipid membrane domains.139

3.30.3.3 Preparation of Cellular Fractions Enriched in Lipid Membrane Domains In Section 3.30.3.2, the relationship between DRM fractions prepared from cultured cells has been reviewed and discussed. In this paragraph, we will discuss the possibility of preparing lipid membrane domain fractions from tissue samples and to separate different subsets of lipid membrane domains.


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3.30.3.4 Preparation of Lipid Membrane Domains from Tissues As discussed in Section 3.30.3.2, the procedure published by Brown and Rose,66 leading to the isolation of a lowdensity insoluble complex from MDCK polarized epithelial cells after detergent extraction, has been subsequently applied to virtually any kind of eukaryotic cell with consistent results. Soon the growing conviction that lipid membrane domains work as signaling platforms in a number of cell events, some of those strictly related to complex pathophysiological events, directed the interest of some investigators toward more complex biological systems, and the Brown and Rose method was applied to various tissues, including chicken gizzard smooth muscle;137 mouse,140 rat,140 and human167,168 cerebral cortex, mouse,139,169–173 rat,138,174–177 and human178 brain; rat cerebellum;175 bovine and mouse brain myelin;147,162,171,179 rat,180 and mouse159 brain synaptosomes; rat,181 mouse,170 and rainbow trout182 liver; rat,183 rabbit,184 and mouse170 lung; rat lung endothelium;185 and pig140,142 and mouse kidney.170 The relatively large number of papers dedicated to the preparation of a lipid membrane domain fraction from brain or brain substructures obviously reflects the interest for the supposed role of lipid membrane domains in neuronal development and in the pathogenesis of neurodegenerative diseases,186 and recently detergent-insoluble membrane fractions were prepared from brains obtained from Alzheimer’s disease patients167,178 or from the Tg2576 transgenic mice with this pathology.173,187 These lines of research are very appealing, but additional care is necessary to evaluate the results obtained analyzing DRM fractions prepared from tissues. The complexity of these biological systems has several implications. Starting from a tissue, usually a more complex sample preparation is required before detergent solubilization (at least a homogeneization step, but in some cases a complex procedure such as myelin or synaptosome isolation). A DRM fraction from tissue would be more likely contaminated by detergent-insoluble extracellular matrix components. Moreover, such a fraction would contain lipid membrane domains originated from heterogeneous cell populations. In this case, the possibility that different lipid membrane domains could artifactually coalesce due to the presence of the detergent is particularly worrying. In the case of rodent brain, it has been shown that, when DRM fractions prepared from rat and mouse brain after solubilization in Triton X-100 are mixed together, rat and mouse Thy-1 were co-purified by immunoaffinity, indicating that fusion of distinct lipid membrane domains did occur with this widely used detergent.139 On the other hand, rat and mouse Thy-1 domains could be immunoseparated when the brains were solubilized in Brij 96. Under these experimental conditions, the glial GPI-anchored protein NCAM-120 (usually recovered in the Triton-resistant lipid membrane domain fraction) was fully solubilized, suggesting that it is indeed possible to discriminate between glial and neuronal lipid membrane domains using detergents with different stringency. The same authors were able to separate domains surrounding different neuronal GPI-anchored proteins (i.e., prion protein, PrP, and Thy-1) from a brain Brij 96-resistant membrane fraction,139 and showed that these domains are structurally and compositionally different.139,174 Remarkably, when a PrP-enriched membrane domain was prepared from rat cerebellar neurons by immunoaffinity purification after lysis in Brij 96, it could not be separated from Thy-1-containing membranes.148 Moreover, in this experimental model, the use of both Triton X-100 and Brij 96 gave very similar results, suggesting that fusion of distinct subdomains does not occur. This suggests that different neuronal populations could bear a radically different pattern of membrane domain subpopulations, or that the same method applied to tissues or to cultured cells originated from the same tissue leads to detergent-resistant fractions that can be compared with difficulty. An unexpected concern about the use of detergents for the isolation of lipid membrane domain fractions from tissues was raised by a recent paper. Heffer-Lauc et al.188 showed that, in brain tissue sections treated with low Triton X-100 concentration at 4 C, extensive redistribution of gangliosides from the gray matter to the white matter occurs. Moreover, when exogenous gangliosides were added to mouse brain sections in the presence of Triton X-100 at 4 C, they were trapped by white matter areas. This indicates that some yet-unknown myelin component is likely able to efficiently sequester gangliosides derived from other brain portions. This phenomenon could not be restricted to brain, but as well be present in other tissues. Thus, the application of detergent-based methods for the preparation of lipid membrane domains from tissues still require a careful evaluation. In particular, only in a few cases a partial characterization of the lipid composition of DRM obtained from tissues has been carried out.167,168,174 This is of crucial importance, since lipid membrane domains are defined on the basis of their peculiar lipid enrichment with respect to the whole cell or cell membranes. Thus, to validate the use of a method for the preparation of a lipid membrane domain fraction, it is essential to quantitatively analyze the complete profiles of cholesterol, glycerolipid, and sphingolipid of the fraction. As discussed elsewhere, the methods for the preparation of lipid membrane domains fractions based on the insolubility in detergents are very sensitive to the specific experimental condition used (preparation of the sample, temperature, type and concentration of the detergent used, detergent/protein ratio). Taylor et al.147 accurately evaluated the protein composition of DRM

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fractions prepared from mouse myelin at 4 and 37 C using four different detergents (Triton X-100, CHAPS, Brij 96, and Triton X-102), clearly showing that each detergent was characterized by a different effectiveness in solubilizing myelin proteins, thus concluding that detergent insolubility is just one of the criteria that need to be fulfilled to conclude whether a protein is associated with a lipid membrane domain. This lesson is valid not only for DRM from tissues, but in this case should be kept in mind very well, considering the lack of comparative studies. For Triton X-100, the effect of various protein/detergent ratios and of temperature on the isolation of a detergent-resistant fraction from mouse brain was systematically evaluated. The results obtained by Parkin et al.142,168 showed that a light-scattering fraction can be separated by sucrose gradient centrifugation after solubilization of mouse cerebral cortex with a fixed 1% Triton X-100 concentration at different protein/detergent ratios, ranging from 15 to 2mg of protein/ml. Regardless of the protein/ detergent ratio, this fraction was always enriched in two lipid membrane domain marker proteins, alkaline phosphatase and flotillin. However, enrichment of lipid membrane domain marker proteins (flotillin, prion protein, and F3) in the detergent-resistant fraction increased when the protein/detergent ratio in the sample was decreased. Conversely, proteins usually excluded from the lipid membrane domain were found in it at high protein/detergent ratios. Another important piece of information is related to the effect of temperature on the detergent resistance of lipid membrane domain protein markers. In the case of mouse cerebral cortex, the lipid membrane domain markers flotillin, F3, prion protein, and alkaline phosphatase were detergent insoluble at both 4 and 37 C. This is in contrast to most reports on cultured cells, where lipid membrane domains seem to easily disaggregate at 37 C.66 Unfortunately, no indications are given on the lipid composition of the detergent-resistant fraction prepared under different conditions. On the other hand, the effect of temperature on Triton X-100-resistant membrane fractions prepared from bovine brain myelin has been shown to be dramatic. When a Triton X-100 extraction procedure was applied to purified myelin at 20 C, and the detergent extract was fractionated over a continuous sucrose density gradient, two distinct low density fractions were isolated.162 Both fractions were characterized by higher cholesterol/phospholipid and GalCer/ phospholipid ratios than the starting myelin preparation (however, quantitative data on lipid contents are not available), but could be differentiated on the basis of their GM1 content (again, only qualitatively) and enrichment in specific protein markers. In both fractions, myelin associated glycoprotein (MAG) was present, even if with different enrichments. It would be extremely interesting to study lipid membrane domains from cultured oligodendrocytes with the same method. In whole brain, MAG (whose expression is restricted to the periaxonal myelin membrane) was completely solubilized in Triton X-100 (in agreement with previous findings),147but it was almost entirely recovered in a Lubrol WX-resistant fraction.171 However, when a Lubrol WX-resistant fraction was prepared under the same experimental conditions from rat oligodendrocytes, a much lower amount of MAG remained detergent insoluble.171 A detailed lipid analysis would clarify in this case whether the different detergent solubility of MAG in oligodendrocytes and myelin under different experimental conditions is due to a different lipid membrane domain localization, or to factors that do not depend on a lipid-enriched membrane environment.

3.30.3.5 Isolation of Heterogeneous Membrane Subdomains Several lines of evidence obtained using artificial membrane models, isolated lipid membrane domain fractions, or intact cells support the notion that lipid membrane domains are not homogeneous entities, but that they rather exist as separate entities at the same time in the same or in different cellular membranes, or behave as dynamic entities evolving during time. Many attempts have been made to separate and characterize different subpopulations of lipid membrane domains. Different experimental protocols used for the preparation of lipid-enriched, low-density membrane fractions could represent tools to separate biochemically distinct lipid membrane domains or to dissect different levels of order within lipid membrane domains (another aspect of the possible heterogeneity of these structures). This concept is based on the assumption that different lipid membrane domains can be discriminated on the basis of their different solubility under defined experimental conditions. Studies with fluorescent antiglycolipid probes showed that a ‘classical’ Triton X-100-resistant membrane fraction prepared from rat cerebellum includes separate substructures positive for GM1, GD3, and GalCer.57 Relying on their different solubilities in two nonionic detergents, Triton X-100 and Brij 96, two different lipid membrane domains were separated from rat brain, containing two functionally unrelated neuronal GPI-anchored proteins, Thy-1 and PrP, characterized by a different lipid composition.139,174 Differential solubilization in Brij 96 and Triton X-100 allowed to separate lipid membrane domains with distinct features from rat basophilic leukemia cells189 and to isolate a novel P-glycoprotein-rich lipid membrane domain from a multidrug-resistant ovary cell line, distinct from caveolae and Triton X-100-insoluble lipid membrane domains.190 Different nonionic (Triton X-100, Brij 96, Triton X-102, Lubrol WX) or zwitterionic (CHAPS) detergents were used as a tool to separate distinct detergent-resistant domains from myelin, brain, and cultured oligodendrocytes.147,171 Again from myelin, two different populations of glycolipid- and cholesterol-enriched lipid membrane domains (likely


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representing caveolae-containing and noncaveolar lipid membrane domains) were discriminated, exploiting their differential solubility in Triton X-100 at 4 and 20 C.162 Different low-density membrane domain populations can be separated mouse brain synaptosomes in the presence of Triton X-100 or sodium carbonate.159 Alternatively, the availability of antibodies toward specific components of lipid membrane domains was sometimes exploited to develop highly specific methods for the immunoisolation of lipid membrane domains.59,129,141,153,155,191–193 In some cases, this approach allowed to demonstrate that low-density DRM fraction can be further fractionated into different subpopulations with strikingly different structure and function. Since caveolins are probably the best-studied proteins belonging to a lipid membrane domain, it is not surprising that anticaveolin antibodies were used to discriminate between plasmalemmal vesicular fraction corresponding to caveolar membrane domains124,154,158,161,194–196 and immunoaffinity-purified noncaveolar membrane domains. Immunoisolated caveolae and detergent-resistant, lowdensity caveolar membrane domains show a closely overlapping molecular architecture and similar functional properties (discussed in Section 3.30.3.7). A lipid membrane domain-rich subpopulation of microvillar vesicles was isolated from enterocytes using anti-galectin-4 (a member of the galectin family of b-galactoside-binding proteins) antibodycoupled magnetic beads, and galectin-4 is quantitatively recovered in the Triton X-100-resistant membrane fraction from these cells.197 Since a high enrichment in glycosphingolipids is a general feature of lipid membrane domains, particularly interesting are the immunoaffinity isolation methods relying on the use of antiglycolipid antibodies. Antiglycolipid-antibody immunoisolated DRMs have been demonstrated to be involved in various aspects of signal transduction, and hence have been termed GSDs.198 Anti-GM3 ganglioside monoclonal antibody DH2 was used to immunoisolate GM3-enriched DRM from melanoma196 and neuroblastoma cells.133 Anti-GD3 ganglioside monoclonal antibody R24 was used to isolate a DRM fraction from differentiated rat cerebellar neurons,161 as well as from human fibroblasts (A. Prinetti and S. Sonnino, unpublished results). Anti-LacCer monoclonal antibody Huly-m13 was used to isolate LacCer-enriched GSD from human neutrophils. Two membrane subfractions were separated from lowdensity Triton X-100-resistant membrane fractions from B16 melanoma cells by anti-GM3 ganglioside monoclonal antibody DH2 and by anticaveolin antibody, respectively. The anti-GM3-immunoisolated subfraction was enriched in GM3 and contained SM, cholesterol, c-Src, and Rho A, but not caveolin, while the anticaveolin-immunoisolated subfraction did contain caveolin, glucosylceramide, SM, and a large amount of cholesterol, but not GM3, c-Src, or Rho A.196 The GM3-enriched subfraction, but not the caveolar subfraction, was involved in cell adhesion-dependent signal transduction in these cells. Immunoaffinity isolation was used to separate from mouse brain two low-density DRM domain subpopulations, containing two functionally different neuronal GPI-anchored proteins, Thy-1 and PrP, respectively. Thy-1 DRM and PrP DRM significantly differ in their sensitivity to detergent solubilization, thus probably reflecting a different degree of ordered packing of lipids.

3.30.3.6 Lipid Membrane Domains in Natural Membranes The existence of lipid membrane domains in natural cell membranes was suggested by the observation that glycosphingolipids at the cell surface form clusters, which have been visualized by immuno-electron microscopy using antiglycosphingolipid antibodies.198 Glycosphingolipid clustering in cell membranes was showed for globoside in human erythrocytes,199 polysialogangliosides in fish brain neurons,200 and GM3 ganglioside in peripheral human lymphocytes and Molt-4 lymphoid cells.122 Several approaches, relying on more advanced technologies, are now available allowing the detection and the study of lipid membrane domains in intact cell membranes.201,202 These techniques are very heterogeneous and include single-particle tracking or single fluorophore tracking microscopy,203–205 fluorescence recovery after photobleaching,206 fluorescence resonance energy transfer,207 and atomic force microscopy,208 and data obtained with different approaches are sometimes conflicting. As an example, there is no agreement on their average size that ranges from 26nm to about 2mm.205–209 In addition, from these data, no information on what composes the lipid membrane domains can be inferred. As discussed in Sections 3.30.3.2 and 3.30.3.3, low-density, detergent-insoluble fractions enriched in lipid membrane domains were isolated and characterized from a wide variety of mammalian cells, yeasts,210 protozoans,135 and tissues.137–142 However, still some aspects regarding the nature and role of lipid membrane domains remain obscure. In the next paragraphs, the focus is on two of these aspects.

3.30.3.7 Lipid Membrane Domains, Caveolae and Caveolins, Noncaveolar Domains In the 1990s, the concept of ‘lipid rafts’ as restricted membrane areas specialized in specific functions evolved from the confluence of experimental results coming from very heterogeneous research areas. Knowledge on the segregation or

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clustering of specific membrane components (glycosphingolipids, GPI-anchored proteins) within the cell membrane and on the existence of caveolae,211–213 subcellular structures morphologically defined as spherical or flask-shaped invaginations of the plasma membrane or associated vesicles, seemed to be unified by the observation that both segregated membrane components and caveolae components behave as detergent insoluble complexes. Lipid membrane domains and caveolae share another property: both are sites for clustering of proteins, and this could represent a mechanism for regulating cell signaling and other cellular events (such as endocytosis214–216 and entry of pathogens217). Caveolae were morphologically described over 50 years ago.211,212 More recently, it has been shown that caveolae69,218 are characterized by the presence of 21–24kDa integral polypeptides, termed caveolins219,220 as their main structural protein components. Caveolins form high-mass oligomeric complexes, providing a scaffold for caveolin-interacting proteins (including H-Ras,149 c-Src, heterotrimeric G proteins,149,221 and growth factor receptors),153,222 that can thus be concentrated within caveolae membranes.223–224 Mainly based on this observation, it has been hypothesized that caveolae may act as specialized plasma membrane structures able to assemble and coordinate the function of signal-transducing protein complexes, in which caveolin should play a pivotal role as scaffolding molecule.224–228 The presence of caveolins as specific scaffolding proteins represents the distinctive feature of caveolae membranes.219 Caveolins (at least, caveolin-1 and caveolin-3) are necessary for the formation of caveolae (experiments with knockout mice).229,230 Caveolin-1 transfection in cell lines lacking caveolin and caveolae resulted in the formation of morphologically distinguishable caveolae in some cell types (lymphocytes, Fischer rat thyroid cell)231,232 but not in others (the human prostate cancer cell line LNCaP).232 Caveolin-1 seems thus an essential component of caveolae, even if its presence on the cytoplasmic face of caveolae was somehow challenged by the finding that caveolin-1 is rather present in intramembrane particles interacting with caveolae surface.233 Caveolin-1 is also found at many other intracellular locations, including the trans-Golgi network and in other organelles,234,235 and caveolin-1 antibodies also bind to flat membrane portions.236 As mentioned above, caveolae share several properties with lipid membrane domains, such as a peculiar lipid composition, resistance to solubilization by cold nonionic detergents, and a low buoyancy on sucrose density gradients. In all cell types so far investigated, cell sphingolipids, together with cholesterol and saturated PC, are associated with a low-density membrane fraction that can be prepared by flotation on sucrose gradient after cell lysis in the presence of Triton X-100 or other nonionic detergents.143 As discussed elsewhere in this chapter, this fraction is usually regarded as a lipid membrane domain-enriched fraction. When this procedure has been applied to cells expressing caveolins, caveolins have been found to partition into the same Triton-insoluble, low-buoyancy membrane fractions,120 and these fractions have been often considered to represent isolated caveolae fractions. However, the relationship between caveolae and lipid membrane domains is much more complicated. Detergent-insoluble domains are also found in cells lacking caveolin expression and caveolae structures, including several neuronal cell types (indeed, for many authors, the postulated equivalence between caveolae and lipid membrane domains has been so much emphasized, that terms such as ‘caveolae-like domains’ or ‘noncaveolar domains’ were coined to describe lipid membrane domains in the absence of caveolins or caveolae).37,71,123,129,133,138,161,237,238 Moreover, caveolin-1 may exist in lipid membrane domains without the formation of caveolae: as for example, in MDCK cells, caveolae are present only on the basolateral but not on the apical surface. Caveolin-1 in these cells is present in caveolae at the basolateral membrane, but as detergent-insoluble, cholesterol-dependent complexes at the apical membrane.239 In the human prostate cancer cell line LNCaP, where caveolin-1 transfection did not correspond to the formation of caveolae, detergentresistant lipid membrane domains exist, independently from the expression levels of caveolin (also see Chapter 4.22).232 The immortalized murine gonadotropin-releasing hormone neuronal cell lines GN11 and GT1 represent immature, migrating neurons and nonmigrating fully differentiated cells, respectively.240 The presence of caveolin-1 polypeptide was reported in the GN11 neuronal-like cell line, while, in contrast, GT1 cells are totally devoid of caveolin.241 GT1 cells are characterized by a very high glycosphingolipid level (mainly represented by GM3 ganglioside), if compared with GN11 cells. From both cell lines, it was possible to prepare a Triton-insoluble, low-density membrane fraction similarly enriched in sphingolipids, that, in the case of caveolin-expressing GN11 cells, is also rich in caveolin.241 Thus, it is clear enough that caveolae and lipid membrane domains can exist independently. A growing number of experimental data support this concept. Fluorescence microscopy and EM studies showed that GPI-anchored proteins are not constitutively concentrated in caveolae. Their caveolar clustering only occurs upon multimerization triggered by cross-linking.242 When a plasmalemmal fragment population was obtained by a detergent-free silica coating procedure from rat lung vasculature, and a caveolae fraction was further purified by specific immunoisolation using anticaveolin-coated magnetic beads, this caveolae fraction was devoid of a number of proteins involved in signal transduction that are usually recovered in the detergent-resistant (lipid membrane domain) fraction.194 Caveolae and caveolin-1 are involved in the modulation of growth factor receptors signaling,222 and EGFR is localized within a


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caveolin-rich fraction in A431 cells. However, EGFR-containing membrane fragments can be separated from caveolae.154,243 The noncaveolar EGFR-enriched membrane domain behaves as a classical lipid membrane domain, that is, it is Triton X-100 insoluble, enriched in GM1 ganglioside, and sensitive to the manipulation of cholesterol levels.244,245 The question to be answered is whether caveolae or caveolin-containing membrane domains are lipid membrane domains or not. This question is of course not merely semantic, but involves the possible role of lipids as structural and functional components of caveolae-based signaling complexes within the plasma membrane. Many papers report on the relationship between caveolae/caveolins and cholesterol. Caveolae are disassembled by cholesterol-binding drugs.219 EM experiments with cholesterol-binding probes showed that caveolae are enriched in cholesterol.246 Caveolin-1 binds free cholesterol and cholesterol-containing artificial phospholipid liposomes.247–249 Caveolin oligomerization in cell membranes depends on the cholesteol level.250 Similarly, EM immunogold labeling experiments using antilipid antibodies or cholera toxin suggested that caveolae are enriched in GM1 ganglioside, neutral glycolipids, and SM.236,250,251 However, in the case of neutral glycolipids and SM, caveolar localization only occurs after antibody cross-linking. The presence of SM and ceramide was observed in a Triton X-100-resistant caveolae fraction from human fibroblasts,252 and GM1 was detected by cholera toxin labeling in a purified caveolae fraction prepared from rat lung endothelium.251 Caveolin-1 has been shown to bind photoreactive derivatives of GM1 and GM3 in A431 and MDCK cells.60,253 However, kinetic studies showed that the interaction between GM3 and caveolin-1 in MDCK is a transient process: it occurs shortly after the incorporation of the ganglioside derivative in the plasma, but is lost after a 24 h chase, suggesting that a redistribution of the ganglioside takes place. Indeed, EM experiments showed that in these cells caveolin-1 and GM3 are not localized in the same domain at the steady state.60 Thus, the information about the interaction of specific sphingolipids with caveolin and their presence in caveolae is scant, fragmentary, and somehow contradictory. Indeed, the question raised by Fujimoto in 1996, whether sphingolipids are concentrated in the purified caveola fraction or not, still remains to be answered.236

3.30.3.8 Lipid Membrane Domains from Intracellular Membranes One of the most widely used terms to describe membrane microdomains characterized by the segregation of lipids is certainly ‘lipid rafts’. Van Meer and Simons254 introduced this term in 1988, following the observation that the apical and basolateral membranes of polarized epithelial cells are characterized by a distinct lipid composition. Based on this observation, they hypothesized that the segregation of newly synthesized proteins in microdomains with a distinct lipid composition might be responsible for the sorting of these proteins to the apical or basolateral membrane. A specific role for sphingolipids as organizers during the formation of these traffic-oriented structures (‘rafts’) has been proposed.255 This hypothesis met an enormous success, and a huge number of experimental papers followed along this line (reviewed in Refs. 237 and 256–259). However, only a very limited number of investigations were directed to verify the assumption that segregation of lipid in domains occurs in intracellular membranes, and only a very limited knowledge is available of the lipid composition of putative intracellular domains. It has been recently shown that neither newly synthesized gangliosides nor the enzymes responsible for their synthesis appear to be segregated in microdomains in Golgi membranes.260 Thus, it has to be assumed that sphingolipid segregation and consequent lipid raft formation take place later on along the secretory pathway, or occur at the plasma membrane level. The latter explanation would strongly argue against a role of sphingolipids in a lipid raft-based protein traffic and sorting mechanism. Some experimental data support the notion that sphingolipid segregation indeed occurs in the Golgi distal membranes or in vesicles of the early secretory pathway. A detergent-resistant fraction has been prepared and characterized from Golgi membranes, showing an SM and cholesterol enrichment with respect to glycerophospholipids which reflects that observed for whole cell- or plasma membrane-derived DRM.70,261 Moreover, it has been shown that segregation of SM and cholesterol occurs during the genesis of coat protein I vesicles from the Golgi in the early secretory pathway.262 The existence of lipid membrane domains in terms of detergent solubility, sensitivity to lipid depletion, and marker protein distribution consistent with those of whole cell lipid membrane domains has been shown in the endoplasmic reticulum,263 in the trans-Golgi network/endosomal compartment,264 in membranes derived from intracellular vesicles (platelet alpha granules,265 intracellular vesicles carrying the Glut4 glucose transporter,266 neutrophil granules),267 in lysosomal membranes,268 and it has been suggested by indirect evidence in mitochondrial membranes.269 However, lipid composition and the role of different lipids (e.g., SM vs glycosphingolipids, sphingolipids vs cholesterol) are still largely unknown.

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3.30.3.9 Artificial Manipulation of Lipid Membrane Domain Composition A very promising experimental approach to better understand the involvement of lipids in the structural and functional properties of lipid membrane domains is represented by the artificial manipulation of lipid composition and/or organization within these structures. To this purpose, a wide variety of experimental approaches has been used to modulate the levels of cholesterol and sphingolipids in lipid membrane domains: (1) pharmacological inhibition of cellular cholesterol synthesis; (2) sequestration or perturbation of cholesterol in the plasma membrane by cholesterolbinding agents; (3) removal of cholesterol from cells by cyclodextrins; (4) pharmacological modulation of cellular sphingolipid synthesis; (5) administration of exogenous sphingolipids. The modification of cellular sphingolipid content at the genetic level, by modulating the expression of sphingolipid metabolic enzymes, also represents a promising tool, but it has never been successfully exploited so far.

3.30.3.9.1 Drugs affecting cholesterol availability Among drugs that are able to downregulate the biosynthesis of cell cholesterol, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins, including lovastatin and mevastatin, usually in combination with mevalonate)128,205,270–277 and the squalene synthase inhibitor squalestatin271 have been used to reduce cholesterol levels in lipid membrane domains and to affect lipid membrane domain-dependent cellular functions. Alternatively, lowering of cholesterol levels in the plasma membrane with possible consequences on lipid membrane domain structure and function has been achieved using external ligands127,128,130,273,278–282 that are able to sequester cholesterol in the plasma membrane (filipin, nystatin, digitonin, saponin, perfringolysin, streptolysin, amphotericin B) or to remove cholesterol from the cells.125,196,283–290 In the former case, the mechanism by which membrane cholesterol is affected is often not clear, but a general feature is that these drugs require incorporation into the cell membrane to interact with cholesterol; thus, they likely induce great perturbations in membrane structure. This probably accounts for the high cytotoxicity observed for many of these compounds. At least in the case of filipin, it has been shown that the drug binds indiscriminately to membrane cholesterol, including plasma membrane as well as intracellular membrane cholesterol.125,289 Oxidation of plasma membrane cholesterol by addition of exogenous cholesterol oxidase has been also used. In this case, the question arises, what would be the fate and the effect of oxidized cholesterol (cholestanone) generated on the cell membrane.276,277,290,291 Among these tools, the extraction of cholesterol from cells by treatment with cyclodextrins is certainly the most widely used, as proven by about 160 papers found by a Medline search with the two keywords ‘cyclodextrins’ and ‘membrane microdomains/lipid rafts’ from 1996 to the present day. Cyclodextrins are cyclic 1,4-linked oligomers of D(þ)-glucopyranose.292 Structures with six, seven, and eight glucose units are called a-,-b- and g-cyclodextrins, respectively. D-glucose units form a ring structure with a hydrophobic cavity and an exposed side that is hydrophilic, due to the presence of hydroxyl groups. Cyclodextrins bind an impressive variety of small molecules, including nonpolar aliphatic molecules, amphipatic lipids, and polar compounds such as amines and organic acids. Chemical modification of hydroxyl groups (e.g., methylation) is often used to obtain cyclodextrins with different solubility and binding properties. Cyclodextins have been in use for many years as carriers of lipophilic drugs in pharmacological research, but only relatively recently they has been introduced as a tool in membrane studies. The addition of cyclodextrins to cultured cells is able to induce the release of cholesterol from the cells themselves. The process has been shown to be cyclodextrin concentration, time, and cell type dependent.127,128,130,197,273,278–282,293 The mechanism by which cholesterol is lost by the cells in the presence of cyclodextrins is not clear, but this process probably involves different plasma membrane cholesterol pools,278,280 but not the intracellular cholesterol pools.278 Cyclodextrins bear unique features as cholesterol-removing agents. Unlike almost all the above-mentioned cholesterol-binding agents, cyclodextrins are not incorporated into membranes, but rather they act at the surface and likely extract membrane cholesterol by including it in the central cavity of the oligomer ring.294 Many studies have shown that cyclodextrin-induced removal of cholesterol from cells leads to the disorganization of lipid membrane domains, to the shift of lipid membrane domain constituents into the bulk plasma membrane or to their release in the culture medium, and to changes in the properties of proteins associated with the lipid membrane domain, that can ultimately lead to the blockade of putative lipid membrane domain-dependent cell functions.127,128,130,190,197,273,278–282,289,290,293,295–303 Frequently, the ability of cyclodextrins to interfere with cellular events has been automatically equated with the dependency of these events on cell cholesterol or on ‘cholesterolenriched’ membrane domains. However, several criticisms can be raised against this simplistic assumption and great care should be taken to interpret experimental results obtained using cyclodextrins and other cholesterol-depleting tools. There is no doubt that these treatments are able to effectively reduce cellular cholesterol level or availability. In the case of cyclodextrins, used alone or in combination with inhibitors of cholesterol synthesis, it is possible to reduce


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by 70–90% the cell cholesterol content.128,275,302 In such conditions, it is unavoidable to wonder whether the resulting effects are cholesterol specific or simply due to the loss of the second most abundant membrane component. Again, this probably explains why effective concentrations of these drugs are often very close or overlapping with cytotoxic concentrations. Moreover, the amount of cholesterol removed from the cells under these experimental conditions likely exceeds the cholesterol present in lipid membrane domains.275 Thus, the treatment probably affects as well membrane areas not organized as lipid membrane domains, and can thus hardly be taken as a proof of a ‘raftdependent’ biological event. The sensitivity of lipid membrane domains to cyclodextrins can be different in different cells, and the sensitivity of lipid membrane domains and lipid membrane domain-dependent events to cholesterol depletion can greatly vary depending on the experimental approach used to lower cholesterol levels (e.g., GPIanchored proteins are dispersed at the cell surface upon saponin treatment in BeWo cells284 or inhibition of cholesterol synthesis by mevastatin in MA104 cells,283 but not by lovastatin or squalestatin in Jurkat T-cells).271 This can be envisaged as an indication that different lipid membrane domains exist, with different dependence on cholesterol. However, it can as well depend upon the exact conditions under which the depletion is carried out. The efflux of cholesterol mediated by cyclodextrin has been studied in details, but very poor is the information on the effect of cyclodextrins on the possible removal of other complex lipids from living cells.197,280,281,293 In the case of gangliosides, only a qualitative information on the efflux of GM1 ganglioside from lymphocytes278 is available. However, it is known that cyclodextrins are able to form complexes with sphingolipid monomers304–306 and to disrupt ganglioside aggregates.305 The property of cyclodextrins to disrupt micellar aggregates of GM1 or mixed aggregates of glycerophospholipids and GM1, and their ability to form complexes with ganglioside monomers, have been studied in detail by NMR spectroscopy and biochemical procedures.304–307 Using quantitative analytical approaches, we showed that methyl-b-cyclodextrin, MCD, was able to induce the release of sphingolipids, cholesterol, and glycerophospholipids from rat cerebellar granule cells in culture, with concentration-dependent effects on the existence and properties of lipid membrane domains (similar results have been obtained using (2-hydroxypropyl)-b-cyclodextrin, indicating that they are not specific for a certain type of cyclodextrin).275 The amounts of sphingolipids, glycerophospholipids, and cholesterol released by a 30min treatment with 5mM MCD were 16%, 2%, and 50% of total cell lipid, respectively (Table 8). In the case of this ‘mild’ treatment, the loss of cell lipids induced a deep reorganization of the remaining membrane lipids. In fact, the amount of PC associated with a Triton X-100-insoluble membrane fraction (highly enriched in

Table 8 Efflux from rat cerebellar granule cells differentiated in culture of cholesterol, sphingomyelin (SM), glycosphingolipids (GSs) and glycerophospholipids (GPLs) as a function of methyl-b-cyclodextrin (MCD) concentration and time of treatments Experimental conditions

Quantity in the medium after treatment (% on the total cell content)

Incubation time(min)

MCD concentration(mM)

Cholesterol

SM

GS

GPL

5 5 5 5 10 10 10 10 30 30 30 30 60 60 60 60 120 120 120 120

0 1 5 10 0 1 5 10 0 1 5 10 0 1 5 10 0 1 5 10

1 16 17 64 1 17 25 73 1 18 50 90 1 25 65 91 1 38 66 91

2 7 7 8 2 4 18 19 2 7 18 38 3 7 20 60 7 10 20 61

1 1 2 2 1 3 12 12 1 3 23 33 2 8 14 38 4 13 25 47

0 1 2 12

Blank cells indicate not determined.37,275

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sphingolipids and cholesterol in nontreated cells) was lowered by the treatment. This suggested a reduction of the lipid membrane domain area. However, the cholesterol and sphingolipid enrichment of this fraction remained substantially unchanged, suggesting the existence of dynamic processes aimed to preserve the segregation of cholesterol and sphingolipids in membrane domains. Under these conditions, the lipid membrane domains retained their property to sort signaling proteins such as Lyn and c-Src, but cells displayed deep alterations in their membrane permeability. When MCD treatment was more stringent (30 min with 10mM MCD), a large loss of cell lipids occurred (40%, 10%, and 90% of total cell sphingolipids, glycerophospholipids, and cholesterol were lost, respectively), the lipid membrane domains were much less enriched in cholesterol and lost the property to sort specific proteins. The loss of the integrity and properties of lipid membrane domains was accompanied by severe changes in the membrane permeability, sufferance, and eventually cell death. Thus, the use of cyclodextrins and other drugs affecting cholesterol synthesis, cellular levels, or availability is of potentially great utility for studies related to lipid membrane domains, but the following aspects need to be verified for each experimental model: (1) the quantitative effectiveness of the drug treatment in lowering cholesterol availability; (2) the overall disturbing effect on the integrity of the cell membrane; (3) the effect on membrane lipids other than cholesterol; and (4) the effect of membrane areas other than lipid membrane domains.

3.30.3.9.2 Drugs affecting sphingolipid metabolism As amply discussed, spontaneous segregation of (glyco)sphingolipids in cell membranes is one of the main driving forces responsible for the formation of lipid membrane domains in living cells. For this reason, the availability of experimental approaches able to modulate (glyco)sphingolipid cellular levels would provide tools allowing to manipulate the integrity and dynamics of lipid membrane domains as well as to dissect the possible functional role of specific sphingolipids within these domains. Fumonisins are mycotoxins from Fusarium moniliforme that are common grain contaminants responsible for food poisoning episodes all over the world. Fumonisins are powerful inhibitors of sphinganine N-acyltransferase, that catalyses the synthesis of dihydroceramide as the biosynthetic precursor of ceramide and of all complex sphingolipids,316 and fumonisin B1 has been widely used in the past to inhibit cell sphingolipid biosynthesis,317,318 and more recently to study the effects of sphingolipid depletion on lipid membrane domains’ stability and function. Upon treatment with fumonisin B1, it has been shown that GPI-anchored proteins that usually associate with DRM domains become detergent soluble (e.g., Thy-1 in hippocampal neurons), structure of lipid membrane domains is altered, and putative lipid membrane domain biological functions are impaired.128,319–325 However, incubation with fumonisin B1 will potentially affect the levels of all cell sphingolipids, ceramide, SM, and glycosphingolipids, in a way that is strongly dependent on the experimental conditions used (time and inhibitor concentration). Since a different role for specific sphingolipids has been hypothesized within lipid membrane domains, it is thus necessary to verify (1) that the experimental conditions used are effective in lowering the cell sphingolipid levels; (2) that this reflects on the composition of the lipid membrane domain; and (3) which sphingolipids are affected by fumonisin treatment. This limitation is only partially overcome by the availability of enzyme inhibitors that only affect the metabolism of glycosphingolipids. D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was the prototype of a family of synthetic inhibitors of glusosylceramide synthase, that catalyzes the synthesis of glucosylceramide, as the common precursor of all glucosylceramide-based complex glycosphingolipids.326 Analogs (D-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, PPPP) with higher inhibition specificity and enhanced potency has been developed later on.327 D-PDMP or PPPP treatment was able to deplete glycosphingolipids from a detergent-insoluble lipid membrane domain fraction in Lewis lung carcinoma cells,328 T lymphocytes,329 neuroepithelial cells,330 F9 embryonal carcinoma cells,331 normal and transformed lung fibroblasts,332,333 and bladder cancer cells334 without affecting the cholesterol and SM levels in the domain328,329 nor the overall integrity of the domain itself. However, this treatment strongly reduced the association of Src family kinases with the lipid membrane domain fraction in most of the above-mentioned cell types,330–334 suggesting a specific and essential role of glycosphingolipids in Src-mediated lipid membrane domain functions. The specific role of glycosphingolipids in lipid membrane domain functions is further supported by the fact that effective depletion of gangliosides in neuroblastoma cells by PPPP and another, nonrelated glucosylceramide synthase inhibitor, N-butyldeoxynojiromycin, was not able to alter the lipid membrane domain localization and drug efflux activity of ABC transporters.335 Remarkably, when comparatively studied, the effects of cholesterol depletion and of sphingolipid depletion on the structure and function of lipid membrane domains are not overlapping.329,331 D-PDMP treatment and cholesterol depletion with cyclodextrin had opposite effects on the function of lipid membrane domain-associated GPI-anchored proteins, likely affecting in a different way their organizational status (in T lymphocytes, D-PDMP increased


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susceptibility of GPI proteins to phosphatidylinositol-phospholipase C (PI-PLC) cleavage, while methyl-b-cyclodextrin decreased it).329 In F9 carcinoma cells, both methyl-b-cyclodextrin and D-PDMP inhibited the localization of Src-family kinases in the domain, but methyl-b-cyclodextrin only affects the association of other marker proteins to the detergent-resistant fraction.331 Fumonisin B1 treatment and cholesterol removal differently affects the Triton X-100 insolubility and sorting of GPI-anchored proteins in polarized Fischer rat thyroid cells.320 This indicates that the structural integrity of lipid membrane domains is differently affected by the presence of cholesterol or sphingolipids (possibly indicating the existence of different degrees of order in the lipid segregation within lipid membrane domains), but also suggests that different lipids might specifically interact with other components of the domains. However, the use of glucosylceramide synthase inhibitors hardly allows to dissect the roles of different glycosphingolipids within lipid membrane domains, and anyway requires, as for fumonisin, a careful evaluation on the effects of this inhibitors on the cell lipid composition.328 Selective depletion of cell surface sphingolipids can be achieved by treating living cells with bacterial sphingomyelinases,289,319 able to hydrolyze SM, effectively decreasing its cellular levels, or with endoglycoceramidase, able to hydrolyze cell surface glycosphingolipids.129 Exogenous sphingomyelinase (similarly to methyl-b-cyclodextrin and filipin) treatment on colonic epithelial cells caused activation of the basolateral large conductance Ca2þ-activated Kþ channel and its redistribution to a detergent-insoluble membrane fraction.289 Both bacterial sphingomyelinase and fumonisin B1 treatments were able to reduce the amount of SM in detergent-insoluble membrane fractions without affecting the distribution of GM1, PrP, and caveolin in neuroblastoma cells.319 Endoglycoceramidase inhibited lipid membrane domain-dependent TAG-1 signaling in cerebellar neurons.129 One of the major concerns about the use of these sphingolipid-degrading enzymes is that their application likely affects ceramide levels within the membrane. On the other hand, they could represent a tool to increase ceramide levels (or at least the ceramide/SM ratio) in order to evaluate the specific effect of this sphingolipid in lipid membrane domains. One simple way to affect the cellular levels of specific glycosphingolipids is represented by their exogenous administration to cultured cells. It has been shown in a large number of papers that exogenously administered sphingolipids are incorporated into the plasma membrane, and subsequently enter the traffic and metabolism routes of endogenous lipids, becoming virtually indistinguishable from those. As discussed in Section 3.30.2, treatment with exogenous GM1 and GM3, but not LacCer, was able to induce dissociation of Csk from the DRM domain in neuroblastoma cells, with consequent activation of c-Src and mitogen-activated protein kinases and neuritogenesis.133 On the other hand, exogenous addition of GM3 to highly invasive YST1 bladder cancer cells caused Csk traslocation to the detergent-insoluble fraction and consequent inactivation of c-Src, negatively influencing cell motility.334 This approach is very interesting; however, it is difficult to ascertain the real amount of sphingolipid incorporated in the membrane and reaching the lipid membrane domain. Moreover, the administered sphingolipid can be metabolically converted into other molecules, complicating the evaluation of the real specificity of the observed effects.

3.30.4 Roles of Glycosphingolipids in Lipid Membrane Domain Functions 3.30.4.1 Glycosphingolipids and Lipid Membrane Domains in the Nervous System Detergent-insoluble low density fractions with features corresponding to those of lipid membrane domains (enrichment in cholesterol and sphingolipids, presence of GPI-anchored proteins, and other protein markers for lipid membrane domains (including Src family protein kinases)) can be prepared from cultures of neural cells (neurons, oligodendrocytres, astrocytes and related tumor cell lines, including neuroblastoma cells), from brain, myelin synaptic plasma membranes177,336 (see Section 3.30.3.4). Caveolae and caveolins are rare in the nervous system (even if conflicting reports and opinions exist about the presence and role of both in the nervous system), and it has been suggested that flotillins could have in nervous system lipid membrane domains a scaffold role similar to that hypothesized for caveolins in extranervous lipid membrane domains.337 Neuronal and glial lipid membrane domains are rich in gangliosides, SM, cholesterol, and proteins involved in mechanisms of signal transduction that are relevant for neuronal functions (receptor tyrosine kinases, including neurotrophin receptors Trk A, Trk B, Trk C, c-Ret, ErbB, the ephrin receptor Eph), GPI-anchored receptors (the GDNF family receptor GFRa), G protein-coupled receptors (including cannabinoid receptors and neurotransmitter receptors such as a1-, b1-, b2-adrenergic, adenosine A1, g-aminobutyric acid GABAb, muscarinic M2, glutamate metabotropic mGLUR, serotonin 5HT2), nonreceptor tyrosine kinases of the Src family, adapter and regulatory molecules of tyrosine kinase signaling, heterotrimeric and small GTP-binding proteins, protein kinase C isoenzymes,

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cell adhesion molecules (integrins, Notch1, NCAMs, TAG-1, Thy-1, F3/contactin), ion channels, proteins involved in neurotransmitter relase, proteins belonging to postsynaptic density complexes).59,71,129,133,138,152,161,196,228,238,337–360 Lipid membrane domains in the nervous system cells have been involved in neurotrophic factor signaling,337–340 cell adhesion and migration,338,347,354 axon guidance, synaptic transmission,338,346 neuron–glia interactions,355,356 and myelin genesis.357 The involvement of lipid membrane domains in neuronal and glial signal transduction includes several different ways. 1. Receptors and effector proteins permanently resident in lipid membrane domains can be activated, giving rise to signal propagation that involves other components intrinsically present in the lipid membrane domain. Examples are neurotrophin receptors of the trk family, EGFR, PDGFR, p75NTR, GFRa,337–340 and the neural cell adhesion molecule TAG-1.129,358,359 Src family tyrosine kinases are among the effector signaling proteins that are most commonly engaged in these cases. 2. The activation of membrane receptors is followed by the recruitment to lipid membrane domains of receptors themselves or effector signaling proteins that are not located in lipid membrane domains under basal conditions, or the activation of receptors that are associated with lipid membrane domains under resting conditions determines their translocation outside lipid membrane domains. Examples of the former are the receptor tyrosine kinase c-Ret, recruited into lipid membrane domains by its GPI-anchored co-receptor GFRa,337,338,340 and the neuronal adhesion receptor NCAM, recruited into lipid membrane domains by cis- or trans-interaction with its membrane-bound, GPIanchored ligand, prion protein.354 Both modes imply changes in the reciprocal interactions of lipid membrane domain components. One question that remains to be answered is regarding the role of lipid molecules in these events. Are lipids active players in the functional dynamics of lipid membrane domains? This question is particularly relevant in the nervous system. Glycosphingolipids, particularly gangliosides, are crucial in the control of the functional properties of neurons and other cell types of neural origin. They have been implicated as modulators of various aspects of neural cell function (see Chapter 4.18).312,313 Glycosphingolipid patterns in the nervous system undergo deep qualitative and quantitative modifications during development, differentiation, and neoplastic transformation.361,362 Their biosynthesis is necessary for the differentiation of neurons in culture,317 and induced expression of GD3 synthetase is able to switch neuroblastoma cells to a differentiated phenotype.363 Exogenously added gangliosides exert neuritogenic, neurotrophic, and neuroprotective effects on a variety of cell systems of neural origin.25,312,364 The role of glycosphingolipids in the maintenance of neuronal structure and function can be at least in part explained by their ability to interact with specific proteins (including growth factor receptors and neuronal adhesion molecules) at the level of the plasma membrane and to modulate their activity. Possible interactions with functional significance between gangliosides and proteins at the level of the plasma membrane have been intensively studied in the past.313,365–367 A new key to understand glycosphingolipid-mediated neural events is represented by the co-localization of glycosphingolipids and regulated proteins within lipid membrane domains. Changes in the lipid and/or protein composition of lipid membrane domains and in the interactions of the lipid (in particular, glycosphingolipid) components with specific proteins of functional relevance could thus be very relevant during the process of neuronal adhesion, survival, migration, differentiation, and senescence. The lipid composition of a lipid membrane domain fraction prepared by lysis in cold 1% Triton X-100 and sucrose density gradient centrifugation from rat cerebellar granule neurons during spontaneous differentiation has been characterized in detail37,71,359 (Tables 6 and 7). In fully differentiated rat cerebellar granule cells, 40% of the total cell cholesterol, 60% of the gangliosides, 67% of the SM, and 50% of the ceramide, but only about 8% of total cell glycerophospholipids were located in the lipid membrane domain fraction. All the gangliosides present in the cell homogenate were associated in a similar extent with the lipid membrane domain fraction. We calculated the molar ratio between glycerophospholipids, cholesterol, SM, ceramide, and gangliosides that were 42.8:6.1:1.3:0.3:1 and 5.6:4.0:1.4:0.2:1 in the cell homogenate and in the lipid membrane domain fraction, respectively. These data indicate that this fraction is highly enriched in sphingolipids. However, the major lipid components of these domains still were glycerophospholipids and cholesterol.71 Among glycerophospholipids, dipalmitoylphosphatidylcholine was particularly enriched in these domains, representing their main lipid component. We studied the properties of lipid membrane domains from rat cerebellar granule cells at different stages of development in culture.37 The surface occupied by these structures increased during development, with the maximum ganglioside density in fully differentiated neurons. On the other hand, a high content of ceramide was found in the domains of aging neurons. The sphingolipid/ glycerophospholipid molar ratio was more than doubled during the initial stage of development, corresponding to axonal sprouting and neurite extension, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased


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during in vitro differentiation. Phosphorylated phosphoinositides were very scant in the domains of undifferentiated cells and dramatically increased during differentiation and aging. Proteins were minor components of the domains (0.1–2.8% of all domain components) at all the stages of in vitro development we investigated. Src family protein tyrosine kinases (c-Src, Lyn and Fyn) known to participate in the process of neuronal differentiation were associated with the lipid membrane domains in a way specific for the type of kinase and for the developmental stage of the cell.37 Co-segregation of Src family proteins with sphingolipids in specialized domains in the membrane of cultured neurons seems to be a general feature of neuronal lipid domains. Within the lipid membrane domains, ganglioside GM3 has been found closely associated with c-Src and Csk in neuroblastoma Neuro2a cells,133 and GD3 associated with Src-family kinase Lyn and the neural cell adhesion molecule TAG-1 in rat brain138,358 and cerebellar granule cells. In these cells, a complex lipid environment characterized by the presence of many ganglioside species and other membrane lipids (mainly cholesterol and dipalmitoylphosphatidylcholine) is essential for the interaction with the domain of c-Src, Lyn, Fyn, TAG-1, and prion protein.37,148,161,238 The presence of Src family nonreceptor tyrosine kinases in lipid membrane domains of neurons is particularly interesting, because many lines of evidence indicate that c-Src and other kinases of this family are important in the process of neuronal differentiation. The expression and the activation of c-Src and Lyn are correlated with the stage of neuronal differentiation in neuroblastomas, neuroblastoma and embryonal carcinoma cell lines.368–372 The expression of a neuron-specific form of c-Src is increased during the differentiation of cultured neurons from rat embryo striatum,373 and c-Src activation is involved in the control of synaptic transmission mediated by N-methyl-Daspartate receptors374 and in the induction of long-term potentiation in hippocampal neurons.375 c-Src in PC12 cells interacts with synaptosomal and cytoskeletal proteins.376 Another tyrosine kinase, Csk, is a negative regulator of Srclike kinases, responsible for their C-terminal phosphorylation, keeping them in a repressed form.377 As mentioned above, in neuroblastoma Neuro2a cells, c-Src and Csk are associated with GM3 ganglioside within lipid membrane domain, and neuritogenic concentration of gangliosides is able to induce c-Src activation followed by mitogenactivated protein kinases’ activation.133 In these cells, anti-GM3 antibody is also able to induce differentiation.372 In rat cerebellum and cerebellar neurons, GD3 ganglioside is associated with Lyn and the neural cell adhesion molecule TAG-1, and antibody-mediated cross-linking of TAG-1 or GD3 induces Lyn activation.138,358 Glycosphingolipids were essential for TAG-1-dependent signaling via Lyn, and for the maintenance of the differentiated neuronal phenotype, since incubation of cerebellar neurons with the glycosphingolipid-degrading enzyme endoglycoceramidase in the presence of its activator protein reduced the levels of cell surface glycosphingolipids, caused the redistribution of TAG-1 from nondomain membranes to the lipid membrane domain fraction, abolished TAGmediated Lyn activation and consequent phosphorylation of p80, and induced neurite retraction.358 A possible role of lipid membrane domains in the pathogenesis of spontaneous and transmissible neurodegenerative diseases was recently highlighted by the discovery that a number of molecules causally connected to such diseases are associated with these domains. The most prominent examples are represented by the amyloid precursor protein (APP) in Alzheimer’s disease and by the prion protein. In both cases, the generation of the aberrant forms of these proteins, which are responsible for the onset of the disease, seems to be localized in the lipid membrane domains and/or dependent from the structure of the domain itself.378,379 APP is a transmembrane protein normally cleaved by a-secretase yielding soluble APP. Alternatively, APP is processed with the production of the Ab amyloid peptide, which accumulates in the brain lesions (senile plaques) that are commonly thought to cause Alzheimer’s disease.380 The physiological function of APP remains poorly understood. However, several studies suggest that APP is able to signal via the plasma membrane.381 Thus, understanding its molecular function and processing is critical to elucidate the molecular basis of the disease. It has been demonstrated that the APP is enriched within lipid membrane domains.273,382,383 Within these domains, APP interacts with Gao, and APP stimulation by a specific antibody inhibits the basal Gao GTPase activity.383 Since a mutated form of APP, associated with familiar Alzheimer’s disease, constitutively activates Gao,384 the regulation of Gao by APP within lipid membrane domains is likely to be relevant for the physiopathological functions of APP itself. In nonneuronal cell lines, nonamyloidogenic a-secretase processing of APP is localized within lipid membrane domains. In these cells, caveolin-1, a principal component of caveolae-like lipid membrane domains, is reported to be physically associated with APP, and a-secretase processing was dependent on the expression levels of caveolin-1 itself.382 On the other hand, lipid membrane domains from mouse brain are enriched in active g-secretase, and are the main cellular site where the production of Ab amyloid occurs.172 Moreover, disturbance of lipid membrane domain structure by cholesterol depletion resulted in the reduction of APP association with the domains, and inhibited the generation of Ab amyloid peptide.273 Prions containing prion proteins (PrP) are implicated in transmissible neurodegenerative disorders.385 The scrapie prion protein, PrPSc, is a disease-specific isoform of a normal cellular protein of unknown function (even if some data

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indicate that it might be necessary for normal synaptic function in hippocampal slices),386 denoted PrPC (cellular prion protein). The process by which PrPSc is formed post-translationally from a protease-sensitive precursor remains uncertain. Interestingly, the bulk of the covalent post-translational modifications of PrP is represented by N-linked glycosylation387 and the GPI (glycosylinositol phospholipid) anchor.388 A novel feature of the structures from PrP is the presence of sialic acid residues on the GPI anchors.389 The identification of sialic acid on GPI anchors presents the possibility for participation of GPI anchors in the regulatory functions served by sialic acid on glycoprotein and glycolipids. As with many other GPI-anchored proteins, both PrPC and PrPSc are associated with lipid membrane domains in normal and scrapie-infected neuroblastoma cells.131 The glycan core of their GPI-anchor showed some structures similar to those reported for rat brain Thy-1,389 which is also a typical component of neuronal lipid membrane domains. The efficient conversion of PrPC into PrPSc occurs after PrPC reaches to the plasma membrane, strictly requires targeting PrPC by GPI-anchors to lipid membrane domains,390 and, in scrapie-infected neuroblastoma cells, is confined in these specific subcellular domains.389 Within the domains, other proteins seem to associate with PrP, likely representing functional partners of PrP.391 We studied the membrane environment of PrPC in differentiated rat cerebellar neurons.148 In these cells, about 45% of total cellular prion protein (corresponding to a 35-fold enrichment) is associated with a lipid membrane domain fraction. Several proteins were found in the PrPC-enriched membrane domains that were separated by immunoprecipitation (i.e., the nonreceptor tyrosine kinases Lyn and Fyn and the neuronal glycosylphosphatidylinositol-anchored protein Thy-1). The PrPC-rich membrane domains contained as well about 50% of the sphingolipids, cholesterol, and PC present in the sphingolipid-enriched membrane fraction, and the presence of these lipids was essential for the integrity of protein–protein interactions within the domain. All main sphingolipids, including SM, neutral glycosphingolipids, and gangliosides, were similarly enriched in the prion protein-rich membrane domains. Thus, prion protein plasma membrane environment in differentiated neurons resulted in a complex entity, whose integrity requires a network of lipid-mediated noncovalent interactions. Moreover, the localization of PrPC to lipid membrane domains and PrPSc formation are inhibited by lovastatin, which reduces cell cholesterol content, presumably disrupting the domain structure.272 All these data suggest that lipid membrane domains might represent the cellular site where prions are propagated, and seem to imply that other components (proteins or lipids) of this compartment might participate to the propagation of prions. As mentioned above, c-Src activation is involved in the control of synaptic transmission374 and in the induction of long-term potentiation in hippocampal neurons.375 Since it has been demonstrated that hippocampal slices from PrP null mice have impaired synaptic inhibition and long-term potentiation,375,392 a further link between PrP and lipid membrane domains might be represented by Src family protein tyrosine kinases. By means of cross-linking experiments with ganglioside derivatives and immunoprecipitation experiments with anti-Src family protein antibodies, or antiganglioside antibodies, it has been shown that these proteins and glycosphingolipids are directly interacting with each other as a part of the same membrane domain, suggesting that a highly organized lipid environment is essential to maintain domain structure and functional status.238

3.30.4.2 Lipid Membrane Domains in Oncogenic Transformation: GM3 and the Glycosynapse Very much attention has been paid in the past to the aberrant glycosylation of glycolipids in tumor cells as an essential mechanism in determining the features of neoplastic transformation and the outcome of tumor progression in patients.393,394 To this regard, in-depth studies showed that altered GM3 ganglioside expression plays a multiple role in the control of tumor cell motility, invasiveness, and survival. Adhesion of B16 melanoma cells (expressing high levels of GM3) to endothelial cells, that express LacCer and Gg3, is mediated by GM3–LacCer or GM3–Gg3 interaction and leads to enhanced B16 cell motility and thereby initiates metastasis.59,395 This probably represents the best characterized example of a GSLs–GSLs interaction involved in the control of the motility and metastatic potential of tumor cells. GM3 is highly expressed in noninvasive, superficial bladder tumors compared with invasive bladder tumors, where the activities of glycosyltransferases responsible for GM3 synthesis were consistently upregulated.396,397 Enhanced GM3 expression achieved by pharmacological treatment with brefeldin A,396,398 or the exogenous administration of GM3397 suppressed the tumorigenic activity and/or the invasive potential of human colonic and bladder tumor cell lines, and the stable overexpression of GM3 synthase in a mouse bladder carcinoma cell line reduced cell proliferation, motility, and invasion with concomitant increase in the number of apoptotic cells.399 High expression levels of GM3 with concomitant expression of the tetraspanin CD9 in colorectal400,401 and bladder 396 cancer cell lines inhibited Matrigel and laminin-5-dependent cell motility. At the molecular level, GM3 control on the properties of tumor cells requires a complex supermolecular membrane organization that defines highly specialized detergent-insoluble lipid membrane domains. The term ‘glycosynapse’ has


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been proposed by S. Hakomori394,402,403 to generally describe a membrane microdomain involved in carbohydratedependent adhesion. Carbohydrate-dependent adhesion in glycosynapse, occurring through GSL–GSL interactions or through GSL-dependent modulation of adhesion protein receptors (such as integrins) leads to signal transduction events reflecting in deep changes in the motility and invasiveness of tumor cells. In the case of GM3-dependent adhesion of melanoma cells, it has been shown that GM3 is closely associated with c-Src, Rho, and Ras within glycosphingolipid-enriched membrane domains, and binding with Gg3 or anti-GM3 antibody stimulates focal adhesion kinase phosphorylation and c-Src activity.59 This molecular assembly defines a classically Triton X-100 insoluble GSL-enriched microdomain (‘glycosynapse 1’), that can be isolated and separated from a caveolin-containing lowdensity membrane fraction in B16 cells.196 A similar association between a sialoglycolipid and c-Src and other related signaling molecules was observed for GM3 also in neuroblastoma cell,133 for disialylgalactosylgloboside in renal carcinoma cells,404 and for monosialyl-Gb5 in breast carcinoma cells.405 Tetraspanin CD9 and integrin a3 or a5 also are co-localized within a distinct low-density, Brij 98-insoluble glycolipid-enriched domain (‘glycosynapse 3’). The presence of GM3 (endogenously synthesized or exogenously added to cells) positively modulated CD9/integrin association. In fact, association between CD9 and integrin in the Chinese hamster ovary mutant cell line ldlD14 (deficient in UDP-Gal-4-epimerase) has been shown by co-immunoprecipitation experiments when cells were grown in the presence of galactose, allowing GM3 synthesis, or supplementing cells with exogenous GM3. In the latter case, the amount of a3 or a5 integrin associated with anti-CD9 immunoprecipitate was quantitatively dependent on the concentration of added GM3. Co-localization of CD9, a3, and GM3 in intact cells was observed by laser scanning confocal microscopy in ldlD14 in the presence, but not in the absence, of galactose.406 The formation of a3/CD9/GM3 complexes strongly inhibited the laminin-5-dependent motility in ldlD14 cells. On the other hand, it has been shown that CD9/GM3 complexes are essential for the regulation of integrin-mediated cell adhesion and signal transduction in oncogenic transformation, suggesting a crucial role for GM3 complexed with CD9 and integrin a3b1 or a5b1 in the control of tumor cell motility and invasiveness. v-Jun-transformed mouse and chicken embryo fibroblasts were characterized by lower GM3 levels and downregulated GM3 synthase mRNA levels respect to the nontransformed counterparts.407 Reversion of oncogenic phenotype of v-Jun-transformed cells to normal could be achieved by enhanced GM3 synthesis through its gene transfection. When v-Jun-transformed cells were transfected with GM3 synthase expression plasmid, leading to increased GM3 synthase activity and GM3 cellular levels, their ability anchorage-independent growth in agar was strongly reduced. During phenotypic reversion induced by GM3 synthase transfection, the association of CD9/a5b1 complex (shown by co-immunoprecipitation and confocal microscopy experiments) was increased. Remarkably, the N-glycosylated form of b1 integrin was preferentially associated with the complex in GM3 synthase gene transfectants.407 GM3 levels were 4–5 times higher in the noninvasive KK47 cell line (originated from superficial human bladder cancer) than in the invasive YTS1 human bladder cancer cell line. Knockdown of CD9 or pharmacologically achieved GM3 depletion in KK47 cells induces the phenotypic conversion to invasive variants. On the other hand, exogenous GM3 addition induces the phenotypic reversion of the highly invasive and metastatic cell lines YTS1 to low-motility variants. The changes in cell motility were strictly correlated with the association of CD9 with a3 integrin. This interaction was higher in noninvasive than in highly invasive cells, and was modulated by the cellular levels of GM3: CD9/a3 integrin association was reduced by GM3 depletion in KK47 and conversely enhanced by exogenous GM3 addition in YTS1 cells. GM3 levels in glycosynapse controls not only CD9/a3 integrin association, but also the activation state of c-Src. c-Src is present in higher amount in the glycosynapse fraction in YTS1 cells, and it is activated in cells with low GM3 levels and high invasive potential (YTS1 or GM3-depleted KK47). On the other hand, exogenous addition of GM3 to YST1 cells caused Csk traslocation to the detergent-insoluble fraction and consequent inactivation of c-Src, influencing cell motility.334 Integrin/tetraspanin/GM3-containing glycosynapse participate as well to the cross talk between integrins and fibroblast growth factor (FGF) receptor.333 Pharmacological GM3 depletion in normal or transformed human embryonal fibroblasts caused enhanced association of FGF receptor with integrins, FGF receptor activation followed by Akt phosphorylation and MAPK activation, and enhanced laminin-5- or fibronectin-dependent cell proliferation. Thus, GM3 organized as glycosynapse negatively modulated FGF receptor activity, giving a novel molecular interpretation to the known GM3 effect as inhibitor of tyrosine kinases associated with growth factor receptors. Glycosynapses bear distinctive properties respect to other types of membrane microdomains. 1. Glycosynapses can be separated as DRM fractions, but the behavior of different glycosynapses toward different detergents is radically different. GM3-enriched membrane domains from B16 melanoma or from Neuro2a neuroblastoma cells133 are classically insoluble in 1% Triton X-100. GM3/CD9/integrin-containing membrane domains (glycosynapse 3, e.g., from human bladder cancer cells) are insoluble in 1% Brij 98408 but soluble in 1% Triton X-100. In particular, integrins and tetraspan membrane proteins are excluded from the detergent-resistant

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low-density fraction when cells where lysed in the presence of 1% Triton X-100, but they became at least in part associated with this fraction when Triton X-100 concentration was decreased to 0.5% or 0.25%.409 2. Glycosynapses can be separated from other detergent-resistant microdomains that co-purify in the same lowdensity membrane fraction. GM3-enriched membrane domains from B16 melanoma and caveolar membranes are separable by immunoaffinity using anti-GM3 or anticaveolin antibodies.196 3. Glycosynapses have a different lipid composition from other detergent-resistant microdomains (i.e., caveolar membranes). The GM3-enriched membrane fraction, separated by anti-GM3 monoclonal antibody DH2, contained GM3, SM, and cholesterol. In contrast, the caveolar membrane fraction, separated by anticaveolin antibody, contained no GM3, only a very small quantity of SM, glucosylceramide, and a very large quantity of cholesterol.196 This different lipid composition has a profound functional significance, as indicated by the fact that glycosynapse-dependent biological events (e.g, GM3-dependent adhesion and consequent c-Src and FAK activation in B16 melanoma cells) are not affected by cholesterol-sequestering drugs. On the other hand, they are disrupted by compounds structurally related to GM3, such as lyso-GM3 and sialyl a2-1 sphingosine.410,411 4. The presence of a specific glycolipid is often essential for the biological function of the glycosynapse. Increased association of a3 integrin and CD9 with consequent inhibition of motility in YTS1 bladder cancer cells induced by exogenous GM3 addition was not reproduced by addition of GM1.334 Activation of c-Src and FAK with enhanced motility and invasiveness was induced in MCF-7 breast carcinoma cells by anti-monosialyl-Gb5 monoclonal antibody, but not by antibodies to other glycosphingolipids (anti-Gb3, anti-Gb5, anti-GM2).405 The minimal requirements for glycosynapse structural and functional integrity has been assessed by experiments on reconstituted membranes.410 Reconstituted membranes containing GM3, SM, PC, and cholesterol with proportions closely resembling those observed in a glycosynapse fraction separated by anti-GM3 antibody from a total detergentresistant fraction from B16 cells were prepared, and mouse recombinant c-Src was incorporated into the reconstituted membranes (in a molar ratio of c-Src to GM3 of 1:1000). When reconstituted membranes were allowed to adhere on Gg3- or anti-GM3-coated dishes (‘GM3-dependent adhesion’), c-Src phosphorylation was observed, as it occurred with natural glycosynapses separated from melanoma cells. Activation occurred in reconstituted membranes even in the absence of PC and cholesterol (even if the presence of PC or cholesterol increased the phosphorylation response), but not when GM3 was replaced with equimolar concentrations of GM1, GD1a, or LacCer. c-Src phosphorylation response upon GM3-dependent adhesion in reconstituted membranes depended on the amount of GM3 present in the system, and was maximal when the composition of the reconstituted membrane was identical to that of natural glycosynapse. Remarkably, the c-Src phosphorylation response in reconstituted membranes was sensitive to lyso-GM3 and lyso-GM3 analogs, as it was in natural glycosynapse.

3.30.5 Perspectives, Critiques and Conclusions Sphingolipids are components of the membranes of all living organism cells. Theoretical considerations about the peculiar physicochemical properties of sphingolipids and experimental data suggest that they play an active role in the biogenesis and maintenance of lipid membrane domains, zones of the membrane with reduced fluidity, where proteins involved in processes of cell signaling are segregated and can carry out their biological functions. Moreover, the ability of sphingolipids to directly interact with membrane proteins, modulating their biological functions, suggests that sphingolipids, in general, and glycosphingolipids, in particular, play a dual, additional role in determining the dynamics and functions of lipid membrane domains. As discussed in this chapter, still many questions remain to be answered. At present, most investigators involved in this field feel the urgent need of reliable and innovative techniques allowing to image and study lipid membrane domains in living cells. However, there is still a lot of analytical work left behind to validate several aspects of the lipid membrane domain working hypothesis, a fascinating but not yet solid one.

Glossary glycosynapse A term proposed to generally describe a membrane microdomain involved in carbohydrate-dependent adhesion. lipid membrane domains Areas of a cell membrane with a peculiar lipid composition, different from the bulk bilayer composition. lipid rafts A term introduced to describe microdomains with a distinct lipid composition involved in the sorting of newly synthesized proteins to the apical or basolateral membranes of polarized epithelial cells. The hypothetical role of lipid rafts implies that segregation of lipid in domains already occur in intracellular membranes.


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liquid-crystalline(lc) or gel phase Physical phase of lipid bilayers, characterized by a extended and ordered disposition of the lipid acyl chains with minimal lateral mobility. liquid-disordered(ld) phase Physical phase of lipid bilayers, characterized by a highly disordered disposition of the lipid acyl chains and by high fluidity. liquid-ordered(lo) phase Physical phase of lipid bilayers, characterized by a highly ordered disposition of the lipid acyl chains and by low. Acyl chains of lipids in the lo phase have properties that are intermediate between those of the gel and ld phases. They are extended and ordered, as in the gel phase, but have high lateral mobility in the bilayer, as in the ld phase.

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Lynch, S. A.; Brugge, J. S.; Levine, J. M. Science 1986, 234, 873–876. Mellstro¨m, K.; Bjelfman, C.; Hammerling, U.; Pa˚hlman, S. Mol. Cell. Biol. 1987, 7, 4178–4184. Bjelfman, C.; Meyerson, G.; Cartwright, C.; Mellstro¨m, K.; Hammerling, U.; Pa˚hlman, S. Mol. Cell. Biol. 1990, 10, 361–370. den Hertog, J.; Pals, C. E. G. M.; Peppelenbosch, M. P.; Tertoolen, L. G. J.; de Laat, S. W.; Kruijer, W. EMBO J. 1993, 12, 3789–3798. Chakraborty, M.; Anderson, G. M.; Chakraborty, A.; Chatterjee, D. Brain Res. 1993, 625, 197–202. Cartwright, C. A.; Simantov, R.; Kaplan, P. L.; Hunter, T.; d’Eckhart, W. Mol. Cell. Biol. 1987, 7, 1830–1840. Yu, X.; Salter, M. W. Proc. Natl. Acad. Sci. USA 1999, 96, 7697–7704. Lu, Y. M.; Roder, J. C.; Davidow, J.; Salter, M. W. Science 1998, 279, 1363–1367. Foster-Barber, A.; Bishop, J. M. Proc. Natl. Acad. Sci. USA 1998, 95, 4673–4677. Cooper, J. A.; Howell, B. Cell 1993, 73, 1051–1054. Hooper, N. M. Biochem. Soc. Trans. 2005, 33, 335–338. 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Biographical Sketch

Sandro Sonnino was born in 1949 in Milan (Italy), and obtained an M.Sc. in chemistry in 1973 at the University of Milan. He became professor of biochemistry at the School of Medicine, University of Milan, in 1990, where he is at present. His scientific activity is all related to studies on sphingolipids. He has solved the primary structure of 11 glycosphingolipids and has studied the conformational, dynamic, geometrical, and aggregative properties of the main gangliosides from the nervous system, the role of gangliosides in the organization of sphingolipid-enriched membrane microdomains, the interaction processes between gangliosides and soluble or membrane proteins, and the metabolism and trafficking of gangliosides and their catabolites.

Alessandro Prinetti was born in 1965, in Milan (Italy). He obtained an M.Sc. in pharmaceutical chemistry at the University of Milan in 1992 and a Ph.D. in biochemistry in 1997. From 2002, he is associate professor at the School of Medicine, University of Milan. He studies sphingolipid metabolism in neural differentiation, the functional role of sphingoid molecules in the control of growth and differentiation of neural cells, the structure of sphingolipid-enriched membrane domains and their functional role in neural cell signaling and in tumor cell invasiveness, and the functional interaction between sphingolipids and Src family proteins.


Laura Mauri was born in 1970, in Monza (Italy), and obtained an M.Sc. in chemistry in 1999 and Ph.D. in biochemistry in 2003 at the University of Milan. She became research scientist at the School of Medicine, University of Milan, in 2005, where she is at present. She is developing studies on the chemistry of gangliosides.

731

Vanna Chigorno was born in 1949, in Milan (Italy), and obtained M.Sc. in biological sciences in 1973 and Ph.D. in applied biology in 1982 at the University of Milan. She became Professor of Clinical Biochemistry at the Medical School of the University of Milan in 2001, where she is at present. She developed research in the field of ganglioside biochemistry and is now mainly involved in research in the field of ganglioside biomedicine.

3.31 Receptor Modifications in Glycobiology J. Inokuchi and K. Kabayama, Tohoku Pharmaceutical University, Sendai, Japan ß 2007 Elsevier Ltd. All rights reserved. 3.31.1 Effects of Exogenous Gangliosides on the Activities of Growth Factor Receptors

733

3.31.2 Effects of Endogenous Ganglioside Manipulation on GFR Functions

735

3.31.3 Localization of GFRs in Detergent-Resistant Membranes (DRMs)

735

3.31.4 Modulating the Microdomain Localization of GFRs by Manipulating Endogenous Ganglioside Levels

738

3.31.5 Ganglioside GM3 and IR

738

3.31.5.1 Ganglioside GM3 Is an Inducer of Insulin Resistance

738

3.31.5.2 Caveolae Microdomains and Insulin Signaling

739

3.31.5.3 Insulin Resistance as a Membrane Microdomain Disorder

740

3.31.6 Perspective

741

3.31.1 Effects of Exogenous Gangliosides on the Activities of Growth Factor Receptors The biological effects of glycosphingolipids (GSLs) on modulating the functions of membrane proteins are well known. In particular, the effects of gangliosides on growth factor receptors (GFRs) have been extensively investigated. One of the most frequently used approaches in such studies is to observe changes in the ligand-dependent autophosphorylation of GFRs and the resulting signals following the addition of gangliosides into culture medium. A summary of the effects of exogenously added gangliosides on epidermal growth factor receptor (EGFR),1–8 platelet-derived growth factor receptor (PDGFR),9–13 fibroblast growth factor receptor (FGFR),14–17 nerve growth factor receptor (NGFR),18,19 and insulin receptor (IR)20–22 are presented in Table 1. Although specific and direct binding have been demonstrated for the ganglioside GM1 to cholera toxin and for GT1b (GQ1b) to botulinus toxin, the mode of interaction between gangliosides and GFRs is yet to be determined. The inhibitory effect of gangliosides, including GM3, GD1b, and GT1b, on ligand-dependent EGFR autophosphorylation has been examined in several laboratories.1–6 Generally, of the gangliosides, GM3 has been the most well studied. Interference in ligand-induced EGFR dimerization23 and the suppression of EGFR expression8 have been proposed as mechanism(s) responsible for the inhibitory effect of exogenous GM3 on EGFR autophosphorylation; however, this has not yet been fully verified experimentally. On the other hand, Ladisch’s laboratory recently reported that exogenous GD1a, but not GM3, activates EGFR.7 Whether such differences in the effects of gangliosides on EGFR are due to characteristics of the cell lines employed or to experimental conditions remains to be clarified. Literature describing the effect of gangliosides on PDGFR function has suggested an inhibitory effect of a- and b-series gangliosides on the ligand-dependent receptor.9,10,12 The exception to this is the effect of the simple ganglioside GM3. Interestingly, GM3 has no effect on PDGFR autophosphorylation, a contrast to its actions toward EGFR.9 Specific effects of individual gangliosides on FGFR autophosphorylation, particularly the inhibitory effect of GM314 and the stimulatory effect of GM1, have been clearly established.15–17 Although the mode of action of these effects has not been fully elucidated, Rusnati et al. have demonstrated that GM1 suppresses FGF signaling through direct binding to FGF but not to FGFR in GM7373 fetal bovine aortic endothelial cells.15 GM1 has been receiving attention for its beneficial effects on neurological functions. For instance, an interaction between NGFR and GM1 has been reported.18,19 Notably, Mutoh et al. observed strong complex formation between NGFR and GM1, even after sodium dodecyl sulfate (SDS) treatment for detection by Western blotting.18 It will be interesting to explore whether such a tight complex is formed by direct binding of GM1 to NGFR or by aggregation through an unidentified intermediating substance. Nojiri et al. initially examined the effect of gangliosides on IR. Using the human lymphoma IM9 cell line, they demonstrated that sialylparagloboside and, to a lesser extent, GM3 inhibit autophosphorylation of IR.20 Adipocytes express GM3 as a major ganglioside and GD1a as a minor one. Recently, Tagami et al. reported that GM3, but not

733

734

GFRs

Gangliosides added

Type of responses

Cell lines

Reference [Signaling molecules investigated]

EGFR

GM3

Receptor autophosphorylation #

H125, A431 A431 A431, SCC12 NBL-W

1,2 3,4 5 6

GD1a

Receptor autophosphorylation "

NHDF

7

GM3

GM3 binds extracellular domain of EGFR in vitro

GM3 GM3, GD1b, GT1b

4

GM3

Suppression of receptor protein expression

HC11, MG1361

8

PDGFRb

GM1,GM2, GD1a,GD1b, GD3, GT1b GM1, GM2, GD1a, GT1b GM1 GM1,GM2, GD1a, GT1b GM1


U-1242MG, Swiss 3T3 U-1242MG U-1242MG SH-SY5Y Swiss 3T3

9 10 11 12 13

FGFR

GM3


WI38, VA13

14

GM1


GM 7373 Primary mixed glial cultures containing astrocytes CHO-K1

15 16

17

NGFR (TrkA)

GM1


PC12

18,19

IR

2 ! 3SPG>GM3 GM3>GD1a


IM9 HL-60 3T3-L1 adipocytes

20 21

GM3>GD1a

Inhibition of insulin stimulated cell growth

FUA169

22

H125, human lung adenocarcinoma cells; A431, human epidermoid carcinoma cells; RPTPs, isotype of transmembrane protein-tyrosine phosphatase; SCC12, human keratinocytederived cells; HC11, mouse normal epithelial cells; MG1361, mouse adenocarcinoma cells; NBL-W, human neuroblastoma cells; U-1242MG, human glioma cells; NHDF, normal human dermal fibroblasts; SH-SY5Y, human neuroblastoma cells; PC12, rat pheochromocytoma cells; IM9, human lymphocytes; HL-60, human promyelocytic leukemia cells; Swiss 3T3, mouse fibroblast; WI38, human embryonal lung diploid fibroblast; VA13, SV40-transformed WI38; FUA169, a subclone of FM3A/F28-7 mouse mammary carcinoma cells expressing high level of GM3.

Receptor Modifications in Glycobiology

Table 1 Effects of exogenous gangliosides on GFRs


735

GD1a, inhibits IR autophosphorylation.21 Gangliosides produced in various cells and organs are secreted into body fluids, and, with plasma levels of approximately 1mM, these secreted gangliosides might affect physiologic cellular functions. However, since many studies cited here employed abnormally high ganglioside concentrations (10–100 mM) in culture medium, it has been difficult to form conclusions regarding the physiological role(s) of gangliosides on GFR functions based on these studies. Thus, as mentioned in the next section, the trend of ganglioside research has shifted to manipulating endogenous ganglioside levels.

3.31.2 Effects of Endogenous Ganglioside Manipulation on GFR Functions The molecular cloning of nearly every glycosyltransferase gene, including the ganglioside synthase genes, has now been accomplished.24 This has enabled the use of genetic approaches to manipulate ganglioside expression in cells and experimental animals, which has become a powerful strategy in identifying and understanding the functions of gangliosides in vivo. Indeed, we are getting much a clearer picture of the roles of individual gangliosides by using this technology to control the expression of a specific ganglioside. Another approach being employed to evaluate the functions of endogenous gangliosides is utilizing inhibitors of GSL biosynthesis, like the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its analogs.25–27 We used D-PDMP to deplete the GSLs in TNF-a-treated 3T3-L1 adipocytes, and found that insulin resistance was partially normalized21 (discussed in Section 3.31.5; TNF-a ¼ tumor necrosis factor-a). As summarized in Table 2, both approaches have yielded valuable information. Since the exogenous addition of GM3 resulted in the inhibition of EGFR autophosphorylation, understanding the roles of endogenous GM3 on EGFR function has attracted much interest. Subsequent studies have reported that enhanced EGF signaling was observed following the suppression of GM3 expression by transfection with gangliosidespecific sialidase cDNA28–30 or by depletion of all glucosylceramide-derived GSLs by D-PDMP.31 In addition, applying sense or antisense oligodeoxynucleotides for GM2/GD2 synthase cDNA, to decrease or increase endogenous GM3 levels, resulted in up- or downregulation of EGFR function, respectively.29,30,32 More direct genetic studies of GM3 influence have also been performed. Uemura et al. generated a GM3reconstituted cell line by transfecting the GM3 synthase (SAT-I) gene into a subclone of 3LL Lewis lung carcinoma cells, which are normally GM3 deficient, and compared the GM3-positive cells to the corresponding mocktransfected, GM3-negative cells.33 They found significant reduction in PDGFRa levels in the GM3-reconstituted cells, yet there was no difference in the cell proliferation rate. Interestingly, the GM3-positive cells exhibited a more malignant phenotype, including an increased ability to form colonies in soft agar, suggesting complementary amplification of growth factor signaling other than signaling via the PDGFRa.33 Genetic studies have also provided more definitive information regarding the effects of gangliosides on the PDGFRb. Mitsuda et al. have reported that PDGF signaling was suppressed in Swiss 3T3 cells expressing high levels of GM1 following transfection with both GM2/GD2 synthase and GM1 synthase cDNAs.34 This result is supported by the observation that exogenous addition of GM1 resulted in the inhibition of PDGF signaling via the PDGFRb.9,13 Similarly, in PC12 cells transfected with the GM1 synthase gene and expressing high levels of GM1, autophosphorylation of the NGF receptor TrkA and MAPK signaling were suppressed. Nishio et al. speculated that the increased GM1 might interfere with the dimerization of TrkA by decreasing membrane fluidity.35 The ambiguity surrounding the mechanisms engaged by exogenously added gangliosides has long been a topic of discussion, partly because of the experimental limitations in proving trans- or cis-type actions of gangliosides toward GFRs. Nevertheless, since GFR functions are definitely affected by the expression levels and profiles of endogenous gangliosides, it is reasonable to accept the physiological importance of the interactions between gangliosides and GFRs. Vital information acquired from genetically manipulated animals expressing altered ganglioside production will definitely contribute to the critical understanding of the roles of gangliosides in GFR signaling.

3.31.3 Localization of GFRs in Detergent-Resistant Membranes (DRMs) In view of the diverse effects of gangliosides on members of the GFR family, one might ask what are the basic principles behind the mechanisms of ganglioside actions? To address this issue, understanding the structure and functions of membrane microdomains (lipid rafts) in relation to GFRs will be a most important key. Within the past decade, data have emerged from many laboratories implicating microdomains as critical for proper compartmentalization of various cellular signals. Biochemical analyses have proved that microdomains are detergent insoluble and

736


Table 2 Effects of endogenous gangliosides remodeling on GFR functions

GFRs

Expression levels of gangliosides

Type of response

Cell lines and treatment

References

EGFR

GM3 #


A431 Sialidase(Neu2) cDNA transfection A431 D-PDMP treatment SCC12 Sialidase(Neu3) cDNA transfection SCC12 GM2/GD2 synthase antisense oligodeoxynucleotide treatment SCC12 GM2/GD2 synthase cDNA transfection

28

GM3 # GM3 # GM3 "


GT1b "

31 29 30 29 30 32 32

GM3 # GD3 "


CHO-K1 GD3 synthase cDNA transfection under the control of the human elongation factor promotor

55

GM3 ! GD3 "


CHO-K1 GD3 synthase cDNA transfection under the control of an ecdysone-inducible promotor

55

GM1 " GD1a "

HB2 CD82 cDNA transfection

56

GD3 "

Increase of EGFR and ErbB2 in DRM. GD1a selectively localize with EGFR on the plasma membrane Receptor autophosphorylation #

57

Gangliosides #


Gangliosides #


CHO-K1 GD3 synthase cDNA transfection CHO-K1 D-PPPP treatment NHDF D-PPPP treatment

PDGFRb

GM1 "

Suppression of cell growth Downregulation of receptordependent ERK1/2 activation

Swiss 3T3 GM2/GD2 synthase and GM1 synthase cDNAs transfection

34

PDGFRa

GM3 "

The selective downregulation of PDGF-dependent ERK1/2 activation. Decreases of receptor mRNA and protein

J5 SAT-I synthase cDNA transfection

33

NGFR (TrkA)

Ganglioside #


PC12

19

D-PDMP

IR

GM3 #

Prevention of TNF-a-induced defect in tyrosine phosphorylation of IRS-1

57 58

treatment

3T3-L1 adipocyte treatment

21

D-PDMP

HB2, human mammary epithelial cells; PDMP, glucosylceramide synthase inhibitor, threo-1-phenyl-2-decanoylamino-3morpholino-1-propanol; PPPP, glucosylceramide synthase inhibitor, threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino1-propanol; J5, a subclone of mouse 3LL Lewis lung carcinoma cells expressing high level of lactosylceramide and low level of GM3.

highly enriched in cholesterol and GSLs, but they lack phospholipids. Thus, following sucrose density gradient centrifugation, these subdomains are found in the low-density, detergent-insoluble fractions, so-called detergentresistant membranes (DRMs). Alternatively, microdomains can be obtained as low-density fractions by a nondetergent method such as alkaline treatment of cells (e.g., with 500 mM sodium carbonate buffer, pH 11). Microdomains recovered in DRMs are often called caveolae, caveolae-like domains, glycolipid-enriched microdomains (GEMs), and lipid rafts,


737

depending on cell types and areas of research study. Gangliosides have been recognized not only as useful markers for microdomains (e.g., GM1 can be detected by FITC-conjugated cholera toxin B subunit), but also as indispensable molecules for regulating microdomain functions. On the other hand, whether GFRs are localized in microdomains is still a confusing issue, because biochemical isolation of DRMs generally uses detergents that are incompatible with proteins. The literature reporting the localization of GFRs in microdomains with detergent (DRM) and nondetergent alkaline (low-density fraction) methods is summarized in Tables 3 and 4, respectively. It has generally been accepted that the concentration of detergents like Triton X-100 needed to isolate DRMs in a sucrose density flotation assay is 1%. At this concentration, most GFRs are not able to localize in the DRMs and are present in non-DRM fractions. On the other hand, GFRs can be recovered in DRMs when other, relatively weak detergents such as 1% Brij98 or 1% Brij35 are employed (Table 3). In addition, GFRs can be partitioned into low-density fractions by nondetergent alkaline treatment (Table 4). We demonstrated for the first time the localization of IR in DRMs using very low concentrations of Triton X-100 (C18

2. Gb4, glycophorin on human RBCs

≤C16

3. Polysialoganglio-side in fish brain neuronal junction O H O CH H C C CH O N CH O H CH2

(a)

H

O H O R O H O R

C=O O

O

C

4. GM3 on T-cells

CH2 O CH CH2 O R

(b)

Figure 6 GSL cluster domains are formed by GSL-to-GSL cis-interaction. a, GSLs (left) have many hydroxyl groups and one N-fatty acyl group. Therefore, GSLs act as both hydrogen bond donors and acceptors. In contrast, phospholipids (right) have no hydroxyl group, and act as hydrogen bond acceptors but not donors. GSL-to-GSL cis-interaction takes place to form GSL clusters in phospholipid bilayer. b, (1) Extensive clustering of GM1 on phospholipid liposome surface, even in the absence of cholesterol; (2) Gb4 and glycophorin clustering on human erythrocytes; (3) Polysialoganglioside in brain neuronal junction; (4) GM3 on T-cells. a, Modified from Pascher, I. Molecular arrangements in sphingolipids: Conformation and hydrogen bonding of ceramide and their implication on membrane stability and permeability. Biochim. Biophys. Acta 1976, 455, 433–451.34 b-1, From Rock, P.; Allietta, M.; Young, W. W. J.; Thompson, T. E.; Tillack, T. W. Ganglioside GM1 and asialo-GM1 at low concentration are preferentially incorporated into the gel phase in two-component, two-phase phosphatidylcholine bilayers. Biochemistry 1991, 30, 19–25.165 b-2, From Tillack, T. W.; Allietta, M.; Moran, R. E.; Young, W. W. J. Localization of globoside and Forssman glycolipids on erythrocyte membranes. Biochim. Biophys. Acta 1983, 733, 15–24.166 b-3, From Rahmann, H.; Ro¨sner, H.; Ko¨rtje, K.-H.; Beitinger, H.; Veybold, V. Ca2þ-ganglioside interaction in neuronal differentiation and development. In Biological Function of Gangliosides (Progress in Brain Research, Vol. 101); Svennerholm, L., Asbury, A. K., Reisfeld, R. A., Sandhoff, K., Suzuki, K., Tettamanti, G., Toffano, G., Eds.; Elsevier: Amsterdam, The Netherland, 1994; pp 127–145.167 b-4, Sorice, M.; Parolini, I.; Sansolini, T.; Garofalo, T.; Dolo, V.; Sargiacomo, M.; Tai, T.; Peschle, C.; Torrisi, M. R.; Pavan, A. Evidence for the existence of ganglioside-enriched plasma membrane domains in human peripheral lymphocytes. J. Lipid Res. 1997, 38, 969–980.40

4.15.3.2 Types of GSL-Enriched Microdomains Based on the stronger side-by-side interaction of GSLs compared to that of phospholipids, GSL clusters separated from phospholipid bilayer have been observed by scanning or transmission electron microscopy (Figure 6b). Clustered sphingolipids together with cholesterol, in many cases, are insoluble in aqueous media containing a low concentration (0.5–1.0%) of nonionic detergent.36 Since disulfide-dependent molecular complex with very high molecular mass in extracellular matrix (ECM) displayed detergent-resistant properties,37 GSL/sphingolipid complexes with other membrane molecules were initially termed ‘detergent-insoluble (or detergent-resistant) microdomain’,36,38,39 or ‘GSL-enriched microdomain (GEM)’.40,41 Similar membrane properties were found for morphologically distinct ‘caveolae’ enriched in caveolin.42–45 Later, microdomains with similar composition and solubility were termed ‘raft’, or ‘lipid raft’, since they were regarded as quickly moving, floating signaling platforms.46,47 However, properties of ‘lipid raft’, which are highly dependent on cholesterol, are distinct from those of a subfraction of GEM, which is cholesterol independent but dependent on tetraspanin (TSP), that is, classically known proteolipid protein (PLP).48,49 Such microdomain controls GSL-dependent cell adhesion and

274

Structure and Functional Interaction of Glycosphingolipids

Gp

1

2

GSL patch

4

3

Integrin or GFR

PLP/TSP TDa

TDb

TDa

TDb

Figure 7 Proposed organization of GSL-enriched microdomain. (1) Cluster of glycoproteins. (2) Cluster of GSLs. (3) Cluster of GSLs with cytoplasmic signal transducers (TDa, TDb). (4) Cluster of GSLs with TDa, TDb, integrin, or growth factor receptor (GFR), and proteolipid protein (PLP) or tetraspanin (TSP). PLP/ TSP facilitates association of GSLs with TDa, TDb, and regulates GSL- or integrin-dependent cell adhesion or GFR-dependent cell growth.

Table 1 Contrasting properties of ‘lipid raft’ vs ‘glycosynapse’

Mobility Essential function Cholesterol dependence Tetraspanin dependence Size

Lipid raft

Glycosynapse

Highly mobile, floating Nonadhesive signaling platform Highly dependent (disrupted by chol-binding reagent) None 10–100nm

Nonmobile Adhesion platform through GSL or glycopeptide, with concurrent signaling Independent (relatively insensitive to chol-binding reagent) Highly dependent 100–1000nm

motility. The term ‘glycosynapse’ was therefore proposed,50–52 in analogy to ‘immunological synapse’ which controls immunocyte adhesion and signaling.53,54 Glycosynapse contains GSLs, TSPs (PLPs), integrins, GFRs, and cytoplasmic protein kinases (cSrc, Src family kinases, small G-proteins)55–58 (Figure 7). Ceramides, when released (mainly from sphingomyelin), activate ‘ceramide-activated protein kinases’.59 Sphingosine released by ceramidase activates ‘sphingosine-dependent kinases’.60,61 The effects of GSLs, as well as their degradation products, therefore create signaling to effect various types of phenotypic changes. Contrasting properties of lipid raft versus glycosynapse are summarized in Table 1. Either raft or glycosynapse provides a basis for functional significance of GSLs in control of cell adhesion, motility, and growth, particularly degree of malignancy in tumors, as described in Section 4.15.7.

4.15.4 GSL Antigens, Markers, and Receptors Various types of GSL antigens, markers, and receptors are listed in Table 2. Many of their structures were originally established through GSLs rather than glycoproteins, since GSLs were relatively easy to isolate to homogeneity and to determine their structures. 1. Structures of four types of histo-blood group ABH antigens (lacto types 1–4) are shown in Figure 8. Type 3, showing A1/A2 distinction, was originally found as repetitive and nonrepetitive A. The A1 repetitive A is expressed only as GSL, not in glycoprotein.62,63 Type 4 (globo-ABH) is expressed highly in urogenital epithelia. Expression of globo-H occurs only in nonsecretors, and is correlated with urogenital infection by Escherichia coli.64 Four types of Lewis antigens (for review, see Ref. 65) and biochemical and genetic basis of histo-blood group P1P2Pkp (for review, see Ref. 66) underlie genetic heterogeneity within a given species, and are therefore regarded as ‘allogeneic’ antigens. They are thought to provide an important basis of immunological incompatibility, although the biological significance of such heterogeneity is still unknown.


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Table 2 GSL antigens, markers, and receptors 1. Allogeneic antigens (blood group antigens) a. ABH (types 1, 2, 3, 4) (structures of four types of ABH: see Fig. 6). b. Lewis (Lea, Leb, Lec, Led)65 c. P1P2Pkp66 2. Heterogenetic antigens a. Forssman antigen Positive: Horse, sheep, goat, cow, dog Negative: Human, marmoset, rabbit, pig b. Gala3Galb3GlcNAc (di-Gal) antigen70,151 Positive: All mammals except as below, including New World monkeys Negative: Human, Old World monkeys 3. Cell-type specific antigens a. Lex (CD15) for myeloid cells;152 for human hypothalamic area153,154 b. Sialyla2-6 poly-LacNAc for B cells (siglec-2, or CD22)83 c. LacCer for myeloid cells (CDW17)155,156 d. Sulfated glucuronyl paragloboside (HNK1 antigen) for peripheral nerves, human NK cells (CD57) 144,145 e. Myeloglycan and its analogs as E-selectin ligands. A long-chain poly-LacNAcCer having terminal NeuAca2-3 and multiple internal Fuca1-380,85 f. siglec-4 (MAG) showing binding specificity to some gangliosides; see text with references g. anti-GM1 or other antibodies to ganglio-series gangliosides, causing Guillain–Barre´ syndrome; see text with references 4. Developmentally regulated antigens Stage-specific embryonic antigen-1 (SSEA-1),157 Lex, SSEA-3,158 SSEA-4,159 etc. 5. Tumor-associated antigens See 4.15.7, and Tables 3–5

Type 1 (lacto type 1) (Note 1)

GalNAcb 3Galb 3GlcNAcb 3Galb -R 2 Fuca

Type 2 (lacto type 2) (Note 2)

GalNAcb 3Galb 4GlcNAcb 3Gal-R 2 Fuca

Type 3 (repetitive A): A1 specific epitope (Note 3) GalNAcb 3Galb 3GalNAca 3Galb 4GlcNAcb 3Galb 4Glcb Cer 2 2 Fuca Fuca A A Gb5

Type 4 (globo-A/B/H) (Note 4)

GalNAcb 3Galb 3GalNAcb 3Gala 4Galb 4Glcb Cer 2 Gb4 (globoside) Fuca Notes 1, 2: Present in gastrointestinal, pulmobronchial, epithelial, salivary epithelia, and secretions therefrom. Epitope A as shown by yellow can be replaced with or B Gala 3Galb H Galb 2 2 Fuca Fuca

Note 3: Present in A1 erythrocytes as GSL, not as glycoprotein. A epitope cannot be replaced. Note 4: Present in urogenital epithelia. A epitope can be replaced with B or H.

Figure 8 Four types of histo-blood group A1, A2, B, H structures.

2. Heterogenetic antigens expressed in some mammalian species but not others (Table 2, item 2). Examples: (a) Forssman antigen (GalNAca3GalNAcb3Gala4Galb4GlcbCer), controlled by expression of its synthase a3GalNAc-T and its gene, is positive in some mammalian species but negative in others, including human.67 (b) ‘a3diGal’, Gala3Galb4GlcNAcb3Galb-R68,69 (negative in humans and Old World monkeys; positive in all other mammalian species).70 (c) Hanganutziu–Deicher antigen, NeuGc (positive in many mammalian species including apes and other nonhuman primates; negative in humans and birds) (see Section 4.15.2). 3. Various antigens whose expression is highly variable, depending on cell type, stage of development, and oncogenic transformation (Table 2, items 3–5).

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4. GSLs were considered some time ago as receptors for many bioactive components, including serotonin, lymphokines, fibronectins, autoantibodies, glycoprotein hormones, gonadotropins, interferons, Sendai virus, etc. (for review, see Ref. 71). However, most of these studies were based on misinterpretations, or lack of proper controls, and the conclusions were not well justified. The only well-justified cases are for bacterial exotoxins, for example, GM1 for cholera toxin, GT1b/ GQ1b for tetanus toxin, GT1b for botulism toxin,71 and more recently Gb5 for butterfly toxin pierisin, causing tumor cell apoptosis.72,73 5. Interaction of bacterial adhesins with defined GSLs as a mechanism for microbial infection has been increasingly well documented; some of these studies are discussed in Section 4.15.7.2. 6. Some brain gangliosides such as GT1a, GT1b, and GQ1ba, but not GM1 or GM3, interact with myelin-associated glycoprotein (MAG),74 suggesting that MAG is the receptor for gangliosides essential for inhibition of neuritogenesis. Further studies indicated that specific gangliosides are nerve cell ligands for MAG, and that their interaction with MAG inhibits neuritogenesis.75 7. The neuromuscular junction, the site where motor cell nerve sends impulses to muscle cells to cause contraction, is enriched in and exposes specific gangliosides such as GM1 (see Section 4.15.7.3).

4.15.5 GSL-Mediated Cell Adhesion GSL clusters in cell surface microdomains may mediate cell adhesion through GSL-binding proteins such as galectins,76,77siglecs,75,78 and selectins.79,80 Extensive studies are in progress on cell adhesion/recognition mediated by various types of carbohydrate-binding proteins, and many reviews on this topic are available.81–84 A schematic version of proposed adhesion mediated by carbohydrate-binding proteins is shown in Figure 9a. In this figure, proteins in each cell bind to GSLs in microdomain on the counterpart cell. Some GSLs are targets of galectins77 or

Figure 9 Model of cell adhesion mediated by GSLs. a, Interaction of GSL with GSL-binding protein (S) mediates cell adhesion. b, GSL-to-GSL trans-interaction mediates cell adhesion. c, In either case, GSL-dependent cell adhesion activates signal transducers present in GSL-enriched microdomain. See text for explanation.


277

siglecs;78 however, in the case of selectins, GSLs have not been clearly identified as targets84 except in human neutrophils and promyelocytic leukemia HL60 cells. In this case, a series of GSLs having terminal sialic acid and multiple internal fucose, termed ‘myeloglycan’, was identified as the target of E-selectin,85,86 particularly under physiological, dynamic flow conditions.80 It is surprising that GSLs with sialyl-Lex or sialyl-dimeric-Lex structure were not found in human neutrophils or HL60 cells.85 In striking contrast to adhesion mechanisms based on GSL-binding proteins, some GSLs expressed and clustered at the cell surface are capable of recognizing themselves to mediate adhesion of homotypic or heterotypic cells, whereby signal transducers are activated at microdomain to create concurrent signal transduction to alter cellular phenotype (see Figures 9b and 9c). Such adhesion was initially observed for autoaggregation of mouse teratocarcinoma F9 cells mediated by Lex-to-Lex interaction.87–90 The mechanism of strong species-specific self-adhesion of sponge cells in the presence of Ca2þ, for example,91 was recently shown to be based on self-recognition of specific oligosaccharide assembled on proteoglycan. Essential oligosaccharides displaying remarkable self-recognition are GlcNAcb3FucaOSer/Thr with or without 3-O-sulfate at GlcNAc, or Galb4GlcNAcb3Fuc having 4,6-cyclic pyruvate substitution at terminal Gal.92,93 Species-specific autoaggregation may reside on different oligosaccharides which show similar self-recognition ability, as described above.94 Since then, various GSL-to-GSL interactions, interactions between clusters of oligosaccharides, and interactions between glycosyl epitopes of glycoproteins have been studied. Such interactions have been verified by cell adhesion studies, for example, adhesion of GM3-expressing melanoma to Gg3-expressing mouse lymphoma L5178V cells, which are NK susceptible. Such GM3/Gg3-dependent adhesion was not observed in Gg3-nonexpressing L5178AV variant, which is NK nonsusceptible. The basic concept of carbohydrate-to-carbohydrate interaction, and experimental studies to support it, have been reviewed repeatedly.95–100

4.15.6 Interaction of GSLs with Growth Factor Receptors and Integrins 4.15.6.1 Interactions of GSLs with TSPs and Integrins Basic phenotypic changes of cells during ontogenic development, differentiation, and oncogenic transformation are associated with changes in signal transduction that induces cell adhesion, motility, growth, and apoptotic processes. Adhesion and motility are defined by combination of adhesive protein components of ECM and their binding receptor integrins. In this system, types of integrins required for recognition of ECM are well defined, for example, integrin a5b1 is for interaction with fibronectin; a3b1 is for laminin-5; a6b4 is for laminin-1 and -2; a1b1 or a2b1 is for collagen, etc.101,102 Integrins are always highly N-glycosylated, and their type of N-glycosylation strongly affects integrinmediated adhesion and motility, as studied extensively by Taniguchi and colleagues (for review, see Ref. 103). Adhesion and motility mediated by integrins are strongly affected by gangliosides, particularly when complexed with TSP. TSP CD9 is a typical chloroform/methanol-soluble proteolipid, and has the ability to form a complex with ganglioside GM3. Such complex strongly inhibits integrin-mediated cell motility.104,105 GM3/CD9 complex interacts with integrin a3, the receptor for laminin-5.57 Further systematic studies on effect of GM3/TSP complex on tumor cell invasiveness and cell motility are described in Section 4.15.7.

4.15.6.2 Effects of GSLs on Cell Growth through Modulation of GFRs In chemically defined medium, growth of fibroblasts is highly dependent on fibroblast growth factor (FGF). In BHK fibroblasts, exogenous addition of GM3 causes refractoriness of cell growth even when high quantity of FGF is added. Various other experiments indicate that FGF receptor (FGFR) function, including its internalization, is inhibited by GM3.106 These findings initiated further studies of ganglioside or GSL effect on receptor function, particularly cytoplasmic tyrosine kinases associated with the receptor. Examples are shown schematically in Figure 10 and explained in its legend. The phenomenon of ‘contact inhibition’, discovered by Abercrombie,107,108 provides an important basis for the mechanism of growth control in normal cells. Human diploid fibroblast WI38 displays clear contact inhibition, that is, cell growth is arrested when cells touch each other. Recently, the phenomenon was reinvestigated to determine (1) the molecular mechanism of cell contact and (2) whether ganglioside involved in cell contact blocks FGFR function, in analogy to previous process as in Ref. 106 It was found that tyrosine kinase of FGFR is strongly inhibited by

278


ss

ss ss

EGF-R

GM3

deNAc GM3

Insulin-R

NGF-R

GM3 SPG

GM1 PLPS

PDGF-R

GD1a GT1b

FGF-R

GM3 CD9

Figure 10 Enhancing or inhibitory effects of GSLs and sphingolipids on tyrosine kinases associated with growth factor receptors. Adapted from figure 3 of Hakomori, S., Igarashi, Y. Functional role of glycosphingolipids in cell recognition and signaling. J. Biochem. (Tokyo) 1995, 118, 1091–1103.168

GM3/CD9/CD81 complex when cells were in contact and growth was arrested. In actively growing cells not in contact, GM3/CD9/CD81 complex does not inhibit FGFR tyrosine kinase. It is important to note that FGFR has preferential binding ability to GM3, but not to other gangliosides or GSLs. An overall mechanism for contact inhibition of WI38 and other cell types is illustrated schematically in Figure 11 and explained in its legend.

4.15.6.3 Functional Interaction (Cross Talk) between Integrins and GFRs In many cells, integrins coexist with GFRs, presumably in the same microdomain. Functional interaction (cross talk) between these two receptors is often observed, and has been found to be a basic mechanism for survival of cells. Reciprocal interaction between b1 and EGFR has been observed in various breast cancer cell lines.109 Application of functional anti-b1 antibody caused functional reversion of malignant breast cancer cells to benign phenotype, which was associated with a decrease of EGF-dependent growth.110 Adhesion of WI38 cells to laminin-5 strongly activated FGFR tyrosine kinase when GM3 was depleted by P4 treatment, or TSP CD9/CD82 was knocked down by siRNA.111 Many other observations indicate that laminin-5-induced EGFR signaling through cSrc/Akt/MAPK is inhibited by GM3/CD9/CD82 complex (Figure 12). Integrin-dependent signaling through adhesion of cells to ECM is essential for cell survival and cell motility. Integrin dysfunction associated with lack of cell adhesion causes apoptosis.112 It is plausible that ganglioside/TSP complex plays an important role in physiological control of cell survival as well as normal cell growth. Absence of ganglioside/TSP complex may cause oncogenic transformation.

4.15.7 Role of GSLs in Disease Development Although the initial mechanisms of disease development are basically different (infection, wounding, poisoning, autoimmune process, cancer, etc.), progression of each disease is highly dependent on interaction of diseased cells with healthy cells in the surrounding microenvironment. Such interaction is generally considered to occur through cell adhesion/recognition between diseased and healthy cells, in which glycosynaptic microdomains may play an essential role. Our knowledge in this area is highly limited and fragmentary. However, significant progress on tumor-associated GSL antigens defined by monoclonal antibodies was made during the 1980s. More recently, the functional significance of GSL changes associated with oncogenic transformation has been clarified to some extent. Significant progress has also been made on the roles of GSL-mediated interaction, adhesion, and signaling to define the outcome of cancer development (see Section 4.15.7.1).


279

W138 cells

2

4 6

3

5 Csk

1

CD9 CD81 FGFR

3

FGFR

GM3

cSrc

MAPK

FGF CD9 CD81 2

Csk

MAPK

5

1

cis-interaction

6

cSrc

GM3

GM3

4

FGFR

cSrc

CD9–CD81

trans-interaction CD9–CD81

FGFR

Csk

VA13 cells FGF

cell-to-cell

GM3

MAPK

FGFR

MAPK

GM3

4

GM3

3

FGFR

FGFR

2

5

FGFR

cSrc

3

FGFR

3 3

1 2

GM3

cSrc

MAPK 5

4

CD9–CD81

Figure 11 Components, and their interactions in glycosynapse of WI38 cells, in contact inhibition of cell growth (a), and those in transformed VA13 cells displaying loss of contact inhibition (b). a, GM3 complexed with TSP CD9/CD81 inhibits FGF-induced activation (tyrosine phosphorylation) of FGFR (cis-interaction). GM3/CD9/CD81 complex also interacts with counterpart surface of FGFR and thereby inhibits FGFR activation (solid line 2) (trans-interaction). Either cis- or transinteraction reduces signaling from GM3 or FGFR to cSrc, and thereby reduces cSrc tyrosine kinase (dotted lines 3 and 4). cSrc activity is further reduced by the presence of Csk, which inhibits cSrc phosphorylation at Y416 through enhanced Y529 phosphorylation (solid line 5). b, Transformed VA13 cells are characterized by loss or great decrease of CD9/CD81, great increase of FGFR and cSrc, and absence of Csk. The inhibitory effect of GM3 on FGFR is much weaker than in WI38, since no GM3/CD9/CD81 complex is formed. Therefore, cis or trans inhibitory signals between GM3 and FGFR are weak (dotted lines 1 and 2). Signaling from FGFR to cSrc, or from GM3 to cSrc, is stronger (solid lines 3 and 4). Since Csk is absent, and amount of cSrc is high, signaling from cSrc is not reduced (rather enhanced) in VA13, and MAPK activation is also greatly reduced (solid line 5). Based on data from Toledo, M. S.; Suzuki, E.; Handa, K.; Hakomori, S. Cell growth regulation through GM3-enriched microdomain (glycosynapse) in human lung embryonal fibroblast WI38 and its oncogenic transformant VA13. J. Biol. Chem. 2004, 279, 34655–34664.58

ECM FGFR Y-P cSrc (Y-416P) Akt MAPK

GM3/ CD9–CD81 complex

a3b1

a5b1

p130Cas

FAK

Rac PI3K/ ERK Rho Akt Integrin-dependent signaling

Figure 12 Functional interaction (cross-talk) between integrins and FGFR is inhibited by GM3/TSP complex. When cells adhere to extracellular matrix (ECM) containing laminin-5 or fibronectin, integrin a3b1 or a5b1 is activated, whereby integrindependent signaling is created, leading to activation of FGFR through enhanced tyrosine phosphorylation (Y-P). cSrc, Akt, and MAPK are sequentially activated to induce cell proliferation. Thus, functional interaction (cross-talk) between integrins and FGFR activates both integrin-dependent cell motility and FGFR-induced cell proliferation. High level of GM3/CD9– CD81 inhibits such cross-talk, and cSrc, Akt, MAPK activation following adhesion of cells to ECM are inhibited. This phenomenon is clearly observed in well contact-inhibited WI38 cells but not in transformed VA13 cells.

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Considerable attention has been paid to the role of glycosylation and GSLs in infection and subsequent disease progression and outcome. This area will be briefly discussed in Section 4.15.7.2. The effect of N-linked glycans in defining functional role of immunoglobulins in arthritis or other autoimmune processes has been a ‘hot topic’ in glycopathology. Little is known regarding the role of GSLs in autoimmune disease, except for ganglio-series ganglioside structures involved in autoimmune antigens in Guillain–Barre´ syndrome (GBS) and Fisher syndrome (FS), as briefly discussed in Section 4.15.7.3. The major subject of glycolipid research in disease development has been the sphingolipidoses. This area was reviewed repeatedly (e.g., see Refs. 113,114). Major progress in recent studies is the discovery of imino sugar inhibitors that block accumulation of many types of GSLs not limited to be deleted, or accumulation of GlcCer in Gaucher’s disease. This topic is not covered in this short chapter.

4.15.7.1 Cancer Dramatic changes of ganglioside pattern associated with oncogenic transformation by DNA virus115,116 and RNA virus (Rous sarcoma virus)117,118 were originally observed. Recently, oncogenic transformation caused by oncoprotein Jun was also found to induce a dramatic deletion of GM3.119 In each case, loss of ganglio-series structure due to incomplete synthesis was observed. Importantly, when lost ganglioside was restored by exogenous addition, or transfection of synthase gene, oncogenic phenotype was reverted to normal phenotype. Jun-induced oncogenic phenotype, associated with loss of GM3, was reverted to normal by transfection of GM3 synthase gene.119 Certain cancer cell lines showing high motility and invasiveness display characteristic GSL pattern as compared to related cell lines with low motility/invasiveness. Some examples are described below. 1. Various colorectal or bladder cancer cell lines show decrease or loss of GM3 in those with higher invasiveness and motility. For example, colorectal cancer SW620 from metastatic deposit had much lower GM3 level than noninvasive SW480 (Balanzino, L., Hakomori, S., unpublished data). Similarly, highly invasive bladder cancer YTS1 had much lower (barely detectable) GM3 level as compared to benign, noninvasive KK47.120,121 Malignancy of each case cannot be correlated solely with ganglioside level, but is more dependent on complex formation with TSP CD9 and integrin a3b1. 2. Many human cancer tissues or cancer cell lines have been characterized by expression of GSL antigens such as Gb3, GD3, GD2, globo-H, dimeric Lea, sialyl-Lex, sialyl-dimeric Lex, sialyl-Lea, Ley, trifucosyl-Ley, GalNAcb4 [Siaa3 or 6]Lc4Cer (Table 3). Expression of these antigens enhances tumor cell malignancy, and their presence in primary tumor is correlated with reduced patient life span.122 In contrast, some GSL antigens expressed in primary tumors suppress tumor progression (Table 4); the mechanism for this malignancy-inhibiting effect is not well known except in the case of GM3/CD9 complex (see Section 4.15.6).

Table 3 Tumor-associated GSL antigens that may promote tumor cell malignancy GSL

Structure

Type of tumor

GD3 Gb3 Globo-H

Siaa8Siaa3Galb4GlcCer Gala4Galb4GlcCer Fuca2Galb3GalNAcb3Gala4Galb4GlcCer Galb3GlcNAcb3Galb3GlcNAcb3Galb4GlcCer 4 4 Fuca Fuca

Melanoma Burkitt lymphoma, ovary, colorectal Breast, prostate

Dimeric-Lea (Lea–Lea) GalNAcb4 disialyl-Lc3

GalNAcb4Galb3GlcNAcb3Galb1R 3 6 Siaa Siaa

Colorectal Renal cell

Sialyl-LeX

Galb4GlcNAcb3Galb4GlcCer 3 3 Siaa Fuca

Gl, breast, lung

Sialyl-Lea

Galb3GlcNAcb3Galb4GlcCer 3 4 Siaa Fuca

Pancreas, Gl, breast, lung


281

Table 4 Tumor-associated GSL antigens that may inhibit tumor progression through complex formation with membrane proteins GSL

Structure

Type of tumor

GM3 Gb3 Gb5 globo-H

NeuAca3Galb4GlcCer (complexed with TSP CD9) Gala4Galb4GlcCer Galb3GalNAcb3Gala4Galb4GlcCer Galb3GalNAcb3Gala4Galb4GlcCer 2 Fuca NeuAca3 Galb4Galb4GlcNAcb3Galb4GlcCer GalNAcb4

Colorectal, bladder cancer (motility inhibition) Seminoma Seminoma Breast, prostate cancer

Sda

Colorectal cancer

Expression of a specific tumor-associated structure could be based on (1) precursor accumulation due to incomplete synthesis or (2) enhanced specific glycosylation. However, many studies along this line did not clarify the mechanism, since a single glycosylation depends on not only expression of a specific glycosyltransferase gene, but also various other factors, for example, (1) organizational arrangement of transferases in Golgi; (2) synthesis of a specific sugar nucleotide; (3) its transporters; and (4) recycling of glycosylated product through trans-Golgi network (TGN), endosomes, and lysosomes.123 The functional notion of these tumor-associated glycosyl epitopes is still largely unknown, except that expression of sialyl-Lea or sialyl-Lex in tumors promotes adhesion of tumor cells to activated endothelial cells through E-selectin interaction with sialyl-Lea or sialyl-Lex; for review, see Ref. 82.

4.15.7.2 Infection 4.15.7.2.1

GSLs as receptors for microbial exotoxins

GSLs are well known to function as receptors for microbial exotoxins. Binding of GM1 to Vibrio cholerae toxin, and of GD1b and GT1b to butolism toxin, are classically well known (for review, see Ref. 71). More recently, it was shown that verotoxin and shigella toxin bind to Gb3124–126 (see also Section 4.15.4).

4.15.7.2.2

GSL interaction with adhesin, and bacterial infection

GSLs are well established as having specific binding profiles to bacteria, through extensive studies by Karlsson and colleagues, as reviewed some time ago.127 Such binding profiles suggest that specific bacteria have GSL-binding lectins (adhesin) that may mediate infectivity, and that specific GSLs may block adhesin-mediated infection. The large amount of basic data presented by Karlsson127 may be useful for future application in prevention of microbial infection. This idea is not limited to GSLs, but applies to oligosaccharides carried by glycoprotein at the cell surface in general. Oligosaccharides as anti-infection agents were proposed some time ago, based on small-scale in vitro experiments;128 however, synthesis of oligosaccharides or GSLs in large quantity to prevent infection is not economically feasible.

4.15.7.2.3

Secretor/nonsecretor difference affecting E. coli infectivity

Incidence of urogenital infection caused by E. coli is 4–5 times higher in nonsecretor women than in secretors. Clinically isolated, infectious E. coli were shown to have adhesin with binding specificity to monosialyl-Gb5 (V3NeuAcGb5Cer), which mediates infectivity at urogenital epithelia.64 Expression of this structure is high in nonsecretors, and much lower in secretors because Gb5 is converted to globo-ABH structure, which has no capability to bind to E. coli.129 Staphylococcus saprophyticus infection to urogenital epithelia is mediated by a glycoconjugate containing GlcNAc, GalNAc, and sialic acid; the epitope structure may well be GSL rather than glycoprotein.130

4.15.7.2.4

Chlamydial infection

GSLs and gangliosides are not known to be involved in chlamydial infection. However, it should be noted that Chlamydia has N-linked glycan, particularly high-mannose type, which is recognized by mannose-binding protein expressed at host cells. Such mechanism as a basis of infection applies to Chlamydia trachomatis as well as Chlamydia pneumoniae, and infection was inhibited in vitro by high-mannose-type oligosaccharide.131

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4.15.7.2.5 Viral infection Currently, considerable attention is being paid to influenza virus infection. In particular, avian-type virus (H5N1) may have a structure similar to the ones that caused the worldwide ‘Spanish flu’ pandemic during 1917–18.132 A common receptor for all influenza viruses is sialic acid of glycoprotein and ganglioside. The virus receptor itself is sialidase, with differing susceptibility to sialidase inhibitors.133 A major focus of current studies is the comparison of virus receptor properties and susceptibility to sialidase (for review, see Ref. 134). Interestingly, human tracheal primary epithelial cells express both sialyl a2-3Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl a6Gal receptor for human influenza virus. Susceptibility to avian influenza virus may be determined by relative abundance of these two receptors.135

4.15.7.2.6 HIV infection and GSLs Regarding the HIV infection mechanism, increasing attention has been paid to the role of host cell GSLs in HIV entry to host cell, besides the well-established interaction between HIV envelope gp120 and T-cell CD4.136 Initially, brain cerebroside (GalCer) was found to be a receptor of gp120 in the infectious process of CD4-negative cell lines of nervous system, since anti-GalCer antibody blocked infection.137 This was strengthened by the observation that recombinant gp120 binds specifically to GalCer or sulfatide, but not to GlcCer, LacCer, or various other GSLs and gangliosides.138 The functional role of GSLs has been widely observed in HIV infection of some types of T-cells in which CD4 expression is minimal but lymphokine receptors CCR5 and CXCR4 act as secondary HIV receptors. Extensive studies by Blumenthal and colleagues clearly indicate that Gb3 or GM3 blocks HIV internalization by blocking gp120 binding to CCR5 or CXCR4 (for review, see Ref. 139). A novel soluble mimetic of Gb3 (adamantylGb3) inhibited binding of gp120 to Gb3 and hence inhibited HIV infection.140 N-Linked high-mannose-type glycan of gp120 affects optimal conformation of gp120 to interact with CD4.141,142 HIV infectivity is therefore controlled by two types of carbohydrate (one, high-mannose type; the other, GSL) depending on the site of the host cell.

4.15.7.3 Autoimmune Processes Little is known concerning the role of GSLs in control of autoimmune processes. A few examples are described below.

4.15.7.3.1 Neuropathy with autoimmune process Some cases of neuropathy initially display sensory nerve disturbance causing abnormal sensation (paresthesia), and later motor nerve paralysis with occurrence of detectable serum antibody directed to MAG.143 Similar observations were reported in a number of subsequent papers. The target of anti-MAG antibody in serum was identified as sulfated glucuronyl nLc4Cer (SO33GlcAb3Galb4GlcNAcb3Galb4Glcb1Cer), which is found as peripheral nerve myelin component.144,145 Therefore, anti-MAG antibody may cause peripheral nerve dysfunction. On the other hand, MAG interaction with gangliosides inhibits neuritogenesis (see Section 4.15.4, (6)). There may be multiple mechanisms by which anti-MAG antibody causes neuropathy. Autoimmune neuropathies such as GBS or FS are considered to be due to antiganglioside antibody, whose production is caused by certain infectious diseases. GBS (acute paralysis of arms and legs) occurs 1–3 weeks after bronchial or gastrointestinal infection by Gram-negative Campylobacter jejuni. The syndrome is associated with presence of anti-GM1 IgG antibodies in many cases,146,147 or with reactivity with different gangliosides (GD1a, GD1b, or their complex) in some cases.148 The GM1 structure is present at the side chain of LPS in C. jejuni, and is therefore considered strongly immunogenic.146 FS is caused by infection of C. jejuni whose LPS side chain includes GQ1b ganglioside, not other gangliosides. FS involves sudden onset of ocular muscle paralysis, and cerebellar dysfunction, due to production of anti-GQ1b IgG antibody with helper T-cells.149,150 Paralysis of arms and legs may occur later, so clear distinction between FS and GBS is sometimes difficult.

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Ono, M.; Handa, K.; Withers, D. A.; Hakomori, S. Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation. Cancer Res. 1999, 59, 2335–2339. 105. Ono, M.; Handa, K.; Sonnino, S.; Withers, D. A.; Nagai, H.; Hakomori, S. GM3 ganglioside inhibits CD9-facilitated haptotactic cell motility: Co-expression of GM3 and CD9 is essential in downregulation of tumor cell motility and malignancy. Biochemistry 2001, 40, 6414–6421. 106. Bremer, E. G.; Hakomori, S. GM3 ganglioside induces hamster fibroblast growth inhibition in chemically-defined medium: Ganglioside may regulate growth factor receptor function. Biochem. Biophys. Res. Commun. 1982, 106, 711–718. 107. Abercrombie, M.; Heaysmann, J. E. M. Observation on the social behavior of cells in tissue culture. Exp. Cell Res. 1953, 5, 111. 108. Abercrombie, M.; Heaysman, J. E. M. 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Gangliosides in DNA virus-transformed and spontaneously transformed tumorigenic mouse cell lines. Proc. Natl. Acad. Sci. USA 1969, 63, 1290–1296. 117. Hakomori, S.; Saito, T.; Vogt, P. K. Transformation by Rous sarcoma virus: Effects on cellular glycolipids. Virology 1971, 44, 609–621. 118. Hakomori, S.; Wyke, J. A.; Vogt, P. K. Glycolipids of chick embryo fibroblasts infected with temperature-sensitive mutants of avian sarcoma viruses. Virology 1977, 76, 485–493. 119. Miura, Y.; Kainuma, M.; Jiang, H.; Velasco, H.; Vogt, P. K.; Hakomori, S. Reversion of the Jun-induced oncogenic phenotype by enhanced synthesis of sialosyllactosylceramide (GM3 ganglioside). Proc. Natl. Acad. Sci. USA 2004, 101, 16204–16209. 120. Satoh, M.; Ito, A.; Nojiri, H.; Handa, K.; Numahata, K.; Ohyama, C.; Saito, S.; Hoshi, S.; Hakomori, S. Enhanced GM3 expression, associated with decreased invasiveness, is induced by brefeldin A in bladder cancer cells. Int. J. Oncol. 2001, 19, 723–731. 121. Mitsuzuka, K.; Handa, K.; Satoh, M.; Arai, Y.; Hakomori, S. A specific microdomain (‘glycosynapse 3’) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9. J. Biol. Chem. 2005, 280, 35545–35553. 122. Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res. 1996, 56, 5309–5318. 123. Gillard, B. K.; Clement, R. G.; Marcus, D. M. Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways. Glycobiology 1999, 8, 885–890. 124. Jacewicz, M.; Clausen, H.; Nudelman, E. D.; Donohue-Rolfe, A.; Keusch, G. T. Pathogenesis of Shigella diarrhea: XI. Isolation of a Shigella toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide. J. Exp. Med. 1986, 163, 1391–1404. 125. Hoey, D. E.; Sharp, L.; Currie, C.; Lingwood, C. A.; Gally, D. L.; Smith, D. G. 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L.; Schumacher-Perdreau, F.; Peters, G.; Heczko, P.; Pulverer, G. Hemagglutination by Staphylococcus saprophyticus and other coagulase-negative staphylococci. Microb. Pathog. 1988, 4, 379–383. 131. Kuo, C.; Takahashi, N.; Swanson, A. F.; Ozeki, Y.; Hakomori, S. An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells. J. Clin. Invest. 1996, 98, 2813–2818. 132. Le, Q. M.; Kiso, M.; Someya, K.; Sakai, Y. T.; Nguyen, T. H.; Nguyen, K. H.; Pham, N. D.; Ngyen, H. H.; Yamada, S.; Muramoto, Y.; Horimoto, T.; Takada, A.; Goto, H.; Suzuki, T.; Suzuki, Y.; Kawaoka, Y. Avian flu: Isolation of drug-resistant H5N1 virus. Nature 2005, 437, 1108. 133. Matrosovich, M.; Suzuki, T.; Hirabayashi, Y.; Garten, W.; Webster, R. G.; Klenk, H. D. Gangliosides are not essential for influenza virus infection. Glycoconj. J. 2006, 23, 107–113. 134. Suzuki, Y. Sialobiology of influenza: Molecular mechanism of host range variation of influenza viruses. Biol. Pharm. Bull. 2005, 28, 399–408. 135. Kogure, T.; Suzuki, T.; Takahashi, T.; Miyamoto, D.; Hidari, K. I.; Guo, C. T.; Ito, T.; Kawaoka, Y.; Suzuki, Y. Human trachea primary epithelial cells express both sialyl(alpha2–3)Galreceptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl(alpha2–6) Galreceptor for human influenza viruses. Glycoconj. J. 2006, 23, 101–106. 136. Poignard, P.; Saphire, E. O.; Parren, P. W.; Burton, D. R. gp120: Biologic aspects of structural features. Annu. Rev. Immunol. 2001, 19, 253–274. 137. Harouse, J. M.; Bhat, S.; Spitalnik, S. L.; Laughlin, M.; Stefano, K.; Silberberg, D. H.; Gonzalez-Scarano, F. Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceramide. Science 1991, 253, 320–323. 138. Bhat, S.; Spitalnik, S. L.; Gonzalez-Scarano, F.; Silberberg, D. H. Galactosyl ceramide or a derivative is an essential component of the neural receptor for human immunodeficiency virus type 1 envelope glycoprotein gp120. Proc. Natl. Acad. Sci. USA 1991, 88, 7131–7134. 139. Viard, M.; Parolini, I.; Rawat, S. S.; Fecchi, K.; Sargiacomo, M.; Puri, A.; Blumenthal, R. The role of glycosphingolipids in HIV signaling, entry and pathogenesis. Glycoconj. J. 2004, 20, 213–222. 140. Lund, N.; Branch, D. R.; Mylvaganam, M.; Chark, D.; Ma, X. Z.; Sakac, D.; Binnington, B.; Fantini, J.; Puri, A.; Blumenthal, R.; Lingwood, C. A. A novel soluble mimic of the glycolipid globotriaosyl ceramide inhibits HIV infection. Aids 2006, 20, 333–343. 141. Scanlan, C. N.; Pantophlet, R.; Wormald, M. R.; Ollmann Saphire, E.; Stanfield, R.; Wilson, I. A.; Katinger, H.; Dwek, R. A.; Rudd, P. M.; Burton, D. R. The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha1!2 mannose residues on the outer face of gp120. J. Virol. 76, 7306–7321. 142. Calarese, D. A.; Scanlan, C. N.; Zwick, M. B.; Deechongkit, S.; Mimura, Y.; Kunert, R.; Zhu, P.; Wormald, M. R.; Stanfield, R. L.; Roux, K. H.; Kelly, J. W.; Rudd, P. M.; Dwek, R. A.; Katinger, H.; Burton, D. R.; Wilson, I. A. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 2003, 300, 2065–2071. 143. Latov, N.; Braun, P. E.; Gross, R. B.; Sherman, W. H.; Penn, A. S.; Chess, L. Plasma cell dyscrasia and peripheral neuropathy: Identification of the myelin antigens that react with human paraproteins. Proc. Natl. Acad. Sci. USA 1981, 78, 7139–7142. 144. Chou, D. K.; Ilyas, A. A.; Evans, J. E.; Costello, C.; Quarles, R. H.; Jungalwala, F. B. Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy. J. Biol. Chem. 1986, 261, 11717–11725. 145. Ariga, T.; Kohriyama, T.; Freddo, L.; Latov, N.; Saito, M.; Kon, K.; Ando, S.; Suzuki, M.; Hemling, M. E.; Rinehart, K. L., Jr. Characterization of sulfated glucuronic acid containing glycolipids reacting with IgM M-proteins in patients with neuropathy. J. Biol. Chem. 1987, 262, 848–853.


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146. Yuki, N.; Taki, T.; Inagaki, F.; Kasama, T.; Takahashi, M.; Saito, K.; Handa, S.; Miyatake, T. A bacterium lipopolysaccharide that elicits Guillain–Barre syndrome has a GM1 ganglioside-like structure. J. Exp. Med. 1993, 178, 1771–1775. 147. Yuki, N.; Susuki, K.; Koga, M.; Nishimoto, Y.; Odaka, M.; Hirata, K.; Taguchi, K.; Miyatake, T.; Furukawa, K.; Kobata, T.; Yamada, M. Carbohydrate mimicry between human ganglioside GM1 and Campylobacter jejuni lipooligosaccharide causes Guillain–Barre syndrome. Proc. Natl. Acad. Sci. USA 2004, 101, 11404–11409. 148. Kaida, K.; Morita, D.; Kanzaki, M.; Kamakura, K.; Motoyoshi, K.; Hirakawa, M.; Kusunoki, S. Ganglioside complexes as new target antigens in Guillain–Barre syndrome. Ann. Neurol. 2004, 56, 567–571. 149. Yuki, N.; Taki, T.; Takahashi, M.; Saito, K.; Tai, T.; Miyatake, T.; Handa, S. Penner’s serotype 4 of Campylobacter jejuni has a lipopolysaccharide that bears a GM1 ganglioside epitope as well as one that bears a GD1 a epitope. Infect. Immun. 1994, 62, 2101–2103. 150. Yuki, N.; Handa, S.; Tai, T.; Takahashi, M.; Saito, K.; Tsujino, Y.; Taki, T. Ganglioside-like epitopes of lipopolysaccharides from Campylobacter jejuni (PEN 19) in three isolates from patients with Guillain–Barre syndrome. J. Neurol. Sci. 1995, 130, 112–116. 151. Galili, U. The a-gal epitope (Gala1–3Galb1–4GlcNAc-R) in xenotransplantation. Biochimie 2001, 83, 557–563. 152. Ito, K.; Handa, K.; Hakomori, S. Species-specific expression of sialosyl-Lex on polymorphonuclear leukocytes (PMN) in relation to selectindependent PMN responses. Glycoconj. J. 1994, 11, 232–237. 153. Koutcherov, Y.; Mai, J. K.; Paxinos, G. Hypothalamus of the human fetus. J. Chem. Neuroanat. 2003, 26, 253–270. 154. Forutan, F.; Mai, J. K.; Ashwell, K. W.; Lensing-Hohn, S.; Nohr, D.; Voss, T.; Bohl, J.; Andressen, C. Organisation and maturation of the human thalamus as revealed by CD15. J. Comp. Neurol. 2001, 437, 476–495. 155. Symington, F. W.; Bernstein, I. D.; Hakomori, S. Monoclonal antibody specific for lactosylceramide (LacCer). J. Biol. Chem. 1984, 259, 6008–6012. 156. Symington, F. W.; Hedges, D. L.; Hakomori, S. Glycolipid antigens of human polymorphonuclear neutrophils and the inducible HL-60 myeloid leukemia line. J. Immunol. 1985, 134, 2498–2506. 157. Kannagi, R.; Nudelman, E. D.; Levery, S. B.; Hakomori, S. A series of human erythrocyte glycosphingolipids reacting to the monoclonal antibody directed to a developmentally regulated antigen, SSEA-1. J. Biol. Chem. 1982, 257, 14865–14874. 158. Kannagi, R.; Levery, S. B.; Ishigami, F.; Hakomori, S.; Shevinsky, L. H.; Knowles, B. B.; Solter, D. New globoseries glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulatedantigen, stage-specific embryonic antigen 3. J. Biol. Chem. 1983, 258, 8934–8942. 159. Kannagi, R.; Cochran, N. A.; Ishigami, F.; Hakomori, S.; Andrews, P. W.; Knowles, B. B.; Solter, D. Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J. 1983, 2, 2355–2361. 160. Sekine, M.; Suzuki, M.; Inagaki, F.; Suzuki, A.; Yamakawa, T. A new extended globoglycolipid carrying the stage specific embryonic antigen-1 (SSEA-1) determinant in mouse kidney. J. Biochem. (Tokyo) 1987, 101, 553–562. 161. Ito, A.; Levery, S. B.; Saito, S.; Satoh, M.; Hakomori, S. A novel ganglioside isolated from renal cell carcinoma. J. Biol. Chem. 2001, 276, 16695–16703. 162. Kannagi, R.; Levery, S. B.; Hakomori, S. Hybrid type glycolipids (lacto-ganglio series) with a novel branched structure: Their presence in undifferentiated murine leukemia cells and their dependence on differentiation. J. Biol. Chem. 1984, 259, 8444–8451. 163. Ostrander, G. K.; Levery, S. B.; Eaton, H. L.; Salyan, M. E. K.; Hakomori, S.; Holmes, E. H. Isolation and characterization of four major neutral glycosphingolipids from the liver of the English sole (Parophrys vetulus): Presence of a novel branched lacto-ganglio-iso-globo hybrid structure. J. Biol. Chem. 1988, 263, 18716–18725. 164. Yamamoto, H.; Iida-Tanaka, N.; Kasama, T.; Ishizuka, I.; Kushi, Y.; Handa, S. Isolation and characterization of a novel Forssman-active acidic glycosphingolipid with branched isoglobo-ganglio- and neolacto-series hybrid sugar chains. J. Biochem. (Tokyo) 1999, 125, 923–930. 165. Rock, P.; Allietta, M.; Young, W. W. J.; Thompson, T. E.; Tillack, T. W. Ganglioside GM1 and asialo-GM1 at low concentration are preferentially incorporated into the gel phase in two-component two-phase phosphatidylcholine bilayers. Biochemistry 1991, 30, 19–25. 166. Tillack, T. W.; Allietta, M.; Moran, R. E.; Young, W. W. J. Localization of globoside and Forssman glycolipids on erythrocyte membranes. Biochim. Biophys. Acta 1983, 733, 15–24. 167. Rahmann, H.; Ro¨sner, H.; Ko¨rtje, K.-H.; Beitinger, H.; Veybold, V. Ca2þ-ganglioside interaction in neuronal differentiation and development. In Biological Function of Gangliosides (Progress in Brain Research, Vol. 101); Svennerholm, L., Asbury, A. K., Reisfeld, R. A., Sandhoff, K., Suzuki, K., Tettamanti, G., Toffano, G., Eds.; Elsevier: Amsterdam, The Netherlands, 1994; pp 127–145. 168. Hakomori, S.; Igarashi, Y. Functional role of glycosphingolipids in cell recognition and signaling. J. Biochem. (Tokyo) 1995, 118, 1091–1103.

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Biographical Sketch

Sen-itiroh Hakomori graduated from Tohoku Univ. School of Medicine, Sendai, Japan. He started his research career in glycoscience at the Dept. of Biochemistry at the same univesity. He earned his Dr. Med. Sci. at the same university in 1956, and conducted postdoctoral research as a Fulbright Fellow at Harvard Medical School, 1956–58. He held a faculty position at Tohoku Pharmaceutical Univ. In 1971, he was appointed Prof. of Pathobiology (School of Public Health) and Microbiology (School of Medicine), Univ. of Washington. He has been Head, Program of Biochemical Oncology, Fred Hutchinson Cancer Research Center (1975–1987); Scientific Director, The Biomembrane Institute, Seattle, WA (1987–1996). He is currently Professor Emeritus at Univ. of Washington (since 2005), and Head, Division of Biomembrane Research, at Pacific Northwest Research Institute (since 1996).

4.16 The HNK-1 Carbohydrate in the Nervous System T. Kawasaki, Ritsumeikan University, Shiga, Japan S. Oka and Y. Kizuka, Kyoto University, Kyoto, Japan ß 2007 Elsevier Ltd. All rights reserved. 4.16.1 Introduction

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4.16.2 Biosynthesis of the HNK-1 Carbohydrate

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4.16.3 GlcAT-P Gene-Deficient Mice

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4.16.4 How Does the HNK-1 Carbohydrate Function?

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4.16.5 Future Perspective

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4.16.1 Introduction The HNK-1 carbohydrate is a negative-charged trisaccharide glycoepitope expressed on glycoproteins and glycolipids in the nervous system. Since this carbohydrate had been identified first as a cell surface antigen expressed on human natural killer cells,1 it was thought to be involved in NK cell functions in the immune system. However, many groups have reported that this carbohydrate is predominantly expressed in the nervous system rather than in the immune system.2 In addition, it has been revealed that the HNK-1 epitope expression is conserved among various species from vertebrates to invertebrates such as Drosophila, Zebra fish, Xenopus, and mammals.3–5 Therefore, the HNK-1 carbohydrate is now considered to have some important roles in the nervous system. The HNK-1 carbohydrate consists of a GlcA attaching to N-acetyllactosamine structure (HSO3-3GlcAb1-3Galb14GlcNAc-) (Figure 1) on a series of cell adhesion molecules, including neural cell adhesion molecule (NCAM), L1, P0, telencephalin,6 and some extracellular matrix (ECM) proteins, including tenascin-R, phosphacan,7 and others. On the other hand, HNK-1 carbohydrate-binding proteins are known to exist in the nervous system, such as sulfoglucuronylcarbohydrate-binding protein (SBP-1), laminin, or brevican.8–10 Expression of the HNK-1 carbohydrate is temporally and spatially regulated during the development in the nervous system and characteristic expression of this epitope is observed in migrating neural crest cells,11 rhombomeres,12 and cerebellum.13 The HNK-1 carbohydrate epitope associates with neural crest cell migration,11 neuron to glial cell adhesion,14 outgrowth of astrocytic processes and migration of cell body,15 as well as the preferential outgrowth of neurites from motor neurons.16 These lines of evidence indicate that the HNK-1 carbohydrate epitope plays important roles in cell–cell and cell–substrate interaction during the development of the nervous system. So far a number of glycosyltransferase genes and related genes have been cloned and targeted deletion of these genes has revealed the roles of glycans in the modulation of cellular interactions at individual level. The similar analysis has been applied to the HNK-1 carbohydrate, and the functional roles of the carbohydrate in the neural network have been revealed. In this chapter, we will describe briefly the biosynthesis and functional roles of the HNK-1 carbohydrate in the nervous system.

4.16.2 Biosynthesis of the HNK-1 Carbohydrate The key step of the HNK-1 carbohydrate biosynthesis is GlcA transfer, since the inner structure, N-acetyllactosamine, is commonly found in a number of glycoconjugates (Figure 1b). Moreover, GlcA, only found in glycosaminoglycan chains, does not exist in N- and O-glycans except for the HNK-1 carbohydrate. The glucuronyltransferases that play a key role in the HNK-1 carbohydrate biosynthesis were cloned and well characterized. Terayama et al. had succeeded in cloning of glucuronyltransferase from rat brain and had named it GlcAT-P.17 Soon after that, the second glucuronyltransferase, GlcAT-S, was cloned.18 By Northern blot analysis using several tissues from adult rat, it is revealed that both GlcAT-P and GlcAT-S mRNA are expressed only in the nervous system. Moreover, these enzymes are able to transfer GlcA to the nonreducing end of N-acetyllactosamine, and when these genes are overexpressed in cultured COS-1 cells, the HNK-1 carbohydrate is actually expressed at the cell surface. However, GlcAT-P, compared with

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The HNK-1 Carbohydrate in the Nervous System

Glycoprotein

Asn

P0

HNK-1 carbohydrate

Glycolipid O HN

SGGL-1 O

(a)

HNK-1 carbohydrate

Ceramide

R N-Acetyllactosamine Glucuronyltransferases (GlcAT-P and GlcAT-S)

N-Acetylglucosamine Mannose Galactose

R Fucose Sulfotransferase (HNK-1 ST) Glucuronic acid R

Sulfate group

(b) HNK-1 carbohydrate Figure 1 Structures and biosynthesis of the HNK-1 carbohydrate epitope. a, Glycans on glycoprotein P035 and glycolipid SGGL-136 are schematically represented. Several neural cell molecules and glycolipids bear the same unique sulfated trisaccharide, HSO3-3GlcAb1-3Galb1-4GlcNAc, which is known as the HNK-1 carbohydrate. b, Glucuronic acid (GlcA) is transferred to the N-acetyllactosamine (LacNAc, Galb1-4GlcNAc) moiety by glucuronyltransferases, GlcAT-P and GlcAT-S. In the synthetic pathway, the sulfotransferase HNK-1 ST, which is expressed ubiquitously throughout brain, is assumed to sulfate the reducing terminal of GlcA to form HNK-1 carbohydrate immediately.

GlcAT-S, has wider expression range in brain and higher enzymatic activity, suggesting that GlcAT-P plays a major role in the HNK-1 carbohydrate biosynthesis. We investigated the detailed substrate specificity using purified native GlcAT-P from rat brain and recombinant soluble form of GlcAT-P and GlcAT-S19 and revealed that these enzymes transfer GlcA not only to a glycoprotein acceptor, asialo-orosomucoid, but also to a glycolipid acceptor, paragloboside. Interestingly, the activity of GlcAT-P toward glycoprotein is markedly enhanced in the presence of sphingomyelin (SM), but that of GlcAT-S does not depend on the presence of phospholipids. In the case of glycolipid acceptor substrate, GlcAT-P essentially requires the presence of phospholipids such as phosphatidylinositol (PI) for its enzymatic activity. PI also enhanced the activity of GlcAT-S toward glycolipids, but its specific activity is much lower than that of GlcAT-P. The activity of these two enzymes toward various kinds of oligosaccharides also revealed interesting results. GlcAT-P strictly recognizes N-acetyllactosamine structure (Galb1-4GlcNAc) as an acceptor substrate, whereas GlcAT-S can use other oligosaccharides such as lacto-N-biose (Galb1-3GlcNAc), lactose (Galb1-4Glc), or Galb1-4Gal as a substrate. Moreover, among bi-, tri-, and tetraantennary N-linked oligosaccharides, GlcAT-P increasingly recognizes these oligosaccharide acceptors in proportion to the number of branches, whereas GlcAT-S strongly prefers triantennary structure. These results indicate that GlcAT-P and GlcAT-S may synthesize functionally and structurally different HNK-1 carbohydrates in the nervous system. The final step of the HNK-1 carbohydrate biosynthesis is sulfation of GlcA at 3-position (Figure 1b). One sulfotransferase involved in this sulfation step was cloned and named as HNK-1 ST.20 The following results suggest


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that sulfation is necessary for HNK-1 carbohydrate functions. One, HNK-1-laminin interaction disappeared after removing the sulfate group from the HNK-1 carbohydrate.9 Two, all the HNK-1 structure so far examined in the nervous system is in the sulfated form, and the nonsulfated form of this epitope has not been identified.21 Curiously, HNK-1 ST mRNA is expressed not only in the nervous system but also in other tissues where the HNK-1 carbohydrate epitope does not exist, suggesting the possibility that HNK-1 ST may play some other roles in these tissues.

4.16.3 GlcAT-P Gene-Deficient Mice As described above, GlcAT-P is a major key enzyme for the HNK-1 epitope biosynthesis; we have generated mice with a targeted deletion of the GlcAT-P gene.22 As expected, GlcAT-P-deficient mice lack almost all the expression of the HNK-1 carbohydrate in the nervous system. Unexpectedly, however, despite the disappearance of the HNK-1 carbohydrate expression, there was no visible abnormality in embryonic development and no significant difference in body weight or brain size between GlcAT-P gene-deficient mice and wild-type mice. The HNK-1 carbohydrate is commonly expressed in a series of cell adhesion molecules (CAMs) such as NCAM, L1, telencephalin, and tenascin-R. These CAMs are important constituents of the synaptic structure and play important and diverse roles in the regulation of synaptic plasticity. Aberrations of long-term potentiation (LTP) in the hippocampal CA1 region occur in mice deficient in NCAM23 and telencephalin.24 Mice lacking ECM molecule tenascin-R are also shown to exhibit aberrant LTP in the CA1 region.25 On this background, we have analyzed LTP in the CA1 region of GlcAT-P genedeficient mice to examine the effect of HNK-1 carbohydrate deficiency on synaptic plasticity. Excitatory postsynaptic potentials (EPSPs) have been evoked by stimulating afferent fibers in the stratum radiatum of the CA1 region, resulting in the significantly lower magnitude of LTP in GlcAT-P gene-deficient mice than that in wild-type mice. In view of the reduced LTP, two types of spatial learning tests have been carried out (Figures 2a and 2b). In the Morris water maze test, the time taken to reach the hidden platform (escape latency) is significantly longer for the GlcAT-P gene-deficient mice than wild-type mice (Figure 2a), and in the water-filled multiple T-maze task, GlcAT-P gene-deficient mice show increased escape latencies to the goal arm (Figure 2b). These results suggest that the differences in performance of GlcAT-P gene-deficient mice in these tasks are related to impaired spatial learning. The GlcAT-P gene-deficient mice also exhibit greater exploratory activity in a Y-maze (Figure 2c). In the elevated plus maze test, GlcAT-P gene-deficient mice spend significantly longer time in open arms than the wild-type mice (Figure 2d). These results suggest that the GlcAT-P gene-deficient mice show reduced anxiety-like behavior. The abnormality in higher brain functions of the GlcAT-P gene-deficient mice such as reduced LTP at the Schaffer collateral-CA1 synapses, defects in spatial memory formation, greater exploratory activity, and modified anxietyrelated behavior is considerably similar to those in NCAM gene-deficient mice.23 This was the first study to demonstrate the involvement of a carbohydrate, notably of only a single nonreducing terminal carbohydrate residue, in higher-ordered brain functions including learning and memory. The detailed molecular mechanisms by which the HNK-1 carbohydrate modulates the higher-ordered brain functions are not clear so far. However, it should be emphasized that only a subpopulation of CAMs expresses the HNK-1 carbohydrate and that the expression is regulated independently from the biosynthesis of the protein backbone of CAMs, indicating that the HNK-1 carbohydrate can be a characteristic functional component in vivo as a fine-tuner that regulates synaptic plasticity or other brain functions. Alternatively, the HNK-1 carbohydrate itself may be involved in LTP via interaction with binding proteins (receptors) on the cell surface or in the cell matrix. However, the association of these receptors or binding proteins with LTP has not been proved. Although the expression of the HNK-1 carbohydrate is almost completely disappeared in GlcAT-P genedeficient mice, a low level of the HNK-1 epitope remained in some limited region that may probably be generated by GlcAT-S.22 In the cerebral cortex, some parts of interneurons seem to express the HNK-1 carbohydrate and such remaining expression is not so significant up to postnatal 2 weeks but seems to increase gradually thereafter. These signals and morphological features observed mainly in cortex layers 3–4 are very similar to so-called perineuronal nets (PNs), which are known to comprise lattice-like accumulation of the ECM on an unidentified subset of neurons.26 Staining with immunofluorescence-labeled WFA lectin, a well-known marker of PNs, revealed that a subpopulation of cortical neurons that are forming PNs is actually HNK-1 positive. These signals appear to be generated by GlcAT-S, but it has not been proved yet. As described above, GlcAT-S may synthesize HNK-1 carbohydrates functionally different from those synthesized by GlcAT-P. However, detailed structure and function of the remaining HNK-1 carbohydrate or carrier proteins of the remaining HNK-1 carbohydrate in GlcAT-P gene-deficient mice remain to be elucidated.

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Figure 2 Impaired spatial learning and behavioral alterations in GlcAT-P / mice. GlcAT-P þ/þ (n¼17, open circles) and / (n¼18, closed circles) mice were tested with spatial learning tasks ((a) and (b)). a, Mice were trained in a Morris water maze for 4 consecutive days. Learning performance is expressed as the mean escape latency of four trials per day. GlcAT-P / mice showed significantly longer escape latency than GlcAT-P þ/þ mice ( p85%) of brain gangliosides in adult mammals and birds. These are biosynthesized stepwise as shown in Figure 3. As brain development progresses, the simple gangliosides GM3 and GD3 give way to more complex species, with GD1a and GM1 becoming the most abundant brain gangliosides.29,30 In the adult, GM1 is found predominantly on myelin, whereas GD1a, GD1b, and GT1b are expressed predominantly on neurons and axons.31–33 Because gangliosides are major molecular determinants on the surface of neural cells, their functions have been the focus of intense interest. Historically, the first functions identified for gangliosides were as binding sites for bacterial toxins, including cholera toxin and tetanus toxin.34,35 Their physiological functions were unknown until recently, and have been revealed through both in vitro experiments and genetic alternations of ganglioside expression in vivo. One of the more striking findings from these studies, given the complexity of gangliosides and the change in ganglioside expression during development, was that complex gangliosides are not required for the development of the vertebrate nervous system.36 Subsequent studies defined more subtle, but nonetheless important roles of gangliosides in nervous system structure and function,37,38 and revealed the devastating results of altered ganglioside expression in humans.39

4.18.3.1 Complex Ganglioside Function in Neuronal Homeostasis and Axon–Myelin Stabilization During development, the conversion of the simple ganglioside structures GM3 and GD3 to more complex structures, that predominate in the adult brain, is accomplished by a series of glycosyltransferases starting with UDP-GalNAc: GM3 b-N-acetylgalactosaminyltransferase (EC 2.4.1.92; GM2/GD2 synthase), which is coded by the Galgt1 gene (new gene symbol B4galnt1). The same enzyme is responsible for transfer of GalNAc from UDP-GalNAc to lactosylceramide

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GM3 GD3 GM2 GM1 GD1a GD1b GT1b GQ1b Increasing ganglioside complexity

Figure 6 Ganglioside expression in liver and brain. a, Sialylated glycoconjugate species in human liver and brain. Proteinbound sialic acid is in equal amount in the two tissues, whereas lipid-bound sialic acid (gangliosides) is much higher in the brain. Data compiled from Tettamanti et al.7 and Alhadeff and Holzinger.93 b, Ganglioside patterns in human liver and brain. Mole percent composition of gangliosides are presented. Whereas essentially all of the ganglioside in liver is the simple structure GM3, brain gangliosides are more varied and more complex. Data compiled from Tettamanti et al.7 and Nilsson and Svennerholm.94 Ganglioside nomenclature is that of Svennerholm.9

(LacCer), GM3, and GD3 to produce GA2, GM2, and GD2 respectively (Figure 3). Mice with a disrupted Galgt1 gene synthesize GD3 and GM3, but not complex gangliosides. The total brain ganglioside concentration is the same in wild-type and Galgt1-null mice, with only the complexity of the saccharide structures differing. These mice provided the first opportunity to thoroughly evaluate the function of complex gangliosides in the brain. Galgt1-null mice are born at the expected frequencies and appear to develop normally despite the complete absence of complex brain gangliosides.36 However, they demonstrate greater susceptibility to seizure and progressive motor neuropathies, revealing roles for complex gangliosides in maintaining neuronal homeostasis and axon–myelin interactions.40,41 Galgt1-null mice are more susceptible to seizure than their wild-type counterparts.40 In a rodent model of status epilepticus induced by systemic infusion of the glutamate receptor agonist kainic acid, adult Galgt1-null mice had markedly more severe seizures compared to wild-type mice. Prior administration of a cell-permeant ganglioside GM1 analog, Liga20, to Galgt1-null mice largely reversed their enhanced seizure susceptibility. Previous studies relating GM1 to calcium homeostasis,42 along with data demonstrating reduced calcium flux into the nuclear envelope of Galgt1-null mice,40 lead to the conclusion that gangliosides regulate intracellular calcium sequestration and protect cells from toxic calcium fluxes, perhaps by direct association with and regulation of a nuclear envelope sodium– calcium exchanger. Greater susceptibility of Galgt1-null mice to excitotoxin-induced seizure is consistent with spontaneous seizure activity observed in mice and humans with deficits in ganglioside biosynthesis (see below). A second phenotype of Galgt1-null mice relates to deficits in axon–myelin interactions. Histological analyses of adult PNS and CNS of Galgt1-null mice revealed marked axon degeneration.41,43 Other neurohistological hallmarks of

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these mutants included reduced axon diameter and reduced axonal neurofilament spacing. These data are indicative of a problem in stable axon–myelin interactions resulting in changes in axon cytoarchitecture and long-term axon instability. As Galgt1-null mice aged, ongoing axon degeneration resulted in significant and progressive behavioral neuropathies, including deficits in reflexes, strength, coordination, and balance, such that 1-year-old Galgt1-null mice demonstrated splayed hindlimbs and walked in small labored movements.41,44 The phenotype of Galgt1-null mice is consistent with a lack of proper cell–cell communication between axons and the cells that ensheathe them with myelin: oligodendrocytes in the CNS and Schwann cells in the PNS. The mechanism for this deficit may involve a lectin – myelin-associated glycoprotein (MAG). Myelination is required for rapid nerve conduction, but myelin also nurtures the axons it ensheathes. When myelin is lost, the underlying axons suffer.45 Axon degeneration associated with dysmyelination is a key factor in the pathology of progressive demyelinating diseases, such as multiple sclerosis.46,47 MAG, which is a quantitatively minor protein in myelin, acts as an axon–myelin stabilizing factor by binding to specific receptors on the axon surface.48 The discovery that MAG is a sialic acid-binding lectin49 (Chapter 3.27) led to the hypothesis that it functions by binding to nerve cell surface gangliosides. Binding studies demonstrated that MAG specifically recognized the ‘NeuAca2-3Galb1-3GalNAc’ terminus of gangliosides GD1a and GT1b.50–52 Since Galgt1-null mice lack that terminus on gangliosides (Figure 3), an axon-degeneration phenotype similar to that of Mag-null mice was predicted and observed.43 If MAG and GD1a/GT1b function via complementary binding to each other, Mag-null, Galgt1-null, and Mag-Galgt1-double-null mice would be expected to have a similar phenotype. To a significant extent, this is the case, with mice of all three genotypes displaying similar PNS and CNS axon degeneration, decreased axon caliber, and decreased neurofilament spacing compared to wild-type mice, leading to similar motor behavioral deficits.41 These observations, combined with prior MAG binding specificity studies,52 are consistent with the conclusion that MAG and complex gangliosides function, in part, via complementary binding to each other. This conclusion gained further support in studies of another function of MAG, regulation of axon regeneration, a topic discussed later in this chapter. Mag-null and Galgt1-null mice both displayed significant hyperactivity, despite their diminished motor behavioral skills.41 Whether this shared phenotype is related to the seizure susceptibility of the Galgt1-null mice (see above) is not yet known. The phenotypes of Galgt1-null mice reveal the functions of complex gangliosides as a group, but does not assign those functions to any particular subset of complex ganglioside structures. In addition, the increased brain concentrations of GD3 and GM3 in Galgt1-null mice may contribute to their phenotype.

4.18.3.2 Functional Roles of Simple Gangliosides and GlcCer-Based Glycosphingolipids in the Nervous System Revealed Using Mouse Genetics Although complex gangliosides are abundant and diverse in the brain, Galgt1-null mice, which lack all complex gangliosides, developed a grossly normal nervous system with subtle rather than devastating neural pathology. To test the roles of simpler gangliosides, additional biosynthetic gene disruptions were engineered. Disruption of Siat8a (GD3-synthase, new gene symbol St8sia1) in mice resulted in loss of the major gangliosides GD1b and GT1b, with compensatory increases in GM1 and GD1a, consistent with the biosynthetic pathway shown in Figure 3. These mice showed no neural pathologies.53 Double-mutant mice with disrupted Siat8a and Galgt1 genes express only ganglioside GM3 in the brain, with the GM3 concentration roughly equivalent to the combined complex ganglioside concentration in wild-type mouse brain.53 Mice expressing only GM3 were viable, but demonstrated a severe neurological phenotype with sharply increased sensitivity to audiogenic seizure, which may be related to the enhanced kainic acid seizure sensitivity in Galgt1-null mice (see above). A second line of Galgt1/Siat8 double-mutant mice was subsequently reported. Although seizure was not noted, the authors observed degenerative changes in peripheral nerves of relatively young mice.54 Together, these findings indicate that mice lacking GD3 and complex gangliosides (GM3-only mice) have phenotypes akin to the Galgt1-null mice, but more severe, suggesting that GD3 partially compensates for the lack of more complex gangliosides in neuronal homeostasis and axon stability. In an effort to generate mice lacking all ganglio-series gangliosides, simple and complex, mice were engineered to lack Siat9, GM3 synthase (new gene symbol St3gal5).55 The resulting mutant mice were viable and showed no overt neuronal pathologies. These mice were much healthier than the Siat8a/Galgt1-double-null mice that express only GM3. Ganglioside analysis in Siat9-null mice revealed the reason. When GM3 synthesis was blocked, the LacCer that accumulated was acted upon by the Galgt1 gene product and other glycosyltransferases leading to the synthesis of the so-called ‘0-series’ gangliosides cisGM1 (GM1b) and GD1a (dashed box, Figure 3). Whereas these structures have been reported as rare brain gangliosides, in Siat9-null mice they became the major species and were expressed at


331

concentrations comparable to the major brain gangliosides in wild-type mice. The overtly normal neural phenotype of Siat9-null mice suggests that cisGM1 and GD1a effectively compensate for the loss of common complex gangliosides. Prior studies demonstrating that GD1a is a potent ligand for MAG are consistent with this interpretation.56 This conclusion was further confirmed when Galgt1 was disrupted in Siat9-null mice (Siat9/Galgt1 double null).57 In these mice, both GM3 synthesis and the pathway from lactosylceramide to the 0-series gangliosides were absent (Figure 3). They lacked all ganglio-series gangliosides in the brain, instead expressing excess lactosylceramide. Although Siat9/ Galgt1 double-null mice were born at expected frequencies, had grossly normal morphology, and developed a functional nervous system, they succumbed within weeks to a severe neurodegenerative disease marked by reduced brain size, white matter vacuolization, axon degeneration, oligodendroglial cytopathology, and perturbed cell–cell interactions at nodes of Ranvier. Most Siat9/Galgt1-double-null mice were dead within 2 months. Whereas gangliosides are evidently not essential for development of the nervous system, they are required for critical cell–cell interactions and cellular homeostasis in the brain. Further evidence that GlcCer-based glycosphingolipids (including gangliosides) are not required for brain development, but are essential to brain function, comes from experiments in which the enzyme responsible for GlcCer synthesis, UDP-glucose:ceramide glucosyltransferase (EC 2.4.1.80), encoded by the Ugcg gene (Figure 3), was disrupted. Since mice engineered to lack the Ugcg gene die at gastrulation, investigators used Cre–Lox technology to disrupt Ugcg selectively in the nervous system.58 The resulting mice had greatly reduced (G (R92G), where arginine 92 is conserved from Homo sapiens to S. pombe. Additionally, the heterozygous patients carried either insertions or deletions, resulting in premature stop codons. The milder case was, on the other hand, homozygous for another mutation (742T>C, S248P), which is not conserved between mammals and yeast. This may explain the discrepancy in phenotypic expression; however, more patients with other mutations need to be identified to validate this subgroup heterogeneity. It is noticeable that an enzyme assay never was performed in the milder case, which allows the speculation that the S248P mutation is a polymorphism. The diagnosis is based on Tf analysis, enzymatic analysis of Dol-P-Man synthase, and a defective LLO pattern with accumulation of Man5GlcNAc2 structures. To exclude the risk of mutations being polymorphisms, an enzyme assay should always be performed. There is no current therapy for CDG-Ie patients, even though the LLO pattern of fibroblasts can be corrected with exogenous addition of Man and Man-1-P derivatives. No animal model of CDG-Ie has been created.

4.19.4.9 CDG-If The underlying defective gene in CDG-If (MPDU-1/Lec35) encodes a protein whose function is not fully understood, but is involved in the utilization of Dol-P-Man and Dol-P-Glc. There is no yeast homolog for MPDU-1/Lec35.

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Phenotypically, CDG-If resembles CDG-Id and -Ie. The patients are usually born at term with normal birth weight and length. They often develop muscular hypotonia, blindness, and severe psychomotor retardation; however, myelination by MRI is usually normal.25 In addition to these symptoms, two patients presented with ichthyosis25,26 and others have shown generalized eczema or patchy desquamation.25 This is the only CDG subtype associated with skin disorders, which may point to a role, other than that in N-linked glycosylation, for MPDU-1 in skin growth and homeostasis. To date, there are six known patients and the diagnosis is based on the Tf pattern, a pathological LLO pattern (accumulation of Man5GlcNAc2 and Man9GlcNAc2 structures), and genetic analysis with complementation of fibroblasts to exclude polymorphisms. No therapy is currently available for CDG-If and no animal model system has been created.

4.19.4.10 CDG-Ig Mutations in hALG12, causing deficiency of the mannosyltransferase that catalyzes the addition of the eighth Man residue to the growing LLO chain, constitute the molecular explanation to this subtype.24,92,93 The patients present with psychomotor retardation, hypotonia, and often inverted nipples and subcutaneous fat pads. Interestingly many of these patients are more prone to infection and show a deficiency in IgG.94 Of the known male patients, all have shown genital hypoplasia, but there is no distinct molecular explanation for this finding.94 Furthermore, facial dysmorphisms with epicantus, large ears, and thin face have been observed in several of the patients.95 In this group, the variation of severity is considerable and a very active intervention with physical, speech, and occupational therapy was suggested to be clearly beneficial.94 There are seven known cases, and 10 published pathogenic mutations. The diagnosis is based on accumulation of truncated LLO (Man7GlcNAc2 structures) and genetic analysis. No current therapy for CDG-Ig is available and no genetic animal model has been brought through.

4.19.4.11 CDG-Ih Deficiency of the second glucosyltransferase in the LLO biosynthesis (encoded by hALG8) causes CDG-Ih.23 Along with CDG-Ib, it is the only CDG subtype that can present without detectable neuronal involvement. The patients often present with a severe syndrome involving the intestine, the kidneys, and the liver, and an early, fatal outcome is often seen (coagulopathies, PLE, kidney failure).96 The lack of severe symptoms from the brain can be explained, in most patients, by the fact that they succumbed before reaching an age where they could be properly tested.96,97 However, the first known case was 3 years at the time of diagnosis, and no neurological findings were present.23 Possibly the lack of CNS symptoms in this case can be explained by the relatively low underglycosylation (Glc1Man9GlcNAc2 structures are transferred relatively well to proteins), whereas the presence of a structure derived from this precursor may be involved in the kidney pathology, not seen commonly in other CDG subtypes. The LLO pattern shows accumulating Man9GlcNAc2 and Glc1Man9GlcNAc2 structures. The presence of Man9GlcNAc2 is likely due to the action on the accumulating Glc1Man9GlcNAc2 structure by an ER glucosidase. This is strengthened by the fact that the Glc1Man9GlcNAc2 structures increase upon treating the cells with castanospermine, an inhibitor of the a-glucosidase.23,96 The LLO pattern along with genetic analysis, diagnoses CDG-Ih, and there are five known cases to date.23,96,97 Since CDG-Ib is treatable, it is vital to exclude this subtype before continuing the diagnosing process of CDG-Ih. There is currently no available therapy of CD-Ih and no animal model exists.

4.19.4.12 CDG-Ii The defective gene is hALG2, encoding the second mannosyltransferase in the LLO pathway. The only patient published with this syndrome was normal at birth, but during the first year developed hypomyelination, mental retardation, seizures, hepatomegaly with coagulation deficiencies, and coloboma of the iris.22 It is noteworthy that on the mother’s side, several cases of early infantile deaths, psychomotor retardation, migraine and seizures were known. The analysis of the small Dol-linked oligosaccharides showed an accumulation of both Man1GlcNAc2-P-P-Dol and Man2GlcNAc2-P-P-Dol. The authors show that this may be caused by alternate pathways in the early steps of the LLO biosynthesis, where the addition of the (a1-3)-linked ‘second’ Man also may be the third mannosylation step.22 Diagnosis was based on the abnormal LLO pattern, an enzymatic assay, genetic analysis, and complementation of a yeast strain. There is no current therapy of CDG-Ii and no animal model system is known.

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4.19.4.13 CDG-Ij In this subtype, the defective gene (DPAGT1) encodes the GlcNAc transferase that catalyzes the addition of the first GlcNAc to the growing LLO chain (GPT). There is only one known case with this defect.21 The patient presented with mental retardation, microcephaly, intractable seizures, muscular hypotonia, and esotropia. The diagnosis was based on LLO analysis (low amounts of LLO formed), enzymatic assays, and genetic analysis. The paternal allele contained a single point mutation (660A>G), and the resulting protein had no GlcNAc-transferase activity. Interestingly, the maternal allele produced a complex pattern of several alternatively spliced bands after reverse transcriptasepolymerase chain reaction (RT-PCR), but no mutations were found on the genomic level. Mice embryos deficient in GPT have been created. They complete the pre-implantation development, but die shortly after implanting in the uterine epithelium around days 4–5 post fertilization.98 There is no current therapy of CDG-Ij.

4.19.4.14 CDG-Ik CDG-Ik stems from deficiency of the first mannosyltransferase (encoded by hALG1) in the LLO biosynthetic pathway. It is a very severe syndrome where only one of four known patients survived into the second year.20,99,100 Main features are intractable seizures, hypotonia, cerebral atrophy, visual impairment, and coagulopathy. One patient developed nephrotic syndrome and hypogammaglobulinemia,100 whereas another had grave dysmorphic features and cardiomyopathy,99 showing the large variability within the CDG subtypes. The diagnosis is based on LLO analysis of short species,101 enzymatic assays, and genetic analysis. No animal model exists and there is no current treatment.

4.19.4.15 CDG-IL This syndrome is caused by mutations in the human ortholog of yeast alg9 (hALG9). This gene encodes an a-1,2mannosyltransferase that catalyzes the addition of both the seventh and the ninth mannose residue onto the growing LLO chain.102 There are two known patients to date.19,103 Both had severe brain abnormalities, including microcephaly,19 diffuse brain atrophy, cerebellar hypoplasia, delayed myelination, and seizures.103 One patient also showed failure to thrive, cystic renal disease, hepatosplenomegaly, pericardial effusion, and inverted nipples.103 Their diagnoses were based on a pathological LLO pattern (accumulating Man6GlcNAc2- and Man8GlcNAc7-structures) and genetic analysis. No animal model exists and there is no current therapy of CDG-IL.

4.19.4.16 CDG-IIa A deficiency in the GlcNAc transferase-II (encoded by MGAT2),104 an enzyme involved in the formation of complex type oligosaccharides,105 is the cause of this syndrome. Four patients are known.18 There is a marked psychomotor retardation compared to CDG-Ia, but no peripheral neuropathy and normal deep-tendon reflexes. The patients develop muscular hypotonia, and seizures have been described. In contrast to CDG-Ia patients, who are usually prothrombotic, CDG-IIa (formerly known as CDGS-II) patients have a tendency to bleed. A possible explanation is that platelets from CDG-Ia patients have an enhanced nonspecific platelet interaction, whereas CDG-IIa platelets are deficient in glycoprotein Ib reactivity with the vessel walls.106 Diagnosis of CDG-IIa is based on oligosaccharide structure analysis and enzymatic assays. An accurate phenocopy animal model has been made,48 but there is no therapy of CDG-IIa.

4.19.4.17 CDG-IIb This syndrome is caused by mutations in GCS1, encoding a-glucosidase-I,36 which constitutes the first processing step of the protein-bound N-linked oligosaccharides. Only one patient has been described, a consanguineous girl with generalized hypotonia, seizures, multiple dysmorphic features, hepatomegaly, feeding problems, and hypoventilation. The outcome was fatal at 74 days. Even though the estimated activity of the patient’s glucosidase was IgG2>IgG3>IgG4 and IgA1>IgA2. Each class or subclass is defined by the amino acid sequence of the heavy-chain constant regions and collectively constitute the nine immunoglobulin isotypes, present in all normal individuals. The sequence homology between heavy chains of different Ig class is 30%, while between subclasses of IgG or IgA it is >90%; reflecting their evolutionary relationships. Each immunoglobulin heavy-chain isotype expresses a unique profile of effector ligand recognition and consequent biological functions; Table 1 summarizes that profile for the IgG subclasses.2–7 The ability of IgG to activate its effector ligands is critically dependent on the IgG-Fc glycosylation, aglycosylated IgG being highly deficient in activities monitored in vitro that are thought to correspond to protective biologic functions in vivo (Table 2).11–17 The demonstration of glycoform heterogeneity for normal polyclonal human IgG-Fc led to the proposition that individual antibody glycoforms might differ, qualitatively or quantitatively, in biologic function. The finding of altered

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Table 2 The ligand binding/activation properties of aglycosylated human IgG1 FcgRI FcgRII FcgRIII C1 MBL/MR FcRn

Activation Binding Activation Activation Binding Catabolism Placental transport

Neutralization Apoptosis Immunogenicity

Reduced100 Abolished Abolished Abolished Abolished Unaffected ?? Unaffected? Unaffected? Reduced?

glycoform profiles for IgG isolated from the serum of patients with inflammatory diseases, initially patients with rheumatoid arthritis (RA), led to speculation that the ‘defect’ may underlie the etiology of the disease. This speculation suggested itself because RA is an autoimmune disease in which a diagnostic feature is the presence of autoantibodies, rheumatoid factors (RFs), specific for epitopes within the patient’s own IgG-Fc. Thus, the rationale was that the ‘abnormal’ IgG glycoform(s) corresponded to altered self-protein that is recognized as non-self by the immune system leading to the production of RF autoantibodies; RF and IgG were shown to be produced within synovial tissue and the RF/IgG immune complexes formed to activate the classical complement pathway, generating a self-perpetuating inflammatory reaction that could contribute to pathology. Subsequently, altered IgG-Fc glycoform profiles have been reported for a number of autoimmune inflammatory diseases in which RF is not present; therefore, a direct role in disease etiology may be dismissed and the impact on immune function must be assessed against our knowledge and understanding of glycosylation in normal polyclonal IgG, monoclonal IgG myeloma proteins, and recombinant monoclonal antibodies of defined antigen specificity.18–22

4.20.2.3 Glycosylation of Normal Human IgG The oligosaccharides released from normal polyclonal human IgG are of the complex diantennary type but exhibit considerable heterogeneity. Potentially, there are 32 different complex diantennary structures that could generate (3232¼1024)/2, that is, 512 IgG-Fc glycoforms, given random ‘pairing’ of heavy chain glycoforms; the apparent total is divided by 2 to account for the symmetry of the molecule. Analysis of oligosaccharides released from normal IgG-Fc and myeloma IgG proteins have defined a ‘core’ heptasaccharide (blue, Figure 3), with heterogeneity arising from variable addition of outer-arm sugar residues (red, Figure 3). The oligosaccharides released from IgG-Fc reveal a paucity of sialylation (0.2ppm for Lys-248 and Val-308 residues;44 this is a very localized change distant from the interaction site for the FcgRIIIa moiety. The finding of a changed environment for these residues is interesting since they were not predicted, from the crystal structure, to make contacts with the (a1-6) arm galactose residue; small perturbations for the predicted contact residues Lys-246, Asp-249, and Thr-256 were also observed. Conflicting data were reported from an NMR study which concluded that glycans with Gal(a1-6) residues had the same relaxation time as the protein backbone, while in the absence of (a1-6) arm galactose the glycan had relaxation rates 30 times slower, indicating high mobility and freedom from interactions with the protein structure.81 Studies of the influence of terminal galactose residues on IgG-Fc effector functions have resulted in conflicting reports, probably because the impact of core fucosylation was not appreciated or analyzed.40,43,79–83 Early studies reported only ‘slight’ differences in binding/adherence to FcgRI, FcgRII, or FcgRIII bearing cells for highly galactosylated anti-D antibody, compared to paucigalactosylated anti-D; however, a consistent reduction in lysis of erythrocytes mediated through FcgRIII on K cells was observed.82,83 No difference in binding or receptor-mediated signaling of FcgRII was reported for G0 and G2 glycoforms of the humanized IgG1 Campath-1H antibody.84 As previously stated, recombinant IgG antibody therapeutics secreted by cell lines adapted for commercial production is hypogalactosylated, relative to normal IgG-Fc. The possible consequences for in vivo activity are extrapolated from in vitro assays and animal experiments. Removal of terminal galactose residues from Campath1H was shown to reduce CDC but to be without effect on FcR-mediated functions.85 Similarly, the ability of Rituximab to kill tumor cells by CDC was shown to be reduced by a factor of 2 for the (G0)2 glycoform in comparison to the (G2)2 glycoform.86 The product that gained licensing approval was comprised of 25% of the G1 glycoform; therefore, regulatory authorities required that galactosylation of the manufactured product be controlled to within a few percent of this value. In the absence of sialic acid and galactose, the terminal sugar residues of IgG-Fc are GlcNAc. The serum protein MBL and the cellular MR can each recognize and bind arrays of GlcNAc.50–54,87–89 It is possible, therefore, that immune complexes comprised of G0 IgG-Fc may engage and activate these lectin molecules. MBL is a structural homolog of the C1q molecule that forms a complex with MASP-1, MASP-2, and MASP-3 molecules that are the homologs of C1s and C1r. The MBL/MASP complex circulates in the blood and when activated triggers the complement cascade through the initial binding and cleavage of C2, as for the C1 complex. A degalactosylated form of IgG1 was shown to bind and activate the MBL/MASP complex and the consequent cascade.50 A G0 glycoform of IgG4-Fc was also shown to bind MBL.51 These findings have been cited to suggest that in inflammatory diseases

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characterized by increased levels of G0 IgG-Fc glycoforms, activation of the lectin pathway may contribute to inflammation.87–89 The MR is a C-type lectin, expressed at the surface of macrophages, endothelial cells, and dendritic cells, that recognizes arrays of mannose and N-acetylglucosamine residues.90 Thus, immune complexes of G0 antibody glycoforms may aid their uptake by these cell types. Of particular interest is uptake by dendritic cells since they are ‘professional’ antigen-presenting cells and may be implicated in the development of autoimmunity. Experimental evidence is provided by the mouse collagen model of arthritis in which monoclonal anticollagen type II antibodies were passively administered as complexes of collagen with G0 or G2 glycoforms of anticollagen type II antibodies. The onset and severity of induced arthritis was greater for complexes formed with G0 glycoform of anticollagen type II antibodies.54 This finding may be relevant to the generation of immune responses to monoclonal antibody therapeutics; thus, the predominance of G0 glycoforms may similarly result in immune complex uptake by dendritic cells and the presentation of non-self-peptides generated from the antibody, for example, mouse V regions and idiotypic determinants. This role for MR has been disputed and there is evidence that enhanced antigen presentation is through the processing of glycoproteins taken up by DC-SIGN, another C-type lectin molecule.91 Cambi and Figdor have reviewed the multiple roles of C-type lectin receptors.92

4.20.4.3 The Influence of Fucose and Bisecting N-Acetylglucosamine on IgG-Fc Activities A comparison of the ability of antibody produced in different cell lines to mediate ADCC showed the product of rat YB2/0 cells to be more active than the product of CHO or SP2/0 cells.93 Analysis of the oligosaccharide profiles of these antibodies showed a possible correlation of ADCC activity with the ability of YB2/0 cells to produce IgG glycoforms bearing a bisecting N-acetylglucosamine residue. This rationale appeared to be vindicated by the demonstration of increased FcgRIII-mediated ADCC for antibody produced in CHO cells that had been transfected with the human b1,4-N-acetylglucosaminyltransferase III (GnTIII) gene and shown to be producing antibody bearing bisecting Nacetylglucosamine residues.94,95 A profound increase in FcgRIII-mediated ADCC was also reported for antibody produced in a mutant CHO cell line that failed to add either bisecting GlcNAc or fucose.96 Comparison of the ability of nonfucosylated, fucosylated, and bisecting GlcNAc glycoforms forms of IgG antibody to mediate ADCC led to the conclusion that it is the absence of fucose rather than the presence of bisecting GlcNAc that accounts for increased FcgRIII-mediated ADCC.97 Further studies showed that in CHO cells the expression of GnTIII competes with the endogenous a-1,6-fucosyltransferase and inhibits addition of fucose.98 Thus, the increased ADCC was in fact due to the absence of fucose. When transfected with genes for chimeric transferases that localize the GnTIII transferase to an earlier Golgi compartment, increased addition of bisecting GlcNAc and increased inhibition of the addition of fucose resulted.99 It is difficult to reconcile these data with the conclusion of Davies et al.95 who credited the improved ADCC to an IgG glycoform bearing bisecting GlcNAc and fucose. A possible explanation for this discrepancy could be that only the major glycoforms were positively characterized in this study and that the ADCC activity observed was due to the presence of a minor but increased population of nonfucosylated glycoforms. Due to the asymmetry of the IgG-Fc/ FcgRIII interface, the lack of fucose on one heavy chain should be sufficient to provide improved binding and activation. It should be noted that the glycoform bearing bisecting GlcNAc in the absence of fucose is not observed for normal human IgG-Fc (Figure 4). Surface plasmon resonance studies show that nonfucosylated IgG-Fc binds soluble recombinant FcgRIII with higher affinity than does the fucosylated form while aglycosylated IgG-Fc shows no evidence of binding.98,99 A similar study of the binding of different glycoforms of FcgRIIIa showed that glycosylation at asparagine 162 of the receptor influenced IgG-Fc binding, with aglycosylation at this site resulting in increased affinity for the normal fucosylated glycoform of IgG-Fc.100 This residue is at the interface between the FcgRIIIa receptor and IgG-Fc, and it was suggested that the presence of fucose on IgG-Fc might result in steric inhibition of glycosylated srFcgRIIIa binding.99 It was concluded that high-affinity IgG-Fc/FcgRIIIa binding requires an interaction of sugar residues attached at Asn-162 with surface structures of the nonfucosylated IgG-Fc glycoform and that due to the asymmetry of the IgG-Fc/ FcgRIIIa interaction nonfucosylation of one heavy chain would be sufficient for tight binding. Interestingly, it was shown that the increased affinity for the nonfucosylated glycoform of IgG-Fc was negated when FcgRIIIa was not glycosylated at Asn-162.99 The presence or absence of fucose was shown not to influence the binding affinity of IgG-Fc for the inhibitory FcgRIIb receptor and it was suggested that IgG antibody glycoform might be a sensitive modulator of FcgRIIIa-mediated ADCC – through tissue-specific production of different Asn-162 glycoforms of FcgRIII.101,102 Although most studies were conducted with IgG1 subclass proteins, increased ADCC was also demonstrated for nonfucosylated IgG3 and IgG4 antibodies, some activity also being observed for IgG2.103 Increased


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ADCC activity was also reported for a nonfucosylated glycoform of CH1/CL-deleted fusion protein and could, presumably, be extended to IgG-Fc fusion proteins.104 As stated previously, crystal structures for IgG-Fc in complex with aglycosylated soluble recombinant FcgRIIIa were initially interpreted to show that there is no direct contact between the oligosaccharide and the FcgRIIIa protein moiety.35,36 However, a subsequent review article61 claimed that, following model refinement, the carbohydrate on IgG-Fc was seen to contribute 100A˚2 to receptor contact (personal communication, P. Sun). These investigators also demonstrated that while the binding of deglycosylated IgG-Fc to aglycosylated srFcgRIIIa was undetectable the binding of deglycosylated whole IgG was only decreased 10–15-fold.105 This serves to remind us to exercise great caution when attempting to extrapolate from in vitro experimental data to predict in vivo biological outcomes. Nonfucosylated oligosaccharides account for 10% of those released from normal polyclonal IgG-Fc. Given random pairing between different heavy-chain glycoforms, a maximum of 10% assembled IgG molecules may be anticipated to be comprised of one nonfucosylated heavy chain, with variable galactosylation. Studies of human IgG myeloma proteins, however, show that antibody producing plasma cell clones can secret predominantly nonfucosylated IgG glycoforms.21 Thus, polyclonal IgG may similarly contain populations of IgG comprised of two nonfucosylated heavychain glycoforms. The significance of fucosylation has been established from studies of monoclonal antibody therapeutics, in vitro; however, improved clinical efficacy for nonfucosylated antibody therapeutics is now being reported.

4.20.5 IgG-Fab Glycosylation It is established that 15–20% of polyclonal human IgG molecules bear N-linked oligosaccharides within the IgG-Fab region, in addition to the conserved glycosylation site at Asn 297 in the IgG-Fc.17,106–110 There are no consensus sequences for N-linked oligosaccharide within the constant domains of either the k- or l-light chains or the CH1 domain of heavy chains; therefore, when present, they are attached in the variable regions of the kappa (Vk), lambda (Vl), or heavy (VH) chains, sometimes both. In the immunoglobulin sequence database, 20% of IgG V regions have N-linked glycosylation consensus motifs (Asn-X-Thr/Ser, where X can be any amino acid except proline). Interestingly, these consensus sequences are mostly not germline encoded but result from somatic mutation – suggesting positive selection for improved antigen binding.107,110,117 The functional significance for IgG-Fab glycosylation of polyclonal IgG has not been fully evaluated but data emerging for monoclonal antibodies suggest that Vk, Vl, or VH glycosylation can have a neutral, positive, or negative influence on antigen binding.17 Analysis of polyclonal human IgG-Fab reveals the presence of diantennary oligosaccharides that are extensively galactosylated and substantially sialylated, in contrast to the oligosaccharides released from IgG-Fc.17,106,107,109 This is somewhat surprising since ‘random’ mutations could result in the generation of a glycosylation motif at many sites within a polyclonal antibody population and, as a consequence, site-specific glycosylation would be observed with varied incorporation of complex diantennary, high-mannose, triantennary oligosaccharides, etc. It could be anticipated that IgG-Fab oligosaccharides would be exposed/accessible and, therefore, readily released by the enzyme PNGase F; however, to date, only contrary experiences have been reported. It might also be anticipated that glycoforms with terminal galactose residues could be subject to accelerated catabolism following binding to the asialoglycoprotein receptor. The stable oligosaccharide composition of IgG-Fab glycoforms suggests that this is not the case; however, we cannot know whether more complex IgG-Fab glycoforms, for example, high-mannose glycoforms, are generated but are rapidly removed and, therefore, not detected on analysis. A similar glycoform profile was observed for human monoclonal IgG1 (myeloma) protein that is glycosylated within the VL region. While both the IgG-Fc and IgG-Fab bore diantennary structures, the IgG-Fc oligosaccharides were predominantly nonfucosylated G0 and G1 whereas the IgG-Fab oligosaccharides were predominantly fucosylated, galactosylated, and sialylated.17 Interestingly, while the IgG-Fc oligosaccharides could be quantitatively released on exposure to PNGase F, the IgG-Fab oligosaccharides were refractory, but could be released on exposure to Endo F, in contrast to the IgG-Fc oligosaccharides that could not be released with this enzyme. A comparable glycoform profile was observed for the therapeutic antibody Cetuximab (Erbitux) that has specificity for the epidermal growth factor receptor and is licensed for the treatment of colon, head and neck cancer, etc. This antibody, produced in Sp2/0 cells, bears an N-linked oligosaccharide at Asn-88 of the VH region; interestingly, there is also a glycosylation motif at Asn-41 of the VL but it is not occupied.112 Different glycoforms predominate at each site; the IgG-Fc bearing fucosylated G0 and G1 oligosaccharides and the IgG-Fab fucosylated G2 and sialylated oligosaccharides. While the IgG-Fc oligosaccharides could be released on exposure to PNGase F, the IgG-Fab

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oligosaccharide linkage was refractory. Thus, this antibody structure and the Sp2/0 cell line appear to replicate the situation observed for polyclonal and monoclonal human IgG produced in vivo. Several other examples of recombinant antibodies bearing IgG-Fab glycosylation have been reported and the presence of a diantennary oligosaccharide resistant to removal by PNGase F has been a repeated observation. Partial occupancy of IgG-Fab but not IgG-Fc has also been observed. A contrary experience has also been reported;113 a detailed analysis of the glycoforms of a humanized IgG antibody, expressed in Sp2/0 cells, bearing oligosaccharides at Asn-56 of the VH and Asn-297, reveals the expected IgG-Fc oligosaccharides profile of predominantly fucosylated G0 and G1; however, 11 oligosaccharides were released from the IgG-Fab, including triantennary, and other oligosaccharides not observed for normal human IgG. Clearance rates, in mice, were independent of the IgG-Fc glycoform and for nine of the IgG-Fab oligosaccharides, however, marginally accelerated clearance was observed for two IgG-Fab glycoforms. All IgG-Fab oligosaccharides were extensively sialylated with N-glycolylneuraminic acid, rather than N-acetylneuraminic acid. The IgG-Fab oligosaccharide linkages were refractory to release by PNGase F. Presentations made by biopharmaceutical companies at meetings have reported similar finding of conserved IgG-Fc oligosaccharide profiles and more heterogeneous IgG-Fab glycosylation;112 the latter showing relatively high levels of galactosylation and sialylation. It is important to emphasize that IgG-Fab glycosylation has not been shown to compromise clearance rates, at least in mouse models.113 The influence of IgG-Fab glycosylation on antigen binding has been the subject of several reports. Three antibodies with specificity for (a1-6)-dextran, differing only in potential N-glycosylation sites at Asn-54, -58, or -60 in the VH CDR2 region, were evaluated for antigen-binding affinity. The Asn-54 and Asn-58 molecules, each of which bore a complex diantennary oligosaccharide rich in sialic acid, were equivalent in antigen binding, and the glycosylated forms had a 10–50-fold higher affinity for antigen compared with aglycosylated forms. In contrast, the Asn-60 molecule bore a high-mannose oligosaccharide and had a lower affinity for antigen.114,115 A significant proportion of IgG-Fab oligosaccharides bore Gal(a1-3)Gal structures.116 By contrast, humanization of a mouse anti-CD33 antibody with concomitant removal of a potential glycosylation site at Asn-73 of the VH resulted in higher affinity for antigen; subsequent deglycosylation of the original mouse antibody similarly resulted in an increased affinity.117 Increased affinity for ovomucoid was reported for the deglycosylated form of a mouse antibody bearing N-linked oligosaccharide in the light-chain CDR2.118 A multispecific human monoclonal antibody, produced in mouse–human heterohybridoma cells, has been reported to bear both diantennary and tetraantennary oligosaccharides attached at Asn-75 of the VH region and to include antigenic N-glycolylneuraminic acid sugar residues.119 IgG-Fab glycosylation can impact differentially on the structural and functional characteristics of IgG. It may be exploited to increase the solubility and stability of antibodies, limiting aggregation and hence immunogenicity. However, given the essential demand for product consistency, it offers an additional challenge to the biopharmaceutical industry. A further intriguing feature of IgG-Fab glycosylation is being revealed in study of human B-cell lymphoproliferative disease. While 10% of normal B-cells bear surface Ig glycosylated within VH or VL, the frequency among patients with sporadic Burkitt’s lymphoma, endemic Burkitt’s lymphoma, and follicular lymphoma is 42%, 82%, and 94%, respectively.111 Sequence analysis revealed multiple glycosylation motifs – one sequence encoding four VH and two VL motifs. These data are evidence of somatic mutation and an apparent selection for VH/VL glycosylation and could be revealing of the etiology of the disease.

4.20.6 IgG Glycosylation and Disease 4.20.6.1 Rheumatoid Arthritis The finding that the serum of 80% of patients with RA contained an antibody specific for their own IgG defined RA as an autoimmune disease.120 These autoantibodies, referred to as RF(s), were shown to be specific for the IgG-Fc region of the patient’s IgG,121 and it was surmised that the immunogen provoking the autoimmune response might be an altered or denatured form of the IgG-Fc. The subsequent demonstration that oligosaccharides released from polyclonal IgG-Fc of RA patient are frequently hypogalactosylated suggested a possible explanation.67 Examination of the crystal structure of IgG-Fc shows that galactose on the (a1-6) arm interacts with the protein surface though a hydrophobic ‘patch’ and makes 27 noncovalent contacts with the protein structure, including a minimum of two hydrogen bonds; in the absence of a Gal(a1-6) residue, this ‘patch’, which may be described as having lectin properties, is exposed. It was hypothesized that terminal galactose residues on other IgG molecules, or other glycoproteins, might bind to the IgG-Fc forming immune complexes that may activate inflammatory reactions and/ or be immunogenic, provoking the formation of RF. 67,121 The site of interaction of the Gal(a1-6) residue is on the


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inner surface proximal to the CH2/CH3 junction, while a predominant epitope recognized by RF is also expressed at the CH2/CH3 junction.121–123 The presence of aggregated IgG in synovial fluid harvested from the joints of RA patients provided supporting evidence and suggested that consequent local complement activation could result in perpetuation of an inflammatory cascade. It is now recognized that RA is a spectrum of similar diseases and has a much more complex etiology, however, whether as a contributing factor or an epiphenomenon hypogalactosylation being still tantalizing – for example, the coincidence of disease remission in pregnant women coinciding with an increased galactosylation of serum IgG.29 Parturition is followed by the re-emergence of hypogalactosylated IgG-Fc and the return of disease symptoms. Hypogalactosylation has now been reported for numerous inflammatory conditions in which RF is not present or implicated and may be regarded as an acute phase reactant, having value in differential diagnosis and patient management.67–74 The specificity and affinity of monoclonal RFs, generated from synovial tissue obtained from RA patients, have been evaluated for differing glycoforms of IgG. While some showed preferential binding to agalactosyl IgG, others either did not discriminate between galactosylated and agalactosylated IgG or showed preferential binding to the former.122 However, the presence and titer of anti-agalactosyl IgG antibody has been developed as a more specific diagnostic and prognostic indicator than RF for RA,124 although also detected in the sera of patients with juvenile arthritis, Sjogren’s syndrome, scleroderma, etc.125 The presence of autoantibodies with specificity for citrullinated proteins is proving to be a more selective diagnostic marker for RA.126 There may be emerging a common characteristic of autoimmune disease – that the autoantibodies have specificity for epitopes, generated by normal posttranslational modification (PTM) processes acting on self-proteins that are not normally subject to the given PTM.127 Unfortunately, hypogalactosylation of human IgG-Fc in RA was frequently reported as ‘abnormal’ glycosylation, while in fact it is an increase in the proportion of G0 oligosaccharides that is observed. Thus, while normal serum IgG may be comprised of 20–30% of G0F oligosaccharides in RA the values could be as high as 50–60%. Paradoxically, at the time that hypogalactosylation was being proposed as a cause of autoimmunity in RA, clinicians were investigating a new therapy that comprised delivery of pooled human IgG at doses of 80g/week; such doses contain 20g of G0F IgG! It is important to appreciate that hypogalactosylation is observed for polyclonal IgG which is comprised of thousands (tens of thousands!) of structurally unique antibodies, each specific for a unique epitope (antigenic determinant). This suggests that either an environmental factor is having a ‘global’ influence on the plasma cells producing and secreting IgG or that galactose residues are being removed, post secretion, by the action of a galactosidase. The demonstration that while IgG-Fc oligosaccharides may be hypogalactosylated IgG-Fab oligosaccharides could be highly galactosylated and sialylated106–110 confirms that the glycosylation machinery and processing within the Golgi is intact and suggests a subtle influence impacting on IgG-Fc processing, as opposed to IgG-Fab processing. Earlier studies proposing that hypogalactosylation resulted from a deficit in the level of b-1,4-galactosyltransferase expressed in peripheral blood lymphocytes of patients with RA are unconfirmed;128,129 a deficit in b-1,4galactosyltransferase activity has been shown to account for the production of hypogalactosylated glycans by LEC 19 CHO cells.130

4.20.6.2 Wegener’s Granulomatosis and Microscopic Polyangiitis Wegener’s granulomatosis (WG) and microscopic polyangiitis (MPA) are autoimmune systemic vasculitic diseases in which antineutrophil cytoplasm antibodies (ANCAs) are characteristically present in the serum. The predominant specificity in WG is for proteinase 3 (PR3), a serine protease present in cytoplasmic azurophilic granules, and in MPA for myeloperoxidase. These antibodies can be useful diagnostic markers since titers correlate with disease activity, falling on remission and rising on relapse.131–133 Indirect evidence for a pathogenic role for PR3-ANCA and MPA-ANCA in vivo is provided by the benefit afforded by plasmapheresis of patients with severe acute disease.134 Analysis of serum-derived IgG from patients with acute disease revealed an extreme example of hypogalactosylation.70 Subsequent analysis of IgG-Fc and IgG-Fab fragments revealed the predominance of G0 glycoforms within IgG-Fc while IgG-Fab oligosaccharides were predominantly galactosylated and mono- or disialylated.109 This study demonstrates, again, that the glycosylation machinery is intact; it is suggested that the microenvironment within the Golgi apparatus is altered with a consequent change in the conformation of the IgG-Fc and its accessibility to the action of b-1,4-galactosyltransferase. The a1-acid glycoprotein (AGP) is a sensitive marker of inflammation and is one of the major acute phase glycoproteins with plasma concentration increasing up to 4 times during acute inflammation.135 A substantial portion of AGP, 45%, bears five complex N-linked glycans and displays a high degree of glycoform heterogeneity and the

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extent of branching, fucosylation, sialylation, etc. During inflammation, the increase in plasma concentration is accompanied by changes in glycoform profiles. These changes vary with the type of inflammation episode and can be used to distinguish between acute and chronic inflammatory episodes. Following acute trauma, such as burns, an increase in diantennary-containing glycoforms (decreased branching) in combination with increased fucosylation is observed while under chronic conditions the increase in AGP concentration is slight; however, the level of fucosylation is increased and diantennary glycoforms are decreased (increased branching).135 It was of interest, therefore, to analyze the glycoform profile of AGP isolated from the sera of the WG and MPA patient groups for whom the IgG oligosaccharide profiles had been determined.70,109 The glycoform profiles of AGP isolated from these sera were consistent with an acute inflammatory condition, in contrasts to the proscribed diagnosis of both WG and MPA as chronic diseases. Thus, the glycoform profiles were highly abnormal and comparable to those observed in acute trauma, such as burns and sepsis (unpublished observations, W. van Dijk, M. Holland, and R. Jefferis).

4.20.7 Recombinant Monoclonal Antibodies for Therapy It is projected that 30–40% of new drugs entering the market over the next 10 years will be based on antibodies and that the market value will be $30 billion in 2010. The efficacy of recombinant antibodies results from their specificity for the target antigen and the biological activities (effector functions) activated by the immune complexes formed. To date, all licensed therapeutic antibodies have been of the IgG class. Numerous parameters impact on the effector function profile, for example, IgG subclass and glycoform, and their definitions offer opportunities for optimizing an antibody for a given disease indication. It is increasingly appreciated that the choice of IgG subclass is a critical decision. In oncology, a major disease indication, it would seem beneficial to maximize the potential to induce ADCC and CDC in order to eliminate targeted cancer cells.136 However, in chronic diseases, neutralization of a soluble target, for example, a cytokine, may be the central objective and excessive effector activity could be detrimental, for instance where the target is also expressed as a membrane protein on certain cells, for example, the targeting of tumor necrosis factor-a (TNF-a) by Infliximab in patients with RA. Consequently, IgG1 may not be the automatic choice and the other IgG subclasses are now being evaluated, both in native and engineered forms. Some companies have selected IgG4 as the preferred alternative subclass and two IgG4 antibodies have been licensed. However, recent studies suggest that IgG4 may not be the best alternative since it is has been shown that it can activate inflammatory reactions through cellular IgG-Fc receptors.17,103,139 It should be noted that the ‘superagonistic’ anti-CD28 antibody TGN 1412, which appears to have induced a ‘cytokine storm’ in healthy volunteers,1 is a humanized IgG4 recombinant antibody. To date, all licensed therapeutic antibodies have been produced in CHO, NSO, or Sp2/0 cells. The glycoform profile of the product can vary widely from clone to clone and is dependent on the mode of production and culture conditions. Under nonoptimal conditions, CHO, NS0, and Sp2/0 cells can produce a number of abnormally glycosylated products, including partial occupancy, high-mannose forms, the addition of Gal(a1-3)Gal and N-glycolylneuraminic acid structures. These glycoforms may compromise potency and have the potential to be immunogenic. The licensed antibody products are characteristically hypogalactosylated, relative to normal polyclonal IgG, and lack the presence of glycoforms bearing bisecting N-acetylglucosamine residues; abnormal glycoforms are deemed to be at an acceptably low level. The glycoform profiles of the currently licensed antibodies were determined rather late in the clone selection process; however, the lessons learned are being applied to clone selection at a much earlier stage and monitored throughout development and production, as an integral element of process analytical technology (PAT).137,138 There have been quite dramatic increases in the productivity of antibody-producing mammalian cells with concentrations of 1–5gl–1 being reported; however, when selecting for high specific protein production, particular attention has to be given to product quality. High levels of antibody production can compromise glycosylation resulting in partial occupancy, unacceptable levels of high mannose, etc.; however, clones producing antibody at high levels and acceptable quality have been achieved.140

4.20.8 Conclusions and Future Perspectives Glycosylation represents the most frequent post-translational modification of natural and recombinant proteins; it is estimated that 50% of genes encode for protein with the potential to be glycosylated. The importance of glycosylation is evident from the fact that defects in a number of genes in the glycosylation pathway result in congenital disorders with serious and/or life-threatening consequences.141 Changes in the glycosylation profiles of specific


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proteins may be recognized as disease markers and be exploited in diagnosis and/or patient management. Hypogalactosylation of human IgG appears to act as a marker for an acute inflammatory episode and G0 IgG may be regarded as an acute phase response. It is relatively nonspecific but combined with other markers may provide discrimination.73,74 The impact of IgG-Fc glycosylation on function has been revealed from studies employing recombinant antibody therapeutics. There is considerable scope for further developments in this area as the glycoform profile can be employed as a functional ‘rheostat’ in order to optimize functional activity for a given disease indication. Production cell lines have been developed that allows for the glycoform profile of an antibody therapeutic to be selected, in advance. A present and future challenge is to develop means of monitoring mechanisms activated by antibody glycoforms in vivo and to anticipate variations in individual patient responses, determined by the interaction of multiple polymorphisms resident in an outbred human population – it is called ‘systems biology’.

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Biographical Sketch

Roy Jefferis did his B.Sc. and Ph.D. in chemistry and was awarded the D.Sc. in immunology. He has been admitted as Fellow of the Royal Society of Chemistry and Fellow of the Royal College of Pathology. He is currently a professor of molecular immunology at the University of Birmingham, UK and has published more than 250 scientific papers. He also acts as consultant on the structure and function of antibody molecules to the biopharmaceutical industry in Europe and USA. A particular focus has been the influence of glycosylation on stability and function of antibody molecules. He is a member of British Society for Immunology, Biochemical Society, American Association of Immunology, and Royal College of Pathology. He has served on the editorial boards of Immunology, Clinical and Experimental Immunology, and Journal of Immunological Methods.

4.21 Diseases Associated with GPI Anchors T. Kinoshita, Y. Murakami, and Y. S. Morita, Osaka University, Osaka, Japan ß 2007 Elsevier Ltd. All rights reserved. 4.21.1 Biosynthesis of GPI-Anchored Proteins in Mammalian Cells 4.21.1.1 Introduction

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4.21.1.2 Biosynthesis of GPI Anchor Precursors

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4.21.1.2.1 4.21.1.2.2 4.21.1.2.3 4.21.1.2.4 4.21.1.2.5 4.21.1.2.6 4.21.1.2.7 4.21.1.2.8 4.21.1.2.9

Step 1 Step 2 Step 3 Step 4 Dol-P-Man synthase Step 5 Step 6 Step 7 Step 8

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4.21.2.1 Introduction

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4.21.2.2 Molecular Genetics of PNH

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4.21.2.2.1 4.21.2.2.2 4.21.2.2.3

Biochemical defects in PNH PIG-A gene Somatic mutation of PIG-A

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4.21.2.3 Mechanism of Clonal Expansion of PNH Cells

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4.21.2.3.1 4.21.2.3.2 4.21.2.3.3 4.21.2.3.4 4.21.2.3.5

A PIG-A mutation is not sufficient for clonal expansion What causes clonal dominance of PNH cells? Immunological selection Benign tumor hypothesis Three-step model for the pathogenesis of PNH

4.21.2.4 Clinical Aspects of PNH 4.21.2.4.1 4.21.2.4.2

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Classification Clinical manifestations of PNH

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4.21.2.5 Recent Topics 4.21.2.5.1 4.21.2.5.2 4.21.2.5.3

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Complement inhibitors Inherited GPI deficiency Future perspectives

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4.21.3 GPI-APs as Receptors of Viruses, Bacteria, and Toxins

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4.21.3.1 Physiological Role of Lipid Rafts

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4.21.3.2 Caveolae and Lipid Raft-Mediated Entry of Viruses and Bacteria

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4.21.3.2.1 4.21.3.2.2 4.21.3.2.3

Entry of viruses Entry of bacteria Binding of toxins

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4.21.4 Infectious Diseases and GPI Anchors

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4.21.4.1 Introduction

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4.21.4.2 Pathogens’ GPI Anchors Crucial for Infection and Survival

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4.21.4.2.1 4.21.4.2.2 4.21.4.2.3 4.21.4.2.4

African sleeping sickness Chagas disease Leishmaniasis Malaria

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4.21.4.2.5

4.21.4.3

4.21.4.3.1 4.21.4.3.2

4.21.4.4

Tuberculosis

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Structural Differences of GPI Anchors between Host and Pathogens Structure of lipids Modification of the glycan core

Metabolic Differences in GPI Anchor Biosynthesis between Host and Pathogens

4.21.4.4.1 4.21.4.4.2 4.21.4.4.3 4.21.4.4.4 4.21.4.4.5 4.21.4.4.6 4.21.4.4.7

Core biosynthesis Inositol acylation Glycerolipid remodeling Ceramide remodeling GPI:protein transamidase complex GPI glycan side-chain modification Membrane topology and compartmentalization

407 408 409

409 409 410 411 412 412 412 413

4.21.1 Biosynthesis of GPI-Anchored Proteins in Mammalian Cells 4.21.1.1 Introduction Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell surface proteins.1–3 This membrane-anchoring structure is ubiquitous in eukaryotes. More than 100 mammalian proteins are anchored in the cell membrane via GPI. Some examples of GPI-anchored proteins (GPI-APs) in mammalian cells are enzymes, adhesion molecules, receptors, complement regulatory proteins, and histocompatibility antigens.4 In addition, GPI also anchors surface proteins in protozoan parasites and in fungi. In all species, GPI-anchoring moieties share a common backbone structure consisting of EtN-P-6Mana1-2Mana1-6Mana1-4GlcN linked a1-6 to an inositol phospholipid, but are variously modified on their side structures depending on organisms and cell types. Structure of the inositol phospholipid also varies in different organisms. Mammalian proteins usually contain 1-alkyl, 2-acyl phosphatidylinositol, while the yeast Saccharomyces cerevisiae uses inositol phosphoceramide or diacylphosphatidylinositol, and Trypanosoma brucei uses dimyristoylphosphatidylinositol at the mammalian bloodstream stage and lysophosphatidylinositol at the insect intestinal stage.5 In lower eukaryotes such as T. brucei, malaria parasites, and yeasts, GPI-APs are abundantly coating the cell surface, and GPI anchors are essential for growth,6,7 although mammalian cells can survive without GPI-APs. GPI anchoring may be an ancient way to link proteins to the cell surface, but there may be reasons why some mammalian proteins retain this anchoring mechanism. On the other hand, many viruses and bacterial toxins use GPI-APs of hosts as their receptors. The core backbone of the GPI anchor is assembled in the endoplasmic reticulum (ER) and is transferred to proteins. The GPI-APs are then transported to the Golgi apparatus where they are modified, and then are targeted to specific microdomains of the cell surface.8 In the following sections, recent progress in molecular cloning and characterization of enzymes involved in mammalian GPI-AP biosynthesis and transport are summarized (Figure 1).

4.21.1.2 Biosynthesis of GPI Anchor Precursors Biosynthesis of GPI anchors occurs in the ER. At least eight reaction steps are required for the generation of a complete GPI anchor precursor.

4.21.1.2.1 Step 1 PI þ UDP-GlcNAc ! GlcNAc-PI þ UDP The biosynthetic pathway of GPI anchors starts with the transfer of N-acetylglucosamine (GlcNAc) from UDPGlcNAc to phosphatidylinositol (PI) by GPI-N-acetylglucosaminyltransferase (GPI-GnT). This enzyme has some selectivity in using various species of PI, because bovine PI is more efficiently used than soybean PI9 and the lipid groups on the glycerol backbone of GlcNAc-PI contain a higher percentage of alkyl chains than in common PI species. A genetic approach revealed that this simple reaction step was regulated by an enzyme complex of seven proteins including PIG-A (phosphatidylinositol glycan class A),-C,-H,-P,-Q (also termed GPI1),-Y, and DPM2.10–12 PIG-A


395

Protein

P

P

P

Lipid raft

Plasma membrane

Golgi

PGAP2 PGAP genes

GDP

P P

P

P

SL15

P

Protein

P

DPM1 DPM2 DPM3

Protein

PGAP1

PIG genes

P

P

P

P

P

P P

PIG-M PIG-X

PIG-W PIG-L

P

P

PIG-V P

P

P

Ac Step 1

P

GPI8 GAA1 PIG-S PIG-T PIG-U

Endoplasmic reticulum PIG-A PIG-C PIG-H PIG-P GPI1 PIG-Y DPM2

P

P

Protein

H2 Step 3

Step 4

H3 Step 5

PIG-O PIG-F

PIG-B

PIG-N P

P

P

P

B

H6

Step 6 Step 7

GPI7 PIG-F P

P

H7

H8

Step 8

Step 2 Glucosamine

Ethanolamine phosphate

P P

P

Phosphatidylinositol

Mannose

Dolichol phosphate

Figure 1 Biosynthesis and transport pathway of GPI anchored proteins. GPI is synthesized and transferred to proteins by the products of PIG genes in the ER, followed by additional modifications carried out by the products of PGAP genes in the ER and Golgi apparatus before being transported to the plasma membrane rafts. H2, H3, B, H6, H7, and H8, GPI intermediates.

encodes a 484-amino-acid ER membrane protein whose large amino terminal portion (400 amino acids) is located on the cytoplasmic side, and contains a region homologous to a bacterial GlcNAc transferase for lipopolysaccharide (LPS) synthesis and binding site for UDP, indicating that it bears a catalytic site.13,14 Functions of the other components of the enzyme complex remain unclear, but the unusual complex structure for a single sugar transferase may be relevant to the regulation of the enzyme.

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4.21.1.2.2 Step 2 GlcNAc-PI þ H2 O ! GlcN-PI þ acetate The next step in the biosynthesis of GPI anchors involves deacetylation of GlcNAc-PI into glucosaminyl-phosphatidylinositol (GlcN-PI). The reaction is mediated by GlcNAc-PI de-acetylase PIG-L, which is an ER membrane protein, most part of which is located on the cytoplasmic side of the cell membrane.15

4.21.1.2.3 Step 3 GlcN-PI þ acyl-CoA ! GlcN-ðacylÞPI þ CoA GlcN-PI is then acylated on position 2 of inositol to form GlcN-(acyl)PI. Palmitoylation is the major reaction. It is catalyzed by the acyltransferase PIG-W. PIG-W is a multiple membrane spanning protein, and is an acyltransferase with no homologous domain to known acyltransferases.16 In contrast to the mammalian and yeast systems, in which inositol acylation precedes the first mannosylation, the reaction order is reverse in T. brucei. This will be described in more details in the next section.

4.21.1.2.4 Step 4 GlcN-ðacylÞPI þ Dol-P-Man ! Mana1-4GlcN-ðacylÞPI þ Dol-P GlcN-(acyl)PI serves as a substrate for addition of the first mannose (Man1) of the three mannosyl residues, all of which are donated by dolichol-phosphate-mannose (Dol-P-Man). Man1, linked to GlcN via an a1-4 linkage, is transferred by GPI mannosyltransferase I (GPI-MT1), which is a complex of the catalytic subunit PIG-M and the regulatory subunit PIG-X.17,18 PIG-M has a functionally essential DXD motif (a motif conserved among the glycosyltransferase family) within the luminal domain, indicating that this step takes place on the luminal side of the ER. Biosynthesis of GlcN-PI occurs on the cytoplasmic side; therefore, GlcN-PI or GlcN-acyl-PI should flip from the cytoplasmic side to the luminal side. Topological study of PIG-W suggested that all conserved domains were facing the ER luminal side, that is, GlcN-PI would flip before inositol acylation. Sequential mannosylation can take place even without inositol acylation, although that would be inefficient. Inositol acylation may function as a trapping mechanism, which prevents GPI intermediates from flipping back. Translocation of GPI would be mediated by a ‘flippase’. This enzymatic activity was shown using microsomes, but the gene remains to be cloned.19

4.21.1.2.5 Dol-P-Man synthase Dol-P þ GDP-Man ! Dol-P-Man þ GDP Dol-P-Man is synthesized on the cytosolic surface of the ER by Dol-P-Man synthase, flipped into the ER lumen, and used as a mannose donor. Dol-P-Man synthases of S. cerevisiae and T. brucei are single-component enzymes encoded by DPM1 genes. They have a hydrophobic membrane-insertion domain at the carboxyl terminus. In contrast, the human enzyme consists of three components including the catalytic subunit DPM1, and the regulatory subunits DPM2 and DPM3.20–22 Human DPM1 lacks a membrane-insertion domain. Instead, it associates with DPM3, and becomes stabilized. DPM2 associates with DPM3 and stabilizes it. Without DPM2 the expression level of DPM1 is severely decreased. As described above, DPM2 also associates with GPI-GnT, suggesting the presence of co-regulation of biosyntheses of GPI and Dol-P-Man.11 For luminal translocation of Dol-P-Man, MPDU1 (SL15), which is probably involved in the translocation reaction, is required.23

4.21.1.2.6 Step 5 Mana1-4GlcN-ðacylÞPI þ Dol-P-Man ! Mana1-6Mana1-4GlcN-ðacylÞPI þ Dol-P This step involves the transfer of the second mannose (Man2) from Dol-P-Man to generate the fifth intermediate, Man-Man-GlcN-(acyl)PI or H3. Man2, linked to Man1 via an a1-6 linkage, is transferred by PIG-V, a GPI mannosyltransferase II.24


4.21.1.2.7

397

Step 6

Mana1-6Mana1-4GlcN-ðacylÞPI þ PE ! Mana1-6ðEtNP-2ÞMana1-4GlcN-ðacylÞPI þ diacylglycerol Step 6 involves the addition of an ethanolamine phosphate (EtNP) side chain to position 2 of Man1, generating the sixth intermediate, Man-(EtNP)Man-GlcN-(acyl)PI or B. This reaction is mediated by PIG-N, a GPI EtNP transferase I, and phosphatidylethanolamine (PE) is the donor substrate. Disruption of the Pig-n gene in mouse F9 cells resulted in a lack of side-chain modification on the first mannose, whereas surface expression of GPI-APs was only slightly affected.25 Therefore, this modification is not essential for later steps in mammalian cells, while MCD4, the yeast homolog is essential for growth.26 PIG-N contains a sequence motif that is conserved in various nucleotide phosphodiesterases and pyrophosphatases. YW3548/BE49385A is a terpenoid lactone produced by a fungus and selectively inhibits GPI EtNP transferase I.

4.21.1.2.8

Step 7 Mana1-6ðEtNP-2ÞMana1-4GlcN-ðacylÞPI þ Dol-P-Man ! Mana1-2Mana16ðEtNP-2ÞMana1-4GlcN-ðacylÞPI þ Dol-P

This step involves the transfer of the third mannose (Man3) to Man2 from Dol-P-Man by PIG-B, a GPI mannosyltransferase III, generating the seventh intermediate, Man-Man-(EtNP)Man-GlcN-(acyl)PI or H6. Man3 is linked to Man2 via an a1-2 linkage.27 Sometimes, the fourth mannose (Man4) is added to Man3 with an a1-2 linkage as a side chain. Man4 is transferred from Dol-P-Man by SMP3, a GPI mannosyltransferase IV. Man4 is essential in S. cerevisiae whereas it is not a common component in mammalian GPI-APs and its biological requirement remains to be clarified.28

4.21.1.2.9

Step 8 Mana1-2Mana1-6ðEtNP-2ÞMana1-4GlcN-ðacylÞPI þ PE ! EtNP6Mana1-2Mana1-6ðEtNP-2ÞMana1-4GlcN-ðacylÞPI þ diacylglycerol

This step involves the addition of EtNP from PE to position 6 of Man3, generating EtNP-Man-Man-(EtNP)ManGlcN-(acyl)PI or H7, one of the complete GPI precursors that are competent for attachment to proteins. This EtNP (bridging EtNP) moiety links GPI to proteins. Transfer of EtNP is mediated by GPI EtNP transferase II, a complex including the catalytic subunit PIG-O and the regulatory subunit PIG-F.29 The H7 can be further modified by the second EtNP-side-chain linked to position 6 of Man2, generating EtNP-Man-(EtNP) Man-(EtNP)Man-GlcN-(acyl) PI or H8. This reaction is mediated by GPI EtNP transferase III consisting of catalytic GPI7 (also termed PIG-G) and regulatory PIG-F. Knockdown of PIG-G by RNAi caused increase of H7 and a concomitant decrease of H8, but no effects on GPI-APs expression. Therefore, modification by the second EtNP side chain is not essential at the cellular level, and its biological significance remains to be determined.30

4.21.1.3 Attachment of GPI to Proteins GPIðH7 or H8Þ þ pro-protein ! GPI-AP þ signalpeptide Attachment of the complete GPI precursors H7 or H8 to proteins is mediated by GPI transamidase. Precursor proteins that are to be GPI anchored contain a carboxyl terminal signal sequence for GPI attachment. GPI transamidase recognizes and cleaves off the GPI attachment signal peptide and forms an enzyme–substrate intermediate linked via a thioester. The intermediate is then attacked by the nucleophilic terminal amino group in GPI, generating GPI-APs. GPI transamidase consists of five subunits: PIG-K (also termed GPI8),31,32 GAA1, PIG-S, PIG-T, and PIG-U.33,34 PIG-K seems to be a catalytic component because it is homologous to members of the cysteine protease family. The conserved cysteine in PIG-K is important for generating a disulfide bond with PIG-T, which was shown to be important for enzymatic activity.35

4.21.1.4 Post GPI-Attachment Processing The first event following attachment of GPI to proteins is inositol deacylation within the ER, which is mediated by the deacylase PGAP1 (Post GPI Attachment to Proteins 1). In PGAP1 mutant, transport of GPI-APs from the ER to

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the Golgi apparatus is significantly delayed, indicating that some carrier protein recognizes inositol-deacylated structure.36 Inositol-deacylated GPI-APs are then transported to the cell surface through the Golgi apparatus via a secretory pathway. One protein termed PGAP2 that is mainly found in the Golgi apparatus was recently identified. Although the exact function of PGAP2 remains to be determined, PGAP2 is likely involved in lipid remodeling of GPI from the unsaturated fatty acid chain at the sn-2 position of PI to the saturated form, facilitating GPI-APs localization in lipid rafts.37

4.21.2 Paroxysmal Nocturnal Hemoglobinuria 4.21.2.1 Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with a prevalence of 1–4 per million, but the unique features of this disorder have attracted the interest of many hematologists and immunologists. PNH was first described by Paul Stru¨bing in 1882 as a distinct clinical syndrome with hemoglobinuria after sleep.38 About 50 years later, Ham demonstrated that hemoglobinuria was due to abnormal sensitivity of red cells to the hemolytic action of the complement system.39 This intravascular hemolysis, from which the disease got its name, is due to intrinsic abnormalities of red cells. In contrast, all other acquired hemolytic anemias are due to extracorpuscular causes. In addition, contrary to other acquired hemolytic anemias, PNH is an acquired genetic disease, and is characterized by clonal blood cells defective in surface expression of GPI-APs such as CD59 and decay accelerating factor (DAF).40 CD59 and DAF are complement regulatory proteins that protect host cells from the destructive action of the complement system. Red cells are protected mainly by CD59 and DAF. Clonal cells defective in GPI-APs are present in all cell lineages, but not in other tissues, and persist for many years, indicating that a clone of hematopoietic stem cells defective in GPI-APs is present in patients with PNH. Such clone is generated by somatic mutation in the PIG-A gene, product of which is essential for the first step in GPI anchor biosynthesis.41 Furthermore, this abnormal clone needs to expand to a certain degree for clinical manifestation. How affected stem cell clone dominates hematopoiesis has still been an issue. In the following section, recent advances in the understanding of the molecular pathogenesis of PNH is summarized and mechanisms of the clonal dominance of PNH cells are discussed.

4.21.2.2 Molecular Genetics of PNH 4.21.2.2.1 Biochemical defects in PNH The biological basis of PNH involves a defect in the biosynthesis of GPI. GPI is synthesized in the ER through at least eight reactions, and is transferred en bloc to proteins by GPI transamidase (Figure 1). If GPI is not transferred, proteins are degraded or secreted into the extracellular compartment leading to defective surface expression of GPI-APs. In fact, blood cells derived from PNH patients are defective in various GPI-APs on their cell surface. Analysis of affected cell lines from patients with PNH showed a defect in the first step of GPI biosynthesis, that is, transfer of GlcNAc from UDP-GlcNAc to PI. Affected PNH cell lines were hybridized for each mutant cells defective in the first step to determine the complementation group of PNH cells. As a result, all PNH cell lines restored GPI-AP expression when fused with PIG-C and PIG-H mutants, but not PIG-A mutants, indicating that PNH-affected cells fall into class A mutants.

4.21.2.2.2 PIG-A gene PIG-A cDNA was isolated in 1993 by expression cloning and using its ability to restore expression of GPI-APs on the surface of JY5 cells: a human lymphoid cell line of class A mutants. The gene was called PIG-A for phosphatidyl inositol glycan complementation group A.42 In fact, PIG-A is mutated in most, and perhaps in all patients with PNH.43 The human PIG-A gene is about 17 kb and consists of six exons while the mRNA contains 3589 nucleotides. The coding region is 1452-nucleotide long for a predicted protein of 484 amino acids, starting at position 86. A hydrophobic region near the carboxyl terminus may corresponds to a transmembrane domain (amino acids 415–442). The hydrophilic carboxyl 42-residue terminal region corresponds to the luminal domain of PIG-A. A region of 92 amino acids spanning residues 304–395 is homologous (27% identity) to bacterial N-acetylglucosaminyltransferase, RfaK,


399

Table 1 Chromosomal location of genes involved in biosynthesis and transfer of GPI Gene

Location

PIG-A PIG-C PIG-H PIG-P PIG-Q (GPI1) PIG-Y PIG-L PIG-W PIG-M PIG-X PIG-N PIG-V PIG-B PIG-F PIG-O PIG-G (GPI7) PIG-K (GPI8) GAA1 PIG-S PIG-T PIG-U DPM1 DPM2 DPM3 MPDU1(SL15)

Xp22.1 1q23.3 14q11–q24 21q22.2 16p13.3 4q22.1 17p12–p11.2 17q12 1q22 3q29 18q21 1p36.11 15q21–q22 2p16–p21 9p13.3 4p16.3 1p22.2–p22.3 8q24.3 17p13.2 20q12–q13 20q11.22 20q13.1 9q33 1q21.2 17p13.1

which is involved in the synthesis of LPSs, suggesting that PIG-A is the catalytic domain of the enzyme, and reaction takes place on the cytoplasmic side of ER. It was shown that the N-acetylglucosaminyltransferase responsible for this reaction consisted of seven proteins: PIG-A, PIG-C, PIG-H, PIG-P, PIG-Y, PIG-Q (GPI1), and DPM29,11,12 (Figure 1). PNH defect is caused by somatic mutation in the PIG-A gene. PIG-A is located on the X-chromosome at Xp22.1. Both male and female somatic cells have only one functional allele of the PIG-A gene through X-inactivation, so one inactivating mutation causes loss of PIG-A function in male and in female cells, resulting in GPI anchor deficiency. More than 20 genes are known to be involved in the biosynthesis of GPI. Among them, PIG-A is the only X-linked gene. In almost all patients with PNH analyzed, PIG-A is responsible for GPI anchor deficiency. This should be due to the X-linkage of the PIG-A gene, and autosomal localization of other genes involved in GPI anchor biosynthesis requiring two inactivating mutations to lead to a loss of function (Table 1).

4.21.2.2.3

Somatic mutation of PIG-A

More than 150 PIG-A mutations have been identified in patients with PNH. They are widely distributed in coding regions and splice sites. Single-base substitutions, deletions, and insertions contribute to 75% of all mutations. The latter mechanisms involve two to several base deletions and insertions, and a few cases involve larger deletions and insertions. The majority of somatic mutations cause frameshift of the coding sequence, resulting in premature stop codons. Base substitutions cause nonsense or missense mutations. Some missense mutations result in partial loss of PIG-A activity which accounts for partial deficiency of GPI-APs.44 These mutations provide indirect information on the critical region of the PIG-A protein for catalytic activity and association with other components. Furthermore, some other regions might be critical for binding to the substrate UDP-GlcNAc, which was recently reported to bind directly to PIG-A, using a photoaffinity-labeling method.14 Somatic mutation in PIG-A occurs in hematopoietic stem cells, so affected granulocytes and lymphocytes from individual patients show the same mutation. The same clones can be maintained for at least 6 years. Many PNH patients have more than one mutant clone, and in most cases, one clone is predominant.45

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4.21.2.3 Mechanism of Clonal Expansion of PNH Cells 4.21.2.3.1 A PIG-A mutation is not sufficient for clonal expansion As mentioned above, a somatic mutation in the PIG-A gene occurs in one or a few of the numerous hematopoietic stem cells. However, GPI-negative cells are dominant within the bone marrow and in the peripheral blood of patients with PNH, indicating that clonal expansion is an essential step in the pathogenesis of PNH. Several lines of evidence support the idea that a PIG-A mutation alone is not sufficient to cause clonal expansion. First, somatic mutation of PIG-A which is found in PNH patients is also found in a very small fraction (average 0.002%) of granulocytes in most healthy individuals, but this percentage does not increase.46 Whether these mutations in healthy individuals occur in hematopoietic stem cells remains unclear. Second, many PNH patients possess more than one clone, but not all clones become equally dominant, while one of them dominates for about 6 years,45 suggesting that some other factors may be necessary for clonal expansion. Third, data from a PNH mouse model support the involvement of additional factors. As GPI-APs are essential for embryogenesis, Piga (mouse ortholog of PIG-A)-disrupted mouse is lethal.47 PNH mice bearing GPI-deficient cells only in the hematopoietic system were generated by a combination of Cre/loxP-mediated disruption of Piga in embryos, and transplantation of fetal liver cells into irradiated hosts. Analysis of these mice revealed that the percentage of GPI-deficient cells did not increase.48 A different group also reported the same conclusion using different Cre/loxP-mediated Piga-disrupted mice.49

4.21.2.3.2 What causes clonal dominance of PNH cells? There are two major hypotheses for possible mechanisms of clonal expansion of PIG-A mutant cells. First, immunological selection by some pathological conditions of the marrow environment has been suggested.50 A second mechanism would involve some genetic changes of the PNH clone, causing benign tumor-like proliferation.51

4.21.2.3.3 Immunological selection This hypothesis is based on the frequent association between PNH and aplastic anemia (AA).50 That is, bone marrow failure is commonly seen in patients with PNH, and a significant percentage of PNH cells can be seen in patients with aplastic anemia.52 AA is most likely caused by autoimmunity against hematopoietic stem cells; therefore, a small fraction of PIG-A mutant cells which is found in normal individuals increases in number by escaping from immunological attacks within the environment of marrow failure.53–55 This notion is now becoming widely accepted, and is supported by several clinical and experimental evidences.

4.21.2.3.3.1 Clinical evidences The effector responsible for AA has not been identified, but there have been reports that CD4þ T-cell clones capable of killing autologous hematopoietic progenitor cells could be generated by culturing T-cells from AA patients in the presence of autologous hematopoietic progenitor cells.56 PNH, like AA, is reported to be strongly associated with the HLA-DR2 allele,57 particularly with the haplotypes DRB1*1501, DQA1*0102, DQB1*0602,58 and skewed usage of T-cell antigen receptor Vb genes;59 suggesting that a CD4þ cytotoxic T-cell clone which recognizes autoantigen presenting MHC class II of a specific haplotype is proliferating within certain patients with PNH. Recently reported clinical cases of myelodysplastic syndrome (MDS) and AA also strongly support this notion. Refractory anemia (RA), one subtype of MDS, and patients with AA can be divided into two groups according to the presence of DAF CD59 PNH-type cells. Both RA patients and AA patients with increased numbers of PNH-type cells possess HLA-DRB1*1501, and show a better response to immunosuppressive therapy, indicating that the presence of PNH-type cells suggests a benign type of bone marrow failure due to immunological mechanisms.60,61 According to these findings, antigenic proteins reacting with an HLA-DRB1*1501 antibody were screened in sera of AA patients possessing HLA-DRB1*1501 and PNH-type cells. Results showed that diazepam-binding inhibitorrelated protein 1 (DRS-1), which is expressed in CD34þ cells derived from normal individuals, presented on HLADRB1*1501 and elicited T-cell responses,62 suggesting that DRS-1 is a candidate protein which serves as an autoantigen eliciting immune attack against hematopoietic stem cells. 4.21.2.3.3.2 Experimental evidence Based on clinical evidence, we established an experimental system to prove that PNH cells could escape immunological attacks.63 Assuming that CD4þ T-cells are cytotoxic effectors that inhibit hematopoiesis in normal stem cells, we


CD4+ T-cell

CD4+ T-cell

TCR

TCR

TCR MHC II

MHC II

(a)

GPI+

401

GPI−

(b)

GPI−

GPI+

NK cell or CD8+ T-cell

NKT cell

TCR

NKG2D GPI ULBP

CD1d

(c) GPI+

GPI−

(d) GPI+

GPI−

Figure 2 Hypothetical mechanisms of resistance of GPI-deficient cells to cytotoxic effecter cells. a, Peptides derived from GPI-anchored proteins are autoantigens presented on MHC class II and recognized by CD4þ T-cells. b, Some GPIanchored protein is important for effecter–target interaction. c, NK cells or CD8þ T-cells are activated through NKG2D binding with its ligand ULBP, which is a GPI-anchored protein. d, The GPI anchor itself is an antigen presented on CD1d and recognized by NKT cells.

currently propose two models (Figures 2a and 2b). First, when the autoantigen recognized by CD4þ T-cells was derived from GPI-APs, GPI-deficient cells were insensitive to CD4þ T-cell-mediated cytotoxicity because GPIdeficient cells could not present the antigenic peptide on MHC-class II. Second, when the autoantigen was not from GPI-APs, CD4þ T-cells recognized GPI-deficient cells less efficiently, probably because GPI-APs co-stimulated antigen presentation by enhancing cell–cell interactions. For example, CD2-expressing T-cells bound GPI-AP CD48 on antigen-presenting cells. To provide in vivo evidence for the second model, we also showed that multipotential hematopoietic GPI-deficient cells escaped from attack by cytotoxic CD4þ T-cells and expanded in a mouse model. Another study assumed that natural killer (NK) cells were the effectors64 (Figure 2c). Sensitivities of GPIþ and GPI cells to NK cells were examined in three different human cell lines. Experimental results showed that PIG-A mutant cells had a certain advantage in surviving NK cell-mediated killing. Nagakura et al. further investigated the mechanisms involved in this hypothesis, and reported that survival advantage of GPI-deficient cells was due to a lack of expression of stress-inducible GPI-APs, UL16 binding protein (ULBP) 1 and 2, which activate NK cells and T-cells through binding to stimulatory receptor NKG2D.65 It is an attractive idea that expression of ULBPs is induced on hematopoietic stem cells by some marrow-damaging incident, which has already been reported.66 Naturally existing GPI-deficient cells escape from the attack of NK cells or T-cells expressing their receptor NKG2D and expand. Another group also reported similar evidences showing that T-cells (CD3þ) from patients with PNH expressed the activated form of IRS (inhibitory receptor superfamily), and showed NK-like cytolytic activity to which GPI– cells were less sensitive than GPIþ cells.67 Another possible hypothesis is that GPI itself is an autoantigen, and is present on a family of MHC class I-related molecules CD1a, CD1b, CD1c, and CD1d, which possess lipids and glycolipid antigens, and is recognized by T-cell or NKT cells. Indeed, CD1d binds GPI in its antigen-binding groove.68 If hematopoietic stem cells present GPI on the CD1d molecule, GPI-deficient stem cells will escape from an attack of NKTcells and will expand (Figure 2d).

4.21.2.3.4

Benign tumor hypothesis

Evidences mentioned above strongly support the idea that immunological selection plays a critical role in clonal expansion of PNH cells. However, several lines of evidence suggest that immunological selection alone cannot

402


account for the so-called florid PNH, a fully developed state of PNH usually showing a very high percentage of GPI anchor-deficient cells. First, percentages of GPI-deficient cells in AA patients are generally low, and do not increase to near complete dominance, even after 10 years. Second, some patients possess several GPI-deficient clones, but one clone is dominant and others are minor after 6 to 10 years and the dominant clone does not always show severe deficiency compared to the minor ones. This evidence suggests that additional factors may be necessary. We propose that the additional factor involves a genetic change in the PIG-A mutant clone itself to allow growth as a benign tumor. The early growth response gene (EGR-1) encoding one of the zinc-finger transcription factors69 and the Wilms tumor gene (WT1) have been reported58 to be upregulated in all PNH cases. Since these transcriptional factors regulate cell growth, differentiation and apoptosis, and would rather function as tumor suppressors, upregulation of these genes may result from proliferation rather than cause proliferation. The supportive evidence of the benign tumor theory is based on the analysis of 2 PNH patients who showed fully expanded GPI-deficient clones with similar chromosomal abnormalities. The high-mobility group protein A2 (HMGA2) is localized at the breakpoint and 30 UTR of HMGA2 is truncated in both patients.69a The truncated form of HMGA2 is ectopically expressed in tumor tissues, unexpressed in normal state, and makes cells grow like a benign tumor.70,71 We suggest that ectopic expression of this truncated HMGA2 or upregulation of this target gene together with a PIG-A mutation results in expansion of GPI-deficient stem cells. It is well known that some patients with PNH eventually develop acute myelogenous leukemia from the PNH clone. Further accumulation of genetic abnormalities in a benign tumor-like subclone leads to the generation of a leukemic clone.

4.21.2.3.5 Three-step model for the pathogenesis of PNH The two previously described hypotheses are not mutually exclusive. Rather, it is possible that both mechanisms operate together. Figure 3 shows a schematic model for the pathogenesis of PNH. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. At this step, GPI-deficient cells survive and proliferate much more frequently than usual to compensate for anemia. Elevated proliferation would increase the chance of additional mutations. Step 3 involves the generation of a subclone bearing the growth phenotype.

4.21.2.4 Clinical Aspects of PNH The International PNH Interest Group proposed the following classification with regards to patient management.72

4.21.2.4.1 Classification Classic PNH. Patients show clinical evidence of intravascular hemolysis, but have no evidence of another defined bone marrow abnormality. PNH in the setting of another specified bone marrow disorder (e.g., PNH/AA or PNH/refractory anemia-MDS). Patients show clinical and laboratory evidence of hemolysis, and concomitantly have or have had a history of a defined marrow abnormality. Subclinical PNH (PNH-sc) in the setting of another specified bone marrow disorder (e.g., PNH-sc/AA). Patients have no evidence of hemolysis, but have a small population of GPI-deficient hematopoietic cells as seen by sensitive flow cytometric analysis, usually associated with bone marrow failure syndrome.

4.21.2.4.2 Clinical manifestations of PNH 4.21.2.4.2.1 Hemolytic anemia Among proteins deficient from the surface of PNH cells are the complement regulatory proteins CD55 (DAF) and CD59. Increased complement sensitivity of PNH cells leads to hemolysis. DAF inhibits C3 and C5 convertases, whereas CD59 is the membrane regulator of membrane attack complex (MAC) assembly. Studies of two kinds of patients with inherited DAF deficiency (Inab phenotype)73 and CD59 deficiency74,75 suggested that hemolysis and thrombosis were mainly caused by a lack of CD59.


403

GPI− cell Somatic mutation of PIG-A

Immunological selection

Additional abnormality

Step 1

Step 2

Step 3

Hematopoietic stem cell

Figure 3 Three-step model for pathogenesis of PNH. Step 1, Somatic mutation of PIG-A occurs in a hematopoietic stem cell. Step 2, GPI cells are selected resulting in expansion by escaping from immunological attack to hematopoietic stem cells. Step 3, The second somatic mutation occurs in a GPI cell, leading to further expansion.

4.21.2.4.2.2 Thrombosis Thrombosis is the leading cause of mortality in PNH. In most cases, the venous system is involved. In contrast to hemolysis, much less is known about the mechanisms of thrombosis. It is considered that absence of CD59 renders platelets susceptible to attack by the complement system. When this occurs, platelets release vesicles or microparticles that are procoagulant in vitro. Indeed, platelet-derived microparticles are present at significantly elevated levels in patients with PNH.76 Recent clinical studies suggested that a thromboembolic event was directly related to the size of the PNH clone. However, prevalence of thromboembolism among patients from Japan, China, and Mexico appears to be lower than in patients from the United States and Europe. 4.21.2.4.2.3 Bone marrow failure As mentioned above, PNH is often associated with marrow failure diseases, making the symptoms of PNH more complicated. Anemia is caused either by hemolysis or marrow failure. Management is also different according to causes.

4.21.2.5 Recent Topics 4.21.2.5.1

Complement inhibitors

Recently, blockade of complement activation by a monoclonal antibody against complement C5 (eculizumab) has been shown to be a safe and effective strategy to prevent hemolysis in PNH.77,78 However, two problems can arise. One is that systemic and prolonged blockade of complement C5 function may render patients susceptible to infections like neisserial infection. The other is that prolonged inhibition of the complement system increases proportions of PNH erythrocytes, raising risks of severe hemolysis if therapy is interrupted. Another strategy is reported to block the complement system using membrane-targeted recombinant soluble CD59.79

4.21.2.5.2

Inherited GPI deficiency

Two families with autosomal recessive GPI deficiency were recently found.80 The patients were characterized by severe portal vein thrombosis and neurological dysfunction. Hematopoietic cells and fibroblasts derived from affected members of these families were partially deficient in the surface expression of GPI-APs, and expression levels of GPIAPs were different among lineages, indicating a defect in some regulatory mechanism, but not in the coding part of the affected gene. In fact this disease is caused by the point mutation in the SP1 binding site of the promoter region of PIG-M, GPI mannosyltransferase I. These patients have grown almost normally in spite of the fact that GPI is essential for embryogenesis. To our surprise, these patients were suffering from severe thrombosis although surface expression of CD59 or DAF on erythrocytes and platelets was only slightly decreased, and there was no evidence of hemolysis. These observations do not agree with a report that a thromboembolic event is directly related to the size of the PNH clone.

4.21.2.5.3

Future perspectives

PNH is a rare disease with a multitude of features. It results from a loss of function of one gene which results in a lack of expression of many proteins. It is clear that PNH is caused by a somatic mutation of the X-linked gene PIG-A.

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Normal people can also show a PIG-A mutation in hematopoietic cells, which is probably the case with many other genes. If the mutated gene gains a self-proliferating character, pathogenesis is simple like with other oncogenes. However, PIG-A mutation alone does not cause clonal dominance. PIG-A-mutated cells increase in number within a marrow-suppressed environment, the cause of which remains to be clarified, waiting for a second genetic change to activate full proliferation. We hypothesized that the second genetic change contributed to proliferation in combination with the PIG-A mutation. It remains unclear how this combination of events leads to cell proliferation. While several clues have been identified, investigations are still underway.

4.21.3 GPI-APs as Receptors of Viruses, Bacteria, and Toxins 4.21.3.1 Physiological Role of Lipid Rafts In the plasma membrane, specialized structural microdomains exist, and are commonly referred to as caveolae and lipid rafts. These are enriched in cholesterol, glycosphingolipids, and GPI-APs. Rafts control numerous protein– protein interactions at the cell surface. At the cellular level, rafts have been implicated in protein sorting, membrane trafficking, and signal-transduction events including proliferation, apoptosis, migration, or adhesion. Rafts have a central role in complex physiological processes such as the immune response.81 As mentioned above, GPI-APs are selectively transported to rafts. A specific reason must exist for necessity of their localization in rafts. Proteins attached to the plasma membrane through GPI linkage are diverse, but some GPI-APs, which transduce signals to inside the cell, seem to use common signaling pathways. Being a GPI-AP, a protein lacks a transmembrane domain to span the lipid bilayer. By being localized in rafts, where many signaling molecules such as the Src family protein tyrosine-kinases (PTKs) and G-proteins assemble, GPI-APs may transduce signals by indirect association with them presumably through transmembrane adaptor proteins.82

4.21.3.2 Caveolae and Lipid Raft-Mediated Entry of Viruses and Bacteria Whereas some viruses enter host cells via endocytosis through clathrin-coated pits, many viruses and bacteria exploit host membrane microdomains as entry points, utilizing non-clathrin-mediated (caveolae and/or lipid raft-mediated) endocytosis pathway. The former pathway leads to fusion with lysosomes which provide the acidic pH environment needed for virus uncoating and entry into the cell cytoplasm.83 These two pathways are equally available in host cells such as phagocytic cells, but many pathogens prefer to enter through rafts.84 One advantage to enter through rafts is avoidance of the intracellular degradative pathway. Lipid rafts are not destined to the endosomal pathway, allowing associated molecules to avoid entering the degradative compartment. The other advantage of using rafts is that it triggers cell signaling that leads to membrane ruffling, and rearrangement of the cytoskeleton to support uptake of bacteria.85

4.21.3.2.1 Entry of viruses A polyomavirus, simian virus 40 (SV40) has been reported to enter through caveolae after binding to its receptor, the major histocompatibility complex (MHC) class I, and is then delivered to ER, by avoiding the traditional endosome/ lysosome system.86 Echovirus 1 (EV1) also uses caveolae to enter, but its receptor is a2b1 integrin.87 A number of other enteroviruses including hemoagglutinating echoviruses,88 enterovirus 70,89 coxsackie B viruses,90 and coxsackievirus A21 (CAV21)91 use DAF as receptors. These viruses bind to different short consensus repeat regions (SCR) of DAF. However, most of these viruses require additional cofactor proteins for infection such as ICAM-I for CAV21, and coxackie and adenovirus receptor (CAR) for coxsackievirus B3.92 Infection by these viruses is inhibited by raft destructive reagents. Immunodeficiency virus type I (HIV1) is also known to enter through rafts.93 Marburg (MBG) and Ebola (EBO) viruses, which cause lethal hemorrhagic fever are also reported to utilize a GPI-AP, a folate receptor, as mediator of entry.94 Prion diseases such as Creutzfeldt–Jakob disease in humans, are caused by self-aggregation of a pathogenic form (PrPsc) of the host prion protein (PrP). PrPsc interacts with PrP, leading to the transformation of PrP into the pathogenic form. This prion protein itself is a GPI-AP and interactions are localized in rafts.95

4.21.3.2.2 Entry of bacteria The phagosome-containing Mycobacterium spp. actively binds to TACO (tryptophan-aspartate-containing coat protein), a protein that associates with the cell membrane in a cholesterol-dependent manner, and prevents delivery


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of internalized bacteria to lysosomes.96 FimH adhesion-expressing Escherichia coli and Dr/afa-expressing E. coli enter host cells through the GPI-APs CD48 and DAF, respectively, within a caveola and this pathway is distinct from the classical pathway.97

4.21.3.2.3

Binding of toxins

Toxins are soluble proteins that bind target membrane using specific cell receptors, many of which are raft components. It is well known that the cholera toxin associates with the ganglioside GM1, and enters through caveolae.98 Rafts not only provide binding sites, but also mediate oligomerization of pore-forming toxins such as Aeromonas hydrophilia aerolysin99 and Clostridium septicum a toxin.100 The former binds GPI anchor and N-glycan parts of the target protein, and GPI anchor only is not sufficient. The latter binds GPI anchors with high affinity.101 It was also reported that cell sensitivity of Helicobacter pylori VacA toxin depended on a GPI-AP.102 Binding of LPS to GPI-AP CD14 induces raft recruitment of several proteins including Toll-like receptors (TLR4), and triggers the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-1, and IL-6. Outcomes of LPS signaling are quite different according to doses of LPS, from optimal-response, LPS tolerance, to septic shock.103 Signaling mediated by rafts regulates LPS sensitivity. Thus, investigation of molecular mechanisms by which microorganisms succeed in entering the host cell via rafts will provide important information for the development of new therapeutic methods.

4.21.4 Infectious Diseases and GPI Anchors 4.21.4.1 Introduction GPIs play critical role in the survival of pathogens in mammalian hosts. Eukaryotic pathogens such as protozoan parasites and fungi possess GPIs, which appear to be involved in pathogenesis. Most bacteria do not synthesize PI, and thus GPIs are generally absent in bacteria. However, one notable exception is mycobacteria, such as the tuberculosis pathogen, in which PI is a major plasma membrane phospholipid and the bacterial GPIs termed PI mannosides (PIMs) and lipoarabinomannan (LAM) are abundantly present and are thought to play important roles in infection. Importance of GPI anchors in the biology of protozoan parasites is clearly evident by the abundance of GPIs in the membranes of these organisms. The GPI anchor structure was first determined in T. brucei, an etiologic agent of African sleeping sickness. The structural determination of T. brucei GPI was facilitated partly due to the abundant occurrence of the GPI-anchored variant surface glycoprotein (VSG) which constitutes approximately 10% of the total protein in this parasite.5 Since the establishment of T. brucei GPI structure, it has become clear that many functionally important surface proteins of a wide variety of protozoan parasites are anchored to the cell membrane through GPI. These observations lead to an assumption that GPI biosynthesis is essential for the survival of these microorganisms. This assumption was validated by the demonstration that GPI anchor biosynthesis was indeed essential for the bloodstream form of T. brucei, which is the proliferative form of the parasite dwelling in the host bloodstream.6 Subsequently, a number of studies have shown that the biosynthesis of GPI anchors is critical for the survival of protozoan parasites and in the case of T. brucei it has been shown that enzymes involved in GPI biosynthesis can be effective drug targets. In this section, we describe the structure and biosynthesis of GPI anchors found in protozoan parasites, fungi, and mycobacteria. We also illustrate the structural differences of GPIs in these organisms as compared to mammalian GPI anchors. Finally, based on the available information on the structures and functions of GPIs in various pathogenic microorganisms, we attempt to pinpoint specific targets for chemotherapies.

4.21.4.2 Pathogens’ GPI Anchors Crucial for Infection and Survival Here, we describe the best-studied GPI anchors of some of the pathogenic organisms and illustrate their crucial roles in pathogenesis. For a more complete coverage of GPI-anchored molecules in various organisms, refer to the published review articles.3,5,104–108

4.21.4.2.1

African sleeping sickness

T. brucei causes sleeping sickness in humans in sub-Saharan Africa. It is believed that half a million people are currently infected and 60 million people are at risk of infection.108a As described above, African trypanosomes reside

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in the bloodstream of the mammalian host and are densely covered by a single GPI-AP termed VSG. VSGs are encoded by hundreds of genes, but only one gene is expressed on the parasite’s cell surface at a given time. Periodical switching of surface coat by antigenically variant VSGs represents a key molecular mechanism of immune evasion for African trypanosomes.109–111 A recent study indicated that there was a cell cycle checkpoint monitoring proper formation of the VSG coat, reiterating the importance of VSG for a parasite’s survival.112 The fact that all VSGs are anchored by a structurally conserved GPI and that GPI biosynthesis is essential for parasite survival indicate that the GPI biosynthesis is a good target for chemotherapy against African sleeping sickness. As described later in this section, it has been demonstrated that the GPI biosynthetic pathway is indeed a valid drug target for African sleeping sickness.

4.21.4.2.2 Chagas disease It is believed that 16–18 million people in Central and South America are infected by T. cruzi, the etiologic agent of Chagas disease, resulting in 20000 deaths annually.108a Infection by T. cruzi leads to the production of pro-inflammatory cytokines such as IFN-g, IL-12, and TNF-a. The GPI-anchored mucin-like glycoproteins have been shown to play key roles in the inflammatory response during the early stage of T. cruzi infection. The GPIanchored mucin-like glycoproteins are encoded by a large multigene family, and represent the major component of the parasite cell surface. The GPI moiety of mucin-like glycoproteins from T. cruzi trypomastigotes induces proinflammatory cytokines, and is as potent as bacterial LPS and Mycoplasma-derived lipopeptide.113 The structural determinants of GPI-APs that are responsible for this strong host cytokine response have been studied in great detail. A unique combination of an unsaturated fatty acid in the lipid moiety and modification of GPI core glycan by galactose residues has been shown to be important structural determinants for the pro-inflammatory activity of the parasite GPIs. A recent review summarizes the series of works related to these topics.105 Another well-studied GPI-AP from T. cruzi is trans-sialidase. Trypanosomes cannot synthesize sialic acids, but instead scavenge them from the host. T. cruzi trans-sialidases are the enzymes that transfer sialic acids from the host glycoconjugates to mucin-like glycoproteins. These enzymes are located on the plasma membrane, and are anchored by GPIs. Interestingly, while multiple genes encode trans-sialidases, some of the gene products are not functional enzymes. These non-functional proteins are suggested to be involved in host–parasite interactions.114,115

4.21.4.2.3 Leishmaniasis Leishmaniasis is found worldwide, and it is believed that some 12 million people are currently infected and 350 million people are at risk of infection.108a A major GPI-anchored phosphoglycan molecule called lipophosphoglycan (LPG) covers the surface of Leishmania parasites. LPG lacks a protein component and consists of a structurally divergent GPI anchor, which is hyperglycosylated with linear chains of -6Galb1-4Mana1-P- (Figure 4). The phosphoglycan backbone is modified with glycan side chains, which vary with the parasite’s life cycle stages and species.104,116,117 LPG is essential for the successful infection and invasion of the insect’s midgut or mammalian macrophages by Leishmania major,118,119 and an insect galectin is used by the parasite as a receptor for specific binding to the insect midgut.120 Once the infective forms of the parasite (metacyclic promastigotes) are taken up by host macrophages, parasites differentiate into amastigotes, which downregulate the surface expression of LPG.121,122 Therefore, persistence in macrophages after differentiation to amastigotes is independent of LPG. In contrast to L. major, LPG is dispensable for the infection of mice by L. mexicana,123 suggesting an evolutionary divergence in terms of LPG as a virulence factor.124

4.21.4.2.4 Malaria Malaria is the most devastating disease in the tropical and subtropical regions of the world, killing more than one million people every year.108a Severe malaria and associated deaths occur primarily by Plasmodium falciparum infection. GPI was suggested to be the pro-inflammatory toxin because of its ability to induce the production of pro-inflammatory cytokines by macrophages and cause acute malaria-like symptoms in mice.125 Indeed, human adults who are resistant to clinical malaria contain high levels of anti-GPI antibodies, while susceptible children do not, implying a role for GPIs as a toxin associated with clinical symptoms of malaria.126 In hope of developing antitoxic vaccines, mice immunized with chemically synthesized GPI glycan were challenged with rodent malaria parasites, and were shown to be protected against clinical symptoms of malaria pathogenesis.127 Structural requirement of malarial GPI to induce cytokine response is similar to that found in T. cruzi mucin, in that an effective immune response requires both the glycan and diacylglycerol moieties of an intact GPI. A comprehensive information about the P. falciparum GPI structure and activity is available from a recent review.106

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L. mexicana LPG

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C16:0 (37%) C18:1 (31%) C16:0 (12%) C18:2 (21%) C18:0 (80%)

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O C=O

C18:1 (88%) C18:2 (12%)

Glucosamine

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Ethanolamine phosphate

Mannose Glucose

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Aminoethylphosphonate Galactose Galactofuranose

Figure 4 Structures of GPI-anchored molecules from various pathogenic organisms. Side-chain modifications may vary (see text). The second mannose of Leishmania LPG is a1-3-linked and thus divergent from the ordinary protein-anchoring core glycan. T. cruzi mucin shown is from trypomastigotes. Amino ethylphosphonate can also substitute the third mannose of mucin GPI instead of ethanolamine phosphate and can function to link proteins. Note that Mycobacterium LAM has direct modification of inositol moiety by mannose residues without glucosamine.

4.21.4.2.5

Tuberculosis

Tuberculosis claims millions of deaths every year. The causative agent is Mycobacterium tuberculosis, which infects one-third of the world population. Mycobacteria belong to the order Actinomycetales in the Bacteria domain, and they do not have eukaryotic GPI anchors. However, these bacteria synthesize glycosylated PIs termed PIMs and LAM.128 PIMs and LAM differ structurally from eukaryotic GPIs (Figure 4), but show parallel mechanisms in the general scheme of biosynthetic pathway with eukaryotic GPI biosynthesis (Figure 5). PIMs and LAM are thought to play important roles in the survival of M. tuberculosis in the phagosomes of the host macrophages by blocking the maturation of phagosomes to phagolysosomes.129 LAM has been shown to induce potent anti-inflammatory activity, which has been the topic of many recent studies, and review articles summarizing the results of these studies are available.130–133 Later in this section, we briefly review the structure and biosynthesis of PIMs and LAM as an example of bacterial GPI anchors.

4.21.4.3 Structural Differences of GPI Anchors between Host and Pathogens The core glycan structure of GPI-APs is conserved among eukaryotes including protozoan parasites. One exception is found in Entamoeba histolytica proteophosphoglycans, which consist of Gal1Man2GlcN-myo-inositol as a core GPI structure.134 While the primary function of GPIs is to anchor protein onto cell membranes, some protozoan and fungal GPIs are not attached to proteins, but to large glycan moieties (e.g., Leishmania LPG, see Figure 4). Another important class of GPIs is free GPIs, which anchor neither proteins nor polysaccharides, and are found in wide varieties of organisms.135 Free GPIs can be considered as a pool of excess biosynthetic precursors, but may also play physiologically important roles in their own right. Depending on organisms and protein species, the lipid moiety of GPIs can be diacylglycerol, alkylacylglycerol, or ceramide. The core glycan can be modified by side chains such as ethanolamine phosphates, monosaccharides, and/or oligosaccharides. Below, we describe key structural characteristics that differentiate pathogen and mammalian GPIs and provide rationale for exploiting specific biosynthetic steps as chemotherapeutic targets.

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Protein Saccharomyces cerevisiae

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ER Lumen

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Fatty acid remodeling ER Lumen

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LAM Mycobacterium smegmatis AcPIM4

AcPIM2 Periplasm

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Glycan chain

Arabinomannan

synthesis P

Cytoplasm

Figure 5 GPI biosynthetic pathways in yeast, T. brucei, and mycobacteria. Membrane topology is speculated based on the types of sugar donors each glycosyltransferase reaction is dependent on, and subcellular localization and/or topological arrangements of orthologous GPI biosynthetic enzymes in other organisms. The second mannose of yeast GPI intermediates can be further modified by an ethanolamine phosphate. Early mannosylated intermediates as well as glycolipid A in T. brucei pathway is in equilibrium with inositol-acylated form (not shown). Galactose side chain is modified after VSG-GPI anchor is formed. In M. smegmatis, AcPIM4 is thought to be the branch point at which LAM pathway diverges from AcPIM6 pathway.

4.21.4.3.1 Structure of lipids Mammalian GPI anchors are glycerolipids containing either diacylglycerol or 1-alkyl-2-acyl-glycerol as lipid moieties. Protozoan parasites have a repertoire of GPIs that are structurally distinct from their mammalian host counterparts. For example, in the case of the bloodstream form T. brucei, the VSG GPI anchor consists exclusively of dimyristoyl glycerol (Figure 4).136 In contrast, in the case of procyclic forms of the parasite, which resides in the midgut of insect vectors, the GPI anchors of major surface glycoproteins known as procyclins are lyso glycerolipids.137 Lyso glycerolipids are also found in Leishmania LPGs.138–140 Ceramide-based lipid anchors are found in GPI-APs and free GPIs of T. cruzi, and appear to be developmentally regulated.141–144 Ceramide-based GPIs are also found in S. cerevisiae145,146 and in other fungal pathogens.147,148 Another structural variation of lipids involves acylation of inositol. In many cases, inositol acylation is an essential step of GPI precursor biosynthesis. However, the acyl group attached to the inositol is often removed before the proteins reach the cell surface (see Figure 5). In some cases, inositol remains acylated, and prominent examples are found in T. brucei procyclin,137 and P. falciparum proteins (Figure 4).149


4.21.4.3.2

409

Modification of the glycan core

In mammalian cells, the glycan core is modified by ethanolamine phosphate moieties attached to the first and second mannose residues in addition to the one attached to the third mannose residue, which is linked to proteins.150–152 In some proteins such as rat brain Thy-1, the fourth mannose is attached to the glycan core.152 These modifications are also found in fungus S. cerevisiae146,153 as well as in P. falciparum and T. cruzi (Figure 4) (see below). In protozoan parasites, varying degrees of core glycan modifications have been described. Well-investigated examples are found in T. brucei. VSG-GPI anchors are modified by galactose residues, which are added to all three mannose residues, though the degree of modification varies depending on the structural type of VSG (Figure 4).154 Galactosyltransferases are thought to be expressed constitutively, and variable galactose modifications are thought to arise from constraints imposed by different VSG structures that sterically interfere with addition of galactoses. Galactosylation is proposed to fill the spaces beneath the VSG protein layer, and thus to provide an additional glycan layer for greater protection.155 Similar galactosylation of GPI core glycan is also found in E. histolytica proteophosphoglycan, suggesting that several parasites have independently evolved similar strategies for making a surface glycocalyx that employs GPIs.134 A more extensive and totally different modification is found in procyclins, the major proteins covering the cell surface of procyclic parasites. The core glycans of procyclins are substituted by heterogeneously branched polylactosamine chains with terminal sialic acids attached to the galactose residues. This large side chains are proposed to be involved in the formation of dense surface glycocalyx.137 How do these structural differences fit into the biosynthetic scheme? What is the physiological significance of these structural variations? What genes are involved in these enzymatic reactions? Many questions remain to be answered, but studies in the past several years have accumulated evidence clarifying some of these issues. Below, we discuss some of the key issues that have been the topics of recent investigations.

4.21.4.4 Metabolic Differences in GPI Anchor Biosynthesis between Host and Pathogens Differences in metabolic pathways between mammalian hosts and pathogens must account for the structural differences in GPIs of these organisms. In the following paragraphs, we describe the main pathway of GPI head group biosynthesis and illustrate the differences in the main pathway in the host and pathogens. We rationalize how these differences in the biosynthetic steps between the host and pathogen can be exploited for therapeutic purposes. We will also discuss the remodeling of lipid moieties that occurs in the GPIs of mammalian host and in some pathogens and exemplify how the observed structural differences in lipid moieties of GPI anchors of host and pathogens are achieved. Finally, we describe modifications of the glycan core that are specific to pathogens and discuss their physiological significance.

4.21.4.4.1

Core biosynthesis

Genetic approaches to validate that GPI biosynthesis can be exploited as a therapeutic target were first reported by Nagamune and colleagues, who cloned TbGPI10, the ortholog of human PIG-B encoding the third mannosyltransferase, from T. brucei.6 The demonstration that both alleles of the TbGPI10 gene could be knocked out only after introduction of an episomal copy provided the first genetic evidence that GPI anchor biosynthesis is essential and thus can be exploited as a drug target. Another study supported the same conclusion. TbGPI12 was cloned from T. brucei, and its product, GlcNAc-PI deacetylase (ortholog of mammalian PIG-L) involved in the second step of GPI anchor biosynthesis was shown to be essential for parasite growth.156 In a series of studies, Ferguson and co-workers further demonstrated that GPI biosynthesis of T. brucei is a practical drug target. Studies using synthetic analog of GlcN-PI demonstrated different substrate specificities of GPI biosynthetic enzymes between mammalian cells and trypanosomes.157–162 These studies culminated with the development of cell-permeable GPI analog that are toxic to trypanosomes, but can be tolerated by mammalian cells.163 These studies represent the direct chemical validation of the main GPI biosynthetic pathway as a drug target, providing valuable lead compounds for future drug development. The situation is less clear for leishmaniasis in terms of GPI biosynthesis as a drug target. As discussed above, while LPG is important for promastigote stages of L. major to survive during macrophage invasion,119 amastigote stages of L. major mutants deficient in LPG are as virulent as wild type. Furthermore, LPG is totally dispensable for the growth and establishment of infection for L. mexicana.123 To examine broader effects of mannose metabolism, a gene involved in the synthesis of dolichol-phosphate-mannose, the mannose donor of GPI and N-glycan biosynthesis, was knocked out in L. mexicana. The mutants lacked all GPI-anchored molecules, but remained viable, and infectious to animals, suggesting the nonessential nature of GPI.164 Nevertheless, mannose biosynthesis per se is critical for survival in macrophages, and for the establishment of animal infection;164–166 and a factor important for virulence is attributed not

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to GPI anchors, but to intracellular b1-2 mannan oligosaccharides.167 Mannan accumulates upon differentiation of metacyclic promastigotes to amastigotes in macrophages, and is suggested to protect the L. mexicana parasite from starvation and/or stress conditions within macrophage phagolysosomes. Taken together, GPI biosynthesis may not be an effective drug target to combat L. mexicana infection. LPG biosynthesis remains a valid target for L. major infection, but the fact that LPG is naturally downregulated and dispensable in amastigotes119,121,122 makes LPG biosynthesis difficult as a strategic drug target. In other parasites, no direct genetic validation is available. In Plasmodium species, the role of GPI as a malaria toxin implies that GPI biosynthesis is likely to be a good target for the development of antimalarial drugs. Furthermore, inhibition of GPI biosynthesis by mannosamine and glucosamine correlates with attenuation of parasitic growth, suggesting that GPI biosynthesis is essential in Plasmodium.168,169 In the case of T. cruzi, GPI-deficient transgenic parasites were created by heterologous expression of T. brucei GPI-specific phospholipase C, and survival of GPI-deficient amastigotes in mammalian cells was shown to be attenuated.170 More direct evidence is needed to firmly establish the importance of GPI biosynthesis in this parasite, but considering the potent immunomodulatory activity of the GPI-anchored surface molecules, GPI biosynthesis may represent a good drug target to treat Chagas disease. In mycobacteria, the first step of the PIM/LAM biosynthesis is the transfer of mannose from GDP-Man to the 2-OH group of the D-myo-inositol residue of PI (Figure 5). This step is mediated by a mannosyltransferase termed PimA, and the pimA gene has been shown to be essential for viability in M. smegmatis, an experimentally tractable model species.171 AcPIM2 is a major PIM species, which is not only a mature product but also functions as a precursor for polar PIM (i.e., AcPIM6) and LAM synthesis. Biosynthetic pathways of PIMs and LAM share first several steps before diverging to AcPIM6 and LAM biosyntheses,172–174 and several genes involved in the early biosynthesis have been identified.172,175,176 AcPIM4 appears to be the intermediate at the branch point of AcPIM6 and LAM pathways. Metabolic flux through these PIM/LAM pathways appears to be regulated and a lipoprotein involved in the flux control has been identified.177 To determine if the essential component of the pathway was AcPIM2, AcPIM6, LAM, or a combination of these components, an inositol auxotroph defective in the final step of inositol synthesis was generated, and detailed analyses provided evidence that AcPIM6 was the essential component associated with cell viability.178 Taken together with immunomodulatory activities of these molecules, PIM/LAM biosynthetic pathway appears to be a good target for anti-tuberculosis drug development.

4.21.4.4.2 Inositol acylation Inositol acylation occurs in mammalian cells as well as in pathogenic organisms. Inositol acylation takes place in a relatively early stage during core biosynthesis. In many cases, fatty acids attached to inositol are removed before mature GPI-AP reaches the cell surface. However, in some cases, inositol-acylated proteins are expressed on the cell surface. The function of inositol acylation remains unclear. Since acylated inositol is often removed from proteins before reaching the cell surface, it is reasonable to assume that it plays a role in controlling GPI precursor metabolism. However, inositol acylation may also control the way GPI-APs associate with the plasma membrane on the cell surface. In some cases, inositol acylation may be linked to specific host–pathogen interactions during infection. In this regard, it is interesting to note that GPIs from T. cruzi trypomastigote mucin are much more active with regard to proinflammatory response than P. falciparum GPIs, and one prominent difference is that Plasmodium GPI is inositol acylated.106 Inositol acylation/deacylation is best studied in mammalian cells and T. brucei, and therefore we will discuss in details differences between these two organisms. In mammalian cells, inositol is acylated after the formation of GlcN-PI, resulting in the synthesis of GlcN-(acyl)PI. Inositol acylation precedes mannosylation reactions,158,179–181 and is mediated by PIG-W.16 The inositol remains acylated until after anchoring to proteins in mammalian cells, but then it is deacylated by an inositol deacylase encoded by the PGAP1 gene before exiting ER.36 GPI-APs on the mammalian cell surface usually do not carry inositol acylation, suggesting that removal of inositol acyl groups is an important processing step after the attachment of GPI precursors to proteins. One exceptional situation exists in human erythrocytes in which inositol residues of surfaceexpressing GPI-APs remain acylated.182–184 Presumably, PGAP1 enzyme activity is regulated in a tissue-specific manner. The biosynthetic pathway is similar in Plasmodium species in that inositol acylation takes place prior to mannosylation reactions.185 A gene highly homologous to human PIG-W is present in the genome of P. falciparum. In contrast to the mammalian system, inositol remains acylated in mature proteins expressed on the cell surface. Thus, Plasmodium GPI biosynthetic pathway may not require PGAP1. Consistent with such a prediction, the PGAP1 homolog is not obvious in the genome of P. falciparum while it can be easily identified in the T. brucei genome (see below).


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In T. brucei, timing for inositol acylation is different. Mannosylated GPI intermediates are inositol acylated, but GlcN-PI is not. Three mannosylated intermediates (i.e., Man1-GlcN-PI, Man2-GlcN-PI, and Man3-GlcN-PI) are all substrates for inositol acylation, and equilibrium exists between acylated and non-acylated forms at least for some of these intermediates.186,187 Inositol acylation is essential for the addition of ethanolamine phosphate to the third mannose residue.158 Once the ethanolamine phosphate is transferred, the acyl group on the inositol is removed before proceeding to fatty acid remodeling reactions in the bloodstream form of the parasite (Figure 5). In procyclic parasites, inositol-acylated GPIs are transferred to proteins, and are expressed on the cell surface as mature GPI anchors, predicting that inositol deacylase is downregulated in this life cycle stage. In the bloodstream form, the mature GPI precursor termed glycolipid A is in equilibrium with its inositol-acylated form called glycolipid C.186,187 The physiological significance of glycolipid C accumulation remains speculative. It has been suggested that glycolipid C serves as a reservoir of GPI precursors, or is the first step in the catabolism of GPI.188 The PIG-W homolog is not obvious in the genome of T. brucei. Considering the fact that the substrate of trypanosome inositol acyltransferase is a mannosylated GPI intermediate rather than simple GlcN-PI, it may be reasonable to speculate that T. brucei has an evolutionary divergent inositol acyltransferase, which cannot be identified by searching databases with the primary structure of PIG-W. In addition, the fact that PMSF inhibits inositol acylation in T. brucei but not in mammalian cells biochemically supports that enzymes are substantially different between these two organisms.187 An inositol deacylase, termed GPIdeAc, has been purified, and the gene encoding GPIdeAc has been cloned.189 GPIdeAc is not homologous to mammalian PGAP1, but affinity-purified GPIdeAc was shown to be an active inositol deacylase able to convert glycolipid C to glycolipid A. Interestingly, inositol deacylase activity was not abolished but only partially reduced in GPIdeAc knockout mutants, suggesting that there was another enzyme with redundant function. We recently identified a PGAP1 homolog, designated as GPIdeAc2, in the genome of T. brucei.190 GPIdeAc2 is six times more highly expressed in the bloodstream than in the procyclic form. Its higher expression agrees with the fact that inositol becomes deacylated prior to protein anchoring in the bloodstream forms, but not in the procyclic forms. RNAi knockdown of GPIdeAc2 expression resulted in accumulation of inositol-acylated intermediates in bloodstream-form parasites, reduction of surface-expressed VSG, and severe growth defects, suggesting that GPIdeAc2 is a major inositol deacylase of T. brucei. T. cruzi synthesizes a GPI intermediate similar to T. brucei glycolipid C,191 suggesting that inositol is acylated and subsequently deacylated during the biosynthesis of T. cruzi GPI. Consistent with this observation, homologs of GPIdeAc and GPIdeAc2 (PGAP1) exist in the genome of T. cruzi. Inositol acylation has also been documented in L. mexicana.192 However, neither GPIdeAc nor GPIdeAc2 homologs are found in the L. major genome. Inositol acylation/deacylation in Leishmania may be a species-dependent phenomenon, or may be catalyzed by enzymes significantly different from trypanosomal counterparts.

4.21.4.4.3

Glycerolipid remodeling

Fatty acid remodeling of GPI anchors was first described in T. brucei.193 After synthesis of the glycan head group, fatty acids attached to the sn-2 and sn-1 positions of diacylglycerol are sequentially replaced by myristate, a short fatty acid (Figure 5).194 Fatty acid remodeling occurs in the ER prior to attachment of the GPI precursor to VSG. There is a second myristoylation mechanism termed myristate exchange for GPIs attached to VSG proteins in a post-ER compartment.195 A large amount of myristate is needed for myristoylation of VSG GPI, and trypanosomes have a fatty acid synthase designed to supply myristate for GPI anchors.196 Furthermore, myristate analogs have been shown to be toxic to trypanosomes.197 These observations suggest that dimyristoyl GPI anchor is critical for parasite survival. Lipid remodeling has been suggested to occur in related organisms, such as Leishmania. In the case of L. mexicana, sn-1 fatty acid of a free GPI is alkylated, and only the sn-2 acyl group is replaced by myristate.198 T. cruzi trypomastigote mucins contain unsaturated fatty acids at the sn-2 position on glycerol, while epimastigote mucins contain saturated sn2 fatty acids.113,142,199 Epimastigotes appear to synthesize PIs with unsaturated sn-2 fatty acids,200 and it is not clear how epimastigote mucins acquire saturated fatty acids at the sn-2 position. As a likely possibility, sn-2 fatty acid remodeling may take place in this organism as well.105 In mammalian cells, similar fatty acid remodeling has been suggested. The intracellular PI pool is represented predominantly by a species containing an unsaturated chain at the sn-2 position.201 In contrast, the sn-2 position of mammalian GPI-APs is usually stearate.3,104,202,203 A recently identified mammalian gene PGAP2 has been proposed to function in the reaction to replace unsaturated sn-2 fatty acid with stearate.37 Genes with high homologies to mammalian PGAP2 exist in the genomes of T. cruzi and L. major where sn-2 fatty acid remodeling has been suggested. Candidates for the T. brucei ortholog could not be found in the genome through standard bioinformatics searches (unpublished observation). Based on these observations, it appears that T. brucei has evolved a substantially different system for fatty acid remodeling of both sn-1 and sn-2 acyl chains.

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4.21.4.4.4 Ceramide remodeling Inositolphosphoceramide is found as a lipid moiety of GPI anchors in some organisms such as yeast and T. cruzi. In yeast, ceramide remodeling takes place in the ER and Golgi apparatus.204–206 The biosynthetic pathway of GPI precursors is based on PI rather than inositolphosphoceramide. The complete PI-based precursor is attached to proteins, but then the glycerolipid moiety is replaced by ceramide before proteins reach the cell surface.207 In T. cruzi, GPI precursors containing sn-1-alkyl-2-acyl-glycerol as a lipid moiety have been identified.191 Furthermore, recent data suggested that inositolphosphoceramide was not a substrate for the first step of GPI biosynthesis in T. cruzi.208 These data suggest that ceramide remodeling takes place in T. cruzi in a similar way as in yeast remodeling. In a fungal pathogen, Aspergillus fumigatus, GPI-anchored molecules have been structurally characterized, and their lipid moiety was found to be inositolphosphoceramide.147,148 In this fungus as well, the biosynthetic precursors are based on PI,209 suggesting that ceramide remodeling takes place after PI-based GPI has been synthesized. The ceramide remodeling appears to be important in yeast for trafficking of GPI-APs to the cell surface.210 Since ceramide-linked GPIs are not present in mammalian cells, ceramide remodeling may be exploited as a chemotherapeutic target.

4.21.4.4.5 GPI:protein transamidase complex GPI:protein transamidase represents a key step in the biosynthesis of GPI-APs. Consistent with the essential nature of the VSG surface coat, RNAi knockdown of TbGPI8, the catalytic component of the GPI:protein transamidase complex, in the bloodstream of T. brucei, was lethal.211 In contrast, in L. mexicana, GPI8 deletion resulted in viable mutants specifically lacking surface-exposed GPI-APs. The mutant parasites having normal levels of LPG and free GPIs did not show significant growth defect in culture and could infect cultured macrophages and mice indicating that GPI-APs are not essential for the survival of parasites and establishment of infection in hosts.192 GPI:protein transamidase is a cysteine peptidase, and attaches a GPI precursor to a nascent protein using an acyltransferase reaction.212 The enzyme complex has different metabolic requirements in different organisms. The mammalian transamidase complex recognizes a number of different proteins, but the quantity of GPI-APs to be processed is relatively small. In T. brucei, GPI:protein transamidase processes a large quantity of proteins, but the repertoire of proteins to be handled is relatively small. In the mammalian system, GPI:protein transamidase is a complex of five polypeptides: GPI8, PIG-T, GAA1, PIG-S, and PIG-U.33–35 The T. brucei counterpart is also a complex of five polypeptides, of which TbGPI8, TbGPI16, and TbGAA1 are homologous to GPI8, PIG-T, and GAA1, respectively.213,214 PIG-S and PIG-U are not present in the parasite transamidase complex, but two novel components, TTA1 and TTA2, are found as unique components. TTA1 is conserved in Kinetoplastids such as T. cruzi and L. major, but the homologs do not appear to be present in other organisms. Interestingly, PSI-BLAST analysis indicated that PIG-S and TTA1 are distantly related, suggesting that they share a common ancestry.

4.21.4.4.6 GPI glycan side-chain modification Relatively little is known about the biosynthesis of GPI glycan side chains especially in the case of protozoan GPIs. In mammalian cells and yeast, genes involved in addition of the fourth mannose (Smp3) and ethanolamine phosphate substituents (PIG-N, GPI7) have been identified.25,26,28,30,215,216 While ethanolamine phosphate side chains are not found in protozoan parasites, the fourth mannose is found in GPI anchors of Plasmodium and T. cruzi. Smp3 is thought to be the mannosyltransferase, but potential candidates for this enzyme could not be identified by homology searches of the P. falciparum genome217 or T. cruzi. In mammalian cells, addition of the fourth mannose is not an essential step in GPI precursor biosynthesis, and GPI-APs with the fourth mannose modification may be a tissue-specific phenomenon.28 In contrast, in yeast and P. falciparum, the fourth mannose is found in a majority of GPI-APs. In the case of S. cerevisiae, addition of the fourth mannose appears to be a step prerequisite for the addition of ethanolamine phosphate to the third mannose.216 In Plasmodium, a precursor with ethanolamine phosphate attached to the third mannose is synthesized without the fourth mannose, suggesting that the fourth mannose is not required for the addition of ethanolamine phosphate.185 T. cruzi synthesizes GPI precursors that are similar to T. brucei glycolipids A and C. These precursors appear to carry a fourth mannose, and the addition of the fourth mannose is proposed to take place prior to the ethanolamine phosphate transfer.191 In-depth studies on the physiological significance of the fourth mannose modification of GPIs in both host and pathogens are necessary to determine whether this enzyme can be an effective target for chemotherapy. Although direct genetic validation remains to be performed, data from several studies strongly support the essential roles of GPI side-chain modifications in T. brucei. Galactose is a major component of GPI side chains in both the bloodstream and procyclic forms of the parasite. When galactose synthesis was blocked, the bloodstream-form


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parasites lost their ability to grow.218 In procyclic forms, galactose is required primarily for the GPI side-chain biosynthesis, and conditional null mutants defective in galactose biosynthesis accumulated truncated procyclins and ceased cell growth. These data are consistent with the possibility that side-chain modification is essential for the survival of the parasite. Another important side-chain modification of procyclic form T. brucei GPIs is terminal sialylation of polylactosamine side chains. Sialylation is mediated by GPI-anchored cell surface enzymes, the trans-sialidases, that transfer sialic acids from the host glycoconjugates to the parasite’s GPI side chains.219–221 Procyclic cell mutants deficient in sialic acid modification on the cell surface could not survive in the tsetse fly midgut, suggesting critical roles for the sialylated GPI side chains.222

4.21.4.4.7

Membrane topology and compartmentalization

It is well established that the GPI biosynthesis starts in the cytoplasmic side of the ER membrane in mammalian cells,223 and at some point during biosynthesis, the GPI precursor flips from the cytoplasmic side to the luminal side. The first glycosyltransferase reaction that involves the addition of GlcNAc to the PI is dependent on sugar nucleotides on the cytoplasmic side. The second step, deacetylation of GlcNAc-PI, also takes place on the cytoplasmic side, but the third step mediated by PIG-W inositol acyltransferase probably occurs on the luminal side.16 The subsequent mannosylation reactions are dolichol phosphate mannose-dependent, and take place in the luminal side as suggested by the active site orientations of mannosyltransferase enzymes.17,24 Similar topological arrangements are probably conserved in all eukaryotic systems. In mouse thymoma cells, the second reaction mediated by PIG-L GlcNAc-PI de-N-acetylase, is enriched in a specific domain of ER membrane that is associated with mitochondria.224 In contrast, in HeLa cells, the de-N-acetylase activity as well as epitope-tagged PIG-L protein are not enriched in the mitochondria-associated domain of ER, suggesting that compartmentalization of GPI biosynthesis in mammalian ER membrane is dependent on cell types.225 In L. mexicana, GPI biosynthesis is enriched in an ER sub-compartment that is clearly different from the localization of a general ER marker, BiP, as determined by sucrose gradient fractionation.226,227 A similar compartmentalization of PIM biosynthesis in mycobacteria has been demonstrated, indicating that the initial and later steps of the biosynthesis were differentially enriched in distinct domains of the plasma membrane.228 The molecular mechanisms and physiological significance of flipping GPI intermediates at specific biosynthetic steps are largely unknown. It is also not clear why the GPI biosynthesis is compartmentalized. Such topological arrangements may be important to control the metabolic flux of the pathway, allowing regulation of the chemical reactions by physical parameters. Understanding of these topological issues is important for both identifying novel chemotherapeutic targets, and for evaluating specific drug targets in a broader view of the biosynthetic mechanism as a whole.

Glossary X inactivation This is a process by which one of the two copies of the X chromosome present in the female is inactivated randomly. It occurs so that the female, with two X chromosomes, does not have twice as many X chromosome gene products as the male. It occurs during early embryogenesis and once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell. variant surface glycoprotein (VSG) The major GPI-anchored protein that coats the surface of the bloodstream from Trypanosoma brucei dwelling in the mammalian host bloodstream. procyclin the major GPI anchored surface coat protein of the procyclic form Trypanosoma brucei that proliferates in the midgut of tsetse fly vector. phosphatidylinositol mannoside (PIM) A major class of free GPI present in mycobacteria. Dimannosides (such as AcPIM2) and hexamannosides (such as AcPIM6) are the major products of the PIM biosynthesis.

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EMBO J. 1999, 18, 3643–3654. 227. Mullin, K. A.; Foth, B. J.; Ilgoutz, S. C.; Callaghan, J. M.; Zawadzki, J. L.; McFadden, G. I.; McConville, M. J. Mol. Biol. Cell 2001, 12, 2364–2377. 228. Morita, Y. S.; Velasquez, R.; Taig, E.; Waller, R. F.; Patterson, J. H.; Tull, D.; Williams, S. J.; Billman-Jacobe, H.; McConville, M. J. J. Biol. Chem. 2005, 280, 21645–21652.

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Biographical Sketch

Taroh Kinoshita studied biochemistry of the complement system in the Department of Bacteriology at Osaka University Medical School and received his Ph.D. in 1981. After a one year post-doctoral fellowship supported by the Japan Society for the Promotion of Science in the same department, he got a position of Research Associate in the same department. He worked as a postdoctoral research associate in the Department of Pathology, New York University Medical School from 1982 to 1985 and studied structure and function of complement receptors and complement regulatory proteins, such as complement receptor type1 and decay-accelerating factor. He continued to work on complement receptors and regulatory proteins after his return to Osaka University. Later he was appointed as a Professor in the Department of Immunoregulation in the Research Institute for Microbial Diseases, Osaka University in 1991. Since then, he has been studying biosynthesis of GPI-anchored proteins in mammalian cells and African trypanosomes, and molecular pathogenesis of GPI deficiency, and paroxysmal nocturnal hemoglobinuria. Major contributions to these research areas are molecular cloning of more than 20 genes involved in GPI-anchor biosynthesis and identification of the gene defective in paroxysmal nocturnal hemoglobinuria. From October 2003 till date, he has been serving as a Director of the Research Institute for Microbial Diseases, Osaka University.

Yoshiko Murakami graduated from Osaka University Medical School in 1984 and worked for 10 years at the general hospital as a pediatrician. She worked as a research fellow in the Department of Immunoregulation at Research Institute for Microbial Diseases, Osaka University studying the pathogenesis of paroxysmal nocturnal hemoglobinuria and got a Ph.D. in 2001. Since then she continued studying on the same subject together with biosynthesis of GPI-anchored proteins in mammalian cells. From 2005, she is woking as a Research Associate in the same department.


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Yasu S. Morita obtained B.A. from International Christian University in Tokyo, Japan, in 1992. He has two years of research experience in Northwestern University, Chicago, U.S.A. He enrolled in the Biochemistry, Cellular and Molecular Biology Graduate Program at the Johns Hopkins University School of Medicine, Baltimore, U.S.A., in 1994 and was awarded a Ph.D. in 2000. He worked with Paul Englund on GPI and fatty acid synthesis in Trypanosoma brucei during the Ph.D. thesis research. He was a summer course student for the Biology of Parasitism Course offered by the Marine Biological Laboratory in Woods Hole, U.S.A. in 1996. He also worked as post-doctoral fellow in the Department of Biochemistry and Molecular Biology in the University of Melbourne, Australia from 2000 to 2003. He worked with Malcolm McConville on PIM biosynthesis in Mycobacterium smegmatis. He was an International Human Frontier Science Program post-doctoral fellow from 2001 –to 2004. He moved to the Research Institute for Microbial Diseases, Osaka University, Japan, in 2003. He is currently a Research Associate, and continues to work on PIM/LAM biosynthesis in mycobacteria as well as GPI biosynthesis in protozoan parasites.

4.22 GnT-V and Cancer S. Ihara, RIKEN Center for Developmental Biology, Kobe, Japan E. Miyoshi, Osaka University Graduate School of Medicine, Osaka, Japan N. Taniguchi, Osaka University, Osaka, Japan ß 2007 Elsevier Ltd. All rights reserved. 4.22.1 Introduction

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4.22.1 Introduction A long-term discussion is underway whether aberrant glycosylation is a result or a cause of malignant transformations.1,2 Although metastatic spread of cancer cells is associated, not only with aberrant glycosylation changes, but also with many other factors, recent studies indicate that some, if not all, aberrant glycosylations are the result of an initial oncogenic transformation, as well as a key event in inducing invasion and metastasis.3–6 One of the most common modifications in transformed or malignant cells is the presence of larger N-glycans, due primarily to a combination of increased GlcNAc branching, sialylation, and poly-N-acetyllactosamine content.3,6 Studies of glycosylation-dependent tumor progression have been conducted in parallel with clinicopathological experiments. High expression of some glycol-epitopes is related to invasion and metastasis, leading to shorter 5–10 year survival rates of patients.7,8 The category of these glyco-epitopes includes b1-6 GlcNAc branching in N-linked structures; sialyl-Tn in O-linked structures; sialyl-Lex, sialyl-Lea, and Ley in N,O-linked, or lipid-linked structures. The hypersialylation of the cell surface is a common signal in transformed and malignant cells, as well as leukemia cells.9–11 Although normal cells rarely express sialyl-Tn antigen, this structure is increasingly seen in cancer cells and is associated with a poor prognosis.12,13 Cell adhesion molecules that recognize glyco-epitopes may play an important role in the inflammatory process, the trafficking of lymphocytes, and other cell–cell interactions. Sialyl-Lex and related antigens act as receptor of E-, L-, and P-selectins and as tumor-associated antigens.14 Human epithelial cancer cells express ligands for E-selectin (ELAM-1).15,16 Human epithelial cancer cells and human leukemic cell lines have been studied for their ELAM-1-mediated adherence to the vascular epithelium. These cancer cells show adhesion to vascular endothelial cells, which is dependent on ELAM-1.16 This type of binding may be important for metastasis of cancer cells in the process of tumor progression. In addition, it was also reported that tumor cells’ migration and invasiveness were modulated by N-glycosylation on integrin/tetraspanin complex in low-density membrane fraction.17–19 Clinically, the expressions of sialyl-Lex and sialyl-Lea are inversely correlated with the postoperative survival rate of patients.20–22

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Our group has focused on the underlying mechanism of the biosynthesis of N-glycan branching in glycoproteins, and reported on various N-glycan branching enzymes that play a pivotal role in various steps.23 The characteristics of the N-acetylglucosaminyltransferase involved in the synthesis of N-glycan antennas have been reviewed.24,25 Rous sarcoma virus and H-ras-transformed cells have shown to significantly increase the extent of b1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), a key structure that is associated with malignant transformations and cancer metastasis.26–31 An increased b1-6 GlcNAc branching of N-glycans is now established as a consistent finding in many animal models, and human melanomas and carcinomas of the breast and colon. Several interesting experiments concerning relationships between the b1-6 branched structures of N-glycans and cancer metastasis have been conducted by many authors.3,30 However, before the successful cloning of the GnT-V enzyme which catalyzes the formation of b1-6 branching , it was not clear whether or not b1-6 branching was actually related to the metastatic potential of cancer cells. The enzyme was independently purified from rat kidney by Pierce’s group and from human cancer cells by our group,32,33 and both groups subsequently cloned the corresponding cDNAs.34,35 Using the cloned genes, several studies have provided evidence for the presence of a strong association between b1-6 GlcNAc branching and cancer metastasis.36–38 Tumor growth and metastasis are suppressed in GnT-V-deficient mice with polyoma middle Tantigen transgenic mice.39 It is established that GnT-V modifies the N-glycan of glycoproteins with diverse roles in cell adhesion, migration, and proliferation, including matriptase, lamp-1, N-cadherin, b1-integrin, and epidermal growth factor receptor (EGFR).36,40–44 N-cadherin is a calcium-dependent glycoprotein that mediates cell–cell adhesion and cell motility.45 Lamp-1 is expressed on cell and lysosome membranes and is important in lysosomal trafficking, matrix degradation, and cell adhesion.46 The b1-integrin subunit associates with several different a-subunits forming a heterodimeric protein involved in cell–cell and cell–matrix binding.47 Focal adhesion formation by mouse embryonic fibroblasts (MEFs) is impaired in GnT-V null cells. Recently, Guo et al. reported that the deletion of MEF GnT-V resulted in enhanced integrin clustering and activation of a5b1 transcription by protein kinase C signaling, which, in turn, upregulated the levels of cell surface a5b1 fibronectin receptors, ultimately resulting in increased matrix adhesion and inhibition of migration.48 EGFR is a tyrosine kinase receptor that binds to several EGF-related growth factors and promote cell proliferation. Our recent findings clearly demonstrated a novel pathway of GnT-V-mediated metastasis via the upregulation of matriptase, an epithelial and integral membrane serine protease.43,44 This protease was initially reported to be as a trypsin-like serine proteinase secreted by human breast cancer cells.49 Protein purification and cDNA cloning revealed that matriptase is a type II transmembrane serine proteinase with multiple extracellular domains.50–52 This proteinase actually activates two important cancer invasion effectors, the membrane-bound activator of urokinase-type plasminogen activator (uPA) and hepatocyte growth factor (HGF), also designated as a scatter factor on the surface of cancer cells.53,54 The expression of both matriptase and lamp-1 is enhanced by GnT-V glycosylation where the addition of b1,6 GlcNAc branches prevents the degradation of both proteins.55,43 In contrast, the overexpression of GnT-V has not been shown to alter the expression of EGFR, b1-integrin, or N-cadherin.41 More recently, we investigated the molecular basis of GnT-V and matriptase expression in thyroid cancer cells, and the clinical significance of their expression was investigated in correlations with the histology and biological aggressiveness of thyroid cancers.56 In this section, GnT-V, its target glycoprotein relative to cancer metastasis, and the unique function of an angiogenesis inducer are reviewed, with emphasis on the roles of b1-6 GlcNAc branching on glycoprotein in terms of the metastasis of cancer cells.

4.22.2 Molecular Properties of GnT-V UDP-N-acetylglucosamine:a-mannoside b-1,6-N-acetylglucosaminyltransferase (GnT-V) catalyzes the formation of the b1-6 GlcNAc branch on asparagine-linked oligosaccharides (Figure 1a) (EC 2.4.1.155).57,58 This enzyme requires the prior action of b-1,2-GlcNAc transferase-2 (E.C. 2.4.1.143), which in turn requires the prior action of b-1,2-GlcNAc transferase-1 (E.C. 2.4.1.101). GnT-V cannot act on substrates that contain a bisecting GlcNAc, the formation of which is catalyzed by b-1,4-GlcNAc transferase-3 (E.C.2.4.1.144). The presence of a core Fuc(a1-6) residue on substrate glycoproteins reduces enzyme activity to a minimal level and the GlcNAc-terminal triantennary compound is a better substrate than the diantennary compound.32 The Leu residue at position 188a-a.59 is required for Golgi localization without affecting enzymatic activity.59 Supporting these data, our laboratory reported that the region comprised of 45-184 amino acid residues in the stem region serves as the functional domain responsible for Golgi localization but is not associated with enzymatic activity.60 The C-terminal portion of rat GnT-V comprising 213–740 amino acid sequences is required for catalytic activity61 (Figure 1b).

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4.22.3 Purification and Cloning of GnT-V We and another group independently succeeded in the purification and cDNA cloning of GnT-V from rat kidney35,33 and human lung cancer cells,32,34 respectively. GnT-V has been purified from a Triton X-100 extract of a rat kidney acetone powder and from culture medium of human lung cancer cells. The rat kidney enzyme was purified 450000-fold , with a 26% yield, to a specific activity of 19 mmol/mm/mg using only two affinity chromatography steps, UDP-hexanolamine-sepharose and an inhibitor column (synthetic oligosaccharide inhibitor-spacer-bovine serum albumin-sepharose). The pure enzyme showed two major bands (69 and 75kDa) on SDS-PAGE. The human lung cancer enzyme was purified 20000-fold from the conditioned medium without the use of a detergent. Two bands 73 and 60kDa were seen on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE); tryptic peptide maps from these two bands were almost identical indicating that the 60kDa protein was probably a proteolytic product.

4.22.4 b1-6 GlcNAc Branching and Malignant Transformation Structural changes in N-glycans are one of the critical steps in cellular transformations and malignant transformation. The level of b1-6 GlcNAc branched N-glycan has been observed to increase in some cancers, and is associated with a poor prognosis. The gene transcription of GnT-V is regulated by proto-oncogenes such as the Ets family,62,63 src,64 and erbB2.65 The 50 flanking region of the GnT-V gene contains the functional binding sites for the Ets family.63 Although increased expression of GnT-V in MuLv epithelial cells resulted in a loss of contact inhibition, morphological transformation, and tumor formation in athymic mice, the modification of specific glycoproteins which contain b1-6 GlcNAc-branching has not yet been clarified.36 The gene expression in mouse mammary carcinoma cells with GnT-V enhances invasion and metastasis.66,67 In an opposite approach to identify

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factors related to the tumor metastasis, tumor cell mutants selected for leukoagglutinating phytohemagglutinin (L4-PHA) resistance and found to be deficient in GnT-V activity are also associated with a decrease in metastasis.68 These experiments suggest that b1-6 GlcNAc-branching produced by GnT-V regulates autonomous tumor cell regulation. In a clinical study, a high level of expression of the GnT-V gene in human colorectal and breast cancer is correlated with distant or lymph node metastasis with a poor prognosis 69,70,38 On the other hand, the expression of GnT-V is an early event in rat hepatocarcinogenesis, and the level of GnT-V expression in patients with hepatomas did not correlate with the prognosis of the patient after an operation.71 In fact, hepatoma cells with high levels of GnT-V, such as Huh7 and HepG2 cells, showed no metastasis in experiments using athymic mice experiments (unpublished data). This discrepancy between colon cancer and hepatoma could be attributed to differences in the target glycoproteins of GnT-V in various tissues. To directly identify the functions of GnT-V in cancer metastasis in vivo, GnT-V-deficient mice were crossed with transgenic mice expressing the polyomavirus middle T oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-PyMT) in breast epithelium. PyMT transgenic mice develop multifocal breast tumors, which then metastasize to the lung. The PyMT viral oncogene activates c-Src, as well as the Shc/RAS and PI3K/PKB pathways, each of which contributes to rapid carcinoma formation.72–74 Breast carcinomas develop in GnT-V knock-out mice with a longer latency and the tumors grow more slowly. In addition, the incidence of lung metastases is reduced by 20-fold. The experiment directly indicated that cancer progression and metastasis require b1-6 GlcNAc branching mediated by GnT-V. The mechanisms associated with this suppression involving b1-6 GlcNAc branching stimulated membrane ruffling and phosphatidilinositol 3 kinases-protein kinase B activation, leading to focal adhesion signaling.39 However, these pathways are not always associated with the late stage of cancer, but, rather, are involved in its early progression. Although lung metastasis was dramatically suppressed in GnT-Vdeficient mice, the size of the original tumors was also small. Therefore, the detailed mechanism of tumor metastasis in terms of b1-6 GlcNAc branching remains unclear and the phenotype of GnT-V-expressing tumor cells should be compared with their original cells from the standpoint of metastasis. These recent studies on targeted mutations of glycosyltransferase genes in mice and the ectopic expression of glycosyltransferases in cell lines suggest that tissue-specific and disease-associated glycosylation processes may regulate the activity of certain receptors, and thereby cell behavior. Matriptase is a tumor-associated type II transmembrane serine protease, which positively regulates carcinoma metastasis by activating latent forms of the hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), and extracellular matrix (ECM) degradation.53,75,54 We recently demonstrated that the overexpression of GnT-V in gastric carcinoma cells prolonged the degradation of matriptase and accelerated the peritoneal dissemination of these cancer cells in athymic mice.43,44 These experimental data indicate that the b1-6 GlcNAc branching of N-glycans of matriptase plays a pivotal role in its stabilization, and provides some new insights into the relation between b1-6 GlcNAc branching and glycoproteins.

4.22.5 Increased b1-6 GlcNAc Branching on Glycoproteins in Proteins Secreted into Conditioned Medium, as Judged by L4-PHA Lectin Blot In order to better understand detailed mechanisms of metastatic potential upregulated by GnT-V, we established GnT-V transfectants using a gastric cell line, MKN45, and a colon cancer cell line, WiDr. After transfection of the GnT-V gene followed by G418 selection, several types of positive transfectants of GnT-V were established. These transfectants showed high levels of GnT-V activity. To better understand changes that occur in oligosaccharide structures of glycoproteins, lectin blotting was performed, using L4-PHA (leukocyte phytohemagglutinin). L4-PHA binds preferentially to GlcNAc residues on b1-6 branches of tri- or tetraantennary sugar chains. Secreted proteins from mock and GnT-V transfectants of MKN45 cells were highly reactive to L4-PHA under reducing conditions (Figure 2a). In contrast, membrane proteins from the mock and GnT-V transfectants showed almost the same results in L4-PHA lectin blots. Although enzymatic activity of GnT-V was increased, no changes were found in total proteins secreted from mock and GnT-V transfectants. These results suggested that the overexpression of GnT-V in MKN45 cells leads to an increase in b1-6 branches on the N-glycan of glycoproteins, especially in conditioned medium. However, when we performed two-dimensional electrophoresis followed by a lectin blot, increases in L4-PHA lectin binding were observed for certain types of proteins. This result indicates that the addition of b1-6 GlcNAc branching by GnT-V on membrane glycoproteins would be relatively selective.

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Figure 2 Experimental metastasis of GnT-V transfectants of MKN45 cells. a, Analysis on oligosaccharide structure of the GnT-V transfectants. L4-PHA lectin blot analysis of the conditioned media from GnT-V transfectants showed an abundant secretion of protein(s) carrying b1-6 GlcNAc branching. Coomassie brilliant blue staining was shown as controls. b, Overexpression of GnT-V in MKN45 cells resulted in severe cancer metastasis in athymic nude mice. Arrowheads indicate metastatic lesion of tumor. c, Gelatin zymography of 10-fold concentrated conditioned media from GnT-V transfectants showed increases in the activity of a metal-dependent serine protease of 70–80kDa. Reproduced from Ihara, S.; Miyoshi, E.; Ko, J. H.; Murata, K.; Nakahara, S.; Honke, K.; Dickson, R. B.; Lin, C. Y.; Taniguchi, N. Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching. J. Biol. Chem. 2002, 277, 16960–16967.

4.22.6 Experimental Metastasis To investigate the metastatic potential of the GnT-V transfectants, we focused on tumor formation in various organs after the injection of tumor cells into the peritoneum of athymic mice. The results of these experiments are summarized in Figure 2b. A marked dissemination of metastatic cancer cells was observed in the GnT-V transfectants compared to parental cells and negative transfectants (mock). The incidence of tumor metastasis in the liver and lymph nodes was significantly higher in the GnT-V transfectants than in the parent or mock cells. The expression of GnT-V mRNA, protein expression, and the enzymatic activity in these metastatic lesions were dramatically increased. Similar phenomena were observed in GnT-V-transfected colon cancer cells WiDr (unpublished data). These results indicate that glycoprotein(s) containing b1-6 GlcNAc branching in MKN45 and WiDr cells might be involved in cancer metastasis to visceral organs, including lymph nodes. Demetriou et al. reported that GnT-V plays an important role in T-cell activation by modifying oligosaccharides on the T-cell receptor.76,77 In our experiments, T-cell systems did not appear to be involved in GnT-V-mediated metastasis, since athymic mice which are devoid of T-cells were used.

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4.22.7 Analysis of Protease Secreted by GnT-V-Transfected MKN45 Cells To better understand the mechanisms underlying increased metastatic potential produced via b1-6 GlcNAc branching, gelatinolytic activity in conditioned medium of mock and GnT-V transfectants MKN45 cells was assayed by gelatin zymography. We detected some protease activities in the conditioned medium of mock and GnT-V-transfected MKN45 cells. Serum-free conditioned medium by these transfectants were assayed for gelatinolytic activity by gelatin zymography. When the gelatin gel was incubated in a reaction mixture containing 5mMCa2þ, a strong negative staining band with an apparent molecular mass of about 80kDa was present in the conditioned medium of both mockand GnT-V-transfected MKN45 cells. The intensity of this band for the GnT-V transfectants was significantly higher than that of control cells. These results indicate that GnT-V activity leads to an increase the 80kDa protease activity in conditioned medium. To characterize this 80kDa protease, a gel was incubated with various kinds of protease inhibitors. The gelatinolytic activities of all proteases were eliminated by the addition of ethylenediamine-tetraacetic acid (EDTA), an inhibitor of metal-dependent proteases (Figure 2c), suggesting that these proteases require metal ions for the activity. Specific metalloprotease inhibitor BE16627B77,78 failed to inhibit the activity of 80kDa protease. However, the gelatinolytic activity of about 92, 72, and 62kDa bands in conditioned medium and tumors were completely inhibited by BE16627B. These results strongly suggest that 92, 72, and 62kDa proteases correspond to MMP9 and MMP2. Treatment with pepstatin, an inhibitor of aspartate protease, had no discernible effects on the gelatinolytic activity of the 80kDa protease. The proteolytic activity of the 80kDa protease of condition medium was completely blocked by aprotinin, a serine protease inhibitor (Figure 2c). These results indicate that the 80 kDa protease is a metal-dependent serine protease.

4.22.8 The 80 kDa Protease in Condition Medium Was Identified as Matriptase The results of gelatin zymography, including inhibitor experiments, suggest that the 80kDa protease is a divalent cation-dependent serine protease. The protease specificities indicate that it is very similar to a protease secreted from human breast cancer cells, T47D.49 It is known that T47D cells secrete about an 80 kDa serine protease, matriptase, also called membrane-type serine protease 1.50–52 In order to identify the protease, the conditioned medium from MKN45 cells was analyzed by immunoblotting analysis with antibody against human matriptase, mAb21-9.50 Western blot and immunodepletion experiments revealed that the protease activity in the conditioned media is matriptase. Matriptase is expressed in normal epithelial tissues such as the skin, stomach, colon, kidney, breast, ovary, and pancreas, but not in mesenchyma.80–82 Matriptase-deficient mice showed that it plays a critical role in epidermal barrier function, hair follicle development, and thymic homeostasis.83,84

4.22.9 Analysis of Molecular Property of Purified Matriptase We performed a pulse chase experiment to examine the degradation of matriptase in cell lysates. As expected, the degradation of matriptase was dramatically delayed in cell lysates from GnT-V transfectants. Even after 300min, approximately 80% of the matriptase was not degraded in the case of the GnT-V transfectants (Figure 3a). Similar phenomena were observed in GnT-V-transfected colon cancer cells, WiDr. Pulse chase studies for matriptase showed that the half-life of matriptase remained unchanged in synthesis rate and secretary pathway, but was markedly prolonged in the conditioned media of GnT-V transfectants (Figure 3b). When we investigated the level of matriptase in conditioned media, high levels of matriptase in GnT-V transfectants was observed after 60h of culture (Figure 3c). We conclude that the upregulation of matriptase by b1-6 GlcNAc branching is due to the resistance of matriptase to degradation. The next question to be raised is why proteins that contain b1-6 GlcNAc branching (such as matriptase) are resistant to degradation. Three scenarios are possible. 1. Matriptase with b1-6 GlcNAc branching could be resistant to degradation by proteases. 2. Certain inhibitors that serve to reduce the degradation of matriptase could be induced/activated by b1-6 GlcNAc branching. 3. In GnT-V transfectants, certain proteases that degrade matriptase are inhibited. To address this question, we purified and characterized matriptase from mock and GnT-V transfectants (referred to as matriptase and b1-6 GlcNAc matriptase, respectively). The b1-6 GlcNAc matriptase purified from the conditioned medium of GnT-Vtransfected MKN45 cells has a great amount of b1-6 GlcNAc branching on N-glycans. In the purification of

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Figure 3 Increases in the protein expression of matriptase due to prolonged degradation. a, Degradation of matriptase was dramatically reduced by addition of b1-6 GlcNAc branching. b, Turnover of cell surface matriptase and release of matriptase from mock and GnT-V transfectants were determined by a pulse chase study. c, Accumulation of matriptase in the conditioned media from mock and GnT-V transfectants was evaluated by Western blot. Reproduced from Ihara, S.; Miyoshi, E.; Ko, J. H.; Murata, K.; Nakahara, S.; Honke, K.; Dickson, R. B.; Lin, C. Y.; Taniguchi, N. Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding b1-6 GlcNAc branching. J. Biol. Chem. 2002, 277, 16960–16967.

matriptase and b1-6 GlcNAc matriptase from the culture medium of MKN45 cancer cells, the crude protein was isolated by precipitation with ammonium sulfate, followed by column chromatography using an antimatriptase column. These purification procedures are simple but very effective for purifying the enzyme. Purified proteins were visualized at approximately 70–80 kDa by silver staining and Western blot analysis. Eighty microgram of control matriptase and 170mg of b1-6 GlcNAc matriptase were obtained from 2 l of conditioned medium from the mock and GnT-V transfectants, respectively. A convenient purification method established by Lin et al. in 1999 enabled us to easily prepare large amounts of matriptase.50 To analyze the oligosaccharide structures on purified matriptase, lectin blotting with L4-PHA was performed. As expected, a marked increase in b1-6 GlcNAc matriptase levels was observed in GnT-V transfectants, indicating that GnT-V attached b1-6 GlcNAc branching to the N-glycans of matriptase.

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4.22.10 b1-6 GlcNAc Branching Enhances Stability of Matriptase To evaluate the effect of b1-6 GlcNAc branching on matriptase, we investigated its autodegradation. It is most likely that matriptase is self-degraded in the conditioned medium, because a secreted type of matriptase from cancer cells has enzymatic activity as an active protease. Although control matriptase was degraded after 24h, this autodegradation process was markedly delayed in the case of b1-6 GlcNAc matriptase. We next investigated the resistance of b1-6 GlcNAc matriptase to proteolysis by various proteases. Control and b1-6 GlcNAc matriptases were treated with trypsin, elastase, and chymotrypsin, followed by observation of the degradation profile by Western blotting. The b1-6 GlcNAc matriptase showed a higher resistance to degradation by trypsin than the control. In contrast, elastase and chymotrypsin led to no changes in the degradation profiles of either the control or b1-6 GlcNAc matriptase (Figure 4a). Importantly, the strong resistance mediated by b1-6 GlcNAc branching was abolished by treatment with N-glycosidase-F which digests all types of N-glycans. These results indicate that the resistance to degradation and stabilization of b1-6 GlcNAc matriptase are specifically due to the presence of b1-6 GlcNAc branching on its N-glycans. The specific activities of matriptase, b1-6 GlcNAc matriptase, and deglycosylated matriptase were next measured using various synthetic fluorescent peptide substrates. The enzymatic activities of control matriptase, b1-6 GlcNAc matriptase, and deglycosylated matriptase were almost the same, as shown in Figure 4b. Thus, the addition of b1-6 GlcNAc branching changes the stability of matriptase but not its specific enzymatic activity toward synthetic substrates. The b1-6 GlcNAc matriptase could be more continuously active for ECM degradation and the activation of growth factors such as HGF. In terms of their molecular mechanisms, we speculated that the b1-6 GlcNAc branching could mask a portion of matriptase that confuses susceptibility to trypsin acts or autodegradation.

4.22.11 N-Glycan of Asn 772 is Important for the Stabilization of Matriptase Matriptase contains four potential Asn-linked oligosaccharide linkage sites (N109, N302, N485, and N772).51,52 To determine which of these sites are involved in the stabilization of matriptase, we prepared three mutants of putative glycosylation sites (N302Q, N485Q, and N772Q) using a site-directed mutagenesis method. After matriptase is synthesized in a latent form, it is immediately cleaved at the Gly149 residue by proteolytic processing.85,86 Therefore, the Asn109 putative N-glycosylation site cannot be involved in the stabilization of active matriptase induced by the addition of b1-6 GlcNAc branching. Although all potential sites contain oligosaccharides, N772Q in the protease domain was more important for the stabilization of matriptase induced by adding b1-6 GlcNAc branching. Furthermore, the N772Q matriptase mutant profoundly inhibited the formation of the matriptase-hepatocyte growth factor activator inhibitor-1 (HAI-1) complex (active form). The N302Q matriptase mutant also dramatically reduced the formation of the matriptase-HAI-1 complex. In contrast, the N109Q and N485Q matriptase glycosylation mutants did not reduce the formation of the matriptase-HAI-1 complex.86 These results indicate that the N-glycosylation of N772 regulates the activation mechanism and stabilization by b1-6 GlcNAc branching. Based on our findings, we propose a model for the molecular behavior of b1-6 GlcNAc branching and matriptase, which is shown in Figure 5.

4.22.12 Immunohistochemistry Analysis of GnT-V and Matriptase b1-6 GlcNAc branching is increased in atypical hyperplasia and carcinomas of breast compared with normal and benign lesions, as evidenced by L4-PHA assays. Similar results were observed from colon cancers.69,38 Our previous studies have shown that an elevated expression of GnT-V is a poor prognosis factor in patients with colonic adenocarcinoma.87 We investigated whether the relationships between GnT-V and matriptase exist in patients with tumors.56 A high GnT-V level of expression was found in 113 (52.1%) nonsmall cell lung cancers (NSCLCs), and a low GnT-V expression was found in 104 (47.9%) NSCLCs. Patients with tumors having a low GnT-V expression had a significantly shorter survival time than patients with tumors having a high level of GnT-V expression in 103 patients with pStage I NSCLCs (5-year survival rates, 49% and 86%, respectively; P¼0.0009), as well as in 59 patients with pStage I nonsquamous cell carcinomas (5-year survival rates, 54% and 89%, respectively; P¼0.007). A low GnT-V expression was a significantly unfavorable prognostic factor in pStage I NSCLCs (hazard ratio, 2.86; P¼0.002) and in pStage I nonsquamous cell carcinomas (hazard ratio, 3.02; P¼0.02). These results indicate that GnT-V expression has an opposite effect in some cancer cells. We, next, investigated whether the co-localization of immunohistochemical staining of GnT-V and matriptase in target glycoprotein in thyroid neoplasm tissues, to determine the clinical

GnT-V and Cancer

Trypsin concentration (kDa)

1.2 0.0012 (mg ml−1)

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b1-6 GlcNAc matriptase


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Control matriptase 728.5 96.2 454.4 − −

b1-6 GlcNAc matriptase Deglycosylated matriptase 787.7 718.9 89.3 84.8 425.1 410.0 − − − − 4

(×10 pmol/h/mg protein)

(b)

−, No cleavage activity was detected.

Figure 4 b1-6 GlcNAc branching increases the stability of matriptase, but does not change its enzymatic activity. a, Upper panels indicate degradation patterns of various concentrations (0.001 2–1.2 mg ml1) of trypsin and elastase, respectively. The lower panel indicates time dependency for the degradation in control and b1-6 GlcNAc matriptase on treatment with 0.012 mg ml1 of trypsin. b, Specific activities of control, b1-6 GlcNAc matriptases and deglycosylated matriptase. The cartoon indicates the method used to measure enzymatic activities toward synthetic peptides.

significance of the co-expression of these two proteins in thyroid cancer. While neither GnT-V nor matriptase was expressed in normal thyroid tissue, positive staining for matriptase and GnT-V was observed in 52/68 and 66/68 cases of papillary carcinoma, 3/23 and 10/23 cases of follicular carcinoma, 5/13 and 9/13 cases of follicular adenoma, and 11/28 and 6/28 cases of anaplastic carcinoma,56 respectively. When the expression of GnT-V and matriptase was investigated

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109

302

485

772

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855 aa G149 Activation step +++

Catalytic sites Activation step +++ Roles of N-glycosylation

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LDL receptor class A domain

N485

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− ; Dispensability, + ; Moderate effect, +++ ; Indispensability Figure 5 Predicted functions of N-glycosylation in matriptase.

in a variety of cancer cell lines by Western blotting, their levels of expression were highly correlated with each other in gastric cancer, colon cancer, and thyroid cancer cell lines, but not in hepatoma and pancreatic cancer cell lines (unpublished data). On the other hand, the expression of matriptase mRNA was not correlated with its protein levels, suggesting that the enhancement in matriptase expression is likely regulated by a post-translational modification such as glycosylation through b1-6 GlcNAc branching. In the case of papillary carcinoma, the levels of expression of both GnT-V and matriptase were significantly higher in tumors that were 1cm or less in size (microcarcinoma) and in those without poorly differentiated lesions, and the two proteins were significantly correlated. In contrast, the prognosis for thyroid carcinoma after surgery was neither correlated with GnT-V expression nor matriptase, because the levels of their expression were quite low in anaplastic (undifferentiated) carcinomas. These results suggest that the prolonged stability of matriptase is due to GnT-V-mediated glycosylation in vivo, thus extending its halftime and permitting it to play a role in the early phases of papillary carcinoma, but not in its later phase progression.

4.22.13 Reduction of Cell Adhesion to Fibronectin by GnT-V In other experiments, we found that GnT-V enhanced the attachment of colon cancer cells to vascular endothelial cells by inducing sialyl Lewisx, a ligand for E-selectin of the endothelium. On the other hand, GnT-V reduced the attachment to fibronectin in the same cells.87 These data are consistent with a previous report showing that adhesion to fibronectin and collagen type IV was reduced in mink lung epithelial cells expressing GnT-V.36A possible mechanism for this reduction was recently proposed for human fibrosarcoma HT1080 cells.40 The clustering of integrin a5b1, a fibronectin receptor, was inhibited by GnT-V transfection, and the inhibition was caused by b1-6 GlcNAc branching on N-glycans of the b1 subunit of integrin but not the a5 subunit. This phenotype is advantageous early in the metastatic cascade, but later at distant organs, cancer cells must attach tightly to endothelial cells for metastasis. According to these data, these demonstration of changes in cell adhesion potentials to ECM or endothelial cells is a topic of considerable interest in cancer biology. It was reported that transfection of sense GnT-V affected upregulation of a5 and a6, but not b1 subunit, while antisense GnT-V decreased a5 and b1 but not a6.88 These results might suggest that GnT-V activity not only modulates the function of integrin mediated by N-glycosylation, but also affects the expression.

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4.22.14 A Novel Function of GnT-V as an Inducer of Angiogenesis and GnT-V Transfectants Induces Hypervascularization in Athymic Mice Angiogenesis is known to be a critical step in cancer progression.89,90 We recently found the angiogenesis effect of GnT-V itself via the direct release of FGF-2 from heparan sulfate proteoglycan (HSPG) on the cell surface and/or extracellular matrix.91 These data suggest that GnT-V is a bifunctional protein and that a secreted type of the GnT-V protein itself plays a pivotal role in tumor angiogenesis, acting as an angiogenic cofactor for FGF-2. To determine which domain of GnT-V contains the human umbilical vein endothelial cell (HUVEC) growth-stimulating activity, we analyzed several types of deletion mutants of GnT-V and found that HUVEC growth-stimulating activity is located in the region corresponding to amino acids 234–436 of GnT-V, which does not encompass the glycosyltransferase activity of the protein. There is a markedly basic region, corresponding to amino acids 254–269, of human GnT-V, the sequence of which is KSVRGKGKGQKRKRKK, and which is very similar to the sequence of amino acids 142–157 of vascular endothelial growth factor 189 (VEGF189). In addition, the context of basic amino acids in this region is conserved in placenta growth factor-2 (PlGF-2) and heparin-binding type epidermal growth factor-like growth factor (HB-EGF) and serves as a heparin-binding motif. The effect of the basic peptide, KRKRKK, which corresponds to amino acids 264–269 of GnT-V, and a nonbasic control peptide, FSGGPL (corresponding to amino acids 291–296 of GnT-V), were examined on the growth of HUVEC. GnT-VD1–73 and peptide KRKRKK induced the release of FGF-2, whereas GnT-VD1–436 and peptide FSGGPL had no effect. Both GnT-VD1–188 and D233, as well as GnT-VD1–73, also induced the release of FGF-2. The peptide KRKRKK promoted the growth of HUVEC. These results suggest that the KRKRKK region is sufficient for HUVEC growth-stimulating activity, and that the GnT-V protein stimulates angiogenesis by releasing FGF-2 from HSPG on endothelial cells via the action of the basic region of the protein. These experimental data are summarized in Figure 6.

4.22.15 Discussion Since our group, as well as other groups, have succeeded in the purification and cDNA cloning of GnT-V, a number of studies have been reported in terms of GnT-V and tumor metastasis. Although a recent study of GnT-V knockout mice demonstrated the biological significance of GnT-V on tumor growth and metastasis, the mechanisms were not investigated in detail, except for the finding of membrane ruffling and phosphatidylinositol 3 kinase (PI3 kinase) activation.39 In C.elegans and mammals, the GnT-V gene is conserved, suggesting specific roles in cellular and extracellular processes. We speculate that b1-6 GlcNAc branching on certain glycoproteins might function in a common pathway in developmental or pathological stages of multicellular organisms.92 Since glycosyltransferases are known to modify many glycoproteins, it is quite difficult to identify a target glycoprotein(s) with b1-6 GlcNAc branching that is a specific contributor to tumor growth and metastasis. The poor

Secretion

GnT-V KRKRKK (basic amino acid cluster)

Stimulate releasing of basic FGF-2 from heparan sulfate proteoglycan

FGF signaling

Angiogenesis

Figure 6 Proposed model of a secreted type of GnT-V and angiogenesis.

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prognosis of GnT-V in certain types of cancers might have a similar profile of organs which express matriptase. Our study indicated one possible pathway of metastasis through the upregulation of matriptase by addition of b1-6 GlcNAc branching. Matriptase is widely expressed in many organs and a high level of expression is observed in the gastrointestinal tract, especially the intestine.52 A breast cancer cell line, T47Dco, with a phenotype of high metastatic potentials expresses large amounts of matriptase.49 The involvement of matriptase in tumor metastasis has been reported in prostate cancer.52 We found that overexpression of matriptase in gastric cancer cells promoted peritoneal dissemination in athymic mice.43 Furthermore, List et al. have reported that transgenic mice of matriptase induced spontaneous squamous cell carcinomas, possibly though activation of the tumor-promoting PI3K–Akt pathway.93 Matriptase is a glycoprotein that contains four potential sites for Asn-linked oligosaccharides. The addition of b1-6 GlcNAc branching to these oligosaccharides was observed in matriptase that had been purified from GnT-V transfectants. Post-translational modification such as the suppression of proteolysis or degradation might be associated with the upregulation of matriptase by GnT-V. Matriptase is also involved in the activation of PAR254 which is associated with cell adhesion through the G protein. A clinical study of GnT-V in colon cancer showed that GnT-V-positive cases underwent lymph node metastasis at an early stage, resulting in a poor prognosis after an operation. Our previous study demonstrated that GnT-V is a tumor marker of poor prognosis after the surgical removal of a tumor, although the detailed mechanism of this remains unknown. When we performed a combinational immunological study of GnT-V and matriptase, their expression completely coincided in 3 cases of 19 colon cancer tissues, indicating that GnT-V and matriptase systems act in the in vivo system (unpublished data). In addition to colon cancer results, we performed a combined immunohistochemical study of GnT-V and matriptase in thyroid cancer cells.56 The findings showed that stabilized matriptase of GnT-Vmediated glycosylation was observed in vivo. However, a positive correlation between GnT-V expression and a poor prognosis was not observed in thyroid cancer. Specific sugar chains are implicated in various roles of glycoproteins. These various phenomena might be cell specific, because the target proteins would be different for each type of cells. While the identification of these glycoproteins is a difficult task, recent advances in biotechnology have overcome some of these difficulties, since glycobiology and cancer cell biology can be used together with their development. In summary, our recent findings of relationship between b1-6 GlcNAc branching and glycoproteins may provide useful insights into malignant transformation and tumor progression.

Glossary GnT-V UDP-GlcNAc, a-mannoside b-1,6-N-acetylglucosaminyltransferase. This enzyme is involved in a synthesis of b1-6 GlcNAc branching on N-glycans. Until recently, approximately 80 papers about GnT-V and tumor metastasis have been reported. matriptase A membrane-type of serine proteases. Two to three groups independently succeeded the cloning of these proteases in 1999. target glycoprotein(s) A glycoprotein whose oligosaccharides are selectively modified by a specific glycosyltransferase.

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45. Nieman, M. T.; Prudoff, R. S.; Johnson, K. R.; Wheelock, M. J. N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression. J. Cell Biol. 1999, 147, 631–644. 46. Carlsson, S. R.; Fukuda, M. The polylactosaminoglycans of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Localization on the peptide backbones. J. Biol. Chem. 1990, 265, 20488–20495. 47. Hynes, R. O. Integrins: Bidirectional, allosteric signaling machines. Cell 2002, 110, 673–687. 48. Guo, H. B.; Lee, I.; Bryan, B. T.; Pierce, M. Deletion of mouse embryo fibroblast N-acetylglucosaminyltransferase V stimulates alpha5beta1 integrin expression mediated by the protein kinase C signaling pathway. J. Biol. Chem. 2005, 280, 8332–8342. 49. Shi, Y. E.; Torri, J.; Yieh, L.; Wellstein, A.; Lippman, M. E.; Dickson, R. B. Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells. Cancer Res. 1993, 53, 1409–1415. 50. Lin, C. Y.; Anders, J.; Johnson, M.; Dickson, R. B. Purification and characterization of a complex containing matriptase and a Kunitz-type serine protease inhibitor from human milk. J. Biol. Chem. 1999, 274, 18237–18242. 51. Lin, C. Y.; Anders, J.; Johnson, M.; Sang, Q. A.; Dickson, R. B. Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity. J. Biol. Chem. 1999, 274, 18231–18236. 52. Takeuchi, T.; Shuman, M. A.; Craik, C. S. Reverse biochemistry: Use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue. Proc. Natl. Acad. Sci. USA. 1999, 96, 11054–11061. 53. Lee, S. L.; Dickson, R. B.; Lin, C. Y. Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease. J. Biol. Chem. 2000, 275, 36720–36725. 54. Takeuchi, T.; Harris, J. L.; Huang, W.; Yan, K. W.; Coughlin, S. R.; Craik, C. S. Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates. J. Biol. Chem. 2000, 275, 26333–26342. 55. Fukuda, M. Lysosomal membrane glycoproteins; structure, biosynthesis, and trafficking to lysosome. Seikagaku 1990, 62, 1225–1240. 56. Ito, Y.; Akinaga, A.; Yamanaka, K.; Nakagawa, T.; Kondo, A.; Dickson, R. B.; Lin, C. Y.; Miyauchi, A.; Taniguchi, N.; Miyoshi, E. Co-expression of matriptase and N-acetylglucosaminyltransferase V in thyroid cancer tissues; its possible role in prolonged stability in vivo by aberrant glycosylation. Glycobiology 2006, 16, 368–374. 57. Brockhausen, I.; Carver, J. P.; Schachter, H. Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V, and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes. Biochem. Cell Biol. 1988, 66, 1134–1151. 58. Cummings, R. D.; Trowbridge, I. S.; Kornfeld, S. A mouse lymphoma cell line resistant to the leukoagglutinating lectin from Phaseolus vulgaris is deficient in UDP-GlcNAc: Alpha-d-mannoside beta 1,6 N-acetylglucosaminyltransferase. J. Biol. Chem. 1982, 257, 13421–13427. 59. Weinstein, J.; Sundaram, S.; Wang, X.; Delgado, D.; Basu, R.; Stanley, P. A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A Chinese hamster ovary glycosylation mutant. J. Biol. Chem. 1996, 271, 27462–27469. 60. Sasai, K.; Ikeda, Y.; Tsuda, T.; Ihara, H.; Korekane, H.; Shiota, K.; Taniguchi, N. The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction. J. Biol. Chem. 2001, 276, 759–765. 61. Korczak, B.; Le, T.; Elowe, S.; Datti, A.; Dennis, J. W. Minimal catalytic domain of N-acetylglucosaminyltransferase V. Glycobiology 2000, 10, 595–599. 62. Kang, R.; Saito, H.; Ihara, Y.; Miyoshi, E.; Koyama, N.; Sheng, Y.; Taniguchi, N. Transcriptional regulation of the N-acetylglucosaminyltransferase V gene in human bile duct carcinoma cells (HuCC-T1) is mediated by Ets-1. J. Biol. Chem. 1996, 271, 26706–26712. 63. Ko, J. H.; Miyoshi, E.; Noda, K.; Ekuni, A.; Kang, R.; Ikeda, Y.; Taniguchi, N. Regulation of the GnT-V promoter by transcription factor Ets-1 in various cancer cell lines. J. Biol. Chem. 1999, 274, 22941–22948. 64. Buckhaults, P.; Chen, L.; Fregien, N.; Pierce, M. Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene. J. Biol. Chem. 1997, 272, 19575–19581. 65. Chen, L.; Zhang, W.; Fregien, N.; Pierce, M. The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products. Oncogene 1998, 17, 2087–2093. 66. Seberger, P. J.; Chaney, W. G. Control of metastasis by Asn-linked, beta1–6 branched oligosaccharides in mouse mammary cancer cells. Glycobiology 1999, 9, 235–241. 67. Seberger, P. J.; Scholar, E. M.; Kelsey, L.; Chaney, W. G.; Talmadge, J. E. N-linked oligosaccharides and metastatic propensity in in vivo selected mouse mammary adenocarcinoma cells. Clin. Exp. Metastasis 1999, 17, 437–444. 68. Lu, Y.; Pelling, J. C.; Chaney, W. G. Tumor cell surface beta 1–6 branched oligosaccharides and lung metastasis. Clin. Exp. Metastasis 1994, 12, 47–54. 69. Fernandes, B.; Sagman, U.; Auger, M.; Demetrio, M.; Dennis, J. W. Beta 1–6 branched oligosaccharides as a marker of tumor progression in human breast and colon neoplasia. Cancer Res. 1991, 51, 718–723. 70. Murata, K.; Miyoshi, E.; Kameyama, M.; Ishikawa, O.; Kabuto, T.; Sasaki, Y.; Hiratsuka, M.; Ohigashi, H.; Ishiguro, S.; Ito, S.; Honda, H.; Takemura, F.; Taniguchi, N.; Imaoka, S. Expression of N-acetylglucosaminyltransferase V in colorectal cancer correlates with metastasis and poor prognosis. Clin. Cancer Res. 2000, 6, 1772–1777. 71. Ito, Y.; Miyoshi, E.; Sakon, M.; Takeda, T.; Noda, K.; Tsujimoto, M.; Ito, S.; Honda, H.; Takemura, F.; Wakasa, K.; Monden, M.; Matsuura, N.; Taniguchi, N. Elevated expression of UDP-N-acetylglucosamine: alphamannoside beta1,6 N-acetylglucosaminyltransferase is an early event in hepatocarcinogenesis. Int. J. Cancer 2001, 91, 631–637. 72. Bronson, R.; Dawe, C.; Carroll, J.; Benjamin, T. Tumor induction by a transformation-defective polyoma virus mutant blocked in signaling through Shc. Proc. Natl. Acad. Sci. USA. 1997, 94, 7954–7958. 73. Guy, C. T.; Muthuswamy, S. K.; Cardiff, R. D.; Soriano, P.; Muller, W. J. Activation of the c-Src tyrosine kinase is required for the induction of mammary tumors in transgenic mice. Genes Dev. 1994, 8, 23–32. 74. Webster, M. A.; Hutchinson, J. N.; Rauh, M. J.; Muthuswamy, S. K.; Anton, M.; Tortorice, C. G.; Cardiff, R. D.; Graham, F. L.; Hassell, J. A.; Muller, W. J. Requirement for both Shc and phosphatidylinositol 30 kinase signaling pathways in polyomavirus middle T-mediated mammary tumorigenesis. Mol. Cell Biol. 1998, 18, 2344–2359. 75. Satomi, S.; Yamasaki, Y.; Tsuzuki, S.; Hitomi, Y.; Iwanaga, T.; Fushiki, T. A role for membrane-type serine protease (MT-SP1) in intestinal epithelial turnover. Biochem. Biophys. Res. Commun. 2001, 287, 995–1002. 76. Demetriou, M.; Granovsky, M.; Quaggin, S.; Dennis, J. W. Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation. Nature 2001, 409, 733–739.

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77. Morgan, R.; Gao, G.; Pawling, J.; Dennis, J. W.; Demetriou, M.; Li, B. N-acetylglucosaminyltransferase V (Mgat5)-mediated N-glycosylation negatively regulates Th1 cytokine production by T cells. J. Immunol. 2004, 173, 7200–7208. 78. Naito, K.; Kanbayashi, N.; Nakajima, S.; Murai, T.; Arakawa, K.; Nishimura, S.; Okuyama, A. Inhibition of growth of human tumor cells in nude mice by a metalloproteinase inhibitor. Int. J. Cancer 1994, 58, 730–735. 79. Naito, K.; Nakajima, S.; Kanbayashi, N.; Okuyama, A.; Goto, M. Inhibition of metalloproteinase activity of rheumatoid arthritis synovial cells by a new inhibitor [BE16627B; L-N-(N-hydroxy-2-isobutylsuccinamoyl)-seryl-l-valine]. Agent. Action 1993, 39, 182–186. 80. Oberst, M.; Anders, J.; Xie, B.; Singh, B.; Ossandon, M.; Johnson, M.; Dickson, R. B.; Lin, C. Y. Matriptase and HAI-1 are expressed by normal and malignant epithelial cells in vitro and in vivo. Am. J. Pathol. 2001, 158, 1301–1311. 81. Oberst, M. D.; Johnson, M. D.; Dickson, R. B.; Lin, C. Y.; Singh, B.; Stewart, M.; Williams, A.; al-Nafussi, A.; Smyth, J. F.; Gabra, H.; Sellar, G. C. Expression of the serine protease matriptase and its inhibitor HAI-1 in epithelial ovarian cancer: Correlation with clinical outcome and tumor clinicopathological parameters. Clin. Cancer Res. 2002, 8, 1101–1107. 82. Oberst, M. D.; Singh, B.; Ozdemirli, M.; Dickson, R. B.; Johnson, M. D.; Lin, C. Y. Characterization of matriptase expression in normal human tissues. J. Histochem. Cytochem. 2003, 51, 1017–1025. 83. List, K.; Haudenschild, C. C.; Szabo, R.; Chen, W.; Wahl, S. M.; Swaim, W.; Engelholm, L. H.; Behrendt, N.; Bugge, T. H. Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis. Oncogene 2002, 21, 3765–3779. 84. List, K.; Szabo, R.; Wertz, P. W.; Segre, J.; Haudenschild, C. C.; Kim, S. Y.; Bugge, T. H. Loss of proteolytically processed filaggrin caused by epidermal deletion of matriptase/MT-SP1. J. Cell Biol. 2003, 163, 901–910. 85. Cho, E. G.; Kim, M. G.; Kim, C.; Kim, S. R.; Seong, I. S.; Chung, C.; Schwartz, R. H.; Park, D. N-terminal processing is essential for release of epithin, a mouse type II membrane serine protease. J. Biol. Chem. 2001, 276, 44581–44589. 86. Oberst, M. D.; Williams, C. A.; Dickson, R. B.; Johnson, M. D.; Lin, C. Y. The activation of matriptase requires its noncatalytic domains, serine protease domain, and its cognate inhibitor. J. Biol. Chem. 2003b, 278, 26773–26779. 87. Murata, K.; Miyoshi, E.; Ihara, S.; Noura, S.; Kameyama, M.; Ishikawa, O.; Doki, Y.; Yamada, T.; Ohigashi, H.; Sasaki, Y.; Higashiyama, M.; Tarui, T.; Takada, Y.; Kannagi, R.; Taniguchi, N.; Imaoka, S. Attachment of human colon cancer cells to vascular endothelium is enhanced by N-acetylglucosaminyltransferase V. Oncology 2004, 66, 492–501. 88. Guo, P.; Zhang, Y.; Zhao, J. H.; Wang, L. Y.; Guo, H. B.; Zhang, X. Y.; Chen, H. L. Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V. Biochem. Biophys. Res. Commun. 2003, 310, 619–626. 89. Baird, A.; Schubert, D.; Ling, N.; Guillemin, R. Receptor- and heparin-binding domains of basic fibroblast growth factor. Proc. Natl. Acad. Sci. USA. 1988, 85, 2324–2328. 90. Folkman, J. Tumor angiogenesis: Therapeutic implications. N. Engl. J. Med. 1971, 285, 1182–1186. 91. Saito, T.; Miyoshi, E.; Sasai, K.; Nakano, N.; Eguchi, H.; Honke, K.; Taniguchi, N. A secreted type of beta 1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation: A novel function of GnT-V distinct from the original glycosyltransferase activity. J. Biol. Chem. 2002, 277, 17002–17008. 92. Warren, C. E.; Krizus, A.; Roy, P. J.; Culotti, J. G.; Dennis, J. W. The Caenorhabditis elegans gene, gly-2, can rescue the N-acetylglucosaminyltransferase V mutation of Lec4 cells. J. Biol. Chem. 2002, 277, 22829–22838. 93. List, K.; Szabo, R.; Molinolo, A.; Sriuranpong, V.; Redeye, V.; Murdock, T.; Burke, B.; Nielsen, B. S.; Gutkind, J. S.; Bugge, T. H. Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation. Genes Dev. 2005, 19, 1934–1950.

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Biographical Sketch

Naoyuki Taniguchi graduated from the Hokkaido University School of Medicine, Sapporo (Japan), in 1967, and obtained his M.D. degree and then Ph.D. at the same university in 1972. He was a visiting associate professor at the Department of Biochemistry Cornell University Medical College, New York, and then became associate professor at the Biochemistry Laboratory Cancer Institute, Hokkaido University, in 1980. He became a professor and chairman at the Department of Biochemistry, Osaka University Medical School, from 1986 to 2006. He is now endowed chair at the Department of Glycomcis, Research Institute for Microbial Diseases, Osaka University, and professor emeritus of Osaka University. His research interest is mainly focused on the disease-related functional glycomics using glycosyltransferase genes and nonenzymatic glycosylation.

Shinji Ihara received his baccalaureate and master’s degrees from Kobe University, and his doctorate in biochemistry from the Graduate School of Medicine, Osaka University, in 2003. He has been working as a research scientist in cell migration at RIKEN Institute. In the Ph.D. course, he studied the function of GnT-V in malignant transformation using culture cells and mouse. He identified a target glycoprotein of GnT-V and their function of b1,6 GlcNAc branching in the glycoprotein. He is currently studying the function of N-glycosylation of ADAM protease (A disintegrin and metalloprotease) that direct cell migration in C. elegans.

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Eiji Miyoshi graduated from Osaka University Graduate School of Medicine in 1986 and obtained an M.D. degree. He worked at Osaka University Hospital and Osaka National Hospital as a medical physician and got a special license of gastroenterology and hepatology. He started basic research about cancer and biochemistry since 1990. From 1990 to 1994, he was a graduate student at the Department of Biochemistry Osaka University (Prof. Naoyuki Taniguchi), and a postdoctoral fellow during 1994–95. He was research fellow at the Department of Clinical Laboratory in Osaka University Hospital (1995–98), research resident of Japan Foundation for Aging and Health (1999–2000), assistant professor at the Department of Biochemistry, Osaka University Graduate School of Medicine (2000–02), associate professor at the Department of Molecular Biochemistry & Clinical Investigation, Osaka University School of Medicine Allied Health Science (2002–03). He is presently an associate professor in the Department of Biochemistry, Osaka University Graduate School of Medicine (since 2004) and visiting associate professor at the Liver Research Center at Brown University (since 2005). His major research areas are as follows: cancer, gastroenterology and glycobiology.

4.23 Glycoconjugates and Bacterial Infections: Helicobacter pylori M. Kobayashi, Shinshu University School of Medicine, Matsumoto, Japan M. Fukuda, Burnham Institute for Medical Research, La Jolla, CA, USA J. Nakayama, Shinshu University School of Medicine, Matsumoto, Japan ß 2007 Elsevier Ltd. All rights reserved. 4.23.1 Introduction

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4.23.1.1 Impact of Helicobacter pylori Discovery

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4.23.1.2 Specialized Traits of H. pylori

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4.23.1.3 Epidemiology of H. pylori Infection

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4.23.1.4

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Helicobacter pylori and Associated Diseases

4.23.1.4.1 4.23.1.4.2 4.23.1.4.3 4.23.1.4.4

Chronic gastritis Peptic ulcers Gastric adenocarcinoma MALT lymphoma

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4.23.1.5 Virulence Factors 4.23.1.5.1 4.23.1.5.2

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CagA VacA

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4.23.2 Glycoconjugates Associated with H. pylori

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4.23.2.1 Glycan Structure of H. pylori Lipopolysaccharide

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4.23.2.2 Putative Consequence of Lewis Expression by H. pylori

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4.23.2.3 Adhesion of H. pylori to Gastric Epithelial Cells

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4.23.2.4

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H. pylori Adhesins

4.23.2.4.1 4.23.2.4.2

BabA SabA

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4.23.3 Induction of PNAd in Gastric Mucosa Infected by H. pylori

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4.23.3.1 Role of PNAd in Secondary Lymphoid Organs and Chronic Inflammatory Sites

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4.23.3.2 HEV-Like Vessels Are Induced in H. pylori-Induced Inflammation

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4.23.3.3 Increased Formation of HEV-Like Vessels is Correlated with Progression of Inflammation

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4.23.3.4 Formation of HEV-Like Vessels Requires Continuous H. pylori Infection

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4.23.3.5 HEV-Like Vessels in NSAID-Induced Gastritis

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4.23.4 Antibiotic Function of GlcNAc(a1-4)-Capped O-Glycans against H. pylori

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4.23.4.1 Two Types of Mucins Present in Gastric Mucosa

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4.23.4.2 Effect of GlcNAc(a1-4)-Capped O-Glycan on H. pylori Growth

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4.23.4.3 GlcNAc(a1-4)-Capped O-Glycan-Mediated Reduction of a Cell Wall Component, Cholesteryl a-D-Glucopyranoside

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4.23.4.4 Role of CGL in H. pylori Viability

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4.23.4.5 Antibiotic Function of GlcNAc(a1-4) Residues

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4.23.1 Introduction 4.23.1.1 Impact of Helicobacter pylori Discovery Spiral microorganisms in the stomach had already been observed in the 1930s and 1940s,1,2 but little attention was paid to gastric microorganisms. In 1983, Marshall and Warren in Australia first isolated and succeeded in culturing the bacterium Helicobacter pylori (H. pylori), originally named Campylobacter pyloridis, from the gastric mucosa of patients with chronic gastritis.3,4 Their epoch-making discovery revealed that H. pylori is associated with various gastric diseases such as chronic gastritis, peptic ulcer, and malignant tumors, and the eradication of this microorganism prevents gastric disorders. For their achievement, Marshall and Warren won the Nobel Prize in Physiology or Medicine in 2005.

4.23.1.2 Specialized Traits of H. pylori Helicobacter pylori is a spiral-shaped, Gram-negative, and microaerophilic bacterium, measuring approximately 3–5mm in length. Helicobacter pylori is a member of a genus of bacteria that have adapted to the ecological niche provided by gastric mucus, where there is little competition from other microorganisms. The specialized traits allowing this organism to flourish in the harsh environment of the stomach include the following: 1. Elaboration of urease. Helicobacter pylori elaborates a large amount of urease (10–15% of total proteins by weight), which produces ammonia and carbon dioxide resulting from hydrolysis of endogenous urea, thereby buffering (neutralizing) gastric acid in the immediate vicinity of the organism. 2. Motility. Helicobacter pylori possesses numerous long flagella, the flailing movements of which allow them to swim through viscous gastric mucus with forceful screw-like movements, much like the spinning of a drill bit.5 3. Binding of H. pylori to gastric epithelial cells via bacterial adhesins. The bacterium colonizes the gastric mucosa by adhering to mucous epithelial cells and the mucus layer lining the gastric epithelium. Helicobacter pylori possesses adhesins that enhance adhesion with gastric epithelial cells by recognizing specific carbohydrate structures, such as the Lewis b blood group antigen and sialyl dimeric Lewis X (see Section 4.23.2.3 for detail).

4.23.1.3 Epidemiology of H. pylori Infection Helicobacter pylori infection occurs worldwide and affects over 50% of the world’s population, but the prevalence of infection varies greatly from country to country. The overall prevalence is highly correlated with socioeconomic status measured by household crowding and parental income.6,7 Prevalence among adults is approximately 80% in many developing countries and 50% in industrialized countries.8 The prevalence of infection increases with advancing age. In some populations, a disproportionately high rate of H. pylori infection is observed in people over 40. This seems to reflect a birth cohort effect, that is, transmission of this chronic infection was more common in the past than it is today.9–11 The mode of transmission has not yet been fully defined; however, it is widely believed that the organism is transmitted directly from person to person by human feces (fecal-oral spread) or gastric contents (gastric-oral spread). It is now generally accepted that most individuals acquire H. pylori infection in childhood.12 Once the stomach is colonized and left untreated, the organism persists for decades, if not for a lifetime.13 Frequently, children are infected by a strain with a genetic fingerprint identical to that of each parent. Husbands and wives do not exchange strains, and infection is rarely transmitted to an uninfected partner.14

4.23.1.4 Helicobacter pylori and Associated Diseases 4.23.1.4.1 Chronic gastritis Following H. pylori infection, a chronic, usually lifelong, mucosal inflammation (gastritis) develops with concomitant appearance of serological responses against the bacterium. However, H. pylori is resistant to innate and acquired immune responses, and the immune system fails to remove the organism effectively.15 Chronic gastritis leads eventually to mucosal atrophy characterized by a decrease in the proper glands and intestinal metaplasia marked by the replacement of gastric epithelial cells with epithelial cells such as columnar absorptive cells and goblet cells of intestinal morphology.16


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Peptic ulcers

Peptic ulcers are chronic, often solitary lesions that occur in gastroduodenal mucosa exposed to aggressive action of acid-peptic juices. These lesions appear to be produced by an imbalance between mucosal defense mechanisms and damaging forces. The pathogenesis of peptic ulcers appears to be multifactorial,15 and the apparent role of H. pylori in peptic ulcers cannot be overemphasized. However, H. pylori infection is present in virtually all patients with duodenal ulcers and about 70% of those with gastric ulcers. Furthermore, antibiotic treatment of H. pylori infection promotes healing of ulcers and tends to prevent their recurrence.16

4.23.1.4.3

Gastric adenocarcinoma

Gastric adenocarcinoma can be divided into two distinct histological subtypes,17 each with different epidemiological and clinicopathological features. One subtype is intestinal-type adenocarcinoma, which usually occurs at a late age and progresses through a relatively well-defined series of histological steps, namely, chronic gastritis, atrophy of pyloric glands, intestinal metaplasia, and dysplasia.18,19 The other subtype is diffuse-type adenocarcinoma, which more commonly affects younger people and is not associated with intestinal metaplasia.19

4.23.1.4.4

MALT lymphoma

Most lymphomas of the stomach are mucosa-associated lymphoid tissue (MALT) lymphoma, a low-grade B-cell lymphoma. This type of lymphoma arises in MALT, hence the name. B-cells that give rise to MALT lymphomas normally reside in the marginal zones of lymphoid follicles and are generated in response to various types of chronic inflammation, including chronic gastritis due to H. pylori infection.20 It is generally accepted that chronic infection with H. pylori leads to generation of H. pylori-reactive T-cells, which, in turn, activate a polyclonal population of B-cells by secreting soluble factors. In time, a monoclonal but T-cell-dependent population of proliferating B-cells emerges. Presumably, such monoclonal B-cell proliferation subsides when the antigenic stimulus for T-cells is removed by antibiotic treatment. However, if untreated, genetic mutations accumulate in these proliferating B-cells, and they eventually become T-cell independent.21

4.23.1.5 Virulence Factors 4.23.1.5.1

CagA

In the industrialized world, 60–70% of H. pylori strains possess the cytotoxin-associated antigen CagA, a 120–145kDa protein.22 The cagA gene is localized at one end of the cag pathogenicity island (cag-PAI), a 37-kb genomic fragment containing 31 genes.23,24 Several of these are homologous to genes encoding the type IV secretion apparatus.14 Upon direct contact of H. pylori with gastric epithelial cells, CagA is injected from the bacterium into the host cell via the type IV secretion system.25–28 After entering an epithelial cell, CagA is phosphorylated and binds to Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2), leading to a growth factor-like cellular response and cytokine production.29 Deregulation of SHP-2 by CagA is an important mechanism by which CagA promotes gastric epithelial carcinogenesis.

4.23.1.5.2

VacA

Vacuolating toxin (VacA) is a major virulence factor secreted by H. pylori and is a key component in the pathogenesis of gastric diseases.30 Approximately 50% of H. pylori strains express the VacA protein, and that expression is correlated with expression of CagA. The most established activity of VacA is cellular vacuolation in mammalian cells.30–32 Although the precise mechanism of VacA-induced vacuole formation is not fully understood, it involves binding and internalization of toxin. It has been proposed that vacuolation is a consequence of anion-selective channel formation in late endosomal compartments.33–36 In addition to its vacuole formation activity, VacA causes numerous cellular events, including depolarization of the membrane,35,37 apoptosis,38–41 interference with epithelial cell attachment,42 and inhibition of T-lymphocyte activation.43

4.23.2 Glycoconjugates Associated with H. pylori 4.23.2.1 Glycan Structure of H. pylori Lipopolysaccharide The cell wall of all Gram-negative bacteria is composed of two phospholipid bilayers with a peptidoglycan layer sandwiched between them. Lipopolysaccharide (LPS) is a structural component of the outer cell wall

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(Chapters 1.17, 2.15, 2.16, and 2.17). LPS is composed of a long-chain fatty acid anchor called lipid A, a core sugar chain, and a variable carbohydrate chain designated O antigen, which is attached to the core sugar.44 Thus, the O antigen has the potential to exhibit enormous structural variability and is the domain determining the serological specificity of LPS.45 Clinical isolates of H. pylori produce O antigen of a relatively constant chain length.46 It is this region of H. pylori LPS that shares structural homology with Lewis blood group antigens in the gastric mucosa, predominantly Lewis X and Lewis Y antigens bearing type 2 blood group determinants. Serologically, 80–90% of H. pylori strains have been found to contain Lewis X and/or Lewis Y epitopes (Chapter 3.19).

4.23.2.2 Putative Consequence of Lewis Expression by H. pylori Lewis blood group antigens are present in normal human gastric mucosa and in human gastric carcinoma, and the expression of these antigens on H. pylori LPS has important biological implications. Molecular mimicry mediated by H. pylori LPS has been suggested to camouflage the bacterium and facilitate initial colonization.47 Additionally, H. pylori Lewis antigens undergo phase variation; specifically, random, reversible high-frequency switching of phenotype contributes to virulence. The molecular mechanisms involved in phase variation are slippedstrand mispairing in poly-C tracts and translational frameshifting by ribosomal slippage.48 At least five glycosyltransferase genes are involved in generating phase variants: the genes encoding a3-fucosyltransferase (of which there are two similar but nonidentical copies), a2-fucosyltransferase, b3-galactosyltransferase, and b3-N-acetyl-D-glucosaminyltransferase.49 Each of these genes can be either ‘on’ or ‘off ’, and thus, in any H. pylori cell population, at least 32 different glycosyltransferase gene ‘on–off ’ combinations and potentially the same number of LPS phenotypes are present.49 Thus, any H. pylori strain can potentially express any LPS Lewis phenotype. This antigenic mimicry may result in immune tolerance against antigens of the pathogen or in induction of autoantibodies that recognize gastric epithelial cells, which is frequently observed in patients with chronic active gastritis.

4.23.2.3 Adhesion of H. pylori to Gastric Epithelial Cells Attachment is a prerequisite for microbial colonization of epithelial surfaces and is mediated by molecules on the bacterial surface, adhesins, which recognize proteins or glycoconjugates on the surface of eukaryotic cells (Chapter 3.21). The specificity of this interaction and the limited distribution of receptors often result in a restricted range of hosts and tissues utilized for colonization, a phenomenon known as tropism. Bacteria unable to adhere to epithelium tend to be rapidly removed by shedding from surface cells and the mucus layer. Helicobacter pylori expresses adhesins that confer intimate adherence to the gastric epithelium where the bacteria can gain easy access to nutrients from host tissues.50 These adherence properties protect the bacteria from the extreme acidity of the gastric lumen and displacement from the stomach by forces such as those generated by peristalsis and gastric emptying.51 Two carbohydrate structures in surface mucous cells serve as specific ligands for H. pylori adhesins: Lewis b, which binds to the adhesin BabA, and sialyl dimeric Lewis X-bearing glycosphingolipid, which binds to the adhesin SabA. In addition, attachment of H. pylori to gastric epithelial cells can induce pedestal formation.52 Pedestal formation describes the creation of an upright support, constructed of host cell material, beneath an attached bacterium.

4.23.2.4 H. pylori Adhesins 4.23.2.4.1 BabA The best defined H. pylori adhesin–receptor interaction characterized to date is that between blood group antigenbinding adhesin (BabA), a member of a family of H. pylori outer membrane proteins, and Lewis b, H, and related ABO antigens.51 These fucose-containing blood group antigens are found on red blood cells and in the gastrointestinal mucosa. Blood group O individuals suffer disproportionately from peptic ulcer disease,53 suggesting that bacterial adherence to H and Lewis b antigens influences severity of infection. BabA has 2 isoforms – babA1 and babA2. The product of babA1 gene, in contrast to that encoded by the babA2 gene, cannot interact with Lewis b; thus it does not enhance H. pylori colonization of the surface epithelium.51,54

4.23.2.4.2 SabA The SabA gene encodes a 651-amino-acid protein of 70 kDa and belongs to the large hop family of H. pylori outer membrane protein genes, which also includes the babA gene.55 Sialyl dimeric Lewis X glycolipid is rarely expressed in


443

normal gastric mucosa. However, the gastric mucosa infected by H. pylori newly expresses this unique glycolipid in surface mucous cells, and its expression level is increased as inflammation progresses. The adhesion mediated by sialic acid-binding adhesin (SabA) binding to sialyl dimeric Lewis X glycolipid contributes to persistent H. pylori infection. Sialyl dimeric Lewis X is also expressed in leukocytes, but an ‘on–off’ frameshift mutation of the SabA gene allows H. pylori to escape intimate contact with these inflammatory cells. Such adaptive mechanisms play an important role in the extraordinary chronicity of H. pylori infection in human gastric mucosa.

4.23.3 Induction of PNAd in Gastric Mucosa Infected by H. pylori 4.23.3.1 Role of PNAd in Secondary Lymphoid Organs and Chronic Inflammatory Sites In chronic inflammatory states, L-selectin and its ligands (Chapter 3.26) are implicated in lymphocyte recruitment in those diseases in which peripheral lymph node addressin (PNAd) is induced on high-endothelial venule (HEV)-like vessels.56,57 Such HEV-like vessels have been observed in rheumatoid arthritis, lymphocytic thyroiditis, and inflammatory bowel diseases.58–61 In these studies, the induction of PNAd is detected by the MECA-79 antibody,62 which decorates PNAd on HEV-like vessels. MECA-79-positive HEVs in secondary lymphoid organs play a major role in lymphocyte circulation.56 The MECA-79 epitope has been shown to be 6-sulfo-N-acetyllactosamine attached to extended core 1 O-glycans, Gal(b1-4)GlcNAc6S(b1-3)Gal(b1-3)GalNAc(a1-O)Ser/Thr.63 Moreover, MECA-79 antibody can also bind to its sialylated and fucosylated form that constitutes PNAd.63 Structural studies also show that 6-sulfo sialyl Lewis X on core 2 branched O-glycans, Sia(a2-3)Gal(b1-4)[Fuc(a1-3)]GlcNAc6S(b1-6)[Gal(b1-3)]GalNAc(a1-O)Ser/Thr, is present as a major L-selectin ligand on HEVs.63,64

4.23.3.2 HEV-Like Vessels Are Induced in H. pylori-Induced Inflammation Because it has been reported that de novo formation of HEV-like vessels, which express PNAd, is associated with various chronic inflammatory diseases, we determined whether chronic inflammation caused by H. pylori infection is associated with formation of HEV-like vessels.65 To do so, gastric mucosa from patients infected with H. pylori were stained with MECA-79 antibody and HECA-452 antibody, which reacts equally well with sialyl Lewis X and 6-sulfo sialyl Lewis X capping structure on extended core 1 and core 2 branches. Gastric mucosa derived from H. pyloriinfected patients displayed HEV-like vessels expressing MECA-79 and HECA-452 antigens as well as CD31 and CD34, which are markers of vascular endothelial cells. These HEV-like vessels can potentially recruit L-selectinexpressing lymphocytes, because L-selectin-IgM chimeric protein bound to the same vessels in a Ca2þ-dependent manner. These results indicate that H. pylori-induced inflammation is associated with formation of PNAd present on HEV-like vessels. These results demonstrate that 6-sulfo sialyl Lewis X on extended core 1 O-glycans is present on HEV-like vessels, based on positive staining by MECA-79 and HECA-452 antibodies. To elaborate further the chemical nature of L-selectin ligands on these vessels, the NCC-ST-439 monoclonal antibody was used. NCC-ST-439 antibody binding has been verified for sialyl Lewis X-capping structure on Gal(b1-4)GlcNAc(b1-6)GalNAc(a1-R but not on natural core 2 branched O-glycan Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc(a1-R.66 Moreover, it has not been determined whether 6-sulfo sialyl Lewis X is also recognized by this antibody. To test these possibilities, we made CHO cells expressing various types of O-glycans and stained cells with NCC-ST-439 antibody. NCC-ST-439 antibody binds to CHO cells expressing nonsulfated and 6-sulfo sialyl Lewis X on core 2 branched O-glycans but barely to CHO cells expressing those capping structures on extended core 1 O-glycans. NCC-ST-439 antibody can also stain HEV-like vessels formed in the gastric mucosa. These combined results suggest that PNAd induced by H. pylori infection expresses 6-sulfo sialyl Lewis X on both extended core 1 and core 2 branched structures in the same manner as PNAd expressed in secondary lymphoid organs.63

4.23.3.3 Increased Formation of HEV-Like Vessels is Correlated with Progression of Inflammation Based on the updated Sydney system, progression of inflammation initiated by H. pylori infection is ranked in four stages from least to most severe: normal, mild, moderate, and marked.67 In moderate and marked stages, intestinal metaplasia frequently occurs, indicating an advanced stage of the disease.

444


12

HECA-452 % of positive vessels/mm2

MECA-79

MECA-79 HECA-452

***

8 6 ** 4

**

2

NS NS NS

0

(b) MECA-79

10

Normal

Mild

Moderate

Marked

100

HECA-452

% of patients

80

MECA-79 HECA-452

***

60 ** 40

** *

20

NS

NS

0 Normal

(a)

Mild

Moderate

Marked

(c)

Figure 1 Gastric mucosa of different degrees of chronic inflammation and association of HEV-like vessels with progression of inflammation. a, (upper) Gastric mucosa at a mild stage barely expresses HEV-like vessels with minimum recruitment of lymphocytes; (lower) gastric mucosa at a marked stage expresses a significant number of recruited lymphocytes (arrowheads) around HEV-like vessels. b, The number of MECA-79þ or HECA-452þ vessels is positively correlated with the progression of chronic inflammation. Each group consists of 11 (normal), 42 (mild), 67 (moderate), and 23 (marked) patients. c, The number of patients exhibiting greater than 1% MECA-79þ or HECA-452þ vessels is highly correlated with progression of chronic inflammation. *, P

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