Glycoproteins II, Volume 29 (New Comprehensive Biochemistry)

GLYCOPROTEINS I1

New Comprehensive Biochemistry

Volume 29B

General Editors

A. NEUBERGERt L.L.M. van DEENENt

ELSEVIER Amsterdam . Lausanne . New York . Oxford . Shannon . Singapore . Tokyo

Editors

J. Montreuil Universiti des Sciences et Technologies de Lille, Laboratoire de Chimie Biologique (UMR no. 111 du CNRS), 59655 Villeneuve d’Asq Cedex, France

J.F.G. Vliegenthart Bijvoet Center for Biomolecular Research, Department of Bio-organic Chemistry, RO. Box 80.075, 3508 TB Utrecht, The Netherlands

H. Schachter Department of Biochemical Research, Hospital for Sick Children, 555 University Avenue, Toronto, Ont. M5G 1x8, Canada

1997 ELSEVIER Amsterdam . Lausanne . New York . Oxford . Shannon . Singapore . Tokyo

Elsevier Science B.V PO. Box 21 1 1000 AE Amsterdam The Netherlands

L i b r a r y of C o n g r e s s Cataloging-in-Publication

Data

G l y c o p r o t e i n s I1 e d i t o r s , J. M o n t r e u i l , J.F.G. V l i e g e n t h a r t . H. Schachter. p. cm. -- ( N e w c o m p r e h e n s i v e b i o c h e m i s t r y ; v . 2 9 b ) I n c l u d e s b i b l i o g r a p h i c a l r e f e r e n c e s a n d index. ISBN 0-444-82393-X 1. G l y c o p r o t e i n s . I. M o n t r e u i l , J e a n , 192011. V l i e g e n t h a r t . J. F. G. 111. S c h a c h t e r . H. ( H a r r y ) IV. S e r i e s . [ D N L M : 1. G l y c o p r o t e i n s . W 1 N E 3 7 2 2 F v.29b 1997 / QU 55 G5682 19971 Q D 4 1 5 . N 4 8 vol. 2 9 b [QP552.G591 572 s--dc21 [572'.S81 97-40500

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Preface Glycoproteins I/ completes a three volume contribution to the New Comprehensive Biochemistry series of Elsevier; the two previous volumes were entitled Glycoproteins (Vol. 29a, 1995) and Glycoproteins and Disease (Vol. 30, 1996). The Editors hope that these volumes follow in the footsteps of the 1965 and 1972 works entitled Glycoproteins edited by Alfred Gottschalk and published in Elsevier’s B.B.A. Library series. Of course only you, the reader, can decide whether we have succeeded in matching those magnificent milestones in glycobiology literature. In 1973, one year after the appearance of the Second Edition of Gottschalk’s Glycoproteins, one of us (JM) organized an international glycoprotein symposium in Lille, France, that was subsequently named “The Second International Symposium on Glycoconjugates”. The proceedings were published (J. Montreuil, Methodologie de la structure et du mktabolisme des glycoconjugues: glycoproteines et glycolipides, Editions du CNRS, 1974, 2 volumes). Gottschalk attended that conference and passed away a few months later. It is quite remarkable that Gottschalk’s book and his death ended an era in glycoprotein research and that the Second International Symposium was the clear beginning of a new and explosive period in the field. Gottschalk showed an uncanny prescience with the following remark at that conference: “We are not at the end of all progress but at the beginning; we have but reached the shores of a great unexplored continent.” These two eras are reviewed by one of us (JM) in Chapter 1 of Vol. 29a of the present series. The contents of Parts A and B of Gottschalk’s Second Edition filled 1378 pages, indicating that there was no shortage of excellent science at that time. The emphasis was on the physical and analytical chemistry of individual glycoproteins and their constituent sugars. There were only a few chapters with discussions of biological function such as the role of sialic acid in influenza virus cellular receptors (co-written by Gottschalk, an expert in the area), the degradation of glycoproteins by lysosomal enzymes and plasma glycoprotein turnover. The sections on glycoprotein biosynthesis reported work on subcellular fractionation and in uitro glycosyltransferase assays using crude extracts. Gottschalk concluded the book with a discussion of the “present state of glycoprotein research”. He marvelled at the progress in the seven years between his two editions and indeed there had been striking advances. However, one can only imagine the exultation that Gottschalk would have expressed had he been able to review progress in our field since he wrote his little chapter! These rapid advances are the result ‘of many different factors: techniques such as lectin chromatography, high resolution nuclear magnetic resonance spectrometry, mass spectrometry and X-ray crystallography; entirely new approaches to glycoprotein research permitted by advances in synthetic carbohydrate chemistry, molecular biology, genetics, cell biology, immunology and various other disciplines; the analysis of human diseases (accidents of nature), of experimentally produced somatic cell mutations, and of transgenic mice and mice with ‘‘null’’ mutations. We have tried in these three volumes to V

v1

cover many of these exciting aspects of glycoprotein research. Not all topics have been included and coverage of included topics is not always comprehensive. The time has long since passed when comprehensive coverage of the glycoprotein field could be achieved in a single work. Perhaps Gottschalk’s books were the last such tomes. We hope, however, that we have presented a significant portion of the excitement of modern glycobiology and that the reader will be stimulated by these books to delve further into the field and contribute to its continued growth and success.

J. Montreuil J.F.G. Vliegenthart H. Schachter

In Memoriam To our great sorrow, we have to report the death of Professor Albert Neuberger CBE FRS on August 14th 1996. Albert Neuberger was born in 1908 in Northern Bavaria. He studied Medicine in the University of Wuerzburg being awarded an MD summa cum laude, and also studied in Berlin where he was able to work in the biochemistry laboratories of Peter Rona. When Hitler came to power in 1933 he moved to London. He gained a PhD in 1936 for his work at University College Hospital Medical School and remained there for six years. After a period in Cambridge, in 1942 he moved to the National Institute for Medical Research where he remained until 1954. He then became Professor of Chemical Pathology at St Mary’s Hospital Medical School, London, where he remained for 18 years. On retirement he continued his research in the Biochemistry Department of the Charing Cross Hospital Medical School. During these periods he held many other important posts including the Principalship of the Wright-Fleming Institute and the Chairmanship of the Lister Institute. Albert Neuberger was a member of the editorial board of Biochimica et Biophysica Acta from 1968 to 1981 and associate managing editor for the last 13 years of this period. He was also one of the editors of Comprehensive Biochemistry and of New Comprehensive Biochemisq. Throughout his life Albert Neuberger showed a very broad interest in biochemistry and medicine. He made highly significant contributions in the areas of the chemistry and biochemistry of amino acids (particularly of glycine, serine, tryptophan and hydroxyproline), nutrition (with particular regard to amino acids and proteins), porphyrin biosynthesis, the chemistry of sugars (particularly amino sugars), lysozymes, lectins and particularly glycoproteins. His pioneering work on glycoproteins started in 1936. Up until the mid-1950’s most biochemists felt that, with the possible exception of mucins, the carbohydrate which could be detected in protein preparations was just an impurity. Neuberger felt that some of these proteins could have covalently attached sugars and chose to investigate hen eggwhite albumin. Before chromatography had been developed, the only possible purification method was by repeated ( x 7) crystallization until the preparation had a constant ratio of protein to carbohydrate. By proteolytic digestion he was able to isolate a compound with composition Man 4 : GlcNAc 2 : unknown amino acid 1 (suspected asparagine or glutamine). This problem was taken up again in 1955 with Marshall and Johansen and they were able to prove that the linkage between the protein and carbohydrate moieties was between asparagine and N-acetylglucosamine. This has subsequently been shown to be a conserved linkage structure in animals, plants, protozoa and other organisms. Albert Neuberger was an outstanding scientist with an impressive intellect and a range of interests outside science. He was very well liked and respected by his colleagues for his tolerance, good humour and high scientific standards. vii

...

Vlll

Albert Neuberger, 1908-1 996

He was also a devoted family man and we extend our sympathy to his widow and family. London, December 1996 Anthony K. Allen

List of contributors* Merton Bernfield 1 Harvard Medical School, and Joint Program in Neonatology, 300 Longwood Ave., Enders 9, Boston, MA 02115, USA Kenneth J. Clemetson 173 Theodor Kocher Institute, University of Berne, Freiestrasse I , 3012 Berne, Switzerland Robert N. Cole 69 University of Alabama at Birmingham, Schools of Medicine and Dentistry, Department of Biochemistry and Molecular Genetics, 404 Basic Health Sciences Building, I918 University Boulevard, Birmingham, AL 35294-0005, USA Ten Feizi 571 The Glycosciences Laboratory, Northwick Park Hospital, Watford Road, Harrow, Mid&. HA1 3UJ UK. Jukka Finne 55 Department of Medical Biochemistry, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland Hudson H. Freeze 89 The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA Irwin J. Goldstein 403 Department of Biological Chemistq University of Michigan Medical School, 1301 Catherine Rd., Ann Arbol; MI 48109-0624, USA Kenneth D. Greis 33 Department of Biochemistry and Molecular Genetics, UAB School of Medicine and Dentistry, UAB Station, Birmingham, AL 35294, USA Gerald W. Hart 33, 69 University of Alabama at Birmingham, Schools of Medicine and Dentistry, Department of Biochemistry and Molecular Genetics, 404 Basic Health Sciences Building, I918 University Boulevard, Birmingham, AL 35294-0005, USA * Authors’ names are followed by the starting page number@) of their contribution(s).

ix

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R. Colin Hughes 507 National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA. UK. Sadako Inoue 143 School of Pharmaceutical Sciences, Showa University, Hatanodai-I, Tokyo 142, Japan Yasuo Inoue 143 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan Johannis P. Kamerling 123, 243 Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, PO. Box 80,075, 3508 TB Utrecht, Netherlands Robert Kokenyesi 1 Edith Nourse Rogers Memorial Veterans Administration Medical Center, Building 70, 200 Springs Road, Bedford, MA 01730, USA; Department of Rheumatology/lmmunology, Brigham and Women b Hospital, and Joint Program in Neonatology, 300 Longwood Ave., Enders 9, Boston, MA 02115, USA Reiko T. Lee 601 Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA Yuan C. Lee 601 Department of Biology, Johns Hopkins University, 3400 N Charles St., Baltimore, MD 21218, USA Halina Lis 475 Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel Joel Mazurier 203 Universitk des Sciences et Technologies de Lille, Laboratoire de Chimie Biologique (UMR no. 111 du CNRS), Bdtiment C9, 59655 Villeneuve dilscq Cedex, France Jean Montreuil 203 Universitk des Sciences et Technologies de Lille, Laboratoire de Chimie Biologique (UMR no. 111 du CNRS), Bdtiment C9, 59655 Villeneuve d’Ascq Cedex, France Ronald D. Poretz 403 Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ 08903, USA

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Roland Schauer 243 Biochemisches Institut, Christian-Albrechts- Universitat zu Kiel, Olshausenstr 40, 24098 Kiel, Germany Nathan Sharon 475 Department of Membrane Research and Biophysics, The Weizrnann Institute of Science, Rehovot 76100, Israel Jeremiah E. Silbert 1 Edith Nourse Rogers Memorial Veterans Administration Medical Centel; Building 70, 200 Springs Road, Bedford, MA 01 730, USA; Department of Rheumatology/lmmunology, Brigham and Womenb Hospital, and Harvard Medical School, 300 Longwood Aue., Enders 9, Boston, MA 02115, USA Genevikve Spik 203 Universiti des Sciences et Technologies de Lille. Laboratoire de Chimie Biologique (UMR no. I l l du CNRS), BGtiment C9, 59655 Villeneuve d 'Ascq Cedex, France Gerard Strecker 163 Universiti des Sciences et Technologies de Lille, Laboratoire de Chimie Biologique (UMR no. 111 du CNRS), BBtiment C9, 59655 Villeneuve d 'Ascq Cedex, France Johannes F.G. Vliegenthart 123 Bijvoet Centel; Department of Bio-Organic Chemistry, Utrecht University, PO. Box 80.075, 3508 TB Utrecht, Netherlands Harry C. Winter 403 Department of Biological Chemistry, University of Michigan Medical School, 1301 Catherine Rd., Ann Arbor, MI 48109-0624, USA Jean-Pierre Zanetta 587 Centre National de la Recherche Scientijique, Center of Neurochemistry, 5,rue Blaise Pascal, 67000 Strasbourg, France

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

In Memoriam Albert Neuberger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Other volumes in the series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xxv

Chapter 1. Proteoglycans: a special class of glycoproteins Jeremiah E . Silbert. Merton Bernjeld and Robert Kokenyesi . . . . . . . . . . . . .

1

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Linkage region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin . . . . . . . . . . . 2.2.2. Keratan sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Degradation and turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Classification, distribution, and function of proteoglycan . ................... 5.1. Matrix proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Aggrecadversican family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. Leucine-rich core protein family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. Perlecan family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.4. Part-time proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Cell surface proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Syndecan family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Glypican family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. Part-time proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Intracellular proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Serglycin family . . . . . . . . ........................ 5.3.2. Other proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. Part-time proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . Fine structure/function relations of glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 21 21 21 22 22 22 25

Chapter 2. Nuclear and cytoplasmic glycoproteins Kenneth D. Greis and Gerald W Hart . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 33

...

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1

1

3 3 6 6 7 7 12 15 16 16 17 18 19 19 19

XIV

2 . 0-linked N-acetylglucosamine modified proteins . . . . . . . . . . . . . . . . . . . . . 2.1, The enzymes of 0-GlcNAc cycling . . ................ 2.2. Nuclear proteins that contain 0-GlcNAc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Nuclear pore proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Chromatin proteins, transcription factors and RNA polymerase I1 . . . . . . . . . 2.2.3. Estrogen receptor, Aplasia 83 kDa protein and autoantigen p43 . . . . . . . . . . 2.3. Cytoplasmic proteins that contain 0-GlcNAc . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. I . Cytoskeletal glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Eukaryotic initiation factor 2-associated 67 kDa polypeptide ......... 2.4. Viral proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Other cytosolic glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Phosphoglucomutase and parafusin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Glycogenin . . ............................ 3.3. Cytosolic fucosylation . ........................... 3.4. Cytosolic proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. N-linked GlcNAc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Other nuclear glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. High mobility group proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. N-linked glycoproteins of the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34 36 38 38 39 40 40 41 41 42 43 43 44 45 46 47 48 48 48 49 50 50 51

Chapter 3. Carbohydrate units of nervous tissue glycoproteins Jukka Finne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Core structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Terminal sequences of N-linked glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Classical 0-linked oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . 0-mannose linked oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . Poly-N-acetyllactosamine glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . Polysialic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Other structures . . . . . . . . ..................... ..... .... 9 . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55 55 55 56 58 59 60 62 64 65 66

Chapter 4. Glycosyl-phosphatidylinositol anchors: structure. biosynthesis and function Robert N Cole and Gerald K! Hart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Structure of GPI anchors . . . . . . . . . . . . . . . . . . . . ................... 2.1. Common core structure of GPI anchors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Structural diversity of GPI anchors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Identification of a GPI-anchored protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Biosynthesis of GPI-anchored proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Biosynthesis of GPI anchors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 69 70 70 70 72 72 74

xv 3.2. GPI anchor remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. GPI anchor attachment to protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Topology of GPI anchor biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Defects in GPI anchor biosynthesis: paroxysmal nocturnal hemoglobinuria . . . . . . . . 4 . Proposed functions of GPI anchors . . . . . . . . . ........................ 4.1. Lateral mobility . . . . . . . . . . . . . . . . . . . . . ................... 4.2. Protein release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Protein targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Endocytosis and protein turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References (Literature review completed as per 29 May 1995) . . . . . . . . . . . . . .....

78 78 79 80 80 80 81 82 83 84

Chapter 5. Dictyostelium discoideum glycoproteins: using a model system for organismic glycobiology Hudson H. Freeze . . . . . . . . . . . ...... ...................

89

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 2. The life cycle of Dictyostelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Dictyostelium as a single celled organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Development in Dictyostelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Types of protein glycosylation in Dictyostelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. N-linked oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 . 1. Biosynthesis of the lipid precursor and processing . . . . . . . . . . . . . . . . . . 3.1.2. Phosphorylation and sulfation of N-linked oligosaccharides . . . . . . . . . . . . 3.2. Two unusual types of 0-linked protein glycosylation in Dictyostelium . . . . . . . . . . . 3.2.1, Phosphoglycosylation in Dictyostelium . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Another type of 0-linked .............. 3.3. Glycophospholipid anchors . . . ................... 3.4. Cytoplasmic glycosylation in Dic ..... 4 . Antibodies against glycans and mutants in glycosylation . . . . . . . . . . . . . . . . . . . . . . . 4.1, Determinants found on N-linked oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . 4.2. Mutants in 0-linked oligosaccharides-modB . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Antibodies against fucose-mAb 83.5 and MUD62 . . . . . . . . . . . . . . . . . . . . . . 4.4. mAbs 81.8, 40.1 and MUD9 . . . . . . . . . . . . 5 . Glycoproteins in specific aspects of the Dictyostelium li 5.1. Mating types in Dictyostelium . . . . . . . . . . . 5.2. Getting around . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Preparing for development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Glycoconjugates in cell adhesion during development . . . . . . . . . . . . . . . . . . . . . 5.4.1. General features in aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2. The EDTA-sensitive adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3. The EDTA-resistant adhesion molecule . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4. Post-aggregation adhesion system . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.5. The surface sheath . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.5.1. Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.5.2. Glycoantigens and glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . 5.4.6. Pre-spore vesicles and the spore coat . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.6.1. Pre-spore vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.6.2. Spore coat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . Molecular glycobiology in Dictyostelium discoideum . . . . . . . . . . . . . . . . . . . . . . . . .

77 77

89 89 89 89

92 92 92 93 97 97 100 101

102 102 104 104 105 106 106 106 106 107 107 107 108

109 110 111

111

112 112 113 115 115

XVI

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 6. Hemocyanins Johannis I? Kamerling and Johannes FG. Vliegenthart

..

123

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Carbohydrate parameters of arthropod hemocyanins . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Androctonus australis hemocyanin glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Aslacus leptodactylus hemocyanin glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Panulirus interruptus hemocyanin glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Carbohydrate parameters of mollusc hemocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Lymnaea stagnalis hemocyanin glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Helix pomatia hemocyanin glycans . . . . . . . . . . ..... ..... 4 . Synthesis and conformational analysis of xylose-containing elements of mollusc hemocyanin glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Biosynthesis of Lymnaea stagnalis hemocyanin glycans . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

123 123 124 126 127 127 129 129 132

Chapter 7. Fish glycoproteins Sadako Inoue and Yasuo Inoue

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List of abbreviations used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Cortical alveolus glycoproteins (hyosophorins) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. First isolation of polysialoglycoproteins (PSGP) from rainbow trout eggs and their ubiquitous occurrence in salmonid fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Occurrence of a deaminoneuraminic acid residue (Kdn) at the non-reducing end of oligo/poly-Sia chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Diversity in oligo/poly-Sia chains of salmonid egg PSGP . . . . . . . . . . . . . . . . . . 2.4. Fish egg PSGP is a cortical alveolar component . . ..... 2.5. Polyprotein nature of apo-PSGP and the molecular mechanism of fertilization-associated depolymerization . . . . . . . . . . . . . . ......................... 2.6. Molecular cloning of apo-PSGP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Biosynthesis of polysialyl glycan chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Hyosophonns bearing bulky multi-antennary N-glycan chains . . . . . . . . . . . . . . . . 2.9. Biological function of hyosophorin and future perspective . . . . . . . . . . . . . . . . . . 2.9.1. Formation and function of the penvitelline fluid . . . . . . . . . . . . . . . . . . . 2.9.2. Sperm agglutinating properties of hyosophorins . ......... ... 2.9.3. Calcium ion binding properties of hyosophorins . . . . . . . ... 2.9.4. De-N-glycosylation of hyosophorins . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Mucin-type glycoproteins found in the vitelline envelope and ovarian fluid of salmonid fish . 3.1, Isolation and glycan structures of Kdn-gp and Sia-gp . . . . . . . . . . . . . . . . . . . . . 3.2. Biosynthesis and possible functions of Kdn-(Sia-)gps . . . . . . . . . . . . . . . . . . . . . 4 . Glycoproteins related to vitellogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

136 137 140 140

143 143 143 144 144 145 146 146

146 148 148 149 151 152 152 153 153 154 154 156 156 157 I59

xvii

Chapter 8. Amphibian glycoproteins Gerard Strecker . . . . . . . . . . . . . .

....

..............

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key for glycosyltranferase activities, examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Role of the oviducal secretions in mediation of gamete fusion in amphibians . . . . . . . . . . 3 . Carbohydrate chains of egg jelly coat glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 9. Blood glycoproteins Kenneth 1 Clemetson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . ................................. 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Plasma proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1, al -Acid glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Antithrombin 111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. a 1-Antitrypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Apolipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Ceruloplasmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. C1 inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7.1. Complement C3 .................................... 2.8. Factor J complement inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Factor V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10. Factor VII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 1 . Factor VIIl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.12. Factor IX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.13. Factor X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.14. Factor XI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.15. Factor XI1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.16. a-Fetoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.17. Fibrinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 8. Fibronectin . . . . . . . ............................... 2.19. Hemopexin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.20. a2-HS-glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.21. a2-Leucine-rich glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.22. a2-Macroglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.23. Plasminogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.24. a 1 -Proteinase inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.25. Protein C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.26. Protein S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.27. Prothrombin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.28. Transferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.29. Vitronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.30. Von Willebrand factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1, Glycoprotein Ib-V-IX complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Glycoprotein Iba/glycocalicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

163 163 163 163 164 165 169 170

173 173 173 174 174 176 177 178 179 179 180 180 180 180

181 181 183 184 185 185 185 185

186 187 187 188 188 189 189 190 190 190 190 190 191 192 192 192

xviii 3.1.2. Glycoprotein Ibfi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Glycoprotein IX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Glycoprotein V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4.1. Leucine-rich domains . . . . . . . . . . . . . . . . . . . . . . . 3.2. Glycoprotein IIb-IIIa (all&) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1, Glycoprotein IIb . . . . . . . . . . . . ........................ 3.2.2. Glycoprotein IIIa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. fil Integrin family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. CD36 (GPIIIb, GPIV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. PECAM-I (CD31) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Thrombospondin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References

Chapter I0. Transferrin superfamily. An outstanding model for studying biochemical evolution Jean Montreuil. Geneuidue Spik and Joel Mazurier . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . 1 . Definition of transferrin superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.2. Biological importance of iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The transferrin superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1, The saga of transferrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1, Ovotransferrins (conalbumins) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Serotransferrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Lactotransferrins (lactoferrins) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Melanotransferrin (human melanoma-associated antigen p97) . . . . . . . . . . . 2.2. Comparative study of transferrin peptide chains . . . . . . . . . . . . . . . . . . . . . . . . 2.2. 1 . Primary and three-dimensional structure . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Location of glycosylation sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.1. Serotransferrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.2. Ovotransferrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.3. Lactotransferrins . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.4. Melanotransferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Role of transferrins and of their receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Serotransferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. I . 1. Role of serotransferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1.2. Serotransferrin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Lactotransferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. 1 . Role of lactotransferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2.2. Lactotransferrin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3, Ovotransferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4. Melanotransferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Comparative study of transferrin glycan primary structures . . . . . . . . . . . . . . . . . . . . . 3 . I . Normal transferrin glycans . . . . . . . . . . . . . . ................... 3.1.1. Serotransferrin glycans . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Lactotransferrin glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Ovotransferrin glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

i94 194 194 194 195 195 196 196 196 198 198 198 198

203 203 203 204 204 204 205 205 206 207 207 207 210 210 212 213 214 214 214 214 214 215 216 216 218 218 219 219 219 219 222 223 226

XIX

3.2. Physiopathological modifications of transferrin glycan primary structure . . . . . . . . . 3.2.1.Physiological modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1.1. Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1.2. Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1.3. Tissue-dependent glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.Serotransferrin and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.1. Liver diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.2. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.3. CDG syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2.4. Serotransferrin in HEMPAS . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Three-dimensional structure of transferrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Three-dimensional structure of glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Three-dimensional structure of transferrins as glycoproteins . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 11. Chemistry. biochemistry and biology of sialic acids Roland Schauer and Johannis l? Kumerling ..... ............ List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. General characteristics of sialic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Occurrence of sialic acids in biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Screening of biological materials for the presence of sialic acid . . . . . . . . . . . . . . . . . . 5. Isolation and analysis of sialic acids . ........................ 5.1. Liberation . . . . . . . . . . . . . . . . . . . . ..................... 5.2. Colorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.High-performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. Gas-liquid chromatography combined with mass spectrometry . . . . . . . . . . . 5.3.4.Fast atom bombardment mass spectrometry . . . . . . . . . . . . . . . . . . . . . . 5.3.5. H NMR spectroscopy . . . . . . . . ........................ 6. Chemo-enzymatic highlights in sialic acid chemistry . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Free sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Glycosides of sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Sialo-oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conformational aspects of sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Biosynthesis of sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Biosynthesis of CMP-sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Transfer of sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Enzymatic modification of sialic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1.Biosynthesis and functions of N-glycolylneuraminic acid . . . . . . . . . . . . . . 8.4.2.Biosynthesis and functions of 0-acetylated sialic acids . . . . . . . 8.4.3.Biosynthesis of 9-0-lactylated sialic acid . . . . . . . .............. 8.4.4.Biosynthesis of 8-0-methylated sialic acids . . . . . . . . . . . . . . . . . . . . . . 8.4.5.Biosynthesis of 5-N-acetyl-2-deoxy-2,3-didehydr o-neuraminic acid . . . . . . . . 8.4.6.Biosynthesis of 5-N-acetyl-2,7-anhydr o-neuraminic acid . . . . . . . . . . . . . . 8.4.7.Occurrence of 2-keto-3-deoxynononic acid (Kdn) . . . . . . . . . . . . . . . . . . .

'

227 227 227 228 229 230 230 230 231 231 231 232 233 234 234

243 243 244 244 251 262 264 264 268 269 269 270 275

281 282 289 290 299 304 309 311 311 311 314 320 320 324 328 328 328 328 329

xx 9 . Catabolism of sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 . I . Sialate-O-acetylesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1, Types of sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2. Primary structures of sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3. Trans-sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4. Pathophysiological significance of sialidases and trans-sialidases . . . . . . . . . 9.2.4.1. Eukaryotic (trans-)sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4.2. Bacterial sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4.3. Viral sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.5. Sialidase and trans-sialidase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Sialate-pyruvate lyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4. Sialic acid permease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Physiological and pathobiochemical significance of sialic acids . . . . . . . . . . . . . . . . . . 10.1. General physico-chemical effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Sialic acids masking biological recognition sites . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Sialic acids representing biological recognition sites . . . . . . . . . . . . . . . . . . . . . 10.3.1.Sialic acid receptors of microorganisms, plants and lower animals . . . . . . . . 10.3.2. Sialic acid receptors of vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4. Do sialic acids have “specific” functions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5. Medical significance of sialic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

329 329 331 331 333 337 339 339 342 344 344 347 349 352 352 354 360 360 364 368 370 372 372

Chapter 12. Plant lectins: tools for the study of complex carbohydrates Irwin 1 Goldstein. Harry C. Winter and Ronald D. Poretz . . . . . . . . . . . . . .

403

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Isolation. purification and characterization of plant lectins . . . . . . . . . . . . . . . . . . . . . . 3. Structure and carbohydrate-binding specificity of lectins . . . . . . . . . . . . . . . . . . . . . . . 3. I . Mannoseiglucose-binding lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 .1. Concanavalin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Pea lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Lentil lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. I .4. Favin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Monocotyledonous mannose-binding lectins . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. N-acetyl-D-glucosamine-bindinglectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Wheat germ agglutinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Tomato lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Potato lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4. Cytisus sessilijiolius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.5. Datura stramonium lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.6. Griffonia (Bandeiraea) simplicifolia I1 lectin . . . . . . . . . . . . . . . . . . . . . . 3.4. N-acetylgalactosamine/galactose-bindinglectins . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Dolichos bij4orus lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Lima bean lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. Soybean agglutinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. Erythrina lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.5. Peanut lectin . . . . . . . . . . . . . . ........................ 3.4.6. Maclura pomifera lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.7. Winged bean lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

403 403 404 411 413 414 414 414 415 416 417 417 418 418 418 418 419 419 419 420 420 421 421 421

XXI

3.4.8. Jack fruit lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.9. Castor bean lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.10. Griffonia (Bandeiraea) simplicifolia I lectin . . . . . . . . . . . . . . . . . . . . . . 3.5. L-Fucose-binding lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1, Asparagus pea lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. Ulex europaeus lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.3. Trichosanthes japonica lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.4. Griffonia simplicifolia IV lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Lectins with complex binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1. Red kidney bean lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Sialic-acid-binding lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1, Sambucus nigra I lectin . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2. Maackia amurensis leukagglutinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.3. Trichosanthes japonica lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Lectin-based approaches for the isolation and characterization of glycoconjugates . . . . . . . 4 . I . Lectin-based reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Soluble glycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. 1 . Lectin precipitation analysis of glycans . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Lectin affinity adsorptiodchromatography . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Lectin-based blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Lectin affinity electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. Integration of lectin-based methodologies for soluble glycoconjugates . . . . . . 4.3, Cell-bound glycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Agglutination analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Lectin-coated magnetic beads and flasks . . . . . . . . . . . . . . . . 4.3.3. Fluorescence activated lectin-based flow cytometry and cell sorting . . . . . . . . 4.3.4. Lectin histochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . ...........................................

422 422 422 423 423 423 424 424 424 424 425 425 426 426 426 427 427 429 431 434 435 436 438 438 442 443 444 445 455 470

Chapter 13. Microbial lectins and their glycoprotein receptors Nathan Sharon and Halina Lis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

475

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Sialic-acid-specific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Influenza virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Other specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mannose-specific (type I fimbriae) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . I .I . Enterobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Sialic-acid-specific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Streptococcus suis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Streptococcus sanguis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Helicobacter pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5. Mycoplasma pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Gal and GalNAc-specific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Pseudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

475 476 476 476 480 480 480 480 483 483 484 485 485 485 486 486 486

xxii

3.3.3. Actinomyces species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4. Rhizobia . . . . . . . . . . . . . . ......... ..... 3.3.5. Myxobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Fucose-specific . . . . . . . . . . . . . . . ........................ 3.4.1. Helicobacter pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Pseudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3, Vibrio cholerae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Multiple specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. I . Bordetella pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1, Gal and GalNAc-specific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. I . Entamoeba histolytica . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Sialic-acid-specific . . . . . . . . ............................... 5.2.1. Plasmodium faleiparum ............................... 5.3. N-Acetylglucosamine- and chitooligosaccharide-specific . . . . . . . . . . . . . . . . . . . 5.3.1. Entamoeba histolytica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. Plasmodium falciparum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Mannose-6-phosphate-specific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1. Giardia lamblia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Biological roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Viruses . . . . . . . . . . . . . . . . . . . . . . ................... 6.1.2. Bacteria . . . . . . . . . . . . . . . . . . . . . . ................... 6.1.3. Fungi . . . . . . . . . . . . . . . . . . . . . . . ................... 6.1.4. Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Non-opsonic phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

487 488 488 488 488 489 489 489 489 489 492 492 492 492 493 493 494 494 495 495 495 495 495 495 496 498 499 500 501

Chapter 14. Adhesive glycoproteins and receptors R . Colin Hughes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

507

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Cadherin family of adhesive glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Classical cadherins . . . ............................ 2.2. Desmosomal glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Immunoglobulin superfamily of adhesive glycoproteins . . . . . . . . . . . . . . . . . . . . . . . 3.1. Nerve cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. 1. N-CAM: the prototype adhesion molecule of the immunoglobulin superfamily . 3.1.2. L1 glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Myelin glycoproteins MAG and PO . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. The L2/HNK-I carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Non-neuronal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. I . ICAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. VCAM . . . . . .................................... 3.2.3. PECAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Matrix glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Fibronectin . . . . . . . .................................... 4.2. Laminin . . . . . . . . . .................................... 4.3. Nidogedentactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

507 510 510 514 517 517 517 52 1 523 524 525 525 527 528 528 529 535 54 1

xxiii

4.4. Tenascin . . . . . . . . . . . . . . . . . . . 4.5. SPARC/osteonectin . . . . . . . . . . . .

..............

...............

............... ........................

5. Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1, Structure and specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Roles of carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 15. Carbohydrate differentiation antigens Ii. SSEA- 1 (Le") and related structures. Prototype mammalian carbohydrate antigens that serve as ligands in molecular recognition Ten Feizi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . The I and i antigens and their sialyl forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Biochemical nature of Ii antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Anti4 and -i antibodies as reagents in studies of cell differentiation . . . . . . . . . . . . 2.4. Anti-I and -i antibodies as immunosequencing reagents for glycoprotein oligosaccharides 2.5. Roles of Ii and related sequences as ligands . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1. Sialyl Ii as host-cell ligands for a pathogen Mycoplasma pneumoniae . . . . . . 2.5.2. Ii-type sequences as ligands for endogenous carbohydrate-binding proteins? . . 3 . Stage-specific embryonic antigen-], SSEA-I (CDI 5/LeX/L5) and the sialyl and sulfated analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

........................................... 3.2. Biochemical nature of SSEA-1 and related antigens in mouse and human . . . . . . . . 3.3. LeX, sialyl-Le' and related sequences as ligands for endogenous carbohydrate-binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 16. Cell adhesion and recognition mechanisms in the nervous tissue Jean-Pierre Zanetta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Mechanisms of neuronal migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Mechanisms of axonal growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Mechanisms of synaptogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Mechanisms of glial wrapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . ................... References . . . . . . . . . . . . . . . . . . . ...................

541 544 545 545 545 550 554 559

571 571 571 573 573 573 575 575 577 577 578 579 579 580 581 582 582 583

587 587 587 587 590 591 593 596 597

Chapter I 7. Neoglycoproteins Reiko T Lee and Yuan C. Lee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

601

List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . History and definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

601 601

xxiv

.................................. 2. Preparation of neoglycoproteins . 2.1. Modification of primary ami roups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Reductive amination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. I .2. Amidination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Acylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. p-Isothiocyanato-phenyl glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Modification of carboxyl groups . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Modification with glycosylamine derivatives . . . . . . . . . . . . . . 2.2.2. Glycamine derivatives . . . . . ........................... 2.2.3. o-Aminoalkyl glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Modification of tyrosyl group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Modification of cysteinyl group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Conjugation of polysaccharides to proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Enzymatic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1, Use of glycosyl-transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2. Use of glycosidases in transglycosylation . . . . . . . . . . . . . . . . . . . . . . . . 2.6.3. Use of transglutaminase . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Glycoproteins of non-covalent attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Synthetic glycopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Applications of neoglycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. I . Probing carbohydrate-protein interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Use in isolation of carbohydrate-binding proteins . . . . . . . . . . . . . . . . . . . . . . . 4.3. Cytochemical markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Neoglycoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Biomedical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Advantages of neoglycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Other neoglycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60 I 602 602 605 605 605 605 606 607 608 608 608 609 610 610 612 613 61 3 613 614 614 614 614 615 615 615 617 618 618 61 8

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

621

Other volumes in the series Volume 1.

Membrane Structure (1982) J.B. Finean and R.H. Michell (Eds.)

Volume 2.

Membrane Transport (1982) S.L. Bonting and J.J.H.H.M. de Pont (Eds.)

Volume 3.

Stereochemistry (1982) C . Tamm (Ed.)

Volume 4.

Phospholipids (1982) J.N. Hawthorne and G.B. Ansell (Eds.)

Volume 5.

Prostaglandins and Related Substances ( 1983) C . Pace-Asciak and E. Granstrom (Eds.)

Volume 6.

The Chemistry of Enzyme Action (1984) M.I. Page (Ed.)

Volume 7.

Fatty Acid Metabolism and its Regulation (1984) S. Numa (Ed.)

Volume 8.

Separation Methods (1984) Z. Deyl (Ed.)

Volume 9.

Bioenergetics (1985) L. Ernster (Ed.)

Volume 10.

Glycolipids (1985) H. Wiegandt (Ed.)

Volume 1 la. Modern Physical Methods in Biochemistry, Part A (1985) A. Neuberger and L.L.M. van Deenen (Eds.) Volume 1 lb. Modern Physical Methods in Biochemistry, Part B (1988) A. Neuberger and L.L.M. van Deenen (Eds.) Volume 12.

Sterols and Bile Acids (1985) H. Danielsson and J. Sjovall (Eds.)

Volume 13.

Blood Coagulation (1986) R.F.A. Zwaal and H.C. Hemker (Eds.)

Volume 14.

Plasma Lipoproteins (1987) A.M. Gotto Jr. (Ed.)

Volume 16.

Hydrolytic Enzymes (1987) A. Neuberger and K. Brocklehurst (Eds.)

Volume 17.

Molecular Genetics of Immunoglobulin ( 1987) F. Calabi and M.S. Neuberger (Eds.) xxv

xxvi

Volume 18a. Hormones and Their Actions, Part I (1988) B.A. Cooke, R.J.B. King and H.J. van der Molen (Eds.) Volume 18b. Hormones and Their Actions, Part 2 - Specific Action of Protein Hormones (1988) B.A. Cooke, R.J.B. King and H.J. van der Molen (Eds.) Volume 19. Biosynthesis of Tetrapyrroles (1991) P.M. Jordan (Ed.) Volume 20. Biochemistry of Lipids, Lipoproteins and Membranes (1991) D.E. Vance and J. Vance (Eds.)

Molecular Aspects of Transport Proteins (1992) J.J. de Pont (Ed.) Volume 22. Membrane Biogenesis and Protein Targeting (1992) W. Neupert and R. Lill (Eds.) Volume 23. Molecular Mechanisms in Bioenergetics (1992) L. Ernster (Ed.) Volume 24. Neurotransmitter Receptors ( 1993) F. Hucho (Ed.) Volume 25. Protein Lipid Interactions (1993) A. Watts (Ed.) Volume 26. The Biochemistry of Archaea ( I 993) M. Kates, D. Kushner and A. Matheson (Eds.) Volume 27. Bacterial Cell Wall ( 1994) J. Ghuysen and R. Hakenbeck (Eds.) Volume 28. Free Radical Damage and its Control (1994) C. Rice-Evans and R.H. Burdon (Eds.) Volume 29a. Glycoproteins (1995) J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.) Volume 30. Glycoproteins and Disease (1 996) J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.) Volume 2 1.

J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins I1 Elsevier Science B.V

CHAPTER I

Proteoglycans: a special class of glycoproteins Jeremiah E. S i l b e ~ - t ~Merton i ~ ? ~ , Bernfield3>4,and Robert

K~kenyesi’,~,~

’Edith Nourse Rogers Memorial Veterans Administration Medical Center; Building 70, 200 Springs Road, Bedford, MA 01730, USA Department of Rheumatology/lmmunology. Brigham and Womenk Hospital, Haruard Medical School, and Joint Program in Neonatology, 300 Longwood Aue.. Enders 9,Boston, MA 02115, USA

Abbreviations Standard abbreuiations

Other abbreviations

Gal Xyl

galactose xylose

GlcN GlcNS GlcNAc GalN GalNAc

glucosamine N-sulfonylglucosamine N-acetylglucosamine galactosamine N-acetylgalactosamine

PAPS RER ER EGF TGFO aFGF bFGF

GlcA IduA

glucuronic acid iduronic acid

3’-phosphoadenylyl, 5’-phosphosulfate rough endoplasmic reticulum endoplasmic reticulum epidermal growth factor transforming growth factor acidic fibroblast growth factor basic fibroblast growth factor

The abbreviations “PG” and “GAG” are frequently used for proteoglycan and glycosaminoglycan, respectively, but these have not as yet been accepted as standard abbreviations by the major journals or by the terminology committee of the International Union of Biochemistry. Similarly the abbreviations “HA” for hyaluronan, “CS” for chondroitin sulfate, “DS” for dermatan sulfate, “HS” for heparan sulfate and “KS” for keratan sulfate are frequently used but have not as yet been accepted as standard abbreviations.

1. Introduction Proteoglycans consist of complex carbohydrates covalently linked to protein, and can therefore be classified as glycoproteins. Nevertheless, for historical reasons and because they differ substantially from other glycoproteins, they are usually regarded as a separate group of substances. There are excellent detailed articles concerning structure, metabolism, distribution, and function of proteoglycans [l-171, so this chapter is not intended to be a comprehensive review. Rather, the aim is to provide an overall background and then describe and emphasize the glycosaminoglycan portion which is the primary characteristic defining this class of substances. The glycosaminoglycans have structures, metabolism, and actions quite distinct from those of the oligosaccharide portions of other glycoproteins. While some of these distinctions are obvious, there are others which are more subtle and have not received much attention.

Originally proteoglycans/glycosaminoglycans were called “mucopolysaccharides” in order to describe their polysaccharide structure and a viscous or mucinous (from the Latin mucus, “slimy”) characteristic. Only the structure of the polysaccharide portions of these molecules was recognized initially, with no realization that the polysaccharides were covalently linked to protein. Because of this, it is understandable that the “mucopolysaccharides” were considered to constitute a separate group of compounds with no relationship to the glycoproteins. Subsequently it became clear that the “mucopolysaccharides” with the exception of hyaluronan (hyaluronic acid) included a covalently linked protein, and were therefore a special class of glycoproteins. Since the term “mucopolysaccharide” did not reflect the presence of protein, it was considered to be misleading and consequently was replaced. Nevertheless, because of previous widespread use, “mucopolysaccharide” is still occasionally used as an all-inclusive name in referring to the polysaccharide portion alone, and sometimes to the entire polysaccharide-protein. The term is not rigorously defined, and is only rarely found in the more recent literature with the exception of its use for the “mucopolysaccharidoses”, a group of disorders in glycosaminoglycan degradation. The term “glycosaminoglycan” was introduced to be more descriptive of the polysaccharide structure and to clarify some confusion over nomenclature. Thus “glycosaminoglycan” is descriptive of a repeating polysaccharide (glycan) structure containing hexosamines (glycosamino) and does not include the protein portion of the entire molecule. The trivial names for the naturally occurring glycosaminoglycans are, hyaluronan (hyaluronic acid), chondroitin 4-sulfate (formerly called chondroitin sulfate A), chondroitin 6-sulfate (formerly called chondroitin sulfate C), dermatan’ sulfate (formerly called chondroitin sulfate B and earlier called b-heparin), heparin, heparan sulfate (formerly called heparitin sulfate), keratan (poly-N-acetyllactosamine), and keratan sulfate. The non-sulfated analogs, chondroitin, dermatan, and heparan, are generally not found in biological materials except at early stages during biosynthesis or when cultured cells are grown in the presence of high concentrations of chlorate [ 191 which prevents the formation of the sulfate donor, PAPS. Nevertheless, non-sulfated analogs have been produced by enzymatic means and by chemical desulfation, and their use has been important in examination of the intermediary metabolism of the glycosaminoglycans. Chondroitin sulfate and heparan sulfate are produced to a variable extent by most if not all vertebrate cells, while heparin, dermatan sulfate, and keratadkeratan sulfate are less widespread. The above named glycosaminoglycans are the only substances found in vertebrates that satisfy the structural criteria for this designation. There are also substances in lower animals and bacteria that fulfill the structural requirements for being a glycosaminoglycan. These compounds may be distinct from the vertebrate compounds by virtue of different function, location, and so on. The term “proteoglycan” was introduced to describe an entire molecule consisting of one or more glycosaminoglycan chains attached to a core protein by means of a linkage oligosaccharide. For example the general term for an undefined proteoglycan

’

It has been suggested by J.E. Scott [I81 that the term “dermochondran or dermochondan” be used instead of dermatan, since dermatan sulfate is derived from chondroitin sulfate and always contains chondroitin-like regions with glucuronic acid rather than iduronic acid.

3

containing a specific glycosaminoglycan would be described as “chondroitin sulfate proteoglycan” or “proteochondroitin sulfate” when the entire molecule is intact, while the term “chondroitin sulfate” would be reserved for the glycosaminoglycan portion alone or the glycosaminoglycan plus the characteristic linkage oligosaccharide that glycosidically attaches the glycosaminoglycan to the core protein. In addition to the glycosaminoglycan chains, some of the proteoglycans contain oligosaccharide structures identical to those of the general class of glycoproteins. The term proteoglycan is used, independent of the presence of these “glycoprotein” portions. All of the glycosaminoglycans with the exception of hyaluronan are synthesized on a core protein and usually appear in the tissues as intact proteoglycans. However, free glycosaminoglycan chains also appear as products of proteoglycan processing or degradation. Since hyaluronan is synthesized without a core protein and appears only as the glycosaminoglycan, the term proteoglycan should not be applied to this substance. During the last few years the core proteins of many classes of proteoglycans have been characterized through the techniques of molecular biology, so that families with similar core protein structure have now been identified. New terms’ for these families of proteoglycans have been and are still being introduced by those investigators who initially describe the core proteins. These names have generally reflected their structure, or the tissue in which they were initially found, or what is presumed to be a major characteristic or function. In addition there are other proteins which can be considered as “part-time” proteoglycans since they may be found either with or without glycosaminoglycan chains.

2. Structure Proteoglycans are defined by their structure as a type of glycoprotein that has covalently linked large polysaccharide (glycosaminoglycan) chains composed of repeating identical or similar disaccharides. The glycosaminoglycans are all glycosidically attached at their reducing end to specific linkage oligosaccharides which in turn are attached covalently to the core protein. There is no characteristic protein structure that defines the proteoglycan core proteins as a class distinct from that of the protein portion of the general class of glycoproteins. The proteins are widely varied in structure and are only defined as core proteins for proteoglycans because they become substituted with the glycosaminoglycans. Thus only the presence of a glycosaminoglycan moiety provides the separate classification for this special type of glycoprotein. Structures of the various core proteins will be discussed in section 5 together with distribution and functions of the proteoglycans.

2.1. Glycosaminoglycans The glycosaminoglycans are distinguished from the oligosaccharide structures found in all other classes of glycoproteins (with the exception of those that contain poly-

*

Terms for proteoglycan families where the core proteins have been sequenced have become widely used and accepted. However it has been suggested [18] that these terms might be reserved to refer to the core protein alone and not the entire proteoglycan, since there may be considerable variation in the glycosaminoglycans attached to the same protein. For example, the term “serglycin” could be used to describe the core protein without glycosaminoglycans, or after removal of glycosaminoglycans, while “serglycin, H” would be used to refer to heparin-containing serglycin and “serglycin, CS’would be used for serglycin with chondroitin sulfate.

4

DERMATAN SULFATE (Chondroitin Sulfate B)

HYALURONIC ACID

[\+gj--p

r ,

7

FH,OH

HNCOCH,

n

/3 1,3Linkage

CHONDROlTlN 6-SULFATE (Chondroitin Sulfate C)

r

GOOH

0

CH20SqH

HNCOCH3

_In

KERATAN SULFATE

1

OH OH

L

OH

L

HNCOCH,

-

H NCOCH:,

J I I

Pi,4 Linkage

n

Fig. 1. Alternating sugars of hyaluronic acid, chondroitin/dermatan sulfate and keratan sulfate glycosaminoglycans.

N-acetyllactosamine regions) by virtue of the glycan heteropolymer consisting of hexosamine residues (either GlcN or GalN) alternating with another sugar (Gal, GlcA, or IduA) (Fig. 1). The GlcN is either N-sulfated (GlcNS) or N-acetylated (GlcNAc) with rare if any residues containing free amino groups, while the GalN is always N-acetylated (GalNAc). There may be 0-linked sulfate substituents on the hexosamine and/or the uronic acid or Gal. Because the hexosamines alternate with the other sugars, it is convenient to think of the glycosaminoglycans as polymers consisting of repeating identical or similar disaccharide units. Although an individual chain may contain both GlcA and IduA, no glycosaminoglycan chain has been found to contain both GlcN and GalN or Gal and uronic acid as part of the repeating portions of the same polymer. All the glycosaminoglycans are anionic by virtue of their uronic acids and especially their sulfate groups, with heparin being the most anionic organic substance found in living tissues. This extensive sulfation is unique to the glycosaminoglycans so that the high degree of negative charge is an important feature distinguishing proteoglycans from other glycoproteins. Glycosaminoglycan chains on proteoglycans range in size from -15 disaccharide units to several hundred, while hyaluronan may be as much as several thousand disaccharides in length. The chains of all the glycosaminoglycans are linear (unbranched) and have a non-specific termination, so that some chains may end with uronic acid and some with hexosamine [20]. Hyaluronan (hyaluronic acid) is the simplest glycosaminoglycan, since it contains identical alternating GlcA and GlcNAc saccharides throughout, fi- 1,3- and 6- 1,4-linked respectively, has no sulfate substituents, and is not linked covalently to protein. Other

5

glycosaminoglycans are sulfated (with the exception of poly-N-acetyllactosamine) and are synthesized bound to specific core proteins. Chondroitin sulfate glycosaminoglycans consist of alternating GlcA and GalNAc3 also 8-1,3- and p-1 ,Clinked respectively, with variable amounts and location of sulfation, including non-sulfated GalNAc, GalNAc 4-sulfate, GalNAc 6-sulfate, GlcA 2-sulfate, or combinations of sulfate substitutions on the same saccharides. Depending upon the animal and the tissue source, chondroitin sulfates exhibit a wide range of sulfation and differ in the amounts of 6-sulfate and 4-sulfate in the same glycosaminoglycan chain. In general, the sulfation on a single chain is mostly or entirely 6-sulfate or 4-sulfate, so that chondroitin sulfate is not ordinarily found with equal amounts of both types of sulfate in the same chain. Usually a single GalNAc residue will have only one sulfate, either 4 or 6, but disulfated 4,6 GalNAc residues are found. In addition, 2-sulfated GlcA is found alternating with sulfated or non-sulfated GalNAc. Dermatan sulfates are more complex than the chondroitin sulfates. The term defines a glycosaminoglycan similar to chondroitin sulfate but containing va$is amounts of IduA rather than having GlcA as the only uronic acid. This is an important concept since the formation of dermatan residues proceeds from chondroitin residues as precursors when the appropriate uronosyl epimerase is present to convert GlcA to IduA. This results in modified glycosaminoglycans with the same protein core as the precursor proteochondroitin sulfate. The IduA residues are ordinarily only found adjacent to 4-sulfated GalNAc and not adjacent to 6-sulfated or non-sulfated GalNAc, although non-sulfated or 6-sulfated GalNAc residues may be found adjacent to GlcA in the same glycosaminoglycan chain. In addition, dermatan sulfate frequently has IduA 2-sulfate in fairly high amounts. Heparin and heparan sulfate glycosaminoglycans are even more complex. These contain two main types of disaccharide residues, although the proportions differ greatly between heparin and heparan sulfate (Fig. 2). Thus heparan sulfate contains areas of sulfated IduA-GlcNS repeating disaccharides, and areas of non-sulfated GlcA-GlcNAc repeating disaccharides [22]. The sugars are linked @-1,4-(or a-1,4- if the uronic acid is I ~ u A and ) ~ a-1,Clinked respectively (in contrast to the 8-1,3- and B-1,4-linkages of hyaluronan and chondroitin. In addition to the N-sulfation of the GlcN, there are variable amounts of 6-0-sulfate on the GlcNS and 2-0-sulfate on the IduA. Heparan sulfate chains usually contain more non-sulfated disaccharides than sulfated disaccharides, resulting in an overall degree of sulfation that averages less than one sulfate per repeating disaccharide unit. Heparin differs from heparan sulfate by its higher content of IduA, N-sulfate and 0-sulfate with most of the disaccharide units containing 2-sulfated IduA alternating with GlcNS which is partially 6-0-sulfated as well. The overall sulfation averages two to Although the conventional description of the disaccharide repeating units is GlcA and GalNAc, (3-1,3and p-l,4-linked, respectively, chondroitin is actually a modified polylactose structure so that it has been suggested [21] that it would be better to describe the disaccharide repeating unit as GalNAc and GlcA, 8-1,4and p-I ,3-linked, respectively. The IduA is defined as L- rather than D- since the epimerization of GlcA occurs at the 5’ position. The linkage of the lduA in dermatan is therefore actually L-IduA, a-i,3-linked rather than the precursor D-GlcA, b-1,3-linked; in heparin and heparan sulfate it is L-IduA, a-I,4-linked rather than D-GlcA, p-1,4-linked. However, it should be understood that only the terminology has changed while the linkages remain the same.

6

HEPARAN (Heparitin) SULFATE

r

1

HEPARIN

r

1 0-

osofl

n 011,4 Linkage Fig. 2. Typical oligosaccharide structures of heparan sulfate and heparin.

two and a half sulfates per disaccharide. In addition to 6-0-sulfation of GlcNS there is also 3-0-sulfation of occasional GlcNS on a fraction of the glycosaminoglycan chains (as well as on occasional GlcNS of some heparan sulfate). This is highly important because the 3-0-sulfate substituent is necessary for the anti-coagulant activity of heparin and heparan sulfate [23]. The similarity of the heparin and heparan sulfate structures has caused some confusion, so that heparan sulfate has occasionally been described incorrectly as “cell surface heparin”, etc. However, as proteoglycans they are not similar, since heparin is only found attached to the specific intracellular serglycin protein or as the free glycosaminoglycan chain split from this proteoglycan, while heparan sulfate is found on several families of extracellular and cell surface core proteins unrelated to serglycin. Keratan sulfates are polymers composed of alternating Gal and GlcNAc residues p- 1,4and @-1,3-linked,respectively’. The degree of sulfation may vary along the keratan chains, with the first one or two reducing end disaccharides being non-sulfated, the next few monosulfated, while the remainder of the chain consists of disulfated disaccharides [24, 251.

2.2. Linkage region

2.2.1. Chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin These glycosaminoglycans are all attached to a core protein at their reducing end through a tetrasaccharide region consisting of GlcA-Gal-Gal-Xyl[3,5,7] (Fig. 3) with As with chondroitin, keratan is actually a modified polylactose.

7

z

W

Glucuronic Acid

Galactose

Galactose

Xylose

Serine

I-

g0 I

NH 0-CHz-C

’0

I I

c=o OH

OH

OH

I E

W

I-

$ a Fig. 3. Structure of oligosaccharide linkage region.

the Xyl glycosidically linked to the hydroxyl of Ser, usually adjacent to a Gly in the protein core. The first sugar of the glycosaminoglycan chain (linked to the Gal) always is GlcA, but is considered to be a part of the linkage oligosaccharide, since its addition to the linkage region appears to be catalyzed by a specific enzyme different from the enzyme that is involved in the incorporation of GlcA into the rest of the glycosaminoglycan [26]. The Xyl may be phosphorylated [27], and one or both Gal residues may be sulfated [28]. In addition to the glycosaminoglycan substituents on proteoglycans, there may be 0-linked and N-linked oligosaccharides similar or identical to those of various species of glycoproteins.

2.2.2. Keratan sulfate Skeletal keratan sulfate and corneal keratan sulfate chains are attached to core protein through 0-linked oligosaccharides [29] and N-linked oligosaccharides [30,3 11 respectively, identical to 0-linked and N-linked oligosaccharides of the general class of glycoproteins.

3. Biosynthesis Cell-free translation of chondrocyte mRNA from a variety of sources [32-341 has been reported. More recently substantial details concerning the molecular size and the amino acid sequence for core proteins from a great number of different sources (see section 4) have been determined by use of conventional cloning techniques. In a few instances recombinant core proteins such as decorin [35], and syndecans [36-381 have been examined as receptors for the glycosylation reactions. Biosynthesis of the core protein does not appear to differ from that of other proteins except for possible post-translational modifications such as that of the large aggrecan protein core [39,40] which may proceed through an even larger “pro-protein” which then is reduced in size during or after formation of the glycosaminoglycan chains. Although post-translational proteolytic trimming of core protein has also been noted for decorin [41], neurocan [42] and serglycin [43,44], little is known about details, nor is there any knowledge whether or not this is general for all core proteins.

8

Post-translational modifications such as phosphorylation of serine residues on decorin [ 151 and attachment of myristoyl and palmitoyl moieties to perlecan [45] have been described, but these modifications are not distinguishable from similar reactions occurring with the general class of glycoproteins. Moreover the core proteins of many proteoglycans contain N- and/or 0-linked oligosaccharides identical to those of the general class of glycoproteins. It can be assumed that biosynthesis of these oligosaccharides is identical to that of other glycoproteins. Compared to the general class of glycoproteins, which may also contain SerGly dipeptides, only a small number of proteins become substituted with glycosaminoglycans. The mechanism of selection of these proteins for subsequent glycosaminoglycan attachment is unclear, but may be related to some as yet undetermined common structural feature. In contrast to the apparent general nature of the protein core biosynthesis, and some post-translational modifications, formation of the glycosaminoglycans follows pathways distinctly different from those of the oligosaccharides of the general class of glycoproteins. Thus, following formation of the precursor sugar nucleotides and synthesis of the protein core in the RER, some of the next steps in biosynthesis are unique to proteoglycans. These consist of: (A) formation of a specific oligosaccharide linkage region by (1) addition of Xyl to Ser moieties of the core protein followed by addition of two Gal residues and a GlcA residue for chondroitiddermatan, heparan, heparin or (2) addition (not unique) of glycoprotein-like N-Asn, 0-Thr, or 0-Ser linked oligosaccharides for keratadkeratan sulfate, (B) formation of the alternating saccharides of the glycosaminoglycan chains and (C) sulfation and/or epimerization of the glycosaminoglycan chains. A schematic presentation is shown in Fig. 4. Of these steps, only the sugar activation, protein formation, and keratan sulfate linkage formation are shared with the formation of other glycoproteins. In the case of hyaluronan, only the activation of sugars and formation of alternating saccharides are applicable, since hyaluronan has only the glycosaminoglycan component and is not sulfated. In addition to Gal, GalNAc, and GlcNAc, which are found in the general class of glycoproteins, the proteoglycans contain, GlcNS, GlcA, IduA, and Xyl, sugars which are rare or have not been found at all in other glycoproteins. The precursor activation pathway for GlcA is by dehydrogenation of UDP-Glc to UDP-GlcA which can in turn be decarboxylated to form UDP-Xyl. The activated form of sulfate, 3’-phosphoadenylyl 5’-phosphosulfate (PAPS), is formed from sulfate and ATF’. The enzyme involved in formation of UDP-Xyl from UDP-GlcA is membrane-bound (ER or Golgi) [46], while the activation of all the other sugars and sulfate take place in the cytosol with soluble enzymes. The main reactions involved in the biosynthesis of the glycosaminoglycan portions of the proteoglycans have been defined. Formation of the GlcA-Gal-Gal-Xyl linkage appears to take place sequentially in different locations as the core protein moves along the smooth ER and Golgi membrane with identical reactions for the formation of chondroitin sulfate, heparan sulfate, and heparin. Polysaccharide chains are initiated by direct transfer of Xyl from UDP-Xyl to specific Ser moieties of the protein core, followed by transfer of each Gal residue and GlcA with no apparent dolichol or other intermediate [47]. Thus there does not appear to be any pathway (except with keratan sulfate) similar to that for the earlier steps in formation of the N-linked glycoprotein oligosaccharides. Unlike protein

9

GLUCOSE

Jp.P GlcN-6-P
I00 amino acids distributed into three types of contiguous, re-iterated domains. One domain shows a continuous stretch of Ser residues (SSSS), another contains several repeats of SGQ (SGQ) and finally a GSGS repeat (GSGS). One or more of these regions is probably the site for addition of GlcNAc-1-P residues. CP5 also has shorter versions of each region. All four genes have been mapped to different yeast artificial chromosome fragments in Dicvvostelium. The gene for proteinase I has not been identified, but it probably resembles CP4.

100

the known amino acid composition of these proteins. Two cysteine proteinase genes have recently been cloned from a vegetative cell cDNA library in Dictyostelium [63]. They are named cysteine proteases (CP) 4 and 5. The amino acid sequences of both gene products are shown in Fig. 5 and compared with two other Dictyostelium cysteine proteinases, CPl and CP2, that appear only in development along with several plant and animal cysteine proteinases. All four of the Dictyostelium enzymes share the conserved regions seen in other eukaryotic cysteine proteinases, but both CP4 and CP5 are substantially different from the others. CP4 contains a 1 15 amino acid insert that is rich in Ser residues distributed into several distinct, contiguous domains. CP5 contains the same series of Ser-rich regions, but each is much smaller. It is likely that one of the serine-containing regions would be the sites for addition of GlcNAcl-P, and expression cloning of both CP4 and CP5 confirms that they carry GlcNAcl-P [63]. GlcNAcl-P transferase uses a SGSG-containing peptide as an acceptor [65]. Based on protein sequencing, neither CP4 nor CP5 is the gene coding for proteinase I, but the amino acid composition of proteinase I is similar to that deduced for CP4 [56]. A spore coat protein called SP96 also reacts with rabbit antibodies against GlcNAcl-P and also has contiguous poly-Ser regions. Monoclonal antibodies against fucose also recognize both this protein and proteinase I, however it is not known if the Fuc is bound to GlcNAc in proteinase I. Riley et al. have shown that a small oligosaccharide containing GlcNAc and Fuc can be isolated from SP96 following p-elimination [66]. Carbohydrate-specific rabbit antibodies have been purified by affinity chromatography of the antiserum using immobilized UDP-GlcNAc as a ligand [67]. More recently, monoclonal antibodies against GlcNAc 1-P have also been prepared [Mehta and Freeze, unpublished]. Proteinase I is maintained at a high level during the first six hours of development and then appears to be lost. The function of GlcNAcl-P in proteinase I is not known. It must await the isolation of chemically induced or gene knockout mutants. Studies in our laboratory indicate that Man-6-P and GlcNAcl-P are found on mutually exclusive sets of proteins in the lysosomes [G. Souza, unpublished]. Moreover, immunofluorescence confocal microscopy shows that the two modifications are found in separate vesicles. This raises the exciting possibility that the proteins are sorted via their mutually exclusive carbohydrate modifications.

3.2.2. Another type of 0-linked glycosylation No complete structure has been determined for this type of 0-linked oligosaccharide in Dictyostelium. These carbohydrate chains are resistant to @-elimination, suggesting that they are not the standard type of HexNAcaIpThrlSer linkage. This type of modification has been defined by a panel of monoclonal antibodies. Reactivity to this antibody is lost in a series of mutant strains which are collectively said to be mutated at the modB locus. Although many proteins contain the modB carbohydrate, most of the attention has been on the analysis of two of them. One is a cell surface glycoprotein, called PsA (a.k.a. SP29), which has been cloned and sequenced. The carbohydrate modifications are located on a repeated motif, PTVT as shown by Edman degradation [68]. This sequence is typical of many of those in mammalian cell proteins which also contain 0-linked oligosaccharides [69]. Several allelic variants of this protein from related strains of Dictyostelium discoideum contain 3-5 of these glycosylated repeats [70]. This protein

101

PsA-C=O I NH-CHz-CH2-0 I

o=P-0 I

OH OH

0

I I +/- Man(a 1-2)Man(a 1-2)Man(al-2)Man(a 1-2)GlcNH,( a 1-6)Ino- 1-PO,-CH,-CH-CH-CH I I I 0I NH (CH,),, I I I O=P-0 6

I

0 I CHZ

%:I

O=!

cH3

I

Fig. 6 . Structure of the glycophospholipid anchor on PsA in Dicfyosfelium discoideum. (Adapted from ref. [721.)

has a glycophospholipid anchor which is discussed more fully below. The other molecule that has received most of the attention is a cell adhesion protein called by various names including contact site-A (csA), gp80, or the EDTA-resistant cell adhesion molecule [7 11. This developmentally regulated protein is also glycophospholipid anchored and has a PTVT repeat sequence where the 0-linked chain is added [70]. Other studies on PsA suggest that the 0-linked modB oligosaccharides may be a heterogeneous mixture of several species. A glycopeptide shows the presence of equimolar (-1 1) amounts of GlcNAc and organic phosphate that roughly correspond to the amount of Thr (-15) in the analysis. In addition, smaller amounts of Fuc (-3), Gal and Man (-1 each) were found [72]. These bound sugars are all resistant to B-elimination and not part of the glycophospholipid anchor. The presence of phosphate on the GlcNAc may make the sugar resistant to p-elimination or its resistance may be due to the close packing of the carbohydrates chains. Riley et al. determined that a small glycopeptide generated by pronase digestion of slug stage proteins, contains GlcNAc and Fuc; however, it is released by mild base hydrolysis suggesting that it is not the modB glycan [66]. Further progress on understanding modB glycans requires structural analysis.

3.3. Glycophospholipid anchors The cell adhesion molecules gp80, gp130/138 and PsA (SP29) all contain glycophospholipid (GPI) anchors [71,72]. Another, called ponticulin, has both an anchor plus a series of transmembrane regions [73]. The anchor from the pre-spore protein PsA has been partially characterized and the structure is shown in Fig. 6 [72]. It has structural features of anchors found in yeast, protozoa and higher eukaryotes [74]. The lipid moiety in PsA is an inositolphosphoceramide with a C18:O phytosphingosine and a mixture of fatty acids, the most common being C18:l unsaturated fatty acid. The lipid can be removed by a bacterial phosphatidyl-inositol-specificphospholipase C (PI-PLC). Exoglycosidase

102

digestions and nitrous acid deamination showed that the GlcNH2 is bound to the inositol ring. The anchor containing proteins are probably segregated from those in the rest of the plasma membrane, and this in turn influences their turnover rate [75]. 3.4. Cytoplasmic glycosylation in Dictyostelium Cytoplasmic glycosylation is now well established in mammalian cells. The most common type is a single O-linked P-GlcNAc residue which is found on a large variety of proteins, especially those involved in gene regulation and cell growth [76]. This type of glycosylation has not yet been seen in Dictyostelium, but another more complex type was recently identified. The cytosol of vegetative and developing amoebae contains a single protein called FP21 (fucoprotein of 21 kDa) that accounts for most, if not all, of this type of glycosylation. It was first detected by labeling cells with 3H-fucose followed by subcellular fractionation [77]. The protein appears to have one small (-5 glucose equivalents) anionic O-linked oligosaccharide composed of one residue each of fucose, xylose and two residues of galactose [78]. FP2 1 has recently been cloned. The protein lacks a typical signal sequence and MADLI-TOF-MS analysis of the protein shows that it is about 1 kDa larger than the predicted mass of about 19 kDa based on cDNA sequence [78]. The sugar at the reducing terminus has not been identified, and the nature of the anionic group on the oligosaccharide is unknown. When the protein is isolated from normal cells, it is fully fucosylated since it cannot be an acceptor for in oitro fucosylation. However, in a fucosedeficient mutant strain (HL250) that cannot convert GDP-Man to GDP-Fuc, FP21 lacks fucose residues and serves as a substrate for fucose addition [77]. A novel fucosyl transferase appears to originate from the cytoplasm and not be derived from proteolyzed or damaged membrane vesicles. The K , for GDP-Fuc is -0.35yM, which is considerably lower than most glycosyl transferases found in the Golgi [78]. This lower K , probably allows it to effectively compete with the sugar nucleotide transporters that deliver GDP-fucose to the Golgi lumen. The cytosolic fucosyltransferase has an apparent mass of 95 kDa and can be photoaffinity labeled with GDP-hexanolaminyl-azido-['251]salicylate.Paranitrophenyl-lacto-N-bioside (pNP-LNB, K , = 0.6mM) is preferred over a wide variety of other acceptors and is converted into Fuc(al,2)Gal(P 1,3)GlcNAcb-pNP, although this oligosaccharide has not been detected in Dictyostelium. Fucosylation of FP2 1 is inhibited by Fuc(a 1,2)Gal(P1,3)GlcNAc~-pNP but not by recombinant FP21, suggesting that the substrate specificity is based primarily on carbohydrate recognition [C.M. West, unpublished]. This is the most complex form of cytoplasmic glycosylation yet reported. Since both the native acceptor protein and the fucosyl transferase are novel, studying this pair in Dictyostelium should provide functional insights and the necessary tools to probe for counterparts in higher organisms.

4. Antibodies against glycans and mutants in glycosylation Many of the sugar structures in Dictyostelium are quite unusual compared to those found in higher organisms. Not surprisingly, they are highly immunogenic. Several laboratories

103 Table 1 Examples of mAb against carbohydrate determinants Antibodies

N-linked CA 1

Determinant recognized

Mutant strains

Comments

Multiple Man-6-S04

HL240, HL241

Mostly in lysosomes

Ref(s)

[14,79,82,198] [47,48,80,83]

HL242, HL243 HL244

-

~ 3 1

-

Phosphatase digestion increases recognition by antibody

~ 4 1

Same

-

Same

Same

-

Same

CAB14

Same

CAB1 1

9

-

Same Destroyed by phosphatase digestion

Probably a series of related carbohydrate epitopes

[IOI]

d-4 1

-

DLI 18, DLI 19, HL220, HL216 Same

Same

[201,202]

16.1

-

Same

Same

54.1 32.1

-

Same Same

Same

[ 107,108]

-

Same

[ 105,1641

MUD50

-

Same

Same

40-62-5

GlcNAc/Maltose/aMan GlcNAc

Same Same

Same

?

-

>1 O6 binding siteslcell Same

w11

83.5

Fuc

HL250

Defective in GDP-Fucose production

[ 107,1081

MUD62 81.8

GlcNAc?

Same Bound to WGA

[ I 101 [ 107,1081

40.1

-

-

Bound to WGA, not Con A

[I 121

-

-

Found in trails

[ 105,1641

-

-

GlcNAc-6-SOd (?)

-

CA3

?

-

CAB4

XylIFuc?

CAB10 CAB13

CA2

mods E48D8

40- 178-3

[97,98,200]

Same

Adhesion blocking d-47 d-48

Others

MUD9 MUD52 MUD54

~

4

1

104

have generated antibodies and a diverse and elaborate nomenclature to identify them. A few cross-comparisons of specificity have been made as shown in Table 1. The best evidence for relatedness or identity of different antibodies is based on the loss of the antigens in one or several mutant strains. 4.1. Determinants found on N-linked oligosaccharides

The best characterized immunogenic carbohydrate epitope is called common antigen 1 (CAI) [11,18,50,79,80] which reacts with a cluster of Man-6-SO4 residues on N-linked oligosaccharides [14,81]. Two mutants (HL241 and HL243) that lack the determinant have incomplete, under-sulfated N-linked chains resulting from the synthesis of a truncated lipid-linked oligosaccharide precursor [ 141. The loss of specific Man residues leads to loss of the determinant. These mutant strains target their lysosomal enzymes normally, but more slowly [82]. The primary lesions in the two mutants appear to be different from each other, and the loss of CAI itself does not prevent development [79,198]. Another mutant strain that lacks CA1 (HL244) is about 90% deficient in sulfation and has been discussed. CA1 is not synthesized later in development, but CAI antigens are retained and are about equally distributed between pre-spore and pre-stalk regions of the slug [47]. Two other monoclonal antibodies define CA2 and CA3 on N-linked oligosaccharides, but these are less characterized than CAI [83]. Competition studies suggest that CA2 may recognize GlcNAc-6-SO4 residues. CA3 shows little preference for inhibition by different sugars. Another series of monoclonal antibodies recognize determinants found on N-linked oligosaccharides derived from slug stage plasma membranes on pre-spore or pre-stalk cells [24]. There was no consistent variation in the amount of the antigens during development. Alkaline phosphatase digestion of the antigens either increased or decreased binding of the antibodies, suggesting that phosphate was part of the determinant or partially covered it. Several of the antibodies bound to horseradish peroxidase which has fi-xylose and core a-fucose on N-linked chains [84,85]. The first post-translational mutation found in Dictyostelium discoideum was not identified by screening with antibodies. It was found as a partial deficiency in lysosomal a-mannosidase activity [86,87]. This recessive mutation defines the modA locus that determines the activity of the N-linked oligosaccharide processing a 1,3-glucosidase [88]. Because of this, these mutants fail to add the normal complement of phosphate and sulfate residues to the branch containing the unprocessed glucose residues [891. This results in under-secretion of several lysosomal enzymes and their prolonged residence time in the Golgi compartment [90]. This is different than in mammalian cells where the retention of Glc residues causes retention in the ER [91]. One explanation for this difference is that one of the modifications on this branch is important for efficient trafficking of the enzymes through the system. 4.2. Mutants in 0-linked oligosaccharides - modB

One of the most studied carbohydrate determinants is defined by mutations at the modB locus, which is recognized by a panel of monoclonal antibodies (Table 1).

I05

Mutants lacking the epitope have been used to examine its role in EDTA-resistant cell adhesion [92-951. The structure is not known, but based on the compositional analysis of the glycopeptide from PsA, it is likely to be a heterogeneous group of structures having GlcNAc, Fuc, Man and Gal residues [72]. Proteins that contain the determinant bind to WGA, probably through GlcNAc residues. The modB epitope is 0-linked to Thr residues in a restricted domain containing several repeats of the sequence Pr*Thr-Val-Thr near the carboxyl-terminus [96]. Competition experiments with various sugars show that some of the modB antibodies are competed by GlcNAc, maltose and a-methyl mannoside while others are inhibited only by GlcNAc [97]. This panel of antibodies probably recognize an overlapping group of determinants containing GlcNAc, Man and Glc [97,98]. The compositional analysis of PsA also suggests that the modB-type sugar chains could contain GlcNAc, Man, Fuc residues [72]. Mutant strains have been isolated that lack the modB determinant [96,98]. Although the loss of the determinant results in proteolysis of gp80[71,93,95] and its lowered accumulation at the membrane, several other proteins that normally contain the determinant still accumulate and localize to the plasma membrane [ 1001. The mutants proceed through development and make smaller fruiting bodies that have fewer spores than wild-type [ l o l l . Mutant slugs show reduced traction on their substratum [102], and their spore coat proteins appear to be less firmly integrated into the coat since they can be more easily extracted from the spores [103]. These strains also have several other, but more subtle abnormalities. For instance, when wild-type and mutant cells are mixed and allowed to form chimeric slugs, the mutant cells tend to sort out to the anterior portion of the slug [ 1041. Consequently, they preferentially become stalk cells. This sorting out may be physiologically significant since pre-stalk cells from the wild-type strain also show reduced intercellular adhesion and expression of the determinant compared to spore cells. Even though the role of modB determinants in cell adhesion is not direct, it may be involved in other aspects of cell-cell recognition within the aggregate. Monoclonal antibody MUD50 also recognizes a determinant that is absent in the modB mutants; extensive studies on the proteins recognized by this antibody indicate that they are also found preferentially, but not exclusively, in pre-spore cells [ 1051. 4.3. Antibodies against fucose -mAb 83.5 and MUD62

Antibody 83.5 and MUD62 recognize a group of developmentally regulated proteins that carry Fuc [ 106-1 101. The proteins are highly enriched in pre-spore cells and in proteins found in the PSVs [ 1081. A mutant strain, HL250, fails to react with this antibody [ 1 1 11. In keeping with the earlier nomenclature, this defect is defined by the modC locus. The primary lesion in this strain is a failure to synthesize sufficient GDP-Fuc from GDP-Man [l 1 11. The block in antigen synthesis is alleviated simply by adding exogenous Fuc to developing cells. Two other mutant strains at the modD and modE loci also have defects in adding Fuc residues. Preliminary evidence suggests that strains with modD mutation could be defective in GDP-Fuc transport into the Golgi and those with a modE defect are likely to be missing a specific fucosyl transferase activity, since only some fucosylated proteins are affected [ 1 111. In each case, the absence of the determinant has significant

106

consequences for survival. Spores isolated from this strain appear to have more porous coats and show decreased viability upon storage. 4.4. mAbs 81.8, 40.1 and MUD9 These antibodies react with components that bind to WGA and may recognize GlcNAc residues [107,108]. 81.8 is expressed in vegetative cells and is found on several lysosomal enzymes. The glycans recognized by antibody 40.1 are found in vegetative cells and do not bind to concanavalin A. On Western blots, the reactive material runs as a highly diffuse, pronase resistant smear with a PI of about 3.5. Labeling with sugar precursors shows that it contains GlcNH2 and fucose, is extracted into butanol-saturated water and its binding to phenyl-Sepharose is destroyed by nitrous acid deamination [Freeze, unpublished]. These results suggest that it may be glycolipid, perhaps similar to the protein-free glycophospholipid inositols made by protozoa such as Leishmania [73]. 40.1 and MUD9 binding material is absent from spores and low in the pre-spore region of the slugs. It is enriched in pre-stalk cells, but is not part of the stalk tube [112].

5. Glycoproteins in specijic aspects of the Dictyostelium life cycle 5.1. Mating types in Dictyostelium

Dictyostelium has different mating types that undergo fusion to produce diploid cells. Two of these mating types are HMI and NC-4. A 70 kDa membrane protein of HMl cells that binds to Con A was partially purified on immobilized membrane proteins of NC-4. When these cells become fusion-competent, a specific, 138 kDa protein, appears on the cell surface of both NC-4 and HMl strains [I 131. The protein binds to WGA, Con A, and LCA. Fab fragments of an antiserum against the protein(s) cause complete inhibition of sexual fusion between NC-4 and HMI cells. Two genes called gp138A and gp138B, have been cloned. The amino terminal regions of both are very similar and the C-terminal region is highly hydrophobic, proline-rich and has homology to an analogous portion of gp80 and PsA and is glycophospholipid anchored [114]. The mRNA of gp138A is expressed at the time cells acquire fusion competence and antisense mRNA inactivation of gp138 decreases sexual cell fusion. gp138B is also expressed during growth and may be one of the proteins used for adhesion in early development [71]. 5.2. Getting around

The vegetative amoebae produce an unusual transmembrane protein called ponticulin that binds F-actin and nucleates actin assembly [73,115-1 181. It is present during vegetative growth and during the first 8 h of development as cells stream into the fledgling aggregates. The deduced protein sequence contains no typical a-helical membrane spanning regions, but it has several hydrophobic membrane spanning regions. Also, immunological and actin binding studies show that portions of the protein are present on both sides of the membrane. In addition, it is one of the few known transmembrane

107

proteins that also has a C-terminal glycophospholipid anchor [73]. Deletion of the single ponticulin gene decreases actin nucleation by ten-fold, but the cells still grow and pinocytose normally. However during aggregation, cells lacking ponticulin form aggregates faster than normal and then proceed into an asynchronous morphogenesis, suggesting that ponticulin is involved in cell pattern formation.

5.3. Preparing for development Many genes are activated when development begins. cAMP is essential for activation of some of these, but others require a threshold cell density for their expression. The second type of regulation is probably used by cells to determine when they are actually part of an aggregate, rather than in route toward it. A series of protein factors are secreted by developing cells at very low rates. These factors can also accumulate in the medium of starving cells and are called conditioned medium factors (CMFs). One of these is an 80 kDa glycoprotein which is sequestered in vegetative cells and secreted during early development [I 19-1221. Smaller CMFs are also seen, and the specific activity of the 80kDa CMF increases roughly 100-fold upon proteolysis. The large CMF is reported to contain both N- and 0-linked glycans based on PNGaseF and 0-glycanase digestions. This is the only report of an 0-glycanase sensitive glycan in Dictyostelium [ 1211. Because of the strict specificity of this enzyme, it may mean that Dictyostelium makes typical Gal(P1-3)GalNAc-0 glycans. This needs to be confirmed by more specific analytical methods. The gene coding for the 80kDa CMF has been cloned and found to have three potential N-glycosylation sites within its 57 1 amino acid length. However, complete activity is retained when the complete protein or an 88 amino acid fragment is expressed in E. coli, showing that glycosylation is not required for activity but it may influence stability or proteolytic processing of the 80 kDa CMF. 5.4. Glycoconjugates in cell adhesion during development The developmental stages use at least three distinct adhesion systems. The first appears within 2-3 h following the initiation of development. It is blocked by EDTA and is mediated, in part, by a set of closely related, non-glycosylated proteins which are collectively called gp24. A glycoprotein gp130/138 also appears to be involved in this type of adhesion. This is the same gp138B mentioned above. The second system begins to appear at about 6 h. It is resistant to EDTA and is mediated by the homophilic association of a glycoprotein called gp80, contact site A (csA), or sometimes antigen 117. It contains both N- and 0-linked sugar chains and a glycophospholipid anchor. This molecule is turned over and replaced by another adhesion system that appears beyond 12 h of development during slug migration. This is mediated by a glycoprotein called gp 150 which can be genetically and immunologically distinguished from the others. Other glycoproteins have also been implicated in mediating adhesion at this stage of development. 5.4.1. Generalfeatures in aggregation Tunicamycin inhibits chemotactic aggregation when added during the first few hours of development [123-1251. Since these effects can be overcome by adding cAMP [123] it

108

suggests that the loss of N-linked chains is not from a cell adhesion molecule itself, but they may be needed for the CAMP chemotactic signaling system. Other unidentified carbohydrates may be important in aggregation. Bozzaro used monosaccharides linked to polyacrylamide gels to examine cell binding, movement and aggregation [ 126,1271. Gels derivatized with glucose, maltose or cellobiose firmly bind the cells, but those with GlcNAc or Man bind cells to a lesser extent. Binding to GlcNAcand Man-containing gels could not be competed by these sugars, but mild trypsin digestion of the cells decreased binding; the opposite was found for binding to Glc gels. As cells proceed beyond aggregation binding to GlcNAc is lost. The interpretation of these results is that there are three receptors, one for each sugar. Further studies showed that when Glc, maltose or cellobiose, but not Man or GlcNAc is linked to the gels, development is arrested at the aggregation stage in a curious way. The cells initially form streams and aggregates, but just prior to the transformation into tight aggregates, they rapidly disperse into single cells, only to reform into aggregates once again. This futile cycle could continue for up to 30 rounds over a 24 h period [127]. Already formed tight aggregates also disperse when transferred to Glc-derivatized gels, but tipped (sheath enclosed) or slug-stage aggregates do not disperse. The mechanism underlying this unusual behavior is not known, but it clearly suggests that the cell surface must interact with a form of Glc and that this is able to affect further development at a specific point. One suggested explanation for the results, is that glucose is being recognized as a signal that coaxes the aggregating cells again into a feeding mode. Since the Glc is metabolically unavailable, they soon return to the starvation-induced developmental program. Two types of mutant cell lines that do not bind to the sugar-derivatized acrylamide gels have been isolated [128]. One type fails to bind to both Glc- and GlcNAc-derivatized gels, and the other fails to bind only to GlcNAc-derivatized gels. Three out of four of those in the first group aggregated only to the mound stage and the other formed aberrant fruiting bodies. Those in the second group mostly (10/13) formed minute fruiting bodies, tipless mounds, or did not aggregate at all. In addition, those that lack binding to both Glc and GlcNAc or severe deficiencies to GlcNAc alone showed reduced EDTAsensitive adhesion and a lower rate of bacterial phagocytosis. Although the structures of these glycans are not known, the results suggest that glycans with these sugars are physiologically important. One report suggests that secreted polysaccharides may play roles in cell adhesion [ 1291, and a variety of animal and plant polyanionic polysaccharides can partially inhibit aggregation at mg/ml concentration [ 1301. Since none of these molecules has been identified in Dictyostelium, the effects are difficult to interpret in molecular terms.

5.4.2. The EDTA-sensitive adhesion molecules A group of proteins collectively called gp24 is responsible for a portion of the EDTAsensitive adhesion (contact site B) and allows further development [ 131-1341. gp24 is not found in vegetative cells, but appears on the cell surface at the onset of aggregation and remains throughout development [ 1321. Cell adhesion is accomplished by a calcium-dependent homophilic interaction of gp24 molecules [ 1341. Four related and adjacent genes code for the different proteins whose molecular weights are approximately 12kDa [134] [W.F. Loomis, personal communication]. Thus, the name gp24 is

I09

somewhat inaccurate. The deduced amino acid sequences show no signal sequence or N-linked glycosylation sites. It is not anchored by glycophospholipid, so the mechanism by which the protein associates with the cell surface is not clear. Much of the protein is found in the cytoplasm and is probably secreted by a mechanism distinct from the endoplasmic reticulum-Golgi route. No mutant strains have been found that lack gp24. This is not surprising since all four genes are active, and the chances of inactivating all of them would be negligible. Production of antisense mRNA for gp24 under the control of a promoter that is active in vegetative cells until about 6 h of development delays cell adhesion and the appearance of gp24 for two hours [ 1351. Thereafter, transcription of the antisense mRNA ceases and the accumulation of gp24 and normal adhesion resume. Deletion of all the genes will be required to finally determine the functional significance of gp24, and other components in the earliest adhesion system. Another glycoprotein in vegetative cells, gp 130, has been implicated in EDTA-sensitive adhesion. It has the same sequence as gp138B, appears to be N- and 0-glycosylated and glycophospholipid anchored in the membrane [7 I]. Its carbohydrates closely resemble those of csA described below, but have been much less thoroughly studied than csA. Besides the gp24 and gp1301138 group, another protein may also be involved in the EDTA-sensitive adhesion system. Based on the historical nomenclature, this molecule is called contact site C. It differs from contact site B, since it has slightly different developmental regulation compared to gp24 [ 1361, because the inhibition is not seen using EGTA, and EDTA inhibition is Mg2+-reversible.The protein has not yet been identified.

5.4.3. The EDTA-resistant adhesion molecule Contact site A (csA) refers to EDTA-insensitive adhesion which is mediated by a glycoprotein called gp80 [137]. It appears at about 6 h of development, persists until 12 h, disappears, and is found once again on the surface of a subset of cells late in development [138]. gp80 is concentrated in filopodia in regions of cell-cell contact [139]. Increased level of gp80 expression appears to regulate the slug size, since individual cells are more adhesive [140]. The protein has 495 amino acids after removal of the 19 amino acid leader sequence and contains N-linked and 0-linked chains together with a glycophospholipid anchor. The N-linked chain is sulfated and fucosylated while the 0-linked carbohydrate has the modB antigenic determinant, and binds to WGA [71,92,93,96,141,142]. These chains are required for the transport and expression at the cell surface. When they are absent, the cell surface molecules are clipped by a surface protease with the release of large fragments into the medium [71,92]. The 0-linked chains are located near the glycophospholipid anchor and mutation of the protease results in normal accumulation of the protein at the surface and leads to normal cell adhesion [71]. The peptide, not the carbohydrate portion, of gp80 mediates adhesion. An eight amino acid peptide inhibits both cell-gp80 and gp8Sgp80 interaction, and antibodies against the peptide inhibit reassociation of previously dissociated cells [ 143,1441. Site directed mutagenesis to remove all of the N-linked chains does not alter adhesion [71]. Studies by Henderson and associates present another view of adhesion. Their results show that stage-specific EndoH-resistant membrane-derived pronase glycopeptides can partially block EDTA-resistant adhesion [ 1451. The glycopeptides could be derived from

110

another protein involved in some aspect of the EDTA-resistant adhesion. In another study by the same group, two temperature-sensitive mutant strains have reduced adhesion at the restrictive temperature of 27"C, but a revertant strain aggregates normally [ 1461. Cell membrane glycopeptides prepared from wild-type cells -and mutants grown at the permissive temperature partially block adhesion, but those isolated from mutant cells grown at the restrictive temperature do not [ 1471. The structure of the inhibitory glycopeptides has not been established. gp80 is glycophospholipid anchored [96,148,149]. PI-PLC digestion does not release the lipid from this protein, and strong base hydrolysis is needed to release free palmitic acid, suggesting that the lipid is in an amide linkage [149]. Ceramide glycans with mannose and glucosamine were described in Dictyostelium discoideum over 20 years ago [150], before such anchors were discovered. Structures similar to those of gp80 have also been reported in yeast, mycobacteria and plants [ 1491. Substituting the anchor domain of gp80 with a transmembrane domain does not alter its ability to mediate adhesion; however, the normally long-lived gp80 turns over much more rapidly when it lacks an anchor. Normally, the gp80 anchor probably excludes the protein from clathrinmediated pinocytosis, and when the anchor is removed it mingles with the other cell surface proteins and is degraded at a much higher rate [75]. Several studies by Klein and co-workers [138,151-1531 also showed that gp80 (called antigen 1 17 in their reports) is insensitive to PI-PLC digestion, but about 25% of non-lipid bearing protein containing ethanolamine can be released from the cell surface by an endogenous membrane-associated non-proteolytic activity [ 1531. When the protein is released, it exposes epitopes that are recognized by antibodies against the GPI anchor of the variant surface glycoprotein of T brucei. These antibodies will recognize the cryptic determinant only after it is cleaved from the lipid portion by PI-PLC, and suggest that the activity which releases antigen 117 from the surface may also be a phospholipase-type enzyme. These data argue against this release being merely a proteolytic clip as seen in the strains that carry the modB mutation. The fact that only a portion of the antigen 117 is released from the membranes could mean that there are distinct classes of the protein, some being cleavable and others not. The difference may be a matter of availability of the enzymes or may involve differently anchored forms of the protein. Deletion of the gp80 gene [71,199] leads to loss of EDTA-resistant adhesion, but does not abolish development. The other two (or more) adhesion systems can apparently mediate adhesion well enough under laboratory conditions in the absence of shear forces to produce mature fruiting bodies. 5.4.4. Post-aggregation adhesion system

After gp80 has disappeared, a third adhesion system is found at the slug-stage and beyond. Disaggregated slug-stage cells still adhere to each other in the presence of EDTA even when gp80 is not expressed and gp24 is blocked by antibody [ 1321. Antisera prepared against cells developed to this stage do not block the adhesion of cells prepared at earlier times in development, but it will block the third adhesion system [154]. A glycoprotein called gp150, appears to mediate the late adhesion system because antibodies against it can block cell reassociation of post-aggregation cells [ 155,1561. It has also been proposed that this molecule may be important in the differential adhesion of pre-stalk and pre-spore

111

cells to themselves, since anti-gp 150 can inhibit preferential self-association when a disaggregated mixture of cell types is allowed to reassociate. Purified gp 150 neutralized the effects of a rabbit antiserum raised against gel-purified gp 150; '*'I-labeled gp 150 shows saturable binding to intact post-aggregation cells suggesting specific cell surface binding sites [ 1551. Soluble gpl50 blocks the reassociation of dissociated cell aggregates, but it shows no effect on cells at the early aggregation stage. Although gp150 is an important post-aggregation adhesion molecule, other results show that a monoclonal antibody which blocks adhesion recognizes carbohydrates on other glycoproteins of 95, 90, 35 and 30kDa (PsA) [157]. This suggests that the carbohydrate may be involved in this adhesion. In summary, the results of two decades of cell adhesion studies still suggest that unidentified carbohydrates play a part in cell adhesion. The extensive studies on csA prove that its partially characterized glycans are not among these players. The elusive connection cannot be resolved until more decisive structural analysis is done on Dictyostelium glycans. 5.4.5. The surface sheath The surface sheath is required for slug migration and complete morphogenesis. Differential gene expression of stalk and spore cell components can occur in disaggregated cells under the proper environmental conditions, but it is the sheath that allows Dictyostelium discoideum to function as a coordinated organism. It must possess considerable resilience and strength to accommodate all of the complex movements a slug makes over various terrains. Analysis of the surface sheath has been challenging for several reasons. Firstly, it is extremely thin and does not provide much material for biochemical analysis. Secondly, although the sheath can be harvested as trails left behind the migrating slugs, the preparation can be contaminated by sluffed cells or the debris of lysed cells. These points have made it difficult to confidently identify bona fide sheath components. The sheath is stationary relative to the substratum and the cells move over and through it leaving the sheath behind as a collapsed tube. Thus, each region of the slug may make its individual contribution to the sheath as the cells pass by, and this probably explains why the sheath appears to be thicker and have additional components at the posterior of the slug compared to the tip.

5.4.5.1. Cellulose. The surface sheath surrounds the developing aggregate just prior to tip formation and continues to envelope it until culmination. A major structural component of the sheath is cellulose [ 1581, and like the sheath itself, it accumulates only at the surface and not in the area between the cells. Cellulose is much more dense on the ventral side of the slug and it may be deposited on the substratum when the tip of the migrating slug touches down [ 1591. The cellulose appears to mature during development as shown by the increase in crystallinity from sheath isolated from early aggregates compared to the trails left behind migrating slugs [ 1601. This change is probably physiologically important since high crystallinity is correlated with increased rigidity and tensile strength of the cellulose fibrils. How this maturation occurs is not known, but it would appear to be regulated, since cellulose is less crystalline in several mutant strains that lack the single lysosomal enzyme, b-N-acetylhexosaminidase [ 1611. Initially aggregates of the mutant strains form

112

normal-sized slugs, but they are unstable and quickly break up into smaller, stable slugs with lower surface to volume ratios. The low crystalline cellulose apparently does not provide enough strength to withstand the forces generated by a larger migrating slug. Since b-hexosaminidase can be secreted during development, it may play some role in controlling the crystallinity of the cellulose. Particulate membrane fractions prepared from the tight aggregate stage (10 h) onward are active in cellulose synthesis [162]. The product made from UDP[’4C]glucose was shown to be cellulose by its solubility properties, periodate oxidation and methylation analysis pattern. Cellulose can also be made in monolayer cultures by cells induced with CAMP and DIF, a potent inducer of stalk cell properties specific genes [24,163]. 5.4.5.2. Glycoantigens and glycoproteins. Immunoelectron microscopy shows that several monoclonal antibodies against carbohydrates react with components of the surface sheath. These include antibodies 8 1.1, 83.5, 40.1 , MUDSO, MUD62 [ 107-1 09,164-1 661. Immunofluorescence studies show that there is a regular pattern of hexagonal “cell-prints’’ outlining the borders of the ventral cells in contact with the substratum [ 102,159,1671, Coincident patterns can be seen using antibodies MUD50 and Calcofluor, a fluorescent tag for cellulose [ 1591. Cell prints are not seen using antibodies MUD1, MUD3, MUD62 or those against SP29 (PsA), SP70, or SP96, so the pattern is not due to cell lysis. Slugs from mutant cells lacking MUD50 epitope appear to “slip” in their forward migration, suggesting that the components bearing the MUD50 determinant are involved in traction and slug locomotion [ 105,159,1661. Two highly acidic pre-stalk-specific proteins ST3 10 and ST430 [ 1681 appear to be totally extracellular. ST430 occurs in the sheath and ST310 is found in the stalk tube and between the stalk cells [ 1691. Since they are extracellular matrix proteins, their genes are called ecmA (ST430) and ecmB (ST310)[170,171]. The ecmA gene has a series of 23 cysteine-rich repeats with a total of 4-6 N-glycosylation sites [ 1701. ecmB has at least 41 repeats of a similar cysteine-rich 24 amino acid segment with a total of 26 N-glycosylation sites! [ 1711 High cysteine content in re-iterated segments are characteristic of other extracellular proteins in lower eukaryotes [ 172,1731. Homologous disruption of the ecmB gene does not alter development, but disruption of the ecmA gene leads to formation of abnormally long and thin standing slugs [174]. Apparently, sheath that lacks ecmA is weak and slugs lose a large portion of the cells as they migrate. If development is arranged to omit the migrating slug-stage, loss of ecmA gives normal fruiting bodies. 5.4.6. Pre-spore vesicles and the spore coat

Ultimately, Dictyostelium discoideum undergoes development to ensure that a portion of the starving population survives. To accomplish this, the stalk cells provide a tower from which the spores can be dispersed over a wide area, but the long-term survival of the spores, and the species, is dependent upon their success in forming an environmentally resistant casing called the spore coat. Preparations begin early in the slug-stage when most of the coat components are beginning to accumulate in PSVs. At the appropriate time, the contents are jettisoned from the cell to generate a pool of precursors for the assembly of the spore coat. The regulation and synthesis of several spore coat glycoproteins and

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cellulose have received most of the attention. Several of the proteins have been cloned and their regulation investigated [3]. 5.4.6.1. Pre-spore vesicles. Pre-spore vesicles (PSVs) contain all of the known major

components that will later form the spore coat, except for cellulose (Table 2) [106]. PSVs are found only in pre-spore cells [175] and appear to be derived from the Golgi, which is quite prominent in those cells; however, a typical Golgi is essentially undetectable in vegetative and pre-stalk cells using similar techniques [176]. It can be detected in vegetative cells by fluorescence microscopy using tagged WGA lectin [177] and a light membrane fraction has been biochemically characterized as the site of several glycoprotein processing events [39,4 1,5 1,521. PSVs have a characteristic density and can be separated from other organelles including lysosomes, and yet surprisingly, these PSVs also contain several typical lysosomal enzymes [ 178-1 801 (Table 2). It has been suggested that a portion of the lysosomes may transform into PSVs or, alternatively, that lysosomal enzymes are also routed to the PSV because they function within it or later in the assembly of the spore coat. The question is not yet resolved since recent work suggests that the PSVs exist as a tubular network which is continuous with the Golgi [106]. The spore coat proteins SP60, SP70 and SP96 have been cloned and their expression is coordinately regulated [ 181-1 831. They all have typical signal peptides and SP70 and SP96 have potential N-glycosylation sites. SP75 is also modified by Table 2 Components of pre-spore vesicles and spore coatsa Component

PSV

Location in spore coat Inner Middle Outer

mAb

Reference(s)

83.5/MUD62, MUD3

[109,191]

16.1/MUD102, MUDS0

[ 1091

7

83.5/MUD62

[lo91

83.5/MUD62

Soluble matrix

Proteins SP96/p 1 12

+

SP8S/PsB

+

+

SP80 SP75

-

-

SP70

?

?

+ +

?

[ 194,1951

SP60

?

?

+

7

[ 195,1961

Enzymes a-Mannosidase

+

+

Phosphatase

+

+

Polysaccharides Cellulose GaliGalNAc a

Compiled from West and Erdos [ 1061

[I091

114

Fig. 7. Probes for pre-spore vesicle components show a non-random distribution. Pre-spore vesicles have the galactose-containing polysaccharide that binds to lectin RCA-1 (large particles) and the spore coat protein SPWPsB binds to a rnodB-dependent carbohydrate (small particles). The probes clearly show their accumulation in separate regions of the vesicle. x 71 500. Another antibody that recognizes fucose (mAb 83.5) also displays particles in distinct regions of the PSV (From ref. [log], with permission.)

N-linked oligosaccharides since its molecular weight is altered in a strain carrying the modA mutation [ 1061. SP96, SP70 and SP75 are also phosphorylated [ 1841 on serine residues, [ 1851 and fucose containing oligosaccharides are released from SP96 and SP75 by ($elimination [106]. The released glycan runs as a hexasaccharide by gel filtration and probably contains the epitope recognized by antibodies 83.5 and MUD62. SP80 and SP85/PsB contain the carbohydrate epitopes in the modB series [107]. There is no direct evidence that SP60 is glycosylated and no N-linked sites occur, but the calculated molecular weight of the unmodified protein without the signal peptide is only about 47 kDa suggesting post-translational modifications may exist [ 1821. How the components are targeted to or retained by the PSV is not known, but a preformed complex of six proteins is found within the PSV [ 1861. The components are associated by both disulfide and non-covalent forces and include SP85/PsB and SP96/pll2. The composition of the complex changes during secretion and spore coat maturation [ 1871. The PSVs also contain polysaccharide composed of galactose, galactosamine and galacturonic acid that can be recognized by pre-spore-specific antisera [ 1881. Lectins RCA-1 and SBA that preferentially recognize Gal and GalNAc residues also bind to this polysaccharide [106]. The distribution of the various components in the PSV is not homogeneous, instead they tend to be clustered in separate regions of the vesicle [109]. This is shown in Fig. 7. The associations are non-random, but the spore coat is not a preassembled complex since the association of the components within the PSV is different than that in the mature spore coat [106,109].

I15

Since the PSV may be a tubular network rather than a series of distinct vesicles, secretion of the components may not require extensive vesicle fusion with the plasma membrane. Secretion parallels loss of UDP-Gal polysaccharide galactosyl transferase that is localized in the Golgi [ 189,1901 suggesting that multiple secretory pathways may be coordinated to form the spore coat.

5.4.6.2. Spore coat. Table 2 [lo91 lists the known spore coat components from either post-germination spore coats or by immunolocalization of spore preparations prior to germination. The various glycoproteins are recognized by several of the carbohydrateor peptide-specific antibodies or by lectins RCA-I or SBA to identify the galactosecontaining polysaccharide. Cellulose can be detected by the binding of colloidal goldderivatized cellulolytic enzymes. The spore coat has three distinct layers and the spores in the sori are bathed in a soluble “matrix”. The outer layer contains SP75, SP80, and SP96, as detected by antibody 83.5 and shows that they are neatly arranged along either side of the electron dense zone. The inner layer is close to the plasma membrane and contains SP85(PsB), a-mannosidase and the galactose polysaccharide. Cellulose is found only in the middle layer. The soluble matrix is lost during preparation of spore coats, but prior cetylpyridinium chloride treatment preserves it. Analysis of various washes of the sori or of secretions produced by submerged developing aggregates also suggests that the lysosomal enzymes and another group of proteins recognized by antibodies 8 1.8 and 85.2. are also found in the matrix. Although single spores can encapsulate individually, in the natural environment of the maturing sorus the assembly is not cell autonomous. The proteins appear to be part of a pool of precursors for spore coat assembly. In favor of this view are the results of West and Erdos [lo51 who show that mixed development of wild-type and mutant HL250 cells that do not produce antigen 83.5 results in the equal distribution of this determinant among all of the spores. The assembly of the coat seems to begin in the outer layer, but the mechanism is unknown. It is likely that the associations of some of the proteins in the outer layer are autonomous, and there is no evidence that membrane receptors guide the assembly of the coat.

6. Molecular glycobiology in Dictyostelium discoideum Since deleting entire classes of glycans is not lethal to single cells, most of the specific functions of sugar chains are probably occurring at the multicellular level [191]. Previously, our understanding of glycan biosynthesis and function in multicellular organisms relied on the chance discovery of rare mutations showing clear and dramatic phenotypes. The arrival of molecular approaches has provided much more systematic and rational methods. The most popular of these is making gene knockouts in transgenic mice, but the results of total deletions can be quite unpredictable. Sometimes the phenotypes are so subtle that they are undetectable during embryonic development or adult life, while deletion of others arrests early embryogenesis [191]. Useful information is derived between these extremes. It is difficult to predict what the outcome of any one gene deletion will be, especially when deleting glycosyl transferases that service many organ systems,

116

tissues and cell types. Even knowing the specific glycosyl transferase defect does not lead to accurate prediction of the phenotype or the severity of the defects [ 191,1921. Recent advances in targeted gene disruption and insertional mutagenesis in Dictyostelium offer an opportunity to study molecular aspects of glycobiology at the multicellular organismic level [7,9]. Restriction enzyme mediated integration (REMI) is a method to make random gene disruptions in Dictyostelium [8,9,193]. A linearized plasmid with a selectable marker is introduced into a recipient strain and random insertion of the marker disrupts an endogenous gene. Re-isolation of the marker together with flanking regions of the disrupted gene can be used for its isolation. To date most of the transformants have been screened for developmental abnormalities with subsequent characterization of the disrupted gene [7,9]. However, this same technique can be adapted to searching for disrupted glycosyl transferase genes by screening with monoclonal antibodies against the glycans. Identification of the mutated gene could then be correlated with an altered structure of the glycan and the loss of enzyme activity. Once isolated, these genes could be used to complement (correct) the mutant strains or to produce antisense oligonucleotides driven by some of the previously characterized temporal- or cell-type-specific promoters [3,4,7]. Using these promoters, one would be able to fine tune the abrogation of transferases rather than insisting only on a total knockout in all cells. The effects of each complete or selective gene knockout could then be monitored for its consequences during growth, aggregation, morphological development, slug migration, tissue proportioning, spore coat structure, and spore viability. Figure 8 shows a scheme using these methods to study glycan function in Dictyostelium.

I

mutant

I \

I

I overexDression I /I

mutant

Or

I

T I ' Clone gene \

Cell- or temporal-

spccific

Fig. 8. Systematic glycobiology in Dictyostelium. This figure shows how various features and technologies in Dictyostelium can be exploited to investigate glycobiology. Antibodies ( I ) against glycans can be used to screen for biosynthetic mutants created by insertional mutagenesis (REMI) (2). The disrupted genes can be identified using the REMI technology (3) and then be ablated in selected cell types or at different times in development by using antisense mRNA controlled by cell-type- or temporal-specific promoters (4). The glycans can be purified from gram quantities of vegetative or developing cells and used as affinity ligands to isolate putative receptors ( 5 ) or in some cases they could be added exogenously to disrupt normal development (6). In some cases the molecular deletions should alter development in ways that are already predicted by analysis of chemically mutagenized cells identified using these antibodies.

117

In this system, we already know that alterations in sugar chains affect cell growth, adhesion, slug migration, and a variety of developmental aspects. Systematic analysis of these glycoconjugates will ultimately yield useful information and strong encouragement to clone Dictyostelium glycosyl transferase genes.

Acknowledgments Supported by US Public Health Service grants GM32485 and GM94096. The author is indebted to members of his laboratory and to Dr. Chris West and Dr. Keith Williams for sharing their unpublished data and to Dr. Glaucia Souza and Marion Lammertz for critical comments on the manuscript.

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J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins I1 0 1997 Elsevier Science B.V. All rights reserved CHAPTER 6

Hemocyanins Johannis P. Kamerling and Johannes F.G. Vliegenthart Bijvoet Center. Department of Bio-Organic Chemistry3 Utrecht University. Netherlands

Abbreviations Ara

arabinose

Arg CROSREL

arginine

LYs Man

CROSS RELaxation

MD

molecular dynamics

Fuc

fucose

Me-T

methy ltransferase

a2Fuc-T

a- 1,2-fucosyltransferase a- 1,3-fucosyltransferase

3MeMan

3-0-methylmannose

a3Fuc-T

3MeGal

3-0-methylgalactose

Gal

galactose

4MeGal

4-0-methylgalactose

fi3Gal-T

fi- 1,3-galactosyltransferae

NMR

nuclear magnetic resonance

GalNAc

N-acetylgalactosamine

PNGase-F

PGalNAc-T

p- 1,4-N -acetylgalactosaminyl-

peptide-N4-(N-acetylp-glucosaminy1)asparagine amidase F

transferase

lysine mannose

GDP

guanosine diphosphate

Pro

proline

Glc

glucose

ROESY

GlcNAc

N-acetylglucosamine

rotating-frame nuclear Overhauser enhancement spectroscopy

p2GlcNAc-T

p-I ,2-N-acetylglucosaminyl-

UDP

uridine diphosphate

transferase

Xaa

unidentified amino acid

Hc

hemocyanin

HSEA

hard sphere exoanomeric

XYl 02Xyl-T

0-1,2-xylosyltransferase

xylose

1. Introduction In nature, three types of oxygen-transporting proteins can be distinguished on the basis of the active site, namely, hemoglobins, hemerythrins, and hemocyanins (Hcs) [ 11. The latter type is found in the majority of the arthropod and mollusc species. In contrast to hemoglobin and hemerythrin, wherein Fe(I1) is essential for oxygen binding, Hc has a binuclear Cu(1) active site. The binding of oxygen in Hc is dependent on the pH and is accompanied by the formal valence change from Cu(1) to Cu(II), thereby giving the oxygenated Hc its characteristic blue color. Hemocyanin does not occur in blood cells, but is found freely dissolved in the hemolymph. It forms the major protein constituent (9&98%) of this fluid[2] in concentrations up to 120mg/ml [3,4], dependent on species, age and season. The high 123

124

molecular mass of the protein (molecular mass 4.5 x lo2-9x lo3 kDa) is of importance for the generation of a low osmotic pressure in the hemolymph. The protein structures of Hcs have been studied along different routes, including primary structure analysis (e.g. ref. [5]) and three-dimensional analysis using electron microscopy (e.g. refs. [6,7]) and X-ray crystallography (e.g. refs. [8,9]). In terms of quaternary structure, native Hc is built up from a number of structurally related subunits, and the oxygen binding is regulated by the allosteric behavior of Hc, based on complex interactions between subunits [4]. From the various studies, it can be concluded that the protein structures of arthropod and mollusc Hc differ specifically from each other, and an impression of the different Hc sizes is presented in Table 1 [ 101. Arthropod Hc consists of subunits with a molecular mass of about 75 kDa [ 1 11; the subunits have kidney-shaped structures and are built up from three domains[5,8]. Each subunit forms a functional unit, defined as a part of the Hc molecule that contains two Cu atoms and is capable of binding one oxygen molecule, and the subunits are arranged as hexamers (1 x 6) or multihexamers (2x6, 4x6, 6 x 6 or 8~6)[2,12,13].On the other hand, mollusc Hc is built up from subunits with a molecular mass of about 400 kDa [13], arranged as large cylindrical molecules (10 to 20 subunits). In general, each subunit is composed of eight functional units. Under specific conditions the cylinders (with collars) can dissociate into defined smaller molecules. In view of the pronounced differences between the structures of the Hc from both phyla, there is an ongoing discussion with respect to their origin, i.e., have they evolved independently or are they derived from one common ancestor (see e.g. refs. [4,14]). Most Hcs have been shown to be glycoproteins, but so far solid information with respect to the significance of the partly unusual glycosylation is not available. It has been found that there exist remarkable differences in carbohydrate content and monosaccharide composition between Hc from arthropods and Hc from molluscs [lo]. Detailed information, mainly based on monosaccharide analysis, methylation analysis, and 500 or 600 MHz 'H NMR spectroscopy, has become available on the primary structure of the carbohydrate chains of the Hcs of the scorpion Androctonus australis[151, the freshwater crayfish Astacus leptodactylus [ 161, the spiny lobster Panulirus interruptus[17,181, the terrestrial snail Helix pomatia [ 19-21], and the freshwater snail Lymnaea stagnalis[ 19,22,23]. Here, a survey is given of our present-day knowledge concerning the carbohydrate composition of Hcs.

2. Carbohydrate parameters of arthropod hemocyanins In Table 2 a survey is presented of the carbohydrate content and the monosaccharide composition of Hcs from typical species belonging to the four arthropod classes Merostomata, Arachnida, Crustacea and Chilopoda [lo]. As can be deduced from this table, the carbohydrate content of arthropod Hc is usually low. For those species containing > 0.1% carbohydrate, only D-Man and D-GIcNAc are observed, suggesting the occurrence of asparagine-linked glycans, dominated by the oligomannose type [24]. In view of our finding that the carbohydrate content of Eurypelma californicum is < 0.1 %, the reported data for the Hcs from the Araneae E. californicum, Eurypelma helluo

I25

Table 1 Survey of different types of hemocyanin distributed over the various species [ 101 Species

Number of subunits

Schematic structure

Molecular mass (kDa)

n"

4.5~10

Arthropod hemocyanin

Lobster

Crab

l

L

Spider

Scorpion

0

12 (2x6)

G

i

no

24 (4x6)

Centipede

36 (6x6)

Horseshoe crab

48 (8x6)

UU

2

9x10'

3

13x10

Mollusc hemocyanin Octopus, cuttle fish, squid; chiton

10 (2x5) (80 functional units)

% Snail (slug)

w e

20 (2x10) (160 functional units)

a Square represents 6 subunits (functional units):

Section represents 2 subunits (2x8 functional units):

00 =

9x1O3

126 Table 2 Carbohydrate content (“h,w/w) and monosaccharide composition (nmol sugar/mg protein) of Hcs from various arthropod species [ 101

Class

Order

Species

Monosaccharide Man

GlcNAc

%

(w/w)

Merostomata

Xiphosura

Limulus polyphemus (horseshoe crab)

Arachnida

Scorpiones

Androctonus australis (scorpion)

Aranea

Eurypelma calijornicum (tarantula)

Decapoda

Panulirus interruptus (subunit type a) (spiny lobster)

29.6

11.3

0.8

Panulirus interruptus (subunit type b)

39.7

16.1

1 .o

Panulirus interruptus (subunit type c)

35.9

15.2

1 .o

Astacus leptodactylus ( 1 x 6-mer) (freshwater crayfish)

31.9

22.0

1.1

Astacus leptodactylus (2 x 6-mer)

6.0

1.9

0.2

Scutigera coleoptrata (centipede)

182.0

78.0

4.9

Crustacea

Chilopoda

Scutigeromorpha

20 ((DP), = 6) H.46 polyclonal antibody specific to poly(Neu5Ac) 3-deoxy-o-glycero-o-galacto-nononic acid Kdn Kdn-rich glycoprotein isolated from vitelline envelope andor female ovarian fluid of Kdn-gp rainbow trout low molecular mass PSGP isolated from fertilized eggs L-PSGP a precursor of PSGP (i.e. PSGP of low sialic acid content formed at early stages of IowSia-PSGP oogenesis, each glycan chain contains mostly a disialyl group) NeuSAc N-acetylneuraminic acid Neu5Gc N-glycolylneuraminic acid NeuSAcyl N-acylneuraminic acid oligo/poly-Sia a-2,l-linked oligomers or polymers of sialic acid poly-Sia a-2,l-linked polysialic acid poly(Neu5Ac) a-2,l-linked polymers of NeuSAc poly(Neu5Gc) a-2,l-linked polymers of Neu5Gc poly(NeuSAc,NeuSGc) a-2,8-linked random copolymers of Neu5Ac and NeuSGc peptide:N-glycanase or peptide:N4-[N-acetyl-~-o-glucosarninyl] asparagine amidase PNGase (EC 3.5.1.52) polysialoglycoproteins or glycoproteins having polysialyl groups PSGP specific proteinase which is responsible for depolymerization of glycopolyprotein PSGPase (H-PSGP) into glycopeptide (L-PSGP) Sia sialic acid(s) mucin-type sialoglycoproteins isolated from vitelline envelope of cherry salmon Sia-gp containing Neu5Gc instead of Kdn ST sialyltransferase activity CMP-Sia:a-N-acetylgalactosaminide [R-Gal(@1-3)GalNAc(a 1-0-SerlThr] a-2,6-ST a-2,6-sialyltransferase a-2,l-ST CMP-Sia:a-2,6-sialoside Sia(a2-6)[R-Gal(@I-3)]GalNAc(a I-0-SerlThr] a-2,l-sialyltransferase a-2,l-polyST CMP-Sia:a-2,8(6)-sialoside a-2,l-sialyltransferase

1. Introduction The topics treated in this chapter do not cover all the glycoproteins and related substances, including enzymes, so far isolated from fish but are restricted to new findings obtained 143

144

through our 20 year studies on teleost eggs. We have been studying fish egg glycoproteins not to find something special to fish but in the belief that what we find in fish may also occur in higher vertebrates including humans. Fish eggs are obtainable with relative ease and in large amounts in Japan and we found them to be a rich source of glycoproteins.

2. Cortical alveolus glycoproteins (hyosophorins) Cortical vesicles are specialized Golgi-derived secretory organelles found in the peripheral cytoplasm of mature eggs of almost all animal species including humans [I]. Upon fertilization or parthenogenetic activation of the egg, the cortical vesicles fuse with the plasma membrane of the egg and release their contents into the perivitelline space; this process is a prerequisite for normal development of the embryos under natural conditions. These vesicles are about ten times larger ( 2 4 0 y m ) in fish eggs, than those in other animal eggs and are often called cortical alveoli rather than cortical granules, a term used for homologous vesicles found in sea urchin and mammalian eggs. Our studies revealed that each species of fish had homologous cortical alveolar glycoproteins which we named “hyosophorins” after hyosopho (Japanese), meaning cortical alveolus [2,3]. Hyosophorins are a family of glycoproteins ubiquitously found in the cortical alveoli of fish eggs and which obey the following criteria: (i) high carbohydrate content (8&90%, w/w); (ii) apo-hyosophorin comprises tandem repetitions of an identical peptide sequence; (iii) the protein is completely cleaved into repeating units when the dormant unfertilized eggs commence development in response to sperm fusion or a parthenogenetic stimulus.

2.1. First isolation of polysialoglycoproteins (PSGP) from rainbow trout eggs and their ubiquitous occurrence in salmonid fish In 1978, we isolated a new family of highly acidic 200 kDa sialoglycoproteins, which we later designated as polysialoglycoprotein (PSGP), from mature unfertilized eggs of rainbow trout [4,5]. The most striking feature of these glycoproteins was their high content (> 50%, by weight) of sialic acid that has been identified as Neu5Gc. All of the glycan chains were 0-glycosidically linked; the major carbohydrate components were Gal and GalNAc with smaller amounts of Fuc. Sialic acid usually occurs as a single non-reducing terminal residue in glycoproteins and in 1978, no poly-Sia structures were known to occur in glycoproteins [6]. The presence of a-2,g-linked oligo(Neu5Gc) in rainbow trout PSGP was established by methylation analysis coupled with glc-ms [7-91. The presence of polySia units with variable chain lengths up to 20 residues was shown by anion-exchange separation of the oligo/poly-sialylglycan units and the structural analysis of each chain [ 10,l I]. PSGP similar to that isolated from rainbow trout eggs was ubiquitously found in the eggs of other Sulmonidue fish species. We have examined 8 fish species from 3 of the 4 major genera of Salmonidae: Oncorhynchus (0. keta, 0. masou ishikawui, 0. mykiss, and 0. nerka udonis), Salmo (Salmo trutta furio), and Suluelinus (S.fontinalis, S. leucomaenispluuius, and S. namuycush) [12-141. All of the salmonid egg PSGP have 5 common oligosaccharide core glycan structures (a-e in Fig. l), of which 2 different

R( -6)

\

GalNAc(al4)SerRhr

/ Gal@1-3) R( -4

\

GalNAc(a I - O ) S e r m

/

Gal@14)Gal(P 1-3)

R( -6)

\

GalNAc(a l-O)Ser/Thr

/ GalNAc(P1-3)Gal(P 1-4)Gal( pl-3) R( -6)

\

GalNAc(a 1 - O ) S e r m

/ Fuc(al-t3)GalNAc(~l-3)Gal(p1-4)Gal(P 1-3)

GalNAc(Pl-4)

Kdn(a2-

\ GalNAc(al-O)SerRhr /

\ GalNAc(pl-3)Gal(pl-4)Gal(pl-3) / ) or NeuSAcyl(a2-3)

R = Kdn(a2-8)[NeuSAcyl(a2-

)I,

Fig. 1 . Structures of the five distinct types of carbohydrate units of salmonid fish egg PSGP Neu5Acyl indicates Neu5Ac and/or Neu5Gc. R = Kdn(a2-[8NeuSAcyl(a2-)1,.

kinds of core structures (d and e) are the biosynthetically matured forms [ 15-18]. The relative abundance of each core type varies depending on the individual rather than on the species of fish. 2.2. Occurrence of a deaminoneuraminic acid residue (Kdn) at the non-reducing end of oligo/poly-Sia chains

In 1986, we reported the first natural occurrence of a deaminoneuraminic acid, 3-deoxyD-glycero-D-galacto-nononicacid (Kdn), in rainbow trout egg PSGP [ 191. Our subsequent studies on the occurrence and biosynthesis of this residue have established that Kdn is a member of the sialic acid family [2&22]. Kdn has been found in all the PSGPs so far isolated from the eggs of various fish species. In fish egg PSGP, Kdn occurs only at the non-reducing terminal position of oligo/poly-Sia chains. Kdn is also found directly

146

linked to the proximal and penultimate GalNAc residues at the positions where Neu5Acyl residues are usually found (see Fig. 1). The Kdn residues are resistant to the action of the commercially available sialidases so far examined.

2.3. Diversity in oligo/poly-Sia chains of salmonid egg PSGP A species-specific structural diversity has been revealed in oligo/poly-Sia chains of salmonid egg PSGPs. For example, the PSGPs from Oncorhynchus (0.mykiss, 0. keta, 0. masou ishikawai, and 0. nerka adonis) contain exclusively NeuSGc, whereas those from Salmo and Salvelinus species contain both Neu5Ac and Neu5Gc. In these species poly-Sia chains can be either poly(NeuSAc), poly(Neu5Gc) or a hybrid type, poiy(NeuSAc,NeuSGc) [ 18,241. While O-acetyl substitution was not significant in most species, it was extensive in some species and occurred at C-4, C-7, and C-9 [25]. O-lactyl substitution also occurred in one species [24]. In species in which O-acetylation of NeuSAcyl was extensive, O-acetylation was also found at C-9 of Kdn [25]. Only the a2%linkage has been identified in poly-Sia chains of fish egg PSGP, although other linkage types have been reported for oligo/poly-Sia chains of bacteria and lower animals [26,27]. 2.4. Fish egg PSGP is a cortical alveolar component Because of its unusually low antigenicity, no antibody against fish egg PSGP has been obtained. The lake trout (Salvelinus fontinalis) PSGP contained poly(Neu5Ac) chains and was reactive with H.46 antiserum raised against colominic acid [28]. By the indirect immunofluorescence staining method using H.46 and cortical alveoli isolated from lake trout eggs, we showed that PSGP was localized in the cortical alveoli [29]. Furthermore, a cortical alveolus-rich fraction was separated from the rainbow trout egg and 200kDa PSGP was shown to be enriched in this fraction as a water-soluble component. 2.5. Polyprotein nature of apo-PSGP and the molecular mechanism of fertilizationassociated depolymerization

We found that PSGP isolated from fertilized rainbow trout eggs had a molecular mass of 9 kDa (L-PSGP), whereas the average molecular mass of PSGP isolated from unfertilized eggs was 200kDa (H-PSGP). Both the amino acid and carbohydrate compositions of H- and L-PSGP were identical. Apo-L-PSGP was a single tridecapeptide which was sequenced as DDAT*S*EAAT*GPSG (* indicates the glycosylated residue) [30]. Next, we found that H-PSGP was made up of a tandem repeat of the L-PSGP glycotridecapeptide [3 I]. No amino acid or peptide was inserted between the glycotridecapeptide repeats. It is apparent that fertilization-associated depolymerization occurs by proteolytic cleavage of the glycoprotein into glycotridecapeptide repeating units. Insemination was not necessary for this depolymerization. In salmonids, egg activation was induced by immersing the eggs in water without insemination. The depolymerization of H-PSGP following egg activation was catalyzed by a specific proteinase (PSGPase) present in the cortical region of the eggs, which becomes activated following egg activation. The enzyme

147

Lake trout (Salvelinus namaycush) L-PSGP(Sn) Asp-Ala-Thr*-Ser*-G1u-Ala-Ala-Thr*-Gly-Pro-Ser-Asp Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser

Brook trout ( 5 . fontinalis) L-PSGP(Sf) Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser

Japanese common char ( S . leucomaenis pluvius) L-PSGP (Slp) Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp

Brown trout (Salmo trutta falio) L-PSGP(Stf) Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser

Chum salmon (Oncorhynchus keta) L-PSGP(0k) Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser

Land-locked cherry salmon (0. masou ishikawai) L-PSGP(0mi) Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser

Rainbow trout (0. mykiss) L-PSGP(0m) Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly

Kokanee salmon (0. nerka adonis) L-PSGP(0n) Asp-Ala-Thr*-Ser*-Asp-Ala-Ala-Thr*-Gly-Pro-Ser-Asp Asp-Asp-Ala-Thr*-Ser*-Asp-Ala-Ala-Thr*-Gly-Pro-Ser-Gly

Fig. 2. Amino acid sequences of L-PSGPs isolated from fertilized eggs of salmonid fish species where * indicates the 0-glycosylation sites. H-PSGPs isolated from unfertilized eggs are polymerized forms of L-PSGP

is active only at NaCl concentrations below 40 mM and the optimal temperature is about 16°C [32]. In natural fertilization, salmonid eggs are spawned and inseminated in fresh water so that the activation of the eggs occurs immediately after insemination. Under these conditions, water flows into the perivitelline space into which PSGP and PSGPase are translocated by exocytosis from the cortical alveoli. The estimated salt concentration within the perivitelline space is 10-20mM. We compared the structures of unfertilized and fertilized egg apo-PSGP from 8 Salmonidae species [24,30,3 1,33,34] (Fig. 2). The amino acid sequence of PSGP was

I48

highly homologous among the species. The occurrence of tandem repeats of dodeca- or tridecapeptide was found in apo-H-PSGP from all species. Sequence analysis of L-PSGP from each species (Fig. 2) revealed that proteolytic cleavages occur at the position two residues C-terminal to the Pro residue, -P-S-X-D (X is either G, S, or D). 2.6. Molecular cloning of apo-PSGP The PSGP molecule is initially synthesized as pre-pro-apo-PSGP and then posttranslationally modified in the Golgi apparatus by proteolytic cleavages of N- and C-termini, and by core glycosylations. cDNAs encoding apo-PSGP of rainbow trout eggs have been cloned [35]. Nucleotide sequence analysis showed that apo-PSGP mRNA contains tandem repeats, each composed of a 39-base oligoribonucleotide encoding a tridecapeptide, and that the sequences of the repeating units are completely preserved at the nucleotide level. Multiple mRNA species are present that are transcribed from multiple genes for apo-PSGP with different numbers of the repetitive sequence [36]. Expression of mRNAs is stage-specific: they are expressed only in immature ovaries but not in the mature eggs, nor in any other organs. 2.7. Biosynthesis of polysialyl glycan chains In a recent study [37], we showed developmental changes in the level and composition of PSGP during oogenesis of rainbow trout. A sialoglycoprotein family designated IowSiaPSGP was identified in ovaries 6 months prior to ovulation when the ovary weight was as low as 1.2 mg. This stage coincided with the appearance of detectable mRNA for apoPSGP. A second more highly sialylated glycoprotein (highSia-PSGP) appeared 3 months later (3 months prior to ovulation). Compositional analyses showed that the two forms of apo-PSGP differed only in their sialic acid contents while both amino acid and other carbohydrate compositions were identical. Structural studies confirmed that lowSia-PSGP contained mostly disialyl [Sia(a2-8)Sia(a2-6)-] chains whereas highSia-PSGP contained a-2J-linked oligo/poly-Sia chains ranging in length from 2 to 20 sialic acid residues. Biosynthetic studies using CMP-[ 14C]NeuSAc indicated that there are three sialyltransferase activities responsible for the assembly of the polysialylglycan chains of PSGP: (i) a-N-acetylgalactosaminide a-2,6-sialyltransferase (a-2,6-ST), which catalyzes the formation of the Sia residues a-2,6-linked to the proximal GalNAc residues in asialo-PSGP; (ii) a-2,6-sialoside a-2,8-sialyltransferase (a-2,8-ST), which catalyzes transfer of the first a-2,8-Sia residue to the a-2,6-linked Sia residue; and (iii) an a-2,8-polysialyltransferase (a-2,8-polyST), responsible for the synthesis of the a-2J-linked oligo/poly-Sia chains in highSia-PSGP (Fig. 3). These sialyltransferases were located in the cortical alveolus fraction obtained from the immature oocytes and mature eggs of rainbow trout. It was concluded that the core region of the glycan units of PSGP is assembled in the REWGolgi apparatus of oocytes giving rise to asialo-PSGPs, which are then transported from the Golgi apparatus into the cortical alveoli where sialylation occurs. Thus, asialo-PSGP molecules are packaged in immature cortical alveoli along with the sialyltransferases. The fact that both the Golgi complex and the endoplasmic reticulum play important roles in the formation of cortical

149

GalNAc(al-O)Ser/lk

+ CMP-Sia'

\GalNAc(al-O)SerlIhr

+ CMP-Sia*

a-2*wT*

Sia'(aZ-6)

Sia*(aZ-8)Sia(aZ-6)

/ R(4)Gd@1-3)

c R(-%4!31-3)

\ GalNAc(al-O)Ser/lh /

(ii)

Fig. 3. A summary of the results from studies on the biosynthesis of the polysialyl chains in salmonid fish egg PSGP. Three sialyltransferase activities, which are responsible for assembly of the polysialylglycan chains of PSGP, were identified: (i) a-N-acetylgalactosaminide a-2,6-sialyltransferase (a-2,6-ST), which catalyzes formation of the Sia residues a-2,6-linked to the proximal GalNAc residues in asialo-PSGP; (ii) a-2,6-sialoside a-2,8-sialyltransferase (a-2,8-ST), which catalyzes transfer of the first a-2,8-Sia residue to the a-2,6-linked Sia residue; (iii) an a-2,8-polysialyltransferase(a-2,8-polyST), responsible for synthesis of the a-2,8-linked oligo/poly-Sia chains in high Sia-PSGP. R = H, Gal(BIL, GalNAc(B1-3)Gal(f3-, Fuc(a1Sia' represents the 3)GalNAc(P 1-3)Gal(PI-, or Kdn(a2/NeuSGc(a2-3)[GalNAc(~1-4)]GalNAc(~1-3)Gal(~l-. sialic acid residue(s) newly incorporated from CMP-Sia' by the sialyltransferase in question.

vesicles and that cortical vesicles grow or mature during oogenesis of invertebrates and vertebrates have been documented by earlier morphological studies [38,39]. Our study first showed that these biosynthetic reactions occurred continuously in the cortical vesicles until ovulation. Both the a-2,6-ST and the a-2,8-ST can use CMP-NeuSAc and CMP-NeuSGc as activated sialyl donors. This is compatible with our finding that poly(NeuSAc), poly(Neu5Ac,NeuSGc), and poly (NeuSGc) chains were expressed in PSGPs from Saluelinus species such as lake trout and a Japanese common char [ 18,241. We have recently demonstrated the enzyme activity that catalyzes the transfer of Kdn from CMP-Kdn to PSGP in the cortical alveolus fraction of the rainbow trout ovary. Analyses of the reaction product showed that this Kdn-transferase could use the nonreducing terminal NeuSAcyl of poly-Sia chains of rainbow trout egg PSGP as an acceptor substrate but could not transfer either Kdn or NeuSAc from the respective activated donor to the terminal Kdn residues on the poly-Sia chains [40]. These observations coincide with our previous view that Kdn occurs as the capping residue of the poly-Sia chains of rainbow trout egg PSGP [17]. 2.8. Hyosophorins bearing bulky multi-antennary N-glycan chains

Our studies on the distribution of PSGP among fish species revealed that fish belonging to species other than Salmonidae have major N-glycosidic glycoprotein components that satisfy the criteria of hyosophorin. The first N-glycan-type hyosophorin was isolated from the eggs of medaka fish, Olyzias latipes[2]. By using a polyclonal antibody against H-hyosophorin and an indirect immunofluorescence staining technique, H-hyosophorin was localized in the peripheral region of the cortical alveoli. L-hyosophorin is a

150

Flounder (Paralichthys olivaceus)

glycononapeptide: AspAla-Ala-Ser-Asn’-Glu-Th-Val-Ser, where * indicates the glycosylation site. It has to be noted that a single N-glycan chain is attached to every repeating unit, in contrast to other PSGPs where three 0-glycan chains are attached to the repeating unit. The complete structure of a large pentaantennary glycan unit (molecular weight -6000) was recently established by H-NMR spectroscopy and FAB-MS spectrometry [41] (Fig. 4). Each of the antennae has a poly-N-acetyllactosaminyl core highly branched at both GlcNAc and Gal residues. The poly-N-acetyllactosaminyl core was completely resistant to endo-(3-galactosidase. The presence of a (3-galactosylated Lewis x antigenic epitope Gal@1-4)Gal(~1~)[Fuc(al-3)]GlcNAc((31-), and its sialylated form, Gal((3I-4)[Neu5Ac(a2-3)]Gal((31~)[Fuc(al-3)]GlcNAc(~l-), the presence of Gal((31-

’

4)Gal(fk and G a l ( p 1 4 ) G a l ( ~ ) G a l ( ~ l -and , the presence of branched Gal residues, -

4)GlcNAc(~l-3)[Gal(~l4)]Gal(~1are novel and unique features. Prior to the elucidation of this structure, we determined the structure of a tetraantennary sialoglycan unit attached to hyosophorin of Indian medaka, Oryzias melastigma [42]. Interestingly, while the amino acid sequence of hyosophorin is completely conserved between these species, their glycan structures show marked differences. The structures of the peripheral portions that contain mostly digalactosyl (0.latipes) and trigalactosylated (0. melastigma) structures are different (Fig. 4). Moreover, a portion of 0. latipes hyosophorin is fucosylated while 0. melastigma hyosophorin is not. In contrast to these inter-species differences, the glycans of hyosophorins are homogeneous with respect to antenna and branching structures within a species. Thus, hyosophorin molecules are uniquely N-glycosylated in uiuo with no heterogeneity in antenna number while most of the animal glycoproteins show heterogeneity with respect to the antenna structures (di-, tri-, and tetraantennary) of their glycan chains. Despite the large differences in the structures of glycan chains, these N-glycan-type hyosophorins and PSGP are both highly acidic, poly-anionic polymers. We have isolated similar N-glycan-type hyosophorins from other species of freshwater fish, Plecoglossus altiuelis, Tribolodon hakonensis, and Cyprinus carpio [43]. Although the species-specific difference was extensive in the peripheral structures, all of them have a poly-N-acetyllactosaminyl core structure attached to penta- (Plecoglossus) or tetra- (Tribolodon and Cyprinus) antennary glycan units. The presence of sulfate groups in addition to sialic acid makes the structural elucidation difficult. It is noted that in all of these fish eggs, PSGP molecules similar to those isolated from Salmonidae are also expressed as a minor component (less than one twentieth of N-glycosidic-type). Hyosophorin isolated from a sea-water fish, flounder (Paralichthys oliuaceus), had neutral fucosylated pentaantennary glycan chains [3]. This non-anionic nature of flounder hyosophorin may account for the poor histochemical staining of the cortical alveoli of this fish, in contrast to intense toluidine blue staining of cortical alveoli of the eggs of salmonid fish, medaka fish, and Plecoglossus. The salinity of water in which the fish spawn and the ionic properties of hyosophorins are not related as we first anticipated, since our studies revealed that the hyosophorin glycans of some seawater spawning fish, herring (Clupea pallasii) (S. Inoue, unpublished observation) and Fundulus heteroclitus [831, are sialylated. 2.9. Biological function of hyosophorin and future perspective Formation and maturation of Golgi-derived secretory vesicles (cortical vesicles) by developing oocytes of many animal species, and the dramatic discharge of their contents at the moment of fertilization just prior to the initiation of embryonic development have for a long time attracted the attention and interest of many biologists. It is very interesting that polysaccharides or mucopolysaccharides have been implicated as a major component of cortical vesicles of most animals ranging from echinoderms to mammals in these earlier works. We have now isolated and determined the chemical structures of cortical alveolar glycoproteins ubiquitously found in fish eggs. Although our study is still far from complete, the following findings may be relevant to the function of hyosophorin.

152

2.9.1. Formation and function of the perivitelline fluid In teleost fish, the spawned eggs are bathed in a hypotonic (fresh water) or a hypertonic (marine) external medium. The embryo enclosed by the plasma membrane is bathed in the perivitelline fluid formed in the space between the plasma membrane and the outermost envelope (vitelline envelope or fertilization envelope). At fertilization, cortical vesicle exocytosis occurs, and the contents of the vesicles are transported into the perivitelline space. Due to the impermeability of the vitelline envelope to high molecular mass substances such as hyosophorins, they are confined to the perivitelline space and cause intake of water (and small ions) across the envelope from the external medium. The tension of the envelope resulting from the internal hydrostatic pressure created by the osmotic properties of the perivitelline space fluid is thought to produce a cushioned environment for the embryo against external deformation and a decrease in the permeability of the plasma membrane. Being poly-anions, hyosophorins of freshwater fish are, under physiological conditions, negatively charged, and may accumulate cations (Na+, K+, Ca2+, and Mg2+) in excess of their concentrations in the external medium. Osmotic and ionic regulatory functions of the perivitelline fluid are supported by physiological measurements [44]. At the present stage, we cannot answer the question why H-hyosophorins undergo depolymerization to L-hyosophorins at fertilization. For polyanions such as PSGP, an increase in terms of molecular concentration by 20 times does not result in an increase of the calculated osmotic pressure due to the large contribution from the Donnan term. Increase in diffusion coefficient by H- to L-transformation may be significant in allowing a response to rapid formation of the perivitelline fluid. In marine species the mechanism of osmoregulation between the egg and the external medium is more complex. However, the observed values of zero to positive perivitelline potentials obtained for eggs of marine species [44] may suggest the difference in the ionic properties of colloids (may be hyosophorins) in the perivitelline fluid. These results are in accord with our results that showed hyosophorin from a marine species (flounder) bore only neutral carbohydrate units. 2.9.2. Sperm agglutfnating properties of hyosophorins One of the important roles of mammalian cortical granules may be to block polyspermy in the perivitelline space [45]. In fish, fertilizing sperm enters through a special opening in the egg envelope called micropyle. The diameter of the micropyle in most fish is usually just large enough to allow a single sperm. Moreover as soon as the first sperm enters into the egg, an electron-microscopically observable plug is formed at the bottom of the micropyle to prevent the entrance of a second sperm. There may be no chance for sperm to be directly in contact with the perivitelline fluid and therefore L-hyosophorins. And indeed, we found that L-hyosophorins, the forms actually present in the perivitelline space, have only weak, if any, sperm agglutinating activity. Though H-hyosophorins agglutinate sperm, this is a rather general property shown by large poly-anions. On the basis of electron-microscopic observation, some workers have claimed that the cortical vesicle contents of fish eggs are partially translocated to the outside surface of the fertilization envelope and there they function to prevent polyspermy. Our careful analysis could not detect any PSGP outside the fertilization envelope or in the extract of the isolated

153

fertilization envelope of rainbow trout. L-PSGP was found in the perivitelline space fluid throughout embryonic development of rainbow trout until hatching. 2.9.3. Calcium ion binding properties of hyosophorins Recently, we investigated the Ca2+ binding of H- and L-PSGP by equilibrium dialysis and circular dichroism methods [46]. Calcium binding to H- and L-PSGP occurred with apparent binding constants (K,) of 2 . 9 8 ~ lo3, and 1 . 0 0 lo3 ~ M-I, respectively. These values were lower than the value (13.9 x 1O3 M-') for colominic acid (DP = 24) but much higher than the reported values of 121 and 193 M-' for Ca2+-Neu5Ac [47] and Ca2+NeuSGc [48], respectively. Ca2+ binding affinity for L-PSGP was one-third of that for H-PSGP, whereas the number of binding sites (n) did not change on going from H-PSGP to L-PSGP. CD measurements also indicated that H-PSGP was more strongly and preferentially complexed with Ca2+ion than L-PSGP. The removal of oligo/poly(Neu5Gc) from PSGPs by sialidase treatment abolished the Ca2+ binding capacities as examined both by equilibrium dialysis and CD methods. The difference of Ca2+binding affinities between H- and L-PSGP has implications for the calcium release at fertilization, i.e. the Ca2+ions bound to H-PSGP represent a reservoir of calcium for liberation at fertilization. The calcium binding capacity of each sialic acid residue of H- and L-PSGP was larger than that of colominic acid. On average, one calcium ion is bound to two NeuSGc residues of PSGP and three NeuSAc residues of colominic acid. Although no information on the Ca2+ concentration in a cortical alveolus is available and it may be difficult to determine it directly in such a fragile vesicle as the cortical alveolus, now we can estimate the concentration to be 93mM on the basis of the average values of egg and cortical alveolus diameters and the amount of H-PSGP present in an egg with a value of Neu5Gc/Ca2+= 2: 1. The derived value of the Ca2+ concentration in cortical alveoli of rainbow trout compared well with those (30 and 95 mM) for two different species of the sea urchin determined by X-ray micro-analysis [49]. Using the estimated volume of 6.7 x ml, the Ca2+ and NeuSGc concentration in the perivitelline fluid of rainbow trout egg was 4.7 and 9.4mM, respectively. One of the morphological changes after fertilization is the deformation from a soft vitelline envelope to a tough fertilization envelope. It is known that this process called hardening is enzyme-catalyzed and requires Ca2+ions. We have observed that at least 1OmM Ca2+is necessary for hardening of the fertilization envelope of rainbow trout. Thus, Ca2+ions transported from the egg into the perivitelline space with H-PSGP, and there released by depolymerization of H-PSGP into L-PSGP may be used in this process. 2.9.4. De-N-glycosylution of hyosophorins We found that N-glycan chains apparently detached from hyosophorins become accumulated in the embryos of two different species of fish, flounder and medaka [3,50]. In both species hyosophorin-derived free glycans were found neither in unfertilized eggs nor in eggs which were fertilized but which failed to continue embryonic development. We have identified alkaline peptide:N-glycanase (PNGase) (optimal pH 7-9) in the blastodiscs isolated from the blastula stage of medaka embryos (A. Seko, in preparation). The expression of alkaline PNGase activity and the accumulation of hyosophorin-derived free

154

glycan increased in parallel and both reached the maximum level at stage 12. These results strongly suggest the physiological importance of de-N-glycosylation of L-hyosophorins for continuation of embryonic development.

3. Mucin-type glycoproteins found in the vitelline envelope and ovarian fluid of salmonidfish 3.1. Isolation and glycan structures of Kdn-gp and Sia-gp The morphology, chemical nature and nomenclature of the egg coats that enclose ovulated eggs vary in different animal groups and largely reflect the mode by which the egg achieves successful fertilization and embryonic development under a variable environment [51]. The following functions have been attributed to the egg coats: (i) to protect the eggs and assist their movement during ovulation and spawning; (ii) to assist species-specific fertilization; (iii) to prevent polyspermy; (iv) to become a chemically and mechanically tough covering for the embryo; and in some species (v) to fix the embryo to the substratum. The second function includes species-specific egg-sperm recognition and induction of the acrosome reaction for some animal groups. Glycoproteins are reported to be major components of the egg coat in many animal groups and their carbohydrate chains are believed to be functionally important. In fish, the egg coats consist of several layers called the vitelline envelope (before fertilization) which, upon fertilization, is transformed into the fertilization envelope. This transformation involves enzyme-catalyzed reformation of various macromolecule(s). An unusual family of glycoproteins containing -50% (w/w) Kdn and no N-acylneuraminic acid was first isolated by us in 1988 from the vitelline envelope of rainbow trout eggs and designated as Kdn-gp [52]. Kdn-gp contains -15% protein and -85% carbohydrate (Gal/GalNAc/Kdn = 1:2:av.5) which is 0-linked to ThdSer. Apo-Kdn-gp is also unusual in that threonine and alanine account for 40 and 27 residue %, respectively. More than 80% of the threonine residues are involved in a carbohydrate linkage. Kdn-gp is isolated as a family of polydisperse molecules ranging over 700-4000kDa (the peak at -3000 kDa) as estimated by gel filtration chromatography. The structure of oligoKdn-containing 0-glycan chains of Kdn-gp is given in Fig. 5. Only one type of core structure was found in Kdn-gp 0-glycans, though diversity was found in the size of the oligoKdn chains ( n= 1-7) [53]. More recently, Kdn-containing di- and triantennary N-glycan chains were identified in Kdn-gp isolated from rainbow trout vitelline envelope[54] (see Fig. 5). Based on composition and the yield of the carbohydrate chains, about 1000 0-glycans and 60 Nglycans are linked to a 3000 kDa Kdn-gp. While Kdn-gp is a major glycoprotein component of the vitelline envelope of rainbow trout, chum salmon, and kokanee salmon, the vitelline envelope of cherry salmon contains little Kdn-gp. An analogous family of mucin-type glycoproteins (Sia-gp) that contain Neu5Gc instead of Kdn is the major glycoprotein component in this salmon (Fig. 5) [55]. That Sia-gp is a functionally analogous molecule to Kdn-gp is supported by the similar molecular mass and amino acid composition (Thr + Ala M 70 residue %).

155

(a) 0- and N-linked Kdn-glycan chains present in Kdn-gp Kdn(a2-8)Kdn(a2- ) -( -8)Kdn(a2-6)

\

GalNAc(a 1-0)SerRhr

/ Kdn(a2-3)Gal(~l-3)GdNAc(al-3)

Kdn(a2-3)Gal(P 14)GlcNAc(P 1-2)Man(a 1-6)

\

*GlcNAc(Pl4)-Man(P

rtFuc(a 1-6)

I

14)GlcNAc(~l-I)GlcNAc(P1-N)Asn

I

Kdn(a2-3)Gal(pl4)GlcNAc(~l-2)Man(al-3)

Kdn(a2-3)Gal( pl4)GlcNAc(P 1-6), Man(al-6)

\

Kdn(a2-3)Gal(P 1-4)GlcNAc(fi1-2)’

@uc(a 1 4 ) I rtGlcNAc(P 14)-Man(Pl4)GlcNAc(P 14)GlcNAc(P 1-N)Asn

f

I

Kdn(a2-3)Gal(P 14)GlcNAc(P 1-2)Man( a 1-3)

(b) 0-linked NeuGc-glycan chains present in Sia-gp Kdn(a2-6)

\

GalNAc(a 1-0)SerRhr

/ NeuGc(a2-3)Gal(pl-3)GalNAc(al-3) Fig. 5. Structures of (a) Kdn-containing 0- and N-linked glycan chains; (b) Neu5Gc-containing 0-glycan chains of Kdn-gp and Sia-gp isolated from the vitelline envelope of salmonid fish species.

Immunohistochemical methods revealed that Kdn-gp is localized in the second layer of the outer surface of the vitelline envelope [56]. We have found that Kdn-gp was also a component of ovarian fluid of this and other salmonid fish (Kdn-gp-OF) [57].In salmonids, eggs are first ovulated in the body cavity of female fish, and wait for the stimulus for spawning while bathed in ovarian fluid (or body cavity fluid). The ovarian fluid is believed to be originated from the extraovarian cells in the ovary. No difference was found in the chemical nature (including the glycan structure) between Kdn-gp-VE and Kdn-gp-OF. The amount of Kdn-gps isolated from a single 2-year-old female rainbow trout (spawning 100g egg) was 10mg of Kdn-gp-VE and

-

-

156

10-20mg of Kdn-gp-OF. Sia-gp-OF was isolated from those fish species which synthesize Sia-gp-VE. 3.2. Biosynthesis and possible functions of Kdn-(Sia-)gps Kdn-gp appeared to be synthesized during relatively later stages of oogenesis, since it was undetectable in the oocyte 3 months prior to ovulation. Although the cell types that synthesize Kdn-gp have not been identified, it is most likely synthesized under hormonal control in some extraoocyte cells (i.e. follicle cells), secreted and partly incorporated into the second outermost layer of the vitelline envelope just before ovulation. Kdn-gp may thus be a molecule homologous to the oviduct glycoproteins of mammals that are reported to be secreted and partly incorporated into the egg surface [5X-601. Recently, we showed a strong Ca2+ ion binding property for Kdn-gp [46]. Being polyanionic, Kdn-gp may control cation and H+ concentrations in the ovarian fluid in which the ovulated eggs bath. The presence of clusters of anionic carbohydrate chains make Kdn-gp resistant to non-specific proteases such as actinase E. Kdn-gp, like other cell surface mucins, may function in facilitating the movement of cells and in protecting cells from proteolytic degradation and from bacterial invasion.

4. Glycoproteins related to vitellogenesis It is well established that in oviparous vertebrates, vitellogenin is synthesized in the liver under hormonal control, transported into the blood stream, and sequestered via a receptor into the growing oocyte [61]. The vitellogenin polypeptide (-200 kDa) was reported to be glycosylated and phosphorylated at the site of its synthesis. Vitellogenin molecules found in female blood also contain a large percentage of lipid. As soon as vitellogenin is incorporated into the oocyte, it is cleaved into lipovitellins and phosvitins and has not been isolated in its uncleaved state from the oocyte. Phosvitins are highly phosphorylated, relatively small proteins (2-30 m a ) and are often glycosylated. The vitellogenin receptor has been partly characterized in the chicken oocyte. The importance of N-glycosylation of vitellogenin on its uptake by the oocyte has been implicated in frog [62]. The system by which fish vitellogenin is sequestered and processed into yolk is poorly understood. Involvement of the lysosomal system in this process has been shown by electron-microscopic and cytochemical studies [63]. Recently, we found accumulation of relatively large amounts of complex-type free sialoglycans in the unfertilized mature eggs of two species of freshwater fish, Plecoglossus altivelis and Tribolodon hakonensis [64,65]. All of these free glycans possessed typical di-, tri-, or tetraantennary structures and the di-N-acetylchitobiose structure at their reducing termini. We originally speculated that these free glycans may be derived from vitellogenin, because of the large amount -5 x mol, and -25 x mol per g fresh egg of Plecoglossus and Tribolodon, respectively. These amounts are comparable to the amount of vitellogenin-derived proteins in the eggs. We also hypothesized that the enzyme involved may be peptide:N-glycanase, an enzyme not previously reported in an animal system. The speculation that free N-glycan chains in the unfertilized fish eggs may

157

originate from vitellogenin molecules was partly substantiated by our recent isolation of glycophosphoproteins having species-specific N-linked glycans previously found as free glycan chains [66]. In addition to the above fish species, the occurrence of free N-glycan chains and the phosphoprotein bearing the same N-linked unit as the free glycan was also demonstrated in medaka fish (Fig. 6). Most recently, we identified and partially purified a peptide:N-glycanase (PNGase) activity in the early embryos of medaka fish [67]. This was the first report of PNGase from an animal source. This PNGase from a medaka embryo had a low optimal pH of 3.7 (acid PNGase). Acid PNGase activity was also found in the ovary egg; spawned, fertilized eggs are usually used as the enzyme source because of the ease in collecting large amounts of material. Acid PNGase may participate in de-N-glycosylation of vitellogenin during vitellogenesis. N-glycosylation of vitellogenin may be needed in some process of its transportation, recognition by the receptor, or in specifying the site of proteolytic cleavages. De-N-glycosylation, in turn, may be necessary for recycling of the receptor and further processing and/or utilization of yolk proteins. This latter idea is supported by our finding that in the mature eggs of Tribolodon, in which the free N-glycan pool is large, non-glycosylated small phosphopeptides (molecular mass -2000) were found in excess of the amount of the glycophosphoprotein (S. Inoue, in preparation).

5. Epilogue Several new constituents of glycoproteins such as poly-Sia and Kdn have been discovered in fish egg glycoproteins. The occurrence of Kdn, a new member of the sialic acid family, has been reported in one bacterial strain [68], two amphibian species [69,70] and various mammalian tissues [7 1,84-861. Use of monoclonal antibodies [7 1,721 and specific enzymes [73-751 that cleave Kdn linkages may facilitate new discoveries not only in the area of fundamental biology but also in oncology and pathology. We have also described a new catabolic pathway for glycoproteins in animal cells. In eukaryotic cells, the biosynthetic mechanism of protein N-glycosylation has been well established. However, the necessity for removing specific N-glycan chain(s) from certain glycoproteins as a possible prerequisite for intracellular transport and/or for acquisition of a functional structure has yet to be demonstrated in all systems. We identified and partially purified peptide:N-glycanase (PNGase) in the fish egg. PNGase had been previously reported only in plants and bacteria [76-78]. Although the purified PNGases from plant and bacterial sources have been used as tools in structural and functional studies of glycoproteins, almost no attention has been paid to the physiological function of this enzyme in living cells. Subsequent to our first finding of PNGase in fish, we have demonstrated a wide occurrence of PNGase in animals, including mammalian species, and their physiological significance [87,88]. Our discovery of PNGase in fish eggs was based on the finding of an unusually large pool of free N-glycan chains identical to parts of the N-glycan units attached to the glycoproteins, including the di-N ,N'-acetylchitobiosyl structure at the reducing termini. If PNGase-catalyzed de-N-glycosylation occurs in other animal cells, the accumulation of free oligosaccharides would not be observed since they are transported to the lysosomal

Oryzius htipes NeuSAc(a2-3)Gal(P 1 +Gal@ 1-4)GlcNAc@ l-‘L)Man(a14)

\

Man($]-)R

/

Neu5Ac(a2-3)Gal(~14)Gal(P1-4)GlcNAc(pl-2)Man(a1-3)

Tribolodon hukonensis Sia(a2-3)Gal(P 14)GlcNAc(p 1-Z)Man(a1-6)

\

Man(pl-)R

/

Sia(a2-3)Gal(~l-4)GlcNAc(~l-2)Man(al-3) Sia(a2-3)[Gal(pl-4)]Gal(~l~)GlcNAc(pl-Z)Man(a1~/3)

\

Plecoglossus altivelis Neu5Ac(a2-3)Gal(~l~)GlcNAc(P1-Z)Man(a 1-6)

\ /Man(P1-)R Neu5Ac(a2-3)Gal(~l4)GlcNAc(~1-2)Man(al-3) Neu5Ac(&3)Gal(~14)GlcNAc(~I-Z)Man(a 14)

\

NeuSAc(a2-3)Gal(P 1-4)GlcNAc(P 14),

Man@-)R

/

Sia(a2-3)Gal(P 14)GlcNAc(p 1-2)Man(al-3/6)

Sia(a2-3)[Gal(~l-4)]Gal(~l4)GlcNAc(pl-2)Man(a1-6)

\ Sia(a2-3)[Gal(P14)]Gal(P14)GlcNAc(pl-2)Man(a1-3)

Man(pl-)R

/

Man(a1-3) Neu5Ac(a2-3)Gal(pl4)GlcNAc(pl-2)’

Neu5Ac(a2-3)Gal(p I4)GlcNAc(~I-6) ,, Man(a 14) \ Neu5Ac(a2-3)Gal(~l~)GlcNAc(~l-2)’ Man(Pl-)R Neu5Ac(aZ-3)Gal(~l-4)GlcNAc(~l-Z)Man(al-3)’

R = 4GlcNAcpl+ 4GlcNAc Sia = NeuSAc or Neu4,5Ac2

Fig. 6.Structures of free sialoglycans accumulated in the unfertilized eggs of Otyzius lofipes, Plecoglossus ulfiuelis, and Tribolodon hukonensis and N-linked glycan structures determined for glycophosphoproteins isolated from the corresponding fish species.

159

system immediately after liberation from the parent glycoproteins and undergo further degradation. As anticipated in our previous report [79], we have identified PNGase activities in mammalian-derived cells and tissues [80,81] and purified the enzyme from CH3 mouse-derived L-929 fibroblast cells [82]. These results indicate that PNGase may participate in important biological processes. Fertilization and early embryonic development may be an area in which the participation of specific carbohydrate units of glycoproteins in recognition and activation phenomena manifests itself. We believe the fish egg provides a good system for work aimed at solving the molecular mechanism of such phenomena.

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(281 [29] [30]

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J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins I1 Elsevier Science B.V. CHAPTER 8

Amphibian glycoproteins Gerard Strecker Laboratoire de Chimie Biologique, UMR no. 111 du CNRS, Universiti des Sciences et Technologies de Lille. 59655 Villeneuve d ’Ascq, France

Abbreviations GalNAc-ol N-acetylgalactosaminitol

Gal

galactose

NeuAc

N-acetylneuraminic acid

GlcNAc

N-acetylglucosamine

Kdn

3-deoxy-D-glycero-D-gaiacto-nonu~osonic GalNAc acid GlcA fucose

Fuc

N-acetylgalactosamine glucuronic acid

Key for glycosyltranferase activities, examples Kdn:(a 14)FucT: a-1,4-fucosyltransferase with Kdn as acceptor. Fuc(a l-4)Kdn: ( a 1-3)FucT:a- 1,3-fucosyltransferase with Fuc(a 1-4)Kdn as acceptor.

1. Introduction When we started these studies, it was our aim to find new and easily accessible sources of natural carbohydrates of high biological interest. Indeed, some rare carbohydrate structures, such as the core GalNAc(al-3)GalNAc(a 1-O)Ser/Thr found in human rectal adenocarcinoma glycoproteins [ 11 and in meconium [2,3], or the terminal sequence GlcNAc(a14)Gal(B 14)GlcNAc present in human meconium but never detected in adult organism, constitute major sugar components in salivary gland mucins of the Chinese swiflet [4] and pig stomachal mucin [5], respectively. We suggested the possibility that some carbohydrate units, which play a fundamental role during tissue differentiation, cellular recognition and malignant growth, could be isolated, in high quantities, from some lower-placed species of the animal kingdom. This hypothesis was based upon the “theory of recapitulation”, proposed by E.H. Heckel (1834-1919): Ontogenesis recapitulates phylogenesis. Pleurodeles waltl and Ambystoma mexicanum (axolotl), bred in many laboratories, were chosen because of the abundance of mucins which surround their eggs. The first analyses led to the isolation of Lewis’ and LewisY antigenic determinants, which are known to be related to human tumor-associated antigens. For these reasons, the jelly coats of amphibian eggs could represent a valuable model to examine 0-linked carbohydrate structures during evolution, associated with a possible function involving specific markers for the fertilization process. 163

164

Table 1 Content (YO) of carbohydrate in various egg jelly coatsa Species

Fuc

Gal

GalNAc

GlcNAc

NeuAc

Kdn

Total 50. I

Bufo bufo

6.4

14.6

15.1

6.2

6.8

0

Rana temporaria

4.3

16.5

10.1

4.4

0

2.7

38.0

10.1

6.1

12.1

7.6

0

6.7

42.6

8.9

8.9

10.2

3.8

0

3.4

35.2 49.1

Ambystoma mexicanum Ambysfoma tigrinum

10.0

5.8

18.6

10.7

0

6.0

Pleurodeles waltl

7.0

6.6

20.2

8.4

0

9.4

5 1.6

Xenopus laevis

8.3

9.1

13.5

8.1

0.9

1.2

41.1

-

Rana palustris

+ +

Bufo japonicus

+

-

Bufo americanus Rana ufricularia

-

+

+

-

Ambystoma maculatum

Rana dalmafina

a

-

Unpublished results, except for Bufo japonicus [ 101. Also contains sulfate and glucuronic acid. Data obtained with a pool of Xenopus clutches (see text).

2. Role of the ooiducal secretions in mediation of gamete fusion in amphibians The requirement of oviducal secretion surrounding amphibian eggs for fertilization is known since the last century. Although amphibians have been often considered as models for studying the mechanism of gamete interaction, the molecular basis for the jelly requirement has been puzzling. The important literature devoted to this subject (for general reviews, see refs. [6-191) can be summarized as follows. The oviducal jellies deposited around amphibian eggs are secreted by the tubular gland cells lining the oviduct. The number of jelly coats varies among species from as many as six in Rana pipiens to only two for Lepidobatrachus laevis [6]. Chemical analysis of amphibian jelly revealed the presence of glycoproteins as major components [7-91 and the low amount of mannose, as well as the high proportion of N-acetylgalactosamine, serine and threonine allowed to predicate that they are typical mucin-type glycoproteins (Table 1). The most documented reports concerning these glycoproteins describe a high content of carbohydrates (about 80%) and a polydisperse state (MM ranging from lo& 4000 KDa) [lo]. It is generally accepted that the biological functions of the amphibian egg jelly coats include (1) sperm binding, (2) induction of the sperm acrosome reaction, (3) forming a barrier for the sperm penetration (4) a block to polyspermy (anurans) and ( 5 ) provision of a protective environment for the developing embryo (for general reviews see refs. [ 11-13]). Many reports have shown that coelomic eggs cannot be fertilized, but become fertilizable when inseminated in the presence of an appropriate jelly preparation [ 14-1 61. The primary role of jelly layers in fertilization may be related to the induction of sperm

165

acrosome reaction. The role of inner jelly layers in amphibian acrosome reaction may be similar to that of fluids from mammalian oviduct of the uterus, which have been also reported to stimulate introduction of sperm capacitation. Soluble factor(s) obtained by dialysis or after ashing at 600°C retained the biological activity, an observation that excluded the involvement of organic substances [ 171. The finding that Ca2+ and/or Mg2+ are essential factors for supporting fertilization of dejellied eggs, followed by determination of the Ca2+ binding capacity of the egg jelly glycoproteins, showed finally that the jelly keeps the ionic environment required for successful fertilization. These observations argued against the proposal that specific molecular interactions operating between jelly and sperm surface underlie the essential role of egg jelly in the fertilization process. The absence of a species-specificity occurring during these experiments performed in uitro may be explained by these properties of egg jelly factors. Although intergenic fertilization is possible in vivo [ 121, cross-fertilization remains impossible in most of the cases. This blockade of sperm migration observed in heterologous jelly is likely due to the incompatibility of the physical architecture of the egg jelly, as argued by Katagari [ 121. The role of carbohydrate determinants in amphibian egg vitelline envelope (which corresponds to the zona pellucida of mammalians) has been clearly demonstrated in the case of Xenopus laeois. The monospermy observed among anurans, but not in urodeles, involves the reaction of a lectin from egg cortical granules with components of the vitelline envelope and the innermost jelly coat JI surrounding the egg [18]. The action of cortical granule N-acetyl-b-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization [ 191.

3. Carbohydrate chains of egg jelly coat glycoproteins Glycoproteins in amphibians have not been yet extensively analyzed, although it is well known since longtime that egg jellies or skin mucus are carbohydrate-rich materials. The integumentary mucins from Xenopus laeois have been investigated by molecular cloning in order to examine the organization of the polypeptide backbone [20-221. Analysis of carbohydrates in various egg jelly coats (Table 1) showed the presence of Fuc, Gal, GalNAc and GlcNAc, in variable amounts. Whereas NeuSAc occurs in Bufo bufo (personal observation) and Bufo japonicus japonicus [lo], Kdn which has been characterized for the first time in fishes by Inoue [23] (see chapter 7, this volume) appears to be highly representative for amphibians. In Ambystoma and Xenopus, Kdn and Kdn 9-0-Ac are occurring in the molar ratio 1:l (unpublished results). Traces of Kdn 7-0-Ac and Kdn 7,9-0-Ac were found in Pleurodeles (A. Klein, personal communication). The carbohydrate content averages between 35 and 50% of the mass of the crude material, but is in the order of 80% in a pure glycoprotein fraction [lo]. The carbohydrate composition strongly suggested that glycans consist mainly of 0-linked structures. They were released by reductive 6 elimination and analyzed by NMR spectroscopy [10,24-331. As shown in Figs. 1-8, a species-specific structural diversity has been observed in carbohydrate chains of amphibian egg jelly.

166

Kdn(a2-6)

Kdn(a24)

\

\

/ GlcNAc( p1-3)

/ GalNAc(a 1-3)Gal( p 14)GlcNAc(P1-3)

GalNAc-01

GalNAc-ol Fuc(al-3)/

Kdn(a24)

\

Kdn(a2-6)

GalNAc-01

\

/ Gal(p1-3)

GalNAc-ol

/ Fuc(a 1-2)Gal(P 14)GlcNAc(P1-3)

Fuc(a1-2)/ Kdn(a2-6)

Fuc(a1-3)’

\

GalNAc-ol

GalNAc(a1-3),

/ Gal@-3)

Kdn(a2-6) GalNAc(a1-3),

Fuc(a1-2)’

Fuc(a 1-2)/

Kdn(a24)

\ GalNAc-01 /

Gal(pl4)Gl~NA~(pl-3) Fuc(a 1-3f

\

GalNAc-01

/ Gal@1-4)GlcNAc(pl-3) Fuc(al-3)/ Fig. I . Oligosaccharide-alditols released from the jelly coat of Pleurodeles waltl[24]. GlcNAc(P 1-3)GalNAc4 Fuc(al-2)Gal(

Kdn(a24)

\

P1-4)GlcNAc(p 1-3)GalNAc-ol

Gal(a 1-4)Gal(P14)GlcNAc(P1-3)GalNAc-1 Fuc(al-2)/ Fuc(a 1-4)Kdn(a2-6) Fuc(a 1-3/

GalNAc-ol

/ Gal(al4)Gal(pl-4)GlcNAc(pl-3) Fuc(a 1-2)/ Fuc(a 1-4)Kdn(a24)

\

\

/

/

GalNAc-ol

GalNAc-ol

GlcNAc(P1-3)

Gal(a 14)Gal(P1-4)GlcNAc(P1-3f Fuc(al-2)/

Fuc(alA)Kdn(a24)

Fuc(al-3)Fuc(al4)Kdn(a24)

\

Fuc(a 1-3)/

G~NAc-~~ Gal(pl4)GlcNAc(pl-3) Fuc(al-2)/

/

\

GalNAc-ol

/ Gal(al4)Gal(~l-4)GlcNAc(~l-3) Fuc(a 1-2f

Fig. 2. Oligosaccharide-alditols released from the jelly coat of Ambystoma rnexicanum [25]

The mucin of Pleurodeles waltl (Fig. 1) appeared to be an abundant source of LeX, Ley and A Ley antigenic determinants, which can be prepared on a large scale, after removing Kdn by mild acidolysis. The much of Ambystoma maculatum (Fig. 3) contained essentially the determinant GalNAc(P14)[Fuc(a 1-3)]GlcNAc, which has also

167 GalNAc(P 14)GlcNAc(P1-3)GalNAc-d Fuc(al-3)/ Gal(plA)GlcNAc(P 1-3)GalNAc-ol Fuc(al-3)/ GalNAc(pl4)GlcNAc(P 1-6) Fuc(a 1-3)' Gal@1-3)

\

/""""'

GalNAc(PlA)GlcNAc(pl-6) \ Fuc(al-3)/ Fuc(al-3)Fuc(al4)Kdn(a2-3)Gal@l-3)

Fuc(a1-3) \ Fuc(alA)Kdn(a2-6) Fuc(a1-2)' \ Gal@14)GlcNAc(/31-3)

Gal(~l4)GlcNAc(pl-6) Fuc(al-3)/ \ GaWAc-ol Gal(P1-3) GalNAc(Pl4)GlcNAc(P 1-6) Fuc(al-3)/ Kdn(a2-3)Gal(P 1-3)

/

Fuc(al-3)/ Fuc(a1-3) \ Fuc(al4)Kdn(a2-6) \ Fuc(a1-2) / GalNAc-ol GalNAc(P14)GlcNAc(P1-3) Fuc(al-3)/

\

7"""

Fuc(a li3f

Fuc(a 1-2)Fuc(a 14)Kdn(a24)

\

Fuc(a1-3).

/

Fuc(a1-2) /

GaWAc-ol

GalNAc(p14)GlcNAc(Pl-3) Fuc(al-3)/

/

GalNAc-01

/

Fuc(al4)Kdn(a2-3)Gal(~1-3f

Fig. 3 . Oligosaccharide-alditols released from the jelly coat of Ambystoma maculatum [26,27].

been found in Schistosoma mansoni membranes [34], human urokinase [35], sea squirt H allergenic antigen [36] and recombinant glycoproteins expressed in human kidney 293 cells [37]. These data confirmed the hypothesis above stated about the presence of particularly rare human carbohydrate chains in lower animal species. Nevertheless, the glycans observed in the other amphibian species represent novel sequences which reflect a natural polymorphism. The mucin of Ambystoma mexicanum was characterized by the presence of new sequences such as Gal(al4)[Fuc(al2)]Gal(P 14)GlcNAc (hybrid P 1/H blood group determinant) and Fuc(a 1-3)Fuc(a 14)Kdn (Fig. 2). Such a substituted Kdn unit has been also observed in A . maculatum (Fig. 3) and X . laeois (Fig. 5). More surprising was the finding of disubstituted Kdn in A . tigrinum (Fig. 4). New polyfucosyl sequences like the trimer Fuc(a1-2)[Fuc(al3)]Fuc(al4) were also observed in A . maculatum (Fig. 4). Other unusual sequences, namely the dimer H epitope Fuc(a 1-2)Ga1(8 1-3)[Fuc(a 1-2)]Gal, and blood group A determinant terminated with an additional a-1,3-linked GlcNAc unit, were found in X.Iaevis (Fig. 5 ) . The first analyses performed on urodeles species samples pointed to the exclusive presence of Kdn, indicating that this sugar could be considered as the potential marker of this subclass of amphibians. But it was rapidly shown that the presence of Kdn or Neu5Ac among urodeles or anurans was not correlated with one of these amphibian subclasses. X . laeois seems to be an exception among the species analyzed so far, since the analysis

168 Fuc(al-4)Kdn(a2-6)

Fuc(al-2)Gal(pl-3)

\ GalNAc-ol /

Fuc(a1-3) \ Gal(PI4)GlcNAc(P 1-6)

\

Fuc(al-5)\ ICdn(a2-3)Gal(pl-3) Fuc(al-4) /

Fuc(al-4)Kdn(a2-6)

Gal(a1-3)

\

7""""'

Fuc(a1-5) \ Kdn(a2-6) \ Fuc(al-4) / GalNAc-ol / Gal(a1-3f

GaINAc(a1-3) \ Gal(pl-4)GlcNAc(pl-6) Fuc(a1-2) / \ GalNAc-ol Fuc(a1-5) \ / Kdn(al-4)Gal(p1-3) Fuc(al-4) Fuc(al-3)\ Fuc(al-2)Gal(p 14)GlcNAc(p1-6)

\ Fuc(al-5), Fuc(al-%)Kdn(a2-6)

\

Kdn(al-4)Gal(pl-3)

Fuc(ul-4)

'

Fuc(al-Z)Gal(al-3) Fuc(al-5)\ Fuc(a1-5)

Fuc(al-4)

Kdnfa2-6)

\ Kdn(a2-3)Gal(P 1-3)GaINAc-ol /

Fuc(al-4)/

~

'\GalNAc-ol /

Fuc(alJ), Kdn(a2-3)Gal(pl-3f Fuc(al-4)'

Fuc(al-Z)Gal(p1-4)GlcNAc(~1-6)

\\ Fuc(a1-5) \ Kdn(a%3)Gal(pl-3) Fuc(al-4)/

Fig. 4. Oligosaccharide-alditolsreleased from the jelly coat of Ambystoma tigrinurn [28,29].

of a series of clutches showed the presence of NeuSAc, while other samples, produced by other X. laeois specimen contained exclusively Kdn. These discrepancies require further experiments, based on the analysis of clutches obtained under controlled physiological conditions. Perhaps the Xenopus genus, which can display the hybridism phenomenon among numerous species, needs to be better defined from a taxonomic point of view. In Rana temporaria, the anionic charge is carried by Kdn, and also by sulfate and glucuronic acid (Fig. 8). In Rana utricularia, traces of NeuSAc were characterized and the presence of glucuronic acid and sulfate has been confirmed. On the basis of these results, it remains impossible to correlate the presence of these acidic sugars with the taxonomy and phylogeny of amphibians.

I69 GlcNAc(P1-6)

\ GalNAool / Gal(P1-3)

GlcNAc(P1-6)

\

Gah‘Ac-ol

Gal(al-4)Gal(pl-3)Gal(~l-3) / Fuc(al-2)’Fuc(al-2)’

GlcNac(P1-6)

Fuc(al-2)Gal(al-3)

\

NeuAc(aZ4)

\

GalNAc-ol

/GalNAc-ol Fuc(al-Z)Gal(p 1-3)Gal(pl-3) fic(al-2)/

/

Gal(al-4)Gal(pl-3)GalNAc4 Fuc(a1-2)

/

’

Fuc(al-4)Kdn(a2-6)

Fuc(a1-3)

GalNAwl

GlcNAc(al-3)Gah‘Ac(aI-3)Gal(~1-3)GalNAc-ol Fuc(a 1-2)

GlcNac(p1-3)

/

Fuc(al-I)Kdn(a2-6) GlcNAc(P1-6)

\ / \

GalNAc-01

\

GalNAc(~l-3)Gal(~l4)GlcNAc(~l-3) / Fuc(al-2)/

Fuc(al-2)Gal(pl-3)Gal(~l-3)

/ Fuc(al-3)Fuc(al~)Kdn(~-6)

Fuc(a1-2)

\

Fuc(al-2)Gal(pl-3)Gal(~l-3)GalNAc-ol Fuc(a1-2) /

GalNAc-01

/ GaINAc(pl-3)Gal(P I-4)GlcNAc(p1-3) Fuc(al-Z)/

Fig. 5. Oligosaccharide-alditols released from the jelly coat of Xenopus laeuis [30,3 11

NeuAc(a2-6)

GlcNAc(P14)

\

\

/ Gal(pl-3)

/ F~c(~tl-2)Gal( pl-3)

GalNAc-ol

NeuAc(a2-6)

\

GalNAc-ol

Fuc(al-2)Gal( p 1-3 j

GalNAc-ol

Fuc(a 1-2)Gal( pl-3)GalNAc-ol

/

Fig. 6 . Oligosaccharide-alditols released from the jelly coat of Bufo japonicus japonicus [ 101

4. Concluding remarks The starting hypothesis that amphibian egg jellies could be a good source of carbohydrates has been shown to be correct. Moreover, the tissues of the animals and in particular the oviducts could also be the source of new and specific glycosyltransferases. The structures listed in Figs. 1-8 allow us to speculate about the activity levels and specificities of transferases in the glycosylation process of oviducal mucins. In fact, referring only to the fucosyltransferase activities, a minimum of six enzymes such as Kdn:(a I-4)FucT, Fuc(a 14)Kdn:(a l-2)FucT, Fuc(a 1-4)Kdn:(a I-3)FucT, Kdn:(a 1-

170

Fig. 7. Oligosaccharide-alditols released from the jelly coat of Bufo bufo [32].

S)FucT, Gal(a 1-3)Gal:(a I-2)FucT and Gal(a l-3)[Fuc(a l-2)]Gal:(a 1-2)FucT can be postulated. As a further attempt to correlate the structure of the enzyme with the specific activity, such a model should be investigated by molecular cloning of this family of fucosyltransferases. Until now, no experimental results support the hypothesis of a carbohydrate-mediated species-specific sperm binding for amphibians, similar to that demonstrated for mammalians. Further studies will probably indicate if the pattern of glycan structures found in each amphibian species represent the support for a specific recognition of the ovule by the spermatozoon. At least, these findings create a field for the hypothesis, that carbohydrate structures could be used as a specific taxonomic marker. However further confirmation should come through the study of other species. The data also point to the diversity of novel glycosyltransferase activities which remain to be studied with regard to animal evolution.

References [ I ] Kurosaka, A,, Nakajima, H., Funakoski, I., Matsuyama, M., Nagayo, T. and Yamashina, 1. (1983) J. Biol. Chem. 258, 11594-1 1598. [2] Hounsell, E.F., Lawson, A.M., Feeney, J., Gooi, H.C., Pickering, N.J., Stoll, M.S., Lui, S.C. and Feizi, T. (1985) Eur. J. Biochem. 148, 367-377. [3] Capon, C., Leroy, Y., Wieruszeski, J.M., Ricart, G., Strecker, G., Montreuil, J. and Fournet, B. (1989) Eur. J. Biochem. 182, 139-1 52. [4] Wieruszeski, J.M., Michalski, J.C., Montreuil, J., Strecker, G., Peter-Katalinic, J., van Halbeek, H., Mutsears, J.H.G.M. and Vliegenthart, J.F.G. (1987) J. Biol. Chem. 262, 6650-6657.

GalNAc-ol

GalNAc-ol

HS03(6)

/ Gal(p1-3)

Fuc(a1-3) /GlcNAc(P14)

HS03(3)/ Gal(P1-3)

Gal(p1-4)Gal(p1-3) /GalNAcq1

/

Kdn(a2-6)

\

1

Gal(p1-3)/ Fuc(a1-2)

HS03(3)/ Gal@14)Gal(p1-3) / / HS03(3) Fuc(a1-2)

GalNAc-ol

Kdn(a2-6)

GalNAc-ol Gal(pl-4)S;al(pl-3)

\

GalNAc-ol

Gal(a1-3) Kdn(a2-6)

/

/ Gal(P1-3) /GalNAC-O1

I

HS03(3)/Gal(pl-3) Fuc(a1-2)

1

GalNAc-ol Gal(pl-4)Gal(Pl-3) HSO3(3)’Gal@1-3) /

\

1

/

GlcA(P1-3)/

Gal(p1-3{ F W a 1-2)

1

Fuc(a1-2)

I Fuc(a1-2) Fuc(a1-2)

Gal(p1-3)

Fuc(a1-2) /GlcNAc@14)

HS03(6)

\

Kdn(a2-6)

GlcNAc(P 1-6)

\

Gal(a1-3) Fuc(a1-2)

\

GalNAc-ol

GalNAc-ol /

\

Gal(P1-3)

GalNAc-ol Gal(a1-3)

\

/

Gal(p1-3)

Fuc(a1-2)

/

/

Fig. 8. Oligosaccharide-alditols released from the jelly coat of Rana temporaria [33].

Gal(P1-3) Gal(Pl-3)/ / GlcA(P1-3) Fuc(a1-2)

1

1

Fuc(a1-2)

\

172 [5] van Halbeek, H., Dorland, L., Vliegenthart, J.F.G., Kochetkov, N.K., Arbatsky, N.F! and Derevitskaya, V.A. (1982) Eur. J. Biochem. 127, 21-28. [6] Caroll, E.J., Wei, S.H., Nagel, G.M. and Ruibal, R. (1991) Develop. Growth Differ. 33, 3 7 4 3 . [7] Bolognani, L., Bolognani-Fantin, A.M., Lusignani, R. and Tonta, L. (1966) Experientia 22, 601. [8] Jego, P. (1974) Comput. Biochem. Physiol. 47, 435446. [9] Freeman, S.B. (1968) Biol. Bull. 135, 501-513. [lo] Shimoda, Y., Kitajima, K., Inoue, S. and Inoue, Y. (1994) Eur. J. Biochem. 223, 223-231. [ l l ] Jego, P., Jolly, J. and Boisseau, C. (1980) Reproduct. Nutr. Develop. 20, 557-567. [I21 Katagari, C. (1987) Zool. Sci. 4, 1-14. [I31 Hedrick, J.L. and Nishihara, T. (1991) J. Electron Microsc. Tech. 17, 319-335. [14] Katagari, C. (1966) Embryologia 9, 159-169. [15] Barbieri, ED. and Raisman, J.S. (1969) Embryologia, 10, 363-372. [16] Elinson, R.P. (1971) J. Exp. Zool. 176, 415428. [I71 Ishihara, K., Hosono, J., Kanatani, H. and Katagari, C. (1984) Develop. Biol. 105, 435442. [l8] Wyrick, R.E., Nishihara, T. and Hedrick, J.L. (1974) Proc. Natl. Acad. Sci. USA 71, 2067-2071. [19] Prody, G.A., Greve, G.A. and Hedrick, J.L. (1985) J. Exp. Zool. 235, 335-340. [20] Hoffman, W. (1988) J. Biol. Chem. 263, 63104316. [21] Probst, J.C., Hauser, F., Joba, W. and Hoffman, W. (1990) Biochemistry 29, 6240-6244. [22] Hoffman, W. and Joba, W. (1995) Biochem. SOC.Trans. 23, 805-810. [23] Inoue, Y. and Inoue, S. (1997) ch. 7, this volume. [24] Strecker, G., Wieruszeski, J.M., Alonso, C., Michalski, J.C., Boilly, B. and Montreuil, J. (1992) FEBS Lett. 298, 3 9 4 3 . [25] Strecker, G., Wieruszeski, J.M., Michalski, J.C., Alonso, C., Leroy, Y., Boilly, B. and Montreuil, J. (1992) Eur. J. Biochem. 207, 995-1002. [26] Strecker, G., Wieruszeski, J.M., Fontaine, M.D. and Plancke, Y. (1994) Glycobiology 4, 604-609. [27] Fontaine, M.D., Wieruszeski, J.M., Plancke, Y., Delplace, F. and Strecker, G. (1995) Eur. J. Biochem. 231, 424-433, [28] Maes, E., Wieruszeski, J.M., Plancke, Y. and Strecker, G. (1995) FEBS Lett. 358, 205-210. [29] Maes, E., Plancke, Y., Delplace, F. and Strecker, G. (1995) Eur. J. Biochem. 230, 146-156. [30] Strecker, G., Wieruszeski, J.M., Plancke, Y. and Boilly, B. (1995) Glycobiology 5, 137-146. [31] Plancke, Y., Wieruszeski, J.M., Alonso, C., Boilly, B. and Strecker, G . (1995) Eur. J. Biochem. 231, 434439. [32] MoreIle, W. and Strecker, G.(1997) Glycobiology, in press. [33] Maes, E., Florea, D., Delplace, F., Lemoine, J., Plancke, Y. and Strecker, G. (1996) Glycoconjugate J. 14, 127-146. [34] Srivatsan, J., Smith, D.F. and Cummings, R.D. (1992) Glycobiology 2, 445452. [35] BergwerR, A.A., Thomas-Oates, J.E., van Oostrum, J., Kamerling, J.P. and Vliegenthart, J.F.G. (1992) FEBS Lett. 314, 389-394. [36] Ohta, M., Matsuura, F., Kobayashi, Y., Shigata, S., Ono, K. and Oka, S. (1991) Arch. Biophys. Biochem. 290, 474483. [37] Yan, S.B., Chao, Y.B. and van Halbeek, H. (1993) Glycobiology 3, 597-609.

J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins II

0 1997 Elsevier Science B.V. All rights reserved CHAPTER 9

Blood glycoproteins* Kenneth J. Clemetson Theodor Kocher Institute, University of Berne, Berne, Switzerland

Abbreviations AAT

a1-antitrypsin

AGP apo ATIII

a l -acid glycoprotein apolipoprotein antithrombin 111

HPLC LCA

Lens culinaris agglutinin

LCAT MAA

1ecithin:cholesterol acyltransferase Maackia amurensis agglutinin

AZ

Alzheimer’s disease

MCTD mixed connective tissue disease

CEA

carcinoembryonic antigen

high performance liquid chromatography

cs

consensus sequence

Onf PM

CSF

cerebrospinal fluid

RA

Con A DSA EGF ESIMS FJ Fn GBF GP HP

concanavalin A

SCL SDS SF

synovial fluid

SLE SNA

Sambucus nigra agglutinin

Datura stramonium agglutinin

epidermal growth factor electrospray ionization mass spectrometry factor J fibronectin

ss

oncofetal polymyositis rheumatoid arthritis scleroderma sodium dodecyl sulfate systemic lupus erythematosus Sjogren’s syndrome wheat germ agglutinin

gelatin-binding fragment

WGA

glycoprotein

WHHL Watanabe heritable hyperlipidemic

hepatopathies

1. Introduction Blood consists of a liquid, plasma, in which a variety of cells circulate. It comes into contact with the lining of the vasculature, consisting of endothelial cells which normally provide a non-thrombotic surface. The plasma contains a very large number of different proteins of which only one, albumin, is known not to be glycosylated. The cells circulating in blood are erythrocytes, responsible for oxygen and carbon dioxide transport, platelets, with a basic responsibility for haemostasis and maintaining the integrity and function of the vascular system and with less well-defined roles in inflammation and defence against parasites, and leukocytes; these last are divided into several categories: lymphocytes, neutrophils, basophils, eosinophils and monocytes. The leukocytes are all critical in the body’s defence against bacteria, viruses and parasites. The endothelial cells provide the non-thrombogenic coating to the vasculature, secrete factors acting on the other blood cells and have an important repair function. As with other cells, all these blood cells have a plasma membrane, containing glycoproteins with a wide variety of functions essential for a normal physiology. These include adhesive receptors, agonist receptors and molecules with protective or housekeeping functions. ~

~~~

This chapter is dedicated to Prof. R.U. Lemieux who played a major role in awakening a whole generation to the importance of carbohydrate structure in biology. 173

114

Gal(fi14)GlcNAc(fi14)

\

Gal@14)GlcNAc(fi 1-2)Man(a 1-6) Fuc(a 1-3),-3

\

FuW+,

Man(BI4)GlcNAc(fi14)GlcNAc(fiI-N)Asn (B) I

Gal@14)GlcNAc(fi1-2)Man(a 1-3) I Gal(fi14)GlcNAc(fi1-6)

2. Plasma proteins Plasma contains minor amounts of a wide range of glycoproteins, involved in immunology, protease inhibition, transport and other functions, and mostly synthesized in the liver [I]. Some major components are listed below.

2. I . al-Acid glycoprotein al-Acid glycoprotein (AGP), also called orosomucoid, is one of the major plasma proteins and has been shown to bind a number of basic drugs. Nevertheless, its function remains obscure. Despite preparations with confirmed purity at the protein level it was demonstrated fairly early that it showed charge microheterogeneity [2] which could be ascribed to glycosylation differences. It has a high carbohydrate content (40%) with 5 N-linked oligosaccharide chains. The structure of these chains includes di-, tri- and tetraantennary types with and without fucosc residues (Fig. 1, 2A) [3-71. These chains are extensively sialylated, accounting for the acidic PI of this molecule and also give rise to the extensive charge heterogeneity. During the acute-phase immunological reaction the forms with complex glycans arc produced preferentially over the diantennary forms. Glycosylation variations of AGP have been found in various disease states. AGP from ascitic fluid of patients with stomach cancer was separated by chromatography on Con A-Sepharose into Con A-nonbound (AGP-21, 43, 5kDa, 70%) and Con A-bound (AGP-2, 41.5kDa, 24% and AGP-3, 40.0 kDa, 5%) forms, differing in monosaccharide composition [8]. Comparative study of N-glycan structures by HPLC of fluorescence-labelled oligosaccharides showed that the molecular forms differ in the ratio of the di-, tri-, and tetraantennary complex type carbohydrate N-chains. The molecular forms of AGP differ from nAGP in amounts of Lewis X (Lex)-fragments and agalacto-oligosaccharides. Plasma protein glycoforms rich in diantennary complex type N-glycans (type I) increase in acute infections, while in some diseases with chronic inflammatory changes, more branched N-glycans (type 11) increase. In sera from 109 human immunodeficiency

175

virus (H1V)-infected persons, 38 rheumatoid arthritis patients, and 44 healthy subjects, the composition of AGP glycoforms was studied using crossed immunoaffinity electrophoresis with concanavalin A (Con A) as a ligand [9]. In patients with chronic inflammatory changes classifications I, 11, and 111, the distribution of AGP glycoforms was analogous to that in normal subjects. Type I alterations were observed in patients in group IV who had no signs of arthritis. Type I1 changes, analogous to those found in rheumatoid arthritis, were seen in group IV patients who developed arthritis. Most significant type I changes were associated with Pneumocystis carinii pneumonia. Rat plasma glycoproteins, but not human, contain terminal 0-acetyl, N-glycolylneuraminic acids in the carbohydrate side-chains of “acute-phase” glycoproteins (predominantly AGP) [ 101. The microheterogeneity of AGP has been studied in 43 patients with early rheumatoid arthritis (RA) without clinical features of intercurrent infection [ 111. In contrast to previous reports, suggesting a decrease in Con A reactivity in patients with RA, high values of AGP reactivity with Con A were found in patients with a disease of short duration, similar to those in patients with acute bacterial infections. Conversely, normal or decreased values of AGP reactivity coefficients were found in patients with a disease of longer duration. Together with previous findings suggesting that cytokines control the glycosylation of acute phase proteins, these results indicate that differences in the microheterogeneity of AGP in early and longstanding RA reflect differences in cytokine action at different stages of the disease. Acute inflammation as well as cirrhosis induces increases in a-1,3-fucosylated AGP molecules, detected first of all by decreased binding to Con A [12,13] and later by the reactivity of AGP, presumably containing three or more fucosylated N-acetyllactosamine units, towards the hcose-binding Aleuria aurantia lectin in crossed affino-immunoelectrophoresis of human sera [14]. In at least part of these Lewis Xtype glycans (Gal~I-[Fuca1-3]GlcNAc-R) appeared to be substituted also with an a-2,3linked sialic acid residue. The structures of a fucosylated tetrasialyl oligosaccharide from cirrhotic AGP [ 12,131 is shown in Fig. 2B and should be compared with the structure from healthy donors in Fig. 2A. Acute inflammation induces a strong increase in sialyl Lewis Xsubstituted AGP molecules that persist at a high level throughout the inflammatory period. These changes may represent a physiological feedback response to the interaction between leukocytes and inflamed endothelium, mediated via sialylated Lewis X structures and the selectin endothelial-leukocyte adhesion molecule. The reducing oligosaccharides released from AGP by conventional hydrazinolysis have been analyzed [ 151. At least 13 different asialo-N-glycans were detected. The carbohydrate structures were assigned by comparison with the known AGP carbohydrate structures and known N-glycan structures by comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, a number of sulfated N-glycans were also tentatively identified. Serum and synovial fluid have been obtained at the same time from 22 patients with rheumatoid arthntis and analyzed for microheterogeneity of a 1-acid-glycoprotein [ 161. In most samples the glycosylation pattern was similar, a nonreactive variant and two Con A reactive variants (the first and the second). In seven samples of synovial fluid an extra third peak was observed representative of the fraction strongly reactive with Con A.

176

NeuSAc(a2-3)Gal(~l4)GlcNAc(~l 4)

\

NeuSAc(a24)Gal(fi14)GlcNAc(fi1-2)Man(a1-6) \

\

Fuc(a1-3)

Man(filM)GIcNAc(fi14)GlcNAc(fil-N)Asn

I

\ -

Neu5Ac(a2-3)Gal(fi14)GlcNAc(fil-2)Man(a 1-3)

(A)

I

I

NeuSAc(a2-6)Gal(fi14)GlcNAc(fil-6) Fuc(a 1-3)

\

Neu5Ac(a2-3)Gal(fi14)GlcNAc(fil4)

\

NeuSAc(a2-6)Gal(fi14)GlcNAc(fiI-2)Man(a 1-6)

\ Fuc(a 1-3)

Man(PI4)GlcNAc(fil4)GlcNAc(fiI-N)Asn (B) \

NeuSAc(a2-3)Gal(P 14)GlcNAc(fi1-2)Man(a 1-3) NeuSAc(a2-6)Gal(fi14)GlcNAc(fi 1-6)

I

I

Fig. 2. Highly fucosylated tetraantennary glycans from a 1 -acid glycoprotein from a cirrhotic patient.

AGP contains complex di-, tri- and tetraantennary glycan chains and can be fractionated into three molecular variants using Con A-lectin chromatography based on variations in these structures. Standard HPLC profiles have been developed to analyze the percentage and distribution of the glycoforms present at each glycosylation site in AGP and its molecular variants [17]. The proportions of di-, tri- and tetraantennary glycans differ at each site for the three molecular variants. The most strongly retained variant from Con A has diantennary glycans at all five sites, whereas the unretained variant is completely devoid of diantennary structures. Only glycosylation site I1 of the five present is completely diantennary in the retained and weakly retained variants. In addition, the two gene products of AGP were glycosylated differently. The “site-directed” model of processing offers the most consistent explanation for the structures seen at the individual glycosylation sites of AGP.

2.2. Antithrombin III Antithrombin 111 (ATIII) is an a2-globulin with a mass of about 58kDa made up of about 425 amino acids. It has a high degree of sequence similarity with al-antitrypsin suggesting a common origin. Two isoforms occur naturally in human plasma. The a-AT111 isoform has four N-glycans attached to Asn 96, 135, 155, and 192. The p-ATIII isoform lacks carbohydrate on Asn 135 (N135), which is near the heparin binding site, and binds heparin with higher affinity than does a-ATIII. Two isoforms (a’ and are also produced when the normal human ATIII cDNA sequence is expressed in baculovirus-infected insect cells, and the recombinant fi-isoform also binds heparin with higher affinity than the recombinant a-isoform. Consensus sequences (CSs) of the ATIII N-glycosylation sites are N-X-S for 135 and N-X-T for 96, 155, and 192. Database and in oitro glycosylation studies suggest that N-X-S CSs are used less efficiently than N-X-T CSs. The 0-ATIII isoform might result from inefficient core glycosylation of the

B’)

177

N135 N-X-S CS due to the presence of a serine, rather than a threonine, in the third position. ATIIIs with N-X-S, N-X-T, and N-X-A consensus sequences were expressed in baculovirus-infected insect cells. In contrast to the N-X-S sequence, which expressed a mixture of a’ and p’ molecules, the N-X-T variant produced a’ exclusively, while the N-X-A variant produced fi’exclusively. Serine in the third position of the N135 CS is responsible for “partial” glycosylation and leads to production of p-ATIII [ 181.

2.3. al-Antitrypsin

a 1-Antitrypsin (Mr 54 kDa, 394 amino acids) is a major plasma component (290 mg/ml) with an important protease inhibitor activity. The carbohydrate content is about 12% [ 191. This glycoprotein is initially synthesized as a 49 kDa single polypeptide chain with the high mannose core oligosaccharide structure GlqMang GlcNAc linked to Asn at 46, 83, and 247. The mature glycoprotein is 54kDa with complex type glycans. Two types of carbohydrate chains are present in normal individuals, diantennary and triantennary glycans (Fig. 3). Gal(fi1-4)GlcNAc(fi1-2)Man(a1-6)

\ [GlcNAc(fiI

I Man@ 14)GlcNAc(fi 14)GlcNAc

Gal(fi14)GlcNAc(fil-Z)Man(a 1-3)

(A)

I

Gal@ 1-4)GlcNAc(P 1-2) \

Man(a1-3) Gal@ 14)GlcNAc(fi1-4)I

\ Man(fi14)GlcNAc(fi 1-4)GlcNAc

(B)

I

Gal(fi1-4)GlcNAc(~1-2)Man(a1-6)’ Fig. 3. (A) Di- and (B) triantennary glycans from antitrypsin.

A diantennary chain with a bisecting N-acetylglucosamine residue was also detected. The chains are present in 80% diantennary, 14% diantennary chain with a bisecting N-acetylglucosamine residue and 6% triantennary, respectively [20]. In hepatocellular carcinoma a diantennary carbohydrate chain with a fucose residue at the innermost N-acetylglucosamine residue was also detected which had a higher affinity for Lens culinaris agglutinin and appeared characteristic for this disease. Alveolar epithelial cells were shown to produce a 1-antitrypsin (AAT) with a modified glycosylation compared to serum AAT [21]. A monoclonal antibody against a specific epitope on al-antitrypsin was shown to be a valuable diagnostic marker for autoimmune conditions [22]. Evidence suggests that this epitope is influenced by the glycosylation of the molecule implying that this is altered in these disorders. In patients with cystic fibrosis[23] significant glycosylation changes in serum al-AT were only seen with free Con A and WGA; this probably results from a reduced synthesis of the diantennary side-chains or by their increased catabolism. Changes in meconium AAT glycosylation were more pronounced with free Con A and LCA. These differences may be useful in the diagnosis of cystic

178

fibrosis. a 1-Antitrypsin was among those hepatic plasma glycoproteins affected in chronic alcoholics, compared to controls, by differences in glycosylation that could be removed by treatment with sialidase [24,25].

2.4. Apolipoproteins Plasma contains several apolipoproteins, involved in lipid and cholesterol transport. Several have been reported to be glycosylated in low amounts including apoB, apoE and apoC-I11[26]. ApoB contains about 2-2.5% carbohydrate as complex-type N-glycan whereas apoE appears to contain O-linked glycan [27]. ApoE is synthesized in sialylated form but in plasma it is 80% desialylated. Site-specific structural characterization of the glycosylation of human 1ecithin:cholesterol acyltransferase (LCAT) has been carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLUESIMS) [28]. Monitoring of carbohydrate-specific fragment ions during HPLCESIMS located eight different groups of glycopeptides in a human LCAT protein digest. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn 20, Asn 84, Asn 272, and Asn 384) of LCAT contained sialylated triantennary andor diantennary complex structures. Two unexpected O-linked glycosylation sites at Thr 407 and Ser 409 of the LCAT O-linked glycopeptide were identified, each with sialylated Gal(@1-3)N-GalNAc structures. The three additional glycopeptides were from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn 45 and Asn 78. These glycopeptides bear sialylated triantennary oligosaccharides or fucosylated sialylated diantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn 272 converts this protein into a phospholipase [29]. Most apolipoprotein B (apoB) in rat hepatocyte membranes is membrane-bound. Various data based on glycosylation specific monoclonal antibodies and carbohydratespecific labelling suggest that membrane-bound apoB is more glycosylated than plasma lipoprotein apoB [30]. N-glycans of apolipoprotein (apo) B- 100 in Watanabe heritable hyperlipidemic (WHHL) rabbit and fasting Japanese White rabbits are heterogeneous [3 I]. The N-glycans of apo B-100 consist of one neutral (N) and two acidic fractions (A1 and A2). N contained a high mannose type oligosaccharide consisting of MansGlcNAq to Man9 GlcNAcz, while A 1 and A2 contained monosialylated and disialylated complex type oligosaccharides, respectively. The molar ratio varied among the five WHHL rabbits. There was an inverse correlation between the ratio of acidic oligosaccharide fractions (A1 +A2) and serum cholesterol levels (r=-0.971, P less than 0.01) in the five WHHL rabbits. The N-glycosylation of apo B-1 00 is closely related to cholesterol metabolism in WHHL rabbits. ApoD consists of 169 amino acid residues, including five cysteines. Cys41 and Cysl6 are joined by a disulfide bridge. There is an intramolecular disulfide bridge between Cys8

179

and Cysll4 and an intermolecular bridge between Cysll6 of apoD and Cys6 of apoA-I1 [32]. N-glycosylation sites were found at Asn 45 and Asn 78. Apolipoprotein F has a molecular mass of 29 kDa and is composed of 162 amino acids. The cDNA sequence predicts that apolipoprotein F is a proteolytic product of a larger protein [33]. 2.5. Ceruloplasmin Ceruloplasmin is an important copper-containing glycoprotein enzyme (ferroxidase) involved in oxidation of Fe2+ to Fe3+ so that it can be transported by serotransferrin. Some mutations in the ceruloplasmin gene are associated with severe iron metabolism and distribution disorders leading to massive iron accumulation in the liver, brain and pancreas as well as to retinal problems and diabetes. Ceruloplasmin also has an important anti-oxidant function in protecting tissue against damage caused by free oxygen radicals. It is secreted as a holoprotein with six atoms of copper incorporated and has a molecular mass of 132 kDa and 8% carbohydrate content. Seven potential N-glycosylation sites exist but only four of these are occupied by oligosaccharides of the di- or triantennary type on which fucose may be present (Fig. 4) [34,35]. Differences in the oligosaccharide structure are responsible for the microheterogeneity on isoelectric focussing. NeuSAc(a2-6)Gal(fi14)GlcNAc(fi1-2)Man(a 1-3)

\

Fuc(al-6),,-,

Man(fiI4)GlcNAc(fi14)GlcNAc(fil -N)Asn

NeuSAc(a2-6)Gal(fiI 4)GlcNAc(flI-2)Man(a 1-6) Fuc(al-3)

,

(A)

o-l

\

Neu5A~(a2-3/6),_~Gal(fi14)GlcNAc((ll4)

\

NeuSAc(a24)Gal(fi14)GlcNAc(fil-2)Man(a 1-3)

\ Man(fi14)GlcNAc(fi 14)GlcNAc@-N)Asn

(B)

Neu5Ac(a2-6)GaI(fl14)GlcNAc(fiI-2)Man(a14~ Fig. 4.(A) Di- and (B) tnantennary glycans from cemloplasmin

2.6. CI inhibitor

Carbohydrate chains of Cl-inhibitor were identified by a binding assay using different lectins [36]. Lectins from Sumbucus nigru (SNA) and Muuckiu umurensis (MAA), that are specific for sialic acids, were shown to bind to C1-inhibitor. Lectin from Duturu stramonium (DSA) also reacted with the inhibitor indicating complex and hybrid sugar structures. C l-inhibitor was enzymatically desialylated and reexamined for lectin binding. SNA and MAA did not react anymore, but in addition to DSA, peanut agglutinin, which can bind to carbohydrate chains after sialic acids are removed, bound to desialylated C1-inhibitor. C l-inhibitor contains about 30 sialic acid residues per molecule. SDS-

180

NeuSAc(a24)Gal@ 14)GlcNAc@1-2)Man(a 1-3)

\ Fuc(a1-6), 0.3 Man(@14)GlcNAc(P14)GlcNAc(P1-N)Asn

(A)

I

NeuSAc(a2-3)Gal(fi 14)GlcNAc(P1-2)Man(a 1-3)

\

Fuc(a1-6),

0.3

Man(~I4)GlcNAc(@I4)GlcNAc(~I-N)Asn(9) Neu5Ac(a2-3)Gal(f1 14)GlcNAc(@1-2)Man(a 1-6f Neu5Ac(a2-3)Gal(f1-3)GalNAcol

(C)

Fig. 5. (A,B) Diantennary glycans and (C) 0-glycan from CI inhibitor

polyacrylamide gel electrophoresis showed that desialylated C 1-inhibitor had a higher mobility than native C 1-inhibitor. The N-terminal sequence of desialylated C 1-inhibitor was the same as that of native C1-inhibitor and no change in the inhibition of human plasma kallikrein was observed. The structure of the N- and 0-linked glycans of C1 inhibitor have been established by NMR spectroscopy and are shown in Fig. 5 [37]. 2.7. Complement 2.7.I . Complement C3 Of the 30 distinct complement proteins recognized to date, C3 is probably the most versatile and multifunctional molecule known, interacting with at least 20 different proteins. It plays a critical role in both pathways of complement activation and participates in phagocytic and immunoregulatory processes. Structural and functional analysis of C3 from different species, in addition to phylogenetic information, provides insights into the structural elements mediating the various functions. cDNA for human C3 has been cloned and its functional sites analyzed [38]. C3 consists of an a- (992 amino acids) and a fi-chain (645 amino acids) with a calculated molecular mass of about 181.5 kDa. The sequence contains three potential N-glycosylation sites: two on the a- and one on the b-chain of C3. One site on each chain is known to be glycosylated. 2.8. Factor J complement inhibitor Factor J (FJ), a new inhibitor of the complement system is a cationic molecule (PI 3 9.6 under native conditions, or PI = 8.1 under denaturing conditions) with a high carbohydrate content (40%) that is able to interact with different lectins, suggesting complex glycosylation [39]. Digestions with different proteinases did not affect activity. After b-glucuronidase digestion, FJ lost 80% of its initial activity. Consequently, glycosylation plays an important role in the inhibitory activity of FJ. 2.9. Factor V Coagulation factor V is a glycoprotein essential for haemostasis by accelerating the activation of prothrombin. Factor V is secreted as a single-chain polypeptide of 330 kDa

181

and is cleaved by the serine proteases thrombin or factor Xa to release B domain peptides of 71 and 150kDa to give the active species, factor Va. Factor Va contains the aminoterminal 94 kDa fragment associated with the 74 kDa carboxy-terminal fragment held together by divalent cations. The plasma concentration is about 8pg/ml and it is also found in platelet a-granules. The B-domain of factor removed during activation, contains 25 potential N-glycosylation sites. Inhibition of N-glycan addition with tunicamycin prevented secretion of factor V but an inhibitor of complex oligosaccharide addition, deoxymannojirimycin, did not affect secretion, although the specific activity of factor V was slightly increased. Thus, complex oligosaccharide addition was not required for secretion or functional activity of factor V. A23187 treatment inhibited addition of serinehhreonine 0-linked glycans to factor V [40].

2.10. Factor VII Factor VII is a single-chain, multidomain, vitamin K-dependent, plasma glycoprotein (50kDa) involved in the extrinsic pathway of blood coagulation. The plasma concentration is about 2pglml. Single-chain factor VII is converted into the two-chain serine protease factor VIIa by cleavage at Arg 152-Ile 153 by several coagulation proteases such as XIIa, IXa, Xa or thrombin. Factor VII is N-glycosylated at Asn 145 and Asn 322 [41] and 0-glycosylated at Ser 52 and Ser 60 in an EGF-like domain. The Ser 52 linked oligosaccharide was shown to be Xyll-zGlc-. A single fucose residue was found linked to Ser 60. Bovine factor VII is similarly glycosylated [42].

2.11. Factor VIII Factor VIII is a complex, plasma glycoprotein involved in blood coagulation and is processed intracellularly to yield a metal-ion-associated heterodimer of three chains, 85, 89 and 93 kDa, respectively, stabilized through association with von Willebrand factor. The plasma concentration is about 0.2 pg/ml. Deficiency leads to haemophilia A. Factor VIII is also deficient in certain types of von Willebrand’s disease due to the lack of the carrier molecule and may be responsible for some of the symptoms in that disorder. The asparagine-linked carbohydrate chains of blood coagulation factor VIII preparations were released as oligosaccharides by hydrazinolysis and purified from human plasma of blood group A donors [43]. Structural study of the oligosaccharides by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that factor VIII preparations contain mainly high mannose type and di-, tri-, and tetraantennary complextype glycans (Fig. 6). Some of the diantennary complex-type glycans from human plasma factor VIII contain blood group A and/or H determinants. A small number of the triantennary complex-type glycans contain the Gal@ 14)[Fuc(a 1-3)]GlcNAc(fll4)[Gal(~l-4)GlcNAc(fll-2)]Man(a1-3) group. The Asn-linked glycans of factor VIII purified from porcine plasma were released as oligosaccharides by hydrazinolysis [44]. Structural study of each oligosaccharide by sequential exoglycosidase digestion and by methylation analysis revealed that porcine factor VIII, like the human, contains high mannose-type and di-, tri- and tetraantennary complex-type glycans. Sixty-seven percent of the complex-type glycans contained the

182

I

Gal(fil4)GlcNAc(~l-2)Man(al-3)

\

Fuc(al-6)

Man(~l4)GlcNAc(~14)GlcNAc(BI-N)Asn(A)

Neu5Ac(a2-3/6)0-2

/

Fuc(a 1-2)Gal(fi14)GlcNAc(fiI -2)Man(a 1-6) Gal@ I4)GlcNAc(fi I -2)Man(a 1-3)

\

Fuc(a1-6)

\

0-1

Man(BI4)GlcNAc(fiI4)GlcNAc(fiI-N)Asn (B) Gal@ 14)GlcNAc(fil-2)Man(al4)

I

Gal(~l4)GlcNAc(~l-2)Man(al-3) Gal(a 1-3)l-z

\

Neu5Ac(a2-3/6)0-1 Gal@ I4)GlcNAc(B I -2)Man(a 1-6)

I

Man(al-6) Man(a1-3) / Man(al-3)

I Gal@ l4)GlcNAc(fi 1-3)o-1 Neu5Ac(a2-316)&3

\

0-1

(C)

/

\ Man(Bl4)GlcNAc(~l4)GlcNAc(fiI-N)Asn(D)

Man(al-2)o~

1

Fuc(al-6)

Man(fil4)GlcNAc(fi I 4)GlcNAc(BI -N)Asn

/

Gal(fi14)GlcNAc(fi1-2)Man(a I -3/6) Gal@ I 4)GlcNAc(B 1-6)

\ Gal(~l4)GlcNAc(fil-2)i Gal(Bl4)GlcNAc(Bl 4),

\

Fuc(al-6)

1 . 0

\

Man(Bl4)GlcNAc(BI4)GlcNAc(BI-N)Asn

(G)

I

Man(a I -3/6)'

I Gal@ I4)GlcNAc(fi1-2)' Gal((3I4)GlcNAc(fi I-2)Man(al-6/3)

\ Ga1(~14)GlcNAc(~14) \

Man(al-316)

Fuc(al-6)

\

0-1

Man(B14)GlcNAc(B14)GlcNAc(fiI-N)Asn (H) /

I

Gal(B14)GlcNAc(f31-2)

Gal(fil4)GlcNAc(fi1-2)Man(al-6/3)

\

Fuc(al-6) o-l \

Man(fi I 4)GlcNAc(B I 4)GlcNAc(B I -N)Asn Gal(fiI4)GlcNAc(fiI4) / \ / Fuc(a1-3) Man(al-3/6)

(I)

/

Gal@14)GlcNAc@I -2)

Fig. 6 . (A-C,E) Di-, (F,H,I) tn- and (C) tetraantennary complex type and (D) high mannose type triantennary glycans from factor VIII.

183

Gal(a1-3)Gal group and 23% of the diantennary complex-type glycans contained the bisecting GlcNAc residue. These structures were not detected in the glycans of human plasma factor VIII. In uitro competition of von Willebrand factor and anti-Gal antibody for binding to factor VIII revealed that von Willebrand factor prevented antibody binding to Gal(a 13)Gal groups in porcine factor VIII glycans. This suggests that anti-Gal antibody present in human plasma may not interact with the glycans of porcine factor VIII used in treatment of haemophilia. Studies of pharmacokinetic parameters of recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma-derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in uiuo [43]. In transfected mammalian cells inhibition of N-glycan addition by treatment with tunicamycin prevented secretion of factor VIII, whereas treatment with an inhibitor of complex oligosaccharide biosynthesis, deoxymannojirimycin, did not affect secretion, although the specific activity of factor VIII was slightly increased [45]. Thus, the presence of complex oligosaccharide was not required for secretion or functional activity of factor VIII. A23 187 treatment inhibited addition of serinehhreonine (0)-linked glycans to factor VIII. Factor VIII has been expressed in Spodopterufrugiperdu insect cells [46]. The construct retained the native signal sequence to allow secretion of recombinant protein into the culture medium. Initial studies revealed the production of secreted factor VIII with coagulation activity. The presence of N-glycans was demonstrated since the glycosylated molecule is similar in size to that expressed in mammalian cells.

2.12. Factor IX Factor IX (Christmas factor) is a plasma glycoprotein with a critical role in blood coagulation. Deficiency causes haemophilia B or Christmas disease, a severe bleeding disorder. Factor IX has a mass of 55 400 Da and activated IXa 46 500 Da. The normal concentration in plasma is 3 4 y g / m l . 0-Glycans have been identified in the activation peptide of human blood coagulation factor IX [47]. Gal-GalNAc-Thr, NeuNAc-(Gal-)GalNAc-Thr, and NeuNAc-Gal-GalNAc-Thr structures were 0-glycosidically linked to Thr 159 and 169 present on 35% of the total amount of activation peptide in circulating blood. 0and N-linked glycans were also released with hydrazine and analyzed [48]. Glycans were identified as mono- and disialyl Gal@ 1-3)GalNAc by two-dimensional HPLC mapping and as NeuSAc(a2-6)Gal(P 14)GlcNAc@I-3)Fuc by exoglycosidase digestion, methylation analysis, and Smith degradation. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing Ser 61 in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose [49,50]. This structure attached to Ser 61 was also shown by hydrazinolysis of a peptide from this region followed by pyridylamination indicating that the reducing end was PA-Fuc. The results indicated that human factor IX has a novel tetrasaccharide linked to Ser 61 through the Fuc residue. Mass spectrometric analysis indicated that fucose was the attachment sugar

184

residue. The Ser 6 1 tetrasaccharide was not susceptible to a-fucosidase digestion. The complete structure of the tetrasaccharide was obtained by methylation and NMR analysis as NeuAc(a24)Gal(~14)GlcNAc(fll-3)Fuca( 1-0)-Ser6 1. Unusual glycans Xyll-2-Glc were shown to be linked to a serine residue (Ser 53) in the epidermal growth factor (EGF)-like domains of both human and bovine factor IX.

2.13. Factor X Factor X is a plasma protein involved in both the intrinsic and extrinsic pathways of blood coagulation. Factor X has a mass of 55 kDa and the activated Xa of 40kDa. The normal concentration in plasma is 6-8 pg/ml. Post-translational modifications of the protein involve y-carboxylation of specific glutamic acid residues, fl-hydroxylation of one aspartic acid residue, and N- and 0-linked glycosylation. Human blood coagulation factor X has two N-linked oligosaccharides at Asn 39 and Asn 49 and two 0-linked oligosaccharides at Thr 17 and Thr 29 in the region of the factor X activation peptide which is cleaved off during its activation by factor IXa. The structure of the oligosaccharides in the activation peptide region of human factor X has been determined[51]. The content of the neutral oligosaccharides at Asn 39 and Asn 49 residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of di-, tri- and tetraantennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was diantennary without a fucose residue. Two major monosialyl oligosaccharides were also diantennary without fucose and with a Neu5Ac(a2-6) residue. In addition, the structures of 0-linked oligosaccharides at Thr 17 and Thr 29 were suggested to be disialylated Gal(fl1-3)GalNAc sequences by component analysis. The effect of deglycosylation of bovine factor XI has been investigated with factor-Xactivating enzyme from Russell’s viper venom or extrinsic Xase (factor VIIdtissue factor/phospholipid) by examining the activation rates of derivatives of factor X prepared using 0-glycanase, sialidase, andor N-glycanase [52]. The removal of 0-linked carbohydrate resulted in a decrease in the rate of activation. Lectin binding and glycosidase treatment were also used [53] to investigate the functional role of carbohydrates on the activation peptide of factor X. Sumbucus nigru agglutinin, a lectin that binds to sialic acid a(24)-linked to galactose or N-acetylgalactosamine inhibits activation of human factor X in a dose-dependent manner. Inhibition of activation was observed for both intrinsic (factor IXa/VIIIa) and extrinsic (factor VIIa/tissue factor) pathway complexes. In accordance with this, removal of sialic acid residues from the activation peptide of factor X by sialidase also drastically reduces activation of the zymogen by these complexes. Parallel reduction of activity in classical clotting assays (activated partial thromboplastin time and prothrombin time) corresponds with this observation. These results also suggest a possible role of N-glycans in the activation of factor X. Thus, carbohydrate residues in factor X may play an important role in the activation of the zymogen.

185

2.14. Factor XI

Human factor XI is a glycoprotein composed of two identical chains linked by disulfide bonds with a molecular mass of 124 kDa. Normal plasma contains 4 yglml. Activation of factor XI by factor XIIa involves cleavage of both chains to 35 kDa and 25 kDa fragments. The active site serines are in the 25 kDa chains. Factor XIa thus consists of four subunits linked by disulfide bridges.

2.15. Factor XI1 Factor XI1 is a single polypeptide chain with a mass of 80 kDa, fully fucosylated at Thr 90 of the heavy chain in the N-terminal EGF domain [54]. The plasma content is 29 yg/ml.

NeuSAc(a2-6)Gal@ 1 -4)GlcNAc(B 1-2)Man(a 1-6)

\ Man(f3I4)GlcNAc(f314)GlcNAc-Asn (A) NeuSAc(a24)Gal(f314)GlcNAc(f31-2)Man(a 1-3)

1

Neu5Ac(a24)Gal(f314)GlcNAc(f3I-2)Man(a 1-6)

\ GlcNAc(f314)-Man(p 14)GlcNAc(f31 -4)GlcNAc-Asn I Neu5Ac(a24)Gal(f314)GlcNAc(f3l-2)Man(a 1-3)

(B)

Fig. 7. (A) Concanavalin A binding and (B) non-binding di- and triantennary glycans from a-fetoprotein.

2.16. a-Fetoprotein a-Fetoprotein is found in relatively high concentrations in the fetal and neonatal sera and in amniotic fluid of many species but it drops to low levels soon after birth. Distinct molecular variants of rat a-fetoprotein have been detected using Con A affinity chromatography [ 5 5 ] . Each a-fetoprotein variant contains two identical glycans which differ between the Con A reactive (Fig. 7A) and non-reactive forms (Fig. 7B).

2.17. Fibrinogen Fibrinogen is a three-chain protein synthesized in the liver that is essential for haemostasis both in linking together activated platelets in aggregation and in the formation of the insoluble polymer fibrin necessary for the consolidation of the haemostatic plug. Fibrinogen is glycosylated on two sites, one in the b- and one in the y-chain, both on Asn. Both glycans were determined to be diantennary structures (Fig. 8) [56]. The primary structures of two Asn-linked glycans from bovine fibrinogen have been determined by methylation analysis and NMR spectroscopy, and were also shown to be diantennary [57].

186

Gal(fil-4)GlcNAc(~l-2)Man(al-6)

\ Man(fil4)GlcNAc(fi 14)GlcNAc-Asn I

Gal(fil4)GlcNAc(fil-2)Man(a1-3) Fig. 8. Diantennary glycan from fibrinogen.

2.18. Fibronectin The fibronectins consist of isohomodimers of two nearly identical cysteine-linked 225 kDa subunits. They are adhesive proteins and can bind to a range of molecules including denatured collagen, fibrin and DNA. They are also important for cell adhesion. The carbohydrate content of fibronectins varies considerably depending on the source and is thought to be important for certain physical properties [58]. The extracellular matrix adhesion molecule fibronectin exhibits different isoforms caused by alternative splicing as well as by variation in 0-glycosylation, as recently demonstrated. Although fibronectin is widely distributed in normal tissues, the individual isoforms have been found to show restricted tissue distribution and association with malignancies. The monoclonal antibody FDC-6 defines a cancer-associated de nouo glycosylation of a specific Thr residue in the C-terminal region of the fibronectin molecule termed oncofetal fibronectin. Oral squamous cell carcinomas, premalignant lesions, and normal oral mucosa have been studied immunohistologically using the FDC-6 antibody [59]. Selective expression of the oncofetal fibronectin epitope was closely related to the invading carcinoma. Previous studies indicated that the de nouo glycosylation is induced by a novel transferase activity only found in fetal and carcinoma cell lines, placenta and hepatoma tissues. The gelatin-binding region of fibronectin contains three Asn-linked carbohydrate moieties, one on the second type I1 module and two on the eighth type I “finger” module. Carbohydrate groups were enzymatically removed from two non-overlapping gelatin-binding fragments (GBFs), 2 1 kDa GBF (modular composition 18-19) and, with much greater difficulty, 30 kDa GBF (modular composition 16-111-112-17) [60]. Fluorescence and calorimetric analyses indicated that module I8 was strongly destabilized by deglycosylation so that the apoform melts near physiological temperatures. A similar effect was caused by decreasing the pH of the holoform to 6.0, suggesting that one or more histidines are important for stability of module 18. The 21 kDa fragment exhibited an acid-induced change in fluorescence that occurred at higher pH in the deglycosylated derivative, providing further evidence of a stabilizing role for one or both carbohydrate moieties. In contrast, the stability of module I12 was unaffected by removal of its single carbohydrate. To determine if differential glycosylation of fibronectin (Fn) in inflammatory synovial fluid (SF) included expression of an oncofetal epitope (Onf Fn) previously detected only on Fn derived from embryonal or neoplastic tissue [61], Fn was purified from plasma, SF and synoviocyte conditioned medium by affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting using a monoclonal antibody (FDC-6) specific for the Onf Fn. The Onf Fn was not expressed on

187

Fn isolated from normal or rheumatoid arthritis (RA) plasma but was strongly expressed on Fn from RA SF and to a lesser extent osteoarthritis SF. Onf Fn was also detected on Fn secreted by cultured RA synoviocytes. 2.19. Hemopexin Hemopexin has a molecular mass of 57kDa and is a glycoprotein involved in haem disposal present in plasma at levels between 8 and lOrng/lOOml. It contains about 22% carbohydrate. The human glycoprotein contains five N-linked and one 0-linked oligosaccharide chain, the glycans are of the di- and triantennary type. The 0-linked glycan blocks the N-terminal threonine [62]. Porcine hemopexin has been isolated in >99% purity as tested by crossed immunoelectrophoresis. Porcine hemopexin has a molecular mass of 62kDa. Based on carbohydrate and sialic acid analyses, it was proposed that hemopexin contains two diantennary (similar to Fig. 7A) and one triantennary glycan chains [63]. The structure of several rat hemopexin glycans were determined and the main component was shown to be a trisialyl diantennary oligosaccharide (Fig. 9) [64]. NeuSAc(a24) \

NeuSAc(a2-6)Gal(fi 14)GlcNAc(fi1-2)Man(a 1-6)

\ Man(fi 1-4)GlcNAc(fil4)GlcNAc-Asn

NeuSAc(a2-6)Gal(fi14)GlcNAc(fi 1-2)Man(a 1-3)

I

Fig. 9. Trisialyl diantennary glycan from hemopexin.

2.20. a2-HS-glycoprotein a2-HS-glycoprotein is the human equivalent of fetuin in animals. Complex type N-glycosides of bovine fetuin contain comparable amounts of triantennary (Fig. 1OA) and tri’-antennary (Fig. 1OB) as major asialo-structures. A specific galactosyltransferase may exist for the biosynthesis of the tri‘-antennary structure. The N-glycosides of ovine fetuin also have both triantennary and tri’-antennary structures in a ratio similar to that of bovine fetuin. However, the major N-glycoside of porcine fetuin has a fucosyl diantennary complex type structure (Fig. 1OC) and human a2-HS-glycoprotein has an N-glycoside which is almost exclusively a nonfucosylated diantennary structure (Fig. 1OD). This species-specific presence of N-glycosides of fetuins and comparison with N-glycosides of other glycoproteins suggest that the polypeptide sequence of a glycoprotein may affect its N-glycan structure by regulating the activity of specific glycosyltransferases [65]. The A-chain of human plasma a2HS-glycoprotein contains two diantennary N-glycans linked to Asn 138 and 158 (Fig. 10E) and two 0-linked trisaccharides of the types shown in Fig. 10F linked to Thr 238 and 252 [66]. The B-chain has one 0-linked trisaccharide of the same type. The N-glycans are sialylated to about 90%.

188

Gal(@14)GlcNAc(fil-2)Man(a 1 4 )

\ Man@1-4)GlcNAc(fi 14)GlcNAc (A) I

Gal(fi14)GlcNAc(fi 1 -2)Man(al-3) Gal(BI-4)GlcNAc(fi1-4)1 Gal((ll4)GlcNAc(fi 1-6)

\

Gal(fil4)GlcNAc(fiI-2)Man(a1-6)

\ Gal@1-4)GlcNAc(fi 1-2)Man(a 1-3)

Man(Bl4)GlcNAc(fiI 4)GlcNAc (B) I

Gal@ 14)GlcNAc(fi1-2)Man(a 1 4 )

\

Fuc(a 1-6)

\

Man(fi14)GlcNAc(@I4)GlcNAc (C)

Gal(fi14)GlcNAc(fi1-2)Man(a 1-3)

I

Gal(fi14)GlcNAc(fi1-2)Man(a 1-6)

\ Gal(fi14)GlcNAc(B 1 -2)Man(a 1-3)

Man(fil-I)GlcNAc(pl 4)GlcNAc (D) I

NeuSAc(a24)Gal(B 14)GlcNAc(fi1 -2)Man(a 14)

\ Man@ 14)GlcNAc(@1 4)GlcNAc(@I-N)Asn I

(E)

NeuSAc(a2-3)Gal(fi 1-4)Gal(@1-3)GalNAc(a 1-0)

(F)

NeuSAc(a2-6)Gal(~14)GlcNAc(fi1-2)Man(a 1-3)

Fig. 10. (C-E) Di- and (A,B) triantennary glycans and (F) 0-linked tetrasaccharide from a l - H S glycoprotein,

2.21. a2-Leucine-rich glycoprotein a2-Leucine-rich glycoprotein is the prototype of the leucine-rich domain family containing 13 repeats of a 24 amino acid sequence [67]. Plasma contains 2.1 mg per 100 ml; the function is unknown. The consensus sequence is LXXLXLXXNXLXXLPXXLLXXXXX, the first part of which forms an @-fold and the second part an a-helix. Based on the crystal structure of porcine ribonuclease inhibitor it can be predicted that these repeats will pack together in an arc-like form. Four N-glycans and one 0-glycan are present. The 0-glycan is close to the N-terminus. One potential N-site is not glycosylated.

2.22. a2-Macroglobulin a2-Macroglobulin is a major plasma component with nearly 300mg/100ml and a high molecular mass (820 kDa) containing about 10% carbohydrate in 3 1 glycans [68]. It has an important role in controlling protease activity in the blood. No complete oligosaccharide

189

structures have been reported yet. Abnormal glycosylation of a2-macroglobulin, a nonacute-phase protein has been detected in various pathological conditions including autoimmune diseases. The protein was purified from serum samples and cerebrospinal fluids (CSF) from patients with autoimmune diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease (MCTD), scleroderma (SCL), Sjogren’s syndrome (SS), and polymyositis (PM); diseases of probable autoimmune origin: hepatopathies (HP); diseases of suspected autoimmune origin: schizophrenia and Alzheimer’s disease (AZ); and conditions not related to autoimmunity: pregnancy and elevation of the carcinoembryonic antigen (CEA) as well as from normal donors. Con A-reactive fragments on Western blots increased specifically and significantly in samples derived from patients of SLE, SCL, MCTD, and RA [69]. Linkage-specific lectins revealed the presence of Neu5Ac(a2-6)Gal but not Neu~Ac(a2-3)Gal in porcine a2-macroglobulin glycan [70].a2-Macroglobulin was found to be microheterogeneous in plasma samples from alcoholic patients but microheterogeneity disappeared after treatment with sialidase [24]. 2.23. Plusminogen

Plasminogen is the precursor of the serine protease plasmin which is involved in dissolution of fibrin clots and, hence, in removal of thrombi. The plasma concentration is about 12 mg/100 ml and the molecular mass 81 kDa. It contains 17.1% carbohydrate on two sites, one N-linked and one 0-linked oligosaccharide. A variant is also known that is only 0-glycosylated. The structures of the oligosaccharides have been determined [7 I731, and are shown in Fig. l l . NeuSNAc(a24)Gal(fi1-4)GlcNAc(fi1-2)Man(a1-6/3)

\ Man@ 1-4)GlcNAc(fi 14)GlcNAcAsn288 (A) NeuSNAc(a2-6)GaI(fi 1-4)GlcNAc(fi 1-2)Man(a 1-316)

NeuSAc(a2-3)Gal(fi1-3)GalNAcThr345 (B) I NeuSNA~(a2-6)~_,

Fig. 1 1. (A) Diantennary glycan and (B) 0-linked branched tetrasaccharide from plasminogen.

2.24. al-Proteinuse inhibitor al-Proteinase inhibitor is a secreted, monomeric protein glycosylated at Asn 46, 83, and 247. Single, double, and triple mutants have been made by changing the codons specifying these Asn residues to encode Gln. The mutant proteins were transiently expressed in COS-I cells. All variants with altered glycosylation sites are secreted at reduced rates, are partially degraded, accumulate intracellularly, and some form Nonidet P-40-insoluble aggregates. The carbohydrate attached at Asn 83 seems to be of particular importance to the export of both al-proteinase inhibitors M and S from the endoplasmic reticulum. All

I90

mutations affecting glycosylation of a 1-proteinase inhibitor S notably reduce secretion, cause formation of insoluble aggregates and influence degradation of the altered proteins. The variant of al-proteinase inhibitor S lacking all three glycosylation sites is poorly secreted, is incompletely degraded, and accumulates in unusual perinuclear vesicles [74]. Thus, N-linked oligosaccharides in a l -proteinase inhibitor S are vital to its efficient export from the endoplasmic reticulum and subsequent processing. 2.25. Protein C Protein C is a glycoprotein with a molecular mass of 62kDa present in plasma at a concentration of 4 pg/ml. Thr 68 of Protein C in the N-terminal EGF domain is not fucosylated but the Asp 71 is fully 6-hydroxylated [54]. Protein C exists in a- (70%) and 6-forms (30%) in plasma. The f3-form is smaller than the a-form. This difference appears to be due to the presence of four N-glycans on the a-form but only three on the f3-form. The fourth site at Asn 329 (Asn-Ala-Cys) is different from the usual Asn-X-Ser/Thr but corresponds to N-glycosylation sites in von Willebrand factor and bovine Protein C. The likelihood of glycosylation at this site may be related to the speed of formation of the Cys33 1-Cys345 disulfide bond [75].

2.26. Protein S Protein S contains three potential N-glycosylation sites at Asn 458,468 and 489. Mutation studies indicate that each site is glycosylated to the same extent and that the blockage of glycosylation generally enhances the cofactor activity [76]. 2.27. Prothrombin

Prothrombin is the precursor of the serine protease thrombin, essential for normal haemostasis in activation of platelets and in catalysing the polymerisation of fibrinogen to fibrin. It is synthesized in the liver and contains about 8% total carbohydrate in four N-glycans. Three of these lie on the Pro region and only one is present on the mature a-thrombin molecule. The structure of the thrombin glycan has been determined by mass spectrometry and is the same as that shown in Fig. 5A with fucose absent or present [77].

2.28. Transferrin The function and structure of the oligosaccharides on the iron-binding transferrin family of proteins is dealt with in detail in Chapter 10. 2.29. Vitronectin

Vitronectin is a glycoprotein with a mass of 70kDa involved in cell adhesion and spreading. Plasma contains about 200-300 pglnil. Vitronectin contains three potential N-glycosylation sites. The structures of the N-linked oligosaccharides present on human plasma vitronectin have been determined [78]. Oligosaccharides were released from the

191

vitronectin by PNGase F digestion and tagged with 2-aminopyridine. The pyridylaminooligosaccharides were then fractionated by anion-exchange and reverse-phase HPLC. Ten major pyridylamino-oligosaccharides were isolated. The linkages and locations of sialic acid residues were determined by desialylation with Salmonella sialidase in combination with acid. The asialo forms were then analyzed by two-dimensional carbohydrate mapping, component monosaccharide analysis and 400 MHz H-NMR spectroscopy. The major oligosaccharides of human vitronectin were of the diantennary N-acetyllactosamine type, with less of the tri- and a small amount of the mono-antennary type, to which one to three sialic acid residues were linked, mostly a(2-6)-linked, although a(2-3) linkages were also present. Several binding activities of vitronectin may be related to its glycan moiety based on the specific features of the N-glycans. The structures of N-linked oligosaccharides on porcine plasma vitronectin have been similarly determined [79]. Nine major pyridyl-amino-oligosaccharides were isolated. After desialylation, the asialo-forms were analyzed by two-dimensional sugar mapping, component sugar analysis and 400 MHz H-NMR spectroscopy. The major oligosaccharides of porcine vitronectin were of the fucosylated diantennary type, with a small amount of the triantennary N-acetyllactosamine type, to which one to three sialic acids residues were linked. Sialic acids were predominantly a(2-6)-linked, although a(2-3) linkages were also present, and fucose was linked to the innermost N-acetylglucosamine through an a ( 1-6) linkage. Each pyridylamino-oligosaccharide population contained NeusGc and NeusAc in a molar ratio of 1 :2-9, and NeuSGc were located predominantly on the Man(a1-6) antenna.

'

'

2.30. Von Willebrand factor Von Willebrand factor is a very large (1-1 0 MDa) multimeric protein synthesized in endothelial cells and in megakaryocytes. It is constitutively secreted from endothelial cells and is also stored in granules in these cells and in platelets and released when they are activated, for example, by thrombin. Von Willebrand factor (vWf) plays an essential role in haemostasis by acting as the carrier molecule for factor VIII. It also binds to collagen on vascular subendothelium and provides the attachment site for circulating platelets in the first step of primary haemostasis. The receptor for von Willebrand factor on platelets is the GPIb-V-IX complex. Several domains on vWf have been identified as involved in binding to GPIb, collagen and heparin. Six 0-glycans flank the A1 domain cystine loop containing the GPIb binding domain. Removal of sialic acid produces asialo-vWf which binds spontaneously to platelet GPIb. These results indicate that the active structure of the GPIb binding domain (the A1 domain) of vWf is dependent on glycosylation [go]. The structure of some of these oligosaccharides has been determined. A major N-glycan representing about 45% of the carbohydrate chains is a monosialylated monofucosylated diantennary glycan of the N-acetyllactosaminic type (Fig. 12A) [8 11. A tetraantennary glycan of the N-acetyllactosaminic type has also been isolated and its structure determined by methylation analysis and 500 MHz 'H-NMR (Fig. 12B) [82]. The major 0-glycan of human vWf was shown to be a diantennary tetrasaccharide (Fig. 12C) [83].

192

NeuSAc(a24)Gal(fi I 4)GlcNAc(fi I -2)Man(a 1-6)

\ Man(fiI4)GlcNAc(fi14)GlcNAc (A) I

Gal(fiI-rl)GlcNAc(fil-2)Man(aI-2)

I Fuc(a 14)

Gal@ l 4 ) G l c N A c ( f i l 4 ) \ Man(al-3)

\

Neu5Ac(a2-6)Gal(fi 14)GlcNAc(!3 1 -2f Gal(fil4)GlcNAc(fi1-2)~

Gal@ l4)GlcNAc(fi1-6)

Fuc(a 1-6)

\

Man(fil4)GlcNAc(fi14)GlcNAc (B)

Man(a 1-6f I Neu5Ac(a24)

\ NeuSAc(a2-3)Gal(b 1-3)

GalNAcol (C) I

Fig. 12. (A,B) Di- and tetraantennary glycans and (C) 0-linked branched tetrasaccharide from von Willebrand factor.

3. Platelets The basic role of platelets is haemostasis, or stopping bleeding, when tissue is damaged. The platelets adhere to exposed subendothelium on vessel walls, are activated and aggregate to form a thrombus that covers the damaged area or blocks off complete vessels when they are involved. These functions involve a number of receptors, all of which are membrane glycoproteins. Platelets are very rich in glycoproteins and contain some 11 times more sialic acid than erythrocytes.

3.1. Glycoprotein Ib- FIX complex This complex is responsible for the primary adhesion of platelets to von Willebrand factor as well as modulating the platelet response to thrombin. There are four separate subunits, GPIba and f~covalently linked by a disulfide bond, GPIX tightly bound non-covalently, and GPV more loosely bound non-covalently. GPIba contains the primary binding sites but the other chains seem to be necessary for physiological function. Absence of GPIbV-IX for genetic reasons leads to the bleeding disorder Bernard-Soulier syndrome. 3.1.1. Glycoprotein Iba/glycocalicin

Glycoprotein Iba is one of the major glycoproteins on the platelet surface containing a large part of the sialic acid present on the platelet surface. There are at least 25 000 copies per platelet but there may be up to 50 000 if molecules present in the surface-connected canalicular system are included. This chain contains the primary von Willebrand factor binding site and a thrombin-binding site. Glycocalicin is the extracellular domain which is readily split off by several proteases, particularly endogenous calpain and contains 40% carbohydrate by mass, all the glycosylation of GPIba. Glycoprotein Iba contains four

193 Neu5Ac(a2-3/6)Gal(fi 14)GlcNAc(fiI-2)Man(a1-6)

\

Fuc(al-6)

\

Man(fiI4)GlcNAc(fi14)GlcNAc(A) Neu5Ac(a2-3/6)Gal(fi14)GlcNAc(fil-2)Man(al-3)’ NeuSAc(a2-3/6)Gal(fi 14)GlcNAc(fi I-2)Man(a 1-6)

\

Fuc(al-6)

\

Man(/314)GlcNAc(fi14)GlcNAc (B)

Neu5Ac(a2-3/6)Gal(fiI 4)GlcNAc(fiI 4) \

Man(a 1-3)

I

NeuSAc(a2-3,6)Gal(fi 14)GlcNAc(fiI -2; NeuSAc(a2-3)Gal(fi 1 4)GlcNAc(/31-6)

\

Man(a 1-6)

Neu5Ac(a2-3)Gal(fiI4)GlcNAc(fi1-2)/ NeuSAc(a2-3)Gal(fi14)GlcNAc(f114),

\

Fuc(a I”),

\

Man(fi14)GlcNAc(fi14)GlcNAc(C)

NeuSAc(a2-3)Gal(fil4)GlcNAc(fi1-6)GalNAc (D) NeuSAc(a2-3)Gal(fiI -3)

I

Neu5NAc(a2-3)Gal(fiI-3)GalNAc (E)

NeuSAc(a2-3)Gal(fil-3)GalNAc (F) Neu5Ac(a24)

I

Fuc(a 1-2)Gal(fi 1-4)GlcNAc(fi 1-6)GalNAc I Gal(fi1-3)

(G)

Fig. 13. (A) Diantennary, (B) triantennary and (C) tetraantennary glycans and 0-linked (D) hexa-, (E) tri-, (F) tetra-, and (G) pentasaccharides from glycoprotein Iba.

putative N-glycosylation sites two of which are known to be glycosylated from primary sequencing data. The N-glycosylation structures were released by hydrazinolysis, isolated and structure determined by methylation analysis, glycosidase treatment and Smith degradation [84]. Typical di- and trisialylated complex-type structures were found and are illustrated in Figs. 13A and B. A tetrasialylated monofucosylated tetraantennary chain was later detected and its structure determined using NMR spectroscopy (Fig. 13C) [85]. 0-linked oligosaccharide alditols were obtained by alkaline borohydride treatment of glycocalicin and the structure of the major chain was determined by methylation and glycosidase treatments[86] or by NMR[87,88] and found to be a hexasaccharide as shown in Fig. 13D. Minor tri-, tetra- and pentasaccharides with the structures shown in Figs. 13E, F and G were also obtained. Glycocalicin contains a domain that is particularly rich in 0-glycosylation and includes five mucin-like repeats. Within this region is a size polymorphism consisting of a 13 amino-acid sequence [89] that is found either once or repeated twice, three or four times, termed D, C, B and A phenotypes, respectively. Since each repeat contains five putative 0-glycosylation sites the difference in molecular

194

mass of 2kDa between these is explicable. Although these differences are referred to as polymorphism, it remains unclear if there exist differences in function resulting from them. Thus, the function of this highly 0-glycosylated domain, which acts as a semi-stiff rod, appears to be to hold the receptor domain out from the platelet surface so that it is available for interaction with von Willebrand factor and thrombin. The fact that this may be extended more or less into the extracellular space may not be without consequences. The high glycosylation of this domain also functions to protect it against proteolysis which is restricted to small unprotected regions between domains. 3.1.2. Glycoprotein IbB The small subunit of GPIb with a molecular mass of 27 kDa and linked to the a-subunit by a disulfide bond has one leucine-rich repeat containing a single N-glycosylation site. Monosaccharide analysis showed that this was likely to be a diantennary glycan of the N-acetyllactosamine type. The lack of N-GalNAc argued against any 0-glycosylation [90]. 3.1.3. Glycoprotein IX Glycoprotein IX has overall a very similar structure to glycoprotein Ibp and is only slightly smaller with a mass of 22 kDa. It also has one leucine-rich repeat containing a single N-glycosylation site which also appears to be a diantennary glycan of the N-acetyllactosamine type. The lack of GalNAc also indicates that there is no 0-glycosylation [90]. 3.I . 4. Glycoprotein V Glycoprotein V is loosely non-covalently associated with the rest of the GPIb-IX complex, probably in a 1:2 ratio. It has a mass of 82kDa and contains 16 leucine-rich repeats, as well as disulfide-bridged loops that appear to be conserved among all the members of this family. An important feature is a thrombin-cleavage site just under these domains which separates the molecule into a 69 kDa soluble fragment and a 20 kDa membrane bound fragment. There are eight putative N-glycosylation sites of which seven are in the leucine-rich domain and one in the disulfide-bridged loops. Based on the molecular mass estimated from the protein backbone (59 m a ) , it seems likely that most, if not all of these sites are glycosylated. There is also evidence, based on primary sequence data, that some 0-glycosylation sites in the 20 kDa fragment near to the membrane are glycosylated [91]. However, the mucin-like repeats found in GPIba are not present in GPV: 3.1.4.1. Leucine-rich domains. The leucine-rich domains consist of a sequence of 24 amino acids forming a loop consisting of an ag-fold and an a-helix. These loops pack together to form arc-like structures or, in some cases with large numbers of repeats, horse-shoe-like or even circular, spiral structures. As mentioned above, several of the N-glycosylation sites in GPIb-V-IX are in the leucine-rich domains and lie without exception on the outside of the loops, generally on the outside face of the arc-like structure.

195

3.2. GIycoprotein IIb-IIIa

(aI1bp3)

Glycoprotein IIb-IIIa (allt$3), also known as CD41/61, is the major member of the integrin family on platelets and was, indeed, important for working out many of the structural and functional aspects of integrins. Integrins typically consist of two subunits an a and a B and form the link between adhesive proteins, such as are present in the extracellular matrix, and the cell cytoskeleton. Thus, they are often found to play an important role in cell adhesion and locomotion. In the case of platelets, GPIIb-IIIa is involved in secondary adhesion of activated platelets to vascular subendothelium. It is particularly important in platelet-platelet adhesion called aggregation, essential for formation of a stable thrombus in haemostasis, and for clot retraction, necessary to hold together the sides of a wound to allow repair to start. The a-subunits are characterized by the presence of four or more cation binding sites, critical for the conformation of the subunit and to allow interactions with the B-subunit and form the binding site. The b-subunit contains a domain rich in cysteine bridged loops but its function is not yet clear. GPIIb-IIIa belongs to the category of integrins that require activation to bind their ligand. In resting platelets it is to a large extent passive although a minor amount may always be in the active state. Activation occurs via changes in the platelet interior caused by signal transduction from other classes of receptor and seems to involve kinase/phosphatase cascades although phosphorylation of the integrin itself appears not to be necessary. There are about 50 000 copies of GPIIb-IIIa on the platelet surface but many are also present on the surface of a-granules and are exposed after platelet activation. Absence of this integrin for genetic reasons leads to the bleeding disorder, Glanzmann’s thrombasthenia.

3.2.1. Glycoprotein IIb

Glycoprotein IIb has a mass of 120kDa non-reduced and of 110 kDa reduced due to the separation of a large and small subunit held together by a disulfide bond. There are four potential N-glycosylation sites in the heavy chain and one in the light chain [92]. GPIIb contains mainly complex type glycans in contrast to GPIIIa accounting for the difference in binding to lectins [93]. The major types present are shown in Fig. 14.

I Neu5Ac(a2-3/6),

-3

I

I Neu5Ac(a2-3/6)

Gal(fiI4)GlcNAc(B14) \ Man(a1-6) Gal@ 14)GlcNAc(B 1-2) I

\

Fuc(a1-6)

\ -

I

Man(b14)GlcNAc@ 1 -4)GlcNAc

Gal(fi 14)GlcNAc(fi 1-2)Man(a 1-3)

(A)

t

Gal@ 14)GlcNAc(b 1-2)Man(a 1-6)

\

Fuc(a1-6)

\ -

,

M a n ( f i I 4 ) G l c N A c ( ~ l 4 ) G l c N A c (B)

I -2

Cial(fi14)GlcNAc((lI-2)Man(a 1-3)

I

196

Man(a 1 4 )

\

Man(a1-2),_,

Man(a 1-6) Man(a1-3) I \ Man(Bl4)GlcNAc(fi 14)GlcNAc I Man(al-3)

Fig. 15. High mannose type triantennary glycan from glycoprotein 1Ila.

3.2.2. Glycoprotein IIIa Glycoprotein IIIa has an apparent molecular mass on gel electrophoresis of 90 kDa nonreduced and 100 kDa reduced due to the large number of intramolecular disulfide bridges. Before reduction the molecule is very compact and after reduction much more stretched out. There are six potential N-glycosylation sites [94]. N-glycosylation on GPIIIa is predominantly of the high mannose type [93]. A typical structure is shown in Fig. 15. 3.3. D l Integrin family Little is known about the glycosylation on this family which is represented on platelets by GPIIa (CD29). From the lectin binding pattern it seems likely that it also contains principally high mannose-type glycans. The a-chains in this family are GPIa, GPIc and GPIc’ (CD49b, e, f). The structures of N-glycans obtained from human placenta integrin a5g1, corresponding to platelet GPIc-Ha, have recently been investigated [95]. A total of 35 different oligosaccharide structures were identified, 10 neutral, 6 monosialyl, 10 disialyl, 7 trisialyl and 2 tetrasialyl. High-mannose type glycans were only 1.5% of the total, the rest being all complex type and representatives of nearly all structures were found. The most prominent structure was the diantennary di-a-(2,3)-sialyl fucosyl corresponding to that shown in Fig. 5B. The major sialic acid linkage was a-(2,3) and 50% of all oligosaccharides were fucosylated at the reducing end GlcNAc. Tetraantennary structures of the same type as shown in Fig. 13C with and without fucose on the reducing terminus were also common.

3.4. CD36 (GPIIIb, GPIV) CD36 is a family of glycoproteins found on the surface of a wide range of different cells including platelets, endothelial cells and monocytes as well as mammary epithelial cells. The molecular mass varies considerably between tissues and species although the DNA coding for this protein appears very similar, implying large differences in glycosylation[96,97]. In platelets CD36 has been implicated in a large number of receptor functions including those for collagen, thrombospondin, oxidised LDL and for Plasmodium falciparum infected erythrocytes. The role of glycosylation in modulation of these receptor functions remains unknown. Glycosylation of CD36 from bovine mammary epithelial cells has been studied in the greatest detail [98]. Based on hydrazinolysis studies it was estimated that each CD36 molecule has on average six N-glycans. From the DNA sequence ten potential N-glycosylation sites were present [99]. Several types of oligosaccharide were found including several hybrid type chains. High mannose

197

Man(a1-2)Man(a1-3)Man(al-6)

\

Fuc(al-6)

\ -

,

Man(@I4)GlcNAc(fi1-4)GlcNAc (A) Gal(fi14)GlcNAc(~I-2)Man(a 1-3)

I

Man(al-6) \

Man(al-3)

Man(a 1-6) I \

Fuc(al-6)

\ -

,

Man(fi14)GlcNAc(fiI4)GlcNAc (B)

Gal(fiI 4)GlcNAc(fi1-2)Man(a 1-3)

I

Man(a 1-6)

\

Man(a 1-2)

Man(a 1-6) !Man(a 1-3/

Gal(fi14)GlcNAc(fi1-2)Man(a 1-3)

\

Man(a 1-2)Man(a 1-3)Man(a 1-6)

\ GalNAc(fiI-4)GIcNAc(fil -2)Man(al-3)

i

Fuc(a1-6)

(C)

\ -

Man(fi I -l)GlcNAc(fi 1-4)GlcNAc (D) I

GalNAc(fi14)GlcNAc(fiI -2)Man(a 1-6)

\ GalNAc(fi1-4)GlcNAc(fiI -2)Man(al-3)

Fuc(a1-6)> o-l

Man@ 14)GlcNAc(fi1-4)GlcNAc I

Fuc(a1-6)

\ -

I

Man@ 1 4)GlcNAc(fi1-4)GlcNAc I

(E)

Fig. 16. Mixed type mannoselcomplex type (A,D) diantennary and (B,C) tnantennary glycans and (E) diantennary complex type glycans from CD36.

type, hybrid-type, and di-, tri-, and tetraantennary complex type glycans were all found (Fig. 16). Based upon the patterns of N-glycosylation observed here and in comparison with other cell types, the conclusion was drawn that the trimming pathway of high mannose type oligosaccharides to hybrid-type ones may be distinct in different cell types. A proportion (28% of all glycans) of the hybrid and complex type which bound to Wistaria floribundu lectin contained the GalNAc(/314)GlcNAc groups instead of Gal(B14)GlcNAc in the outer chain. Some of these contained NeuSAc(a24)GalNAc. Most of the hybrid-type glycans with the Gal/GalNAc(fi 14)GlcNAc outer chain on the Man(a1-3) antenna contained an unusual Man(a I-2)Man(a 1-3) group on their Man(a 16) antenna (Fig. 16). Less is known about glycosylation of CD36 in human platelets although there has been one study [IOO] indicating the presence of both N- and 0-glycans in overall 28% carbohydrate content and both branched tetra- and disaccharides were found, probably with one or two terminal sialic acid residues.

198

3.5. PECAM-I (CD31) Platelet endothelial cell adhesion molecule- 1 (PECAM-1, CD3 1) is a member of the IgG superfamily found on various cells and may bind to glycosaminoglycans. There are nine potential N-glycosylation sites [ 10 11 but no detailed investigations have been done. 3.6. Thrombospondin Thrombospondin is a high molecular mass (420 kDa) glycoprotein found in the a-granules of platelets but it is also secreted by a range of other cells such as fibroblasts. It seems to play an important role in cellular adhesion and contains fibrinogen, heparin and collagen binding domains [ 1021. There are six potential N-glycosylation sites [ 1031.

Acknowledgements Work carried out at the Theodor Kocher Institute was supported by grants from the Swiss National Science Foundation (3 1-42336.94) and Hoffmann-La Roche Ltd. The supply of buffy coats for the isolation of platelets by the Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross is gratefully acknowledged.

References [ l ] Bowman, B.H. (1993) Hepatic Plasma Proteins: Mechanisms of Function and Regulation. Academic Press, San Diego, New York. [2] Schmid, K., Binette, J.P., Kamiyama, S., Pfister, V. and Takahashi, S. (1962) Biochemistry I , 959-966. [3] Van den Eijnden, D.H., Joziasse, D.M., Dorland, L., Van Halbeek, H., Vliegenthart, J.F.G. and Schmid, K. (1980) Biochem. Biophys. Res. Commun. 92, 839-845. [4] Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J.F.G., Vliegenthart, J.F.G., Binette, J.P. and Schmid, K. (1978) Biochemistry 17, 5206-52 14. [5] Schmid, K., Binette, J.P., Dorland, L., Vliegenthart, J.F.G., Fournet, B. and Montreuil, J. (1979) Biochim. Biophys. Acta 581, 356-359. [6] Van Halbeek, H., Dorland, L., Vliegenthart, J.F.G., Montreuil, J., Fournet, B. and Schmid, K. (1981) J. Biol. Chem. 256, 5588-5590. [7] Cardon, P., Parente, J.P., Leroy, Y., Montreuil, J. and Fournet, B. (1986) J. Chromatogr. 356, 135-146. [8] Shiian, S.D., Nasonov, V.V., Bovin, N.V., Aleshkin, V.A., Novikova, L.I. and Liutov, A.G. (1994) Bioorg. Khim. 20, 1125-1 131. [9] Mackiewicz, A., Khan, M.A., Gorny, A,, Kapcinska, M., Juszczyk, J., Calabrese, L.H. and Espinosa, L.R. (1994) J. Infect. Dis. 169, 1360-1363. [lo] Grootveld, M., Claxson, A.W., Chander, C.L., Haycock, I?, Blake, D.R. and Hawkes, G.E. (1993) FEBS Lett. 322, 26tL276. [ I I] Hrycaj, I?, Sobieska, M., Mackiewicz, S. and Muller, W. (1993) Ann. Rheum. Dis. 52, 138-141. [I21 Biou, D., Konan, D., FCger, J., Agneray, J., Leroy, Y., Cardon, P., Fournet, B. and Durand, G. (1987) Biochim. Biophys. Acta 913, 308-312. 1131 Wieruszeski, J.M., Fournet, B., Konan, D., Biou, D. and Durand, G. (1988) FEBS Lett. 238, 390-394. [I41 De Graaf, T.W., Van der Stelt, M.E., Anbergen, M.G. and Van Dijk, W. (1993) J. Exp. Med. 177, 657-666.

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J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins II 0 1997 Elsevier Science B.V. All rights reserved

CHAPTER 10

Transferrin superfamily An outstanding model for studying biochemical evolution Jean Montreuil, Genevikve Spik and Joel Mazurier Uniuersiti des Sciences et Technologies de Lille, Laboratoire de Chimie Biologique (UMR no. 111 du CNRS), 59655 Villeneuve dilscq Cedex, France

1. Introduction I . I . DeJinition of transferrin superfamily The superfamily of transferrins (for reviews, see refs. [ 1-10]), serotransferrins from blood plasma, lactotransferrins (also called lactoferrins) from mammalian milk, external secretions and polymorphonuclear leukocytes, ovotransferrins from avian and reptile egg-white and melanotransferrins (also called p97 protein) from cell membranes, constitutes a group of well conserved glycoproteins which present the following common physicochemical and biological properties: (i) their molecular mass is around 80 kDa; (ii) they are constituted of a single polypeptide chain organized in two lobes originating from a gene duplication; (iii) each lobe binds reversibly one Fe3+ ion and iron-saturated transferrins develop a salmon-pink color; (iv) the protein moiety of transferrins presents a high degree of homology (about 50%); (v) all transferrins are glycosylated (2.2-1 1.2% total sugars), except some fish transferrins; (vi) they play a key role in iron transport and are recognized by specific membrane receptors; (vii) they are inhibitors of the growth of microorganisms by a mechanism of ferrideprivation. Consequently, the transferrins represent a remarkable model for answering the question posed by Hughes and Butters [ 111: “Glycosylation pattern in cells: an evolutionary marker?”. This explains why we undertook, 30 years ago, a systematic and comparative study on glycan primary structures of transferrin from tissues and biological fluids of different species. In addition, our second aim was to address another important question: “Why are transferrins glycosylated?’. But, we must confess that, on the basis of the determination of the glycan primary and three-dimensional structure of transferrins, we are still unable to establish relationships between primary structure and function of the glycans, on the one hand and, on the other hand, to answer the two aforementioned questions as well as the following one: “How to explain the great microheterogenicity of their glycan moieties?”. At the moment, the mystery thickens, since, as observed in our laboratory and in others [12], the partial or complete deglycosylation of transferrins does not affect the reversible iron fixation, the recognition and binding to the reticulocyte membrane, or the iron transfer into the cell. In addition, as shown by Van Eijk et al. [13], serotransferrin molecules with the same iron content, but with variable antennae number and sialic acid content, i.e. from pentasialo- to asialotransferrins, show the same iron delivery to rat reticulocytes. In the same way, Mason et al. [ 141 have demonstrated that a 203

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non-glycosylated mutant of serotransferrin expressed in BHK cells, after mutation of the two asparagine carbohydrate linkage sites to aspartic acid residues, binds to HeLa S3 cells with the same avidity and to the same extent as the glycosylated protein, so proving that the glycans have no role in this function. Consequently, the present chapter does not deliver any information about the structurefunction relationships of transferrink glycans. However, it is devoted to an interesting study of ‘horizontal evolution’ through the superfamily of transferrins. In this respect, the present chapter is a useful addition to chapters 6 1 2 of volume 29A [15] and to chapters 1-8 of the present volume [16]. In fact, these chapters are devoted to the description of the primary structure of glycoprotein glycans from bacteria to man and are thus relevant to ‘vertical evolution’.

1.2. Biological importance of iron In Aphorisms, Hippocrates asserted that “diseases which are not healed by cures, are healed by iron”. We know now that iron is distributed in all of the living organisms and that all of the cells need iron to survive and to grow. For this reason, Nature has coined the transferrins of which the mission is to furnish this invaluable metal to all living cells (for a review, see ref. [7]). The total iron content of the normal human adult is approximately 3-5g and may be subdivided into four compartments: red blood cell iron, tissue iron, storage iron and transport iron. The largest iron fraction, 1.5-3 g, consists of hemoglobin of the red blood cells. The tissue iron-porphyrin proteins such as myoglobin, cytochromes, as well as ironsulfur proteins, peroxidases and catalases, account for only 0.1-0.3 g of iron. The storage iron fraction (1.0-1.5g) is widely deposited in tissues of the body, the largest fraction being in the liver. Its main form is ferritin which can store 23% of its weight of iron (5000 Fe3+ ions per mole) and has a molecular mass of approximately 480 kDa in its iron-free form of apoferritin. The link between the various forms of tissue and storage iron is serotransferrin. Only 3 4 m g of iron is present as serum iron which is equivalent to about 30% of iron-saturated serotransferrin.

2. The transferrin superfamily In the present chapter, the term ‘transferrin’, introduced forty-nine years ago by Laurel1 and Ingelman [ 171, will concern any protein possessing the characteristics above listed in section 1.1. Consequently, all of the compounds we describe possess, as a trade-mark, this common root in their name, according to Williams [ 181 who wrote on the chaotic nomenclature of the group: “All members of the group should carry the same family name. Such names as conalbumin, lactoferrin, p97 and sciatin are not acceptable”. 2.1. The saga of transferrins

The first transferrin was isolated in 1900 from hen egg-white and the definition of its properties, chiefly its inhibitory power of bacterial growth due to its affinity for

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iron, contributed to the discovery of the other transferrins. This explains the order of presentation of the transferrins we have adopted since it respects the chronology of the events.

2. I . I . Ouotrunsferrins (conalbumins) Although conalbumin, as it was called, was isolated by Osborne and Campbell in 1900 [19] from hen egg-white, almost half a century was to pass before the protein was identified in 1944 as the antimicrobial agent of egg-white whose properties were suppressed by the addition of iron as demonstrated by Schade and Caroline[20]. As related by Schade in a review full of humour[21], this discovery was the issue of a contract signed by Schade with the Medical Corps of the U.S. Army for producing a bacteriophage preparation effective against Shigellu dysenteriae. In order to protect the phages from the effects of lyophilisation, Schade used some additives, including eggwhite and observed that the growth inhibition of Shigellu was proportional to the eggwhite concentration of the medium. On the basis of this observation, Schade obtained by fractional ammonium sulfate precipitation a pure and very active compound which bound ferric iron to produce a salmon-pink color and to which he gave the Greek-rooted name of ‘siderophilin’ [22]. A few months later, Alderton et al. [23] isolated the protein they identified as conalbumin. In this way the bacteriostatic property of egg-white, which was well known for a long time, was explained. In this regard, as Schade wrote in an excellent review on non-heme metalloproteins [24]: “One may wonder whether Shakespeare was aware of this property, for in King Leur, Act 111, Scene VII, just following the excision of the eyes of the Duke of Gloucester and a cut face by the Duke of Cornwall, one servant speaks to a second: ‘I’ll fetch some flax and white of eggs to apply to his bleeding face’ ”. Schade opened the way to the discovery of serotransferrins. He suggested that blood might have a protein component similar to that of conalbumin and capable of so lowering the availability of iron to microbial pathogens that it might serve as a protective factor against infection of the host. Using Cohn’s method of plasma fractionation, Schade confirmed this view by characterizing a fraction which exhibited both the bacterial growth inhibitory and color-producing properties shown by conalbumin [25,26]. Conalbumin comprises 3-16% of the egg-white from various species of birds. It is structurally related to plasma serotransferrin, the polypeptide chain being coded by the same structural gene [27], but the carbohydrate moiety being different (see section 3.1.3). Because of the close relationship of these two proteins, the name ouotrunsferrin is now used in place of conalbumin. 2.1.2. Serotrunsferrins The existence in human plasma of non-heme iron was first demonstrated by Fontes and Thivolle in 1925 [28]. The association of bound iron with the globulin fraction was established by Starkenstein and Harvalik in 1933 [29] and, with the advent of electrophoresis, iron-binding was localized to the 61-globulin fraction by Surgenor et al. in 1949 [30]. In the same year, the same authors, on the basis of the studies of Holmberg and Laurel in 1947 [31] and of the aforementioned works of Schade and Caroline in 1946 [25], isolated and characterized the (3, -iron-binding globulin from Cohn’s fraction IV-3,4 to which Schade et al. [22] gave the name siderophilin. The crystallisation of the

206

serotransferrin was achieved by Koechlin in 1952 [32]. The presence of carbohydrate in serotransferrin was first reported by Surgenor et al. in 1949 [30]. In the meantime, in 1947, Laurel1 and Ingelman [ 171 had independently purified the ‘red protein’ from pig plasma and in the same year proposed the name ‘transferrin’ which has since been adopted as the generic name of the proteins of this family: serotransferrin (instead of siderophilin) present in blood and some external secretions, ovotransferrin (instead of conalbumin) in avian egg-white, lactotransferrin (also called lactoferrin) from milk, external secretions and leukocytes and melanotransferrin (instead of p97) in melanocyte and normal cell plasma membrane. A dozen mammalian and some frog, fish and insect serotransferrins were later isolated and characterized. In humans, the plasma concentration of serotransferrin, which is biosynthesized by the liver, varies from 3 to 3.5 g per liter and it is worthwhile to mention that the protein is only 20-30% iron-saturated, an important fact considering the inhibitory power of bacterial growth provided by aposerotransferrin acting via ferrideprivation (see above). Important variations of the plasma serotransferrin concentration are observed in physiological and pathological situations. They are often accompanied by dramatic modifications of glycan primary structure (see section 3.2).

2.1.3. Lactotransferrins (lactoferrins) Lactotransferrins (also called lactoferrins) were isolated for the first time from human and cow milk in 1960. Isolation of human lactotransferrin was described almost simultaneously by Montreuil et al. [33,34] and by Johansson [35]. By applying a fractionation procedure which associated an ammonium sulfate concentration gradient and a pH gradient, Montreuil et al. obtained a red-salmon coloured glycoprotein. The authors described in detail the physicochemical properties of the compound, demonstrated that it was glycosylated, bound reversibly ferric ions and possessed all the characteristics of a transferrin. However, it did not immunologically crossreact with human serotransferrin and had a 200-fold higher affinity for iron. These specific characteristics led Montreuil et al. to call this compound: lactosiderophilin or lactotransferrin [34]. For unknown reasons, the lactotransferrin is now commonly called lactoferrin. In 1960, Johansson described the isolation of a ‘red protein’ binding iron, devoid of Soret’s band and losing the metal at low pH [35]. In 1961, Blanc and Isliker [36] isolated from human milk by rivanol precipitation, a protein that they called lactoferrin and that they further identified as lactotransferrin. The term lactoferrin was reintroduced by Masson et al. in 1965 [37]. Bovine milk lactotransferrin first reported in 1939 by Serrensen and Serrensen [38], was isolated by Groves [39] and described under the name of bovine milk ‘red protein’. Since these first discoveries, lactotransferrin has been isolated from milk of numerous mammals: goat [40], mare [41], monkey [42], mouse [43,44], rabbit [45] and sow [46,47]. These findings caught the attention of many milk companies since, rightly or wrongly, a nutritional role was suggested for these proteins in iron transport for the mammalian newborn. The discovery of lactotransferrin specific receptors in the brush-border of enterocytes [48] supports this concept. In addition, as suggested by Montreuil et al. [34], human lactotransferrin could intervene in the intestinal defence of breast-fed infants by a

207

mechanism of iron-deprivation identical to that previously demonstrated by Schade and Caroline for ovo- and serotransferrin. Indeed, this biological role of lactotransferrin is now well established and extended to all mucous secretions (see section 2.3.2.1.2). In 1963, using immunoelectrophoresis, Biserte et al. [49] demonstrated that lactotransferrin was present in bronchial secretions. This result was confirmed in 1965 by Masson et al. [37] who, in addition, localized the lactotransferrin in human bronchial glands by immuno-histochemistry. Lactotransferrin has been identified, mainly due to the work of Masson, in most mucous secretions, i.e., bronchial and intestinal mucus and in various biological fluids such as saliva, tears, synovial fluid, seminal plasma, pancreatic juice, bile and, in very small amount, in blood plasma. In milk and mucous secretions, lactotransferrin is associated with secretory IgA (sIgA) and lysozyme and we now know that this association represents a powerful system of defence of mucosae (see section 2.3.2.1.2). The discovery by Masson et al. in 1969 [50,5 13 of the presence of lactotransferrin in the specific granules of neutrophilic leukocytes suggested its participation in the mechanisms of cell-mediated defence and opened the way to active research on this fascinating protein. 2.1.4. Melanotransferrin (human melanoma-associated antigen p97) Melanotransferrin (also called p97) (for a review see ref. [52]), discovered in 1980 by Brown et al. [53,54] and by Dippold et al. [55], is a cell-surface sialoglycoprotein that is present in most human melanomas, in certain foetal tissues, but only in trace amounts in normal adult tissues. This tumor-associated antigen was first identified in human melanoma by using monoclonal antibodies. Analysis of somatic cell hybrids and in situ hybridization have shown that the p97 gene, like the genes for transferrin and transferrin receptor, is located on chromosomal region 3q2 1-3q29 [56,57]. This observation suggested that p97 plays a role in iron metabolism and led Rose et al. [58] to propose “that p97 could be renamed melanotransferrin to denote its original identification in melanoma cells and its evolutionary relationship to serotransferrin and lactotransferrin, the other members of the transferrin superfamily”. In addition to being detected at various levels in other tumours such as lymphomas, melanotransferrin has subsequently been found in a wide range of cultured normal cell types including liver cells, intestinal epithelial cells, foetal intestinal cells, umbilical cord, placenta, sweat gland ducts, capillary endothelium of human brain and reactive microglia of Alzheimer’s disease patients. In contrast to other molecules of the transferrin family, melanotransferrin is the only one so far shown to be connected to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor as demontrated by Alemany et al. [59] and Food et al. [60]. In addition, examination of melanotransferrin peptide sequence has led to the conclusion that the protein possesses only one iron binding site at its N-terminal half [61].

2.2. Comparative study of transferrin peptide chains 2.2.I . Primary and three-dimensional structure Fourteen complete transferrin and transferrin-related amino acid sequences have now

208

been determined, either directly at the protein level, or by translation from the nucleotide sequence of the corresponding mRNA or gene: human [57,62-64], pig [65], horse [66], rat [67], rabbit [68], chicken [69], frog Xenopus Zueuis [70], cockroach [71], flesh-fly Surcophugu peregrinu [72] and tobacco hornworm Munducu sextu [73] serotrunsferrins, human [74-761, bovine [77-791, pig [47,80], goat [8 13 and mouse [82] lactotrunsferrins and human melanotrunsferrin [58] (for reviews, see refs. [ 10,831). On the basis of the following data, it is clear that transferrin evolution was highly conservative. (1) All proteins consist of a peptide chain of 680-700 amino acid residues which can be divided into two homologous halves. The insect transferrin isolated from hemolymph of Surcophugu peregrinu fly is distinct since its molecular mass is 65 kDa only and, unlike the other transferrins, the similarity between its N-terminal and C-terminal halves is only 19% [72]. (2) As shown in Table 1, the sequence homology between eight different transferrins, as well as between N- and C-terminal halves of the molecules, is very extensive. These results favour the hypothesis of Williams [84] according to which transferrins originated by gene duplication. This hypothesis has been verified at the gene level by the study of exon distribution within the genes of hen ovotransferrin and human serotransferrin [85,86]. Melanotransferrin, a cell membrane transferrin-like glycosylphosphatidylinositol-anchored protein, is considered to be an intermediate between transferrins and receptors for transferrins [60]. Finally, the confirmation of the gene duplication hypothesis came from the isolation from the primitive urochordate F‘yuru stolonifera of a transferrin-like molecule of molecular mass 41 kDa with only one high affinity iron-binding site [87]. In experiments described by Bowman et al. [88], hybridization of human serotransferrin cDNA with fragments of Pyuru DNA showed hybridization signals. It was concluded that the intragenic duplication that produced the amplified transferrin gene and enabled transferrin to gain an additional iron-binding site would have occurred at some point in time which has been estimated as some 400 to 500 million years ago, at the evolutionary period wherein prochordates and humans separated. However, according to Escriva et al. [67] who have constructed the phylogenetic tree of the transferrin family, at least three gene duplications have occurred during the evolution of the transferrin sequences. An initial duplication occurred before the separation of arthropods and chordates, as both insect transferrins have duplicated N- and C-termini. A second duplication occurred in the branch leading to vertebrates before the emergence of land animals. This duplication gave rise to human melanotransferrin which can be observed to be re ancient than any other vertebrate transferrin. The third duplication took place b ore the appearance of mammals, and lactotransferrins are the resulting products. (3) The position of disulfide bridges within the two halves of the peptide chain are highly conserved in transferrins. (4) When considering the structure of the iron-binding sites, the amazing conservative evolution of transferrins bursts upon view. In fact, (i) in all transferrins, four ligands are provided by the surrounding protein structure in both iron-binding sites of N- and C-lobes: one carboxylate from aspartic acid, two phenolate oxygens

7

209

Table 1 Percentage homology between N- and C-terminal halves of various transferrins [ 101 Alignment

Percentage homology a

score (SD)a Human

Human N C N

x

C 34.0

pig N

C

Horse N C

Rabbit N C

Rat C

Hen N

C

Frog N C

Hornworm N C

48.6 71.9 45.1 75.7 44.8 81.9 43.5 39.3

53.8

45.8 71.3 45.1 70.6 48.6 75.9 66.9

45.9

54.3 39.7 51.2 30.3 33.2 45.1 43.9 44.7 28.0 32.6

x

x

46.2 42.7 42.4 28.4 31.4

Pig

N 40.5 25.6

45.5 74.9 43.6 73.4 43.0 42.9

54.6

C 22.3 44.3 22.1 x 42.6 73.4 45.1 73.4 60.5 N 67.7 29.3 76.9 22.5 x 43.4 74.2 43.2 41.9 C 24.5 76.8 27.8 61.7 24.7 x 43.8 70.9 65.2

46.2

54.1 38.6 52.3 28.8 30.1

Horse

55.2

44.5 46.9 42.1 29.9 31.5

Rabbit

N 60.8 28.3 75.1 28.9 61.0 15.8

Rat Hen

25.3

x

N 25.3 18.8 28.3 17.8 26.5 18.1 34.3 15.3

0.6

29.9

22.5

C 25.6 35.7 23.4 34.4 26.9 35.9 21.6 32.5 18.0

22.5

53.0 20.7

0.7

12.8

9.2 12.6

0.8

17.7

9.0 12.1 10.1

43.0

52.6 39.0 51.7 27.9 28.6

42.9 39.9

55.8

43.8 44.0 42.4 28.0 29.7

66.3

44.3

53.0 35.9 50.5 30.0 31.3

C 8.6 21.5 6.9 19.9 6.5 26.2 5.0 26.0 x N 30.6 29.7 29.5 28.0 37.1 21.4 34.0 22.8 3.7 C 19.5 31.7 25.2 36.4 21.7 62.4 21.6 47.3 24.8

33.3

48.0 31.3 51.4 25.3 27.8

x

41.4 42.9 44.2 28.2 32.9

x

C 23.6 55.2 28.5 44.9 28.9 59.9 24.5

Frog

Hornworm N 13.0 C a

9.9 10.2

x

7.5 10.2 10.8 14.6 11.5

13.8 11.0 15.8 11.7 14.5

8.5 15.0 10.2

37.2 50.7 27.5 28.0 x

37.1 28.9 26.6 x

8.4

27.9 29.0 x

30.3

8.5

x

N,C: N- and C-terminal part, respectively.

Fig. 1. Schematic representation of the iron- and anion-binding site in lactotransferrin (Anderson et al. [89]).

from tyrosine and one imidazole nitrogen from histidine (Fig. 1) [89]. In addition, one arginine intervenes in the prior fixation of a carbonic acid anion which is a prerequisite for the binding of iron; (ii) the position of these amino-acids along the peptide chain of all of the transferrins is highly conserved as demonstrated in Table 2 [90].

210 Table 2 Structural comparison of the iron binding sites of N-lobe of rabbit serotransferrin. human lactotransferrin and hen ovotransferrin [90] Rabbit serotransferrin

Human lactotransferrin

Hen ovotransferrin

Arg-124

Arg-121

Arg-121

Arp-63

ASP-60

His-249

ASP-60 His-253

Tyr-95,I 88

Tyr-92,192

Tyr-92,191

His-250

The conservative evolution of the primary structure of transferrin peptide chains leads, as a consequence, to a conservative evolution of transferrin three-dimensional structure (for a review see ref. [91]). In fact, X-ray crystallographic studies of human lactotransferrin [89,92-971, rabbit serotransferrin [98] and hen ovotransferrin[99] show (see Fig. 20) that the single peptide chain of these transferrins is folded into two lobes corresponding to the N-terminal and C-terminal halves of the molecules: in human lactotransferrin for example, amino acids 1 to 333 and 345 to 691, respectively. Both lobes are associated by non-covalent hydrophobic amino acid interactions, salt bridges and water molecules as demonstrated by Legrand et al. [loo] for human lactotransferrin and by Ikeda et al. [ l o l l for hen ovotransferrin. Both lobes are joined by a short connecting peptide of about twelve amino acids: eleven in human lactotransferrin (amino acids 334-344), sometimes in an extended conformation, as in rabbit serotransferrin [98] and hen ovotransferrin [99] and sometimes in a three-turn a-helix conformation as in human lactotransferrin [92] (see Fig. 2 1C). The three-dimensional pictures of human seroand lactotransferrin peptide chains are perfectly superimposable [9 11 with very few differences, so demonstrating conservative evolution of transferrins led to a conservative evolution of their three-dimensional structures. 2.2.2. Location of glycosylation sites 2.2.2.1. Serotransferrins. All known serotransferrins contain one or two glycans of the N-acetyllactosaminic type which are located in the C-terminal lobe of the polypeptide chain. Hen [102], rabbit [103,104], pig [I051 and rat [67] serotransferrins contain a single glycan located in a very similar position which does not correspond to Asn-4 13 in human serotransferrin (Fig. 2). As shown in Fig. 3 the two glycosylation sites (Am-413 and 611) of human serotransferrin may be occupied either by di-, tri- or tetraantennary glycans of the N-acetyllactosaminie type [ 106,1071. Concerning the triantennary glycans two positions of the third antenna exist: p-1,4-linked to the a-1,3-mannose residue of the inner-core, namely 2,4-variant, or b-1,6-linked to the a-176-mannoseresidue of the inner-core, namely 2,6-variant. This leads to several human transferrin glycovariants which are separable by Con A-Sepharose chromatography into glycovariants Tf-I, Tf-11 and Tf-111 described in section 3.1.1 [ 1081. The location of 2 4 - and 2,6-variants can be determined by using serial lectin affinity chromatography, fast atom bombardment-mass spectrometry and H-NMR

'

21 1

Human

679

Rat

695 490

Pig

727

497

Rabbit

676 485

Hen

686 473

618

Horse 1

687

Horse 2

687

729

Melano Tf 38

135

515

Fig. 2. Location of glycans in human [106,107], rat [67], pig [105], rabbit [103,104], hen [102], horse (1,2: vanants 1 and 2, respectively [I 14,1151) serotransfernns and human melanotransferrin [52].

spectroscopy. The results obtained show that the ratio of 2,4-variant versus 2,6-variant at the glycosylation site Asn-413 is approximately 5:1, whereas this ratio is 1:l for the glycosylation site Asn-61 1 [ 1091. In addition, on the basis of their different content in sialic acids, the glycovariants have been separated by isoelectric focusing [ 1101 or by high performance pellicular anion-exchange chromatography [ 1 111. The most accurate method for detecting the presence of human serotransferrin glycovariants is electro-spray ionization mass-spectrometry. On the basis of a theoretical mass of 75 143 daltons for the polypeptide chain of human serotransferrin [ 141, the mass values of the different serotransferrin glycovariants which could be found in normal and pathological sera are given in Fig. 3 [ 1121. Horse serotransferrin presents two glycovariants with one (Asn-496) [ 1 131 or two (Asn-496 and -6 19) glycans [ 1141 due to a mutation from Asn-638 to Ser-640 [1 151. Potential glycosylation sites are present in j s h [116], tobacco hornworm [73] and insects [72] serotransferrins, but the position of glycans in their peptide chain has not yet been determined. Figure 2 synthesizes the present data concerning the location of glycans on the peptide chain of different serotransferrins. It is remarkable that, except for melanotransferrin, all of the glycans or potential glycosylation sites are located in the C-domain and in very conserved positions.

212 1

75143

473

1

'?'

,679

N

79260

3SA

79551

4SA 1

2

1

2

1

79916

80207

80863

81519

4SA

1 5SA

'

1

2

1

2

' 1

1

6SA

I

7SA

2

Fig. 3. Location and molecular masses from 79 260 to 81 519 of human serotransferrin glycovariants. Open circle, NeuAc; solid square, Gal; solid circle, GlcNAc; solid diamond, Man; 1 to 679, amino acids of the peptide chain; x SA, number of sialic acid residues [ 1 121.

2.2.2.2. Ouotransferrins. The peptide chain of hen ovotransferrin is identical to that of hen serotransferrin. However, both glycoproteins differ only by the structure of their glycans (see Figs. 16B,D). Two potential glycosylation sites have been identified

213

Human 1

Human 2

138

479

624

138

479

624

Mouse

692

692

689

cow

689 233281

368

476 545

(a) Mare

692

Goat

692

(a) Swine

667 366

472

571

Fig. 4. Location of the glycosylation sites on the peptide chain of human variants 1 [74] and 2 [117], mouse [121], cow [I 181, mare, goat [81] and swine[80,120] lactotransferrins: (a), mapping not determined; solid circles on bars, glycosylated sites; heavy bars, non-glycosylated sites. In cow lactotransferrin, glycans are of the oligomannosidic type at positions 233 and 545, of the oligomannosidic or N-acetyllactosaminic type at positions 368 and 476. Only the glycans of the N-acetyllactosaminic type at position 476 contain one N-acetylgalactosamine residue.

in the C-terminal lobe (Asn-473 and -618) (Fig. 2) but only residue Asn-473 is glycosylated [ 1021. N-glycosylation site mapping of lactotransferrins from different species is described in Fig. 4. The number of N-glycosylation sites present in lactotransferrin polypeptide chains is quite variable and two types of lactotransferrins can be distinguished. Lactotransferrins with N-acetyllactosamine type glycans possess one or two glycosylation sites, while lactotransferrins with both oligomannosidic and N-acetyllactosamine type glycans contain five potential glycosylation sites. Contrary to serotransferrins in which glycans are located in the C-domain only, glycans of lactotransferrins are located in both N- and C-domains. H u m a n lactotransferrin possesses three potential glycosylation sites (Asn- 137, 478 and 624) and only two glycans in positions Asn-137 and Asn-478 [74]. However, Van Berkel et al. [ 1171 have identified a human lactotransferrin glycovariant glycosylated on Asn-624. Bovine lactotransferrin possesses five potential glycosylation sites, but the presence of only four glycans has been demonstrated and Asn-281 has not been glycosy-

2.2.2.3. Lactotransferrins.

214

lated [ 1181. The glycans of bovine lactotransferrin present a high degree of structural heterogeneity (see Fig. 14). The location of the four glycosylation sites is given in Fig. 4. The glycan of the N-acetyllactosamine type containing the GalNAc(fi1-4)GlcNAc unit is linked to Asn-476. This result suggests that a recognition signal for the N-acetylgalactosaminyltransferase should exist in bovine lactotransferrin and that the gene for this enzyme is expressed in bovine mammary gland. During lactation, the relative ratios of the different types of glycans linked to the four glycosylation sites vary significantly. Only oligomannosidic structures are present in cow lactotransferrin, up to one month before calving, whereas three glycans of the oligomannose type and one of the N-acetyllactosamine type have been detected in colostrum [119]. This result suggests either an effect of the hormonal status or of the rate of lactotransferrin biosynthesis on the type of glycosylation. Caprine lactotransferrin has, like bovine lactotransferrin, five potential glycosylation sites (Fig. 4), but the location of glycans has not yet been defined. Porcine lactotransferrin possesses three potential N-glycosylation sites which have been located in the C-terminal lobe (Asn-366, 472 and 571) (Fig. 4) [80] but only one site is glycosylated as recently demonstrated by Coddeville et al. [120]. Murine lactotransferrin polypeptide chain carries two glycans of the N-acetyllactosaminic type (Fig. 4) [121]. 2.2.2.4. Melanotransferrin. Melanotransferrin is a sialoglycoprotein [56] with three potential glycosylation sites located in the N-terminal and C-terminal lobes (Fig. 2) [52]. However, structure and location of glycans have not yet been defined. 2.2.2.5. Conclusion. In conclusion, considering in Figs. 2 and 4 the location of the glycans on the sero- and lactotransferrin polypeptide chains from different species, it is remarkable to note that glycans are located in highly conserved positions of the C-terminal lobe: Asn-490 to 496 and Asn-611 to 619 for serotransferrins and Asn-472 to 479 for lactotransferrins. This observation suggests that these particular glycans should play a crucial role in the biological functions of transferrins probably by interacting with an important peptide sequence. 2.3. Role of transferrins and of their receptors 2.3.1. Serotransferrin 2.3.1.1. Role of serotransferrin. Serotransferrin is now recognized as the protein transporting iron throughout the vascular as well as the lymphatic system (for a review, see ref. [7]). First, from the iron entering through the intestinal mucosa to the storage organs, and, secondly, from these storage organs to all cells requiring iron for growth and maintenance, and, in greatest quantity, for maturation of the developing erythrocytes. The capture of iron by cells is mediated by a specific membrane receptor of which a schematic structure proposed by Trowbridge [122] is given in Fig. 5. Serotransferrin, two thirds of which is iron-free in normal plasma, may intervene in the defence of organisms against bacteria due to its ability to bind iron so tighly under physiological conditions that the concentration of free ionic iron in plasma and lymph

215

671 AMINO ACID

EXTRACELLULARDOWIN

Fig. 5 . Schematic representation of the human serotransferrin receptor. C, cysteine residues; positions 89 and 98, disulfide bridges; position 62, fatty acid chain. From Trowbridge et al. [122].

is very low. Thus, the creation of a nutritional iron lack establishes an inhospitable environment for many pathogens leading to an inhibition of their growth, as demonstrated by Schade et al. [26,123]. This constitutes a defense mechanism against disease which reinforces the immunity system and serves as protection against free-iron toxicity, 2.3.1.2. Serotransferrin receptors. In many cell types, the uptake of iron is mediated by a specific receptor first identified as a placenta brush border membrane glycoprotein in 1979, by Sussman et al. [124] (for reviews, see refs. [125-1281). The number of receptors per cell varies from several ten thousands to almost a million. Investigations in a large number of tissues including tumors have revealed significant increases of serotransferrin receptors in all dividing cells so that the measurement of receptor expression has become a standard procedure to determine the growth potential of in uiuo tumors (for a review, see refs. [129,130]). Serotransferrin receptor genes are encoded on the same chromosome (chromosomal region 3q21-3q29) in humans [13 1,1321 as those for sero- and melanotransferrins (for a review, see ref. [SS]). Receptors of numerous cells have been identified by the use of monoclonal antibodies and appear to have similar structures to that represented in Fig. 5. They are all disulfidelinked dimers consisting of two identical transmembrane glycosylated units (MM: 95 kDa) of 760 amino acids with the N-terminus facing the cytoplasm. The cytoplasmic domain corresponds to the first 61 amino acid residues and is followed by a single hydrophobic

216

transmembrane region of 28 residues. Ser-24 is phosphorylated by a protein kinase C. The serotransferrin receptor, like other type I1 membrane proteins, does not have a cleavable signal sequence and it has been suggested that such transmembrane proteins have appeared early in the course of biochemical evolution [133]. The two subunits are linked by two disulfide bonds. Cysteine at position 62 is the site of acylation by a fatty acid residue. Serotransferrin receptor is a glycoprotein containing both N- and 0-linkages to the peptide chain[134-138]. The location in the extracellular domain of the three N-glycosylation sites Asn-25 1, 3 17 and 727 was deduced from the cDNA sequence of the receptor [131,132]. The sites of N-glycosylation of serotransferrin receptors are highly conserved in evolution. In fact, all of the receptors cloned up to date, including human, chicken, Chinese hamster, mouse and rat, possess three sites of N-glycosylation [ 13 1,132,1391. The first two sites are equivalent to human sites Asn-25 1 and Asn-3 17 [ 139,1401. Rodents have two glycosylation sites [ 1391 and chicken one glycosylation site [ 1411. The presence of one 0-glycan linked through an N-acetylgalactosamine residue to threonine-I04 near the transmembrane domain has been established by Do and Cummings [ 1371, The type of glycan is not randomly distributed along the peptide chain. Asn-251 site contains glycans of the N-acetyllactosaminic type while Asn-727 is entirely of the oligomannosidic type [142,143]. Glycans linked to Asn-317 are a mixture of glycans of the N-acetyllactosaminic, oligomannosidic and hybrid type [ 1443. Serotransferrin receptor N-glycans play a key role in the folding and transport of the receptor to the cell surface. Inhibition of N-glycosylation by treatment of cells with tunicamycin blocks translocation of the receptor to the plasma membrane [ 145,1461 interferes with formation of active dimers [145,146] and leads to the retention of the receptor in the endoplasmic reticulum [ 147,1481. Site-directed mutagenesis led to the same results [ 147-1501. Although all three sites contribute to the correct folding, transport and functioning of the serotransferrin receptor, the glycan conjugated to Asn-727 is the most critical to the structure and function of the receptor [ 143,lSO]. Glycans of sites 25 1 and 317 are the least critical for the folding and transport of the receptor. The serotransferrin-to-cell cycle, simultaneously discovered in 1983 by Dautry-Varsat et al. [151] and Klausner et al. [152], is a shuttle involving a complex pathway of endocytosis and recycling of the serotransferrin-serotransferrin receptor. Endocytosis is initiated by the binding of diferric serotransferrin to the receptor and is followed by the accumulation of the complex in clathnn-coated vesicles. Vesicles fuse with endosomes and progressive acidification to pH 5.5 leads to the release of iron, leaving the apotransferrin molecule still firmly bound to its receptor. Finally, the apotransferrinreceptor complex is recycled back to the plasma membrane and, encountering a neutral pH, the apo-serotransferrin dissociates from its receptor, is released in the circulation and goes off in a renewed search for iron at the level of storage tissues. 2.3.2. Lactotransferrin 2.3.2. I . Role of lactotransferrin. When the first lactotransferrins were isolated from mammalian milks, two hypotheses were proposed about the role they could play [34]: iron nutrition of the new-born and antibacterial defence of the gut due to growth inhibition

217

of microorganisms by iron deprivation (see sections 2.1.1 and 2.1.2). However, the fundamental discovery by Masson et al. in 1969 [50,5 I] of the presence of lactotransferrin in the granules of neutrophilic leukocytes, suggesting the participation of this protein in cell-mediated defence, gave rise to active research in this field. Two events attest to the development of knowledge on the biological role of lactotransferrin. First, the proliferation of literature in this domain in the past five years [7,129,153-1631. Second, the organization every two years of an international symposium on “Lactoferrin, structure and function” in the USA in 1993 and 1995 [159,163] and in France in 1997. We know now that lactotransferrin plays roles in iron transport, in local defence of epithelia, in cell-mediated defence of organisms and as a growth factor. 2.3.2.1.1. Iron transport. Experiments in oitro carried out in 1979 by Cox et al. [164] and using human intestinal biopsies demonstrated that human lactotransferrin can donate iron to intestinal mucosal cells. The characterization of a specific intestinal lactotransferrin receptor in rabbit [48], mouse [ 165,1661, Rhesus monkey [ 1671 and human foetal intestinal brush border membranes [ 1681 reinforces the concept of the role of lactotransferrin in intestinal iron absorption. However, despite these findings, the nutritional activity of lactotransferrin is still a subject of controversy (for a review, see ref. [7]). 2.3.2.1.2. Bacteriostasis and bactericidal effects. The antibacterial effect of lactotransferrin was first demonstrated by Masson and Heremans [169] and by Oram and Reiter [ 1701. This effect could be explained by iron-deprivation, iron being essential for the growth of numerous microorganisms. In fact, only apolactotransferrin is active. However, the mechanism of antibacterial activity is more complex and it is clear that lactotransferrin protects the mucosae in association with, at least, secretory IgA (sIgA) and lysozyme as first demonstrated by Bullen et al. [171] and Spik et al. [172,173]. The association of human sIgA with human (or bovine) lactotransferrin strongly inhibits the growth of pathological bacteria, in contrast to sIgA or lactotransferrin alone. The inhibitory power of the mixture is reinforced by the lysozyme which is present in human milk in a association with lactotransferrin in a molar ratio of 2: 1 [ 1741. The role of this association has been elucidated by Perraudin and Preels [ 1751 who showed that bacteria submitted to lysozyme action were agglutinated by lactotransferrin due to charge-charge interactions. Lactotransferrins also possess a bactericidal effect observed in 1977 by Arnold et al. [ 1761; according to Tomita et al. [ 177,1781 this is due to the liberation of short peptides like lactoferricin B (fragment 1 7 4 1 of bovine lactotransferrin) by partial proteolysis. Lactoferricin B derived from the peptidic segment binding to lactotransferrin receptors. The industrial interest in lactoferricin is evident. 2.3.2.1.3. Cell mediated defence of organism. A concise description of the different mechanisms of cell mediated defence of the organism by lactotransferrin would be an “impossible mission”. Consequently, the reader is referred to a series of excellent books and reviews [7,158-1621. Briefly, lactotransferrin has been shown to be involved in numerous inflammatory events (for reviews, see refs. [179,180]) and in immune response functions such as regulation of granulocyte monocyte colony stimulating factor synthesis with suppression of myelopoiesis by inhibition of the production of IL-1 (for a review, see ref. [lSl]),

218

regulation of interleukin synthesis [ 181,1821, natural killer cell activation and antitumor effects [129,183,184] and maturation of T- and B-cells [lS5-187]. 2.3.2.1.4. Growthfactor actiuity. Recent studies have demonstrated that lactotransferrin may promote growth of intestinal epithelial cells, suggesting that it might play a part in maturation of the intestine in the newborn (for reviews, see refs. [161,162]), of B-lymphocytes [1881 and of PHA-activated peripheral blood lymphocytes [ 189,1901.

2.3.2.2. Lactotransferrin receptors. The existence of a lactotransferrin receptor was first demonstrated by Van Snick and Masson in 1976 [191] at the surface of mouse peritoneal macrophages and lymphocytes. Since this discovery, the presence of lactotransferrin receptors has been demonstrated at the surface of various cells (for reviews, see refs. [156,158,192,193]): rabbit [48], mouse [165,166], monkey [167] and human [168] enterocytes; human HT29 and Caco-2 enterocyte cell lines [ 1941; human monocytes (reviewed in ref. [ 195]), human alveolar macrophages [ 1961, human neutrophils [ 195,1971, human resting lymphocytes [ 1971, human activated lymphocytes [ 1891, human Jurkat T cell line [190], human epithelial mammary cell line [198], human platelets [199,200] and megakaryocytes [201], hepatocytes [202,203] and in bacteria (for a review see refs. [204,205]). Contrary to serotransferrin receptors, little is known about the structure, physicochemical properties and gene expression of lactotransferrin receptors. Their main characteristics, which are totally different from those of serotransferrin receptors, can be summarized as follows: (i) Lactotransferrin receptors constitute a single peptide chain of about 1 10 kDa (100 to 13OkDa for intestinal receptor, 105kDa for lymphocyte and platelet receptors) [48,192,201]. A soluble fraction of 95 kDa is liberated by limited proteolysis [206]. (ii) Receptors are glycosylated but nothing is known about the structure and role of glycans [165,166,199]. (iii) Lactotransferrin receptors do not bind serotransferrin [48]. However, due to the high homology between the peptide chains of lactotransferrins, a given receptor is able to bind lactotransferrins originating from different species [ 1921. This explains the interest of milk companies in bovine lactotransferrin which is prepared in ton amounts and added to the food of young infants. (iv) The receptor binding site of lactotransferrins from different origins is located in the N-terminal part of the molecule covering amino acid residues 4 to 52. The conformation of this peptide segment of human and bovine lactotransferrin is very similar, explaining that bovine lactotransferrin is able to recognize human lactotransferrin receptor [ 1921. (v) Contrary to serotransferrin receptor which binds iron-saturated serotransferrin only, lactotransferrin receptor binds apo- and ferri-lactotransferrin 1481. 2.3.3. Ouotransferrin At the moment, we do not know if ovotransferrin can serve as an antibiotic protecting the egg and embryo development. In fact, if bacteria are present in the oviduct and appear in the egg-white, the bacteriostatic effect of ovotransferrin, which is only in the

219

apoprotein form in egg-white, could inhibit their growth by ferrideprivation. Additionally, it is possible that ovotransferrin plays a role in iron nutrition and in differentiation of the developing chick embryo. 2.3.4. Melanotransferrin The role of melanotransferrin has been recently elucidated by Kennard et al. [207] who demonstrated that this membrane bound iron binding protein is involved in the transferrin-independent uptake of iron in mammals but from iron-citrate and not from iron-transferrin complexes. This alternative iron uptake pathway may not function in the normal recirculation of iron within the body but might play a role during iron overload. On the other hand, rapidly proliferative tumor cells like melanocytes could use the alternative pathway to increase iron uptake. This independent system could also participate in the absorption of iron by intestinal cells that have no transferrin receptor on their lumenal surfaces [208], but express a transferrin-like GPI-linked iron-binding protein at the apical surface of fetal intestinal epithelial cells [209].

3. Comparative study of transferrin glycan primary structures The comparative study of sero-, lacto-, ovo- and melanotransferrins from different species or tissues shows (Table 3) that all are N-glycosylproteins which differ in their carbohydrate content (2.2 to 11.2% total sugars), in the number of glycans (1 to 4), except in serotransferrins of some fishes which are not glycosylated, and of antennae (2 to 4) as well as in the fucosylation rate. All of the glycans are of the N-acetyllactosaminic type, except in bovine, sheep and goat lactotransferrins which contain additional oligomannosidic structures (for reviews see refs. [ 119,2101). In addition, significant modifications of serotransferrin glycan primary structure have been observed in different physiological and pathological situations (see section 3.2.2). 3. I . Normal transferrin glycans

3. I . 1. Serotransferrin glycans As shown in Table 3 and in Figs. 6 to 10, serotransferrins of analyzed species possess 0 to 2 diantennary glycans of the N-acetyllactosaminic type, except human and fish serotransferrins which contain tri- and tetraantennary glycans, but in lower amounts. For example, in human serotransferrin (Fig. 6A-D), the relative proportions of serotransferrin having two triantennary glycans (glycovariant Tf-I), one diantennary and one triantennary glycan (glycovariant Tf-11) and two diantennary glycans (glycovariant Tf-111) (Fig. 6C,D) are 1, 17 and 82%, respectively [lOS]. In this regard, it is interesting to note, as described in section 3.2, that in some physiological or pathological situations, the amount of triand tetraantennary glycans increases significantly. Serotransferrin glycans are generally non-fucosylated except in human cerebrospinal fluid (trace amounts) [246,247], rat (20-30% of the molecules) [221], pig (100% of the molecules) [220] and the serotransferrin-like glycoprotein from mouse milk [2 18,2191 in which this protein co-exists with a lactotransferrin. None of the serotransferrin glycans

w 0 N

Table 3 Characteristics of sero-, ovo- and lactotransferrin glycans from different species Transferrins

Carbohydrate

Number of

Types and number of glycan

content ("h)

glycans

structuresa L

M

Fucosylation a-l,3b

Bisecting

GalNAc

Figure

a-1,6"

Serotransferrins Human

5.80

2

bi + tri +tetra

6A-D

Bovine

2.90 2.85 2.85-5.20 2.90 2.90 3.00 2.90 2.20 3.50

1

bi

6A,B

1

bi

16D 9 6A 7 6A 8 6A

Hen Horse Marsupial Mouse Rabbit Rat Sheep Fishe

1-2

bi

1

bi

1

bi + tetra

1

bi

1

bi

1

bi

1

tri tetra

+ +

+

10

Ovotransferrins Hen Turkey

2.65 3.70

tri bi

+ +

16B 16A continued on next page

Table 3, continued Transferrins

Carbohydrate content (“h)

Types and number of glycan structuresa

Number of glycans

Fucosylation ~

~

M

a-1,3b

a-1,6‘

+

+

Bisecting GlcNAc

GalNAc

Figure

L

Lactotransferrins

Human (milk) Human (1eukocyte)g Mouse Bovine Goat Pig

6.40

2

bi

2

bi

3.50

1

1 1.20

11.00

4 4

3.40

1

bi (4-2) bi(2)

+ + +

bi

+

bi 2 4 2

M, oligomannosidic type; L, N-acetyllactosaminic type; bi, tri, tetra: bi, tri and tetraantennary glycans, respectively. a-1,3, fbcose residue linked in C-3 position to GlcNAc of N-acetyllactosamine residue. a-1,6, fucose residue linked in C-6 position to the GlcNAc residue conjugated to the peptide chain. a

g

11B-D 11A 1 lB,C

+ + +

13-1 5 11B,C; 15B-D

Kangaroo, opossum and wallaby serotransferrin Carp big-head. Agalactoglycan. Polymorphonuclear leukocytes.

N

tl

222

NeuSAc(a2-6)Gal(fi14)GlcNAc(fi I -2)Man(a 14)

\ NeuSAc(a2-6)Gal(fi 14)GlcNAc(fi 1-2)Man(a 1-3)

Man(fi14)-R I

(A)

Gal(fi14)GlcNAc(fi 1-2)Man(a 1-6) \

NeuSAc(a2-6)Gal(fi 14)GlcNAc(fi 1 -2)Man(a 1-3)

han(fi1-4)-~ (B) I

NeuSAc(a2-3)Gal(fi14)GlcNAc(fi14)

\

Man(a1-6)

\

NeuSAc(a2d)Gal(fi 14)GlcNAc(fi 1-2)’

Man(fi14)-R

(C)

I

NeuSAc(a2-6)Gal(B 14)GlcNAc(fi1-2)Man(a 1-3) NeuSAc(a2-6)GaI(fi 14)GlcNAc(fi 1-2)Man(a 1-6)

\ NeuSAc(a2-6)Gal(fi 14)GlcNAc(fi 1-2)

NeuSAc(a2-3)Gal(fiI 4)GlcNAc(P 1 4 )

\

Man(fi14)-R

(D)

I

Man(a1-3) I

Fig. 6. Primary structure of serotransferrin diantennary glycans from human (A,B) [211-213], cow (A,B) [ I 19, 2141, rabbit (A) [103], sheep (A) [ I 19,2151, marsupial (kangaroo, opossum, wallaby) (A) [ I 19,2161. Primary structure of human serotransferrin triantennary glycans (C,D) [I 19-21 71. R, GlcNAc(fi14)GlcNAc(fil-N)Asn.

contains a bisecting residue of N-acetylglucosamine residue, except fish serotransferrin and human cerebrospinal fluid transferrin (Figs. 10A and 17A). The only sialic acid found to date in serotransferrin glycans is N-acetylneuraminic acid, except in horse serotransferrin glycans which contain 4-O-acety1, N-acetylneuraminic acid in addition to N-acetylneuraminic acid (Fig. 9) and in mouse serotransferrin in which only N-glycolylneuraminic acid is present (Fig. 7).

3.1.2. Lactotransferrin glycans Lactotransferrins from human, bovine, porcine, caprine and murine milk are N-glycosylproteins with diantennary glycans of the N-acetyllactosaminic type, a- 1,6-fucosyIated on the N-acetylglucosamine residue linked to the peptide chain. Only human lactotransferrin has a-l,3-fucosylated N-acetyllactosamine residues (Fig. 11). Cow, sheep and goat lactotransferrins contain additional glycans of the oligomannosidic type (Fig. 15). Only human lactotransferrin possesses poly-N-acetyllactosaminic glycans (Fig. 12). Bovine lactotransferrin glycans present a great microheterogeneity (Fig. 13). They are characterized a-1,3-linked galactose residues in terminal position (Fig. 14) and by N-acetylgalactosamine residues replacing galactose residues (Fig. 14) as in caprine lactotransferrin glycans. Interestingly, the glycan primary structure of lactotransferrin extracted from human polymorphonuclear leukocytes is identical to that of the non-fucosylated diantennary

223

Neu5Gc(a2-6,3)Gal(fi14)GlcNAc(fi1-2)Man(a 1-6)

\ Man(fil+)-R I

Neu5Gc(a24,3)Gal(fi14)GlcNAc(fi 1-2)Man(a 1-3) NeuSGc(a2-6)Gal(fi14)GlcNAc(fi1-2)Man(a I d )

\ Man(fi14)-R I

NeuSGc(a2-3)Gal(fi1-3)GlcNAc(fi1-2)Man(al-3)

I (a241

NeuSGc

NeuSGc(a2-3)Gal(fi14)GlcNAc(fi14)

\

Man(a1-6)

\

Neu5Gc(a2-3,6)Gal(fi14)GlcNAc(fi 1-2)’ NeuSGc(a2-6)Gal(fil 4)GlcNAc(fil -2)Man(a 1-3)

Man@ I 4 ) - R I

NeuSGc(a2-3)Gal(fi14)GlcNAc(fil4)

\

Man(al-6)

\

NeuSGc(a2-6)Gal(fi 1 4)GlcNAc(fi 1-2)’ NeuSGc(a2-6)Gal(fi 1 4)GlcNAc(fi1-2)

Man(fil4)-R \

Man(a1-3)

I

I

NeuSGc(a2-3)Gal(fi14)GlcNAc(fi 14)

Fig. 7. Primary structure of the glycans from mouse serotransferrin [ 1 19,2 18,2 191 R, GlcNAc(fil+)[Fuc(a 1-6)10-, GlcNAc(kN)Asn.

glycan of human serotransferrin (Fig. I IA). This data should be taken into consideration when human milk fucosylated lactotransferrin is used in binding experiments with various cells, mainly with cells of the immune system. 3.1.3. Ouotransferrin glycans In contrast to sero- and lactotransferrins, glycans of ovotransferrins from avian egg-white contain a bisecting N-acetylglucosamine residue, like other glycoproteins from oviducts, such as ovomucoid and ovalbumin for instance (Fig. 16A-C). Like all of the avian egg glycoproteins, ovotransferrins are not fucosylated. Concerning the hen sero- and ovotransferrin glycans, it is worthwhile noticing that they present completely different primary structure, whereas both transferrins which derived from the same gene [27]possess exactly the same polypeptide chain [69].In fact, hen serotransferrin glycan has a diantennary sialylated structure (Fig. 16D). This result does not favor the hypothesis that the protein moiety controls the structure of its own glycans, but rather that the glycan primary structure depends only upon the enzyme equipment of secreting cells, serotransferrin being synthesized by the liver, and ovotransferrin by the oviduct.

224 NeuSAc(a2-6)GaI((3 I -4)GlcNAc(p 1-2)Man(a 1-6)

\ Man(p14)-R I

(A)

NeuSAc(a2-3)Gal(fiI -3)GlcNAc(fi 1-2)Man(a 1-3)

I~

4

)

NeuSAc

Neu5Ac(a2-3)Ga1((31-3)GlcNAc(~l-2)Man(a 1-6)

\ Man(fi1-4-R I

(B)

NeuSAc(a2-3)Gal(fiI -3)GlcNAc(p1-2)Man(a 1-3)

I

032-6)

NeuSAc NeuSAc(a2-6)Gal(fi 14)GlcNAc(fi 1-2)Man(a 14)

\ NeuSAc(a2-6)Gal(p 14)GlcNAc@1-2)Man(a 1-3)

Man(fi14)-R I

(C)

[NeuSA~(a2-6)]~-, Gal@14)GlcNAc(fi1-2)Man(a 1 4 )

\ Man(fil4)-R

(D)

I

Neu5Ac(a2-6)Gal(fl1-4)GlcNAc(~l-2)Man(a 1-3) Fig. 8. Primary structures of the glycans from rat serotransferrin (A,B,C) [221] and from rat mammary gland GlcNAc(fi-N)Asn. transferrin (D) [67]. R, GlcNAc(~l-4)[Fu~(a1-6)]~-~

NeuSAc(a2-6)Gal(f314)GlcNAc(~I-2)Man(a 1-6)

\ NeuSAc(a2-6)Gal(fi 1-4)GlcNAc(p 1-2)Man(a 1-3)

Man(p14)-R I

NeuSAc(a2-6)Gal(f~I4)GlcNAc(fi1 -2)Man(a 1 4 )

\ Neu4,5Ac2(a2-6)Gal(fi1 4)GlcNAc(p 1-2)Man(a 1-3)

Man(b14)-R I

Neu4,5Ac2(a2-6)Ga1(@I-4)GlcNAc(fi 1-2)Man(a 1-6)

\ NeuSAc(a2d)Gal(fi14)GlcNAc(~1-2)Man(a1-3)

Man(p1 4 ) - R I

Neu4,5Ac2(a2-6)Gal(fi1 4)GlcNAc(fi1-2)Man(a 1 4 )

\ Neu4,5Ac2 (a2-6)Gal(D 14)GlcNAc@1-2)Man(a 1-3)

Man(fi1-4)-R I

Fig. 9. Primary structure of the four glycans identified in the three horse serotransferrin variants [113,114,119, 222,2231. R, GlcNAc(fi14)GlcNAc(flI-N)Asn.

225 [Gal(fiI 4)GlcNAc(fi 1-6)]o-l

\

[Neu5A~(a2-3)]~_~ Gal(@14)GlcNAc(fiI-2)Man(a 14)

\ (A)

GlcNAc(fi14)-Man(fil-4-R I

[Neu5Ac(a2-3)lo_, Gal(fiIL4)GlcNAc(@1-2)Man(a 1-3) [Gal(fiI 4)GlcNAc(B 14)Io-l NeuSAc(a2-3)Gal(fi 14)GlcNAc(fi1 -2)Man(a 1-6)

\ Man(@I 4 ) - R

NeuSAc(a2-3)Gal(fi14)GlcNAc(fiI-2)Man(a1-3)

(B)

I

Fig. 10. Primary structure of glycans from fish serotransferrins. A, Carp big-head (Aristichlhys nobilis) [224]; B, pike (Esox lucius) [225]. R, GlcNAc(fiI4)GlcNAc(fil-N)Asn.

NeuSAc(a2-6)Gal(fi I4)GlcNAc(@1-2)Man(al-6)

\ Man@14)GlcNAc(fi 1-rl)GlcNAc(@1-N)Asn NeuSAc(a2-6)Gal(fiI 4)GlcNAc(fiI -2)Man(a 1-3)

(A)

I

Gal(fi1-4)GlcNAc(fi1-2)Man(al-6)

\ Man(fi14)GlcNAc(fi 14)GlcNAc(fiI-N)Asn I I @l+) Neu5Ac(a24)Gal(fi14)GlcNAc(fil-2)Man(al-3) Fuc

6'

5'

(B)

4'

Neu5Ac(a2-6)Gal(fil-4)GlcNAc(fi1-2)Man(a1-6)

2 1 \3 Man@I 4)GlcNAc(fi14)GlcNAc(fil-N)Asn (C) I I W+) NeuSAc(a2-6)Gal(fi14)GlaNAc(fiI-2)Man(a1-3) Fuc 6 5 4 1' Fuc

I (at-3)

Gal@14)GlcNAc(@1-2)Man(a1-6)

\ Man@1 -4)GlcNAc(fi 14)GlcNAc(fiI-N)Asn I

NeuSAc(a2-6)Gal(fil4)GlcNAc(fiI-2)Man(al-3)

(D)

I (aid) Fuc

[Ne~5Ac(a2-3)]~_~ Gal(fiI4)GlcNAc(fi1-2)Man(a1-6)

\ Man(fi14)GlcNAc(fiI4)GlcNAc(fiI -N)Asn

Gal(fiI4)GlcNAc(fiI-2)Man(a1-3) [Neu5A~(a2-3)]~_~

I

I

(E)

(U1-h)

[Fuclo-1

Fig. 1 I . Primary structure of the glycans from human leukocyte lactotransferrin (A) [226] and human (B,C,D) [211,227-2301, Rhesus monkey (A) [231], sheep (A) [232], goat (B,C) [I 19,228,2321, and mouse (B,C) [ I 19, 218,2331 milk lactotransferrins. E, human recombinant lactotransferrin expressed in BHK cells [234].

226

Fuc

I (al-3)

Gal@ I--I)GlcNAc(fiI-3)Gal(fiI -4)GlcNAc(fiI -2)Man(a 1-6)

\ Man@ I--I)-R

Gal(fi14)GlcNAc(fl1-2)Man(a 1-3; Fuc

I

(Ul-3)

Gal(fiI4)GlcNAc(fiI-2)Man(a1 4 )

\ Man(fil4)-R NeuSAc(a2-6)Gal(fi 14)GlcNAc(fi 1-3)Gal(fi 14)GlcNAc(fiI-2)Man(a 1-3; Fig. 12. Primary structure of the poly-N-acetyllactosaminic glycans from human lactotransferrin [235]. R, GlcNAc(fl1-4)[Fuc(a 1 -6)]o-l GlcNAc(kN)Asn.

Gal(a 1-3)Gal(fi14)GlcNAc(fi 1 -2)Man(a 1-6)

\ Man@ I --I)GlcNAc(fi 14)GlcNAc(fi 1-N)Asn /

NeuSAc(a24)Gal(fi14)GlcNAc(fiI-Z)Man(a1-3) [NeuSAc(a2-6)]GalNAc(fi14)GlcNAc(fi 1-2)Man(a 1-6)

\ Man(fil--I)GlcNAc(fil4)GkNAc(fiI-N)Asn NeuSAc(a2-6)Gal(fi14)GlcNAc(fiI-2)Man(a 1-3)

I

GlcNAc((3-N)Asn. Fig. 13. Microheterogeneity of cow lactotransferrin [236]. R, GlcNAc(fil~)[Fuc(a14)],~, NeuSAc(a24)Gal( p 1-4) Gal(ald)Gal@ 1 4 ) Gal(P1-4)

Gd(p14)i

NeuSAc(a2-6)Gal(Pl-4) , 1 NeuSAc(a24)GalNAc(Pl-4) GalNAc( p 1 4 )

GlcNAc(pl-2)Man(al-6)

>

GlcNAc(pl-2)Man(al-3)

\

Man(pl-4tR

/

/

Fig. 14. Primary structure of diantennary glycans from cow lactotransferrin with an a-1,3-Gal residue in the terminal position [I 19,2361 and with a GalNAc residue replacing a Gal residue.

3.1.4. Concluding remarks

In conclusion, the characteristics of the carbohydrate moieties of transferrins are specific to each of them on the basis of the primary structure of glycans and of their number and position in the peptide chain as demonstrated in Figs. 2 to 4. In addition, each transferrin presents unexplained microheterogeneity.

221

Man(fi14)-R Man(al-2)Man(a 1-2)Man(a 1-3)

(A)

I

Man(a1-6)

\

Man(a1-6)

\ Man(bI4)-R Man(a 1-3)

(B)

I

Man(a 1-6)

\

Man(a1-6)

\

Man(a1-3)’

Man(fil4)-R

(C)

I

Man(a 1-3) Man(a1-2)Man(a 1-6)

\

Man(a 1-6) Man(alL2)Man(a 1-3)’

\ Man(fil4)-R

[Man(a1-2)J0_,Man(aIL3)Man(a 1-3)

(D)

I

Fig. 15. Primary structure of the glycans of the oligomannosidic type from cow [236,237] (A-D), goat (BD) [I 19,2321, sheep (B-D) [232] and Rhesus monkey (D) [231] milk lactotransferrins. R, GlcNAc(b14)GlcNAc(fi1-N)Asn.

3.2. Physiopathological modijications of transferrin glycan primary structure Human serotransferrin presents a microheterogeneity based on the co-existence of biand triantennary glycans of the N-acetyllactosaminic type leading to three glycovariants. The characterization and quantitation of these glycoforms is easily carried out by immuno-affinity electrophoresis as well as their isolation by affinity chromatography on concanavalin A-Sepharose columns. In this way, serotransferrin (TO isolated from healthy donors is resolved into three glycovariants: Tf-I (less than 1%), Tf-I1 (17 f2%) and Tf-I11 (82 3%), containing, two triantennary glycans, one tri- and one diantennary glycan and two diantennary glycans [ 1081, respectively. There are two “isomers” of the triantennary glycans (ratio 1 : 1) in which the third antenna is either E-l,4-linked to the a - 1,3-mannose residue or fi-1,6-linked to the a-1,6-mannose residue. Variations in the structure and location of serotransferrin glycans were detected in physiological and pathological cases [242].

*

3.2.1. Physiological modijications Serotransferrin is involved in the process of iron transfer to the foetus by a placental receptor-mediated mechanism. The three glycovariants of serotransferrin and their binding to human syncytiotrophoblast microvillar membranes have been studied by Ltger et al. [108]. The results obtained by these authors and others[243,244] showed that in the serum of pregnant women, especially in the last 3.2.1.1. Pregnancy.

228

three months of pregnancy, the serum concentration of serotransferrin reached 4.5-5 g per liter and the relative proportions of the glycovariants Tf-I and Tf-I1 increased from 1 to 6 f 1% and from 17 2 to 26 3~3%, respectively, while that of Tf-I11 decreased from 82 3 to 67 3~3%. In addition, an increase of sialylation has been observed [243]. The binding of the three serotransferrin glycovariants to the receptor of the syncytiotrophoblast plasma membranes has been studied and no difference in the binding affinity has been observed [108].

*

*

3.2.1.2. Embryogenesis. Changes in glycosylation of chicken serotransferrin synthesized during embryogenesis and by primary cultures of chicken embryo hepatocytes have been observed by Jacquinot et al. [241]. In the three transferrins analysed, the glycans were of the diantennary N-acetyllactosaminic type, having several prominent features. In particular, the embryo serotransferrin glycan (Fig. 16C) differed from that of chicken serotransferrin (Fig. 16D) by the presence of a bisecting N-acetylglucosamine residue, suggesting a developmental change in glycosylation. The glycan structure of the transferrin secreted by the embryo hepatocytes in primary culture was marked by the presence of fucose a-1,6-linked to the core N-acetylglucosamine, suggesting that expression of the fucosyltransferase activity is dependent on cell culture conditions. Moreover, comparative analysis of chicken serotransferrin (Fig. 16D) and ovotransferrin (Fig. 16B) glycans reinforces the idea that the glycosylation of two identical polypeptide chains is organ specific. [Gal(fi 14)10-, GlcNAc(fi1-2)Man(a 1-6)

\ GlcNAc(fi14)-Man(fi 14)-R I [Neu5Ac(a2-6)lo_, Gal(fi1-4)GlcNAc(f3 1-2)Man(a 1-3)

(A)

GIcNAc@-2)Man(a 1-6)

\ GlcNAc(fiI--l)-Man(fiI4)-R(B) GlcNAc(fi1-2)

\ Man(a1-3)

I

I

GlcNAc(fl 1 4 )

Gal(pl4)GlcNAc(flI-2)Man(a 1-6)

\ GlcNAc(fiIH)-Man(fil4)-R I Neu5Ac(a2-6)Gal(fi14)GlcNAc(fi1-2)Man(a 1-3)

(C)

[Neu5Ac(a2-6)],-, Gal@ 14)GlcNAc(fi I-2)Man(a 1 4 )

\ Man(fil-4)-R

(D)

Fig. 16. Primary structure of glycans from turkey ovotransferrin (A) [ I 19,2381, hen ovotransferrin (B) [239, 2401, chicken embryo serum (C), and chicken serotransferrin (D) [241]. R, GlcNAc(fiI-4)GlcNAc(fiI-N)Asn. Glycans of transferrin from embryo hepatocytes secreted into culture medium are a-I ,6-fucosylated.

229 GlcNAc(fi1 -2)Man(a 1-6)

\ GlcNAc@4)-Man(fiI 4)GlcNAc(fi14)GIcNAc(fil -N)Asn GlcNAc(fi1-2)Man(a 1-3)

I

I

(A)

(Ul-6)

Fuc

Gal(fl14)GlcNAc(fiI-2)Man(a 1-6)

\ Man(fil4)GlcNAc(fi 1 -4)GlcNAc(fil-N)Asn Gal@ 1 -4)GlcNAc(fi1-2)Man(a1-3)

(B)

I

Fig. 17. Major glycan structures of human cerebrospinal fluid transferrin [246,247]. Part of glycan B is sialylated.

3.2.1.3. Tissue-dependent glycosy lation. 3.2.1.3. I. Human cerebrospinal jluid transferrin. Transferrin is an important protein constituent of human cerebrospinal fluid, some 30% being present in an unsialylated form [245]. The glycan primary structure of the so-called fiz-transferrin or T-globulin, has been recently determined by Hoffmann et al. [246]. The major structure turned out to be an N-acetyllactosaminic type agalacto-diantennary oligosaccharide with bisecting N-acetylglucosamine and proximal fucose (Fig. 17A). Analysis of a second transferrin preparation containing both asialo- and sialotransferrin revealed another major glycan species derived from the sialylated transferrin variant which is galactosylated and lacks fucose and bisecting N-acetylglucosamine (Fig. 17B). The first result supports the hypothesis of a de nouo “brain-type’’ glycosylation of intrathecally synthesized cerebrospinal fluid proteins and the second one is in favor of an enzymatic desialylation of serotransferrin. 3.2.1.3.2. Human seminal transferrin. Human seminal transferrin (MM: 80 m a ) contains 6.1% sugars. The primary structure of the major N-linked glycan is identical to that of the serotransferrin diantennary glycan of Fig. 6A [247]. 3.2.1.3.3. Rat mammary-gland transferrin. Rat milk transferrin contains four glycovariants that differ only in their sialic acid content. The primary structure of the two major variants has been determined by Escriva et al. [67]. As shown in Fig. XD, the glycoforms contain either one or two N-acetylneuraminic acid residues a-2,6-linked to galactose in a conventional diantennary glycan of the N-acetyllactosaminic type. Most contain fucose a-1,6-linked to the proximal N-acetylglucosamine residue. 3.2.1.3.4. Recombinant transferrins. In view of the potential importance of human lactotransferrin for the production of artificial milk and for the pharmaceutical industry, numerous attemps have been made to produce recombinant human lactotransferrin using different cells: BHK cells [234,248], yeast, Aspergillus orizae [249], Saccharomyes cereuisiae [250] and tobacco cells [25 11; or transgenic animals [252]. But production of recombinant glycoproteins represents a formidable challenge. In fact, the yields are often low and, in addition, the glycan primary structures do not conform to the native ones (for reviews, see refs. [253,254]). Legrand et al. [234] have shown that expression in BHK cells of a full-length cDNA coding for human lactotransferrin led to diantennary glycans of the N-acetyllactosamine type, a-2,3-disialylated

230

(80%) and a-2,3-monosialylated (20%) forms. In addition, 70% of total glycans were a-1,6-fucosylated at the proximal GlcNAc residue, as in the native human milk lactotransferrin. The absence of N-acetylneuraminic acid a-2,6-linked to the terminal galactose residue is explained by the absence of a-2,6-sialyltransferase in BHK cells. 3.2.2. Serotransferrin and disease Serotransferrin glycans are highly sensitive to numerous pathological modifications of protein glycosylation. For this reason, serotransferrin is used as a tool to probe changes in N-glycosylation in pathological situations, particularly in liver diseases (for reviews, see refs. [242,255,256]). 3.2.2.1. Liver diseases. By applying the crossed affino-electrophoresis method, Spik et al. [255,256] have demonstrated that the human serotransferrin glycans are profoundly modified in liver diseases such as viral hepatitis and alcoholic cirrhosis with a marked increase of triantennary glycans (see also refs. [257-2591). 3.2.2.2. Cancer. The same kind of alterations have been observed by Yamashita et al. [260] in the serotransferrin of patients with hepatocellular carcinoma: dramatic increase of tri- and tetraantennary structures, a- 1,3-fucosylation of peripheral N-acetyllactosamine residues and, in small amounts, presence of bisected diantennary glycans. Identical results have been obtained from HepG2 cell line transferrin. In fact, profound modifications of glycan structure of serotransferrin synthesized by the human hepatocarcinoma cell line HepG2 have been shown by Campion et al. [261]. A comparative study of normal serotransferrin and of HepG2 transferrin shows the presence of tri-, tetra- and pentaantennary glycans of the N-acetyllactosaminic type, with fucose residues a-1,3-1inked to peripheral N-acetylglucosamine residues (Fig. 18). These results indicate that the increase Gal(@14)GlcNAc(@ 1 4 ) [Fuc(a 1-3)]

\

Man(a 1-6)

I

\

Gal(@14)GlcNAc(@1-2)/ [NeuSAc(a2-3)I2 Gal(fiI4)GlcNAc(@1-2) \

[NeuSAc(a24)], Gal@14)GlcNAc(@1 4 )

Man(al-3) I

Man(@14)-R I

Gal(@14)GlcNAc(@1 4 )

\

[Fuc(a

I

[NeuSAc(a2-3)I2

Gal(@I4)GlcNAc(@14)-Man(a 1-6) Gal(@14)GlcNAc(@1-2)l Gal(@1-4)GlcNAc(/3-2) \

Man(a1-3)

[NeuSAc(a24)I2 Gal(@14)GlcNAc(@1-4)

\

Man(@1 4 ) - R I

I

Fig. 18. Primary structure of tetra- and pentaantennary glycans from human serotransferrin secreted into culture medium of human hepatocarcinoma cell line Hep G2. The a-l,3-linked fucose residue is conjugated to the GlcNAc of one of the antennae [261]. R, Gl~NAc(~14)[Fuc(a1-6)]~~~GlcNAc(~-N)Asn.

23 1

in the number of antennae in transferrin glycans synthesized by the hepatocarcinoma cell line is much more pronounced than in liver diseases such as alcoholic cirrhosis and that, in addition, the malignant transformation of human liver induces fucosylation. These results might be due, at least in part, to the regulation of N-acetylglucosaminyltransferase V activity as has been observed in numerous cancer cells [262]. 3.2.2.3. CDG syndromes. The carbohydrate-deficient glycoprotein (CDG) syndromes are a family of genetic multisystemic diseases with severe nervous system involvement, growth retardation and hepatopathy during infancy which were first reported by Jaeken et al. [263] (for recent revicws, see refs. [264,265]). CDG-type I syndrome [264,266,267] is due to a deficiency in the oligosaccharidyltransferase which transfers “en bloc” onto the nascent protein the oligosaccharide linked to dolichol diphosphate. Later, van Schaftingen and Jaeken [ 1995, FEBS Lett. 377, 3 18-3201 demonstrated that the syndrome was due in fact to a phosphomannomutase deficiency, an enzyme which provides the mannose- 1-phosphate required for the initial steps of protein glycosylation. This leads to four transferrin isoforms: non-glycosylated, glycosylated in Asn-4 13 or in Asn-6 1 1 and in both Asn-4 13 and 6 1 1. CDG-type I1 syndrome [265,268] is a separate variant since it is characterized by a severe decrease in the activity of N-acetylglucosaminyltransferase I1 (UDPGlcNAc: a6-D-mannoside fi- 1,2-N-acetylglucosaminyltransferase).As a consequence, the serotransferrin isoforms contain two truncated monoantennary glycans of which the primary structures are described in Fig. 19. Man(a 1-6)

\ NeuSAc(a2-6)Gal(~14)GlcNAc(P1-2)Man(a1-3)

Man(pl-4)-R i

Fig. 19. Primary structure of the glycan from human serotransferrin isolated from a patient with carbohydratedeficient syndrome (CDG) type I1 [265,268]. R, GlcNAc(PI-4)GlcNAc((ll-N)Asn.

3.2.2.4. Serotransferrin in HEMPAS. Congenital dyserythropoietic anaemia type I1 or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a genetic disease caused by membrane disorganisation of erythroid cells (for a review, see refs. [269,270]). A defect in N-acetylglucosaminyltransferase 11 or in a-mannosidase I1 has been suspected. As a result, the serum glycoproteins, serotransferrin in particular, are taken up by the hepatocytes and the Kuppfer cells. Analysis carried out by Fukuda et al. [269,270] showed the presence of oligomannosidic (M6 to M9 structures) and hybrid type glycans in the HEMPAS transferrin.

4. Three-dimensional structure of transferrins As mentioned above (see section 2.2. l), the three-dimensional structure of the peptide chains of rabbit serotransferrin, of human and bovine lactotransferrins and of ovotrans-

232

ferrin have been defined by X-ray diffraction. However, the X-ray diffraction patterns provided little information on the 3D-structure of the glycans and on their relationships and interactions with the peptide chain. For example, in the case of human lactotransferrin only the disaccharide Fuc(a I-6)GlcNAc linked to asparagine is visible [97]. 4.1. Three-dimensional structure of glycans

The only view we had in the 1970s of the 3D-structure of glycans was mainly speculative. In fact, it resulted from molecular building of the diantennary glycan from human serotransferrin and lactotransferrin whose glycan primary structure had just been determined [211,212]. This approach led to the description of the Y, T, bird and broken-wing conformation (for reviews, see refs. [210,227,253,27 1-2741). This situation was due to the difficulties encountered in the crystallization of glycans and glycoproteins. However, little by little, the application of sophisticated techniques like NMR, EPR and neutron scattering led to results which favoured the emerging concept of conformation interconversions due to the mobility of antennae [227,271-2741. This concept was recently verified on the basis of molecular modelling [275,276], molecular dynamics simulations [277] and X-ray diffraction [278-2831 data concerning human sero- and lactotransferrin. Results obtained from X-ray diffraction are unambiguously demonstrative. In fact, Cambillau et al. (for a review, see ref. [278]) have devised an elegant method of co-crystallisation of the iso-lectins I and I1 of Lathyrus ochrus with glycans and glycoproteins. A free glycan [279] and a so-called N2-peptide [280,283] from human lactotransferrin were co-crystallized with the lectin and X-ray diffraction data from single crystals were collected at 2.3 A resolution. The results are illustrated in Fig. 20 which shows that the 3D-structure of the free glycan (bird-conformation) and of the N2-glycopeptide (broken wing-conformation) are different. Thus, the concept

Fig. 20. Spatial conformation (a) of a free asialoglycan and (b) of the N2-asialoglycan from human lactotransfcrrin, determined by X-ray diffraction [283]. Numbers correspond to the numbering used in structure C of Fig. 1 1.

233

of flexibility of antennae proposed about 20 years ago[227,272] (for reviews, see refs. [2 10,272-2741) is now firmly established by experimental data. .4.2. Three-dimensional structure of transferrins as glycoproteins As mentioned above, X-ray diffraction of transferrin furnishes little information on the 3D-structure of the glycans and the images we have today remain largely speculative since they result from molecular modelling studies. We have represented in Fig. 21 the 3D-structure, determined by molecular modelling on the basis of X-ray diffraction data of rabbit serotransferrin [276] and of human lactotransferrin [89,92]. In rabbit serotransferrin, the single glycan linked to the peptide chain is immobilized into only

Fig. 21, Molecular modelling (A,B) of rabbit serotransferrin glycan and (C) of human lactotransferrin [192, 210,2751: (A) 3D structure of rabbit serotransferrin; (B) interaction of rabbit serotransferrin glycan in a broken-wing conformation with a peptide segment (amino acids 254 to 271) in an a-helix conformation, 7,7', N-acetylneuraminic acid residues (see Fig. 6A). (C) 3D structure of human lactotransferrin. Arrows indicate the position of glycans.

234

one conformation, the broken wing-conformation. In addition, the glycan is conjugated to the Asn-485 residue of the C-lobe while its two antennae interact with a peptide portion (amino acid residues 254 to 271 in an a-helix conformation) of the N-lobe (Figs. 21A and B). This result strongly supports the hypothesis that, in the case of rabbit serotransferrin, the glycan might reinforce the association of the two lobes and contribute to maintain the protein moiety in a biologically active 3D-structure. On the contrary, in human lactotransferrin, the glycans do not seem to interact with the peptide chain (Fig. 21C). They are free in space and could thus play a role as recognition signals. In addition, lactotransferrin glycans are in opposite positions in the N-and C-lobes while serotransferrin glycans (not shown) are both located in the C-domain of the protein and are very near in space.

Acknowledgements The authors are very grateful and want to thank Laurence Onraet and Khadija Khalfa for their skilful assistance in typing this manuscript.

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24 1 [249] Ward, P.P., Lo, J.Y., Duke, M., May, G.S., Headon, D.R. and Conneely, O.M. (1992) Biotechnology 10, 784-789. [250] Liang, Q. and Richardson, T. (1993) J. Agr. Food Chem. 41, 180&-1806. [251] Mitra, A. and Zhang, Z. (1994) Plant Physiol. 106, 977-981. [252] Platenburg, G.J., Kootwijk, E.P.A., Kooiman, RM., Woloshuk, S.L., Nuijens, J.H., Krimpenfort, P.J.A., Pieper, F.R., De Boer, H.A. and Strijker, R. (1994) Transgenic Res. 3, 99-108. [253] Montreuil, J. (1995) In: J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.) Glycoproteins, New Comprehensive Biochemistry, Vol. 29a. Elsevier, Amsterdam, pp. 1-1 2. [254] Montreuil, J. (1993) In: C. Rivat and J.F. Stoltz (Eds.). Colloque INSERM no. 227. John Libley Eurotext, Montrouge, France, pp. 283-292. [255] Spik, G., Debruyne, V. and Montreuil, J. (1983), In: H. Popper, W. Reutter, E. Kottgen and F. Gudat (Eds.), Structural Carbohydrates in Liver. MTP Press, Boston, pp. 477483. [256] Debruyne, V., Montreuil, J. and Spik, G. (1984) Prot. Biol. Fluids 31, 6 3 4 8 . [257] Stibler, H. (1991) Clin. Chem. 37, 2029-2037. [258] Stibler, H. (1993) Acta Neurol. Scand. 88, 279-283. [259] Xin, Y., Lasker, J.M. and Lieber, C.S. (1993) Hepatology 22, 1462-1468. [260] Yamashita, K., Koide, K., Endo, T., Iwaki, Y. and Kobata, A. (1989) J. Biol. Chem. 264, 2415-2423. [261] Campion, B., Ltger, D., Wieruszeski, J.M., Montreuil, J. and Spik, G. (1989) Eur. J. Biochem. 184, 405413. [262] Hahn, T.J. and Goochee, C.F. (1992) J. Biol. Chem. 267, 23982-23987. [263] Jaeken, J., Van Eijk, H.G., Van der Heul, C., Corbeal, L., Eeckels, R. and Eggermont, E. (1984) Clin. Chim. Acta 144, 245-247. [264] Yamashita, K. and Ohno, K. (1996) In: J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins and Disease, New Comprehensive Biochemistry, Vol. 30. Elsevier, Amsterdam, ch. 1 6a. [265] Jaeken, J., Spik, G. and Schachter, H. (1996) In: J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins and Disease, New Comprehensive Biochemistry, Vol. 30. Elsevier, Amsterdam, ch. 16b. [266] Ohno, K., Yuasa, I., Akaboshi, S., Itoh, M., Yoshida, K., Ehara, H., Ochiai, Y. and Tokeshita, K. (1992) Brain Dev. 14, 30-35. [267] Yamashita, K., Ideo, H., Okhura, T., Fukushima, K., Yuasa, I., Ohno, K. and Takeshita, K. (1993) J. Biol. Chem. 268, 5783-5789. [268] Jaeken, J., Schachter, H., Carchon, H., De Cock, P., Coddeville, B. and Spik, G. (1994) Arch. Dis. Child. 71, 123-127. [269] Fukuda, M.N. (1996) In: J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins and Disease, New Comprehensive Biochemistry, Vol. 30. Elsevier, Amsterdam, ch. 8. [270] Fukuda, M.N., Gaetani, G.F., Izzo, P., Scartezzini, P. and Dell, A. (1992) Br. J. Haematol. 82, 745-752. [271] Montreuil, J., Foumet, B., Spik, G. and Strecker, G.(1978) C.R. Acad. Sci. Paris 287, 837-840. [272] Montreuil, J. (1980) Adv. Carbohydr. Chem. Biochem. 37, 157-223. [273] Montreuil, J. (1984) Biol. Cell 51, 115-132. [274] Montreuil, J. (1984) Pure Appl. Chem. 56, 859-877. 12751 Mazurier, J., Dauchez, M., Vergoten, G., Montreuil, J. and Spik, G. (1991) C.R. Acad. Sci. Paris 313, 7-14. [276] Mazurier, J., Dauchez, M., Vergoten, G., Montreuil, J. and Spik, G. (1991) Glycoconjugate J. 8, 390399. [277] Dauchez, M., Mazurier, J., Montreuil, J., Spik, G. and Vergoten, G. (1992) Biochimie 74, 63-74. [278] Cambillau, C. (1995) In: J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins, New Comprehensive Biochemistry, Vol. 29a. Elsevier, Amsterdam, 29a, pp. 29-65. [279] RougC, P.,Bourne, Y. and Cambillau, P. (1992) J. Biol. Chem. 267, 197-203. [280] Bourne, Y., Nesa, M.P., Rouge, P., Mazurier, J., Legrand, D., Spik, G., Montreuil, J. and Cambillau, C. (1992) J. Mol. Biol. 227, 938-941. [281] Bourne, Y., Mazurier, J., Legrand, D., Rougt, P., Montreuil, J., Spik, G. and Cambillau, C. (1994) Curr. Biol. 2, 209-219.

242 12821 Bourne, Y., Mazurier, J., Legrand, D., Rouge, P., Montreuil, J., Spik, G. and Cambillau, C. (1994) Structure 2, 209-219. [283] Bourne, Y., Van Tilbeurgh, H. and Cambillau, C. (1993) Curr. Opin. Struct. Biol. 3, 681-686. [284] Bailey, S., Evans, R.E., Garratt, R.C., Gorinsky, B., Hasnain, S., Horsburgh, C., Thuti, H., Lindley, P.F., Mydin, A,, Sarra, R. and Watson, J.L. (1988) Biochemistry 27, 5804-5812.

J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins II

0 1997 Elsevier Science B.Y All rights reserved CHAPTER 11

Chemistry, biochemistry and biology of sialic acids Roland Schauer' and Johannis P. Kamerling2 'Biochemisches Institut, Christian-AIbrechts-LIniuersilatzu Kiel,Germany, 'Bijuoet Center, Department of Bio-Organic Chemistry, Utrecht Vniversiy, The Netherlands

List of Abbreviations Abbreviations used for sialic acids (Sia) are included in Tables 1 and 13. Alt altrose

GlcA

D-glucuronic acid

GlcNAc

N-acetyl-o-glucosamine

GlcN

o-glucosamine

Ara

L-arabinose

GD3

disialoganglioside

Asn

L-asparagine

GM 1

monosialoganglioside

ASP CDP

L-aspartate cytidine diphosphate

Cer CI

ceramide chemical ionization

CMP

cytidine monophosphate

CoA

coenzyme A

CTP

cytidine triphosphate

Gul

gulose

HeP HPLC

heptose

HPTLC

high-performance thin-layer chromatography

high-performance liquid chromatography

IgG

immunoglobulin G

C7-NeuSAc NeuSAc missing the C8-C9 part C8-NeuSAc NeuSAc missing the C9 part

IgM Leu

immunoglobulin M

Da DFP

Dalton diisopropylfluorophosphate

Man

o-mannose

ManNAc

N-acetyl-D-mannosamine

DIG DMB

digoxigenin 1,2-diamino-4,5methylenedioxybenzene

MS

mass spectrometry

MU

4-methylumbelliferyl

NMK

nucleoside monophosphate kinase

DNA

desoxyribonucleic acid

NMR

nuclear magnetic resonance

EI

electron impact

NOESY

EPR

electron paramagnetic resonance

nuclear Overhauser enhancement spectroscopy

FAB Fuc

fast atom bombardment L-fucose

P, Pi

phosphate pulsed amperometric detection pyruvate kinase

L-leucine

FucN

L-fucosamine

PAD PEP

Gal

D-galactose

PK

GalNAc-ol

N-acetylgalactosaminitol

PPase

inorganic phosphatase

GalANGro

N-galacturonyl-2-aminoglycerol

PPi

pyrophosphate

GalNAc

N-acetyl-D-galactosamine

PYR

pyruvate

GDP Glc GLC

guanosine diphosphate D-glucose gas-liquid chromatography

ROESY

rotating-frame nuclear Overhauser enhancement spectroscopy

QuiNAc

N-acetyl-o-quinovose

243

phosp hoenolpyruvate

244 sulfate SDS/PAGE sodium dodecyl sulfate/polyacrylamide gel electrophoresis Ser L-serine TADH Thermoanaerobium brockii alcohol dehydrogenase trifluoroacetic acid TFA thin-layer chromatography TLC S

Thr TOCSY

L-threonine total correlation spectroscopy

UDP

uv

uridine diphosphate ultraviolet

xYl

D-XylOSe

2D

two-dimensional

I . Introduction Since the discovery of N-acetylneuraminic acid, the most universal sialic acid, at the end of the 1930s [1,2] as well as the structural and stereochemical elucidation of its free and bound forms at the end of the 1960s (reviewed in ref. [3]), there has been a continual increase in the number of sialic acid types (1994: more than 40) recognized to occur in a variety of living organisms. It is now generally accepted that naturally occurring sialic acids are monosaccharides which influence many important biological and pathological phenomena. In previous articles [3-51, the former literature has been extensively reviewed with respect to a number of chemical, biological, metabolic, functional as well as historical aspects. Since 1982 [4,5], the proliferation of the literature on the chemistry, biochemistry and (molecular) biology of sialic acids has accelerated dramatically, and several short reviews dealing with specific aspects of sialic acids have appeared [6-121. The aim of this chapter is to collate a mixture of, what is in our opinion, relevant information published before 1982 and new data appeared since then. Because of the profusion of biochemical and biological data published in the last few years, it was necessary to select those reports which, we believe, reflect potential trends in the future development of sialobiologyr

2. General characteristics of sialic acid In Table 1 a survey of the 43 naturally occurring members of the sialic acid family [ 135 13, together with their abbreviations and typical biological sources, is presented. The general name “sialic acid” is derived from the Greek “sialos”, meaning saliva. Taking into account the Rules for Carbohydrate Nomenclature, as recommended by the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (1999, the mother-molecule neuraminic acid, which does not occur in free form in nature, due to its immediate cyclization to form an internal Schiff base, is systematically named 5-amino-3,5-dideoxy-~-gbcero-~galacto-non-2-ulosonic acid (Fig. 1), and abbreviated as Neu, whereby the D-notation is implied in the trivial name. Chemically, this nine-carbon-containing monosaccharide is a 2-keto-carboxylic acid, a deoxysugar, and an aminosugar. The amino group is generally N-acetylated (5-acetam~do-3,5-d~deoxy-~-g~cero-~-galacto-non-2-ulopyranoson~c acid;

Table 1 Survey of established structures of naturally occurring members of the sialic acid familya Name

Abbreviation

Typical biological sources

Neuraminic acid

Neu

does not occur in free form; only found in gangliosides

N-Acetylneuraminic acid

Neu5Ac

5-N-Acetyl-4-0-acetyl-neuraminicacid

Neu4,5Ac2

5-N-Acetyl-7-0-acetyl-neuraminicacid

Neu5,7Ac2

5-N-Acetyl-8-0-acetyl-neuraminicacid

Neu5,8Ac2

5-N-Acetyl-9-0-acetyl-neuraminicacid

Neu5,9Ac2

oligosaccharides, polysaccharides and glycoconjugates from man, higher animals, and microorganisms; free form in body fluids ungulate, monotreme and guinea pig oligosaccharides and glycoconjugates animal glycoconjugates; bacterial polysaccharides bovine glycoproteins; bacterial polysaccharides human and animal glycoconjugates; bacterial (1ipo)polysaccharides

5-N-Acetyl-4,9-di-O-acetyl-neuraminic acid 5-N-Acety1-7,9-di-O-acetyl-neuraminic acid

Neu4,5,9Ac3

equine glycoproteins

~151

Neu5,7,9Ac3

bovine glycoproteins and bacterial lipopolysaccharides

[ 15J6,I 8,22,23,

5-N-Acetyl-8,9-di-O-acetyl-neuraminic acid 5-N-AcetyI-7,8,9-tri-O-acetyl-neuraminic acid 5-N-Acetyl-9-O-~-lactyl-neuraminic acid

Neu5,8,9Ac3

bovine glycoproteins

Neu5,7,8,9Ac4

bovine glycoproteins

NeuSAc9Lt

5-N-Acetyl-4-0-acetyl-9-0-lactyl-neuraminic acid

Neu4,5Ac29Lt

human and animal glycoproteins; free form in body fluids equine glycoproteins

Reference(s)

[13,15-181

[I 5,16,18-21]

[15,16,18,22-241 [ 15,22-241

[15,16,18,22-261

26,271 [ 15,16,18,22,23] [15,221 [15,28,29] [15,301 continued on next page

N

N m P

Table 1, continued Name

Abbreviation

Typical biological sources

5-N-Acetyl-8-0-methyl-neuraminic acid 5-N-Acetyl-9-0-acetyl-8-0-methyl-neuraminic acid 5-N-Acetyl-8-0-sulpho-neuraminic acid 5-N-Acetyl-9-0-phosphoro-neuraminic acid

NeuSAc8Me

starfish glycoconjugates starfish glycoconjugates

5-N-Acetyl-2-deoxy-2,3-didehydro-neuraminic acid 5-N-Acetyl-9-0-acetyl-2-deoxy-2,3-didehydro-neuraminic acid 5-N-Acetyl-2-deoxy-2,3-didehydro-9-0-lac~l-neuraminic acid 5-N-Acetyl-2,7-anhydro-neuraminic acid

Neu2en5Ac Neu2en5,9Ac2 Neu2enSAc9Lt Neu2,7anSAc

N-Glycolylneuraminic acid

NeuSGc

glycoconjugates from most higher animals

4-0-Acetyl-5-N-glycolyl-neuraminicacid 7-0-Acetyl-5-N-glycolyl-neuraminic acid

Neu4AcSGc Neu7AcSGc

8-0-Acetyl-5-N-glycolyl-neuraminicacid 9-0-Acetyl-5-N-glycolyl-neuraminicacid

Neu8AcSGc Neu9AcSGc

ungulate glycoconjugates glycoconjugates from most higher animals bovine glycoproteins

7,9-Di-O-acetyl-5-N-glycolyl-neuraminic acid 8,9-Di-0-acetyl-5-N-glycolyl-neuraminicacid 7,8,9-Tri-0-acetyl-5-N-glycolyl-neuraminic acid 5-N-glycolyl-9-0-lactyl-neuraminic acid

Neu7,9Ac25Gc Neu8,9Ac25Gc

5-N-glycolyl-8-0-methyl-neuraminic acid 9-0-Acetyl-5-N-glycolyl-8-0-methyl-neuraminic acid 7,9-Di-0-acetyl-5-N-glycolyl-8-O-methyl-neuraminic acid

NeuSGc8Me

Neu5,9Ac28Me NeuSAc8S NeuSAc9P

Neu7,8,9Ac35Gc NeuSGc9Lt

Reference(s)

sea urchin glycolipids biosynthetic intermediate to NeuSAc body fluids and tissues urine and tissues urine and tissues urine, wet cerumen, leech

glycoconjugates from most higher animals bovine glycoproteins bovine glycoproteins bovine glycoproteins porcine glycoproteins

Neu9AcSGc8Me

starfish glycoconjugates starfish glycoconjugates

Neu7,9Ac25Gc8Me

starfish glycoconjugates

[ 15-17,34]

[351 [351 [36-391

[I 3,15,16,18,25, 401 [ 15,16,18] [15,221 [361 [ 15,16,18,22]

Table 1, continued Name

Abbreviation

Typical biological sources

5-N-Glycolyl-8-0-sulpho-neuraminic acid N-(0-Acetyl)glycolylneuraminic acid N-(0-Methy1)glycolylneuraminic acid 2-Deoxy-2,3-didehydro-5-N-glycolyl-neuraminic acid 9-0-Acetyl-2-deoxy-2,3-didehydro-5-~-glycolyl-neuraminic acid 2-Deoxy-2,3-didehydro-S-N-glycolyl-9-0-lactyl-neuraminic acid 2-Deoxy-2,3-didehydro-5-N-glycolyl-8-0-methyl-neuraminic acid 2,7-Anhydro-5-N-glycolyl-neuraminic acid 2,7-Anhydro-S-N-glycolyl-8-~-methyl-neuraminic acid

NeuSGc8S

sea urchin glycolipids

NeuSGcAc

rat thrombocytes

NeuSGcMe

Neu2enSGc9Lt

starfish glycolipids body fluids and tissues urine urine and tissues

Neu2enSGc8Me Neu2,7anSGc

starfish rat urine

Neu2,7anSGc8Me

starfish

2-Keto-3-deoxynononic acid

Kdn

fish, amphibian and mammalian glycoconjugates; bacterial polysaccharides

[46-501

9-0-Acetyl-2-keto-3-deoxynononic acid

Kdn9Ac

fish glycoconjugates

[511

Neu2enSGc Neu2en9AcSGc

Reference(s)

a If possible, typical biological sources have been indicated. In the case of a sialic acid structure being proven by mass spectrometry andor NMR spectroscopy, reference numbers refer in general to such studies or to review articles including this sialic acid.

N

m P

E

COOH

C

I

O

H

I

c=o I p z

H-C-OH-

I

H,N-C-H

I

HO-C-H I

c - OH'

t

H

H-C-OH A ~ H N - &I H

I

I

H-

-

AcH( D-galacto

rH-C > -OH H

0-L-H reference --c H- I - OH

atom

H

I

H-C-OH

I

H- C - OH

I

D-glycero

CH,OH

I

OH

CH,OH

'w 1

OH

HO

H

AcHN

5

OH

qOOH

I

HOH,C

CH,OH

OH

3

?H

1

H

o

H

H

~

~

C

O

O

H

HO OH

Fig. 1. Chemical structures for simple sialic acids in different views. (a) 5-arnino-3,5-dideoxy-o-glycero-o-galacfo-non2-ulosonic acid (Neu, open chain, Fischer projection formula); (b) 5-acetamido-3,5-dideoxy-~-g~ceroa-o-g~~acfonon-2-ulopyranosonic acid (a-NeuSAc, Fischer projection formula, note that C7 is the anomeric reference atom); (c) a-NeuSAc (Haworth projection formula); (d) a-NeuSAc ('C, chair conformation); (e) 3-deoxy-o-glycero-~~-galacto-non-2-ulopyranosonic acid (P-Kdn, 'C, chair conformation). Note that the o-notation is part of the trivial name.

249

0

II

H0 H,

c

>kOH

O-P-O-

HO

AcHN

OH Fig. 2. Chemical structure of cytidine 5’-(5-acetam~do-3,5-d~deoxy-D-g~cero-~-D-g~~~cfo-non-2-ulopyranosylonate monophosphate (CMP-P-Neu5Ac).

N-acetylneuraminic acid; Neu5Ac) (Fig. 1) or N-glycolylated (5-hydroxyacetamido3,5-d~deoxy-~-glycero-~-galacto-non-2-ulopyranoson~c acid; N-glycolylneuraminic acid; Neu5Gc). As is evident from Table 1, the hydroxyl groups may be free, esterified (acetylated, lactylated, sulfated, phosphorylated) or etherified (methylated). In the case where a sialic acid bears a hydroxy group instead of an amino group at C5, so-called deaminated neuraminic acid, it is systematically named 3-deoxy-D-glycero-D-galacto-non2-ulopyranosonic acid (Fig. l), and is abbreviated as Kdn (2-keto-3-deoxynononic acid; also in this case the D-notation is part of the trivial name). Sialic acids are relatively strong acids, e.g. Neu5Ac has a pK, value found in the range of 2.2-3.0 in various studies with an average of 2.6. This strong acidity is responsible for processes such as autohydrolysis of sialic-acid-containing carbohydrate chains. Conformationally, sialic acids adopt the 2Cs chair conformation, having the glycerol side chain in an equatorial orientation [52]. Due to hydrogen bonding of HO7 and H08, leading to a trans-orientation of these groups, the glycerol side chain is not as flexible as one might expect. This has consequences for the course of the mild periodate oxidative degradation of this moiety in the case of substitution at C9[53]. Free sialic acids have mainly the b-anomeric ring structure (>93%), whereas glycoconjugate-bound sialic acids occur specifically in the a-anomeric form [54]. In nucleotide-bound sialic acids, i.e. CMP-Neu5Ac (Fig. 2), a P-configuration for the glycosidic bond is present [55]. Crystalline Neu5Ac occurs specifically in the 0-anomeric form [56]. With respect to 0-acetylation patterns in sialic acids, it is worthwhile to mention that spontaneous migration of 0-acetyl groups can occur between C7, C8 and C9. At pH values at which no significant de-0-acetylation is observed (e.g. physiological pH values), Neu5,7Ac2 can readily transform into Neu5,9Ac2, whereas Neu5,7,9Ac3 yields an equilibrium of Neu5,7,9Ac3 and Neu5,8,9Ac3 in a molar ratio of approximately 1:l; Neu4,5Ac2 does not give rise to 0-acetyl migrations[23]. Also starting from a-Neu5,8,9Ac3 4-aminophenylthio-glycosidea 1:1 equilibrium between the 8,9- and 7,9-di-O-acetylated derivatives is established [57]. 9-0-Acetylated N-acylneuraminic acids have been found in both glycoconjugates and oligolpolysaccharides of different

250 AcHN

OH

I

1

AcHN

y3rcoo

1

OH

HO

H

-

CH,OH

COOH

N ----+-OH

CH OH H

o H

e

AcHN o

O

W

/ o

o

0

H

HO

Fig. 3. Chemical structures of (a) 5-N-acetyl-2-deoxy-2,3-didehydro-neuraminic acid (Ne3en5Ac); (b) 5-Nacetyl-2,7-anhydro-neuraminic acid (Ne3,7an5Ac); and (c) the tautomers of S-N-acetyl-4,8-anhydroneuraminic acid (Neu4,SanSAc).

biological origin (Table 1). The same is true for 7-0-acetylated N-acylneuraminic acids. It should be emphasized that Neu5,9Ac2 and other side-chain-0-acylated sialic acids occur in man[%] (see also sections 8.4.2 and 8.4.3), e.g. in human colon. Ungulates form the major source for 4-0-acetylated N-acylneuraminic acids [ 131, whereas minor sources are monotremes [19], guinea pigs [21] and humans [59]. In an evaluation of the naturally occurring 0-acylation patterns, it is evident that 0-acyl groups are most frequently found at C9 of both NeuSAc and NeuSGc. 0-Methylated and 0-sulfated N-acylneuraminic acids have only been found in lower animals, e.g. in echinoderms [13,31,42]. 5-N-Acyl-2-deoxy-2,3-didehydro-neuraminic acids like Neu2enSAc (S-acetamido-2,6anhydro-3,5-d~deoxy-~-g~cero-~-galacto-non-2-enon~c acid, Fig. 3) occur in free form in nature. Moreover, they can be generated from corresponding CMP-N-acylneuraminic acids in a non-enzymatic elimination reaction, occurring under physiological and, much faster, under alkaline conditions [34,35]. Small amounts of Neu2enSAc are formed by a water elimination side reaction from NeuSAc during influenza-B-virus-sialidase-catalyzed desialylations of sialoglycoconjugates [60]. S-N-Acety1-2,7-anhydro-neuraminic acid (Neu2,7anSAc, Fig. 3), which occurs in free form in nature [36,37], can be generated from NeuSAc(a2-3)Gal(fi 1- containing glycoconjugates using a sialidase isolated from the leech Macrobdella decara [38,6 13.

25 1

It is clear that such a sialidase has a highly unusual specificity (see sections 5.1, 8.4.6 and 9.2). The methyl ester of Neu2,7anSAc is formed as a very minor by-product during methanolysis [62]. 5-N-Acetyl-4,8-anhydro-neuraminicacid (Neu4,8anSAc, Fig. 3), having CH2-COCOOH as a side chain of a pyranose ring with the 7C4 conformation, does not occur as such in nature. Initially, it has been isolated from an acid hydrolysate of edible bird’s nest substance [63]. Furthermore, Neu4,8an5Ac was detected in a sialic acid mixture obtained after acid hydrolysis of equine serum, whereas incubation of Neu4,5Ac2 under alkaline conditions showed, in addition to de-0-acetylation, a partial conversion into Neu4,8an5Ac [41]. Heating of solid sodium Neu5Ac (3 h, 140°C) or a solution of Neu5Ac at pH > 11 or at pH 2.0 (30 min, 80°C) also yielded Neu4,8anSAc (sodium salt) as a major degradation product, probably in two tautomeric forms [64]. Hyperexcretion of free Neu5Ac in urine, defined as sialuria, has been observed in several mentally retarded patients [ 6 5 4 8 ] (see section 10.5).

3. Occurrence of sialic acids in biornolecules Members of the sialic acid family occur mainly in bound form in higher animals, from the echinoderms onwards in evolution, but also in some viruses, various bacteria, protozoa, and pathogenic fungi [8,13,69]. Generally, they are constituents of glycoproteins [20,21,25,40,46-48,5 1,70-I071 (Table 2), glycolipids [13,3 1,44,108-1241 (Table 3), and oligosaccharides [ 16,19,25,76,125-1551 (Table 4), usually occurring as terminal monosaccharide units, and of homo- and heteropolysaccharides [24,26,27,49,156I851 (Table 5). However, glycosylphosphatidylinositol membrane anchors have also been shown to contain sialic acid [ 1861. Neu5Ac- and Neu5Gc-containing glycans frequently occur together, whereby ratio differences reflect species specificity, tissue specificity or physiological fluid specificity. Also Kdn has been found together with NeuSAc/SGc in specific glycoconjugates (Table 2). It should be noted that the presence of Neu5Gc in normal human tissue and soluble glycoproteins has not been established conclusively [ 13,187,1881. As is evident from Table 2, NeuSAc/SGc- and Kdn-containing elements in Nand O-glycoprotein glycans do occur in many different microenvironments, however, with a restricted glycosidic linkage specificity. In general, N-acylneuraminic acids are a-2,3- or a-2,6-linked with D-galactose (Gal), a-2,3- or a-2,6-linked with N-acetyl-Dgalactosamine (GalNAc), a-2,6-linked with N-acetyl-D-glucosamine (GlcNAc), or a-2,8linked with other N-acylneuraminic acids. These types of glycosidic linkages have been firmly established by different analytical methods, including NMR spectroscopy [76,77]. The sialic-acid-containing N-linked carbohydrate chains form part of the complex (Nacetyllactosamine and N,N’-diacetyllactosediamine subtypes; mono-, di-, tri-, tri’-, and tetraantennae) or the hybrid type of structures [ 1891. For the mucin-type 0-glycans, the N-acylneuraminic-acid-containingsequences are extensions of most of the wellestablished core types [ 1901. In addition, terminal NeuSAc(a24)Gal[75], terminal NeuSAc(a24)GlcNAc [96], and internal Fuc( 14)NeuSGc [97] sequences have been reported, and only one example of a terminal NeuSAc(a2-9)NeuSAc(a2- dimer has

252 Table 2 Sialic-acid-containing elements in N- and 0-glycoproteins a Partial structure

N

0 Ref(s).

Neu5Ac(a2-3)Gal(fil-3)GlcNAc(filNeu5Ac(a2-3)Gal(fi1-3)~eu5Ac(a2-6)]GlcNAc(fil-

+

[70,71]

+

NeuSAc(a2-3)Gal(fi 1-3)[Fuc(a 14)]GlcNAc(fiI-

[70,72,73] +

[741 [751 [70,73]

Neu5Ac(a24)Gal(fil-3)[Neu5Ac(a2-6)]GlcNAc(filGal(fi1-3)[Neu5Ac(a24)]GlcNAc(fi 1Neu5Ac(a2-3)Gal(fil4)GlcNAc(fi 1NeuSAc(a2-3)Gal(fi 14)[Fuc(a 1-3)]GlcNAc(fi 1-

+

+ +

[77,78]

NeuSAc(a2-3)[Gal(fiI 4)]Gal(fi 14)GlcNAc(filNeuSAc(a2-3)[GalNAc(fiI 4)]Gal(fi l4)GlcNAc(fi1-

+ + +

[79,80] [77,81]

Neu5Ac(a2-3)[6S]GaI(fil 4)GlcNAc(fil-

+

[821

Neu4,5Ac2(a2-3)Gal(fi1 4)GlcNAc(filNeu4,5Ac2(a2-3)[Ga1((3 14)]Gal(fil4)GlcNAc(fil-

+

Neu5,9Ac2(a2-3)Gal(fi14)GlcNAc(filNeu5Gc(aZ-3)Gal(fil4)GlcNAc(filNeuSAc(a24)Gal(fiI4)GlcNAc(fiI Neu5Ac(a2-6)Gal(fi14)GlcNAc(fiI-

+ +

~ 3 1 [791 [25,84]

Neu5Ac(a24)Gal(fiI -4)[6S]GlcNAc(filNeu5Ac(a24)[GalNAc(a 1-3)]Gal(fil4)GlcNAc(fil-

+

Neu4,5Ac2(a2-6)Gal(fi 14)GlcNAc(fiI-

+

Neu5Gc(a2-6)Gal(~14)GlcNAc(fil-

+

Neu5Ac(a2-3){ Gal(fiI -4)) ,_2Gal(fi14)GlcNAc(fil -

+

fNeu5Ac(a2-3)Gal(fi1-4)[tNeu5Ac(a2-3)]Gal(fil4)[fFuc(aI-3)]GlcNAc(fiI -

+

+ + +

+

[76,77]

[25,84]

+

+ + +

[751 [76,77]

~ 5 1 [861 [20,21,87,88] [401 [80,89]

NeuSAc(a2-3)Gal(fiI -3)GalNAc(a 1Neu5Ac(a2-3)Gal(fi1-3)GalNAc(a 1-O)Ser/Thr

+

~901 [771

+

1771

Neu5Ac(a2-8)Neu5Ac(a2-3)Gal(fi1-3)GalNAc(a l-O)Ser/Thr

+

[911

NeuSAc(a2-3)[GalNAc(fi I4)]Gal(fi 1-3)GalNAc(a 1-O)Ser/Thr

+ +

1771 [771

NeuSGc(a2-3)Gal(fil-3)GalNAc(a I-O)Ser/Thr Neu5Ac(a2-3)GalNAc(fi14)GlcNAc(filNeu5Ac(a2-6)GaINAc(fil 4)GlcNAc(fiINeu5Ac(a2-3)[GalNAc(fi14)]GalNAc(fi 1-3)Gal(fi14)Gal(fiINeu5Gc(a2-3)[GalNAc(fil4)]GalNAc(fil-3)Ga1(fil 4)Gal(fiI -

+

1921 [93,94]

+ +

[951

+

[771

NeuSAc(a2-6)GalNAc(a I-O)Ser/Thr NeuSGc(a2-6)GalNAc(a I-O)Ser/Thr

+

[771 [771

Neu5Ac(a24)GlcNAc(fi1-3)[Neu5Ac(a2-6)]Gal(fil-

+

-Fuc( 14)Neu5Gc(2-

+

+

Neu5Ac(a2-9)Neu5Ac(a2-3/6)Gal(fi 14)GlcNAc(fil-

+

{Neu5Ac(a2-8)},Neu5Ac(a2-3)Ga1(fi14)GlcNAc(~1{Neu5Ac(a2-8)},Neu5Ac(a2-6)GalNAc(al -O)Ser/Thr

+

+

[961 1971 [981 [991 I771

continued on next page

253

Table 2, continued ~

Partial structure

N

0 Ref(s).

{NeuSGc(a2-8)},NeuSGc(a24)GalNAc(a 1-O)Ser/Thr {NeuSAc(a2-8)},- with 4Ac, 7Ac, 9Ac or 9Lt

+

+

[771 [loo]

(NeuSGc(a2-8)},- with 4Ac, 7Ac or 9Ac

+

[51,100]

{Neu5Ac/SGc(a2-8)},- with 4Ac, 7Ac or 9Ac

+

[loo]

{NeuSGc(a2-05)},,-

+

[loll [lo21

Kdn(a2-3)Gal(fiI 4)GlcNAc(fi1Kdn(a2-3)Gal(o1-3)GalNAc(a 1-3)GalNAc(a 1-O)Ser/Thr

+ +

[471

Kdn(a2-3)[CalNAc(fi 14)]GalNAc(fi1-3)Gal(fi14)Gal(~lKdn(a2-6)GalNAc(a I-O)Ser/Thr

+

[771 [48,77,103]

{ Kdn(a2-8)}, Kdn(a24)GalNAc(a I -O)Ser/Thr Fuc(a 1-4)[Fuc(a 1-S)]Kdn(a24)GalNAc(a 1-O)Ser/Thr

+ [471

-Fuc(a14)Kdn(aZ-6)GalNAc(al-O)SeriThr Kdn(a2-6) [Kdn(a2-3)] G alNAc(a 1-0)Thr Kdn(a2-3)GalNAc(al-O)Thr Fuc(al4)[{ Fuc(a 1-5)}0-, ]Kdn(a2-3)Gal(fiI -

+

+ + +

[I041

[I051

+

[lo61 [106a] [I041

(Kdn(a2-8)},- with 4Ac, 7Ac or 9Ac Kdn(a2-8){Ne~SAc(a2-8)}~- with fAc

+ +

[I071 [lo71

Kdn(a2-8){Ne~SGc(a2-8)}~-with f A c Kdn(a2-8){Ne~SAc/SGc(a2-8)}~-with fAc

+

~461 [I071

Kdn9Ac(a2-8) { NeuSGc(a2-8)}, -

+ ~511

+

+

a In the case of a specific fragment being established by ' H NMR spectroscopy, the reference refers to such a study or to a review that includes the fragment. S means sulfate.

been described [98]. In a number of structures not only sialic acid is responsible for the acidic character of the carbohydrate chain, but also sulfate. Here, an unusual example is the Neu5Ac(a2-3)[6S]GaI@ 14)GlcNAc element [82]. The list of sialic-acid-containing sequences, in which N-acylneuraminic acid is replaced by Kdn is continuously growing (Table 2). Interestingly, also structural elements occur which have not been found so far for N-acylneuraminic acids, e.g. Kdn(a2-6)[Kdn(a2-3)]GalNAc(al-O)Thr [ 1061. General structural information with respect to glycoprotein glycans is presented in volume 29a of the New Comprehensive Biochemistry series, and detailed information with respect to poly-N-acylneuraminyl-containing glycoproteins and Kdn-containing glycoproteins in the present volume 29b. Several of the sialic-acid-containing sequences present in N- and 0-glycoprotein glycans, also occur in glycolipids, milk and urinary oligosaccharides, and in (1ipo)polysaccharides of different biological origin. An impression of this overlap in structures can be obtained from an inspection of the structural data in Tables 3-5, summarizing sialic-acid-containing elements of glycolipids, structures of milk and urinary sialo-oligosaccharides, and structures or elements of sialic-acid-containing (lipo)polysaccharides, respectively. It is interesting to note that

v, N P

Table 3 Compilation of sialic-acid-containingfragments in glycolipidsa Structure

References

Neu5Ac(a2-3)Gal(P 1- I’)Cer {Neu5Ac(a2-8)}o~,Neu5Ac(a2-6)Glc(~l-l’)Cer Neu5Gc/5Gc8S(a2-6)G1c(~l-1’)Cer Neu5Ac(a24)Glc(~l-8)Neu5Ac(a24)Glc(~I-l’)Cer Neu5Ac(a2-8)Neu5Ac(a24)Glc(P14)Glc(~l-I’)Cer Neu5Ac/5Gc/4AcSGc/5Gc8Me/5GcMe(a2-3)Ga1(~14)Glc(~1-1 ’)Cer Neu5Ac/5,7Ac2/5,9Ac2/5,7,9Ac3/5Ac8S/5Gc(a2-8)Neu5Ac(a2-3)Gal(~1 -4)Glc(PI -1’)Cer Neu5Ac/5Gc(a2-8)Neu5Gc(a2-3)Gal(P14)Glc(P1-1 ‘)Cer Neu5Ac(a2-8)Neu5Ac(a2-8)Neu5Ac(a2-3)Gal(~l4)Glc(~l-l ‘)Cer Neu5,9Ac2(a2-8)Neu5Ac(a2-8)Neu5Ac(a2-3)Gal(~1 -4)Glc(~l-I’)Cer GalNAc(~14)[Neu5Ac/5Ac8S/5Gc/5Gc8S(a2-3)]Gal(~14)Glc(~ l-l’)Cer l-l’)Cer Gal(PI-3)GalNAc(P 14)[Neu(a2-3)]Gal(~l4)Glc(~ [R-3]GalNAc(P14)[Neu5Ac/5Gc(a2-3)]Gal(P 14)Glc(P 1-1 ’)Cer [R-3]GalNAc(fi4 I)[{Neu5Ac(a2-8)} l-2Neu5Ac(a2-3)]Gal( 1~ 4)Glc(~l-l’)Cerc [R-3]GalNAc(f 1~ 4)[Neu5,9Ac2 (a2-8){ Neu5A~(a2-8)}~-~ Neu5Ac(a2-3)]Gal(P 14)Glc(PI -1 ’)Cer‘ [R-3]GalNAc(PI4)[{Neu5Gc(a2-8)} l_zNeu5Gc(a2-3)]Gal(P 14)Glc(P1-1’)Cerb l-l’)Cer [R-3]GalNAc(fi-4)[Neu5Ac(a2-8)Neu5Gc(a2-3)]Gal(~l4)Glc(~ I Ara( 14)Gal(P 1 4 ) [{Gal@ 1-8))o-1 ]Neu5Gc/5Gc8Me(a2-3)Gal(P 14)Glc(P 1-1 ’)Cer Neu5Gc8Me(2-3)GalNAc( 1-3)Gal( 14)Glc( 1-1’)Cer Neu5Gc8Me(a2-3)[NeuSGc8Me(a24)]GalNAc(~1-3)Gal(~I 4)Glc(Pl-l’)Cer Neu5Ac/5,9Ac2/5Gc(a2-3)Gal(~l-3)GalNAc(~1 {Neu5Ac(a2-8)} ,-2Neu5Ac(a2-3)Ga1(~1-3)GalNAc((3 1Neu5Gc(a2-8)Neu5Gc(a2-3)Gal(flI-3)GalNAc(P 1Neu5Ac(a2-3)[Neu5Ac(a24)]Gal(~1-3)GalNAc(~ 1continued on next page

Table 3, continued Structure

References

Neu5Ac(a2-3)[[R-3]GalNAc(fil4)]Gal(fil-3)GalNAc(filNeu5Gc(a2-3)[GalNAc(!314)]Gal(fil-3)GalNAc(fil{Neu5Ac(a2-8)}o~,Neu5Ac(a2-3)Gal(fi1-3)[{Neu5Ac(a2-8)}~~lNeu5Ac(a2~)]GalNAc(~1 NeuSAc(a2-3)Gal(fil-3)~eu5Ac(a2-6)]GalNAc(fi14)[{Neu5Ac(a2-8)}o~l Neu5Ac(a2-3)]Gal(fi1-4)Glc(fil-l’)Cer NeuSAc(a24)Gal(fil4)GlcNAc(filNeu5Ac/5Gc(a2-3)Gal(fil4)GlcNAc(fi 1{Neu5Ac(a2-8)}1-2Neu5Ac(a2-3)Ga1(fi 14)GlcNAc(fi1 Neu5Ac/5Gc(a2-8)Neu5Gc(a2-3)Gal(fl1 4)GlcNAc(P1 Neu5Ac(a2-3)[GalNAc(~14)]Gal(fil4)GlcNAc(filNeuSAc(a2-3)GaI(fi1 -4)[Fuc(a1 -3)]GlcNAc(fi1Neu5Ac(a2-3)Gal(fi1-3)GlcNAc(filNeu5Ac(a2-3)[GalNAc(fiI4)]Gal(fi1-3)GlcNAc(fllNeu5Ac(a2-3)Gal(fil-3)[Fuc(al4)]GlcNAc(~lNeu5Ac(a2-3)Gal(~1-3)~eu5Ac(a2-6)][R-4]GlcNAc(fil-c Neu5Ac(a2-3)GalNAc(fi1-3)Gal(a 1 NeuSAc(a2-3)GalNAc(fi1-3)Gal(fi1 (NeuSGc8Me(a2-05)},Kdn(a2-3)Gal(fil4)Glc(fi 1-1 ’)Cer

[R-3]GalNAc(pI 4)[Kdn(a2-3)]Gal(fl 14)Glc(fil-l’)Cer Kdn/Kdn9Ac(a2-3)Gal(fi1 -3)GalNAc(fi1Kdn(a2-3)Gal(fi1 -3)[Neu5Ac/Kdn(a2-6)]GalNAc(fi1 a

S, sulfate; Cer, ceramide. R, saccharide.

R, H or saccharide

Table 4 Sialic-acid-containing human milk oligosaccharides Structures

References

Neu5Ac(a2-3)Gal(fl 1-4)Glc a-e Neu5Ac(a24)Gal(~l-4)Glca,b

[ 16,125-1271

Neu5Ac(a2-3)Gal(fi1-3)GlcNAc(~l-3)Gal(fil4)Glc Neu5Ac(a2-3)Gal(~l-3)[NeuSAc(a2-6)]GlcNAc(fil-3)Gal(~14)Glc qf Neu5Ac(a2-3)Gal(~l-3)[Fuc(a 14)]GlcNAc(fll-3)Gal(~l4)Glc

[ 12S,l26,128,129]

Neu5Ac(a2-3)Gal(P 1-3)[Fuc(a 1 4 ) ][NeuSAc(a24)]GIcNAc(PI-3)Gal(fi 1-4)Glc NeuSAc(a2-3)[Fuc(a 1-2)]Gal(P 1-3)GlcNAc(fi 1-3)Gal(P 14 ) G l c

[ 129,1301

Gal(~1-3)[NeuSAc(a24)]GlcNAc(fil-3)Gal(flI4)G1~ a Gal@1-3) [NeuSAc(a24)]GlcNAc(fi1-3)Gal(fi14)Glc

[ 16,125-1 271

[12S,128-130] [ 1 2 83~1,1321

[1331 [12S,126,128]

Fuc(a 1-2)Gal(fi 1-3)[NeuSAc(a24)]GlcNAc(P 1-3)Gal(P 14)Glc NeuSAc(a24)Gal(~14)GlcNAc(~1-3)Gal(fil 4)Glca

[1341 [128,131,13S] [ 125,126,1281

NeuSAc(a2-3)Gal(P 1-3)GlcNAc(@1-3) { Gal(fi14)[Fuc(a 1-3)]GlcNAc(fi 14)}Gal(P 14)Glc Neu5Ac(a2-3)Gal(~1-3)GlcNAc(fil-3)[NeuSAc(a2-6)Gal(fll-4)GlcNAc(fiI4)]Ga1((314)Glc

~361 ~291

NeuSAc(a2-3)Gal(~1-3)[NeuSAc(a2-6)]GlcNAc(~l-3)[Gal(~l4)GlcNAc(~14)]Gal(~I4 ) G l c NeuSAc(a2-3)Gal(~1-3)[NeuSAc(a2-6)]GlcNAc(P l-3)[Fuc(a 1-3)Gal(~l-4)GlcNAc(~l4)]Gal(fi14)Glc NeuSAc(a2-3)Gal(fi1-3)[NeuSAc(a2-6)]GlcNAc(fi l-3){ Gal(fi14)[Fuc(al-3)]GlcNAc(fi14)}Gal(PI -4)Glc NeuSAc(a2-3)Gal(fi1-3)[Neu5Ac(a2-6)]GlcNAc(P 1-3)[Neu5Ac(a24)Gal(~l4)GlcNAc(~l-6)]Gal(~l-4)Glc NeuSAc(a2-3)Gal(fi1-3)[Fuc(a 1-4)]GlcNAc(~l-3)[Gal(fiI4)GlcNAc(fil4)]Gal(~I 4)Glc NeuSAc(a2-3)Gal(P 1-3)[Fuc(a 14)]GlcNAc(P 1-3){ Gal(P14)[Fuc(a 1-3)]GlcNAc(P 1-6)}Gal(@ 14)Glc Neu5Ac(a2-3)Gal(fil-3)[Fuc(a 14)]GlcNAc(fi 1-3)[Neu5Ac(a24)Gal(PI4)GlcNAc(Pl4)]Gal(~14)Glc

~291 [1371 [ 129,130,137]

[1381 [1391 [1391 [1391

Neu5Ac(a2-3)Gal(PI-3)[Fuc(a

IH)]GlcNAc(fl 1-3)[Gal(~I-4)GlcNAc(~ld)Gal(~ 1-4)GlcNAc(~1-6)]Gal(PI4)Glc Neu5Ac(a2-3)Gal(P 1-3)[Fuc(a 14)]GlcNAc(fi 1-3) {Gal@14)[Fuc(a 1-3)]GlcNAc(fl 1-3)Gal(fl14)GlcNAc@ 1-6)}Gal(~14)Glc

[ 1401

Neu5Ac(a2-3)Gal(P 1-3)[Fuc(a 1-4)]GlcNAc(P 1-3)[Gal(B 1-3)GlcNAc(P 1-3)Gal(fi 1-4)GlcNAc(P 1-6)]Gal(P 1 4 ) G l c NeuSAc(a2-3)Gal(P 1-3)[Fuc(a 1-4)]GlcNAc(fi 1-3) { Gal(fi1-3)[Fuc(a 1-4)]GlcNAc(fiI-3)Gal(fl 14)GlcNAc(j314)}Gal(fi1 4 ) G l c

~411

[1411 [1411 continued on next page

Table 4, continued Structures

References

Neu5Ac(a2-3)Gal(fi 1-3)[Fuc(a 14)]GlcNAc(!31-3){ Gal@1-3)GlcNAc(fi 1-3)Gal(P 14)[Fuc(a 1-3)]GlcNAc(P 1 4 ) } Gal@ 1-4)Glc Neu5Ac(a2-3)Gal(P 1-3)[Fuc(a 14)]GlcNAc(fi1-3){ Fuc(a 1-2)Gal(P 1-3)GlcNAc(fi 1-3)Gal(fi 1-4)[Fuc(a 1-3)]GlcNAc(fi 14)}Gal(fi 14)Glc Gal(fi1-3)GlcNAc(fi 1-3)[Neu5Ac(a24)Gal(fi 14)GlcNAc(fi1-6)]Gal(B 1 4 ) G l c

Gal(fiI-3)[NeuSAc(a24)]GlcNAc(fi1-3) { Gal(fiI4)[Fuc(a 1-3)]GlcNAc(P 14)}Gal(fi 14 ) G l c Gal(~1-3)~eu5Ac(a24)]GlcNAc(~l-3)[Gal(~1-3)GlcNAc(~14)]Gal(~l4)Glc Gal(~1-3)[Neu5Ac(a24)]GlcNAc(~l-3)[Fuc(al-2)Gal(~l-3)GlcNAc(~ 14)]Gal@l4)Glc Gal@1-3)[Fuc(a 1-4)]GlcNAc(fi 1-3)[Neu5Ac(a24)Gal(fil-4)GlcNAc(fi1-6)]Gal(PI 4 ) G l c Gal(P1-3)[Fuc(a 14)]GlcNAc(P 1-3){Neu5Ac(a2-3)Gal(fi 14)[Fuc(a 1-3)]GlcNAc(P 1-6)}Gal(P 14 ) G l c Fuc(al-Z)Gal(fi 1-3)GlcNAc(fil-3)~eu5Ac(a24)Gal(fil 4)GlcNAc(fiI4)]Gal(fi14)Glc Fuc(a 1-2)Gal(fi 1-3)GlcNAc(fi 1-3) {NeuSAc(a2-3)Gal(fi 14)[Fuc(a 1-3)]GlcNAc(fi 14)}Gal(fi 1 4 ) G l c Fuc(a 1-2)Gal(fi 1-3)[Fuc(a 14)]GlcNAc(@1-3)[Neu5Ac(a2-6)Gal(P 14)GlcNAc(fi1d)]Gal(B 1-4)Glc Gal@1 4)GlcNAc(P 1-3)[NeuSAc(a24)Gal(fi 1-4)GlcNAc(fiI-6)]Gal(P 1 4 ) G l c

Neu5Ac(a24)Gal(fil4)GlcNAc(~l-3)[Gal(fi 14)GlcNAc(~l-6)]Gal(~14)Glc Neu5Ac(a24)Gal(fi14)GlcNAc(fi1-3){ Gal@ 14)[Fuc(a1-3)]GlcNAc(fi 14)}Gal(P 14)Glc Neu5Ac(a24)Gal(fi1-4)GlcNAc(fi1-3){ Fuc(a 1-2)Gal(fiI 4)[Fuc(a 1-3)]GlcNAc(fi 14)}Gal(fi 1 4 ) G l c Neu5Ac(a24)Gal(fil4)GlcNAc(fil -3)[NeuSAc(a24)Gal(P 14)GlcNAc(fil4)]Gal(fil4)Glc Neu5Ac(a2-6/3)Ga1(fi1-4)[Fuc(a1-3)]GlcNAc(fi1-3)~eu5Ac(a2-3/6)Gal(fil-4)GlcNAc(fi14)]Gal(P14)Glc

Neu5Ac(a2-3)GaI(fi14)[Fuc(al-3)]Glc NeuSAc(a24)Gal(!3 14)GlcNAc(P1-3)Gal(fi 14)[Fuc(a 1-3)IGlc

Neu5Ac(a2-3)Gal(~1-3)[Neu5Ac(a2-6)]GlcNAc(fiI-3)Gal(~14)[Fuc(a 1-3)]Glc NeuSAc(a2-3)Gal(P 1-3) {Gal@14)[Fuc(a 1-3)]GlcNAc(P 14)}Gal(P 14)Glc

Neu5Ac(a2-3)GaI(fi1-3)[Fuc(a l4)]GlcNAc Neu5Ac(a2-3)Ga1((31-3)[Fuc(a 14)]GlcNAc(fi 1-3)Gal

continued on next page

Table 4, notes Also occurring in feces of preterm infants fed on breast milk [147]. Also occurring in bovine colostrum and human (pregnancy) urine [148]. Other sialyloligosaccharides in bovine colostrum are: NeuSAc(a2-3)GaL Neu5Gc(a2-3)Gal(P14)Glc, Neu5Ac(a2-8)Neu5Ac(a2-3)Gal(~1-4)Glc,NeuSAc(a26)Gal(P 14)GlcNAc, Neu5Ac(a2-6)Gal(~14)GlcNAc(a1-P, NeuSAc(a2-6)Gal(P 1-4)[6P]GlcNAc. Other sialyloligosaccharides in human (pregnancy) urine are: Neu5Ac(a2-3)Gal(~14)GlcNAc, Neu5Ac(a24)Gal(Pl4)GlcNAc, NeuSAc(a2-3)GaI(~l4)GlcNAc(al-P, Neu5Ac(a24)Gal(Pl4)GlcNAc(al-F', Neu5Ac(a2-3)Gal(P1-3)[Neu5Ac(a24)]GalNAc, Neu5Ac(a2-3)Gal(~l-3)[Neu5Ac(a24)]GalNAc(alO)Ser, Neu5Ac(a2-3)Gal(PI-3)GalNAc(al -P, Neu5Ac(a2-3)Gal(~1-1 L)-myo-inositol, NeuSAc(a2-3)Gal(PI -)scyllo-inositol. The urinary carbohydrates, except the inositol derivatives and the tetrasaccharide, do also occur in hemofiltrates of patients with end-stage renal disease; in addition NeuSAc(a2-8)NeuSAc, Neu5Ac(a2-3)Gal(fll-3)GalNAc(aI-O)Ser-Leu and Neu5Ac(a2-3)Gal(PlIt)Xyl(Pl-O)Ser have been detected [ 149,1501. For NMR data of Neu5Ac(a2-3)Gal(~l4)Glc, Neu5Ac(a2-3)Gal(~l4)GlcNAc,and Neu5Ac(a24)Gal(PIIt)GlcNAc, see also [ 1511. Urine of patients with aspartylglycosaminuria contains Neu5Ac(a2-3)Gal(~l4)GlcNAc(Pl -N)Asn, Neu5Ac(a24)Gal(~14)GlcNAc(P1-N)Asn, and NeuSAc(a23)Gal(P 14)GlcNAc(~l-3)Gal(~I 4)GlcNAc(PI -N)Asn [76,152]. Urine of patients with P-mannosidosis contains NeuSAc(a24)Man((3l4)GlcNAc [ 1531. ' Neu5Ac(a2-3)[6S]Gal(P14)Glc occurs in rat milk and mammary gland [154]. Neu5,9Ac2(a2-3)Gal(P14)Glc occurs in rat urine (251. Neu4,5Ac2(a2-3)Gal(~14)Glcoccurs in monotreme milk [I 91. Occurs also in human pregnancy urine [155]. a

v h 1 ,

m

259

for both glycoprotein and glycolipid glycans a sequence has been found comprising the oligomerization of Neu5Gc residues through their anomeric centers and N-glycolyl moieties, Neu5Gc(a2-05)Neu5Gc(a2-05)Neu5Gc(a2- [3 1,lO 11; in the case of the glycolipid material the NeuSGc residues are also 8-U-methylated [3 I]. In microbial polysaccharides, besides internal 8- and 9-substituted NeuSAc residues, also internal 4- and 7-substituted Neu5Ac units have been frequently found (Table 5). It should be noted that some of the sialic-acid-containing glycan fragments and polysialic acids are specifically found in lipopolysaccharides and capsular polysaccharides of pathogenic bacteria, leading to severe problems in the development of suitable vaccines. A typical source for sialo-oligosaccharides generated from N- and O-linked glycans is the urine of sialidosis and I-cell disease patients [191-1991, though this is not discussed in detail here (see section 10.5, and volume 30 of the New Comprehensive Biochemistry series). Patients with other inborn errors of metabolism, like aspartylglycosaminuria [76,152] or P-mannosidosis [ 1531 excrete small amounts of structurally unusual sialooligosaccharides, of which the formulae have been included in the footnotes of Table 4. Finally, sequence information of the already mentioned glycosylphosphatidylinositol anchor is available, showing that the glycan core consists of Mana-Mana-Man-[NeuSAcGal-Ga1NAc)Man-GlcN-inositol [ 1861. The phenomenon of intramolecular lactone formation, often reported for polysialic acid [200-2021 and for gangliosides [203-2061, has not been detected so far in glycoprotein sialoglycans. In the case of a NeuSAc(a2-8)NeuSAc sequence, lactonization affords a NeuSAc(a2-8,1-9)Neu5Ac element (Fig. 4), whereby the COOH group of one residue reacts with H 0 9 of an adjacent residue, to give a six-membered ring. Similarly NeuSAc(a2-9)NeuSAc can be converted into Neu5Ac(a2-9,1-8)NeuSAc, and NeuSAc(a2-3)Gal into Neu5Ac(a2-3,1-2)Gal or Neu5Ac(a2-3,1-4)Gal. In a polysialic acid chain of a-2,8-linked NeuSAc residues the a-2,8/ 1,9-lactonization can be effected under relatively mild conditions, like mild acid treatment [200], yielding a water-insoluble polymer. The NeuSAc(a2-9,1-8)Neu5Ac formation in a polysialic acid chain can only be realized by carbodiimide treatment [20 11, illustrating a more difficult condensation with the secondary H 0 8 group. The difference in reactivity between the primary and the secondary OH group has been nicely illustrated for the alternating a-2,8/a-2,9polysialic acid of E. coli K92 (Table 5) [202]. For gangliosides, it has been stated that ganglioside lactones occur also as such in nature[206], and that in this way the negative charge of a ganglioside under physiological conditions can be modulated. Lactonization makes the oligosaccharide chain also more rigid, which may have important biological implications. Treatment with carbodiimide or glacial acetic acid can even convert a Neu5Ac(a2-8)NeuSAc(a2-3)Gal(~ 1- into a NeuSAc(a2-8,1-9)NeuSAc(a23,1-2)Gal(Pl- sequence [204,205]. Over the years NeuSAc has been prepared by a variety of methods. Several biological sources have been explored to isolate this sialic acid in high amounts. Among them are edible birds nest substance [207], urine of sialuria patients [65], colominic acid {Neu5Ac(a2-8)}, produced by E. coli strains [208], and hen’s egg chalaza, egg-yolk membranes and delipidated egg yolk [209,210]. The large scale organic synthesis of Neu5Ac is still complicated (see section 6.1). However, recent biotechnological routes, using sialate-pyruvate lyase (aldolase; see section 9.5), readily yield large amounts of

Table 5 Survey of structures or elements of sialic-acid-containingmicrobial polysaccharides Structwdelement

Species

Reference(s)

Neisseria meningitidis B Escherichia coli K235

~41 ~561

Capsular polysacharides (CPS)

{ -8)Neu5Ac(a2-8)Neu5Ac(a2-),

{ -8)Ne~SAc(a2-8)NeuSAc(a2-}~ (with7Ac or 9Ac) {-9)Ne~5Ac(a2-9)Neu5Ac(a2-}~ (with7Ac or 8Ac) {-8)Ne~5Ac(a2-9)Neu5Ac(a2-}~ {-4)Neu5Ac(a24)Gal(aI -},, (-4)Neu5Ac(a24)Glc(al-},, (withAc) {-3)GalNAc(fi14)Gal(a14)Neu5,9Ac2(a2-3)Gal(fil -}" { -4)Glc(fi14)[NeuSAc(a2-3)Gal(fi1-4)GlcNAc(fi1 -3)]Gal(fi1 - } n { -4)Glc(fiI 4)meu5Ac(a2-3)Gal(fi 1-3)GlcNAc(fi1 -3)]Gal(fi1-},, { -4)GlcNAc(fil-3)[Gal(fil4)]Gal(fil4)Glc(fil-3)Glc(fil-2)[Neu5Ac(a2-3)]Gal(fi 1 -},, {-4)Glc(fil-6)[Neu5Ac(a2-3)Gal(fil4)]GlcNAc(fi 1-3)Gal(fiI-}" { -4)Glc(a 1 4 ) [NeuSAc(a2-3)Gal(fi14)GlcNAc(fi14)]Gal(fi 1-4)Glc(fi1 -}" {-4)Glc(fi14)[Neu5Ac(a2-3)Gal(fil4)GlcNAc(fil4)]Glc(a1-4)[Glc(fil-3)]Gal(fi1-}" { -4)Glc(fil4)[Neu5Ac(a2-3)Gal(~l-3)]Glc(fil-3)Gal(fi1-}n { -4)Glc(fi1-4)[Neu5Ac(a2-3)Gal(fi 1-4)GlcNAc(fi14)]Glc(a 14)Gal(fi1-}n { -3)Glc(fi1-2)[Kdn(a24)][3Ac]GlcA(a1-3)Man(a1-3)Glc(a 1-},,

Pasteurella haemolytica A-2

[1571

Morarella nonliquefaciens Escherichia coli KI

[1581 [24,159]

Neisseria meningitidis C

~41

Escherichia coli K92

[i601

Neisseria meningitidis W 135

~41 ~41 [26,161]

Neisseria meningitidis Y Escherichia coli K9

Group B Streptococcus la Group B Streptococcus Ib Group B Streptococcus 11 Group B Streptococcus I11 Group B Streptococcus IV Group B Streptococcus V Group B Streptococcus VI Group B Streptococcus VII Klebsiella ozaenae K4 (2211)

Lipopolysaccharides (LPS)

{ -3)GalNAc(fi1-7)Neu5Ac(a2-3)[Glc(a1-2)]Glc(fiI-}" { -3)GlcNAc(fi1-7)Neu5Ac(a2-3)[Gal(al-2)]Glc(fiI -}" {-3)GalNAc(fi14)Gal(a14)Neu5,7/8,9Ac3(a2-3)Gal(fil -},, (also with 9Ac)

Escherichia coli 024

[ 166,1671

Escherichia coli 056

[ 166,1671

Escherichia coli 0104

[26,271 continued on next page

Table 5, continued ~

~~

Structurelelement

Species

Reference(s)

{-3)[+6Ac]GlcNAc(fi 1-7)Neu5Ac(a2-3)[CH3(NH)C-2]FucN(a1 -},,

Salmonella arizonae 0 2 1

El681

{ -4GalANGro(al-4)Neu5Ac(a2-3)GalANGro(fi1-3)QuiNAc(fi 1- } n { -3)[Glc(al4)]GalNAc(@14)Neu5Ac(a24)Glc(alM)[Glc(a 1-4)Gal(fi1L6)Gl~((3l-3)]Gal((31-}~

Vibrio cholerae H11 (non-01) Hafnia aluei 0 2

~ 9 t1701

Neu5Ac(a2-3)Gal(@14)GlcNAc(fi 1-3)Gal(@I4)Glc((314)Hep-

Neisseria meningitidis

[ 171-1731

Neu5Ac(a2-3/6)Gal(@ l4)GlcNAc(@l-3)Gal(fi1-4)Hep-

Haemophilus ducreyi Campylobacter jejuni 0 1

[ 1741

NeuSAc(a2-3)[GalNAc(@1-4)]Gal(@ 1-3)[Gal(al-Z)]Gal(fi1-3)Hep-

1

Core regions of lipopolysaccharides (LOS) a

[I751 ~761

NeuSAc(a2-3)Gal(fi 1-3)[Gal(a 1-2)]Gal(fi 1-3)Hep-

Campylobacter jejuni 0 2

Neu5Ac(a2-3)Gal(fil-3)GalNAc(fil-4)pJeuSAc(a2-3)]Gal(fi 1-3)HepNeu5Ac(a2-3)[GalNAc(fil-4)]Gal(fil4)Glc((31-2)Hep-

Campylobacter jejuni 0 4 Campylobacter jejuni 0 2 3 and 0 3 6

[I751 [I751

Gal(a 14)Glc((3l-7)Neu5Ac

Rhodobacter capsulatus 37b4

[I771

a Some other species for which it has been demonstrated that their LPS or LOS contains sialic acid are: Rhizobium meliloti MI IS (LPS, Neu5Ac/Neu5,9Ac2)[178]; Salmonella toucra (LPS, terminal and 4-linked NeuSAc) [179]; Salmonella djakarta (LPS, terminal NeuSAc) [I 801; Salmonella isaszeg (LPS, terminal and 4-linked Neu5Ac) [ 1801; Citrobacter freundii 0 3 7 (LPS, terminal and 7-linked NeuSAc) [ 1791; Pasteurella haemolytica 1 and 5 (LPS, Neu5Ac) [181]; Haemophilus injruenzae (LOS, terminal NeuSAc) [182,183]; Neisseria gonorrhoeae (LOS, terminal Neu5Ac) [184,185]. Note that for E. coli 0 2 4 and 0 5 6 and H. aluei 0 2 also terminal Neu5Ac has been found [179]. GalANGro, N-galacturonyl-2-aminoglycerol.

262

C //O /I

0 AcHN

Fig. 4. Lactonization of a NeuSAc(a2-8)NeuSAc(a2sequence, affording a Neu5Ac(a2-8,1-9)Neu5Ac(a2fragment.

this sialic acid (see section 6.1). Efficient procedures to prepare Neu5Gc from colominic acid via de-N-acetylation, N-acryloylation and reductive ozonolysis, followed by acid or enzymatic hydrolysis of the formed (NeuSGc(a2-X)}, has appeared in refs. [211,212]. Also porcine submandibular gland is a good source for the large scale preparation of NeuSGc, whereas Neu5,9Ac2 can be prepared from bovine submandibular gland [2 13). Besides the occurrence of sialic acids [(0-acetylated) Neu5Ac and Kdn] in microbial polysaccharides (Table 5), some lipopolysaccharides have shown to contain sialic-acidlike monosaccharides. They differ from sialic acids in the presence of an additional amino function at C7, a deoxy function at C9, and in the configuration at the chiral centers: 5,7-diamino-3,5,7,9-tetradeoxy-~-glycero-~-manno-non-2-ulosonic acid and 5,7-diamino3,5,7,9-tetradeoxy-~-g~cero-~-ga~acto-non-2-u~osonic acid [2 14,214al. So far, the two amino groups have been found to be substituted in different combinations, yielding acetamido, formamido, (R)-3-hydroxybutyramido,4-hydroxybutyramido, or acetamidino functions. Also 0-acetylation can occur, whereas the (R)-3-hydroxybutyryl group at C7 can be used to link monosaccharides in a polysaccharide chain. Typical species are Pseudomonas aeruginosa strains [214], Shigella boydii type 7 [214], Vibrio salmonicida [214a], Vibrio cholerae 0 2 [215], Vibrio alginolyticus strain 945-80 [216], Salmonella arizonae 0 6 1 [2 171, Yersinia ruckeri 0 1 [2 181, Legionella pneumophila strain 1 [219,220], and PseudomonasJluorescens ATCC 49271 [220a]. For a review, see ref. [220], but it should be noted that the absolute configuration of 5,7-diamino-3,5,7,9tetradeoxy-~-glycero-~-ga~acto-non-2-u~osonic acid was earlier assigned as D-glycero-Lgalacto- [2 14,2 14a,216-2 19,220al.

4. Screening of biological materials for the presence of sialic acid For the staining of sialic acids in tissues and cells, a great variety of techniques is available. Classical histochemistry of sialic-acid-containing glycoconjugates makes use of either binding of cationized dyes (e.g. Alcian blue, cationized ferritin, ruthenium red) or selective periodate oxidation (derivatization of generated aldehyde groups of sialic acids with e.g. p-dimethylaminobenzaldehyde,dansylhydrazine, rhodamine, biotidferritin-conjugated avidin). The second approach is strongly dependent on sidechain modifications [53]. Comprehensive reviews on this subject have appeared [6,22 11.

263

Nowadays, specific lectins are frequently used to detect the presence of glycosidically bound sialic acids in complex carbohydrates. However, the specificity of lectins is generally broad, and positive information has always to be checked in control experiments using e.g. sialidases in the presence and absence of inhibitors. For the histochemical analysis, lectins may be conjugated e.g. with gold particles, peroxidase, rhodamine or fluorescein isothiocyanate [6]. A large series of lectins which recognize sialic acid have been demonstrated to occur in nature, and most of their biological sources have been summarized in refs. [6,11,222]; see also refs. [223-2271. For updated reviews focusing on lectins, see the present volume 29b of the New Comprehensive Biochemistry series. Generally, lectins have been isolated from lower animals such as prawns, snails, crabs, spiders, scorpions, lobsters, slugs, oysters, but also from plants, rat brain and B cells. While some of these lectins bind to both NeuSAc and NeuSGc, others are specific for NeuSAc. 0-Acetylation may also influence the binding strength of the lectin, both in a positive and in a negative manner (see section 10.3). For introductory glycoprotein analysis, in answering questions like “what monosaccharides are in the glycoprotein glycans?”, two sialic-acid-specific plant lectins, having also a glycosidic linkage specificity, have been included in commercially available kits. These lectins are the agglutinins from Muuckia umurensis and Sumbucus nigra, being diagnostic for NeuSAc(a2-3)Gal and NeuSAc(a2-6)Gal/GalNAc elements, respectively. For screening purposes, the lectins are labelled with digoxigenin-succinyl-E-amidocaproic acid hydrazide (DIG, a spacer-linked steroid hapten digoxigenin). After SDS/PAGE of the (g1yco)protein mixture and Western blotting, sialoglycoprotein bands with a-2,3and/or a-2,6-linked NeuSAc can be labelled by one or both of these DIG-labelled lectins, whereby the detection is carried out in an enzyme immuno-assay using a digoxigeninspecific antibody conjugated to alkaline phosphatase, followed by color development with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium chloride [228,229]. Viruses and antibodies can also be used for the detection of sialic acid in complex carbohydrate systems. The sialic-acid-binding properties of a number of viruses have been established, and it has been shown that influenza C virus, bovine corona virus and encephalomyelitis virus recognize 9-0-acetylated sialic acids (see also sections 8.4.2, 9.1 and 10.3). Microtiter-plate and nitrocellulose-membrane assays have been developed that use the hemagglutinin (receptor) and the receptor destroying activities (sialate 9-0acetylesterase) of the influenza C virus to specifically detect bound 9-0-acetylated sialic acids in glycoproteins [230,23 I]. Although the recognition site for Neu5,9Ac2 and the esterase activity are located on the same viral glycoprotein, these activities can be separated using different temperatures for the binding (4°C) and the enzyme reaction (4-methylumbelliferyl acetate or a-naphtyl acetate as substrates; 20-37°C). Other approaches are based on binding of the virus to immobilized ligands, and detection of the virus with monoclonal antibodies, whereby the esterase was selectively inactivated by the use of diisopropylfluorophosphate (DFP) [232]. Virus particles have also been labelled with radioactive isotopes [232] or biotin [233]. The application of the assay with virus particles for the staining of 0-acetylated sialic acids in tissue sections will be described in section 8.4.2. Recently, a new technique using a soluble chimera of the hemagglutininesterase portion of the hemagglutinin-esterase-fusion-glycoprotein from influenza C virus and the Fc portion of human IgG, has been reported [234,235]. Such a chimera retains

264

both its recognition and enzymatic functions, and also has the binding properties of the Fc portion of IgG. The probing can be carried out on blots and TLC plates taking into account precautions for the recognition and esterase activities (this has to be inhibited by DFP in the test) as discussed above. An interesting electrochemical method for the determination of bound sialic acid has been developed, making use of a potentiometric four-channel thick-film sensor [236]. The sialidase sensor consists of a bilayer of a membrane containing Clostridium perfringens sialidase immobilized in a poly(viny1 acetate)-polyethylene copolymer, which is placed on top of an H+-selective poly(viny1 chloride)-poly(viny1 acetate) indicator membrane. The enzyme-induced release of bound sialic acid leads to a concomitant decrease in pK, of the carboxyl function of sialic acid. This decrease affords a local pH change inside the sialidase-containing sensor membrane, which is monitored by the H+-selective indicator membrane. The pH optimum of the sialidase sensor was pH 4 for sialyllactose, mucin and colominic acid. Finally, TLC analysis may also be of great help in the screening of biological materials for the presence of sialic acids. This item will be discussed in section 5.3.1.

5. Isolation and analysis of sialic acids 5.1. Liberation

The characterization of the type of sialic acid in sialoglycoconjugates is frequently carried out after release and (partial) isolation. The cleavage of sialic acid from sialic-acidcontaining material is mainly performed by two methods, namely, acid hydrolysis and enzymatic hydrolysis. Both approaches have advantages and disadvantages. In the case of acid hydrolysis, problems arise with respect to de-0-esterification, which complicates quantitative analysis procedures. With respect to the enzymatic hydrolysis with sialidases, linkage specificity as well as a reduced or complete lack of susceptibility have to be taken into account. Moreover, in most cases much lower amounts of sialic acids are released by sialidases than by acid hydrolysis, which may be due to the different accessibility of the sialic acids in the biomolecules to be analyzed. Additionally, in work-up procedures and analyses, pH values below 4 and over 6 should be avoided to prevent migration of the 0-acetyl group at C7/C8 and hydrolysis of 0-acetyl groups as much as possible [23,57]. Several approaches have been reported for the effective acid hydrolysis of the labile a-2,3-, a-2,6- and a-2,8-linkages. All these procedures suffer from being not optimal in giving the real spectrum of sialic acids originally present in the sialoglycoconjugate under study, especially in the case of a mixture of (0-acylated) N-acylneuraminic acids. Terminal sialic acids are released in high yield (and low destruction) using a two-step acid hydrolysis procedure comprising treatment with formic acid (pH 2, 1 h, 70°C), followed by HCl (pH 1, 1 h, SOOC). After each step, the liberated sialic acids must be recovered by centrifugation, ultrafiltration or dialysis [I 1,12,213,237]. It should be noted that in the case of a spectrum of (0-acylated) N-acylneuraminic acids, the supernatant, ultrafiltrate or diffusate of the formic acid hydrolysis contains the majority of the 0-acylated N-acylneuraminic acid, whereas that of the HCl hydrolysis contains

265 Table 6 Substrate specificity of commercially available sialidases a Source of sialidase

PH optimum

a-2,3

Arthrobacter ureafaciens Vibrio cholerae

4.4-5.5' 5.0-6.5g

100

100

Clostridium perfingens Salmonella typhimurium

5.5-7.2'

100

5.0-7.01

100

Newcastle disease virus

5.0-6.0

100

Relative rates of cleavage are indicated (1 00 =full activity). Neu5Ac(a2-3)lactose. NeuSAc(a2-6)lactose. a

Neu5Ac(a2-8)Neu5Ac(a2-3)lactose. NeuSGc(a2-3)lactose. Sialyllactose, pH 5.0-5.5, and colominic acid, pH 4.34.5. g Sialyllactose, pH 5.0-5.5.

'

a-2,6C

a-2,Sd

a-2,3e

167 53

53 31

12 25

44

44

20

PI PI

0.4

n.d.k.l

n.d.

[247]

0.2

78

11

Reference

151

r51

High-molecular-mass isoenzyme (P. Roggentin, personal communication) (see also section 9.2). ' Sialyllactose, pH 5.0-5.1 (acetate buffer) and pH 5.8-6.0 (phosphate buffer). Depending on the buffer system used. n.d., not determined. { -8)Ne~5Ac(a2-8)NeuSAc(a2-}~, 0.1% and {-9)Neu5Ac(a2-9)Neu5Ac(a2-),, 0.08%.

J

' '

mostly Neu5Ac and Neu5Gc. In the case of low-molecular-mass substances, isolations can be carried out by gel-permeation chromatography. Although these conditions do not lead to significant de-N-acylation, de-0-acylation has been shown to occur to an extent of about 30-50%. One has to consider that milder acidic conditions result in incomplete release of sialic acids. Acid hydrolysis with acetic acid (2M, 3 h, SOOC) as suggested in ref. [238] did not improve the yield of 0-acylated N-acylneuraminic acids [ l 11. When focusing on sialic acid analysis, the use of H2S04 (0.05 M) is not recommended because of work-up problems. In connection with the HPLC analysis of Neu5Ac and NeuSGc, microwave hydrolysis in 2 M acetic acid has shown to be an interesting alternative [239]. In the methanolysis procedure, as used for the standard quantitative monosaccharide GLC analysis of glycoconjugates [240,241], 1 M methanolic HCl (24 h, 85OC) is applied. However, under these conditions released sialic acids are completely de-N, 0-acylated, which makes this approach unsuitable for the characterization of different types of sialic acid. It is, however, a reliable approach for the determination of the total amount of a mixture of (0-acetylated) N-acylneuraminic acids. When using a milder methanolysis procedure (0.05 M methanolic HCl, 1 h, 80°C) the de-N-acylation but not the de-0-acylation is strongly reduced [242]. In the quantitative determination of N-acylneuraminic acids in poly-N-acylneuraminicacid-containing glycoproteins, the release of free sialic acid was shown to be optimal using a combined mild acid hydrolysis (0.1 M TFA, 3 h, 8O0C)/subsequent mild methanolysis (0.05 M methanolic HCl, 1 h, SOOC) [243]. This method is also advised for the analysis of the Kdn content in Kdn-containing glycoproteins. In contrast, optimal release of Kdn from poly-Kdn-containing glycoproteins is obtained by mild methanolysis with a longer incubation time [243]. In the latter case the standard conditions of methanolysis also give good results.

266

The enzymatic release of N-acylneuraminic acids can be carried out under such mild conditions (low temperature, pH 5 4 ) , that destruction, migration, and de-0-acylation is kept at a minimum. In Table 6 the substrate specificity of commercially available sialidases from Arthrobacter ureafaciens, Yibrio cholerae, Clostridium perfringens, Salmonella typhimurium, and Newcastle disease virus, using simple N-acylneuraminyllactose substrates is compared (for comprehensive reviews on sialidases, see refs. [5,33,244-2461). It is evident that the sialidases show different ratios of cleavage rates for the a-2,3-, a-2,6- and a-2,8-linkages. The finding of a strong preference of the Newcastle disease virus sialidase for a-2,3-linkages holds also for other viral sialidases, such as those from fowl plague virus and influenza A2 virus. The latter enzyme also has a low specificity for a-2,g-linkages [5]. Among the bacterial sialidases, A. ureafaciens sialidase has a certain preference for a-2,6linkages. The S. typhimurium sialidase is the only bacterial sialidase with a viral sialidase-like pronounced preference for a-2,3-linkages [247]. Recently, two sialidases from Bacteroides fragilis having a higher preference for the cleavage of a-2,8linkages, when compared with a-2,3- and a-2,6-linkages, were isolated [248]. The sialidase, recently isolated from Macrobdella leech, cleaves only NeuSAc(a2-3)Gal linkages [61]. In general, Neu5Ac residues are released faster than NeuSGc residues. In a study using different N- and 0-glycoproteins with a-2,3- and/or a-2,6-linked N-acylneuraminic acids as substrates [antifreeze glycoprotein, ovine submandibular gland glycoprotein, a I-acid glycoprotein; Neu5Ac(a2-6)GalNAc(a l-O)Thr/Ser, NeuSAc/SGc(a26)[Gal(b 1-3)]GalNAc(a 1-O)Thr, NeuSAc/SGc(a2-3)Gal(fi 1-3)GalNAc(a 1-O)Thr, Neu5Ac(a2-3)Gal(B 14)GlcNAc(b 1-0)R; Neu5Ac/5Gc(a2-6)Gal(~l-4)GlcNAc(fi 1-O)R] and sialidases from A. ureafaciens, V cholerae, C. perfringens, Newcastle disease virus, fowl plague virus, and influenza A2 virus, roughly similar patterns of substrate specificity as for sialyllactoses were found. However, it was demonstrated that the core oligosaccharide andor the protein structure may also influence the rate of release for different glycosidic linkages[249]. In the case of S. typhimurium sialidase, also N-glycoprotein a-2,3-sialoglycans were susceptible to efficient cleavage, but not mucin 0-glycoprotein sialoglycans [247]. The most recently discovered sialic acid, NeuSGcAc, could not be released with V cholerae sialidaseE431. A comparison of the different commercially available sialidases shows that the C. perfringens sialidase iso-enzyme with a molecular mass of about 63 kDa has the broadest specificity. It should be noted that C. perfringens in fact produces two sialidases, the larger of which (63kDa) is commercially available (P. Roggentin, personal communication) (see also section 9.2). As described for the acid hydrolysis procedure, the work-up of enzymatically released sialic acids can be achieved employing various methods, depending on the starting sialoglycoconjugate material. 4-0-Acetylated neuraminic acids in any glycoconjugate are resistant to most sialidases tested so far; only viral sialidases show a low but significant activity. With the exception of Streptococcus sanguis sialidase [250], the presence of 0-acetyl substituents at C7-C9 leads to a reduced rate of cleavage by all sialidases, so that prolonged incubations are necessary for an efficient release [251]. More information has been collected in a detailed study with bacterial and viral sialidases and 4-methylumbelliferyl (MU) a-glycosides of 4-, 7-, and 9-0-acetylated N-acetylneuraminic acids as substrates [252]. In contrast to

267

the other sialidases tested, the fowl plague virus sialidase catalyzes a slow release of Neu4,5Ac2 from a-Neu4,5Ac2-MU. The recent finding of {Neu5Gc(a2-05)}, elements in a glycoprotein [ l o l l initiated a kinetic study of the enzymatic and non-enzymatic hydrolysis of Neu5Gc(a2-05)Neu5Gc and Neu5Gc(a2-8)NeuSGc [253]. It turned out that at pH < 3.8 the rate of acid hydrolysis of the unusual a-2,05-linkage was greater than that of the normal a-2,8-linkage. However, at pH > 3.8 reverse results were obtained; NeuSGc(a2-8)NeuSGc released a small but detectable amount of Neu5Gc even at pH 6. The a-2,05-linkage was only partially cleaved by C. perfringens and! l cholerae sialidases, and was essentially resistant to A . ureafaciens sialidase. The detection of sialidases that can release Kdn is so far highly limited. The liver of the loach Misgurnus fossilis was found to contain a sialidase capable of releasing both Neu5Ac and Kdn from sialoglycoconjugates [39]. While the sialidases investigated so far in detail require an NH-acyl group at C5 for full activity, the loach enzyme can handle both NH-acyl and OH functions at C5. The rainbow trout also turned out to be a useful source for the isolation of a sialidase, active in releasing both Neu5Ac and Kdn [254]. Recently, a sialidase was isolated from Sphingobacterium multiuorum that specifically released a-2,3-, a-2,6- and a-2,8-linked Kdn; Neu5Ac and Neu5Gc were not liberated [255]. Before fractionation, pools of free sialic acids can be freed from contaminants by several methods, including ion-exchange chromatography, cellulose chromatography, reversed-phase chromatography, preparative TLC [6,11,121. Generally, one of the purification procedures for the pool of sialic acids comprises Dowex ion-exchange chromatography at low temperature. After passage through a cation-exchange resin (Dowex SOW-XS, H+-form), the pool of sialic acids is adsorbed to an anion-exchange resin (Dowex 2-X8 or 1-X8, HCOO--form). Elution from the anion-exchange resin is generally carried out with &2 M formic acid. The ion-exchange chromatography should be carried out rapidly, as prolonged contact of acylneuraminic acids with the resin or the solvent systems used may result in degradation, 0-acetyl migration and/or de-0-acetylation. After rotary evaporation or lyophilization, the sialic acid pools are stored at -20°C. In general, mixtures of sialo-oligosaccharides from {Neu5Ac(a2-8)},, (NeuSAc(a2{Neu5Gc(a2-8)},, and (Kdn(a2-8)},, are 9)},, {-8)Neu5Ac(a2-9)Neu5Ac(a2-),, generated by limited acid hydrolysis [211,256,257]. Depending on the polysialic acid, different conditions have been applied. Also attention has been paid to the intramolecular self-cleavage of polysialic acids such as {Neu5Ac(a2-8)}, [258]. Adjacent COOH groups with a high pK, (3.9-5.5) act as proton donors for general acid catalysis. The lability is seen under mild acidic conditions, that can be encountered in various physiological situations. {Neu5Ac(a2-8)},3 is substantially more unstable than {Neu5Ac(a2-8)}2-3. A highly useful enzyme for the depolymerization of polysialyl carbohydrate chains, yielding oligosialyl compounds, is endo-sialidase (endo-N) produced by infection of E. coli K1 with a lytic bacteriophage [9,259,260]. The enzyme is specific in cleaving a-2,8-linkages, and requires at least five Neu5Ac or Neu5Gc residues for activity. A limited digestion of {NeuSAc(a2-8)},-R affords mainly {Neu5Ac(a2-8)}4, with some {Neu5Ac(a2-8)} 1-3. Alternating a-2,8/a-2,9-polysialyl chains, as present in some

268

bacterial polysaccharides, are also cleaved, but a-2,9-linked polysialyl chains are resistant. Poly-Kdn and {Neu5Gc(a2-05)}, are not substrates for endo-N. A similar endo-sialidase associated with phage particles, namely, E. coli bacteriophage $92, has been reviewed in ref. [261]. 5.2. Colorimetry One of the oldest methods used to detect and to quantitate sialic acids is colorimetry [3,6,12,237,262]. When carried out on non-purified samples, the influence of contaminants interfering with the assays has to be taken into account. Greatest problems are encountered when using cells or tissue extracts, as the level of contamination is inevitably high. Moreover, factors such as non-identical reactions of different sialic acids in the same assay and the non-specificity of the reactions for the sialic acids are important. Although several colorimetric methods have been developed in the past, only two main procedures are currently routinely applied, namely the orcinol/Fe3+/HC1assay, known as the “Bial” reaction, and the periodic acidhhiobarbituric acid assay. For microadaptations of these two different tests, see ref. [l 11. In the first assay, the sample is mixed with orcinol, FeC13 and concentrated HCl and heated at 96°C. The formed purple to red-violet chromophore is extracted with isoamyl alcohol and its absorbance measured at 572 nm. Because of the use of HCl, the method can be used to quantitate the total amount of both free and glycosidically bound sialic acids. Due to the strongly acidic conditions, ester groups are released. As the assay does not discriminate between bound and free sialic acids, it is widely used to monitor the presence of sialic acids in either form during fractionation of biological material. It should be noted that other monosaccharides, especially pentoses, hexoses and uronic acids interfere with the assay, which is of importance when small amounts of sialic acid are present. In the second assay, only suitable to quantitate free sialic acids, sialic acid is oxidized by periodate at 37°C under strongly acidic conditions. The oxidation leads to the formation of a prechromogen, a six carbon aldehyde, which then yields the chromogen fi-formyl pyruvic acid by aldol cleavage between C4 and C5. The chromogen reacts with thiobarbituric acid to give a red chromophore, the absorbance of which is measured at 549 and 532nm. In principle two approaches can be followed, called the Warren method and the Aminoff method. Differences between the methods lie in the acidity of the initial periodate oxidation and in the solvent used for the extraction of the pigment (cyclohexanone, Warren; acidic 1-butanol, Aminoff). It should be noted that substituents in the glycerol side chain severely influence the periodate oxidation. Therefore, in the case of ester substituents, a prior saponification is necessary (0.1 M NaOH, 37°C). Types of free sialic acid which do not yield the chromogen are negative in this test. Several compounds have been shown to interfere with the periodate/thiobarbituric acid assay, most especially 2-deoxyribose, 2-keto-3-deoxyaldonic acids other than Kdn, disaccharides such as lactose and maltose, and unsaturated fatty acids. The greatest errors arise in the quantitation of sialic acids from cellular extracts or homogenates containing membrane and nucleic acid material. Therefore, prior ion-exchange column chromatography and removal of lipids

269

by ether extraction are of advantage. Special attention to the periodic acidthiobarbituric acid assay of Kdn has been paid in ref. [243]. When both tests are used in combination, a differentiation between total and free sialic acid is possible, allowing the calculation of the amount of glycosidically bound sialic acid. In a new approach for the direct determination of free and bound sialic acid, an acidic ninhydrin assay has been proposed [263]. Heating of solutions of sialic-acid-containing material with ninhydridacetic acid/37% HCl at 100°C yields a stable chromophore, the absorbance of which can be measured at 470nm. In view of the comments made in section 5.1 with respect to the release of sialic acid by sialidases, a quantification procedure for bound sialic acid based on the enzymatic analysis of pyruvate, formed after sialidase/aldolase treatment [6,264], should be handled with care. Of the various fluorimetric assays available for sialic acid analysis, the method which allows the discrimination between sialic acids with or without 0-acyl groups at C8 and/or C9 may be of interest [6,265]. After mild periodate oxidation, formaldehyde, derived from C9 in the case of non-substituted H 0 9 and H 0 8 is derivatized with acetylacetone in the presence of ammonium acetate, leading to the fluorigen 3,Sdiacetyl1,4-dihydr0-2,6-dimethylpyridine (4 10 nm excitation, 5 10nm emission). It is evident that all contaminants producing formaldehyde under the influence of periodate will interfere with this sialic acid analysis. Finally, for the quantitative estimation of the 0-acyl content of sialic-acid-containing samples (Hestrin assay), also a colorimetric assay is routinely used. The method is based on the reaction with hydroxylamine in alkaline medium yielding hydroxamates, which form with Fe(C104)3 red chromophores, the absorbance of which is measured at 520 nm [262].

5.3. Chromatography 5.3.1. Thin-layer chromatography

From the beginning of free sialic acid analysis, TLC has played a major role in screening and tentative assignment procedures, and over the years several solvent systems for both cellulose and silicagel plates have been reported [6,237,262]. One of the most popular TLC methods for the analysis of (substituted) N-acylneuraminic acids comprises the use of plastic HPTLC plates precoated with cellulose and 1-propanol/l-butanol/O. 1 M HCl (2:1:1, v/v/v) as solvent system. It shows the best and most reproducible separation of different sialic acids, and is less sensitive to interfering substances when compared with other systems. Generally, the visualization of the different sialic acids is carried out by spraying with the orcinol/Fe3+/HC1reagent [237], yielding typical purple bands. For quantitative purposes densitometry is also used. It should be noted that due to differences in cellulose quality (impurities), even after pre-washing of the plates, the reproducibility of Rf values is relatively low. Therefore, analyses should be carried out in the presence of an appropriate reference sialic acid mixture on a separate lane. To give an impression of the separation capacity of cellulose plates, Table 7 summarizes the Rf values of a series

270 Table 7 Thin-layer chromatographic migration rates (Rf) of sialic acids on 0.1 mm cellulose plates using 1-propanol/lbutanol/O.l M HCI (2:1:1, v/v/v) [6] Compound Neu5Ac Neu4,5Ac2 Neu5,7Ac2 Neu5,9Ac2 Neu5,7,9Ac3 Neu5,8,9Ac3 Neu5,7,8,9Ac4 Neu5Ac9Lt

Rf

0.45

Compound

0.60 0.54 0.63 0.70 0.75

NeuZen5Ac Neu5Gc Neu4Ac5Gc Neu9Ac5Gc Neu7,9Ac25Gc Neu5GcAc

0.80

NeuZenSGc

Rf 0.55 0.35 0.65 0.55 0.70 0.51 0.45

0.56

of N, 0-acyl-neuraminic acids from one experiment [6]. In the case of radio-labelled sialic acids, the bands can also be traced by radio-TLC-scanning. Application of the TLC method in a two-dimensional procedure with intermediate ammonia treatment gives information about the type of N-acylneuraminic acid of the constituting NO-acylneuraminic acids. In this way a differentiation is possible for example between co-migrating Neu9AcSGc and Neu5,7Ac2 [6]. In principle, de-0-acetylations can also be carried out by sialate 0-acetylesterases [266]. For the analysis of oligomers of a-2,g-linked NeuSAc, NeuSGc or Kdn, a TLC procedure on silicagel with the solvent system 1-propanol/25% ammonia/water (12:2:5, v/v/v) has shown good results [267]. In this way, mixtures of {Neu5Ac(a2-8)}2 to {Ne~SAc(a2-8)}~4,{Ne~5Gc(a2-8)}~to {Neu5G~(a2-8)}~~, or (Kdn(a2-8)}2 to {Kdn(a2-8)}7 are well separated. It should be noted that (NeuSAc(a2-X)}, and {Neu5Gc(a2-8)}, can be visualized also with a resorcinol spray reagent, whereas for { Kdn(a2-8)}, the orcinol spray reagent is needed. 5.3.2. High-performance liquid chromatography Initially, the separation of sialic acids was mainly carried out by cellulose chromatography at low temperature [6,237]. However, nowadays HPLC fractionations using different column materials, elution protocols and detection techniques have replaced this approach [6,11,268]. The application of HPLC has also introduced a rapid method for tentative assignments of sialic acids in complex mixtures, based on elution times of known standards, being more reliable when more than one HPLC procedure is followed. Moreover, a rapid method for quantification of released sialic acids has become available. Due to the relatively short HPLC runs, also fast transitions between members of the sialic acid family due to migration of substituents, introduction of substituents, cleavage of substituents, or other (enzymatic) modifications can easily be monitored. First detailed reports on the separation of non-derivatized sialic acids deal with the application of Aminex A-28 or A-29 anion-exchange chromatography using 0.75 mM [269, 2701 or 0.5 mM [268] Na2S04 as eluting system and UV monitoring at 200 nm (nanomole range). In a different approach, fluorigenic derivatives of sialic acids, prepared by reaction

27 1

with 1,2-diamin0-4,5-methylenedioxybenzene(DMB) in the presence of 2-mercaptoethanol and sodium hydrogensulfite, have been separated by C reversed-phase HPLC [27 1-2731, using acetonitrile/methanol/water (9:7:84, v/v/v) as solvent system and fluorescence monitoring at 373 nm excitation and 448 nm emission wavelengths. An appropriate cutoff filter may be used instead. The fluorescence labelling makes a relatively specific and highly sensitive (femto- to picomole range) detection possible. However, using radiolabelled sialic acids, it was found that the derivatization reaction is not quantitative [268]. For an adapted protocol, see ref. [l 11. Another interesting approach is the conversion of NeuSAc/SGc into chemiluminescent quinoxalinone derivatives using 4,s-diaminophthalhydrazide dihydrochloride (a-keto acid derivatization) [274]. These derivatives are analyzed by reversed-phase HPLC (femtomole range), whereby the chemiluminescence detection follows the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) in alkaline solution. Other conversions of NeuSAc, useful for HPLC separations, include the derivatization with 4,4'-dicarboxy-2,2'-biquinoline [275], 2-cyanoacetamide [276], periodatethiobarbituric acid [277], benzoic anhydride [278], 4'-hydrazino-2-stilbazole [279] and 1,2-diamino-4,5-dimethoxybenzene[280]. Taking advantage of the separation capacity of the anion-exchange resin CarboPac PA- l, fractionation of non-derivatized sialic acids at neutral pH, using sodium acetate as eluent and pulsed amperometric detection (PAD) following postcolumn addition of alkali, has shown excellent results in terms of the wide array of sialic acids that can be separated, the sensitivity of the detection method (picomole range), and the relative ease of use for preparative work (without PAD detection) [268]. The general problem of quantification in PAD analyses, due to differences in detector response attributed to differences in the number of free hydroxyl groups of the various components separated, holds also for sialic acids. So far, only for a limited number of sialic acids relative detector response factors have been calculated (e.g. NeuSAc, 30 500; Neu5,9Ac2, 14 500; NeuSGc, 35 400). It is important to note that pH values > 11, as usually applied in CarboPac-PAD analyses of oligosaccharides (for such a NeuSAc/NeuSGc separation, see ref. [28 l]), will lead to rapid de-U-acylation of the 0-acylated sialic acids [282]. This phenomenon can even occur between the point of postcolumn alkali addition and the entry into the PAD detector [268]. In an evaluation of five different HPLC methods, it turned out that no single method is adequate to completely separate and quantitate complex mixtures of sialic acids [268], and the use of multi-dimensional HPLC is advised. As a clear illustration of this statement, Table 8 is included. This evaluation also compares a series of essential features of the five HPLC methods, namely, sensitivity, specificity of detection, separation by number of hydroxyl groups or substituents, separation of isomers, preparative use, avoiding of ester migration during purification, and avoiding of ester loss during purification. In this comparison [268,270,271,283,284] the HPLC systems I (CarboPac PA-I) and 111 (TSK-ODS 120T, DMB derivatives) gave the highest averaged scores in terms of applicability. A major advantage of HPLC system V (Aminex A-29) is the short running time, only 5 4 min, which makes this approach highly attractive for studying enzymatic conversions. In order to obtain information about the structure of sialic acids, HPLC is a very useful

272 Table 8 High-performance liquid chromatographic elution times of sialic acids in five different HPLC systems I to V, relative to Neu5Ac [268] Compound

la

IIb

Neu2Me Neul ,2Me2

0.17

0.13

0.40

0.85

IV

IIIC

.oo '

*

Ve

0.23 0.46 1 .oo

1 .oo 1.41

1.98

I .22 0.95 1.30 1.17 1.62

1.39

2.04

Neu5Ac

1 .oo

1 .oo

1 .OO/l

Neu4,5Ac2

0.76

0.39

1.6811.71

'

Neu5,7Ac2

0.74 0.95

0.36 0.35

1.06 1.5711.62

'

Neu5,7(8),9Ac3 Neu5,7,8,9Ac4

0.74

0.23

1.90

Neu5AcZMe

0.73

I .02

Neu4,8an5Ac

2.15

0.58

Neu2en5Ac

2.21

0.66

1.54

1.68

Neu5Gc

1.17

1.50

0.84/0.78'

1.20

1.33

Neu4Ac5Gc

0.86 1.06

0.54 0.49

1.53 1.30'

1.59 1.57

1.69

0.82 0.67 0.59

0.75 0.36 0.29

1.03/0.98 1.70 2.20

0.94 1.21

0.89

Neu5,9Ac2

Neu9AcSGc Neu5Gc8Me Neu9AcSGc8Me Neu7,9Ac25Gc8Me

0.62

0.73

Kdn Acetic acid

0.34

0.08

0.87 1.47 1.76 2.05 0.73

1.70

1.47

' I .70

System 1: Anion-exchange chromatography on CarboPac PA-I with PAD detection at room temperature; 5 mM sodium acetate for 5 min, then a linear gradient to 50% 5 mM sodium acetate/50% 5 mM acetic acid in 30 min; mixing of the column effluent with 300mM NaOH; running time 18-24 min [268]. System 11: Amine adsorptiodion suppression chromatography on Micropak AX-5 with UV monitoring (200 nm) at room temperature; acetonitrile/water/0.25 M sodium dihydrogenphosphate (72: I8:6, v/v/v); running time 21-24 min [283]. System 111: Reversed-phase chromatography on TSK-ODS 120T and fluorescence monitoring (373 nm excitation and 448 nm emission) at room temperature; DMB derivatives; acetonitrile/methanoI/water (9:7:84, v/v/v); running time 12-13 min [271]. System I V Anion-exchange chromatography on Aminex HPX-72s with UV monitoring (200 nm) at 40°C; 0.1 M sodium sulfate; running time 15-1 7 min [284]. System V Anion-exchange chromatography on Aminex A-29 with UV monitoring (200nm) at room temperature; 0.75 mM sodium sulfate; running time 5 4 min [36,270]. Taken from [ 1 I], RP-18 column. a

'

technique to be applied in combination with mild chemical or enzymatic degradation methods. For instance, HPLC before and after alkaline treatment of a mixture of 0-acylated sialic acids can give information about the de-0-acylated sialic acids present, e.g. in terms of their N-acyl substituents. Also the linkage specificity of sialidases in releasing sialic acids from sialoglycoconjugates (see section 5.1) can be monitored by HPLC. Furthermore, the specificity of enzymes involved in sialic acid metabolism can be studied in this way. In this respect, interesting results have been obtained

213

with aldolase, cleaving sialic acids to N-acylmannosamine derivatives and pyruvate, and with sialate 9-O-acetylesterase, hydrolyzing 0-acetyl groups from C9 of sialic acids. The aldolase degrades Neu5Ac faster than Neu5Gc; a slow degradation has been observed for 0-acylated sialic acids, not affecting 4-0-acetylated sialic acids at all [5]. A typical example of the HPLC analysis of enzyme reactions, in which esterase and aldolase are involved, and including the non-enzymatic conversion of an 0-acetyl group from C7 to C9, is presented in Fig. 5 [6,7,36]. Other examples are the determination of sialidase activity (sialyllactose as substrate, Neu5Ac as product, and Neu2en5Ac as inhibitor), CMP-Neu5Ac synthase activity (disappearance of NeuSAc, appearance of CMP-NeuSAc), CMP-Neu5Ac phosphodiesterase activity (appearance of NeuSAc, disappearance of CMP-Neu5Ac) [36]. Recently, 5-N-acetyl-9-0-acetyl2-(N-dansyl-4-aminophenylthio)-a-neuraminicacid has been proposed as a highly sensitive fluorescent substrate for the HPLC measurement of sialate 9-0-acetylesterase (334 nm excitation, 564 nm emission) [285]. As a thioglycoside, the compound is very stable in acidic aqueous solution and towards enzymatic hydrolysis by sialidases. In this context, it is also worthwhile mentioning that a sensitive HPLC assay has been developed for the tracing of sialyltransferase activity, making use of the synthetic fluorigenic acceptor lactose 2-[(2-pyridyl)amino]ethyl glycoside [286]. Details for a HPLC separation of CMP-NeuSAc, CMP-NeuSGc and CMP-Kdn on a DC-613 cation-exchange column are reported in ref. [ 1071. In addition to the fractionation procedures described for free sialic acids, several approaches have been reported for the separation of sialyl-oligomers. These compounds with a degree of polymerization up to 16 have been fractionated with varying results using conventional gel-filtration, TLC, DEAE-Sephadex A-25, and HPLC methods (see section 5.1 for preparation; see section 5.3.1 for TLC). A survey of literature has been included in ref. [287]. In general, mixtures of sialo-oligomers from (NeuSAc(a2-8)},, {NeuSAc(a2-9)},, {Neu5Gc(a2-8)},, or { Kdn(a2-8)}, can be isolated on a preparative scale via convential DEAE-Sephadex A-25 [256,257] or DEAEToyopearl 650M [267] anion-exchange chromatography. HPLC procedures comprise anion-exchange and adsorption-partition chromatography. A mixture of { NeuSAc(a28)}2-16 has been efficiently separated on a Zorbax SAX anion-exchange column using 0.2-1 M NaCl in lOmM phosphate buffer pH 3.5 [287]. Also adsorptionpartition chromatography on polystyrene DC-6 13 using mixtures of 0.02-0.025 M sodium phosphate buffer pH 7.4 and acetonitrile as solvent system, has shown good results [267]. On Mono Q anion-exchange columns, excellent results were obtained in the separation of { N e u S A ~ ( a 2 - 8 ) ) ~ -{Ne~5Gc(a2-8)}~_lo, ~~, or {Kdn(a2-8)}~_~after conversion into alditols with NaBH4 (or NaBT4), and using a NaCl gradient in Tris-HC1 buffer pH 8 as elution system [267,288]. In this context, several studies have focused on the determination of the chain lengths of sialo-oligomers and -polymers (for a review of methods currently employed in the analysis of polysialic acids, see ref. [289]), and recently a highly detailed adapted methodology for the analysis of a-2,8-linked sialooligomers and -polymers has appeared [290]. Using three variable assay procedures, providing overlapping information, details could be provided with respect to the degree of polymerization, the simultaneous identification of NeuSAc, Neu5Gc and Kdn when present in a single preparation, and the ability to distinguish qualitatively between

N

-4 P

0.03

lyase

-

__c

(C)

0.02

(i

-

0.01 -

z

8

16

8

16

8

16

Fig. 5. HPLC profiling (Aminex A-28, 0.75 mM NazSO,) of the chemo-enzymatic conversion of Neu5,7Acz into pyruvate and N-acetylD-mannosamine (ManNAc). (a) Intramolecular migration of the 0-acetyl group from C7 to C9 under slightly alkaline conditions, yielding Neu5,9Acz, accompanied by some de-0-acetylation; (b) enzymic release of the 0-acetyl group at C9 with the aid of sialate-9-0-acetylesterase, yielding Neu5Ac; (c) aldolase (1yase)-catalyzed degradation of NeuSAc, yielding pyruvate and ManNAc.

275

reducing and non-reducing polymers. The developed approach may include mild periodate oxidation (degradation of non-reducing terminal unit) in combination with reduction (degraded glycerol side chain yielding C7-sialic acid; reducing unit if present affording the corresponding stereoisomeric alditols), whereas monomer analysis is carried out after sialidase or acid hydrolysis on CarboPac PA-1 with pulsed amperometric detection. In the structural analysis of glycoprotein-derived N- and 0-linked sialic-acid-containing carbohydrate chains, fractionation procedures based on HPLC play a major role. As this aspect is outside the scope of this chapter, no details are included. Typical examples, making use of anion-exchange chromatography (Mono Q, CarboPac), and normal (e.g. Lichrosorb-NH2) or reversed-phase chromatography, are found in refs. [25,81,84,133,291-3001. In this context, it is also worth noting the recent use of highperformance capillary electrophoresis for the separation of glycoprotein-derived N-glycan chains [301] and 0-glycan chains [302].

5.3.3. Gas-liquid Chromatography combined with muss spectrometry As discussed in section 5.1, methanolysis of free and glycosidically bound sialic acids gives rise to the formation of methyl ester b- and a-methyl glycosides. Using the conditions of the standard quantitative monosaccharide analysis of glycoconjugates, de-N-acylatiodde-esterification takes place, which means that NO-acylneuraminic acid residues are converted into neuraminic acid methyl ester methyl glycoside (8, -96%; a, -4%). For the characterization by GLC the sialic acid methyl ester methyl glycoside is derivatized via N-acetylatiodtrimethylsilylation or pertrifluoroacetylation [241]. It should be noted that during the N-acetylation step H 0 9 of NeuSAc, when not substituted, is partially 0-acetylated (-4%). Using milder methanolysis conditions [242], an N-acetylation step is not necessary, yielding a method to determine both Neu5Ac and Neu5Gc by GLC. In principle, the latter approach is also suitable to determine sialic acids bearing only 0-alkyl groups. GLC analysis is generally carried out on SE-30 type column materials. Starting from free sialic acids (purified or as a pool), mainly present in their 8-anomeric forms, volatile sialic acid derivatives are generated using mild derivatization procedures such as esterification with diazomethane followed by trimethylsilylation [ 15,3031 or pertrimethylsilylation [ 11,3041. With respect to silylation cocktails, it should be noted that N-methyl-N-trimethylsilyl-2,2,2-trifluoroacetamide/pyridine leads to the formation of N-trimethylsilyl derivatives, yielding two different peaks for each sialic acid [36,305]. Subsequent GLC analysis is generally carried out on SE-30 or OV-17 type column materials. Both types of derivatives are highly suitable for MS analysis, and GLC coupled with electron impact (EI) MS formed the basis for the development of a highly reliable mass spectrometric method for the identification of sialic acids. Originally set up for the GLC-EI MS analysis of mixtures of NO-acylneuraminic acids [303], the method has also proved to be useful for the analysis of other naturally occurring sialic acids, of (partially) 0-methylated sialic acid methyl ester methyl glycosides as obtained in methylation analyses, and of synthetic sialic acid(s) (derivatives) [6,11,15]. In the following the principles of the EI MS identification procedure will be explained. Typical derivatives are trimethylsilylated methyl ester or pertrimethylsilylated derivatives of N, 0-acylneuraminic acids or of N-acyl-0-alkylneuraminic acids, acetylated N-acyl-

216

I

CHORg

I

CH2ORs

J cH=6Rs

I

CHZORg

I

R40

- CHOR~CHOR~CH~OR~ - NHRgRs' H

I

H

- RzOH - R40H

ICI

Fig. 6 . Survey of the selected fragment ions A-H worked out for the following derivatives: trimethylsilylated methyl ester or pertrimethylsilylated derivatives of NO-acylneuraminic acids or of N-acyl-0-alkylneuraminic acids, and trimethylsilylated/methylated N,N-acy1,methyl-neuraminic acid methyl ester methyl glycosides [ 151.

0-alkylneuraminic acid methyl ester methyl glycosides, trimethylsilylatedrnethylated N,N-acy1,methyl-neuraminic acid methyl ester methyl glycosides (methylation analysis), and acetylatedmethylated N,N-acy1,methyLneuraminic acid methyl ester methyl glycosides (methylation analysis). The determination of the type, number, and position of the 0-acyl or 0-alkyl groups as well as the type of the N-acyl group in neuraminic acids is facilitated by the highly specific EI mass spectra of the derivatized compounds. In Fig. 6, a schematic survey is depicted showing the selected fragment ions A-H, which furnish the information (abundances and mlz values of the ions) necessary to deduce the complete structure of the sialic acids. Fragments A and B indicate the molecular mass of the sialic acid derivatives and thereby the type and the number of substituents. Fragments C-H can be used for the determination of the positions of the different substituents. Fragment A is formed from the molecular ion by the elimination of a methyl group originating from a trimethylsilyl substituent in trimethylsilylated (0-acylated0-alkylated) N-acylneuraminic acid derivatives. When RSI = CH3 (methylation analysis), the eliminated methyl group can

277

also originate from the NN-acy1,methyl group. Fragment B is formed by elimination of the C l part of the molecule. Eliminations of OCOCH3 in 0-acylated sialic acid derivatives and of NH2COCH3 in N-acetylneuraminic acid derivatives, which in principle give rise to the same mlz value as fragment B in the case of R1 =CH3, can be neglected. For 0-trimethylsilylated N 0-acylneuraminic acids (B-anomers) it holds that, when compared to their methyl esters, in their trimethylsilyl esters the intensity of fragment A decreases relative to B. Fragment C is formed by elimination of C8-C9, with localization of the charge on position 7. In general, cleavage occurs between two alkoxylated carbon atoms, or between an acetoxylated and an alkoxylated carbon atom, rather than between two acetoxylated carbon atoms. In accordance with the fragmentation rules for partially methylated alditol acetates [306], the charge is preferentially located on an ether oxygen instead of on an ester oxygen. Therefore, fragment C has only significant abundance if C7 bears an ether group. When an ester group is present at C7, this fragment ion is absent or hardly observable. Fragment D is formed from fragment C by consecutive eliminations of R20H and R40H. It is evident that the occurrence of this fragment ion is dependent on the presence of fragment C. Fragment E is formed by elimination of the whole side-chain C7-C8-C9 and the substituent at C5. This fragment ion is not observed if an 0-acyl group is attached to C4, illustrating that the transition state in the McLafferty rearrangement is more favored when the substituent at C4 is an ether group rather than an ester group. For 0-trimethylsilylated N, 0-acylneuraminic acids (B-anomers) it holds that, when compared to their methyl esters, in their trimethylsilyl esters the intensity of fragment E is much reduced but still present; instead, an additional fragment derived from fragment E by loss of Me3SiOH is clearly present. Fragment F contains C8-C9. Based on the same fragmentation rules as mentioned above for fragments C and D, this ion can only readily be formed if an ether group is attached to C8. Fragment G consists of the C4-CS part of the molecule. Fragment H, necessary to use for derivatives containing only 0-alkyl substituents, is formed by elimination of the C9 part, followed by elimination of R4OH and R70H. For instance, this fragment is useful to discriminate between an OSiMe3 group at C8 or C9 in trimethylsilylated partially methylated N-acylneuraminic acids. Finally, for quadrupole analyzers, in the high mass range the fragment ions A, B and C often are of low intensity, especially when only small amounts of material are available. In Table 9 a survey is presented of GLC retention times and of characteristic EI MS fragment ions for a series of naturally occurring sialic acids, analyzed as their trimethylsilylated methyl ester or as their pertrimethylsilylated derivatives [6,11,15,3 1,38,43,307]. Although the sialic acids predominantly occur in the B-anomeric form, the a-anomer could occasionally be detected separately from the B-anomer, in most cases as a small shoulder. As a typical example, in Figs. 7a,b the EI mass spectra of the trimethylsilylated methyl ester of B-NeuSAc and of the pertrimethylsilylated derivative of P-NeuSAc, respectively, are depicted. Additional spectra have been published in refs. [ 15,43,304,307]. For a detailed survey of fragment ions of other derivatives mentioned, including mass spectra, and those obtained from periodate-oxidized sialic acids (C7-NeuSAc, C8-NeuSAc, C7-NeuSGc and C8-NeuSAc), see refs. [15,308]; for EI MS data of Neu4,8anSAc, see ref. [63]; for EI MS data of permethylated Kdn, see ref. [46]. In additional studies, the suitability of chemical ionization (CI) for the GLC-MS analysis of pertrimethylsilylated N, 0-acylneuraminic acids has been investigated. Isobu-

Table 9 GLC and characteristic EI MS fragment ions (70 eV) of (i) trimethylsilylated methyl ester (TM) and (ii) pertrimethylsilylated (PT) derivatives of naturally occurring sialic acids (o-anomers) a Sialic acid

RN~~sA~ TM

Neu5Ac

1.00

Neu4,5Ac2

1.18 1.04

Neu5,7Ac2 Neu5,8Ac2

1.05

Neu5,9Ac2 Neu4,5,9Ac3

1.13 1.31

Neu5,7,9Ac3 Neu5,8,9Ac3 Neu5,7,8,9Ac4

1.14 1.19 1.15

Neu5Ac9Lt

2.55

Neu4,5Ac29Lt Neu5Ac8Me

3.01

Neu5,9Ac28Me Neu2en5 Ac Neu2,7an5Ac Neu5Gc Neu4Ac5Gc Neu7Ac5Gc Neu9Ac5Gc Neu7,9Ac25Gc Neu8,9Ac25Gc

1.09

PT 1.00 1.05

Fragment (mlz) A

1.04

0.98 1 .oo 1.01

1.81 2.02 1.83

1.19

2.04 2.01 I .99

1.21

Reference(s)

D

E

G

F

TM

PT

TM

PT

TM

PT

TM

PT

TM

PT

TM

PT

TM

PT

668 638 638

726 696

624 594 594

624 594

478 448

536 506

298 298

356 356

317

375

173 143 173

[11,15]

-

205 205

173

-

143

[11,15]

638 1.02

C

B

594

638 608

696

594 564

594

608

666

564

608 578

666

740 710

798

-

317

478

298

317

-

478 448

536

564

-

564 534

564

478

696 666

696

668 638 578

-

205 205 205

298 298

356

-

-

-

536

298

356

478 448

536 536

566 536

636 564

-

756 726 726

814 784

712 682 682

712 682

726 696 696

784

682 652 652

682

-

298 298

388

462 566 536 566

298

624 594

386 386

356

173 143

173

317

375

175

175

173

317 317

375

-

-

173 173

173 173

[151 [6,15] [11,15]

317

375

277

173 143

173

[11,151 [15,307]

356 444 444

227

-

-

566

386

285

205

317

375

-

-

317 317 317

375

205 205

-

205 205 205

205 205

261 231 261

175 175

175

261 261 261

-

-

~ 5 1 [11,31] [11,31] [11,15]

173 261

[6,38] [11,15]

231

[6,15]

261

~ 5 1 [11,15]

173 173

147 117

-

317 444

277 277

315 375

-

386

-

-

-

624

175

356 356

446 374

-

175 175

317 -

536

375

~ 5 1 ~ 5 1 [11,15]

173

~ 5 1 u51 continued on next page

4 N W

Table 9, continued Sialic acid

Fragment ( m / z )

RNeuSAc

TM

PT

Neu7,8,9Ac35Gc Neu5Gc8Me Neu9Ac5Gc8Me Neu5GcAc Neu2en5Gc

1.93

B

A TM

PT

666

TM

D

C PT

TM

PT

-

622

Reference@)

TM

E PT

-

TM

F PT

TM

G PT

-

317

TM

PT

1.14 1.17

756 726

654 624

624 624

444 444

375 375

147 117

26 1 261 261

~ 5 1 [11,31] [11,31]

1.21

784

682

594

414

375

205

231

[43]

724

-

534

444

285

205

-

[61

The RNeuSAc values of the TM derivatives on 3.8% SE-30 at 215°C are given relative to the TM derivative of P-NeuSAc. The RNeuSAc values of the PT derivatives on CP-Sil 5 (capillary column), using the program 5 midl40"C; 2Wmin up to 220°C; 15 mid220"C, are given relative to the PT derivative of P-NeuSAc. For the preparation of TM derivatives, see 1151; for the preparation of PT derivatives, see [304]. For an explanation of the minus signs, see text. a

-4 N

W

280

100-

(A)

%

I

E

317

50-

n b 00

L.h 200

100

h .

3 00

LOO

A 668

B 62L

I

L

L

L

L

t I,

J

L

I

G 173

A 726

+--%+--

Fig. 7. (A) EI mass spectrum (70 eV) of the trimethylsilylated methyl ester of b-NeuSAc; (B) EI mass spectrum (70 eV) of the pertrimethylsilylated derivative of P-NeuSAc.

28 1

tane[304], as well as methane and ammonia[ll] were used as reactant gases. The CI mass spectra are characterized in the high mass range by [M+H]+ pseudomolecular ions, and typical major fragment ions derived from [M+H]+ by loss of R20H (fragment I), &OH (fragment I'), and RzOH+&OH (fragment 11). It was found that methane in particular gave CI spectra that also include several of the typical fragment ions observed in the EI spectra. In addition to GLC-MS, HPLC-CI MS with Aminex A-29 as column material and ammonium formate in water/acetonitrile as solvent system has been explored for the analysis of underivatized N , 0-acylneuraminic acids [6,309]. Although the positive-ion mass spectra allow the discrimination between different N-acylneuraminic acids (NeuSAc, NeuSGc) and the determination of the degree of 0-acetylation (Neu5,9Ac2, Neu5,7,9Ac3, Neu5,7,8,9Ac4), the position of the 0-acetyl groups (Neu4,5Ac2, Neu5,7Ac2, Neu5,9Ac2) could not be established. For the latter assignment, the combination with specific elution positions of standards on the HPLC column is advised. In the interpretation of the various fragment ions, the open-chain structure of the sialic acids has been generally used. NeuSAc and NeuSGc have also been converted into phosphatidylethanolamine dipalmitoyl derivatives, and after separation by HPTLC and subsequent isolation, the sialic acid derivatives were analyzed by liquid secondary ion MS [281]. In both cases intense [M - HI- ions together with sodium attachment ions were detected. For the detection of Neu5Ac on human tumor mucin, after liberation with sialidase, electrospray MS has been used [310]. 5.3.4. Fast atom bombardment mass spectrometry Free sialic acids, isolated after cleavage from glycoconjugate starting material, have been investigated, without derivatization, by FAB MS using 5% aqueous acetic acid solutions for loading into glycerol on the FAB target [4 I]. The positive and negative FAB mass spectra of each sialic acid showed clear [M+H]+ and [M-HI- pseudomolecular ions, respectively. Sialic acid mixture analysis (pg range) made the recognition of subgroups of sialic acids with the same molecular mass possible (e.g. NeuSAc(Ac)l, NeuSAc(Ac)z, NeuSAc(Ac)3, NeuSGc(Ac)3). However, a differentiation between positional isomers was not possible. Sialic acids were also studied after derivatization, which improves the sensitivity [4 I]. Direct peracylation failed to produce suitable derivatives, but reduction under acidic conditions followed by peracylation (perdeuteroacetylation or perpropionylation) gave good results. Generally, the sialic acids give rise to two major pseudomolecular ions, corresponding to the peracylated open chain form and an open-chain-derived lactone form, and a minor pseudomolecular ion corresponding to an open-chain-derived anhydrofonn (2,6 and/or 4,8). As the lactone peak is markedly reduced in the spectrum of Neu4,5Ac2, H 0 4 seems to be mainly involved in the lactonization. In the case of sialic acid mixtures, a fast sialic acid subgroup analysis based on molecular masses is possible; again, a differentiation between positional isomers cannot be achieved. Careful analysis of the negative FAB spectra of reduced and perpropionylated sialic acids in mixtures demonstrated that these spectra could also be used for quantitative purposes. As worked out for mixtures of NeuSAc, Neu5,9Ac2 and NeuSGc, an estimate of the relative amounts of these sialic acids can be given with an error of 1&I 5%, when the sum of the intensities

282

of the [M + HIf ions of the linear and the lactone forms of each component is compared, taking into account that the molar response of Neu5Gc is approximately 50% of that of Neu5Ac. In order to generate sialic-acid-derived compounds, which can be used to differentiate between positional isomers, use has been made of the rather difficult periodate oxidation under mild or more rigorous conditions [53,308]. Under both conditions, the resulting aldehyde groups were derivatized with p-amino-benzoic acid ethyl ester, reductively introduced at acidic pH without loss of the native 0-acetyl functions [41]. Sialic acids treated in this way were additionally reduced and peracylated, and then analyzed by FAB MS. Mixtures of products with different ring sizes (original, lactonized, anhydro) and/or open chain forms, depending on the substitution pattern, are often obtained. Of the mono-0-acetylated N-acylneuraminic acids Neu4,5Ac2, Neu5,7Ac2, Neu5,9Ac2, Neu4Ac5Gc and Neu9Ac5Gc were investigated, however, no attention was paid to the behavior of Neu5,8Ac2. Neu5,7(8),9Ac3 and Neu7,9Ac25Gc were also included in these investigations. Although not discussed in this chapter, FAB MS is widely used in the characterization of glycoprotein-derived N- and 0-linked sialic-acid-containing carbohydrate chains. Typical information can be found in refs. [311,312].

5.3.5. ' H NMR spectroscopy Since the introduction of high-resolution H NMR spectroscopy for the structural analysis of glycoprotein-derived glycans, a huge amount of NMR data have been generated, and highly detailed reviews on N-linked [76] and 0-linked [77] carbohydrate chains have appeared. The continuous expansion in the amount of data has made it necessary to develop computerized search programs, and, in connection with the still growing Complex Carbohydrate Structural Database (CARBBANK), attention has been paid to the development of a NMR-spectroscopic data base of carbohydrate structures, called SUGABASE [3 131. Sialic-acid-containing oligosaccharides/glycopeptides constitute a considerable majority of the glycoprotein glycans. In addition to the two reviews mentioned above, a specific review focusing on the NMR spectroscopy of sialic acids has also been published [ 161. Free as well as glycosidically bound sialic acid give rise to highly characteristic 'H NMR parameters. The 'H NMR spectra are generally recorded in D20, and because of the pH dependency of the proton chemical shifts, the spectral data are standardized at pD 6-7. The choice of the pH is also of importance in view of the earlier discussed de-0-acylation, 0-acyl migration, and autohydrolysis. As a typical example of a free sialic acid, in Fig. 8 the 500MHz 'H NMR spectrum of Neu5Ac in D20 at pD 7 is depicted. The spectrum shows a minor and a major set of protons, reflecting the subspectra of the a- and p-anomer of NeuSAc, respectively (a$ = 7:93), and especially the H3e,3a signals, resonating outside the bulk signal, can be used for the differentiation between both anomers. The effect of pH on the proton chemical shifts is clearly illustrated by the positions of the Neu5Ac H3e,3a resonances. At pD 1.4, the H3e and H3a signals of B-Neu5Ac resonate at S 2.313 and 6 1.880, respectively, whereas these values are 6 2.208 and 6 1.827 at pD 7. In the case of a-NeuSAc, at pD 1.4, the resonances are found at 6 2.718 and S 1.705,

'

SAC

H5

rSAc

HO

H7

I!

,

4.1

4.0

3.9

3.8

3.6

n30

3.5

2.2 ppm

2.1

2.0

Fig. 8. Resolutionenhanced 500MHz 'H NMR spectrum of Neu5Ac dissolved in D20recorded at pD 7 and 27°C.

1.9

1 .I

1.7

1.6

284

respectively, and at pD 7.0 at 6 2.730 and 6 1.621, respectively. Over the years, a large number of (naturally occurring) sialic acids and related derivatives have been analyzed, and in Table 10 a survey of the chemical shift values of a selected group of compounds, including naturally occurring sialic acids and CMP-sialic acids, is presented [16,31,38,51,64,107,308,314,315]. Inspection of these data reveals the typical influences of the substituents on the chemical shift values of the skeleton H-atoms. Comparison of the H3a,3e resonances of NeuSAc and NeuSGc indicate more downfield positions for those of NeuSGc (A6 M + 0.02). Additional data for NeuSAc, Neu5,7Ac2, Neu5,9Ac2, Neu5,7,9Ac3, and Neu5,8,9Aq in 0.1 M sodium phosphate/D20 at 37°C and pD 7-7.5 are reported in ref. [23]. The 'H NMR studies of free NO-acylneuraminic acids have shown that the anomeric equilibrium of a 7-0-acetylated sialic acid differs strongly from that of the sialic acids not substituted at C7. In contrast to the normal equilibrium values of a$ M 7:93, both Neu5,7Ac2 and Neu5,7,9Ac3 have an equilibrium ratio of about 23:77. Additional ' H NMR data of methyl glycosides and methyl ester methyl glycosides of N,0-acylneuraminic acids and some sialyllactoses can be found in ref. [16]. The 'H NMR data of the (methyl ester) methyl glycoside of (3-Kdn have been reported in ref. [46], and those of CMP-9amino-NeuSAc and CMP-9NAc-NeuSAc in ref. [316]. ' H NMR spectroscopy has shown to be an excellent method to monitor chemical and biochemical conversions of sialic acids, directly in the NMR-tube or by analysis of isolated reaction products. A typical example is the demonstration of the release of a-NeuSAc as the primary product of bacterial and viral sialidase action on Neu5Ac(a2-glycosides and oligosaccharides [3 17-3201. The initial formation of a-NeuSAc, as traced by 'H NMR spectroscopy, formed an excellent probe to investigate the kinetics of the mutarotation of NeuSAc by means of 'H NMR analysis in dependency of the pH [321]. At pD 5.4 the establishment of the equilibrium of mutarotation turned out to be rather slow, but at higher and lower pD values a more rapid establishment was observed, so that at pD 1.3 and pD 11.7 mutarotation was too fast to be measured. With the ability to generate a-NeuSAc in situ, the aldolase-catalyzed degradation of NeuSAc to pyruvate and N-acetylmannosamine (ManNAc) could also be investigated in more detail using ' H NMR spectroscopy [322]. Using sialidase (pH optimum 5.4) and aldolase acid (pH optimum 7.2) from C. perfringens and N-acetyl-2-azido-2-deoxy-a-neuraminic as substrate at pH 5.4, only released a-NeuSAc was found to be consumed by the aldolase, yielding specifically a-ManNAc followed by a fast mutarotation to a,P-ManNAc. These findings confirmed earlier work using Neu5Ac(a2-3)lactose as a-Neu5Ac-generating system and crystalline b-NeuSAc [319]. In the reversed reaction a-ManNAc is the substrate [322]. For more details about the aldolase-catalyzed degradation, see section 9.3. In connection with these studies, it has to be noted that under comparable conditions the activities of sialidase and aldolase in D20 are only about 50% of those in H20. With respect to 0-acetyl migrations, also the earlier mentioned (see section 2) spontaneous conversion at physiological pH of Neu5,7Ac2 into Neu5,9Ac2 and of Neu5,7,9Ac3 into Neu5,8,9Ac3 has been monitored by H NMR spectroscopy [23]. Furthermore, the NMR approach has shown its value in the determination of substrate specificities of various sialidases using substrates with differently linked sialic acid residues [3233251.

'

Table 10 'H Chemical shifts for different types of sialic acids. Chemical shifts are given in ppm relative to internal acetone in D 2 0 (6 2.225) at 300K, unless indicated otherwise Sialic acid

uD

Chemical shift H3a

H3e

H4

H5

H6

H7

H8

H9

H9'

Ref 4Ac

SAC

7Ac

8Ac, 8Me

B-NeuSAc

2

1.880

2.313

4.067

3.93 1

4.056

3.556

3.750

3.841

3.619

2.053

-

-

a-NeuSAc

2

1.705 2.718

n.d. a

3.85

3.684

3.53

3.75

3.85

3.62

2.036

-

-

B-NeuSAc

7

1.827 2.208

4.024

3.899

3.984

3.514

3.753

3.835

3.608

2.050

-

-

a-NeuSAc

7

1.621 2.730

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

2.030

-

-

P-Neu4,5Ac2

7

1.951

2.249

5.274

4.15

4.15

3.570

3.775

3.844

3.619

1.992

-

-

~-Neu5,7Ac2

4

1.905

2.236

3.950

3.767

4.246

5.045

3.91 1

3.629 3.444

a-Neu5,7Ac2

4

1.649 2.757

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

P-Neu5,9Ac2

7

1.833 2.221

4.024

3.913

3.99 1

3.571

3.977

4.365

a-Neu5,9Ac2

7

1.624 2.720

n.d.

n.d.

n.d.

n.d.

n.d.

fi-Neu5,7,9Ac3

2

1.924 2.303

3.978

3.775

4.293

5.162

a-Neu5,7,9Ac3

2

1.686

n.d.

n.d.

n.d.

n.d.

B-Neu5,8,9Ac3

7

1.838 2.189

3.978

3.903

3.780

3.838

0-NeuS, 8,9Ac3

2

1.862 2.250

Neu2enSAc

6

-

Neu2,7an5AcC

7

Neu4,8an5Acd

7

2.751

1.976b 2.144b

-

1.947b 2.12gb

-

4.187

2.057

-

-

n.d.

n.d.

n.d.

-

-

4.140

4.106

4.106

1.981b 2.134b

-

n.d.

n.d.

n.d.

1.956b n.d.b

-

5.114 4.528

4.287

2.057

-

2.089

4.006

3.912

3.830

3.866

5.1 15 4.545

4.287

2.059

-

2.091

5.690 4.470

4.051

4.213

3.601

3.936

3.885

3.646

2.068

-

-

2.167

2.007

3.953

3.919

4.543

4.434

3.537

3.592

3.755

2.035

-

-

2.983

2.844

4.188

4.333

3.852

3.496

3.363

3.814

3.734

2.041

-

-

2.143

1.723

n.d.

4.249

n.d.

3.439

3.268

3.690

n.d.

2.041

--

-

9Ac

5Gc

Table 10, continued Sialic acid

PD

Chemical shift H3a

P-Neu5Ac2P

7

H3e

1.548 2.403

CMP-P-Neu5Ac

8

CMP-P-NeuSAc9Ac

7

1.64

C8-P-Neu5Ac

6

1.814 2.203

1.639 2.484 2.48

Ref

H4

H5

H6

H7

H8

H9

H9'

4.093

3.888

4.239

3.386

4.028

3.883

3.581

4.066

3.92

4.141

3.456

3.92

3.90

3.622 n.d.

4Ac

5Ac

7Ac

8Ac, 8Me

9Ac

2.045

-

2.054

-

5Gc

-

[I61

-

[I61

n.d.

3.96

n.d.

3.51

4.07

n.d.

2.05

2.08

-

[314]

3.998

3.892

3.772

3.722

3.655'

-

2.055

-

-

[308]

-

-

2.044

-

-

I3081

[308]

3.586' C7-P-NeuSAc

6

1.821 2.238

3.992

3.710

3.800

3.671g 3.6349

C7-a-Neu5Ac

6

1.599 2.630

n.d.

n.d.

n.d.

n.d.

-

-

-

2.026

-

-

P-Neu5Gc

7

1.840 2.243

4.127

4.002

4.106

3.549

3.777

3.821

3.613

-

-

4.143

[16]

n.d.

n.d.

n.d.

n.d.

n.d.

-

4.12

[16]

a-Neu5Gc

7

1.644 2.749

n.d.

n.d.

P-Neu9AcSGc

7

1.842 2.234

4.14

4.006

4.109

3.570

3.970

4.365

4.183

-

2.115

4.144

[I61

a-Neu9Ac5Gc

7

1.649 2.751

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

-

n.d.

4.123

[I61

P-NeuSGc8Me

7

1.863 2.219

4.017

3.559

3.432

3.932

3.652

3.425

-

4.133

[31]

a-Neu5GcSMe

7

1.643 2.55 1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

-

4.125

[31]

3.60

-

-

4.11

[314]

-

4.144

[308]

4.1 10 3.983 n.d.

n.d.

CMP-P-Neu5Gc

7

1.64

2.48

3.92

4.03

n.d.

3.42

3.60

3.87

C8-P-Neu5Gc

6

1.832 2.218

4.097

3.976

3.887

3.709

3.657'

-

3.589' continued on next page

Table 10, continued ~

Sialic acid

Chemical shift

PD H3a

C8-a-NeuSGc C7-P-Neu5Gc

6 6

Ref.

H3e

H4

H5

H6

H7

H8

H9

H9'

4Ac

5Ac

7Ac

8Ac, 8Me

9Ac

5Gc

1.622 2.665

n.d.

n.d.

n.d.

n.d.

n.d.

-

-

-

-

-

-

-

4.129

[308]

-

-

-

-

-

-

-

-

4.133

[308]

-

-

-

-

-

-

-

-

4.116

I3081

-

-

-

1.840 2.253

4.096

3.798

3.906

3.6749 3.6329

C7-a-NeuSGc

6

n.d.

2.650

n.d.

n.d.

n.d.

n.d.

6-Kdn

7

1.837 2.265

4.015

3.603

3.995

3.881

3.772

3.883

3.675

-

-

-

[I061

o-Kdn9Ac

7

1.768 2.158

3.94

3.575

3.94

3.91

3.91

4.376

4.238

-

-

-

-

2.124

-

[51]

CMP-P-Kdnh

7

1.568 2.379

3.968

3.538

4.046

3.863

3.896

3.731

3.618

-

-

-

-

-

-

[I071

n.d., not determined. On one line values may have to be interchanged. ' Sialic acid occurs in 5C2 conformation. Sialic acid occurs in 'C4 conformation and is present in two tautomeric forms; chemical shifts are relative to HOD at 6 4.750; H3a= H3 and H3e = H3'. Values are assigned relative to the HOD signal at 6 4.81 at 296 K.

a

H8 and H8'. H7 and H7'. Personal communication Y. Inoue; values are assigned relative to 2,2-dimethyl-2-silapentane-5-sulphonatein D 2 0 (set to 0 ppm). Note that in CMP-6-Kdn the H8 and H9 signals have been interchanged when compared with ref. [3 151.

g

N m 4

288 Table 11 'H-Chemical shifts for the H3e and H3a signals of sialic acids as part of N- and 0-linked glycoprotein glycans. Chemical shifts are given in ppm relative to internal acetone in D 2 0 (6 2.225) at 300 K, or relative to internal 2,2-dimethyl-2-silapentane-5-sulphonate in D 2 0 set to 0 ppm (marked ') Structural element

H3a

H3e

NeuSAc(a2-3)Gal(fiI -

1.78-1.81

2.75-2.78

Neu4,5Ac2(a2-3)Gal(P 1-

1.93

2.77

Neu5,9Ac2(a2-3)Gal(fi1 Neu5Gc(a2-3)Ga1((31-

1.80

2.76

1.81-1.82

2.77-2.79

Kdn(a2-3)Gal(fi14)GlcNAc(flIKdn(a2-3)Gal(fi1-3)GalNAc(al-3)GalNAc-ol Neu5Ac(a2-3)[Gal(fil-4)]Gal(fi1NeuSAc(a2-3)[GalNAc(fi 14)]Gal(fl1 -

1.75-1 .76a

2.11-2.72

1.72a

2.69a

1.82a 1.92-1.94 1.70-1.72

2.73 a 2.66-2.68 2.67

Gal(~l-3)[Neu5Ac(a24)]GlcNAc(fil-2)Man(a1-

1.85 1.71 1.73-1.74 1.71-1.72

2.68 2.67 2.69-2.70 2.73-2.74

Gal(fi1-3)[Neu5Ac(a24)]GlcNAc(fi 1-4)Man(a 1NeuSAc(a24)GalNAc(fi 1 -

1.76-1.77 1.70-1.72

2.72-2.73 2.66

Neu5Gc(a2-3)[GalNAc(fil-4)]GalNAc(fi1-

1.85-1.86

Kdn(a2-3)[GalNAc(fiI 4)]GalNAc(Bl-

2.55-2.56 2.48 a

~~~

~

Neu5Ac(a2-6)Gal(flINeu4,5Ac2(a2-6)Gal(fi 1Neu5,9Ac2(a2-6)Gal(fi1 NeuSGc(a24)GalbI -

Neu5Ac(a24)GalNAc-ol

1.79a 1.69-1.71

Neu5Gc(a24)GalNAc-oI

1.71-1.72

2.74-2.75

Kdn(a2-6)GalNAc-01

1.64-1.66

2.66-2.68

a

2.72-2.74

Another interesting pH phenomenon, for the first time demonstrated by 'H NMR spectroscopy, is the complete replacement of H3a by a D-atom, when free Neu5Ac is kept in an alkaline D20 solution at pD 9.0 [326]. In fact, H3a can be exchanged in the pH range 6.5-9.0, and the H-D exchange is reversible. In the 'H NMR spectrum the H3a signal disappears, and the coupling patterns of H3e and H4 alter. At pD 12.4, H3e can also be replaced by a D-atom [327]. On the basis of this finding, the exchange experiments were also carried out in T20, yielding T-labelled NeuSAc, which was converted enzymatically into T-labelled CMP-Neu5Ac [326]. In glycosidically linked NeuSAc the H3 atoms are not exchangeable, rendering this specific labelling technique suitable for sialyltransferase experiments. In the H NMR structural-reporter-group concept, developed for the structural analysis of glycoprotein N- and 0-glycans, advantage is taken of the fact that a number of the H-atoms of the constituting monosaccharides, pccurring in special microenvironments, resonate outside the bulk-signal region [76,77,328]. In the case of glycosidically linked a-sialic acids the structural reporters are the H3a- and H3e-atoms, and the N-acetyl or N-glycolyl groups. The positions of the H3a,3e signals reflect

289

not only structural information with respect to the type of sialic acid present, but also with respect to the coupled monosaccharide in terms of type of linkage and type of monosaccharide. Furthermore, 0-acyl substituents induce additional shifts for other H-atoms. In Table 11 a survey is presented of chemical shifts of H3e and H3a signals of sialic acid residues occurring in different linkage types as part of N- and 0-glycans. For a further fine-tuning of the chemical shifts within the presented ranges, influenced by the different microenvironments wherein the sialic acid residues occur, see refs. [76,77] and the references cited in Table 2. It should be noted that the presence of a certain sialic acid in a certain linkage also influences the structural-reportergroup signals of other monosaccharide residues [76,77]. More detailed information with respect to the H3e,3a chemical shifts of sialic acids in sialo-oligomers and -polymers can be obtained from refs. [31,47,77,329,330]. A series of H3a,3e signals of sialic acids in milk- and glycolipid-derived oligosaccharides have been included in ref. [77]. More general NMR data of glycosidically linked sialic acids in glycolipids, milk and urinary oligosaccharides and (lipo)polysaccharides, if available, can be found in the references cited in Tables 3-5 (see also ref. [331]). For a series of sialocarbohydrates, it has been shown that H6 of NeuSAc, easily traced from the TOCSY H3a,H6 correlation, also has potential value for discriminating between a-2,3- (6 3.63 & 0.01 1) and a-2,6- (6 3.70 0.017) linked NeuSAc in NeuSAc(a2-3/6)Gal(~l-O)R/NeuSAc(a26)GalNAc(al-O)Ser/Thr [33 13. However, for branched oligosaccharides, this rule is not valid if the monosaccharide in the branching position is in the alditol form [332]. The H3a,3e/NAc structural reporters of Neu5Ac have proved to be suitable in studying sialylation reactions in terms of positional specificity and branching (N-linked carbohydrate chains) specificity, using different sialyltransferases and CMP-Neu5Ac as donor [333-3351 (see also section 6.3). In more biophysical studies, several aspects of sialic acids have been investigated by NMR spectroscopy. Although so far mainly a-forms of bound sialic acid have been detected, good differentiation systems for a- and p-forms are essential, and a number of empirical rules have been reported [336]. In a heteronuclear 2D-approach it could be demonstrated that the determination of the geminal C,H coupling constant 2J(C2,H3a) offers a unique criterion for the anomeric assignment in sialic acid glycosides (a, -8 Hz; p, -3 to -4Hz) [337]. Also the values of the vicinal C,H coupling constants 3J(C1,H3a) can be applied for this differentiation (a, -6 Hz; 0, -1 Hz) [55,336,338]. More details with respect to anomeric determinations have been reviewed in ref. [339]. 13C NMR data of sialic acids and glycoprotein-derived sialocarbohydrates have been reviewed in ref. [ 161. In several sialic-acid-related investigations, e.g. synthetic studies, I3C NMR spectroscopy forms part of the analysis techniques, and will not be reviewed in detail. For some additional data, more directly related with the glycoprotein glycan character of this chapter, see refs. [48,63,64,103,105,152,33 1,332,337,340-3431,

*

6. Chemo-enzymatic highlights in sialic acid chemistvy During the last ten years, activity in sialic acid chemistry has grown exponentially. Synthetic as well as biosynthetic routes for the preparation of sialic acids, sialic acid

290

derivatives, analogues, glycosides and sialoglycoconjugates have been explored. The main reason for this considerable interest in preparing sialic acids and sialic-acidcontaining compounds lies in the fact that sialic acids were found to be among the most biologically important carbohydrate units in glycoconjugates. The progress in organic synthetic protocols and the availability of relevant enzymes in suitable amounts made it realistic to develop the sialic acid field from a preparative synthetic side. Initially, the main tools were to prepare suitable derivatives to study the properties of sialic acids, or to prepare substrates and inhibitors for sialidases, sialyltransferases or for sialic acid converting enzymes. Although these tools are still highly relevant, the preparation of sialo-oligosaccharides using strictly organic synthetic or enzymatic methods, or a mixture of both, are also receiving considerable attention. For relevant reviews on preparative (bio)synthetic aspects, see refs. [339,344-3531. 6.1. Free sialic acids

Several protocols have been followed for the organic chemical synthesis of NeuSAc. At first, the approaches were based on condensation reactions of (derivatives of) N-acetylD-mannosamine (ManNAc) or N-acetyl-D-glucosamine (GlcNAc) with (derivatives of) oxaloacetic acid (for a review, see ref. [3]), however, the yields were very low. In this context, a procedure was worked out, that allowed modifications at Cl-C3 [354]. A total synthesis of NeuSAc from non-carbohydrate precursors has been reported in ref. [355]. Using a protocol for indium-mediated allylations of aldehydes, NeuSAc was prepared in good yields from a ManNAc precursor [356]. Furthermore, synthetic routes for NeuSAc have been proposed, based upon 1-deoxy- 1-nitro-sugar chemistry, that should also allow the preparation of NeuSAc analogues, modified at several carbon atoms of the skeleton [357-3591. A separate route also yielded NeuSAc [360]. A synthesis starting with the aldol condensation of D-glucose (Glc) and oxaloacetic acid, followed by adaptation of the substituent at C5 has been described in ref. [361]. More recently, another approach for the organic synthesis of NeuSAc and NeuSAc derivatives, based on the cis-selective Wittig reaction of benzoyl 2,3-O-isopropylidene-a-~-lyxo-pentodialdo1,4-furanoside with [(3S)-3,4-(isopropylidenedioxy)butyl]-triphenylphosphonium iodide as a first step, has been reported in ref. [362]. Comments on the acetylation of NeuSAc and its methyl ester have been published in ref. [363]. Starting from NeuSAc, in some derivatization reactions 1,4- as well as 1,7-lactone formation has been observed [349]. For the organic synthesis of Kdn, several routes were employed [364-3681, among them procedures starting from NeuSAc or from D-mannOSe (Man). NeuSAc-aldolase-catalyzed condensations of ManNAc and pyruvate [3,33], initially only investigated to understand sialic acid metabolism, have been optimized for preparative purposes. In principle, ManNAc can be generated from the cheaper GlcNAc in an alkaline epimerization process, yielding an epimeric mixture of which only the monosaccharide with the D-manno-configuration is recognized by the aldolase [369,370]. However, ManNAc can also be generated from GlcNAc in a GlcNAc-epimerasecatalyzed isomerization [37 I]. A multigram-scale enzymatic synthesis based on the aldol condensation of ManNAc and pyruvate in the presence of phosphate, catalyzed by immobilized microbial NeuSAc-aldolase, has been reported [372]. A similar approach,

29 1

using the aldolase enclosed in a dialysis membrane instead of being immobilized, has also been described [373]. Although in uiuo, the conversion of Neu5Ac into Neu5Gc occurs exclusively on the level of activated sialic acid (see section 8.4.1), Neu5Gc can be prepared in uitro by incubating a mixture of N-glycolyl-D-mannosaminelN-glycolylD-glucosamine and pyruvate with immobilized aldolase [374]. Interestingly, a series of other sugars also turned out to be accepted by the aldolase, and Man and 2-deoxyD-glucose in particular are excellent substrates [372,375-3771. In the case of Man as starting material, relatively moderate amounts of Kdn have been prepared [254,376]. A series of free 0-acetylated sialic acids, i.e., Neu4,5Ac2, Neu5,9Ac2, Neu4,5,9Ac3, and Neu5,7,8,9Ac4, together with the benzyl ester a-glycosides of Neu5,7Ac2 and Neu5,7,9Ac3, have been synthesized by organic synthetic routes using protecting group techniques [378,379]. Partially 0-acetylated sialic acid derivatives have also been prepared using more simple synthetic routes. @-Neu5,9Ac21,2Me2, fi-Neu4,5,9Ac31,2Me2, and @-Neu4,5,8,9Ac41,2Me2 were obtained from @-NeuSAcl,2Me2 by using N-acetylimidazole [53]. To realize 9-O-acetylations, also other acetylating reagents were applied, such as trimethyl orthoacetate [3 80,38 I], acetyl chloride [378], and dimethylacetamide dimethyl acetal[382]. In a recent comprehensive study, in particularly the use of trimethyl orthoacetate and dimethylacetamide dimethyl acetal was explored using the 4-aminophenylthio, 4-nitrophenylthio, and 4-nitrophenyl glycosides of COOH-esterified a-Neu5Ac as acceptors, and depending on the acetylating reagent a range of partially 0-acetylated derivatives could be generated [57]. One of the naturally occurring 0-acetylated sialic acids, Neu5,9Ac~,has also been synthesized in an enzymatic way [383,384] on a gram-scale [372]. After the enzymatic acetylation of 0 6 of ManNAc, using isopropenyl acetate and protease N as a catalyst, 2-N-acetyl-6-O-acetyI-~-mannosamine was condensed with pyruvate as catalyzed by the aldolase. These two enzymatic steps turned out to be highly regio- and stereoselective. Following another route, Neu5,9Ac2 has been synthesized enzymatically by incubating Neu5Ac with trichloroethyl acetate in pyridine using porcine pancreas lipase as a catalyst [385]. An enzymatic synthesis of Neu5Ac9Lt has also been worked out [370,384]. For the study of biochemical pathways, several isotopically labelled sialic acids and sialic acid derivatives have been prepared, both by enzyme-catalyzed synthesis and by organic synthesis. A survey of labelled sialic acids is presented in Table 12 [107,344,386]. In enzymatic procedures, use is generally made of the aldolase-catalyzed condensation of N-acybmannosamines and (phosphoenol)pyruvate, suitably labelled in one or both of the two synthons. For the preparation of N-[l-'4C]acetyl- and N - [ I-'4C]glycolylneuraminic acid, as well as O-['4C]acetylated sialic acids, surviving slices of submaxillary salivary glands incubated with [ 1-I4C]acetate, followed by isolation of the glycoprotein fraction and mild acid hydrolysis, have been used. A number of these labelled sialic acids have been converted into their CMP-glycosides, and subsequently incorporated into glycoconjugates (see section 6.3). Of course, labelling of glycoconjugates can also be carried out by periodate oxidatiodtritiated borohydride reduction, thereby converting sialic acids, if chemically possible, into their radiolabelled C7 and C8 analogues. Using the latter approach, fluorescent probes (dansylhydrazine, dansylethylenediamine, fluoresceinamine) and EPR spin labels can also be incorporated (see references cited in ref. [344]). The same holds for glycine [387].

292 Table 12 Survey of radiolabelled sialic acidsa Sialic acid

Reference

N-[3H]Acetylneuraminic acid N - [ 1-'4C]Acetylneuraminic acid N-A~etyl-[3-~H]neuraminicacid N-Acetyl-[9-) Hlneuraminic acid N-Acetyl-[ l-14C]neuraminicacid

N-A~etyl-[4-'~C]neurarninic acid N-A~etyl-[2-~~C,9-~H]neuraminic acid

S-N-AcetyI-9-azido-9-deoxy-[ 1-14C]neuraminicacid 5-N-A~etyl-4-O-methyI-[3-~ Hlneuraminic acid 5-N-[1-'4C]Acetyl-2-deoxy-2,3-didehydro-neuraminicacid

N-[l-14C]Glycolylneuraminic acid N-Glycolyl-[ 1-14C]neuraminicacid

N-Gly~olyl-[2-~~C,9-~H]neuraminic acid [14C]-2-Keto-3-deoxynononic acid a

For specific references, see [344].

Several interesting sialic acid variants and sialic acid derivatives have been synthesized, and a list is presented in Table 13 [3 16,344,347,349,357-359,368,370,376-380,384,3884461. A number of these compounds have been surveyed in ref. [3SO]. Both organic synthetic and aldolase-catalyzed routes have been followed. The major part of these compounds were prepared to study sialic acid metabolism (aldolase, CMP-NeuSAc synthase), sialic acid transfer (sialyltransferases), sialic acid release (sialidases), inhibition phenomena, or hemagglutinin-sialic acid interactions, and biological details are presented in sections 8-10. Compounds reported up to 1982 have been reviewed earlier [344]. Of special interest are the fluorescent and photoactivatable sialic acid derivatives [390,4 191, which can be applied, after conversion into their corresponding CMP-glycosides, to detect enzyme activities or to follow biological processes (see sections 6.2 and 8.2). In the context of sialic acid variants, the following compounds are also of interest. In view of the similarity in acidity of a tetrazoie group and a carboxyl function, a variant of Neu5Ac has been prepared containing a CN4H instead of a COOH group [447]. Also the synthesis of a series of Neu5Ac derivatives with specifically introduced tert-butyldimethylsilyl groups have been reported [400,448]. Furthermore, variants of 2d-2Ha-NeuSAc and 2d-2He,NeuSAc, in which the carboxyl function has been replaced by a phosphono (PO3H2) group [449], and a phosphonic acid analogue of Neu2en5Ac [450], have been synthesized. In addition to 6-amino-2,6-dideoxy-sialic acids, as mentioned in Table 13, the preparation of 2-C-hydroxymethyl derivatives [45 I], and C6 and C7 analogues [452] have also been reported.

Table 13 List of sialic acids, prepared along organic chemical or aldolase-catalyzed routes for use in biochemical studiesa Compound

Abbreviation

N-Acetylneuraminic acid

NeuSAc

5-N-Acetyl-2-deoxy-2,3-didehydro-neuraminic acid

NedenSAc

2-Deoxy-2,3-didehydro-neuraminicacid

Neu2en

N-Glycolylneuraminic acid

Neu5Gc

2-Deoxy-2,3-didehydro-5-N-glycolyl-neuraminic acid

Ne3enSGc

5-Azido-neuraminic acid

NeuSN,

5-Azido-2-deoxy-2,3-didehydro-neuraminic acid

NeuZenSN,

2-Keto-3-deoxynononic acid

Kdn

2,3-Didehydro-2,3-d~deoxy-~-g~cero-~-ga~acfo-non-2-ulopyr~oson~c acid

Kdn2en

N-Acetyl-[ 1-’3C]neuraminicacid

[ l-”C]NeuSAc

N-A~etyl-[3-~H]neuraminic acid

[3-*H]NeuSAc

N-A~etyl-[6-~H]neuraminic acid

[6-2H]Neu5Ac

Reference@)

N-Aminoacetyl-neuraminic acid

NeuSAcNH2

N-Thioacetyl-neuraminic acid

NeuSAcSH

5-N-Acetyl-2,7-anhydro-neuraminic acid

Neu2,7an5Ac

5-N-Acetyl-2-deoxy-2-Hax-neuraminic acid

2d-2Ha,-Neu5Ac

[392-3941

5-N-Acetyl-2-deoxy-2-HCq -neuraminic acid 5-N-Acetyl-2-deoxy-2-Heq-4-oxo-neuraminic acid

2d-2HCq-Neu5Ac

[393-3951

2d-2Heq-40x0-Neu5Ac

W I

5-N-Acetyl-2-deoxy-4-epi-neuraminicacid

2d-4epi-NeuSAc

5-N-AcetyI-2-deoxy-7-epi-2-H,,-neuraminicacid

2d-7epi-2HCq-Neu5Ac

[3971 [394,398]

5-N-Acetyl-2-deoxy-8-epi-2-Heq-neuraminic acid 5-N-Acetyl-2-deoxy-7,8-diepi-2-H,,-neuraminic acid

2d-8epi-2Heq-Neu5Ac

[394,398]

2d-7,8epi2-2Heq-Neu5Ac

[394,398] continued on next page

N

rg

w

N

W P

Table 13, continued Compound

Abbreviation

Reference(s)

5-N-Acetyl-3-fluoro-neuraminic acid

3F,, -Neu5Ac

[344,392]

5-N-Acetyl-3-hydroxy-neuraminic acid

30Heq-Neu5Ac

[344,399]

5-N-Acetyl-4-0-acetyl-neuraminic acid

Neu4,5AcZ

[378,380]

5-N-Acetyl-4-O-acetyl-4-epi-P-neuraminic acid methyl ester methyl glycoside

4epi-PNeu4,5Ac21 ,2Me2

5-N-Acetyl-4-deoxy-neuraminicacid

4d-Neu5Ac

[4001 [358,4014031

5-N-Acetyl-4-deoxy-4-iodo-neuraminic acid

41-Neu5Ac

[4041

5-N-Acetyl-4-deoxy-4-(R)-C-methyl-neuraminic acid

[4051

5-N-Acetyl-4-deoxy-4-(S)-C-methyl-neuraminic acid

[d051 [357,4061

5-N-Acetyl-4-epi-neuraminicacid

4epi-Neu5Ac

5-N-Acetyl-4-epi-4-0-methyl-neuraminic acid ethyl ester

4epi-Neu5Ac 1Et4Me

5-N-Acetyl-4-0-methyl-~-neuraminic acid (ethyl esterkthyl glycoside)

Neu5Ac4Me

[3441 [344,407]

5-N-Acetyl-4-0x0-neuraminic acid (methyl P-glycoside or ethyl a-glycoside)

40x0-Neu5Ac

[408,409]

5-N-Acetyl-7-deoxy-neuraminic acid

7d-Neu5Ac

[377,410]

5-N-Acetyl-7-epi-neuraminic acid 5-N-Acetyl-7-0-methyl-neuraminic acid

7epi-Neu5Ac

[411,412]

Neu5Ac7Me

P771

5-N-Acetyl-7-0x0-fi-neuraminic acid methyl glycoside

7oxo-fiNeu5Ac2Me

[4081

5-N-Acetyl-8-deoxy-neuraminicacid

8d-Neu5Ac

5-N-Acetyl-8-epi-neuraminicacid

8epi-Neu5Ac

[4 101 [411,412]

5-N-Acetyl-8-0-methyl-neuraminic acid

Neu5AcBMe

5-N-Acetyl-8-0x0-neuraminicacid methyl a- and P-glycoside

8oxo-Neu5Ac2Me

W41 [394,408,413]

5-N-Acetyl-9-0-acetyl-neuraminicacid

Neu5,9Acz

see text

5-N-Acetyl-9-S-acetyl-9-thio-neuraminic acid

[4141 continued on next page

Table 13, continued Compound

Abbreviation

Reference(s) ~~

S-N-Acetyl-9-amino-9-deoxy-neuraminic acid

9amino-NeuSAc

acid (methyl a-glycoside) S-N-Acetyl-9-azido-9-deoxy-neuraminic

9azido-NeuS Ac

S-N-Acetyl-9-(4-azidobenzamido)-9-deoxy-ne~uaminic acid S-N-Acetyl-9-(4-azidosalicylamido)-9-deoxy-neuraminic acid 5-N-Acetyl-9-benzamido-9-deoxy-neuraminic acid

9NBz-NeuSAc

5-N-Acetyl-9-(4-benzoylbenzamido)-9-deoxy-neuraminic acid

5-N-Acetyl-9-cyano-9-deoxy-a-neuraminic acid benzyl glycoside S-N-Acetyl-9-O-(N-dansylglycyl)-neuraminicacid 5-N-Acetyl-9-deoxy-neuraminic acid (methyla-glycoside)

9cyano-aNeuSAcZBn

5-N-Acetyl-9-deoxy-9-(3-fluoresceinylthioureido)-neuraminic acid

9d-NeuSAc 9fluoresceinyl-NeuSAc

S-N-Acetyl-9-deoxy-9-fluoro-neuraminic acid

9F-NeuSAc

S-N-Acetyl-9-deoxy-9-iodo-neuraminic acid

91-NeuSAc

S-N-Acetyl-9-deoxy-9-thioacetamido-neuraminic acid 5-N-Acetyl-9-O-(dimethylphosphinyl)-neuraminicacid 5-N-Acetyl-9-O-glycy1-neuraminic acid methyl ester

NeuSAc9GlylMe

S-N-Acetyl-9-hexanoylamido-9-deoxy-neuraminic acid

5-N-Acetyl-9-0-lactyl-neuraminicacid 5-N-Acetyl-9-0-methyl-neuraminic acid

9NHx-NeuSAc NeuSAc9Lt NeuSAc9Me

5-N-Acetyl-9-0-phosphoro-neuraminic acid

NeuSAc9P

~~

~

[316,390, 415 4 171 [316,384,390, 417,4181 [4191 [4191 [316,390,415, 4171 [4191 13941 [4201 [394,410,421] [390,415,422, 4231 [384,4244261 [344,427] [390,3911 [3W 13441 [316,390,4171 [370,384] [3471 [3441 continued on next page

W N

N m W

Table 13, continued Compound

Abbreviation

5-N-Acetyl-9-thio-neuraminic acid

5-N-Acetyl-4,9-di-U-acetyl-neuraminic acid

Neu4,5,9Ac3

5-N-Acetyl-2,7-dideoxy-2-Heq-neuraminic acid

2,7dz-2Heq-Neu5Ac 2,8dz-2Heq-Neu5Ac 4,7dz-Neu5Ac 7,9dz-Neu5Ac 7,8epi2-Neu5Ac

5-N-Acetyl-2,b-dideoxy-2-Heq-neuraminic acid

5-N-Acetyl-4,7-dideoxy-neuraminic acid 5-N-Acetyl-7,9-dideoxy-neuraminic acid 5-N-Acetyl-7,b-diepi-neuraminic acid (methyl a-glycoside)

5-N-Acetyl-7,7-dimethoxy-P-neurarninic acid methyl glycoside

5-N-Acetyl-4,7,9-trideoxy-neuraminic acid

5-N-Acetyl-4-azido-2,3-didehydro-2,4-dideoxy-neuraminic acid 5-N-Acetyl-9-S-(4-azido-2-nitrophenyl)-2,3-didehydro-2,9-dideoxy-9-thio-neuraminic acid 5-N-Acetyl-2-deoxy-2,3 -didehydro-4-epi-neuraminicacid (methyl ester) 5-N-Acetyl-2-deoxy-2,3-didehydro-4-oxo-neuraminic acid 5-N-Acetyl-2-deoxy-2,3 -didehydro-6-thio-neuraminicacid 5-N-Acetyl-2-deoxy-2,3-didehydro-7-epi-neuraminic acid 5-N-Acetyl-2-deoxy-2,3-didehydro-8-epi-neuraminic acid

5-N-Acetyl-2-deoxy-2,3-didehydro-7,8-diepi-neura~nic acid

[4101

[4211 [394,411,412]

Neu5,7,8,9Ac4 4,7,9d3-Neu5Ac

[3791 [4211

4amino-Neu2en5Ac 4azido-Neu2enSAc

~4281 [428430] [430,431]

5-N-Acetyl-4-allylamino-2,3-didehydro-2,4-dideoxy-neuraminicacid 5-N-Acetyl-4-amino-2,3-didehydro-2,4-dideoxy-neuraminic acid

[4141 P781 [394,398] [394,398]

[4081 [394,408,413]

5-N-Acetyl-8,8-dimethoxy-neuraminic acid methyl a-and P-glycoside 5-N-Acetyl-7,8,9-tri-U-acetyl-neuraminic acid

Reference(s)

4epi-NeuZenSAc 4oxo-Ne3en5Ac

14321 [344,433,434] [409,433,435] [4361

7epi-Neu2enSAc bepi-Neu2enSAc 7,8epi2-Neu2en5Ac

[3501 [4371 [3501

5-N-Acetyl-2-deoxy-2,3-didehydro-8,8-dimethoxy-neuraminic acid continued on next page

Table 13,continued Compound

Abbreviation

Reference(s)

5-N-Acctyl-2,3-didehydro-2,4-didcoxy-4-dimcthylamino-neuram~nic acid 5-N-Acetyl-2,3didehydro-2,4-dideoxy-4-gu~idinyl-neuraminic acid

4guanidino-Neu2enjAc

[428,429]

[4281

5-N-Acetyl-4-(N-hydroxy-N-allylamino)-2,3-didehydro-2,4-dideoxy-neuraminic acid

[4281

5-N-Acctyl-4-( 2-hydroxycthylamino)-2,3-dide~~dro-2,4-dideoxy-neuraminic acid

[4281 [4381 P501

5-N-acetyl-2,3-didchydro-2,4-didcoxy-ncuraminic acid 5-N-acetyl-2,3-didehydro-2,8-dideoxy-neuraminic acid 5-N-acetyl-2,3 didehydro-2,9-dideoxy-neuraminic acid

4d-NeuZenSAc 7d-Neu2enSAc 8d-NedenSAc 9d-Neu2en5Ac

5-N-acetyl-2,3didehydro-2,4,7-trideoxy-neuraminic acid

4,7d2-Neu2en5Ac

5-N-acetyl-2,3-didehydro-2,7-dideoxy-neuraminic acid

4-Acetamido-5-N-acetyl-2,3-didehydro-2,4-dideoxy-neuraminic acid

13501 [350]

13501 14281

5-N-Acetyl-6-amino-2-dcoxy-2-H,,-neuraminicacid 5-N-Ace~yl-6-amino-2-deoxy-2-H,,-4-epi-neuraminiti atiid 5-N-Acetyl-6-amin0-2-deoxy-2-H,~-4-epi-neuraminic acid 5-N-Acetyl-6-thio-neuraminic acid 5-N-Acetyl-5-epi-6-thio-neuraminic acid 5-N-Acetyl-4,5-diepi-6-thio-neurarninic acid 9-U-Acetyl-5-N-glycolyl-neuraminic acid

Neu9Ac5Gc

4-Acetamido-5-N-aetyl-4-deoxy-neuraminic acid

4NAc-Neu5Ac

4-Acelamido-3,4-~deoxy-n-g~~ero-~-~~~u~f~-non-2-ulopyranosonic acid

iso-Neu4Ac 2d-2F,-3Fq-Neu5Ac

5-Acetamido-2,6-anhydro-3,5-dideoxy-2,3-dIfluoro-~-urubino-~-~u~o-nononic acid 5-Acetamido-2,6-anhydro-3,5-dideoxy-4-C-met~yl-~-e~~~~o-~-u~~~o-nononic acid 5-Acetamido-2,6-anhydro-3,5-dideoxy-4-C-methyl-~-erythro-~-munno-nononic acid

conrinued on next page

W N 4

N W

Table 13,continued Compound

Abbreviation

Reference(s)

5-Acetamido-2,6-anhydro-4-C-methyl-3,4,5-tndeoxy-D-eryfhro-L-u~fro-nononic acid

W I

5-Acetamido-2,6-anhydro-4-C-methyl-3,4,5-trideoxy-~-eryfhro-~-mu~~o-nononic acid

t3961 [3961 [442,443]

5-Acetam~do-2,6-anhydro-l-C-methylene-3,4,5-tndeoxy-~-glycero-~-gulacfo-nonon~c acid 5-Acetamido-2,5-dideoxy-2,3-difluoro-~-eryfhro-~-gZuco-non-2-ulopyranosonic acid 5-C-Acetamidomethyl-5-deamino-neuraminic acid

9-Acetamido-5-N-acetyl-9-deoxy-neuraminic acid

9NAc-NeuS Ac

[4441 [3 16,390,415, 4171

5-N-Glycolyl-9-0-phosphoro-neuraminic acid

Neu5Gc9P

[W

5-Bromo-3,5-d~deoxy-~-glycero-~-gulucfo-non-2-ulopyranoson~c acid

5Br-Kdn 5epi-Kdn 4epi-Kdn 5epi-5F-Kdn 5d-Kdn 7d-Kdn 7epi-7,9F2-Kdn

3-Deoxy-D-g~cero-D-gu~o-non-2-u~opyranosonic acid 3-Deoxy-D-g~cero-D-fa~o-non-2-u~opyranosonic acid 3,5-Dideoxy-5-~uoro-D-g~cero-D-gu1o-non-2-u~opyra~0sonic acid 3,5-Dideoxy-~-g~cero-~-ga~acfo-non-~-u~opyranosonic acid

3,7-Dideoxy-D-g~cero-D-gu~acfo-non-2-ulopyranosonic acid 3,7,9-Tndeoxy-7,9-difluoro-D-glycero-~-alfro-non-2-ulopyranosonic acid a

For literature references before 1982 and additional lists of sialic acid(?.)

(derivatives), see ref.[344].

[376,384,445]

299

6.2. Glycosides of sialic acids The organic synthesis of a long series of alkyl and aryl a-glycosides of N-acylneuraminic acids has been previously reported (for reviews, see refs. [339,344]). One of the famous condensation reactions (classical Koenigs-Knorr method) comprised the silver carbonate-promoted condensation of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy(3-neuraminic acid or the corresponding methyl ester (Fig. 9, structure A) with the appropriate alcohol, followed by removal of the protecting groups. In order to improve the yields, much attention has been paid to better catalysts. In these glycosidation reactions typical side reactions are the formation of unsaturated sialic acid derivatives (elimination of HCl) and of (3-glycosides(see section 6.3). For the preparation of simple (3-glycosides, N-acylneuraminic acids are often heated with the appropriate alcohol in the presence of an acid catalyst, followed by saponification of the formed ester. However, complex alcohols give rise to problems (for a review, see ref. [344]). A mild and efficient Raney nickel-catalyzed deuteration procedure has been reported for Neu5Ac glycosides, with a rate of exchange at C8 > C9 > C7 >> C4 [453]. Attention has also been paid to the synthesis of N-, S- and Se-glycosides, which are sialidase stable [339,454,455]. Specific S-glycosides are used as sialic acid donors in sialoglycoconjugate organic synthesis (see section 6.3). Early examples are the syntheses of the 4-nitrophenyl N- and S-glycosides of a-Neu5Ac [456]. Of special interest are the syntheses of 5-N-acetyl-2-azido-2-deoxy-a- and p-neuraminic acids [322,457,458]. The azides can readily be converted into the corresponding 2-amino derivatives, and used in e.g. N-acylation reactions [459]. For a preparation of the 6-thioanalogue of 2azido-a-NeuSAc, see ref. [436]. In further investigations, a series of S-glycosides of a-Neu5Ac was synthesized (thiophenyl, 4-nitrothiophenyl, 4-aminothiophenyl, 2-mercaptopyridyl), starting from 5-N-acetyl-4,7,8,9-tetra-O-acetyl2-chloro-2-deoxy-~-neuraminic acid methyl ester and using triethylbenzylammonium chloride as a phase transfer catalyst 114541. These compounds turned out to be effective sialidase inhibitors. For the detection of sialidase activity both (naturally occurring) oligosaccharides and simple a-glycosides are used. In these assays, two approaches can be followed, namely, determination of the released (modified) sialic acid or identification of the released aglycon. In the case of a-glycosides, in which the released aglycon concentration is measured spectrophotometrically or detected on solid supports, substrates with synthetically introduced aglycons having specific chromogenic properties, are used. Among such substrates, released aglycons can be detected directly or after condensation with specific reagents. One of the oldest substrates is the 4-nitrophenyl glycoside of a-NeuSAc [456], whereby released 4-nitrophenol is estimated by absorption at 400nm. In an adapted synthetic version, the compound has been prepared by coupling of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-(3-neuraminic acid methyl ester with sodium-nitrophenoxide in N,N-dimethylformamide, and subsequent deprotection [460]. Another suitable substrate is the 3-methoxyphenyl glycoside of a-NeuSAc, synthesized by coupling of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2deoxy-(3-neuraminic acid with 3-methoxyphenol in the presence of silver carbonate, followed by de-0-acetylation. Liberated 3-methoxyphenol is determined after coupling

300 X

OAc

I

(A)

1

AcOH z AcHN OAc X = OAc. F, CI, Br Y = H, Me, Bn. All

ACOH 2CW AGO C

O

O

M

e

AcoH 2 A ccd

w

c

o

AcHN

AcHN OAc

o

M

e

OH OAc

X=F; Y=SePh X=Br; Y=SPh X=CI: Y = S P h X = F ; Y=SPh Z = Me or Bn I

0-P(

COOMe

OAc

('3

1

Acd AcHN

AcHN

OAc

OAc

X Et, Bn Y = Me. Bn

Y = Me, Ph, C-OEt

II

S

COOMe

OAc

I

1

COOMe Acd AcHN

SPh

OAc AcHN OAc

Fig. 9. Frequently used NeuSAc donors in the organic synthesis of sialo-oligosaccharides.

ox

ox

301

with the diazonium salt of 4-amino-2,5-dimethoxy-4'-nitroazobenzene(red colored product) [461] or with 4-aminoantipyrine in the presence of the oxidizing agent potassium ferricyanide (colored quinone) [462]. The most popular fluorigenic substrate is the 4-methylumbelliferyl glycoside of a-NeuSAc, which is prepared by different methods [344]. A convenient synthesis is the condensation of 5-N-acetyl-4,7,8,9-tetraO-acetyl-2-chloro-2-deoxy-~-neuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in N,N-dimethylformamide, followed by deprotection [463]. Released 4-methylumbelliferone is measured at 360 nm (excitation)/440 nm (emission). Although the 4-methylumbelliferyl glycoside of a-Kdn has been synthesized starting from Neu5Ac [445], also a direct route using the glycosyl chloride of peracetylated b-Kdn methyl ester and the sodium salt of 4-methylumbelliferone has been explored [254]. In addition, several 4-methylumbelliferyl a-glycosides of sialic acid variants and substituted sialic acids, including partial 0-acetylated ones, have been synthesized (e.g. refs. [252,350,399,445,464]. To develop a sensitive assay for the analysis of the linkage specificity of bacterial and viral sialidases, Neu5Ac(a2-3)- and NeuSAc(a2-6)Gal(P 1O)C6H4N02 were synthesized enzymatically by using a-2,3- and a-2,6-sialyltransferase, respectively, CMP-NeuSAc (see section 6.3), and p-nitrophenyl-P-Galp [465]; after cleavage of NeuSAc, p-nitrophenol can be released by additional treatment with b-galactosidase. For the localization of sialidase on electropherograms or for histochemistry, the chromogenic 5-bromo-indol-3-yl glycoside of a-NeuSAc has been synthesized by coupling of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-~-neuraminic acid methyl ester with 1-acetyl-5-bromo-3-hydroxyindole,and subsequent deprotection [466]. The unstable intermediate 5-bromo-indoxyl, released by sialidase, is readily transformed into insoluble blue-green 5,Si-dibromo-indigo, which marks the sites of enzyme activity. To facilitate the screening of bacterial colonies or plaques for sialidase activity, the 5-bromo-4-chloro-indo1-3-yl glycoside variant has also been synthesized [467]. Using the same sialic acid synthon as starting product, the 4-azido-2-nitrophenyl S-glycoside of a-Neu5Ac has been prepared, which is a potential photoaffinity probe reagent for the screening of sialidases in tissues and the purification of sialic-acid-binding proteins [468]. The sialidase-resistant thioglycosyl linkage also makes the incorporation of 35 S possible. In order to detect sialate 9-0-acetylesterase activity, a highly sensitive fluorescent substrate, 5-N-acetyl-9-O-acetyl-2-[4-(dansylamino)phenylthio]-a-neuraminic acid, has been synthesized (see also sections 5.3.2 and 9.1) [285]. The regioselective acetylation at 0 9 of the dansylated S-glycoside was carried out with trimethyl orthoacetate. Other useful fluorescent substrates for sialate 0-acetylesterase assays comprise 5-N-acetyl-7,8,9-tri-O-acetyl-2-[4-(dansylamino)phenylthio]-a-neuraminic acid and 5-N-acetyl-4-O-acetyl-2-[4-(dansylamino)phenylthio]-a-neuraminic acid [469], and the 4-[3-(fluoresceinyl)thioureido]phenyl S-glycoside of a-Neu5,9Ac2 [57]. In all cases the fluorescent groups have been coupled to the glycosidic 4-aminophenylthio group of 0-acetylated NeuSAc derivatives. In connection with the generation of a monoclonal antibody to free Neu5Ac for the purpose of establishing a simple and specific assay of NeuSAc in serum and urine, a broad series of sialic acid a- and P-glycosides have been synthesized using substituted glycerol, substituted sphinganine and cholesterol as aglycons [470].

3 02 NaOH/H20

GlcNAc

pyruvate

ManNAc

Neu5Ac

C Neu5Ac aldolase

CTP

pyruvate

CMP-P-Neu5Ac

PEP

Fig. 10. One-pot synthesis of CMP-6-NeuSAc from ManNAc and CMP [473]. PEP, phosphoenolpyruvate; PPi, pyrophosphate.

The synthesis of CMP-sialic acids is generally carried out enzymatically using CTP and CMP-sialic acid synthase as a catalyst [33,3 14,370,47 1,4721. A multigram-scale one-pot synthesis of CMP-@-NeuSAchas been reported in ref. [473]. ManNAc, prepared by basecatalyzed epimerization of GlcNAc, was reacted with sodium pyruvate in the presence of NeuSAc-aldolase to yield NeuSAc (see section 6.1). For the formation of CMPNeuSAc, CTP was generated in situ from CMP by using adenylate kinase, pyruvate kinase, and phosphoenolpyruvate, and reacted with NeuSAc in the presence of CMPNeuSAc synthase (Fig. 10). Instead of a one-pot synthesis, for practical reasons it is easier to generate and store crude solutions of NeuSAc and CTP. For the use of GlcNAc in combination with GlcNAc-epimerase, see ref. [474]. Experiments with cloned CMPNeuSAc synthases from E. coli systems with NeuSAc and Kdn showed a high specificity for NeuSAc, thereby suggesting that in this case the 5-acetamido group is critical [384]. Chemical syntheses of CMP-NeuSAc, applying the phosphoramidite method [475] or using sialyl phosphites [476], have also been described. Furthermore, a synthetic approach for the preparation of CMP-NeuSGc based on the phosphite method has appeared [477]. In addition to CMP-p-NeuSAc, CMP-@-Neu5,9Ac2,CMP-b-NeuSGc, and CMP-8-Kdn, a large series of artificial CMP-sialic acids have been prepared biochemically on microscale starting from the corresponding sialic acid (see references cited in Table 13) and CTP. Among them are CMP-9azido-NeuSAc, CMP-9amino-NeuSAc, CMP9NAc-NeuSAc and other C9-modified CMP-sialic acids, CMP-NeuSAcNH2, CMPNeuSAc4Me, and CMP-4d-NeuSAc [33,3 14,350,390,407,419,4781. The CMP-sialic acids have found a broad application in enzymatic sialylations using different sialyltransferases (see sections 6.3 and 8.3). Several of the artificial CMP-sialic acids turned out to be suitable donors for asialo-a, -acid glycoprotein as acceptor with Gal(@lH)GlcNAc a-2,6-sialyltransferase from rat liver as a biocatalyst [3 16,402,4171. The transfer of CMP-9amino-NeuSAc is of considerable interest, as a-linked 9amino-NeuSAc in sialoglycoconjugates is not a substrate for bacterial, viral or mammalian sialidases tested so far. CMP-9amino-NeuSAc and CMP-NeuSAcNHz have also been used as synthons

303

to prepare fluorescent and photoactivatable analogues [419]. Because of the defined acceptor specificity, sialyltransferases in combination with fluorescent or photoactivatable donor CMP-sialic acids are excellent tools for selective introduction of a fluorescent or photoactivatable substituent to a distinct glycoconjugate. The latter reference [4 191 also includes kinetic data and information concerning the fluorimetric sialyltransferase assay. Typical fluorescent products comprise the CMP-sialic acids of S-N-acetyl-9-deoxy-9(3-fluoresceinylthioureido)-neuraminicacid (CMP-9fluoresceinyl-Neu5Ac), 5-N-acetyl9-(7-amino-4-methylcoumarinyl)acetamido-9-deoxy-neuraminic acid (CMP-9AMCANeuSAc), 5-N-acetyl-9-deoxy-9-(fluoresceinylaminomonochlorotriazinyl)amino-neuraminic acid (CMP-~MTAF-N~USAC), and N-(3-fluoresceinylthioureido-acetyl)neuraminic acid (CMP-NeuSfluoresceinyl). In the preparation of photoactivatable derivatives the NH29 group of CMP-9amino-NeuSAc has been substituted with a 4-azidobenzoyl, a 4-azidosalicyl, a 4-benzoylbenzoyl or a 4-azido[T]benzoyl group. In a similar way, CMP-NeuSAcNH2 has been labeled with a 4-azidobenzoyl group. Of special interest is the recently reported chemical synthesis of CMP-{NeuSAc(a2-8)NeuSAc} [479]; an attempt to prepare this compound biosynthetically with NeuSAc(a2-8)NeuSAc and CMP-sialic acid synthase failed so far [472]. In addition to the preparation of regular CMP-sialic acids, synthetic approaches have been worked out for the organic synthesis of a S-(N-acetylneuraminy1)nucleoside analogue [480,48 11 and other CMP-sialic acid variants [482]. For the immobilization of sialic acids on Sepharose solid supports, which provides potentially useful affinity materials, see the references cited in ref. [344]. The preparation of an affinity adsorbent with immobilized sialic acid through a thioglycosidic linkage has been described in ref. [483]. Synthetic sialidase-stable a-Neu5,9Ac2 p-aminophenylthio glycoside has been immobilized directly or by a six-carbon long spacer group to agarose for lectin isolations [382]. The allyl glycoside of a-NeuSAc has been applied as a starting material for the synthesis of NeuSAc-neoglycoproteins and pseudopolysaccharides. These polymers containing multivalent sialic acid are in principle useful for various applications related with recognitionhindinglinhibition processes. Reductive ozonolysis of the allyl then MezS), followed by coupling of the formed aldehyde to protein carriers group (03, (E-aminogroup of lysine) by sodium cyanoborohydride-mediated reductive amination, yielded neoglycoproteins with varying amounts of NeuSAc [484,485]. Copolymerization of the allyl glycoside with acrylamide generated a water-soluble pseudopolysaccharide [484]. In order to create a longer spacer arm for copolymerization with acrylamide, the allyl glycoside was converted into a 3-(2-aminoethylthio)propyl glycoside by reaction with cysteamine hydrochloride, after which the amino function was N-acryloylated [486]. The same principle of conjugation or copolymerization via N-acryloyl groups was also used for the preparation of sialo-oligosaccharide-neoglycoproteins and copolymers of sialo-oligosaccharides and acrylamide [487]. Using the strategy of reductive amination, p-formylphenyl glycoside of a-NeuSAc was also conjugated with proteins [488], and starting from the p-nitrophenyl 0- and S-glycosides, p-N-acryloylamino analogues were synthesized, which could be copolymerized with acrylamide, yielding water-soluble pseudopolysaccharides with Neu5Ac and acrylamide in different ratios [489], or directly coupled with polylysine [490]. Using trimethyl orthoacetate, the NeuSAc residue of the

3 04

S-glycoside-containing polymer was converted into Neu5,9Ac2 [489]. Finally, a series of interesting NeuSAc and Neu5,9Ac2-based dendrimers have been synthesized [49 11.

6.3. Sialo-oligosaccharides The organic synthesis of oligosaccharides having terminal a-linked sialic acid has proved to be highly complex. The specific difficulties arise from three factors inherent in the sialic acid molecule. First, the carboxylic acid function at the anomeric center (C2) electronically disfavors oxonium ion formation. Secondly, from a steric point of view, the carboxyl function restricts the glycoside formation. Thirdly, the presence of a neighboring methylene group in the ring (C3), instead of a substituted carbon atom, eliminates the possible assisting andor directing effect of an adjacent substituent [346]. This means that side reactions can be relatively important, mainly the thermodynamically favored fi-glycoside and 2,3-dehydro-derivative formation, and low yields are quite often obtained. Initially, the synthesized glycosidic linkages comprised mainly NeuSAc(a26)Gal(fi 1-, NeuSAc(a24)GlcNAc((31-, NeuSAc(a2-6)Glc(fll-, NeuSAc(a2-3)Gal((3 1-, NeuSAc(a2-3)GlcNAc(fl1- [344], and over the years these glycosidic linkages, together with NeuSAc(a24)GalNAc(al-, still receive most of the attention. In view of the desire to prepare biologically relevant carbohydrate chains, this is understandable. In Fig. 9 a series of typical NeuSAc donors, introduced by different research groups with the aim of increasing both the glycosidation yield and the a-stereoselectivity, is depicted [339,34&348]. For each class of donors, some further information is presented in the following paragraphs. The oldest approach of synthesizing sialo-oligosaccharides is the one starting from 2-deoxy-2-halo-fi-NeuSAc derivatives (Fig. 9, structure A). Methyl S-acetamido-4,7,8,9tetra-O-acetyl-2-ch~oro-2,3,5-t~deoxy-~-g~cero-fi-~-ga~acto-non-2-u~opyranosonate, X= C1, Y = Me, turned out to be a particularly useful donor [492], and typical promoters are silver and mercury salts. Due to poor stereoselectivity, a$-glycoside mixtures are generally obtained, and HC1-elimination from the donor is a major side reaction. The reaction with secondary hydroxy groups in particular gave rise to problems. In the case of the aim to prepare NeuSGc-containing oligosaccharides, also the N-glycolyl group in the donor analogue is 0-acetylated [493]. For the synthesis of NeuSAc(a2-9)NeuSAq see ref. [494]. In another approach, a series of 3-substituted NeuSAc donors was prepared, starting from peracetylated [495] or perbenzylated [496,497] Neu2enSAc methyl ester, thereby making use of the highly reactive 2,3-double bond to form adducts (Fig. 9, structures B-F) (see also ref. [498]). In the case of structure B as donor with silver triflate as a promoter, only fi-glycosidic linkages were created, and among several products, the NeuSAc(P2-8)NeuSAc linkage was synthesized [495]. Structure C yielded mainly (3-glycosidic linkages. From structure D, only the bromo variant is effective, although a$-glycoside mixtures are still formed. The bromo variant of structure D with silver triflate as a promoter has been applied in the synthesis of NeuSAc(a2-8)NeuSAc and NeuSAc(a2-9)NeuSAc linkages [499,500]. Structures E and F form another series of donors, and E with X = Br and Y = SPh (mercury salts as promoter) has been shown to give particularly high glycosidation yields and a-stereoselectivity.

305

A third type of donor involves the use of S-methyl or S-phenyl a-glysosides (Fig. 9, structure G) [498,501-5031. Initially developed to synthesize S-glycosides, making use of sodium salts of the peracetylated Neu5Ac methyl ester a- or 8-thioglycosides and suitable protected bromides [504-5061 (see also refs. [507,508]), this type of donors has shown to be highly attractive in 0-glycosidation reactions. In these couplings, frequently used promoters are dimethyl(methy1thio)sulfonium triflate or N-iodosuccinimideltriflic acid [347]. The choice of the solvent system is very important, as it greatly influences the stereoselectivity; e.g. acetonitrile gives mainly a-glycosidation. In addition to reports dealing with the synthesis of many monosialo-oligosaccharides, including those with a NeuSAc(a2-2)Glc, a NeuSAc(a2-3)GlcNAc, and a NeuSAc(a2-3)GalNAc sequence [509], typical examples are the creation of NeuSAc(2-9)NeuSAc [5 10,5 1 11 and NeuSAc(a2-8)NeuSAc [512] linkages, as well as sialyl LeX sequences (native and variants) (ref. [513] and references cited therein). In this context, it is also interesting to note that several syntheses of sialo-oligosaccharides include the use of a separately prepared disaccharide donor with a terminal a-linked sialic acid [514]. Although in general the donors contain a N-acetyl group at C5, other examples have been reported with the phthaloyl (benzeneselenenyl triflate as a promoter [5 151) or the tert-butoxycarbonyl function as N-protecting group, e.g. in the case of the synthesis of Neu-containing glycoconjugates [5 16,5171. Following similar routes, Kdn-containing oligosaccharides have also been synthesized [5 181. As a variation on this theme, the application of S-sialyl xanthates (SCSOEt) as donors in sialo-oligosaccharide synthesis has led to interesting results, including a high a-stereoselectivity [5 19-5221. Here, also the use of 0-benzoyl protection instead of 0-acetyl protection has been proposed [523,524]. Furthermore, the preparation of sialic acid S-glycosyl donors employing S,S’-bis( 1-phenyl- 1H-tetrazol-5y1)dithiocarbonate should be mentioned [525]. Another efficient donor combines parts of the structures F and G, yielding an a-thioglycoside with a SPh substituent at C3 (Fig. 9, structure H) [526]. The high stereoselective a-sialylation was obtained using either methyl sulfenyl bromide/silver triflate or N-iodosuccinimide/triflic acid as promoters. Sialyl phosphites with trimethylsilyl triflate as a promoter have additionally been shown to be of practical use (Fig. 9, structure I), affording good yields and a-stereoselectivity [527-5291, and examples include the synthesis of sialyl LeX sequences [528]. For a detailed study on the evaluation of different sialyl phosphites, see ref. [530]. For the organic synthesis of sialo-oligosaccharides with di- or trimeric Neu5Ac elements, specific glycosyl donors have been prepared directly from NeuSAc(a28)NeuSAc or Neu5Ac(a2-8)Neu5Ac(a2-8)Neu5Ac [53 1-5331. Treatment of the free oligosaccharides with H+-resin in methanol, followed by 0-acetylation and subsequent replacement of the anomeric acetoxy group by a phenylthio function yielded the corresponding peracetylated methyl ester phenyl 2-thioglycosides, in which Neu5Ac residues are linked via a (a2-8,l-9) lactone ring (Fig. 9, structure J for a disialosyl donor). For additional data with respect to the preparation of dimeric donors with structure A at the reducing site, see ref. [534]. In terms of preparative chemistry, the use of CMP-8-Neu5Ac as a glycosyl donor and (cloned) a-2,3/6-sialyltransferasesas biocatalysts have achieved a permanent position in the planning of synthetic routes for sialo-oligosaccharide chains; see e.g. refs. [3 14,342, 345,351,472,473,535-5441. Especially in the field of the preparation of sialyl LeX frag-

306

PEP Neu5Ac(a2-3)Gal(P1-4)[Fuc(al-3)]GlcNAc(P1-O)R GDP-Man

GDP-FUC

NADP

Neu5Ac(a2-3)Gal(PI -4)GlcNAc(pI-0)R

lranslerase

GlcNAc(p1-0)R

Gal(pl-4)GlcNAc(p1-O)R

x

4-epimerase UDP-Gal

(

c%pEp PYR

CMP-Neu5Ac

XCTP

"yKkPEpI

p1,4-Gal translerase

UDP-Gal

IM PPi

Neu5Ac

P Pase

PYR

UDP-Glc

2 Pi

ManNAc

XUTP UDP-Glc pyrophosphorylase

2 Pi-

P Pase

PPi

Glc(a1-O)P

Fig. 1 1. Chemo-enzymic synthesis of the sialyl Le" sequence [541]. NMK, nucleoside-monophosphate kinase; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PPase, inorganic phosphatase; Pi, phosphate; PPi, pyrophosphate; PYR, pyruvate; TADH, Thermoanaerobium brockii alcohol dehydrogenase.

ments, excellent results have been obtained. To illustrate the enzymatic conversions, a series of typical examples will be presented. A first comprehensive example comprises the enzymatic sialylation on a microscale of oligosaccharides containing Gal(fil-3)GlcNAc, Gal(P14)GlcNAc, Gal(pl-4)Glc, and Gal(P1-3)GalNAc sequences by using different purified mammalian sialyltransferases [342], creating NeuSAc(a24)Gal, NeuSAc(a23)Gal, or NeuSAc(a2-6)GlcNAc linkages, as verified by 'H and 13C NMR spectroscopy (see section 5.3 3. The generation of the Neu5Ac(a24)GaINAc(a 1-0) sequence on a microscale has been described in ref. [536]. A second example (Fig. 11) is the chemo-enzymatic synthesis on a preparative scale of NeuSAc(a2-3)Gal(p 14)[Fuc(ctl3)]GlcNAc(P l-O)CH2CH=CH2 (and analogues) using P- 1,4-galactosyltransferase and recombinant a-2,3-sialyltransferase and a-1,3-fucosyltransferase with in situ regeneration of UDP-Gal, CMP-Neu5Ac and GDP-Fuc [541]. A third example is the one-pot enzymatic synthesis of Neu5Ac(a24)Gal(~l-4)GlcNAc (and analogues) based on a P-galactosidase-catalyzed galactosylation, using lactose as a donor and GlcNAc as

307

an acceptor, and a pig liver a-2,6-sialyltransferase-catalyzed sialylation with in situ regeneration of CMP-NeuSAc [542]. A fourth example is the enzymatic synthesis of NeuSAc(a24)Gal(fi 14)GlcNAc(fi l-O)pent-4-ene, a precursor for the organic chemical synthesis of higher oligosaccharides [472]. The trisaccharide was synthesized starting from GlcNAc(fi l-O)pent-4-ene, UDP-Gal (in situ generated from UDP-Glc catalyzed by UDP-Gal 4-epimerase), and NeuSAc in a one-pot reaction employing fi- 1,4-galactosyltransferaseand a-2,6-sialyltransferase in a complete cofactor regeneration system. The availability of specific sialyltransferases will certainly contribute to a further expansion of this area. In this context, the recent finding of a novel sialyltransferase which catalyzes the transfer of Kdn from CMP-Kdn to the non-reducing termini of oligo/polysialyl chains, thereby capping a further elongation of a (NeuSGc(a2-8)}, chain, is of interest [545]. In the framework of the finding that two NeuSAc(a2-6)Gal(fil4)GlcNAc units are the receptor determinants for the influenza virus hemagglutinin, these elements have been systematically anchored on a Gal residue in order to design structures capable of bimodal viral binding, and along chemo-enzymatic routes heptasaccharides with the general formula NeuSAc(a2-6)Gal(fi 14)GlcNAc(fil-x)[NeuSAc(a2-6)Gal(fi 14)GlcNAc(fil-y)]Gal(fi1-O)(CH~)~COOMe, where x and y are 2 and 3, 2 and 4, 2 and 6, 3 and 6, and 4 and 6, respectively, have been synthesized[546]. The concept of preparing compounds with a multivalent presentation of NeuSAc(a2-6)Gal(fi 14)GlcNAc((31- fragments on a linear or branched (via lysine) peptide backbone has been nicely worked out in ref. [547]. After the organic synthesis of a large series of peptide backbones in which GlcNAc((31-N)Asn units were incorporated, the oligosaccharide extensions were performed enzymatically by using fi- 1,4-galactosyltransferase and a-2,6-sialyltransferase. Neu5,9Ac2-containing oligosaccharides have been prepared along both organic chemical and enzymatic routes. In a reaction with trimethyl orthoacetate, NeuSAc(a2-6)Gal(fi14)Glc could be readily converted into Neu5,9Ac2(a2-6)Gal(filL4)Glc [380]. Employing CMP-Neu5,9Ac2 and immobilized porcine liver Gal((3lL4)GlcNAc a-2,6-sialyltransferase, Neu5,9Ac2(a2-6)Gal(fi lL4)GlcNAc has been synthesized [478]. The NeuSAc4Me-, Neu5Ac9Me-, and 8epi-NeuSAc-thioglycoside donors have been used to synthesize the corresponding sialoglycoconjugates [548], whereas also thioglycoside donors of 4d-, 7d-, 8d-, and 9d-NeuSAc have been prepared [549]. In other enzymatic approaches, use has been made of a fi-D-gaiactoside a-2,3-transsialidase from Trypanosoma cruzi as biocatalyst (see also section 9.2.3). This enzyme catalyzes the reversible transfer of NeuSAc from a donor-substrate of the sequence NeuSAc(a2-3)Gal(fi 1-O)Rl to virtually any Gal@ l-O)R2 acceptor substrate, affording a product NeuSAc(a2-3)Gal(fil-O)R2 [550-5521 (Fig. 12). A frequently used donor substrate is Neu5Ac(a2-3)Gal(fi1-4)Glc. In the case of Gal(fil-3/4)GlcNAc(fil-O)R sequences, the GlcNAc unit should not be substituted with a Fuc residue, as in LeX or Lea determinants [551]. The trans-sialidase reaction has been used in the chemo-enzymatic preparation of a water-soluble polyacrylamide, bearing multivalent NeuSAc(a2-3)Gal(filL4)GlcNAc elements [553]. To solve the problem of the poor enzymatic a-2,3-sialylation of Gal 2-(trimethylsily1)ethyl (3-glycoside using all known a-2,3-sialyltransferasesand CMP-NeuSAc, attention has been paid to the development of a sequence of enzymatic reactions, including cloned a-2,3-sialyltransferase and

308

b

z

I 0

r

0

8

I

0

-5

0

I

P

'

P

z

g

23 x

309

CMP-NeuSAc synthase, yielding an alternative active sialyl-donor-substrate in situ (e.g. NeuSAc(a2-3)1acto-N-tetraose), which can be used by trans-sialidase [554]. In this way it was possible to convert Gal 2-(trimethylsi1yl)ethyl P-glycoside into NeuSAc(a2-3)Gal 2-(trimethylsily1)ethyl 13-glycoside, a sialodisaccharide that can readily be transformed into a disaccharide donor, of interest for additional organic syntheses. In another study 4-MU-NeuSAc was tested as a donor with lactose as acceptor[555]. Starting from periodate treatedreductive aminated 4-MU-NeuSAc derivatives, interesting possibilities for the inclusion of fluorescent or photolyzable groups were demonstrated. Bacterial sialidases have also been explored in synthetic approaches. In a reverseenzyme reaction with A . ureafaciens sialidase, incubation of a concentrated solution of NeuSAc and lactose yielded NeuSAc(a24)Gal(P 1 4 ) G l c and Gal@14)[NeuSAc(a26)IGlc [556]. Similar experiments were carried out with immobilized K cholerae sialidase, using NeuSAc p-nitrophenyl a-glycoside as a donor. In this way NeuSAc(a2-x)Gal and NeuSAc(a2-x)Glc linkages could be produced, in which the a-2,6-linkage dominated over the a-2,3-linkage [557]. Transglycosylation of a NeuSAc unit using NeuSAc(a28)NeuSAc as a donor to Gal(P14)GlcNAc and Gal(b14)Glc was performed using sialidases of various origin [558]. Although the yields were low, a high regioselectivity was observed. The C. perfringens, A . ureufaciens and c! cholerae sialidases generated a-2,6-linkages, and the Newcastle disease virus sialidase a-2,3-linkages with the terminal Gal residue. For detailed information with respect to the synthesis of C-glycosides of sialic acids, see refs. [559-5631. As an example, the synthesis of a multivalent material that consists of the C-glycoside of NeuSAc, which is resistant to viral sialidase hydrolysis, should be mentioned [562].

7. Conformational aspects of sialic acids Earlier studies have appeared on the X-ray crystallography of both crystalline P-NeuSAc. H20 and B-NeuSAclMe*lHzO[56,564]. In an additional study, the crystal and molecular structure of a-NeuSAc1,2Me2 was also analyzed[565]. The C=O bond of the COOH function is approximately coplanar with the ring C-0 bond in a-NeuSAcl,2Me2, whereas in both P-NeuSAc and P-NeuSAclMe the C=O bond is found to be nearly eclipsed with the anomeric C-0 bond. In all three derivatives, the N-acetyl group is essentially planar, adopting the Z-conformation of a peptide bond. For a-NeuSAc 1,2Me2 and P-NeuSAclMe a hydrogen bond between the H-atom of H 0 7 and the carbonyl 0-atom of AcNHS was observed. The overall conformation of the glycerol side chain is the same for all three derivatives, as far as non-H atoms are concerned. In a-NeuSAc1,2Me2 a hydrogen bond between the H-atom of H 0 8 and the carbonyl 0-atom of the COOMe group is detectable. One of the oldest NMR studies on the conformation of NeuSAc is that focused on the spatial structure of aNeuSAc2Me in D20 [566,567]. On the basis of 'H-'H coupling constants in combination with 13C spinlattice relaxation times ( T I ) , a model could be constructed in which the amide H-atom of AcNHS is hydrogen-bonded to 0 7 , and the H-atom of H 0 8 is hydrogen-bonded to the ring-oxygen. A third hydrogen bond between the carbonyl 0-atom of AcNHS

310

and the H-atom of H 0 4 was suggested on the basis of molecular model building. In this model, apparently, the anomeric center is not involved in any hydrogen bonding, leading to the same conformation for a- and p-anomers. Independent of the models discussed above, the results fit the observation made by 'H NMR spectroscopy that H 0 7 and H 0 8 usually occur in a tuuns-orientation[53]. An NMR (H2O-suppressed 1D TOCSY, ROESY, NOESY) study, carried out on NeuSAc(a2-3)Gal(~14)GlcNAc and NeuSAc(a2-6)Gal(B 14)GlcNAc in 85% H20/15% (CD3)2CO, and aimed to detect hydroxyl and amido protons, indicated that in both compounds, thus irrespective of the type of linkage, the H-atom of H 0 8 of NeuSAc is involved in a strong intramolecular hydrogen bond [568]. In view of the fact that 7epi-NeuSAc and 7Jepi2-NeuSAc are substrates for CMP-sialic acid synthase (see section 6.1), but not 8epi-Neu5Ac [411], a conformational study on the side chain conformation of these sialic acids and NeuSAc itself (all Mana6(3)Man

Rab

Reference(s)

Mannose-specific group

Allium ascalonicum A. satiuum A. ursinum Cymbidium hyb. Epipactis helleborine

monomer

Mana3ManOR > Mana6[Mana3]ManOR Mana6[Mana3]ManOR > Mana3ManOR 25

12.5

a2

Man, ASiaFetuin= Thyroglobulin

Rab

25

12

a2

Man, ASiaFetuin = Thyroglobulin

Rab

a4

Mana6[Mana3]Man > Mana3Man > (Mana6)3

Rab,

(Ma11a6)~> Mana6[Mana3]ManOR

Galanthus niualis Hippeastrum hyb.

50 56

14.3

a4

Leucojum aestiuum L. uernum Listera ouata

25

13

a2

Narcissus lobularis N pseudonarcissus Tulipa gesneriana I

13

RabT

25

13

25

12.5

25

13

a2

(Mana6), > Mana6[Mana3]ManOR

67

17

a4

(Mar1a6),,~ > (ManaZ),,,

47

a13.3; p11.9

azp2

Man,_,GlcNAc

106

26.5 a13; PI4

a4

MeaMan > aMan > Glc > GlcNAc

ABO

a4

aMan > aGlc

Rab

azpz

aMan > aGlc > GlcNAc

ABO

a2

Thyroglobulin

W T ,

a2

(Mana3),_, > Mana3ManOR > (Mar1a3)~

Rab

(Mana6 > 2)2

Rab, Yeast

Mannose/glucose group

Bowringia mildbraedi Canaualia ensformis Cliuia miniata Cratylia mollis (1 & 2) Dioclea grandiflora

100

Lathyrus odoratus

52

25

ab Rab>AO>>B

31(1); 60(2) 25 a5.8; g20

continued on next page

Table A 1, continued ~

Lectin

Molecular weight

Subunit

Cells agglutinatedb

Reference(s)

aMan > aGlc > GlcNAc aMan > aGlc MeaMan >Man > aGlc, GlcNAc

ABO

[71 [71

aMan > aGlc 3-OMeGlc > 2.3-diOMeGlc > MeaMan > Man > Glc

Cat >> Rab

structure

Native KDa

Subunit KDa

L. sativus L. tingitanus Lens culinaris

49 50 46

a4.4; 819 a5.0; 820 (15.7; 817.5

Onobrychis viciifolia Pisum satiuum

53 50

26.5 a5.7; 817

Vicia cracca (Man) Y ervilia

a5.8; 817.5 a4.7; 821

Yfaba

44 53 52.5

a5.6; 820.7

a262

Man,Glc,Fruc,MeaMan,Mal,aa’Tre 2,3-diOMeMeaGlc > 3-OMeGlc > 3-OBzGlc > Man

T sativa

40

a 6 ; 814

a282

Melz > MeaMan > Man > Glc

Brachypodium syluaticum Chelidonium majus

36 23

18

a2

(GlnNAc(34), > GlcNAc

a9.5; 81 1.5

aB

(GlcNAc(34)4,3,, >> GIcNAc

Coccinea indica Codium fragile C. tomentosum

64 >60

32

a2

Cyphomandra betacea Cytisus sessilifolius

50

a282

a282 a282 a2

a282 a2B2

a282

ABO ABO ABO ABO ABO ABO (Sase) Rab > A > BO

N-Acetylglucosamine group

ABO ABO

GalNAc > GlcNAc Rab, H,

16.3; 15.6 (GlcNAc(34)4 > (GlcNAc(34)3 GlcNAc(34GlcNAc > ~-Fuca2Gal~4GlcNAc

Rab 0

monomer

(GlcNAc[34)3 > (GlcNAc(34)z > GIcNAc (Gl~NAc(34),,~= LacNAc > GlcNAc

a4

GlcNAca3GallGlc > GlcNAc~3Gal,GlcNAca6Gal

ABO ABO Tk

25

a2

a8

110

Datura stramonium

86

a40; (346

Ficus cunia‘ Griffonia simplicifolia I1

3.5 1 I3

3 .5 30

Hordeum uulgare Lufa acutangula

36

18

48

24

~~~~~~

Specificitya

a2

(GlcNAcfM), > GlcNAc

a2

(G1cNAc(34)5>4,3>2

ABO Rab

[71 [71 [71 [71 [71 [71 ~71

Table A l , continued Lectin

Molecular weight Native KDa

Subunit

Specificitya

monomer

Lycopersicon esculentum

71

71

Oryza satiua

38 19

18-19 -

monomer

40

monomer

Secale cereale

40 36

(G1cNAc(341)4>3>2 (GlcNAc(34)"> GlcNAc (GlcNAc(34)" GlcNacfi3Gal(34GlcNAc > GlcNAc!36GalppNC~

a2

(GlcNAc(34)">> GlcNAc

Solanum tuberosum

100

Triticum uulgaris

43.2

Wlex europaeus I1

105 8.5

Phytolacca americana Psathyrella uelutina

Urtica dioica

GalactoselN-acetylgalactosarninegroup 135

Abrus precatorius (Agglutinin)

Cells agglutinated

Subunit KDa

18 50 21.6 23-25 8.5

a33; 036; p'37.5

a2

a2 a2

(GlcNAC(34),_5 (GlcNAc(34), > GlcNAcP > NeuSAc

monomer

(GicNAc(34)4>3,2 GlcNAc oligomers

a2w

fiGal> GalNAc

a4

O>AB ABO 0

ABO ABO ABO 0 >AB Rab, ABO

A . precatorius (toxin A)

62.5

a31; a'33; 836

A. precatorius (toxin C) Adenia digiiata A . digitata (6B)

67 5743

a29; 636 a28; p35 a27; 031

Gal > Me1 > Lac,Raf,o-Fuc >> L-FUC

Rab > ABO

Aegopodium podagraria

480 58.5

60(a,a')

GalNAc >Lac > Fuc > Gal > Me1 Galf33GalNAc-T,S

ABO ABO

Amaranthus caudatus

62

A . cruentus

66

33-36 35

(NeuSAc)Galfl3GalNAc> GalNAca3Gal Fetuin >> GalNAc; GalOR = 0

ABO Rab,ABO

A . leucocarpus

66

3346

ASiaFetuin > Fetuin > Human IgG > >GalNAc

H~ > H ~

65.8

37

ASiaFetuin > Fetuin >> GalNAc

Rab, ABO

Agaricus bisporus

A. spinosus

57

pGal> GalNAc

Reference(s)

Table A l , continued Lectin

Molecular weight Native KDa

Subunit KDa

Amphicarpa bracteata

135

Arachis hypogaea

110

a28.5; 836; y32 27

A. integrifolia Bauhinia purpurea

54 195

a12; PI5 44

Boletus satanas Botiytis cinerea

63 34 61

63 17

Subunit

a4

18; 14.5

Artocarpus altilis

Biyonia dioica Caragana arborescens Ceratobasidium cornigerum

103

a32; 830 26

Clerodendron irichotomum

56

28

Codium fragile (see under GlcNAc group) Colchicum autumnale

100

a15; 810 31

Cratylia mollis (3)

Specificitya

Cells agglutinatedb

GalNAca3GalNAc > GalNAca3Gal Galb3GalNAc > a,fiGal; Lac > PGal

A1 T cells

Galp3GalNAc

a2

Gal83GalNAc; MeaGal >> MePGal Galfi3GalNAc > a,BGalNAc > a,PGal Gal MeaGal > GalNAc, Gal, Me1

aP

GalNAc

P

a3 a4

monomer

a4

GalNAc >Gal, eomplex GalNAc

H, Rat Rab, ABO 0 >AB

GalNAcOR (a@= !3@ > Me > OH) > Lac > Gal

0

a484

Lac > GalN > GalNAc >Gal (Ho only) Gal

ABO, Rab ABO

a4?

Lac,Mel> GalNAc >Gal

ABO (Sase)

[591

A,

>> A2

[71

120 75-90?

31 31

Dolichos biflorus

110

27.3; 27.7

a282

GalNAca3GalNAc >> GalNAc

>50 5648

a30; 828 26-33 30.9

aP

GalNAc

dimer dimer

Galfi4GlcNAc > GalNAc > Gal Lac,Gen,MeOGal> MeaGal,Gal> GalNAc

Erythrina spp. g Euphorbia marginata

ABO Rab > ABO

a2

Crotalaria juncea Cyiisus scoparius Echinocystis lobata Eranthis hyemalis

[581 [81 171

a4

ABO

52.5

Reference(s)

Structure

ABO ABO, Rab

[601 [61,621

[71 ~ 3 1 continued on next page

$, W

P m 0

Table A l , continued Lectin

Molecular weight Native KDa

Subunit KDa

Falcata japonica

125

Galactia tashiroi

90 120 114 I20

34 24

Glycine max Grifonia simplicifolia I d Hura crepitans Iris germanica

30 a32; 033 31 35; 30 a21; 634

Iris x hollandica

60

Lactarius deliciosus

37 37

a18; B19

4046 108

a10; p12

L. deterrimus Maclura pomifera Macrotyloma axillare’ Moluccella laeuis Momordicn charantia Mucuna deeringiana Phaseolus lunatus’ Phaseolus mungo Pleurotus ostreatus

130 129

110 124 137

18

a,1327 26-46 a29; 836

Subunit

Specificitya

Cells agglutinatedb

Reference(s)

GalNAc(a@)O@>> GalNAc > Gal >> GlcNAc,GalN

A, > A 2 > A ,

[64,651

GalNAc >> Gal,MeGal,Lac GalNAc > Gal a4,GalNAc >Gal; @,Gal

ABO > Rab A>>B

[661 [71

A(a4), B(B4) Shp > ABO AB>B

[71 [71 ~671

R4T) ABO ABO O>A>B

[681 ~ 9 1 [701 17~71,721

Structure

GalNAc > Ga1,MepGlc GalNAc >Lac > Gal GalNAca3Gal> GalNAca6Gal> GalNAcOMe GalB3GalNAc > GalB3GlcNAc, GalNAc-1 P Galfi3GalNAc pN@aGalNAc > pN@aGal >> GalNAc > Gal GalNAc GalNAcaOR > GalNAcBOR >> GalOR Lac >> MeflGal> a,BGal> GalNAc

A, [71 BONN> AMM> OMM [73,74] ABO

[71

RabT, Shp

[751

A , >A2 RabT

[71 [761

GalNAc >Lac > Gal, Me(a,P)Gal> Raf > Me1

ABO>Rab

[771

29(a,B,v) a30; 634

Gal > GalNAc; Lac > Me1 BGal> aGal >> GalNAc a,pGal> GalNAc GalNAc, human a-glycoproteins

ABO (t. as) ABO ABO

[71 [71 [71 [78-811

a,a’,P,3 1 66 a44; @ l

GalNAca3GalNAc > GalNAc >Gal GalNAca3[~-Fuca2]Gal>GalNAc MeaGal> pN@aGal> Me1 > Gal > GalNAc

54

Psophocarpus tetragonolobus

87 52

Ricinus communis I

120

R. communis 11 (Ricin) Robinia pseudoacacia I11 Sambucus nigra I1

63 105

a30; 034

51

30

29; 30.5

GalNAca3/6Gal> GalNAc > GalNAcfi6Gal

Rab > ABO

[34,821 continued on next page

Table A l , continued ~~

~

Lectin

Molecular weight Native KDa

Subunit KDa

Sarothamnus welwitschii

64

Sclerotinia miyabaena

34

21.5 17-18

132.8

32.5(a,b)

Sophora japonica

Specificitya

Cells agglutinatedb

a3

GalNAc > MeaGal> Lac,Mel

A,>A>BO Rab,

a2B2

MeaGal> GalNAc > Lac > Gal; AS-Much > M u c h GalNAc > Gal

Subunit Structure

26

Sphenostylus stenocarpus

GlcNAc > Lac > Raf,Gal MeBGal >Lac > GalNAc,MeaGal,Mel

Telfairia occidentalis

180

Tetracarpidium conophorum 1

70 34

a,(330 34 34

64

a33; (329

aB

Fuca2Galp4GlcNAc > (Galp4GlcNAc)4,3,2

56 114, 125

a26; (330 33

aB

Lac > BGal

105

T conophorum I1 Trichosanthes japonica (11) L kirilowii Vicia cracca I , I1

(a(3)3 as-Sa monomer

GalNAc > Me(3Gal > Lac > Ga1,MeaGal Lac > Me(3Gal> Gal,MeaGal,GalNAc

B > A >> O(H) 0 >AB ABO A A O>>B > A A, >A2

a4

a4

(R = T or S peptide) (G~IB~G~INAc-OR),

A B O ~> A B O ~

an(34-n

GalNAc >> Gal

At(a4); Tn((34)

a29; (334

a(3

Gal >> GalNAc

ABO, animal

60

30

a2

GalNAca6Gal> aGalNAc > BGalNAc

ABO

Grifonia simplicifolia IV

72 56.6

31 a27; (329

aB

Mea-L-Fuc > Lea,Leb ~-Fuca2Gal(33[~-Fuca4]GlcNAc

ABO Leb

Lotus telragonolobus A

120

a4

Fuca6GlcNAc > Fuca2Ga1~4[Fuca3]GlcNAc

O(H)

Lotus tetragonolobus B

58

27.8 27

Ulex europaeus I

60

a29; 831

aB

!l graminea F uillosa

110-

26 a34; (336

Viscum album

120 60

Wistaria floribunda

Reference(s)

L-Fucose group

Aleuria aurantia

a2

OW)

a2

Fuca2Gal@GlcNAc~> FucaZGal(34Glc> MeaFuc

O(H) continued on next page

P

0'

tR N Table A l , continued Lectin

Molecular weight

Subunit

Specificitya

Cells agglutinated

Reference(s)

a2P2

NeuSGc >NeuSAc > NeuSAca3'Lac > GalUA

Pig > A,O > B

~ 9 1

a4

NeuSAca3Galp4GlcNAc

[ 12,90-941

34.5; 37.5

a2P2

Neu5Aca6Gal/GalNAc >> Lac

[12,93-971

a38; 832

a6

Neu5Aca6Galp4GlcNAc, S03-6Gal@GlcNAc

[981

Native KDa

Subunit KDa

Hericium erinaceum

54

a1S; 616

Maackia amurensis

130

33

Sambucus nigra

140

Trichosanthesjaponica (I)

70

Sialic acid group

Complex group (not inhibited by monosaccharides)

Alocasia indica Boodlea coacta (A-D)

55 14-20

13 14-20

a4

ASiafetuin (3 Galfi3GalNAc?)

Rab, GP, Rat

monomers

High mannose glyconjugates, mannan

Rab, ABO(P, BO>A2,H,

43

26

a2

Human IgM, bov. fetuin

126.7

3s

a4

Gala3[Fuca2]LacNAc > Fuca2LacNAc

Marchartia polymorpha

20

18

monome1

Subs. A, B; IgG, lactalbumin

Phaseolus coccineus (I, 11)

120

f! coccineus

112

Cicer arietinum Euonymus europaeus

'

0 > Rab, Bov. ABO ABO

a4

Phaseolus vulgaris Erythroagglutinin

11s

30

a4

Tetraantennary octasaccharide

0

Leucoagglutinin

115

30

a4

(Galp4Gl~NAc)~P2,6Man

Leucocytes

145,175

34.5, 37, 39

?

ASiaFetuin > Thyroglob. > fetuin > orosomucoid

Rab, ABO

Pinellia terneata

40

10-1 1

a4

Mannan, thyroglobulin

Rab

Robinia pseudoacacia I

59

34

a2

Ovomucoid

40

a26; 614

k? vulgaris'

Sclerotium rolfsii Tulipa gesneriana I1

45

Hog gastric mucin > bov. submax. ASiamucin Thyroglobulin > mucin, orosomucoid

E. coli Mouse, rat

continued on next page

Table A l , notes Abbreviations for oligosaccharides and substituents: Gen, gentibiose; Lac, lactose; Mal, maltose; Melz, melezitose; Raf, raffinose; Tre, trehalose; Asia, asialo-; Bz, benzyl; Me, methyl; NCP, nitrophenyl; CP, phenyl. Abbreviations for blood cells: Rab, rabbit; RabT, trypsinized rabbit cells; H, human, type unspecified; ABO, human, type specified; Bov. bovine; Shp, sheep; Sase, sialidase-treated. Ficus semicordata (J.E. Smith) is preferred over E: cunia (Hamilton ex Roxburgh). Griffonia is accepted over Bandeiraea. Synonymous with Amaranthus hypochondriacus. Synonymous with Artocarpus heterophyllus. g The following species of Erythrina possess nearly identical lectins: arborescens, caffra, corallodendron, cristagalli, Jadelliformis, latissima, litosperma, lysistemon, perrieri, stricta, suberra, zeyheri. Falcata japonica (Oliver) V Komarov = Amphicarpa edgeworthii Benth. i Macroiyloma axillare (E. Mey) Verde, formerly Dolichos axillaris E. Mey. Synonymous with I? limensis. Nearly identical lectins are found in S. minor. S. trifoliorirm, and 3 isolates of S. sclerotiorum. Euonymus has been incorrectly spelled and indexed as “Euonymus” in some literature and data bases; europaeus (not europaea) is the correct form of the specific epithet. Variety ’Xlubia”. ” Variety Red Kidney Bean. O Variety “Great Northern”. a

*

J

’

464 Table A2 Representative applications of lectins to biomedical research Lectin

Application

Reference(s)

Mannose/glucose group Bowringia mildbraedi

Effect on infectivity of HIV

Cymbidium hyb.

Inhibitor of HIV and CMV replication in uitro

Epipactis helleborine

Inhibitor of HIV and CMV replication in vitro

Galanthus nivalis

Effect on infectivity of HIV Purification of HIV and SIV envelope glycoproteins Glycosylation of rat spermatids Purification of mouse and human glycoproteins Typing 6-hemolytic Streptococci

Lens culinaris

Analysis of human a-fetoprotein

Leucojum aestivum

Effect on infectivity of HIV

Listera ouata

Separation of a-o-mannans

Characterize strains of Neisseria gonorrhoeae

Inhibitor of HIV and CMV replication in uitro Narcissus lobularis

Effect on infectivity of HIV

N-Acetylglucosamine group Codium tomentosum

Interaction with intestinal parasites

C. fragile

See C. tomentosum

Datura stramonium

Purification of poly-LacNAc glycopeptides

Griffonia simplicifolia I1

Detect blood-group related poly-LacNAc in tissues Characterize strains of Neisseria gonorrhoeae Detect human colonic carcinoma glycoproteins

Lycopersicon esculen turn

Agglutinate type B streptococci type Staphylococcus aureus strains Identify pathogenic trypanosomes

Psathyrella velutina Solanurn tuberosum

Study of glycosylation abnormalities of rheumatoid IgG Agglutinate type B streptococci Characterize strains of Neisseria gonorrhoeae

Triticum uulgaris

Numerous

Wlex europaeus I1

Trace maturation of rat kidney cells

Wrtica dioica

Induction of human IFN-g in lymphocytes Activate specific T-cells Inhibit HIV and CMV replication in vitro

GalactoselN-acetylgalactosamine group Aegopodium podagraria

Typing P-hemolytic Streptococci

Arachis hypogaea

Numerous

[341 [120,125-1331 continued on next page

465

Table A2, continued Lectin

Application

Artocarpus integrifolia

Separation of 0-linked glycoproteins

Bauhinia purpurea

Isolation of specific B- and T-cell populations

Reference(s)

Identify pathogenic trypanosomes Boletus satanas

Release of IL-la and IL-2 Protein synthesis inhibitor

Biyonia dioica

Typing B-hemolytic Streptococci

Canavalia ensiformis

Numerous

Ceratobasidium cornigerum

Typing 0-hemolytic Streptococci

Clerodendron trichotomum

Screening of hematopoietic cells

Dolichos bgorus

Numerous

Eranthis hyemalis

Protein synthesis inhibitor

Erythrina cristagalli

Distinguish embryonic & mature rat kidney cells

Glycine m a

Remove T cells from bone marrow

Griffonia simplicifolia I

Detect Tn antigen in cancer cells

Differentiate types of alveolar macrophages Identify alveolar macrophages in respiratory disease Maclura pornifera

Rat T cell studies

Moluccella laeois

Typing of NM blood groups

Ricinus communis I

Trace development of chick thymus Assay for sialidase

Robinia pseudoacacia 111

Study of cyclic nucleotide phosphodiesterase in peripheral blood lymphocytes

Sambucus nigra 1

Typing fi-hemolytic Streptococci

Sclerotinia spp.

Typing 6-hemolytic Streptococci

Sophora japonica

Identify alveolar macrophages in respiratory disease Identify pathogenic trypanosomes

Sphenostylus stenocarpus

HIV infectivity studies

Tetracarpidium conophorum

Effect on HIV infectivity

Vicia graminea

Identification of polyagglutinating erythrocytes in hemoglobin variant

!I oillosa

Separate T-cell types Stain cancer cells Identify pathogenic trypanosomes

Viscum album

Specific staining of microglial cells Stain amyloid plaques in Alzheimer’s

L-Fucose group

AIeuria aurantia

Characterize fucosylated glycoproteins in brain

[1511 continued on next page

466

Table A2, continued Lectin Lotus tetragonolobus

Application

Reference(s)

Purify fucose-containing glycoconjugates Identify pathogenic trypanosomes Development of chick thymus

Ulex europaeus 1

Identify secretors Identify alveolar macrophages in respiratory disease Determine epithelial cell differentiation Characterize strains of Neisseria gonorrhoeae Identify pathogenic trypanosomes

Sialic acid group Maackia amurensis

Glycosylation of rat spermatids Characterization of sialyltransferases in Ehrlich ascites cells Characterization of sialidases Typing 6-hemolytic Streptococci

Sambucus nigra I1

Glycosylation of rat spermatids Characterization of sialyltransferases in Ehrlich ascites cells Characterization of sialidases Sialylation of neoplastic colon tissue Isolation of sialylated glycoconjugates Typing &hemolytic Streptococci

Complex group Euonymus europaeus

Purify human complement fractions

Phaseolus vulgaris

Trace development of optic tectum

Pinellia terneata

Motor nerve terminal studies Typing 6-hemolytic Streptococci

Robinia pseudoacacia I

~1541 [ 155,1561 [ 108,109]

[341

467 Table A3 Classification of species listed in Table Al Order and Family

Genus and species

Tissue used

Agaricaceae

Agaricus bisporus

Fruiting body

Boletaceae

Boletus satanas

Fruiting body

Coprinaceae

Fruiting body

Polyporaceae

Psathyrella veluiina Pleurotus ostreatus

Russelaceae

Lactarius spp.

Carpophore

Hericium erinaceum

Fruiting body

Botrytis cinerea

Mycelia, sclerotia

Algae & fungi (Thallophyta)

Agricales

Fruiting body

Aphyllophorales Hydnaceae Helotiales Sclerotinaceae

Sclerotinia spp.

Mycelia, sclerotia

Sclerotium roljsii

Culture fluid

Aleuria aurantia

Fruiting body

Pezizales Pezizaceae Siphonales Codiaceae

Codium spp.

Siphonocladiales Valoniaceae

Boodlea coacta

Tulasnellales Ceratobasidiaceae

Ceralobasidium cornigerum

Mosses & liverworts (Bryophyta)

Marchartiales Marchartiaceae

Marchartia polymorpha

Gametophyte

Brachypodium sylvaiicum Oiyza saliva Secale cereale

Germ Seed Germ

Triiicum vulgaris

Germ

Tulipa gesneriana

Bulb

Alliuma spp. Colchicum autumnale

Tuber

Seed plants (Spermatophyta) Monocotyledoneae

Glumiflorae Gramineae

Liliiflorae Liliaceae

Bulb

continued on next page

468

Table A3, continued Order and Family

Genus and species

Tissue used

Clioia miniata

Leaf

Galanthus nioalis

Bulb

Liliiflorae (cont a) Amaryllidaceae

lridaceae

Leucojum spp.

Bulb

Narcissus spp.

Bulb

Hippeastrum hyb.

Bulb

Iris germanica

Rhizome

Iris x hollandica

Bulb

Listera ovata

All

Microspermae (Orchidales) Orchidaceae

Cymbidium hyb.

All except root

Epipactis helleborine

All

Alocasia indica

Tuber

Pinellia terneata

Rhizome

Spathiflorae Araceae

Dicotyledoneae Urticales Moraceae

Artocarpus spp.

Seed

Ficus cunia

Seed

Maclura pomifera

Seed

Urtica dioica

Rhizome

t7iscum album

Vegetative

Amaranthaceae

Amaranthus spp.

Seed

Phytolaccaceae

Phytolacca americana

Root (all)

Eranthis hyemalis

Tuber

Chelidonium majus

Seed

Abrus precatorius

Seed

Urticaceae Santalales Loranthaceae Centrospermae

Ranales Ranunculaceae Rhoeadales Papaveraceae Rosa1es Leguminoseae (Fabaceae)

Amphicarpa bracteata

Seed

Arachis hypogaea Bauhinia purpurea

Seed Seed continued on next page

469

Table A3, continued Order and Family

Genus and species

Rosales

Bowringia mildbraedii

Seed

Canavalia ensifarmis Caragana arborescens

Seed

Cicer arietinum

Seed

Cratylia mollis Crotalaria juncea

Seed

Cytisus spp.

Seed

Dioclea grandiflora Dolichos biforus

Seed

-

Leguminoseae

(cant 3)

Seed

Seed

Seed

Erythrina spp. Falcata (=Amphicarpa) Galactia tashiroi

Seed

Glycine max

Seed

Seed Seed

Griffonia simplicifolia

Seed

Lathyrus spp. Lens culinaris Lotus tetragonolobus

Seed Seed

Maackia amurensis

Seed

Macrotyloma axillare

Seed

Seed

Mucuna deeringiana

Seed

Onobrychis uiciifolia Phaseolus spp.

Seed Seed

Pisum satiuum

Seed

Psophocarpus tetragonolobus

Tuber, seed

Robinia pseudoacacia

Seed

Sarothamnus welwitschii

Seed

Sophora japonica

Seed

Sphenostylus stenocarpus Ulex europaeus

Seed Seed

Vicia spp.

Seed

Kstaria floribunda

Seed

Geraniales Euphorbiaceae

Euphorbia marginata

Latex

Hura crepitans

Seed

Ricinus communis Tetracarpidium conophorum

Seed Seed

Euonymus europaeus

Seed

Sapindales Celastraceae

continued on next page

470

Table A3, continued Order and Family

Genus and species

Tissue used

Parietales Passifloraceae

Adenia digitata

Root

Umbelliflorae Umbelliferae (Apiaceae)

Aegopodium podagraria

Rhizome

Clerodendron frichotomum

Fruit Seed Fruit

Tubiflorae Verbenaceae Labiatiae Solanaceae

Moluccella laeois Cyphomandra betacea

Solanum tuberosum

Seed Fruit Tuber

Rubiales Caprifoliaceae

Sambucus nigra

Bark

Cucurbitales Cucurbitaceae

Bryonia dioica

Root Fruit (phloem) Seed Fruit (phloem) Seed Tuber Root

Datura stramonium Lycopersicon esculenturn

Coccineu indica Echinocystis lobata Luffa acutangula Momordica charantiu Trichosanthesjaponica

T kirilowii a

Allium is placed in Amaryllidaceae by some taxonomists. Some classifications elevate the three subfamilies of Leguminoseae to families in the Order Leguminales

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J. Montreuil, J.F.G. Vliegenthart and H. Schachter (Eds.), Glycoproteins 11 0 1997 Elsevier Science B.V. All rights reserved

CHAPTER 13

Microbial lectins and their glycoprotein receptors Nathan Sharon and Halina Lis Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel

1. Introduction Numerous microorganisms appear to have selected carbohydrates as the preferred attachment sites on their host cells and tissues [l]. This is not surprising, in view of the abundance of carbohydrates on the surfaces of eukaryotic cells, in the form of glycoproteins and glycolipids. Binding to the carbohydrates is mediated by microbial surface lectins, members of a large group of microbial adhesins, that play a major role in the initiation of infection and in non-opsonic phagocytosis [2-51. Host range, tissue tropism and target cell specificity demonstrated by a particular microbe are determined, at least in part, by a stereochemical fit between microbial lectins and complementary carbohydrate receptors on host cell surfaces [6]. Adhesion to insoluble carbohydrates may also play an important role in the biofouling of marine surfaces and in ecological phenomena such as biodegradation, as well as in the monitoring by bacteria of the nutrient status of the environment [7]. Although a considerable number of these lectins are well defined, the existence of many more has been inferred from experiments on the effect of carbohydrates on the interaction of intact microorganisms with different target cells. The oldest, and perhaps most thoroughly studied system of this type is the interaction between the hemagglutinin of influenza virus A and N-acetylneuraminic acid' on cell surfaces. It is responsible not only for the adhesion of the virus to the cells, but also for fusion of the viral membrane with the host cell membrane and is also the viral component to which protective antibody is directed. Virtually all bacterial species and genera express lectins or lectin-like activities, frequently of more than one type and with different specificities. However, it is usually not known whether individual cells coexpress multiple types of lectin or each lectin is confined to a distinct cell population. Many Gram negative bacteria (for example, Escherichia coli and Salmonellae spp.) and a few Gram positive ones (e.g. certain actinomyces), produce surface lectins that are often in the form of submicroscopic hairlike appendages known as fimbriae (pili) that protrude from the surface of the cells. The best characterized bacterial surface lectins with respect to their molecular properties, carbohydrate specificity and genetics are the type 1 fimbriae specific for mannose and the type P fimbriae specific for galabiose, [Gal(al+Gal], produced by many strains of E. coli. Other examples are S fimbriae of E. coli, specific for NeuAc(a2-3)Gal, and type 2

'

All sugars are of the D-configuration, except for fucose which is L475

476

fimbriae of oral actinomyces, specific for Gal(fi1-3)GalNAc or GalNAc fi- that are not so well characterized. Non-fimbrial lectins that are components of the cell surface have also been described. The case of Pseudomonas aeruginosa is unusual in that certain strains of the organism produce intracellular lectins, at least one of which appears also on the bacterial surface. Among the fungal and protozoal lectins only a few have been studied in detail. One of these is the galactose-specific lectin of the protozoa Entamoeba histolytica. It mediates adhesion of the parasite to human colonic mucin glycoproteins and has a central role in the contact-dependent cytolysis or histolysis for which the parasite is named. A sialicacid-specific lectin has been isolated from merozoites of the human malarial parasite, Plasmodium falciparum. An unusual lectin is that of the protozoan Giardia lamblia, specific for mannose-6-phosphate, which is activated by trypsinization. The carbohydrates to which the microbial lectins bind are in the form of glycoproteins and glycolipids (Table 1). Many lectins interact with both classes of glycoconjugate; this is not unexpected, since these compounds frequently contain identical oligosaccharides. Identification of the receptors for microbial lectins on animal cells (or in secretions) is based on methods such as binding of intact bacteria or of the isolated lectin either to blots of electrophoretically. separated cell membrane glycoproteins [8] or to thin layer chromatograms of glycolipids extracted from the cells. Glycoproteins can also be analyzed by affinity chromatography of cell membrane extracts on immobilized microbial lectins. The finding that a glycoprotein or glycolipid interacts with the bacteria (or isolated lectin) in such tests does not prove, however, that it serves as a functional receptor in uiuo. Most importantly, it should be obtained from cells susceptible to infection by the organism studied. Also, antibodies to the presumed receptor should inhibit binding of the organism to the cells and cells devoid of the receptor (as seen sometimes in mutants) should lack the ability to bind the microorganism. Glycolipids that serve as native receptors for microbial lectins have been identified in a large number of cases, but information on glycoproteins that play such a role is still scarce. In the following we shall discuss mainly those lectins of viruses, bacteria, protozoa and fungi that are known to interact with either glycoproteins alone or with glycoproteins as well as glycolipids. We shall also deal with their role in infection and lectinophagocytosis. For bacterial lectins that appear to bind exclusively to glycolipids, see recent reviews on the subject [9-1 I].

2. Viruses 2. I . Sialic-acid-specijic 2.1.1. Influenza uirus The ability of the virus to agglutinate erythrocytes has been first reported in 1941. It took more than a decade before it was shown that influenza virus binds to erythrocytes and other cells via N-acetylneuraminic acid residues present on the cell surface and that this binding is a prerequisite for initiation of infection [24,25]. Other viruses, such as Sendai, Newcastle disease, polyoma and rotavirus also exhibit an affinity for sialic

477 Table 1 Carbohydrates as attachment sites for infectious agents a Organism

Target tissue

Reference( s)

Carbohydrate Structure

Form’

Respiratory tract

NeuAc(a2-6)Gal

GP

[I21

Respiratory tract

NeuAc(a2-3)Gal

GP

1121

type c Parvovirus B 19

Respiratory tract

9-0-AcNeuAc(a2-3)Gal

GP

[I21

Erythroid cells

GalNAc(@1-3)Gal(a 1-4)Gal(@1 4 )

GSL

[I31

Polyoma virus

Epithelial cells

NeuAc(a2-3)Gal

GP

[I41

Urinary tract

Man(a 1-3)[Man(a 1-3)[Man(al4)]

Urinary tract

Gal(a 1-4)Gal

GP GSL

[4,81 [9]

GP GSL

PI

GP GP

[81 [I61

Viruses Influenza type A type B

Bacteria

E. coli type 1 type p type

s

Neural

NeuAc(a2-3)Gal(fi 1 -3)GalNAc NeuGc(a2-3)Gal

type CFAI/II

Intestine

NeuAc(a2-3)Gal

type KI

Endothelial cells

GlcNAc(fi14)GlcNAc

type K88ac

Intestine

Gal(fi1-3)GalNAc

type K99

Intestine

NeuGc(a2-3)Gal(fi14)Glc

Oral

Gal(@14)Glc

Actinomyces naeslundi

GalNAc(B 1-3)Galfi

Neisseria gonorrhoea

Genital

Gal@14)Glcfi NeuAc(a2-3)Gal(fi 1-4)GlcNAc

Streptococcus sanguis

Oral

NeuAc(a2-3)Gal(fi1-3)GalNAc

GP GSL GSL GP

[I51

[17] [I81 [I91 [20]

GSL GP GP

[9]

GSL

[22]

GP

[23]

[21]

Fungi

Candida albicans

Skin and mucosa Gal(@14)Glc Fuc(a I-2)Gal

a

For protozoa, see Table 3.

’ Predominant; GP, glycoproteins; GSL, glycolipids. acid [ 121. The hemagglutinin (lectin) of the influenza virus, responsible for its attachment to cells, was purified, crystallized, and studied in detail, culminating in the elucidation of the three-dimensional structure of its complex with N-acetylneuraminic-acid-containing oligosaccharides at the atomic level [26-281. The subunit of the lectin is composed of two polypeptides, HA1 and HA2 (with molecular masses of 36 and 26kDa, respectively), covalently linked by a single disulfide bond, and it associates non-covalently to form trimers that are located on the surface of the viral membrane [27]. It is a glycoprotein, with six N-linked oligosaccharide chains attached to HA1 and one to HA2. Except for one oligomannose unit, all are complex, bi- or triantennary structures, with three of them containing sulfated galactose [29]. The carbohydrate binding site is located in a pocket of the HA1 polypeptide chain, in a domain of the lectin protruding from the membrane,

478

and is composed of amino acids that are largely conserved in the numerous strains of the virus [27]. Other conserved residues are found behind the pocket and seem to stabilize the architecture of the site without interacting with the carbohydrate. Over 100 strains of influenza virus, mostly of the A- and B-types, were examined for their ability to bind to enzymatically modified erythrocytes carrying terminal N-acetylneuraminic acid attached to galactose either by an a2-3 or a2-6 linkage [ 12,261. Differences in their specificity with respect to this linkage were correlated with the species origin of the virus. Thus, human isolates preferentially agglutinated resialylated erythrocytes containing the NeuAc(a24)Gal sequence, while the avian and equine isolates exhibited preference for NeuAc(a2-3)Gal. Strains of influenza C virus (as well as coronaviruses) do not bind N-acetylneuraminic acid at all, but only recognize its derivative, 9-0-acetyl-N-acetylneuraminic acid; the 9-0-acetyl group is critical for mediating cellular attachment [30]. Comparison of the primary sequences of hemagglutinins of the human virus with those of mutants showing decreased affinity for NeuAc(a2-6)Gal and markedly increased affinity for NeuAc(a2-3)Gal, revealed that they differ in a single amino acid substitution, Leu226 in the parental strains being replaced by glutamine in the mutants. Similar studies with avian isolates and their variants showing the reverse change in specificity (from a23 to a 2 4 linked N-acetylneuraminic acid), again revealed a substitution only at position 226 - from glutamine to leucine. This illustrates that a single amino acid substitution can alter the sugar specificity of a lectin. Although residue 226 is located in the carbohydrate binding site of the hemagglutinin, it is not in direct contact with the bound sugar, as shown by crystallographic studies of wild-type influenza virus hemagglutinin complexed with NeuAc(a2-6)Gal(fi14)Glc [sialyl(a24)lactose] and of a mutant hemagglutinin complexed with NeuAc(a2-3)Gal(fil-4)Glc [sialyl(a2-3)lactose]. The suggestion has therefore been made that the change in specificity is due to conformational differences between the mutant and wild-type proteins. X-ray crystallographic analysis of the complex of the hemagglutinin with bound sialyl(a2-6)lactose, [NeuAc(a24)Gal(fi 1-4)Glc], placed the sialic acid in the binding pocket with one side of the pyranose ring in tight contact to the protein and the other side facing the solvent [28] (Fig. 1). It also permitted to predict potential hydrogen bonds and van der Waals contacts of sialic acid atoms with amino acids in the binding site based on their proximity to each other. The validity of these predictions was tested by evaluating the affinity for the hemagglutinin of a series of synthetic analogs of N-acetylneuraminic acid with modified functional groups, by determining: (i) the ability of the analogs to inhibit viral attachment to cells [3 11 and (ii) the equilibrium dissociation constants for the binding of these analogs to the hemagglutinin [32]. It was thus confirmed that the two carboxylate oxygens of N-acetylneuraminic acid receive hydrogen bonds from the hydroxyl group of Szr-136 and from the main chain amide group at Asn-137, respectively, and that both bonds are necessary for binding. These studies also provided additional evidence on the critical importance of the hydrophobic contact between the acetamido group at C-5 of N-acetylneuraminic acid with the indole ring of Trp- 153. On the other hand, the hydroxyl at C-9 does not seem to participate in ligand binding, contrary to what has been proposed from the crystal structure. The structures of complexes of the hemagglutinin with four sialic acid analogs, with

479

\7%

Thr 155

I, Trp 153

n

Fig. 1. Model for the position of sialic acid in the binding pocket of influenza virus hemagglutinin. This model for the best fit has been deduced from the difference electron density maps of X-ray crystallographic studies. Some of the hydrogen bonds proposed in this model are shown by dashed lines. (Taken from ref. [31]; with permission from the authors.)

affinities 10- to 100-fold higher than that of N-acetylneuraminic acid, were determined by high-resolution X-ray crystallography [33]. In these analogs, the sialic acid core was substituted at the 4 or 6 positions with spaced naphthyl or dansyl groups. In each of the complexes, the sialic acid moiety was equivalently positioned in the binding site of the hemagglutinin, while the substituent groups that differentiate the high-affinity analogs frclm each other interacted with hydrophobic patches and polar residues adjacent to the binding site. In addition to the binding site discussed above, the hemagglutinin possesses a secondary site, at the interface between HA1 and HA2 polypeptide chains [34]. However, crystallographic studies have shown that sialyl(a24)lactose does not bind to the secondary site at all, while sialyl(a2-3)lactose binds to this site with at least 4 times lower affinity than to the first one. Another virus specific for sialic acid is murine polyoma virus. Two types of strains are known that differ in their specificity for sialic acid oligosaccharides: those that form large plaques bind to oligosaccharides terminating in NeuAc(a2-3)Gal, whereas the small plaque strains also tolerate branched structures with a second, a2-6 linked, sialic acid, e.g. NeuAc(a2-3)Galp3 ~ e u A c ( a 2 4 ) ] G a l N A c These . strains also differ in their ability to form tumors in mice - the small plaque strains produce few, if any, tumors, while

480

the large plaque strains are highly tumorigenic. The critical difference in the structure of the viral protein of these strains is in residue 91, which is glycine in the small plaque strains and glutamic acid in the large plaque strains. Crystallographic studies at 3.65 A resolution of the viral protein from small plaque strains in complex with sialyl-3-lactose have shown that the combining site is in the form of a shallow groove and that both the sialic acid and the galactose form contacts with the protein [14]. This is in contrast to influenza virus hemagglutinin, where only sialic acid makes such contacts. 2.2. Other specijicities Information on the existence of viral lectins with specificities other than for sialic acid is scant. Thus, parvovirus B 19 binds GalNAc(P 1-3)Gal(a14)Gal(fi1-4) [ 131, Simplex virus binds heparan sulfate [35] and HIV recombinant envelope glycoproteins interact specifically with certain N-linked carbohydrate units of glycoproteins, e.g. of the oligomannose type, with mannose-6-phosphate and sulfated polysaccharides, such as heparin and dextran sulfate [36,37].

3. Bacteria The largest number of microbial lectins characterized to date are from bacterial sources (Table 1).

3.1. Mannose-specijic (type I jimbriae) 3.1.1. Enterobacteria Type 1 fimbriae are expressed by most strains of E. coli, as well as by other enterobacteriaceae, such as Klebsiella pneumoniae [38,39] (Fig. 2). They are heteropolymeric organelles, about 7 nm in diameter and 100 to 200 nm in length, consisting of helically arranged subunits (pilins) of several different types, assembled in a well defined order[5,40]. The bulk of the fimbrial filament (shaft) is made up of polymers of a major subunit, FimA, mw 17 kDa. In addition, fimbriae contain a cassette of three minor ancillary subunits, FimF, FimG and FimH. The latter is the only subunit that possesses a

Fig 2. Type I fimbriated Eschenchia coh; magnified 24 OOOx (courtesy Dr. A. Gbarah)

48 1

carbohydrate binding site and is thus responsible for the sugar specificity of the finibriae. Although FimH is present both at the distal tip and at intervals along the length of the filament, only the subunit at the tip appears to be able to mediate mannose-sensitive adhesive interactions; the subunits at the other positions are inaccessible to the ligand. In other types of fimbriae (e.g. type P) the carbohydrate binding subunit is exclusively located to the tip. Isolated FimH binds mannose-containing glycoproteins and adheres in a mannosespecific manner to human neutrophils [41]. Moreover, it triggers an oxidative burst in a manner that mimics the activity of type 1 fimbriae. In addition, inert microspheres coated with FimH, but not with bovine serum albumin, are phagocytosed by neutrophils. Mutants lacking t h e j m H gene, but not genes encoding other fimbrial subunits, fail to bind to eukaryotic cells. Two proteins, FimC and FimB, are involved in the biogenesis of type 1 fimbriae, without being part of the final structure. The former acts as a periplasmic chaperon that stabilizes fimbria subunits in the periplasm through the formation of distinct complexes. The subunit-chaperon complexes are targeted to FimB, an outer membrane protein, which organizes their ordered secretion into an extracellular polymer [42]. Chaperons are also required for the assembly of other types of fimbriae, as well as of non-fimbrial lectins [43451. The expression of fimbriae is phase variable, i.e. bacteria shift periodically between a fimbriated and non-fimbriated state [38]. As a result, a given bacterial population will always contain cells of both phenotypes. The on- and off-phase variation is controlled at the transcriptional level and involves the inversion of a 314-base pair DNA segment harboring the promoter of t h e j m A gene [46]. Remarkable differences are found in the size and antigenic properties of the structural subunit of type 1 fimbriae (FimA) among different species of enterobacteria[47]. In contrast, a high degree of conservation is found between FimH proteins from type 1 fimbriae expressed by various species of the Enterobacteriaceae family, although they differ in their fine sugar specificity [48]. Thus, the combining sites of type 1 fimbriae of E. coli and K. pneumoniae correspond to the size of a trisaccharide and are in the form of a depression or pocket on the surface of the lectin [49]. In the case of the E. coli lectin, there are probably three adjacent subsites, each of which accommodates a monosaccharide residue. In the proximity of the combining site there is a hydrophobic binding region, as indicated by the finding that aromatic a-mannosides are significantly more powerful inhibitors than methyl a-mannoside. In contrast, several Salmonella species examined bind aromatic a-mannosides, as well as the trisaccharide Man(a I-3)Man(fi I-4)GlcNAc, weaker than methyl a-mannoside, indicating that the combining site of the Salmonella lectin is probably smaller than that of E. coli and K. pneumoniae, and is devoid of a hydrophobic region. Although very similar in their carbohydrate specificity, the lectins from E. coli and K. pneumoniae differ in their affinity for aromatic mannosides. Thus, 4-methylumbelliferyl a-mannoside is about 10-fold more effective than p-nitrophenyl a-mannoside in inhibiting yeast aggregation by type 1 fimbriated E. coli, while it is only 4 times more effective than p-nitrophenyl a-mannoside as inhibitor of K. pneumoniae [50]. Since the FimH subunits of the two organisms exhibit 88% homology, the possibility has been

482 Table 2 Glycoprotein receptors for microbial lectins Organism

Source of glycoprotein

Designation

Human granulocytes, peritoneal macrophages

CD1 l/CD18 integrin

Human granulocytes

NCA-50

Colonic mucosa Urine

IgA Tamm-Horsfall glycoprotein

Sialic acid specific E. coli CFA/I I? fakiparum

Human epithelial cells

26 kDa gp

Human erythrocytes

Glycophorin

P aeruginosa, PAK

Human oral epithelium

82 kDa, 40-50 kDa gp’s

Mannose specific E. coli type 1

Reference( s)

Mouse corneal cells S. sanguis

Human saliva

400 kDa gp

Human oral epithelium

160 kDa gp

Human saliva

180 kDa gp

GaVGalNAc specific

A . naeslundii type 2

B. pertussis toxin

CHO cells

165 kDa gp

Human T cells, Jurkatt

43 kDa gp; 70 kda gp

considered that the differences in specificity are due to differences in the presentation of the combining sites of these subunits in the fimbriae. To test this hypothesis, two types of hybrid fimbriae were genetically generated: in one of these the E. coli FimH was presented on a filament of K. pneumoniae structural subunits (EcFimH-KpFimA); in the other K. pneumoniae FimH was presented on a shaft of E. coli FimA (KpFimH-EcFimA) [50]. It was found that the specificity of the EcFimH-KpFimA hybrid with respect to aromatic a-mannosides was similar to that of native K. pneumoniae lectin, whereas that of KpFimH-EcFimA was like that of the E. coli lectin. These results indicate that the shaft on which FimH is presented plays a role in modulating the specificity of type 1 fimbriae lectins, probably by imposing conformational constraints on the carbohydrate binding subunit. The interspecies heterogeneity of the FimA subunit of enterobacteria thus ensures significant diversity in their sugar specificity and as a result, in the function of their lectins, as reflected by their ability to mediate adhesion to a particular type of animal cell. The notion that the fimbrial filament can influence the specificity of the carbohydrate binding moiety is novel and contrasts with the P fimbriae system, in which PapG appears to be the sole determinant of binding specificity. Recently a form of type 1 fimbriae has been described which, in addition to binding carbohydrates, interacts also with non-glycosylated regions of proteins in a mannoseinhibitable manner [51]. It is not clear whether this interaction occurs via the carbohydrate binding site proper and how it is inhibited by mannose. The difference between the two

483

functional forms of the fimbriae may be due to subtle variations in FimH or to quantitative or qualitative differences in the assembly of one or more of the subunits. In this context it should be noted that concanavalin A, a mannose/glucose-specific plant lectin, has also been shown to bind peptides in a carbohydrate-inhibitable manner [52,53]. Type 1 fimbriated E. coli or the isolated fimbriae bind to glycoproteins from diverse sources [8]. These include a 65 kDa glycoprotein from guinea pig erythrocytes [54], the carcinoembryonic antigen, normally localized at the apical border of epithelial cells of the large intestine, secretory IgA and IgA myeloma proteins, especially those of the IgA2 subclass [55] Tamm-Horsfall glycoprotein (often referred to as uromucoid) [56], as well as several constituents of mucous layers [57] (see also section 6.1.2). The fimbriae bind to three glycoproteins derived from the cell membrane of human granulocytes (or neutrophils)[58] (Table 2). Two of them have been identified as components of the integrin superfamily CD1 l/CD18 (also known as leukocyte adhesion molecules). The fimbriated bacteria bound in a mannose-specific, dose-dependent and saturable manner to the isolated integrin in wells of microtiter plates and on SDS-PAGE gels; this binding was inhibited by monoclonal antibodies to integrin 1591. Monoclonal antibodies to CDI 1/CD18, but not to other granulocyte surface antigens, inhibited binding of the bacteria to the granulocytes. The same molecules serve also as receptors for type 1 fimbriae on human peritoneal macrophages [60]. In addition, a glycoprotein called NCA-50, which belongs to a family of non-specific cross-reacting antigens associated with the granulocyte membrane, was reported to specifically bind type 1 fimbriae [61]. 3.2. Sialic-acid-specijic

3.2.I . Escherichia coli Certain strains of E. coli isolated from humans and farm animals express fimbrial lectins specific for glycoconjugates containing sialic acids [3-51. This conclusion is based primarily on the observation that hemagglutination caused by these organisms is decreased or completely abolished by treatment of the erythrocytes with sialidase. Examples of such lectins are type S fimbriae of bacterial strains causing sepsis and meningitis in newborn infants, CFA (Colonization Factor Antigen) I and I1 of human enterotoxigenic E. coli isolates, as well as E. coli K99 fimbriae of enterotoxigenic strains isolated from piglets, calves and lambs suffering from diarrhea. The structure of S fimbriae is very similar to that of type 1 fimbriae. They are composed of a major subunit and several minor components, of which only one binds sialic acid. In contrast, both in the CFAs and K99 fimbriae the major subunit also contains the carbohydrate binding site [72,73]. Comparison of the amino acid sequence of the carbohydrate binding subunit of type S fimbriae with those of the major CFA 1 and K99 subunits revealed the presence of a common motif, rich in basic amino acids (Fig. 3). Site-specific mutagenesis experiments showed that a lysine and an arginine residue in this region play a part in ligand binding [74]. S-fimbriated E. coli combine with a2-3 linked sialic acid residues on integral membrane glycoproteins. Limited trypsinization of human erythrocytes completely abolished binding of such bacteria, indicating that glycophorin A is their sole receptor on the erythrocytes[75]. S-fimbriated E. coli also bind to sialic acid on gangliosides,

484

Organism

Amino acid residues

Reference

Escherichia coli

SfaS

-

K99

LYS”~ -

-

LysSh

-

CFAiI Helicobacter pyIori

Lys”‘

-

Lys134 -

Ala

~~

-

Arg

Ala

&I

Ser

-

Lys

[74]

LYS -

Asp

-

Asp

Arg

[74]

Val

Lys

[74]

&

Glu

Lys -

[89]

Lys -

Val

Arg -

Thr

Fig. 3. Sequence homology in the binding region of sialic-acid-specific bacterial lectins. Identical or functionally identical residues are underscored. Gaps have been introduced for optimal alignment.

preferentially NeuGc(a2-3)Gal and NeuAc(a2-8)NeuAc [ 151, as well as to sulfated glycolipids [76]. The latter binding apparently occurs through a different fimbrial subunit than that which interacts with sialic acid. The specificity of CFA I is not well defined, beyond the fact that it recognizes sialic acid [77,78]. A sialoglycoprotein with an apparent molecular weight of 26,000 is the only glycoprotein from the human erythrocyte membrane that binds CFA I fimbriated E. coli [621. K99-bearing E. coli bind to sialylated mucus glycoproteins [79]. The binding is not inhibited by sialic acid nor by other simple sugars, but by glycopeptides isolated from glycoproteins of bovine plasma, suggesting that the lectin recognizes complex carbohydrate structures. Glycopeptides bearing the terminal NeuGc(a2-3)Gal sequence strongly inhibited hemagglutination caused by E. coli K99, demonstrating the specificity of these bacteria for N-glycoloylneuraminic acid [SO]. The physiological receptor for K99 on intestinal epithelial cells of pig [Sl] and horse [82] appears, however, to be the glycolipid N-glycoloylneuraminyllactosyl ceramide [NeuGc(a2-3)Gal(P14)Glc~lCer]. 3.2.2. Streptococcus suis The Gram positive bacterium S. suis, a common cause of sepsis, meningitis and other serious infxtions in young piglets and also of meningitis in humans, agglutinates human erythrocytes, but not after treatment with sialidase. Resialylation of the desialylated erythrocytes with a2-3 sialyltransferase resulted in strong agglutination of the cells by the bacteria, whereas resialylation with sialyltransferases having different specificities gave cells that were poorly agglutinated [83]. Blotting experiments revealed binding to band 3, band 4.5 and glycophorin (as well as to polyglycosyl ceramides) of human erythrocyte membranes. The involvement of glycophorin as a ligand for the bacteria on intact cells is however excluded by the finding that trypsinization of the cells does not affect their agglutination by the bacteria and by the agglutinability of En(a-) erythrocytes which are defective in glycophorin A. The ligands for S. suis are thus the sialylated polyN-acetyllactosamine glycans carried by band 3 and band 4.5.

485

3.2.3. Streptococcus sanguis S. sanguis, an oral microorganism, adheres to saliva-coated tooth surfaces by binding

to salivary glycoproteins. The binding to these glycoproteins on SDS-PAGE blots was abolished by their treatment with sialidase, as well as with hydrofluoric acid, but was not affected by peptide-N-glycanase F (PNGaseF), indicating that S. sanguis binds to sialic acid on 0-linked chains of the glycoprotein(s) [84]. A 23 kDa membrane glycoprotein from human buccal epithelial cells bearing 0-linked NeuAc(a2-3)Gal(@ 1-3)GalNAc chains was implicated as receptor for S. sanguis OMZ9 on these cells [21]. The cDNA of the sialic-acid-specific lectin of S. sanguis codes for a polypeptide of 1435 residues (calculated mw of 158.4kDa) with three unique domains, two of which consist of repetitive amino acid sequences [66]. The third, which resides near the carboxy terminus, contains 48% proline. The lectin bound to a single salivary glycoprotein of mw 400 kDa [66]. Binding was inhibited by sialic acid and was abolished by desialylation of the glycoprotein; the best inhibitor was N-acetylneuraminyllactose.

3.2.4. Helicobacter pylori H. pylori is a pathogen which colonizes the mucus layer of human gastric tissues and is associated with gastritis and peptic ulcers (and possibly also with gastric carcinoma). It exhibits several specificities, one of which is for sialic acid. H. pylori agglutinates erythrocytes [85] and binds to mouse adrenal gland cells [86] in a sialic-acid dependent fashion. Furthermore, specific binding of H. pylori to acid glycosphingolipids extracted from human gastric mucosa, such as the ganglioside GM3, has been reported[87]. In addition, H. pylori has an affinity for fucose (see section 3.4.1) and interacts with glycolipids such as cerebroside and sulfated lactosylceramide [88], which lack both sialic acid and fucose. The inhibitory activity of lactosylceramide sulfate and GM3 ganglioside on hemagglutination induced by H. pylori was additive, consistent with the possibility that two distinct lectins are involved in the binding to sialic acid and to sulfated glycolipids, respectively. The sialic-acid-specific lectin is a fibrillar surface protein with a mw of 20 kDa. Its cDNA has been cloned, sequenced and expressed in E. coli [89]. A sequence of the lectin (residues 134-139) was found to be homologous to a region that forms part of the carbohydrate binding sites of the sialic-acid-specific lectins of S-fimbriae, K99 and CFA I (cf. Fig. 3). An antibody against a synthetic peptide containing the above sequence blocked hemagglutination of human erythrocytes by H. pylori, suggesting that in this lectin, too, it is part of the carbohydrate binding site. 3.2.5. Mycoplasma pneumoniae M. pneumoniae, a well-established pathogen of the human respiratory tract, is another organism specific for sialic acid. It adheres to animal cells primarily through a lectin, known as P1 protein (mw 170kDa), which is densely clustered at the tip of the organism [90]. The lectin is specific for N-acetylneuraminic acid linked a2-3 to terminal galactose residues of the poly-N-acetyllactosamine sequence of blood type I/i antigen, as shown by binding experiments with sialidase-treated human erythrocytes that have been resialylated by specific sialyltransferases [9 11. The preference for sialic acid a2-3 linked, rather than a2-6 linked, was confirmed by the finding that the oligosaccharides and glycoproteins containing the former linkage were more inhibitory than those containing

486

the latter one. The most potent inhibitors were glycopeptides derived from bands 3 and 4.5 of human erythrocytes, and the bovine erythrocyte glycoprotein GP-2, all rich in poly-N-acetyllactosamine chains. Further evidence for the importance of such sequences in binding the M. pneumoniae lectin was provided by experiments with human blood type i erythrocytes, whose linear poly-N-acetyllactosamine chains are susceptible to digestion with endo 6-galactosidase. Following treatment with the enzyme, the binding of M. pneumoniae to the erythrocytes decreased by 85% [92]. 3.3. Gal and GulNAc-specijk

3.3.1. Escherichia coli CS3, a subcomponent of CFA I1 of enterotoxigenic E. coli binds specifically to GalNAc(6 1-4)Gal[93]. This was demonstrated by inhibition studies, using well-characterized antibodies and glycoconjugates of defined structures. Support for these findings was provided by electron microscopic experiments showing that the disaccharide, 0-linked to bovine serum albumin via a spacer, localized around bacteria expressing CS3 but not around CS3-negative mutants. Enterotoxigenic E. coli producing K88 fimbriae occur in three serological variants: ab, ac or ad, that differ in their fine carbohydrate specificity. All are galactose-specific, but whereas K88ab fimbriae recognize the sequence Gal(al-3)Gal[94], K88ac fimbriae appear to bind preferentially to Gal@-3)GalNAc and Fuc(al-2)Gal(~1-3/4)GlcNAc[ 171. These carbohydrate structures were shown to be present in two porcine brush border glycoproteins of 210 and 240kDa that bind K88ac fimbriae, but not K88ab and K88ad fimbriae. They were not detected in glycoproteins from brush borders of piglets that do not bind K88 fimbriated E. coli. 3.3.2. Pseudomonas aeruginosu I! aeruginosa, an opportunistic pathogen, capable of causing infections of eye, lung, skin and other parts of the body, produces two well-Characterized intracellular lectins; one is specific for galactose (PA-I), the other for fucose (PA-11, see section 3.4.2) [95]. PA-I exhibits a preference for a-galactosides, especially those with a hydrophobic aglycone[96]. The cDNA of PA-I was isolated and shown to encode a chain of 121 amino acids (mw 12.7kDa) with a predominant central hydrophilic core between two hydrophobic domains [97]. PA-I agglutinates papain-treated human erythrocytes independent of blood type. However, it exhibits preferential affinity for the branched oligosaccharides bearing both A and B blood group determinants that are present in the saliva of AB secretors [98]. Although PA-I is located mainly intracellularly, evidence has been obtained that it is also exposed on the cell surface [99]. This could explain the finding that injection of the purified lectin into mice protected the animal against lethal doses of the live bacteria [951. I? aeruginosa appears to bind largely to glycolipids [l], but binding to human respiratory mucins has also been reported. The binding is sensitive to periodate oxidation of the mucins, suggesting the involvement of the carbohydrate chains of the mucins and a (putative) surface lectin(s) on the bacteria [ 1001. Inhibition studies have shown that the

487

organism recognizes Gal(@1-3)GlcNAc and Gal(fl14)GlcNAc, but has no affinity for sulfated glycopeptides [loll. This lectin(s) has, however, not yet been isolated and its relation to PA-I is not known. 3.3.3. Actinomyces species Actinomyces naeslundii and Actinomyces viscosus are prominent oral bacteria that colonize tooth and mucosal surfaces by binding to epithelial cells or other bacteria, such as S. sanguis. These interactions are mediated by-galactose/@-N-acetylgalactosaminespecific lectins, associated with type 2 fimbriae [ 19,20,102]. Although isolated type 2 fimbriae alone do not agglutinate either S. sanguis or sialidase-treated erythrocytes, lactosespecific agglutination occurred when the cells were incubated with multivalent complexes, formed by crosslinking the fimbriae with small amounts of specific antibody. The actinomyces lectin has not yet been purified. A fimbrial subunit gene,jmA, has been cloned, but the protein it encodes is apparently not involved in the interaction with S. sanguis. The carbohydrate specificities of the lectins of A . viscosus T14V and A. naeslundii WVU45 were defined using galactose-containing oligosaccharides as inhibitors of coaggregation with S. sanguis 34. The most effective disaccharide inhibitor was Gal(fi1-3)Gal, which was more than 10 times as active as lactose and also more active than any galactose disaccharide tested. Receptors for the actinomyces lectins have been isolated and extensively characterized from four streptococcal strains. All are linear cell wall polysaccharides, composed of repeating hexa- (or hepta-) saccharide units linked by phosphodiester bonds to the 6-carbon of the non-reducing terminal sugar of the repeating unit and all contain N-acetylgalactosamine. Two of them contain the sequence Galf((31-6)GalNAc(fl1-3)Gal(al-),in the other two it is Galf(P1-6)Ga1((3 13)GalNAc(al-) [102a]. This region is considered to be important in determining the recognition of the streptococci by the actinomyces lectins. The binding of A . naeslundii WVU45 to sialidase-treated monolayers of epithelial cells was inhibited by pretreatment of the latter with peanut agglutinin and Bauhinia purpurea lectin [103]. Although these two lectins differ in their fine specificity, both react well with Gal@ 1-3)GalNAc, and similar to the actinomyces lectins, their binding to epithelial cells is enhanced by treatment of the cells with sialidase. In contrast, Erythrina cristagalli lectin, specific for Gal@ 14)GlcNAc, failed to inhibit bacterial binding. These and other experiments with lectins led to the conclusion that the receptor for the actinomyces lectin on epithelial cells is most likely 0-linked GaI((31-3)GalNAc. A. naeslundii binds to a glycoprotein of mw 160 kDa extracted from oral epithelial cells [67]. A. naeslundii WVU45, as well as some other strains of actinomyces expressing type 2 fimbriae, bind also to sialidase-treated polymorphonuclear leukocytes, resulting in the activation of the latter cells, phagocytosis and destruction of the bacteria. The interaction of A . naeslundii WVU45 with the leukocytes was inhibited by the same lectins that inhibited the binding of these bacteria to oral epithelial cells. The receptors for the organism on the surface of sialidase-treated polymorphonuclear cells were identified as a 130 kDa glycoprotein as well as asialoganglosides with Gal((31-3)GalNAc termini [ 1041. Certain actinomyces species, such as A. naeslundii 12104 and A. viscosus 19246 and 147, have an affinity for GalNAcfi-terminating oligosaccharides. These strains exhibit heterogeneous receptor specificities and bind to different salivary and submaxillary

488

glycoproteins on blots [20]. A 180 kDa salivary glycoprotein that binds A. naeslundii has been isolated and characterized [68]. A special type of coaggregation of oral bacteria is that resulting from the bridging between one cell type and its partner by a third organism. This happens with Prevotella loescheii PK1295, which can serve as a bridge between Streptococcus oralis 34 and Actinomyces israeliz PK14, two Gram positive oral bacteria that are otherwise unable to coaggregate. Coaggregation of P loescheii PK1295 with S. oralis 34 is inhibited by lactose, while that with A. israelii PK14 is not, indicating that two different kinds of adhesins are involved, at least one of which is a lectin [105]. The latter has been isolated and purified to electrophoretic homogeneity, the first from oral bacteria. It is a fimbriaassociated protein with a molecular weight of 450kDa and consists of six identical subunits. In its oligomeric form it agglutinates S. oralis 34 and a variety of sialidasetreated erythrocytes in a lactose-sensitive manner, while the individual protomers blocked coaggregation between P loescheii PIC1295 and S. oralis 34 [ 1061. 3.3.4. Rhizobia A lectin specific for galactose has been isolated from Bradyrhizobium japonicum [ 107, 1081. It is a protein of 38kDa that binds lactose about 15 times better that galactose and does not recognize N-acetylgalactosamine. The lectin is localized at one pole of the bacterial cell surface, which is coincident with the site of cell-cell contact in homotypic aggregation of the bacteria and in their adhesion to the cultured soybean cell line SB-1. Such topological distribution is consistent with a role for the lectin in the polar binding of the organism to soybean roots.

3.3.5. Myxobacteria Myxobacteria differ from other bacteria in being social organisms. They tend to maintain close contact with each other and to aggregate into swarms. Aggregation is a developmentally regulated process that occurs as the cells differentiate from the vegetative form into mature spores. In Myxococcus xanthus, high hemagglutinating activity was found in extracts of the mature cells, but not in early vegetative cells. A lectin was purified from extracts of the aggregated stage of this organism. In solution it exists as a monomer with an apparent molecular weight of 28 kDa. The hemagglutinating activity of the lectin was not inhibited by simple sugars, only by glycoproteins such as fetuin, glycophorin and rabbit IgG, all of which contain the 0-linked tetrasaccharide NeuAc(a23)Gal(fl1-3)[NeuAc(a2-6)]GalNAc.The penultimate galactose was directly implicated in the affinity of the lectin for the saccharide, since inhibition by asialofetuin was diminished to one-fifteenth by fl-galactosidase treatment. The lectin was detected on the surface of developmental, but not vegetative cells, localized in distinct patches at one or both of the cell poles. This localization suggests that the lectin may function in end-to-end cellular interactions during aggregation. 3.4. Fucose-specific 3.4.1. Helicobacter pylori A specificity exhibited by H. pylori (in addition to that for sialic acid) is for fucose.

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The putative lectin discriminates between closely related fucosylated epitopes as well as carbohydrate core chains, and only binds H and Lewisb antigens expressed on lacto-series type 1 but not type 2 chains [109]. To test the hypothesis that Leb antigen functions as receptor for H. pylori and mediates its attachment to gastric pit/mucous cells [ 1 101, mice that normally do not synthesize this carbohydrate structure were genetically engineered to produce it by transfection with human ~ ~ 1 , 3 1fucosyltransferase 4 [I 1 I]. Expression of Leb in the transgenic mice was associated with acquisition of the ability to bind clinical isolates of H. pylori. Binding was blocked by pretreatment of the bacteria with soluble Leb-serum human albumin conjugates.

3.4.2. Pseudomonas aeruginosa PA-11, an intracellular lectin of P aeruginosa, is specific for fucose with an unusually high affinity ( K , = 1.5x lo6 M-') and interacts weakly also with mannose ( K , = 3.1 x lo2 M-2) [112]. 3.4.3. Vibrio cholerae Agglutination of human group 0 erythrocytes by K cholerae and adhesion of the organism to brush borders are specifically inhibited by fucose, and to a lesser extent by niannose. It has been suggested that structures on eukaryotic cell surfaces containing fucose may function as receptors for a vibrio lectin and may therefore be an important determinant of host susceptibility to these bacteria. A soluble lectin specific for fucose, produced by ci cholevae strain CA401 was purified to apparent homogeneity and was found to focus at three different PI: 6.3, 5.3 and 4.7 [I 131. Thus, there are apparently three distinct PI isotypes of the lectin that exist as non-covalently associated polymers of 32kDa subunits. The lectin possessed proteolytic activity, which likewise focused at pH values 6.3, 5.3 and 4.7. It was therefore concluded that the soluble lectin is a bifunctional molecule, capable of mediating hemagglutination and proteolysis. 3.4.4. Others Recently, a fucose-specific lectin associated with the bacterial surface of Rhizobium lupinii has been purified, but its role in the interactions between the bacteria and the lupin root has not been established [ I 141. A fucose-specific lectin has also been isolated from the cell walls of Agrobacterium tumefaciens, a bacterium belonging to the Rhizobiaceaea family which infects dicotyledonous plants and forms crown gall [ 1 151. 3.5. Multiple specijcities 3.5.1. Bordetella pertussis Pertussis toxin, produced by virulent strains of B. pertussis, the etiological agent of whooping cough, is a classical A-B type toxin comprised of an A subunit that possesses ADP-ribosyltransferase activity and is responsible for most of the biological effects of the toxin, and a B subunit with affinity for carbohydrates. The B subunit of the pertussis toxin is a pentamer composed of four different subunits (S2-S5). The toxin acts as a hemagglutinin and exhibits dual carbohydrate specificity, due to

490

Selectins

Toxin subunits S 2 P l 9 Y G R c A N K T R A L T = S3

419

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P N C; T R A

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