Advances in Macromolecular Carbohydrate Research, Volume 1

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ADVANCES IN MACROMOLECULAR CARBOHYDRATE RESEARCH

Volume 7

1 997

This Page Intentionally Left Blank

ADVANCES IN MACROMOLECULAR CARBOHYDRATE RESEARCH Editor:

ROBERT J. STURGEON Department of Biological Sciences Heriot- Watt University Edinburgh, Scotland

VOLUME 1

1997

@) jAI PRESS INC. Greenwich, Connecticut

London, England

Copyright © 1997 by JAI PRESSINC 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESSLTD. 38 Tavistock Street Covent garden London WC2E 7PB England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-323-2 Manufactured in the United States of America

CONTENTS

LIST OF CONTRIBUTORS PREFACE

Robert J. Sturgeon

vii

ix

ARABINANS AND ARABINAN DEGRADING ENZYMES

G. Beldman, H.A. Schols, S.M. Pitson, M.J.F. Searle-van Leeuwen, and A.G.J. Voragen

STRUCTURAL ELUCIDATION OF THE N-LINKED OLIGOSACCHARIDES OF GLYCOPROTEINS USING HIGH pH ANION-EXCHANGE CHROMATOGRAPHY

Kevin D. Smith, Elizabeth F. Hounsell, John M. McGuire, Moira A. Elliot, and Heather G. Elliot

NEOGLYCOCONJUGATES AS ARTIFICIAL ANTIGENS: CHEMICAL ASPECTS

Nikolay K. Kochetkov and Anatoly Ya. Chernyak

65

93

GLYCOSYLATION PATTERNS IN MUCUS GLYCOPROTEINS

Amalia Slomiany, Chinnaswamy Kasinathan, and Bronislaw Slomiany

INDEX

177 213


LIST OF CONTRIBUTORS

C. Beldman

Department of Food Science Wageningen Agricultural University Wageningen, The Netherlands

Anatoly Ya. Chernyak

N.D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences Moscow, Russia

Heather G. Elliot

Department of Pharmaceutical Sciences University of Strathclyde Glasgow, Scotland

Moira A. Elliot


Elizabeth F. Hounsell

Department of Biochemistry and Molecular Biology University College London London, England

Chinnaswamy

Research Center University of Medicine and Dentistry of New Jersey Newark, New Jersey

Kasinathan

Nikoiay K. Kochetkov

N.D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences Moscow, Russia

John M.

McGuire


LIST OF CONTRIBUTORS

SM. Pitson


HA. Schols


M.j.F. Searle-van Leeuwen


Amalia

Research Center

Slomiany

University of Medicine and Dentistry of New Jersey Newark, New Jersey Bronislaw

Slomiany

Research Center University of Medicine and Dentistry of New Jersey Newark, New Jersey

Kevin D. Smith


A.G.j. Voragen


PREFACE

The decision to introduce a new series, Advances in Macromolecular Carbohydrate Research, was taken as a result of the remarkable advances which have been made in recent years in the understanding of the chemistry, biochemistry, and biology of carbohydrates. By far the most significant advances have been made in knowledge of the structure and function of carbohydrates in the macromolecular state, whether polysaccharides, glycoproteins, or glycolipids. New terms created to describe such work—^for example Glycobiology, Glycotechnology, and Glycoengineering—now form part of the everyday vocabulary of those interested in and/or working in the field of carbohydrates. Many of the advances achieved arise from the design and implementation of new analytical techniques, such as nuclear magnetic resonance spectroscopy and mass spectrometry, which allow the study of minute amounts of material. This new knowledge has already been applied, for example, in the synthesis of carbohydrate therapeutics, in the engineering of proteins such as carbohydrate hydrolases, for the conversion and modification of polysaccharides, and in the use of recombinant DNA technology to produce glycoproteins of importance in health and disease.

X

PREFACE

In compiling this volume, authors were invited to submit chapters which are not necessarily exhaustive reviews, but which address those areas of the subject in which the authors believe the most important advances are being made. The chapters therefore reflect the interests and views of the individual authors and do not attempt to provide exhaustive citations of early literature. This volume contains four chapters. In "Arabinans and Arabinan Degrading Enzymes", Beldman et al. review an area which is becoming more and more important to plant biochemists, food technologists, and nutritionists. The chapter deals with the isolation of arabinans together with their structural and physical characteristics. Current understanding of the mode of action of arabinan degrading enzymes is of importance in food technology. "Structural Elucidation of the A^-Linked Oligosaccharides of Glycoproteins" is covered by Smith et al. The authors demonstrate the value of high pH anion-exchange chromatography in the study of the heterogeneity of 7V-glycosylated structures in glycoproteins. Functional studies have demonstrated that, in addition to other properties, the oligosaccharide chains of glycoproteins are important determinants of the overall biological activity of these macromolecules. It was established a number of years ago that many disease-creating microorganisms contain polysaccharides or glycoconjugates on their outer cell surfaces, and that these molecules can be used to prepare vaccines for the protection of an animal species against those diseases. Kochetkov and Chemyak discuss the chemical aspects of the "Production of Neoglyconjugates as Artificial Antigens". The increasing use of neoglycoconjugates has led to a rapid development of new synthetic approaches to their production. Mucus glycoproteins (mucins), unlike the more commonly studied animal glycoproteins which bear mainly //-linked oligosaccharides contain almost exclusively 0-glycosidically-linked carbohydrate. Slomiany et al., in reviewing the gylycosylation patterns, concentrate on addressing the area of research relating to apomucin synthesis and co-translational modifications. It is clear that research initiatives will continue to influence advances in carbohydrate research for many years to come. The chapters presented here of necessity focus on a relatively small window of what is a vast subject, and provide valuable insight into a number of aspects

Preface

xi

of a rapidly developing field that will undoubtedly continue to increase in importance in the future. Robert J. Sturgeon Editor


ARABINANS AND ARABINAN DEGRADING ENZYMES

G. Beldman, H.A. Schols, S.M. Pitson, MJ.F. Searle-van Leeuwen, and A.G.J. Voragen

Note: This chapter was originally accepted for publication in 1992. An Appendix at the end of the chapter contains updated material from 1992-1997. I. Introduction II. Substrates A. Arabinans in General B. Arabinoxylans C. Arabinogalactans D. Isolation of Arabinans E. Pretreatment of Raw Material F. Extraction G. Purification

Advances in Macromolecular Carbohydrate Research Volume 1, pages 1-64. Copyright © 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-323-2

1

2 3 3 5 5 8 8 9 9

:

BELDMANETAL.

H. Characterization of Isolated Arabinans 11 III. Enzymes 20 A. Classification of Arabinanases 20 B. Cooperative Effects during Enzymic Degradation of Arabinose Containing Polysaccharides . . . 33 C. Purification and Assay Methods for Arabinanases . . 36 IV. Concluding Remarks 40 V. Appendix: Review of the Literature 1992-1997 41 A. Substrates 41 B. Enzymes 42 Acknowledgments 55 References 56

I. INTRODUCTION Polysaccharides containing L-arabinose residues are important in many plant tissues. They occur as either homoglycans, generally associated with pectins or, in a number of plant tissues, in a genuine, natural form, or as heteroglycans such as arabinoxylans, arabinogalactans, and arabinogalactan proteins [1-7]. Here we have reviewed arabinans and arabinan degrading enzymes. This subject is becoming more and more important to plant biochemists, food technologists, and nutritionists. Plant biochemists are interested in the biosynthesis of arabinans, their functions as a cell wall constituent in cell wall cohesion, cell growth, metabolic exchanges between plant tissues and soil, and in germination [8-11]. Arabinans have also attracted the attention of food technologists because of their role in fruit and vegetable processing and their occurrence in by-products of some processed plant crops [12—19]. Recently, nutritional aspects of arabinans as component of dietary fiber and their possible physiological activities are mentioned [20-22]. Medical research shows that some arabinans exhibit biological activities in humans [23-25]. The food industry is also interested in arabinan degrading enzymes where their application in fruit and vegetable processing are explored in upgrading of arabinan-rich by-products and in bioconversion of plant biomass [12,14,15,18,19,26-28].

Arabinans and Arabinan Degrading Enzymes

3

Arabinan degrading enzymes also have become essential analytical tools in the elucidation of the fine structure of arabinans [19,29,30]. In this review we have collected information on the occurrence of arabinans and arabinan degrading enzymes. The isolation of the arabinans and their structural and physical characteristics are considered. Also, the isolation, purification, and characterization of enzymes able to degrade these arabinans are covered. To this end, the mode of action of arabinan degrading enzymes is related to their substrate specificity. The possible role of arabinans and the arabinan degrading enzymes in food technology are discussed. Earlier reviews related to this subject are from Whitaker [31] and Kaji [32]. IL SUBSTRATES A. Arabinans in General Arabinans, mainly composed of a-L-arabinofuranosyl residues, are generally arranged in (l->5)-linked chains with varying n u m b e r s of r e s i d u e s s u b s t i t u t e d with other a-Larabinofuranosides at the 0 2 and/or 0 3 position. In some sources, they have been isolated by nondegrading extraction as homopolysaccharides. Other arabinans contain small amounts of additional sugar residues of uncertain significance. Another class of arabinans, containing other constituent sugars, including galacturonic acid, and isolated under degrading conditions, probably originates from the residual stubs of pectins [1-6]. A schematic structure of arabinan is shown in Figure 1. Studies on pectins from many sources clearly reveal that pectin is not a homopolysaccharide, but is constituted of a backbone of (axialaxial) a-(l~>4)-linked D-galacturonopyranosyl units interrupted at intervals by the insertion of a-(l->2)-linked L-rhamnopyranosyl residues in adjacent or alternate positions [1,6,31]. Other constituent sugars are attached in side chains to this rhamnogalacturonan backbone. They comprise D-galactose, L-arabinose, and D-xylose—^the more common ones—ând D-glucose, D-mannose, L-fucose, and D-glucuronic acid which are found

BELDMANETAL

HOCH.

Figure 1. Schematic structure of a highly branched arabinan.

less frequently. Most of these sugars occur in short side chains, one of three units long, glycosidically linked to 04 and 03 of L-rhamnopyranose or 02 or 03 of some of the galacturonosyl residues. The main sugars, D-galactose and L-arabinose, are present in more complex chains with structures similar to genuine arabinans and arabinogalactans and with chain lengths which can be considerable [2-6]. Pectins with arabinans attached have been described for apples [19,20,22,33-35], sugar beets [36-40], suspension-cultured sycamore cell walls [41], rape-seed [42], apricots [43,44], tomatoes [45], carrots [46,47], cabbage [21,48], cell walls of mung bean hypocotyls [8], horsebean roots [10], onions [49,50], pears [12], and angelica [51]. By degrading extracted pectins specifically in the galacturonan backbone by Pelimination [52—54] or enzymatically with endo-ôXygalacturonase, e«ô-pectin lyase or e/i^fo-pectate lyase [35,55— 61], oligogalacturonic acid fragments, and rhamnogalacturonan fractions of higher molecular mass in which virtually all of the neutral sugars were concentrated could be isolated. From these results it was concluded that there is an intramolecular distribution in which the neutral sugars are concentrated in blocks of more highly substituted rhamnogalacturonan regions ("hairy"), separated by unsubstituted regions ("smooth") containing almost exclusively D-galactopyranosyluronic acid residues [62].


5

Rhamnogalacturonans rich in neutral sugars were also isolated from other cell walls using pectolytic enzymes [6,49,63-68] and this can be seen as a confirmation of this concept. B. Arabinoxylans

Arabinose also occurs in considerable amounts in plant cell walls as a constituent of arabinoxylans and arabinogalactans. Arabinoxylans have a homopolymeric backbone of (l->4)linked p-D-xylopyranosyl residues substituted with (1-^2)linked and/or (l->3)-linked a-L-arabinofuranosyl residues as in the endosperm of annual plant cereals and together with (1-^2)linked a-D-glucuronic acid residues and/or its 4-0-methyl ether in soft woods, cereals, brans, hulls, corn cobs, straw, etc. In addition, the xylopyranosyl residues may be acetylated on 02 and/or 03 [2,4,5,69]. Arabinoxylans often contain ferulic and j!?-coumaric acids which are bound to C5 of the arabinofuranosyl side groups. Through oxidative dimerization of these ferulic acid residues arabinoxylan chains can be cross-linked and rendered insoluble [70,71]. C. Arabinogalactans

Arabinogalactans have been grouped by Clarke et al. [7] into three main structural types: the arabino-4-galactans (type I), the arabino-3,6-galactans (type II), and polysaccharides with arabinogalactan side chains like pectic substances and gum exudates (type III). Arabino-4-galactans have a linear chain of (l-^4)-linked P-D-galactopyranosyl residues with 20 to 40% aL-arabinofuranosyl residues (1^5)-linked in side chains connected in general to 03 of D-galactopyranosyl residues [2,6]. Arabinogalactan type II is a highly branched polysaccharide with ramified chains of P-D-galactopyranosyl residues joined by (l->3)- and (l->6)-linkages mainly in the exterior chains. Most of the side chains are terminated with L-arabinofuranosyl residues and to some extent L-arabinopyranosyl residues occur [2,4,5,6].

D O to D O

I o c 03

c ID v_ D

2
3)- and (l->5)-linked arabinosyl residues which are present in beet arabinan. The nomenclature of Rombouts et al. [89] will


23

Table 5. Some Properties of a-L-Arabinofuranosidase A from A. niger and S. purpurascens M^(Da) Optimum pH K^ (mMol/L) ^cat(în~')

pl Reference

A. niger

S. purpurascens

83,000 4.1 0.6' 15.7x10^' 6-^.5 89,138

62,000 6.5 0.082^ 5.5x10^*^ 3.9 90

Notes: "pH 5.0; 30 °C; /7-nitrophenyl-a-L-arabinofuranoside. V H 6.5; 30 °C; p-nitrophenyl-a-L-arabinofuranoside.

be used, referring to this type of enzyme as arabinofuranosidase B (Arafur B). Properties of the enzyme has been reviewed by Kaji [32] and Whitaker [31]. Its appearance in nature is extensive, including fungi, bacteria, actinomycetes, yeasts, protozoa, and plants. However, in order to compare the different arabinofuranosidases B properly, discussion will be restricted to those enzymes which have been purified and characterized in more detail. Arabinofuranosidases type B have been isolated from the microbial sources: A, niger, Cortitium rolfsi, Rhodotorula flava, Streptomyces massasporeus, Streptomyces sp. 17-1, Bacillus subtilis, Ruminococcus albus, Dichomitus squalens, and Trichoderma reesei. The enzyme has also been purified from plant sources including: Scopolia japonica, Lupinus luteus, and Daucus carota (carrots). Table 6 summarizes some properties of arabinofuranosidase B. Molecular weights of arafur B are generally below 100,000. The native form of the enzyme from R, albus is probably a tetramer with a molecular weight of 305,000-310,000 [101]. Most of the purified enzymes are optimally active at a pH between 3.7 and 6.0. An optimum pH for bacterial arabinofuranosidases appears to be more in the neutral region. C. rolfsii and R. flava produce an enzyme which is active at extreme acidic conditions. K^ values of all purified arabinofuranosidases are of the same order of magnitude. Abberative values have been obtained when phenyl-a-L-arabino-

24

BELDMANETAL

furanoside was used as substrate instead of j!?-nitrophenyl- aL-arabinofuranoside. The two—^probably different—strains of ^. niger produce arabinofuranosidases with different properties, especially with respect to molecular weights and isoelectric points (Table 6). The best characterized enzyme has been isolated from^. niger. From several lines of evidence, it has been proven that this enzyme hydrolyzes a-(l~>3)- as well as a-(l->5)-linkages in beet arabinan. The rate of hydrolysis of a-(1^3)-linkages of the side chains is much higher than of the a-(l-->5)-linkages in the back bone. This is mainly caused by the relatively higher concentration of accessible a-(l->3)-linked arabinosyl residues present in the side chains. However, the affinity, expressed as \/K^ (mol/L)~^ for both linkages is in the same order of magnitude [89]. The K^ for a branched arabinan isolated from apple juice and expressed as terminally linked arabinosyl residues appeared to be 3.7 X 10"^ (mol/L). After enzymic removal of the branches this value was 2.9 x 10"^ (mol/L) which indicates even a slightly higher affinity for the a-(l->5)-linkages. The ability to hydrolyze both linkages in beet arabinan is not only demonstrated for arafur B from A. niger, but is also observed for the enzymes from C. rolfsii, R. flava, S. massasporeus, Streptomyces sp. 17-1, B. subtilis, M, fructigena, D. squalens, and T. reesei, as well as from carrot cells. Recently, we made a detailed study of the linearization of sugar beet arabinan by arafur B. Sugar beet arabinan (0.2%) in 0.05 M sodium acetate buffer (pH 4.0) was incubated with arafur B. (60 U/50 ml) and the release of arabinose was followed by measuring the increase in reducing sugars after set time intervals. The residual polymeric arabinan remaining after dialysis and freeze-drying of the reaction mixture was analyzed by methylation studies. Part of the results are summarized in Figure 3, which shows the percentage of degradation and the relative proportions of the various glycosidic linkages in sugar beet arabinan plotted against incubation time with arafur B. These relative proportions are expressed in percentages of the total of glycosidic linkages present in the starting material. From this figure it can be concluded that the proportion of terminal

Table 6. Some Properties of a-L-Arabinofuranosidase B from Different Sources Source Aspergillus niger Aspergillus niger Corticium rolfsii RhodotorulaJava Streptomyces massasporeus Streptomyces sp. No 17-1 Bacillus subtilis Ruminococcus albus Monilinia fmctigena Dichomitus squalens Trichoderma reesei Scopolia japonica Lupinus luteus Daucus carota

M, (Da)

Optimum pH

PI

Kma (mMol/L)

kcoP

(min-')

Reference

26

BELDMANETAL

^

40

C

c

O -z

a c c

o

4 5 6 incubation time (h) Figure 3. Debranching of highly branched sugar beet arabinan by arabinofuranosidase B. Relative proportions of the various glycoside linkages are plotted against incubation time (a t-ara/; +1,5 araf; O 1,3-ara/; A 1,3,5-ara/; o 1,2,5-ara/; v 1,2,3,5-ara/).

arabinofuranosyl residues decreases rapidly and reflects the increase in reducing sugars. It also can be seen that the proportion of (l-^5)-linked a-L-arabinofuranosyl residues doubly branched at 02 and 03, declines while the proportions of residues singly branched, at either 02 or 03 remained almost constant. However, it must be realized that new single-substituted residues are formed by the debranching of the double-substituted residues. The proportion of a-(l->3)-linked arabinofuranosyl residues did not change. Based on these results it can be concluded that arafur B does not discriminate between single and double substitution nor between sites of substitution (02 or 03) of arabinofuranosyl residues in the backbone. a-(1^3)-Linkages in longer side chains appear to be more resistant towards enzymic degradation. Heteropolysaccharides, like arabinoxylan and arabinogalactan, have been used as substrates for arafur B. Several enzymes mentioned above are able to release arabinose at least partially


27

from these substrates. Neukom et al. [109] and Tagawa and Kaji [29] have found an almost complete release of arabinosyl residues from wheat arabinoxylan using the A. niger enzyme, while arafur B from T. reesei liberates only 50% of arabinose groups from wheat straw arabinoxylan [105]. Andrewartha et al. [110] investigated an enzyme which was isolated from the commercial preparation, Pectinol 59-L. Only 18% of the arabinosyl residues of wheat arabinoxylan could be removed. The enzyme was called an arabinofuranosidase and cited as such in the literature [32], but its activity on/7-nitrophenyl-a-L-arabinofuranoside was not reported. Based on reaction rates, beet arabinan is a better substrate than wheat a r a b i n o x y l a n for a r a b i n o f u r a n o s i d a s e B [29,89,99,104]. Taking into account the proportion of arabinosidic branches in beet arabinan (33%) and in wheat arabinoxylan (30-50%), this means that the a-(l->3)-linkages in the former substrate are more easily hydrolyzed than the a-(l-^2)and a-(l->3)-linkages in the latter polysaccharide. a^'Arabinofuranohydrolase, Specific for Arabinoxylans Several enzymes capable of releasing arabinosyl residues, present as side chains in arabinoxylans, are described in the literature. Mainly, two groups can be distinguished: the arabinofuranosidase type B and a type of enzyme belonging to the group of nonspecific xylanases which can hydrolyze the xylan backbone as well as release the arabinosyl residues [111]. While investigating the enzymic degradation of arabinoxylans, Kormelink et al. [88] found a new enzyme in the culture filtrate of Aspergillus awamori which did not fit this classification. It was highly specific for arabinosidic linkages in arabinoxylans from oat spelts, wheat, or barley and therefore called l~>4-pD-arabinoxylan arabinofuranohydrolase (AXH). Some properties of this enzyme are listed in Table 7. Neither jc^-nitrophenyl-aL-arabinofuranoside, nor a whole range of synthetic substrates could be hydrolyzed. Neither branched nor linear arabinans were degraded by this enzyme. Also other heteropolysaccharides, containing arabinose, were resistant to hydrolysis.

28

BELDMANETAL

Table 7. Some Properties of 1 ->4-p-L-Arablno-D-xylan Arabinofuranohydrolase from Aspergillus awamori [88,120] i^(Da) pH-optimum Temp.-optimum (°C) Activity towards:^ /7-Nitrophenyl-a-L-arabinofuranoside Arabinoxylan from oat spelts Arabinoxylan from wheat Arabinan from sugar beet UFR from apple*' Linear arabinan Arabinogalactan from citrus Arabinogalactan from potato Arabinogalactan from coffee Arabinogalactan from larch wood

32,000 5.0 50 + + -

Notes: ^+ Release of arabinose; — no release of arabinose. \jltrafiltration retentate isolated according to Voragen et al. [19].

A comparison has been made for the activity of AXH and arabinofuranosidase B from A. niger on arabinoxylan from oat spelts. On short incubation time arafur B (at a higher protein concentration) could not release a measurable amount of arabinose from this substrate, while 1,4-P-D-arabinoxylan arabinofuranhydrolase readily hydrolyzed 43% of the arabinosidic linkages. Similar results have been obtained with wheat arabinoxylan. On this substrate the specific activities of AXH and arabinofuranosidase B were 22 U/mg and 0.9 U/mg, respectively. Kormelink et al. [88] suggest different specificities of these two types of enzymes for the different types of glycosidic linkages present at the xylan backbone as a-(l-^3)- and a(l~>2)-linked arabinosyl residues. exo-a-i-Arabinanases^ Not Active on p-Nitrophenyl-a-i-arabinofuranoside Using beet arabinan as a substrate Kaji and Shimokawa [112] isolated an arabinanase from Erwinia carotovora lAM 1024 with


29

unusual properties. The enzyme produced arabinotriose from beet arabinan in an ^xo-fashion. It was not active towards aryl arabinosides and heteropolysaccharides containing arabinose as side chains. It was considered not to act on the (l->3)-arabinofuranosyl side chains of the branched beet arabinan and to be specific for a-(l-^5)-linked arabinofuranosyl residues. In contrast, a linear arabinan was not attacked, thus establishing that this enzyme is not an ^wt/o-arabinanase. It is probable that an exo-aXtdick occurs on side chains of beet arabinan, which most likely contain three arabinose residues. Small amounts of arabinobiose in the reaction product also suggest the ability of the enzyme to split off dimeric side chains. In recent work, Lahaye and Thibault [113] claim the existence of an exo-arabinanase as the product of ^. niger (var. aculeatus). The enzyme appeared to be a glycoprotein with properties as listed in Table 8. The major product from beet arabinan was arabinobiose, together with the formation of a small amount of trisaccharide. Considering these products, together with the low activity of the enzyme on carboxymethylarabinan, these investigators conclude that this arabinanase is an exo-acting enzyme. Table 8. Some Properties of exoArabinanase from Aspergillus niger (var. aculeatus) [113] M^ (Da) pH-optimum Temp.-optimum (°C) pl Activity (nKat/ml) towards: Reduced arabinan from sugar beet Arabinogalactan from potato Arabinogalactan from larch wood Polygalacturonic acid /?-Nitrophenyl-a-L-arabinofuranoside /7-Nitrophenyl-P-D-galactopyranoside Carboxymethylarabinan Gum arabic

67,000 4.0 60 2.85 28.8 0 0 0 1.2 0 2.0 0

30

BELDMANETAL

^'I'Arabinopyranosidase So far, only enzymes which are able to release arabinose of the a-L-furanose form, have been discussed. This chapter deals with another type of arabinanase, namely P-L-arabinopyranosidase. Commonly p-L-arabinopyranosides as well as a-D-galactopyranosides and a-D-fucopyranosides are good substrates for a-galactosidases because of the similarity of the glycosidic linkage and the orientation of hydrogen and hydroxyl groups at C2, C3, and C4. Dey [114] discovered an activity, not being an a-galactosidase, but yet able to hydrolyze P-L-arabinopyranosides. The enzyme was isolated from Cajanus indicus seeds, until now the only source for this enzyme mentioned in the literature. Characteristics of P-L-arabinopyranosidase were given in this first publication and supplemented in a next paper [115]. Some of the properties are compiled in Table 9. Probably a carboxyl group and a histidine imidazolium group are involved with the enzyme substrate complex and influence the activity [114]. There is a cooperative catalytic action of a proton donor, being the imidazolium ion, and a nucleophile in the form of a carboxyl group. Probably this is a two-step mechanism which includes the formation of a glycosyl—enzyme intermediate. The enzyme also catalyzes a ^ra«5-glycosylation Table 9. Some Properties of P-L-Arabinopyranosidase from Cajanus indicus Seeds [114,115] M^(Da) pH-opt pi ^.(M) Activity towards:^ />-Nitrophenyl-p-L-arabinopyranoside /7-Nitrophenyl-a-D-galactopyranoside p-Nitrophenyl-P-D-galactopyranoside p-Nitrophenyl-a-D-fucopyranoside Note: ^+ activity; - no activity.

25,900 3-4.6 4.4 0.83 X 10"^ + -

Arab!nans and Arabinan Degrading Enzymes

31

reaction in which the anomeric conformation of the product is retained. One could speculate on the function of p-L-arabinopyranosidase in plant tissue. Maybe the enzyme is involved in the breakdown of highly ramified polysaccharides such as larch arabinogalactan. Arabinosyl residues are present in branches of this polysaccharide in both the a-L-furanose and the p-L-pyranose forms [4]. endo' 1-^5Hi'L'Arabinanases

The existence of an e«(io-arabinanase was first noticed in 1963 as a product of Clostridium felsineum (var. sikokianum) [116]. Since then, the enzyme has been purified from other bacterial sources, including Bacillus subtilis F-11 [30,117] and B. subtilis IFO 3022 [118]. An enzyme from this last source is able to disintegrate potato tissue in the absence of a pectinase, which is commonly necessary to macerate this type of tissue. An enzyme, with similar properties, has been isolated from B. subtilis IFO 3134 [119]. Considering its ability to release protopectin from beet pulp, this enzyme has been called "protopectinase-C". However, according to its properties one has to conclude that it is actually an e«(io-arabinanase with the same characteristics as the e«ô-arabinanases from the B. subtilis strains mentioned before. ewrfo-Arabinanase was also found in a fungal preparation from A. niger [89] in 1988. In previous investigations on arabinofuranosidase B from A. niger [91,92] the existence of this enzyme remained unnoticed because a heat treatment was introduced into the purification procedure. ^«Jo-Arabinanase as well as arafur A appeared to be relatively more sensitive to heat in activation than arafur B. Arabinanase from B. subtilis F-11 was active towards linear (l->5)-a-L-arabinan and branched beet arabinan, but inactive towards phenyl-a-L-arabinofuranoside, arabinoxylan, arabinogalactan, and gum arable. With respect to this substrate specificity, the other purified enJo-arabinanases resembled the B. subtilis F-11 enzyme. In general linear (l->5)-a-L-arabinan ap-

32

BELDMANETAL

peared to be a much better substrate than the branched beet arabinan. For instance, after prolonged incubation (120 h) Kaji found a hydrolysis limit of the first substrate of 23.3% while the branched polysaccharide was only hydrolyzed by 3.3% [117]. The activity of ^«Jo-arabinanase on arabinans declines during the course of the reaction as the (l-^5)-a-L-arabinofuranosyl sequences become shorter or more highly substituted [19]. Some properties of the purified e«Jo-arabinanases mentioned in the literature, including protopectinase-C, are summarized in Table 10. Yoshihara and Kaji [118] concluded that the enzyme from B. subtilis IFO 3022 is evidently the same as the endoA-^5-a-harabinanase from B. subtilis F-11. We already mentioned that this is also true for the e«Jo-arabinanase from B. subtilis IFO 3134. However, the fungal enzyme firom^. niger showed somewhat different properties, not only with respect to molecular weight, pH-optimum, and isoelectric point, but also with respect to the product formation. The bacterial e«Jo-arabinanases produce arabinose and arabinobiose as end-products, while arabiTable 10. Some Properties of Purified endo-Arabinanases B. subtilis

M^ (Da) pH-optimum pl Activity towards: Linear (l->5)-a-L-arabinan Beet arabinan /7-Nitrophenyl-a-LArabinofuranoside Products after prolonged incubation^ Reference

F-11

IFO 3022

IFO 3134

A. niger

32,000 6.0 9.3

33,000 6.0 7.9; 9.7'

30,000 6.0 9.0

35,000 5.0 4.5-5.5

+ + —

+ + —

+ + —

+ + —

A1+A2

A1+A2

A,+A2

A2+A3

30,117

118

119

19,89

Notes: ^Reference 32. Âi arabinose; A2 arabinobiose; A3 arabinotriose.


33

nobiose and arabinotriose accumulate in the reaction mixture using the fungal enzyme. B. Cooperative Effects during Enzymic Degradation of Arabinose Containing Polysaccharides Arabinans

It has been shown that A. niger produces arabinofuranosidases A and B, as well as e«t/o-arabinanase. These enzymes can act in concert during the degradation of branched arabinans [19]. Combinations of these arabinanases were made according to Table 11 and their activities were expressed as a percentage of the sum of activities of single enzymes. ewrfo-Arabinanase shows a strong synergistic interaction with both arabinofuranosidases. The mechanism of both synergistic effects, however, is different. Arafur A, on its own, is unable to degrade the polymeric substrate. Its synergistic interaction is only expressed when endoarabinanase forms oligo-arabinosides, which are a good substrate for the furanosidase. The synergism between arafur B and e«t/o-arabinanase can be explained as follows: arafur B liberates linear l->5-a-L-arabinosyl sequences, which are better substrates for the endo-enzymt. In turn, the oligomeric products are new substrates for the furanosidase. As could be expected from their substrate specificities, there was no synergism between the two arabinofuranosidases. In the experiments where the enzyme is combined with itself, resulting Table 11. Relative Activities of Arabinanases Towards Branched Arabinan (UFR) Separately and in Combinations [19]^ Arabinofuranosidase Enzyme

A

B

endO'Arabinanase

Arabinofuranosidase A Arabinofuranosidase B e«t/o-Arabinanase

80 109 191

63 143

79

Note: *Activities of combinations are expressed in % of the sum of activities of single enzymes.

34

BELDMANETAL.

OH

"

""'

OH

L-arabi no-oligosaccharides

Figure 4. Mode of action of the arabinanases of A. niger in the breakdown of highly branched arabinan.

in a double dosage, the increase of activity is less than proportional. The authors ascribe this to the relatively high enzyme dosage and reaction time. The conclusion from these observations is that the arabinanase complex of ^. niger can completely break down branched L-arabinans to arabinose, according to a reaction scheme presented in Figure 4. Most likely this model is also valid for the degradation of such a branched substrate by the arabinanase complex of 5. subtilis, composed of a similar mixture of enzymes. Arabinoxylans

Kormelink et al. [120] studied the degradation of arabinoxylan by combinations of enzymes. Arabinose releasing enzymes, be-


35

Table 12. Relative Activities of Arabinoxylan Degrading Enzymes Towards Wheat Arabinoxylan Separately and in Combinations [88]^ Enzyme AXH' Arabinofiiranosidase B ewflfo-Xylanase

AXH 109 103 133

Arafur B^

endo-Xylanase

88 142

100

Notes: Âctivities of combinations are expressed in % of the sum of activities of single enzymes. **! ,4-p-D-Arabinoxylan arabinofuranohydrolase. 'â-L-Arabinofuranosidase B.

ing a substrate-specific 1,4-p-D-arabinoxylan arabinolfuranohydrolase (AXH) from A. awamori [88] and an arabinofiiranosidase B fi'om A. niger, were combined with an ^«Jo-xylanase ft*om A. awamori and incubated with wheat arabinoxylan. The degradation of arabinoxylan was measured by a reducing sugar assay. Clear synergism was found for the combination of endoxylanase and an arabinose releasing enzyme (Table 12). This is valid for AXH, as well as for arafur B. Similar cooperative effects between arabinofuranosidase and e«5)-a-L-arabinan was incubated with ewrfo-arabinanase from A. aculeatus and at several times samples were taken and analyzed by HPLC (Figure 5). The chromatographic conditions will be published elsewhere. Monomer and oligomers could be detected up to a degree of polymerization of 23. The relatively high peaks represent the linear oligosaccharides, whereas smaller peaks in between these peaks probably originate from branched oligomers. However, this has still to be proven by structural analysis such as NMR. The method described was used to study the intermediary reaction products of linear arabi-

40

BELDMANETAL.

nan in various stages of the breakdown (Figure 6). At an inital stage of reaction, relatively small amounts of low molecular weight products are present in the reaction mixture. As the reaction proceeds more and more small oligomers accumulate, with arabinotriose, -biose and -tetraose being the most important products. An accurate evaluation of peak area's in the region of DP 9-16 (Figure 6, insertion) showed us a 'valley point' at DP 13, especially for a reaction time of 45 and 60 minutes. Apparently the enzyme has a relatively higher affinity for oligomers of that size. We do not know whether this is caused by structural features of the enzyme (i.e. the number of sub-sites) or of the substrate (i.e. its 3D structure in solution). This phenomenon can only be investigated further with kinetic experiments using purified oligosaccharides.

IV. CONCLUDING REMARKS In this contribution, we have tried to bring together the present knowledge on arabinans and arabinan degrading enzymes. This knowledge is far from complete. In recent years particularly, food technologists have shown an urgent need for more information. This need arises from problems in fruit juice processing like fouling of membranes in ultrafiltration of fruit juices and haze formation of fruit juice concentrates, as well as interest by the food industry to find applications for arabinan-rich byproducts. Recently, McCleary et al. [127] succeeded in preparing an enzymically-debranched arabinan from sugar beets which can gel to a creamy paste and which has potential food applications. It is necessary to obtain more knowledge on the chemical structure of pectic arabinans and their enzymic modification and degradation, and to have more insight in the relation between chemical structure and physical behavior. This is also of importance to understand their role as a dietary fiber. For the degradation and tailored modification of arabinans, the active and most efficient enzymes have to be identified and their action patterns studied at a molecular level. This will boost the production of these enzymes and improve their properties by DNA technology.


41

V. APPENDIX: REVIEW OF THE LITERATURE 1992-1997 The preceding sections of this review were prepared and accepted for publication in 1992. Since there has been considerable research into arabinans and arabinan degrading enzymes in the last few years, at our request, the editor allowed us to review the recently published findings which are summarized below. A. Substrates

Although the structural features of a number of arabinose-rich polysaccharides have been published in the last few years, a relatively small number of these studies give additional information to that already discussed in the previous sections. While not discussed further here, arabinan-rich pectic molecules have also been briefly investigated recently in relation to their role in the processing of fruits and vegetables [129] and their possible application in foods [130]. Recently, an arabinan fragment isolated from the water-soluble fraction of dehuUed rapeseed has been described in detail [131]. However, since this fraction still contained about 16% galactose and relatively high amounts of 1,2,5-linked arabinose, it would appear to resemble the highly branched arabinogalactan from rapeseed [132] rather than the free rapeseed arabinan described by the same researchers [16]. Cooper et al. [133] described in detail the physical properties of an enzymically-debranched arabinan from sugar beet, isolated according to the patent of McCleary [127]. It was demonstrated that these arabinans form gels that show the progressive structural breakdown and flow characteristics of fat-continuous spreads, rather than the abrupt fracture typically found for normal polysaccharide gels. Furthermore, the possible use of debranched arabinans as a fat-replacer was demonstrated in a number of products, including low fat spreads, ice cream, and chilled and frozen desserts [127,133]. This same debranched arabinan fraction motivated Chandrasekaran et al. [134] to study the molecular architecture using

42

BELDMANETAL.

X-ray diffraction and computer modeling methods in order to explain the functional properties of these polysaccharides. They concluded that the arabinan fraction was able to form microcrystallites and exists in at least three crystalline allomorphs differing in the conformation of the twofold helical structure. The conformational flexibility of the arabinofuranose ring was studied by molecular mechanics software by Cros et al. [135]. In a following study [136], the same authors used high resolution NMR and computerized molecular modeling to study the conformational behavior of arabinobiose. They also extrapolated their findings to regular polymeric arabinans generating arabinan chains displaying right- and left-handed chirality and a wide range of repeating units per turn of helix. Advanced NMR spectroscopy techniques on a 600-MHz apparatus was used by Eriksson et al. [131] to characterize the above mentioned arabinan fraction from rapeseed. Using HMQC, NOESY, and TOCSY experiments, they were able to assign most peaks in the rather complex ^H and ^^C NMR spectra of the highly branched arabinan. In conclusion, it can be still stated that freely occurring arabinans are not frequently extracted from plant materials, and it is considered that they are, in general, an integrated part of pectic molecules or arabinogalactans. Their role in the plant matrix, in plant-derived products, as well as food ingredients should be studied in more detail to enable a better understanding of their structure-function relationships. B. Enzymes Classification of Arabinanases

A considerable number of arabinanases from a variety of microbial and plant sources have been purified and characterized in the last few years (1992-1997). Most of these recently isolated enzymes appear to fit broadly into the classification system we have proposed based on their mode of action and substrate specificity. Some of these arabinanases are discussed below. Again, only enzymes that have been adequately purified and


43

characterized well enough to allow their classification have been included in this appendix. a-i-Arabinofuranosidase A (Not Active Towards Polymers; Arafur A). Several recently isolated arabinofuranosidases appear to be of the arafur A type since they can hydrolyse aryl-a-L-arabinofuranosides, arabino-oligosaccharides, and arabinoxylan oligosaccharides, but have very little, or no activity against arabinofuranosyl linkages in polymeric substrates such as branched arabinan, arabinoxylan, or arabinogalactan. Some physicochemical properties of these arabinofuranosidases are given in Table 14. Some arafur A's are monomeric proteins with molecular weights of around 40-80 kDa, while the native form of the enzymes from B. polymyxa [139], B. stearothermophilus [141], S. lividans [144], and B. xylanolyticus [143] are much larger, being composed of several subunits. The Table 14. Some Properties of Arabinofuranosidase A's from Different Sources Source Aspergillus aculeatus Ara2 Aspergillus niger N400 Bacillus polymyxa Bacillus stearothermophilus LI Bacillus stearothermophilus T-6 Bacillus subtilis Bacteriodes xylanolyticus Streptomyces lividans Wheat Ara I

MW(Da)

Optimum pH pl

37,000

4.0-4.5

83,000 163,000' 110,000^

3.4 6.5 7.0

256,000' 5.5-6.0

137 3.3 4.7

0.68 1.19 0.22

2.74 X 10^ 3.49 X 10^ 1.21 X 10^

138 139 140

6.5

0.42

4.80 X 10^

141

0.5

9.46 X 10^

142 143

0.6

1.24x10^

7.0 61,000 366,000^^ 5.5-6.0 69,000' 49,000

6.0 4.5

^m

(mM) feat (mirf ) Reference

4.6

Notes: **Composed of two subunits of 65,000 Da and one subunit of 33,000 Da. ''Composed of two subunits of 52,500 and 57,500 Da. *^Composed of four subunits of 64,000 Da. '^Composed of six subunits of 61,000 Da. *Gelfiltrationresults in a MW estimate of 380,000 Da.

144 145

44

BELDMANETAL

characterized fungal arafiir A's have quite acidic pH optima (pH 3.0-4.1), while that of their bacterial counterparts generally appear at higher pH (5.5-7.0). AT^^ values for arafUr A's with;7-nitrophenyl-a-Larabinofiiranoside are similar and appear to be slightly lower than for most arafiir B 's examined, possibly indicating a general higher affinity for this substrate. Recent studies with the arafur A's fromv4. niger and B. subtilis, the two most extensively characterized enzymes of this type, have contributed important information regarding the substrate specificity of this enzyme class. During studies of the enzymic degradation of alkali-extractable wheat-flour arabinoxylan, Kormelink et al. [146] examined the action of the A. niger arafur A on isolated arabinoxylan-derived oligosaccharides. While this enzyme was not active against polymeric arabinoxylan, it readily cleaved all (l-^3)-a-linked arabinofuranosyl groups from singly substituted xylopyranosyl residues in arabinoxylan oligosaccharides, irrespective of whether the substituted xylopyranosyl residue was in a terminal or nonterminal position. Further recent work in our laboratory has also suggested that this enzyme has similar activity against (1^2)-a-linked arabinofuranosyl groups of singly substituted xylopyranosyl residues (K.M.J. Van Laere, unpublished work). In contrast, no hydrolysis of arabinofuranose from doubly substituted xylopyranosyl residues was observed [146]. Similar action patterns on small arabinoxylan oligosaccharides were also found for the B. subtilis arafur A [142]. Kaneko and Kusakabe [147] have shown recently, using chemically synthesized arabinobiose derivatives, that the B. subtilis arafur A effectively hydrolyzes all possible linkages occurring between two a-L-arabinofuranosyl residues (i.e., ( 1 ^ 2 ) - , ( 1 ^ 3 ) - and (l->5)-a-L-linkages). Perhaps surprisingly, due to it's less frequent occurrence in natural substrates, highest activity was shown against the (l->2)-a-L-arabinofuranosyl linkage, while the lowest hydrolysis rate was seen against the more commonly occurring (l->5)-a-L-linkage. Further examination of the action of this enzyme on a small branched arabino-oligosaccharide derivative (methyl 3,5-di-O-a-L-arabinofuranosyla-L-arabinofuranose) also suggested that the (1^3)-a-L-


45

arabinofuranosyl linkage in this substrate was cleaved in preference to the (l-->5)-a-L-linkage [147]. a-i-Arabinofuranosidase B (Active Towards Polymers; Arafur B). The majority of new arabinofiiranosidases examined in the last few years appear to be of the arafur B type. Physicochemical properties of these enzymes, summarized in Table 15, appear similar to those of previously characterized arafur B's discussed earlier (Table 6). While all arafur B's, by definition, have activity against aryla-L-arabinofuranosides and a-L-arabinose containing oligo- and polysaccharides, some of the recently isolated enzymes appear to show some diversity in their substrate specificities. For example, most arafur B's hydrolyze branched arabinan and also have at least some activity against linear arabinan, arabinoxylan, and arabinogalactan, although the relative rate of hydrolysis of these substrates varies markedly between different enzymes. However, an arafur B recently isolated from soybean seedlings was reported to have no activity against arabinoxylan or arabinogalactan, even though it hydrolyzed both linear and branched arabinans [165]. A similar substrate specificity was also seen with an arafur B from C. acetobuylicum [155], while other Arafur B's from P. capsulatum [158] and B. fibrisolvens [154] were reported to show no activity against arabinogalactan, although branched arabinan and arabinoxylans were hydrolyzed rapidly. Two other arabinofuranosidases with more unusual substrate specificities were recently isolated from G. candidum [157]. Like typical arafur A's, these enzymes hydrolyzed pnitrophenyl-a-L-arabinofuranoside but had no activity against branched beet arabinan. However, irrespective of this, they are probably best considered as arafur B's due to their ability to hydrolyze arabinogalactan and arabinoglucuronoxylan. An arafur B recently isolated from A. awamori [148] is also unusual since it appears capable of releasing feruloyl and pcoumaroyl arabinose residues from arabinoxylans. An arafur B from T. emersonii may also have this ability [160], although other arafurs so far examined do not [154,158]. Although details of the action pattern of A. niger arafur B on branched arabinans have been known for some time (see

46

BELDMANETAL.

Table 15. Some Properties of Arabinofuranosidase B's from Different Sources Source

MW (Da)

Optimum pH

pi

Km (mM)

kcdX

(mirT^)

Reference

Aspergillus Ara I 37,000 3.0-3.5 137 aculeatus Aspergillus 4.6 3.6,3.2 1.39 64,000 148 awamori Aspergillus 4.0 3.3 0.679 6.46x10^ 65,000 149 nidulans A. niger 5-16 4.0 3.5 67,000 150 (intracellular) ^. «/gerN400 138 3.8 3.5 0.52 1.58x10' 67,000 A. niger 151 4.6 3.0 1.03 52,900' Aspergillus 152 5.0 3.9 34,300 sojae 153 Aspergillus 4.0 Ara I 39,000 7.5 terreus 4.0 Aral! 59,000 8.3 4.0 Ara III 59,000 8.5 154 0.7 3.38 X 10' 248,000'' 6.0-6.5 6.0 Butyrivibrio flbrisolvens 155 5-5.5 8.15 4.0 3.42x10' Clostridium ace94,000 tobutylcum 156 Clostridium 5.0 52,000' stercorarium 157 Ara I 80,000 4.00 0.63 1.68x10' Geotrichum 4.1 Aral! 69,000 3.85 0.83 2.00 X 10^ candidum 4.1 158 4.15 0.18 2.06 X 10^ Ara I 64,500 Penicillium 4.0 4.54 1.3 4.82 X lO'* capsulatum Ara II 62,700 4.0 7.34 1.3 1.26x10^ 159,160 55,000 Phanerochaete 2.5 chrysosporium 161 8.8 10 Ara I 38,000 4-7 Streptomyces 8.3 12.5 Ara II 60,000 diastaticus 162 3.5 0.16 6.3 X 10' 210,000^ 3.2 Talaromyces emersonii 163 0.1 92,000' Thermomono6.0 sporafusca 164 3.8 Daucus carota Ara i f 80,000 5.6 0.22 1.47x10^ 165 0.53 Soybean 4.8 87,000 166 2.2 68 Spinach 4.8 Ara II 68,000 4.2 Notes: "Gel filtration. ^Composed of eight subunits of 31,000 Da. ''Gel filtration results in a MW estimate of 195,000 Da. '^Composed of two subunits of 105,000 Da. ^Composed of two subunits of 46,000 Da. 'Ara I appears in Table 6.


47

earlier [137]), recent examination of the hydrolysis of arabinoxylan and isolated arabinoxylan-derived oligosaccharides by this enzyme has provided new insights into its substrate specificity. Against arabinoxylan oligosaccharides this enzyme is quite specific, only removing arabinofuranosyl residues from singly substituted, nonreducing terminal xylopyranosyl residues of these substrates. No activity is shown against arabinofuranosyl residues of singly substituted nonterminal, or doubly substituted xylopyranosyl residues in these substrates [146]. This type of action pattern on arabinoxylan oligosaccharides would appear common for arafur B's, since similar results have been described for an intracellular A. niger arafur B [150] and an arafur B from B. fibrisolvens [154]. Furthermore, since this action is different from that of arafur A-type enzymes on these substrates (see earlier), Kaneko et al. [142,147,150] have proposed that this is one way of easily differentiating between these two enzyme classes. However, in an apparent contradiction, there are indications that A. niger arafur B primarily removes arabinofuranosyl residues from doubly substituted xylopyranosyl residues during action on polymeric arabinoxylan [146], although detailed studies have not been performed. Arabinoxylan Arabinofuranohydrolase (a-i-Arabinofuranosidase Specific for Arabinoxylans; AXIH), Enzymes with similar specificity for arabinofuranosyl linkages in arabinoxylans as the A. awamori AXH [88] have been recently isolated from Pseudomonas fluorescens [167], wheat [145], Bifidobacterium adolescentis [168], and T, reesei (R. Kavitha, unpublished work). Two further similar enzymes have been isolated from B. polymyxa, although in addition to hydrolyzing arabinoxylans, these enzymes also have very low, but detectable activity against aryl-a-L-arabinofuranosides, but no other arabinose containing substrates [169]. Physicochemical properties of some of these enzymes are given in Table 16. Our examination of the action patterns of A. awamori AXH on arabinoxylans and arabinoxylan-derived oligosaccharides [146,170] have revealed its high specificity for both (l->2)and (l->3)-a-L-arabinofuranosyl residues of singly substituted xylopyranosyl residues in these substrates. No activity was

48

BELDMANETAL

Table 16. Some Properties of (1 ->4)-p-D-Arabinoxylan Arabinofuranohydrolases from Different Sources Source Bacillus polymyxa Bifidobacterium adolescentis Pseudomonas fluorescens Wheat

AF64 AF53'

MW(Da)

Optimum pH

64,000 53,000 100,000

6.5 6.5 6.0

pl 8.7 9

Reference 169 168

59,000

167

40,000

145

Note: ^This protein probably arises from the proteolytic cleavage of ca. 100 amino acids from the C-terminus of AF64 [169].

shown by this enzyme toward arabinofuranosyl residues of doubly substituted xylopyranosyl residues in these substrates [146,170]. Our recent analysis of the B. adolescentis AXH [168] indicated that this enzyme had a different specificity, since it only hydrolyzed (l-^3)-a-L-arabinofuranosyl residues of doubly substituted xylopyranosyl residues of arabinoxylans and arabinoxylan oligosaccharides. No activity was shown against the (l-»2)-a-L-arabinofuranosyl residues of these doubly substituted xylopyranosyl residues, or against arabinofuranosyl residues of singly substituted xylopyranosyl residues of these substrates [168]. The discovery of such highly specific enzymes raises the possibility that other AXH's with different specificity may also exist. In light of this, we have proposed a nomenclature for these enzymes that more accurately indicates their individual specificities [168]. AXH's that remove arabinofuranosyl residues specifically from singly (mono) substituted xylopyranosyl residues are given a postfix m, while a d postfix indicates those AXH's acting specifically on arabinofuranosyl residues of doubly substituted xylopyranosyl residues. Furthermore, AXH's acting on only one type of a-L-arabinofuranosyl linkage (i.e., either ( 1 ^ 2 ) - or (l->3)-a-L-linkages) are given a number to indicate the linkage cleaved, while the number is omitted for enzymes acting on both linkage types. Thus, the A. awamori enzyme


49

[88] is designated AXH-m, while the enzyme from B. adolescentis is designated AXH-(i3 [168]. endo- 1-^5-a-i-Arabinanase. e«(io-Arabinanases have been recently described from SQWQval Aspergillus species, namely^, aculeatus [137], A. nidulans [149], and a few further A. niger strains [138,171,172]. The physicochemical properties of these enzymes, summarized in Table 17, are comparable to those of the previously examined ewtio-arabinanases (see Table 10). The action patterns of all recently purified Aspergillus endoarabinanases examined appear similar to that of the A. niger enzyme previously discussed [19,89] since arabinobiose and arabinotriose are the major final hydrolysis products released from linear arabinan [137,151,171,173]. However, comparison of the initial (transient) hydrolysis products released from this substrate by the A. niger and A. aculeatus ewrfo-arabinanases revealed some distinct differences in their apparent action patterns [137]. Our further recent studies on the action of these enzymes against linear arabino-oligosaccharides have shown that these observed differences result, at least in part, from variations in the substrate binding regions of these two enzymes. Table 17. Some Properties of endo-0 -^5)-a-L-Arabinanases from Different Sources A. aculeatus A. nidulans MW (Da) Optimum pH pl Activity towards: Linear (l->5)-a-L-arabinan Beet arabinan p-Nitrophenyl a-Larabinofiiranoside Products after prolonged incubation* Reference

A. niger N400

A. niger

45,000 5.5

40,000 5.5 3.25

43,000 4.6 3.0

42,500 4.8 2.9

++ + —

++ + —

++ + —

++ + —

A2 + A3

Note: Â2 arabinobiose; A3 arabinotriose.

137

A2+A3

149

138, 149

172,173

50

BELDMANETAL.

with the A. niger enzyme having five binding subsites and the A. aculeatus enzyme six [174]. The extent and rate of branched arabinan hydrolysis by all ewrfo-arabinanases appears affected by the degree of substitution on the arabinan backbone by (l->3)- or (l->2)-a-L-arabinofuranosyl residues [19,30,117,137,173]. ^^C-NMR studies of hydrolysis products released from branched arabinan by the A. niger ^«(io-arabinanase [175] indicated that the smallest branched oligosaccharide released was a tetrasaccharide with an (1^3)-a-L"arabinofuranosyl residue linked to the central residue of (l-^5)~a-L-arabinotriose [i.e., 3^-a-L-arabinofuranosyl (l-»5)-a-L-arabinotriose]. This suggested a requirement for this enzyme of at least one unsubstituted a-L-arabinofuranosyl residue on either side of the substituted residue before hydrolysis can occur. Comparison of the hydrolysis products formed by action of each of the Aspergillus e«(io-arabinanases on branched arabinans gave similar distribution of branched oligomeric products [137], possibly indicating that the two ^«rfo-arabinanases have a similar tolerance for branched sites, although further work is needed to confirm this. Multifunctional Enzymes with Arabinanase Activity. A variety of multifunctional enzymes have been described with apparent arabinanase activity. Enzymes with both endo-xyldinase and arabinofuranosidase activity, the so-called "arabinose-releasing endo-xylanases", have been proposed to exist for many years [111,176-178]. In arecentstudy [179], ewô-xylanase 1 from Fibrobacter succinogenes was found to first remove arabinofiiranosyl residues from arabinoxylan before acting in an endo manner on the remaining debranched xylan backbone. Several other enzymes have been reported with similar action, although the contamination of these enzymes by an arabinofuranosidase, or more likely an AXH, has long been suspected. As Coughlan [178] suggested, the question regarding the true existence of these enzymes could finally be answered if cloned enzymes are shown to exhibit the same combined activities. Enzymes with both P-D-xylopyranosidase and a-L-arabinofuranosidase activity have also been reported. Again, the purity of such enzymes has been questioned, although in contrast to


51

the "arabinose-releasing e«(io-xylanases", firm evidence to support the existence of these enzymes has been reported in the form of multifunctional cloned enzymes [180-182]. The properties of some of these enzymes with high arabinofuranosidase activity are shown in Table 18. From this data it can be seen that these enzymes generally appear to have much higher affinity for p-nitrophenyl-P-D-xylopyranoside than /?-nitrophenyl-a-Larabinofuranoside, and thus are often considered as P-D-xylosidases with the catalytic flexibility to also hydrolyze a-L-arabinofuranosides [159,160,176]. Inhibition studies [184] have suggested that the bifunctional activity of these enzymes may reside in a single catalytic site, a proposal that some workers [180,182,184] have rationalized on the basis of the structural similarity of P-D-xylopyranosyl and a-L-arabinofuranoysl residues around CI, C2 and C3, similar to that discussed earlier to account for arabinopyranosidase activity seen with some galactosidases [145,185].

Table 18. Some Properties of Bifunctional P-D-Xylosidase/a-L-Arabinofuranosidases from Different Sources Clostridium Thermoanaerobacter T. reesei ethanolyticus stercorarium MW (Da) Optimum pH pl pNPA' K^{mM) ^cat ("în ')

Radish

Spinach (Ara I)

64,000 4.5 4.7

118,000 4.8 4.2

212,000' 7.0

170,000*^ 5.0-5.2

100,000 4.0 4.7

17.6 8.85 X 10^

4.6 9.10 X 10^

>2.5

9.7 1.2 4.67 X 10^ 73.2

2.5 3.13 X 10^ 182

0.038 1.56 xlO"^ 181

0.08

0.95 3.56 X 10^ 184

pNPX^ ^m ^cat ("în ' )

References

Notes: ^Composed of four subunits of 53,000 Da. ''Composed of two subunits of 85,000 Da. *^;7-Nitrophenyl-a-L-arabinofuranoside. p-Nitrophenyl-p-D-xylopyranoside.

183

166

52

BELDMANETAL

Classification ofArabinanases Based on Amino Acid Sequence Similarity, Traditionally, glycosyl hydrolases have been classified on the basis of substrate specificity and action patterns [186]. However, generally this classification gives little information regarding the structure and catalytic mechanisms of these enzymes, nor was it intended to. Therefore, to compliment this traditional classification and better reflect the structural features and evolutionary development of these enzymes, a classification system for glycosyl hydrolases based on amino acid sequence similarity has been recently devised [187—189]. Currently 60 glycosyl hydrolase families are recognized, although more families will undoubtedly arise as the number of known enzyme sequences increase. Members of those families to which enzymes with arabinanase activity have been classified are given in Table 19. This classification system has already proved useful in predicting the catalytic mechanisms of arabinanases [202], and is sure to become invaluable in identifying key residues and predicting active site structures when more detailed information is obtained on a few members of these families [203]. Catalytic Mechanisms ofArabinanases

Enzymic hydrolysis of glycosidic linkages can occur via two major mechanisms which result in either net retention, or inversion of anomeric configuration [204]. Both hydrolytic mechanisms involve general acid catalysis and require two critical residues: a proton donor and a nucleophile/base. Inverting enzymes operate by a single displacement reaction [205], involving protonation of the glycosidic oxygen, followed by nucleophilic attack on the anomeric carbon by water. With retaining enzymes a double displacement mechanism operates [205], with the hydrolysis proceeding through either a covalent glycosyl-enzyme intermediate, or an oxocarbonium ion intermediate stabilized electrostatically by enzyme carboxylate(s) [204]. It is now recognized that enzymes with similar substrate specificities and action patterns do not necessarily catalyze hydrolysis with the same stereochemical outcome. However, members of a given glycosyl hydrolase family do exhibit the


53

Table 19. Classification of Arabinanases Based on Amino Acid Sequence Similarity [187-189] Accession Numbers Glycosyl Hydrolase Family 43

Source Aspergillus niger Bacillus polymyxa Bacillus pumilus Bacillus subtilis Bacillus subtilis Bacillus sp. K-17 Bacteriodes ovatus

Enzyme Endo-arabinanase {abnA) Xylanase D^ {xynD) p-Xyl {xynB) Endo-arabinanase {abnA) ORF (J3A) p-Xyl p-Xyl/Arafur {xsa) p-Xyl/Arafur ixylB) Arafur

Butyrivibrio flbrisolvens Butyrivibrio flbrisolvens Clostridium p-Xyf cellulolyticum Clostridium p-Xyl/Arafur stercorarium ixylA) Prevotella ruminicola p-Xyl {xynB) 51

54

Ax2if\xr A(abfA) Aspergillus niger Streptomyces lividans Arafur {abfA) Bacteriodes ovatus Arafur I (asdl) Bacteriodes ovatus Arafur II (asdll) Bacillus Arafur'^ stearothermophilus Bacillus subtilis Arafur (abfA) Arafur (asd) Bacillus subtilis

SWISS- EMBL/ PROT Genbank References P42256 L23430

190

P45796 X57094

191

P07129 X05793 Z75208

192 193

P42293 D31856 P49943 U04957

194 195 196

P45982 M55537

180

U55187 197 P48790 D13268

182

P48791 Z49241

198

P42254 L29005 P53627 U04630 U15178 U15179

199 144 141

Z75208 Z75208

193 193

Arafur B (abJB) P42255 X74777 Aspergillus niger Synechocystis sp. Arafur (abfB) D64004 Trichoderma koningii Arafur/Xyl {xyll) P48792 U38661 Trichoderma reesei Arafur (abfl) Z69252

200

Non-classified^ Pseudomonas fluorescens

AXH (xynC)

P23031

201 167

Notes: ^Corresponding genes are shown in parentheses. ^This enzyme was originally considered to be an e« Jo-xylanase [191], however, recent flirther analysis has indicated that is probably an AXH-type enzyme [169]. '^Classification based on partial (N-terminal) amino acid sequence. No similar sequences currently known to allow classification.

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same stereoselectivity [206,207], which is not surprising since they share a common fold and active-site topology [208—210]. Therefore, mechanistic data for glycosyl hydrolases can best be interpreted in terms of membership in these glycosyl hydrolase families. Glycosyl hydrolase family 43 contains enzymes catalyzing hydrolysis with inversion of anomeric configuration, as determined for the A. niger endo-amhinanasQ [202] and P-xylosidases from B. pumilus [211] and C cellulolyticum [197]. In contrast, members of glycosyl hydrolase families 51 and 54 act with retention of anomeric configuration, determined by following the stereochemistry of hydrolysis of the A. niger arafur A and arafur B, respectively [202]. The stereochemistry of hydrolysis of several other unsequenced arabinanases not currently classified into glycosyl hydrolase families are also known. All a-L-arabinofuranosidases examined to date act with retention of anomeric configuration, including those from A. aculeatus, A. awamori, Humicola insolens, P. capsulatum, B. subtilis [202], and also Monilinia fructigena [212] for which detailed mechanistic data have been determined [see ref 204]. As discussed earlier, the C. indicus P-L-arabinopyranosidase also acts with retention of configuration [115]. Like its A. niger counterpart, the A. aculeatus endo-arabinanase catalyzes hydrolysis with inversion of anomeric configuration [202]. However, the 5 . subtilis IFO 3134 ewJo-arabinanase (protopectinase-C) has apparent glycosyl transferase activity [213,214], which would suggest that this enzyme may act with retention of configuration [204], although a detailed examination has not been performed. New Substrates and Assays for Arabinanases

The last few years have seen considerable advancement in the understanding of arabinanase action and specifity, with a major reason for this being the recent availability of pure, welldefined arabinose-containing oligosaccharides for use as substrates for these enzymes. For example, purified (l->5)-a-L-


55

arabino-oligosaccharides (up to arabinooctaose) are now commercially available [124], as are a number of aryl-a-L-arabinofuranosides [215]. Several recent studies have reported the isolation and structural characterization of a variety of arabinoxyIan-derived oligosaccharides [216-218], many of which have been used in combination with HPLC and NMR analysis to provide valuable information on arabinanase specificity [146,147,168]. Other useful substrates for arabinanase characterization have been recently synthesized, including arabinobiose derivatives with different linkages (i.e., methyl 0-a-L-arabinofuranosyl-(l-^2)-a-L-arabinofuranoside, methyl 0-a-L-arabinofuranosyl-(l->3)-a-L-arabinofuranoside, and methyl 0-a-L-arabinofuranosyl-(l->5)-a-L-arabinofuranoside) [219], and a branched arabinotriose derivative (methyl 3,5-di(9-arabinofuranosyl-a-L-arabinofuranoside) [220]. Finally, some recently developed arabinanase assays are worth mentioning. A specific endo-arabinansiSQ assay kit is now commercially available [124], employing a dyed and cross-linked substrate (Azurine-cross-linked linear arabinan). This substrate is reported to be very specific for e«Jo-arabinanase and can be used to detect activity in solution in the presence of large excesses of other pectic enzymes, including arabinofuranosidases, or to localize activity in electrophoresis gels [124]. Similarly, Yoshida et al. [221] have developed a sensitive method for detecting arabinofuranosidase activity in isoelectric focused gels based on the use of a colorimetric substrate, 6-bromo-2naphthyl-a-L-arabinofuranoside. Dunkel and Amado [222] have also recently developed a rapid, semiquantitative cujy-plate (diffusion) assay useful for detecting ewîo-arabinanase activity in column chromatography fractions based on the use of Procion Brilliant Red arabinan (discussed earlier) as substrate.

ACKNOWLEDGMENTS We would like to thank Dr. Gerhard A. de Ruiter for critically reading the manuscript, particularly the section dealing with NMR analysis, and Mrs. Helga Belling and Mrs. Gerda van Laar-Engelen for typing the manuscript. We would also like to thank Dr. Bemard Henrissat for kindly providing

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information relating to the classification of arabinanases by amino acid sequence similarity.

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[136] Cros, S., Imberty, A., Bouchemal, N., Herve du Penhoat, C. and Perez, S., Biopolymers, 34 (1994) 1433-1447. [137] Beldman, G., Searle-van Leeuwen, M.J.F., De Ruiter, G.A., Siliha, H.A. and Voragen, A.G.J., Carbohydr. Polym., 20 (1993) 159-168. [138] Van der Veen, P., Flipphi, M.J.A., Voragen, A.G.J, and Visser, J., Arch. Microbiol., 157 (1991) 23-28. [139] Morales, P., Madarro, A., Flors, A., Sendra, J.M. and Perez-Gonzalez, J.A., Enzyme Microb. TechnoL, 17 (1995) 424^29. [140] Bezalel, L., Shoham, Y. and Rosenberg, E., Appl. Microbiol. BiotechnoL, 40 (1993)57-62. [141] Gilead, S. and Shoham, Y, Appl. Environ. Microbiol., 61 (1995) 170-174. [142] Kaneko, S., Sano, M. and Kusakabe, I., Appl. Environ. Microbiol., 60 (1994) 3425-3428. [143] Schyns, P.J.YM.J., de Frankrijker, J., Zehnder, A.J.B. and Stams, A.J.M., Appl. Microbiol. BiotechnoL, 42 (1994) 548-554. [144] Manin, C , Shareek, F., Morosoli, R. and Kluepfel, D., Biochem. J., 302 (1994) 443^49. [145] Beldman, G., Osuga, D. and Whitaker, J.R., J. Cereal Sci., 23 (1996) 169-180. [146] Kormelink, F.J.M., Gruppen, H. and Voragen, A.G.J., Carbohydr. Res., 249 (1993)345-353. [147] Kaneko, S. and Kusakabe, I., Biosci. Biotech. Biochem., 59 (1995) 2132-2133. [148] Wood, T.M. and McCrae, S.I., Appl. Microbiol. BiotechnoL, 45 (1996) 538545. [149] Ramon, D., van der Veen, R and Visser, J., FEMS Microbiol. Lett., 113 (1993) 15-22. [150] Kaneko, S., Shimasaki, T. and Kusakabe, I., Biosci. Biotech. Biochem., 57 (1993)1161-1165. [151] Schopplein, E. and Dietrich, H., Dtsch. Lebensm-Rundsch., 87 (1991) 212219. [152] Kimura, I., Sasahara, H. and Tajima, S., J. Ferment. Bioeng., 80 (1995) 334-339. [153] Luonteri, E., Siika-oha, M., Tenkanen, M. and Viikari, L., J. BiotechnoL, 38 (1995)279-291. [154] Hespell, R.B. and O'Bryan, RJ., Appl. Environ. Microbiol., 58 (1992) 10821088. [155] Lee, S.R and Forsberg, C.W., Can. J. Microbiol., 33 (1987) 1011-1016. [156] Schwarz, W.H., Bronnenmeier, K., Krause, B., Lottspeich, F. and Staudenbauer, W.L., Appl. Micobiol. BiotechnoL, 43 (1995) 856-860. [157] Golubev, A.M., Kilimnik, A.Y, Rodionova, N.A., Kaprelyants, L.V. and Neustroev, K.N., Biochemistry (Russia), 58 (1993) 124-128. [158] Filho, E.X.R, Puis, J. andCoughlan, M.R, Appl. Environ. Microbiol., 62 (1996) 168-173. [159] Coughlan, M.R, Tuohy, M.G., Filho, E.X.R, Puis, J., Claeyssens, M., Vrsanska, M. and Hughes, M.M., In Coughlan, M.R and Hazelwood, G.R (eds.), Hemicellulose and Hemicellulases, Portland Press, London, 1993, pp. 53-84.

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STRUCTURAL ELUCIDATION OF THE N-LINKED OLIGOSACCHARIDES OF GLYCOPROTEINS USING HIGH pH ANION-EXCHANGE CHROMATOGRAPHY

Kevin D. Smith, Elizabeth F. Hounseil, John M. McGuire, Moira A. Elliott, and Heather G. Elliott

I. Introduction II. The Occurrence of A^-Glycosylation Sites


65

....

66 67

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SMITH ETAL

III. IV. V. VI. VII.

The Structure of A^-Glycosylation The Glycoform Concept The Chromatography of A^-Linked Chains High pH Anion-Exchange Chromatography The Diversity of HPAEC A. Monosaccharide Composition B. Sialic Acids C. Oligosaccharides VIII. Applications of HPAEC References

68 71 73 75 76 76 78 80 87 89

I. INTRODUCTION Protein glycosylation is a major modification which occurs during and after translation and is characterized by extensive structural heterogeneity. Functional studies have concluded that the oligosaccharide chains of glycoproteins are important determinants of overall biological activity through influencing both physiochemical (solubility, folding, conformation, stability, and protease resistance) and intermolecular (circulating half-life, immunogenicity, intracellular trafficking) properties [1]. Thus, the knowledge of the oligosaccharide structures of a glycoprotein, in addition to protein primary structure, is a prerequisite for the study of structure—function relationships. Oligosaccharide chains are ordered structures composed of various monomer units called monosaccharides (commonly Lfucose, D-mannose, D-galactose, A^-acetyl-D-galactosamine and A^-acetyl-D-glucosamine) and charged molecules (sialic acids) in a specific sequence. The latter are normally present as Aâcetylneuraminic acid (Neu5Ac) and its partially acetylated forms, such as 4-0-acetyl-7V^-acetylneuraminic acid, which in turn may be substituted with lactyl, methyl, or phosphate groups [2], or as A^-glycolylneuraminic acid (NeuSGc) and its 0-acetylated derivatives. Oligosaccharide chains consist of the aforementioned monosaccharide residues linked by glycosidic bonds, from the CI hydroxyl group (C2 in the case of the sialic acids), in either a or p anomeric configuration, to any other hydroxyl group of the adjacent residue. A typical oligosaccharide chain

N-Linked Oligosaccharides

67

contains from 2 to 20 residues arranged in a branched structure and covalently linked to the protein through the reducing end of one of the chain residues. Thus, the seven common monosaccharides found in the oligosaccharide chains of the mammalian cell surface may be assembled into many different structures by means of different linkages, by chain branching, and by the inclusion of substituents such as sulfate, phosphate, and acetyl groups. The oligosaccharide to protein linkages of glycoproteins occur either through the formation of an amide bond with asparagine (A^-glycosylation) or through a hydroxyl side chain of serine or threonine (0-glycosylation). Both A^- and O-glycosylation can occur on many different types of protein but the former predominate on plasma glycoproteins and the latter on glycoproteins of epithelial origin. This article will concentrate on the heterogeneity of A^-glycosylated structures between glycoproteins and review the use of high pH anion exchange chromatography (HPAEC) with pulsed electrochemical detection to detect these differences.

II. THE OCCURRENCE OF N-GLYCOSYLATION SITES The amino acid sequence of a glycoprotein can be used to predict the possible locations of A^-glycosylation sites: the presence of the consensus sequence asparagine-X-serine/threonine (Asn-XSer/Thr), where X is any amino acid except proline, normally indicates an A^-glycosylation site [3]. However, examples of A^glycosylation at asparagines outwith a consensus sequence exist in egg phosvitin [4], human von Willebrand factor [5] and the heavy chain of bovine protein C [6]. Earlier research [7] concluded that only one-third of sites possessing the consensus tripeptide were A/^-glycosylated. Komfeld and Kornfeld [8] provided an explanation stating the requirement for effective A^glycosylation of proteins as being "a sufficient pool of completely assembled and glycosylated lipid-linked oligosaccharide donor, an adequate activity of oligosaccharyltransferase, and a properly oriented and accessible Asn-X-Ser/Thr sequence in the acceptor". The critical point concerns the attainment of

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SMITH ETAL

a favorable conformation for glycosylation to occur which has been predicted as being a p-turn [9,10] or loop structure. These are generally located at the surface of proteins, thus being accessible to glycosyltransferases. Additionally the p-turn or loop represents a spatial arrangement favoring the formation of a hydrogen bond between the amide group of asparagine and the oxygen from the hydroxyl group of serine or threonine in the consensus signal. This is viewed as critical [11] to TV-linked glycosylation, perhaps due to the mechanism of the biosyntheses enzymatic reactions. Proline cannot equal X in the tripeptide sequence (in Asn-X-Ser/Thr) since it cannot achieve the conformation necessary for the occurrence of the aforementioned hydrogen bonding. Bause et al., [11] reported the formation of a disulfide bond near or around a consensus sequence limits the adoption of the required information. Yet and Wold [12] indicated that A^-linked oligosaccharide chains may interact with aromatic but not charged amino acid side chains within the three amino acid residues on either side of the glycosylated asparagine. This explains the observation that chymotryptic cleavages at the carboxy terminals of leucine, tyrosine, phenylalanine, tryptophan, and histidine residues, but not tryptic cleavages at the carboxy terminals of arginine or lysine residues, within this region are inhibited.

III. THE STRUCTURE OF N-GLYCOSYLATION So far we have dealt with A^-glycosylation at the level presented in most biochemical textbooks; however this secondary modification is not as simplistic as it seems, The A/-linked oligosaccharide structures of the same glycoprotein are capable of exhibiting a high degree of subtle variation in structure. Individual chains can be heterogeneous in terms of the number of outer chain branches or antennae [normally mono- (1), bi- (2), tri- (3), or tetra- (4) antennary], monosaccharide composition and sequence, the intrachain linkages, and degree of sialylation. All A^-linked oligosaccharides have a common pentasaccharide


69

core (Structure I) linked to asparagine, since they originate from a common precursor. a-Man-( 1 ->3)-|3-Man-( 1 -»4)-p-GlcN Ac-( 1 -^4)-p-GlcN Ac-( 1 ^ Asn 6

t

1 a-Man I The biosynthesis of A^-linked oligosaccharides is initiated by the transfer of a lipid-linked intermediate to the forming polypeptide in the endoplasmic reticulum (ER). Passage through the ER and the Golgi expose the immature chain to a variety of synthesizing and cleaving enzymes. This results in three distinct classes of TV-linked oligosaccharide chains which can be categorized according to their branch constituents: (a) mannose only (high mannose iV-glycans); (b) alternating 7V-acetylglucosamine and galactose residues terminated by sialic acid (a sialyllactosamine sequence), with the possibility of intrachain substitution with A^-acetylglucosamine or fucose or sialic acid (complex A^-glycans; Table 1); or (c) attributes of both high mannose and complex chains (hybrid A^-glycans). The heterogeneity of A^-linked oligosaccharide chains occurs in the monosaccharides linked to the a-(l->3)- and a-(l->6)linked mannose residues in the core region furthest from the asparagine. It is normally manifested as differences in the number of branches, the presence of peripheral sugars such as fucose, or the number of sialic acids, all of which affect the relative hydrophobicity and charge of the moiety. Complex and hybrid chains can exist as biantennary, triantennary, tetraantennary, and pentaantennary structures, and even in nonmammals, hexaantennary structures. The elongation of branch structures through substitutions of peripheral residues is most prominent in complex chains. In addition to those shown in Table 1, Kornfeld and Kornfeld [8] summarize the most common linkages as being a-Fuc-(l->2) or a-Gal-(l->3) or [a-Neu5Ac-(2->8)]„-aNeu5Ac-(2->3) [n = 8-12] to the terminal galactose residue on the lactosamine branch sequence; and a-Fuc-(1^3) or a-

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SMITH ETAL

Table 1. A Generalized Structure for the N-Glycosidically Linked Chains of Glycoproteins a-Neu5 Ac-(2-^3/6).p-Gal-( 1 ->3/4)-p-GlcNAc-1 ^ 6 a-Neu5 Ac-(2-^3/6)-p-Gal-( 1 ^3/4)-p-GlcNAc-( 1 ^2)-a-Man-1

C

i ^ 6 p-Man-(1^4)-R 3 ^ a-Neu5 Ac-(2->3/6)-p-Gal-( 1 ^3/4)-p-GlcNAc-( 1 ^6)-a-Man-1

B

A

t a-Neu5 Ac-(2->3/6)-p-Gal-( 1 ^3/4)-p-GlcNAc-1 R = -p-GlcNAc-(l->4)-p-GlcNAc-(lÂsn A complex chain consisting of branches: (i) A and B is biantennary and capable of being de-, mono-, or bisialylated; (ii) A, B, and D is Type I triantennary and capable of being de-, mono-, bi-, or trisialylated; (iii) A, B, and C is Type II triantennary and capable of being de-, mono-, bi-, or trisialylated (iv) A, B, C, and D is tetraantennary and capable of being de-, mono-, bi-, tri-, or tetrasialylated

Neu5Ac-(2-^6) to the A^-acetylglucosamine on the lactosamine branch sequence. A well-known substitution in high mannose chains is the addition of one or more phosphate groups on the outer mannose residues (mannose-6-phosphate) which occurs in mammalian lysosomal hydrolases and allows binding to the mannose-6-phosphate receptor. Occasionally the sialyllactosamine branch structures of complex A^-linked glycoproteins are incompletely processed as illustrated by Montreuil [13] for ovotransferrin and ovomucoid and by Yamashita et al. [14] for ovalbumin. A large number of nonconforming A^-linked structures occur as a result of disease such as the absence of one A^-acetyglucosamine residue from the pentasaccharide core in


71

human myeloma IgM [15]; or agalactosylation (the absence of both sialic acid and galactose residues) on the outer antennae of IgG in diseases such as rheumatoid arthritis, Crohn's disease, and tuberculosis [16].

IV. THE GLYCOFORM CONCEPT A property common to A^-linked glycosylation is the presence of glycosylated variants (glycoforms) of a single protein which have identical amino acid sequences and thus identical glycosylation sites but different glycosylation patterns. This heterogeneity can be explained by the fact that different cell types have different complements of processing enzymes which act upon the precursors of oligosaccharide chains in different ways. These variations reflect both the source of the molecule (cell or tissue) and the particular physiological and biochemical conditions existing at the time of release [17]. In particular, the presence of disease may alter the structures of the oligosaccharide chains giving rise to novel glycoforms of the same glycoprotein. The glycosylation pattern of a disease specific glycoform can be completely different from the normal pattern, or variation may be confined to one particular site. Additionally the basis of heterogeneity could be the presence or absence of a single monosaccharide which would be the difference between a structure being antigenic or not (Table 2). It is not within the scope of this article to document every published instance of novel A^-linked structures in disease since many excellent articles are already in print [16-18]. We will confine our interest to bovine fetuin, a-1-acid glycoprotein (AGP), and epidermal growth factor receptor (EGFR), for which HPAEC analytical data will be presented later. Bovine fetuin is an a-globulin present in fetal calf serum at a high concentration which diminishes gradually during the first weeks of life. It is a single polypeptide which is 22% glycosylated, shared unevenly between three 0-linked [19,20] and three A^-linked sites [19,21]. The considerable heterogeneity of structure at each A^-linked glycosylation site [22] appears to be due to sialylated variants of three structures. The majority of

72

SMITH ETAL

Table 2. Common Oligosaccharide Antigens

A—B- —c

t f

D Antigen

A

B

E

c

Blood group a-GalNAc- |3-Gal-(l-> — A (l->3) — a-GalBlood group (3-Gal-(l-> B (1^3) — Blood group a-Fucp-Gal-(l-> H (l->2) — p-Gal-(l-^4) (3-GlcNAcLewis X

D

E

a-Fuc(l->2) a-Fuc(l->2) —

—

—

Sialyl Lewis X Lewis A

a-NeuAc- p-Gal-(l->4) p-GlcNAc(2^3) (1^ — p-Gal-(l-^3) p-GlcNAc-

—

Sialyl Lewis A

a-NeuAc- p-Gal-(l->3) p-GlcNAc(2^3) (1^

—

—

— — a-Fuc(1^3) a-Fuc(1^3) a-Fuc(l->4) a-Fuc-

the A^-linked chains are triantennary with P-Gal-(l->4)-PGlcNAc-( 1^2/4) lactosamine antennae [23] with a type I branching pattern (Table 1); i.e., one antenna on the a-Man(l->6) and two on the a-Man-(l-^3) linked residues. Other minor populations consist of biantennary chains with Gaipi-4GlcNAc (lactosamine) sequences and triantennary chains with p-Gal-(1^3)-p-GlcNAc-(l->2/4) (lactosamine) sequences. Sialylated variants of the biantennary and the two triantennary structures have resulted in the identification of approximately 23 oligosaccharide chains in bovine fetuin. This glycoprotein therefore provides an excellent standard for HPAEC. AGP (orosomucoid) is a major serum glycoprotein that is classified as one of the positive acute phase reactants since its plasma concentration becomes elevated two- to fivefold in some disease states [24]. Human AGP is a tightly folded, monomeric

N'Linked Oligosaccharides

73

polypeptide, glycosylated with five asparaginyl-linked complex oligosaccharide chains accounting for approximately 45% of the molecular weight, of which 11% is sialic acid [25]. In certain diseases, both the total concentration of the glycoprotein and the relative proportions of the glycoform variants are altered markedly [17,26]. In cancer and other acute phase inflammatory conditions there is an increase in the proportion of glycoforms with biantennary chains, while in pregnancy, liver damage, and chronic inflammatory disorders such as rheumatoid arthritis the proportion of the biantennary glycoforms is decreased [17,26]. Recently, it has been reported that the absolute amount of sialyl Lewis X (SLX) determinants (Table 2) substituted AGP molecules and the number of SLX per molecule were enhanced under various acute inflammatory conditions and in patients with rheumatoid arthritis [27]. Epidermal growth factor receptor (EGFR) is the membrane bound mediator of the effects of EOF. The mature cell surface receptor, molecular weight 175 kDa and 20% glycosylation [28], is overproduced by cancer cells such as the A431 human epithelial cell line. The latter system also produces a soluble 115kDa protein with a sequence corresponding to the extracellular domain of the membrane receptor [29]. EGFR is one of the few membrane receptors in which glycosylation is an essential requirement for the interaction with its ligand [30]. Within the glycosylated population of EGFR, possession of the terminal blood group A specific GalNAc residue correlates with a low affinity for EGF [31].

V. THE CHROMATOGRAPHY OF N-LINKED CHAINS A complete structural characterization of individual A^-linked chains, in terms of its oligosaccharide structures, requires the isolation of each oligosaccharide chain together with the determination of the composition and anomeric nature of the constituent monosaccharides and their sequence and linkage positions. This would normally require significant (microgram) quantities of each oligosaccharide which may not be practically feasible. The heterogeneity of the oligosaccharides of glyco-

74

SMITH ETAL

proteins requires that chromatography be an essential part of any structural elucidation procedure. Several chromatographic methods are used for the analysis of the released oligosaccharides [32-34], which together with specific enzyme digestion greatly extend the amount of structural information capable of being elucidated. Classically this has been carried out for A^-linked chains of glycopeptides by Bio-Gel P4 chromatography and stepwise exoglycosidase digestion [22,35-37]. The susceptibility of an oligosaccharide to purified exoglycosidases (specific for anomeric configuration and nonreducing terminal sugar residue) is indicated by changes in the GPC elution profile. This technique is very time-consuming, only separates according to size, and problems may arise with the purity and specificity of the glycosidases. However, the recent introduction of an automated version of this technique results [38] in the ability to sequence picomole amounts of oligosaccharide structures. HPLC modes such as gel permeation, metal (lithium, barium or potassium) loaded cation-exchange, normal (aminopropyl bonded silica) phase, and reverse (octadecylsilica) phase have all been utilized extensively. However the optimum resolution of oligosaccharides is difficult to achieve. The differing chromatographic behavior of oligosaccharides on reverse-phase and amino-bonded (or amino-modified) columns allows a combination of these two systems which can often separate oligosaccharides not resolved by one system alone [39,40]. Reverse phase chromatography has also proved useful for the separation of oligosaccharides, for example as the pyridylamino or benzoyl derivatives [41—43]. The utilization of a two-column HPLC technique provides a 2D map of oligosaccharide which can be interpreted analytically [44-47], particularly when pyridylamino derivatives are used [48—51]. The highly sensitive fluorescence detection of pyridylamino derivatives is counterbalanced by the loss of sialic acid from sialylated oligosaccharides; however the use of l-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP) in labeling reactions has been shown not to cause desialylation [52]. Recently, a porous graphitized column support has been shown not only to exhibit similar properties to reverse phase


75

but also to separate closely related oligosaccharide isomers [53,54]. Anion exchange chromatography using alkali-susceptible silicabased columns can also be used for separation at acidic pH. For example, the separation of bi-, tri- and tetra-sialyl A^-linked oligosaccharides can be achieved on columns of AX-5 ion-exchange HPLC resin with an increasing potassium phosphate gradient [55]. Additional separations of isomeric oligosaccharides which differ only in the linkage position of the terminal Nacetylneuraminic acids, e.g., a-Neu5Ac-(2^3/6) residues linked to P-Gal-(l->4)-p-Glc [35], are achieved on these columns and amine-bonded phases such as aminopropylsilica when eluted in the presence of acetonitrile. Ion suppression amine absorption chromatography (ISAA-HPLC) introduced by Baenziger's group [39,40] also resolves oligosaccharide isomers which differ only in NeuAc linkage. ISAA-HPLG utilizes amine-bearing bonded phase columns from which oligosaccharides are eluted with glacial acetic acid/acetonitrile titrated to pH 5.5 with triethylamine. The technique overcomes the main disadvantage of normal ionexchange chromatography in that size as well as net charge contribute to the separation. Earlier work by Green and colleagues [58] demonstrated that the addition of borate to the eluent in the analysis of neutral sugars resulted in the formation of anionic oligosaccharide—borate complexes and subsequent separation by anion-exchange chromatography.

VI. HIGH pH ANION-EXCHANGE CHROMATOGRAPHY One of the most significant developments in the chromatogaphy of A/'-linked oligosaccharides has been the emergence of anionexchange separation using quaternary-ammonium-bonded pellicular resins (CarboPAc PA-1 or PA-100) at high pH elution. The use of strong alkali exploits the weakly acidic property of the hydroxyl groups of monosaccharides (and thus oligosaccharides) at pH>12. The resulting oxyanions can be chromatographed as anions, without pre-derivatization or the use of additives in the mobile phase, on the basis of molecular size, monosac-

76

SMITH ETAL

charide composition, and linkage of monosaccharide residues [59-61]. Excellent separations of the oxyanion derivatives of isomers and structurally closely related neutral and acidic oligosaccharides have been achieved [59-69]. The spherical pellicular resin contributes towards the high resolution of the technique by locating all the ionic groups on the surface resulting in the elimination of diffusion [60]. The technique is further enhanced by using pulsed amperometric detection (PAD), to give additional potential for 2D oligosaccharide mapping with sensitivity in the picomole range [70]. It utilizes the principle of triple amperometry to overcome the problem of electrode contamination by the oxidized product of carbohydrates associated with normal amperometry [71]. For many years the major disadvantage of HPAEC was the inability to further analyze eluted fractions due to the high salt content of the eluent. This problem has been largely overcome by the introduction of suppressor systems.

VII. THE DIVERSITY OF HPAEC HPAEC has the capacity to supplement and extend the existing techniques of structural elucidation of A^-linked oligosaccharides, particularly with regard to the rapid analysis of the monosaccharide, sialic acid, and oligosaccharide contents of small amounts of A^-linked glycoproteins without structural derivatization. A. Monosaccharide Composition

The ability to determine the monosaccharide composition for an individual glycoprotein is extremely important. Apart from verifying that the protein is glycosylated, it can indicate the type of glycosylation present and the identity and quantity of each monosaccharide present. The presence of A^-acetylgalactosamine usually indicates (9-glycosylation; while significant amounts of mannose are indicative of A^-glycosylation. The technique is useful as a mechanism to monitor the qualitative and quantitative changes in glycosylation which may occur during


77

disease, or to establish the consistency of the product after the recombinant production of a glycoprotein. The most cost-effective method for obtaining monosaccharides from intact oligosaccharide structures is by acid hydrolysis; for example incubation with 2 M trifluoroacetic acid for 4 h at 100 °C [71]. These conditions are optimal for the cleavage of all intrachain linkages in oligosaccharides and, with the exception of NeuSAc (which is destroyed), liberate the monosaccharide components in quantitative amounts [71]. Although direct analysis of the hydrolysate is possible, better profiles are obtained after ion-exchange chromatographic purification to remove the peptide and amino acid components of the digest. Monosaccharide compositional analysis using HPAEC is a rapid and economical method with the generation of profiles from one microgram of glycoprotein in less than 15 minutes [72]. The mobile phase of 160 mM sodium hydroxide ensures that the monosaccharides are present as their oxyanions and the order of elution is then correlated with their individual pK^ values. Additionally, since each of the hydroxyl groups in an individual monosaccharide has a slightly different pK^ value, the loss of a specific hydroxyl group, for instance in the formation of a glycosidic bond, alters the elution position of a monosaccharide by changing its pK^ value. Thus, a mixture of galactose, 2-deoxygalactose, and 6-deoxygalactose (fucose) can be separated to baseline resolution (Figure 1). The advantages of HPAEC for monosaccharide composition, namely its rapidity and reproducibility with no requirement for sample derivatization, has resulted in the examination of glycan chains derived from a wide variety of glycoproteins [73 and references therein]. The compositional profiles for standard monosaccharides and the recombinantly produced extracellular domain of EGFR are illustrated (Figure 2). In the standard profile the order of elution is largely correlated to the pK^ value although the positions of galactose and 7V-acetyl-D-glucosamine are interconvertable with concentration [60]. The amounts of A^-acetyl-D-glucosamine, mannose, and galactose in the oligosaccharide chains of EGFR are consistent with the presence of complex bi- or triantennary

78

SMITH ETAL

50 H

I/)

LL

>

E

c

Q UJ Q_

Figure 1. The HPAEC separation of (1) fucose, (2) 2-deoxygalactose, and (3) galactose by HPAEC. The chromatography was carried out as described in Smith et al. [75].

A^-linked structures (Table 3). The presence of fucose could signify the appearance of an antigenic determinant on the outer chain. Fucose (a-Fuc) and A'^-acetyl-D-galactosamine are present in amounts which suggest that one-third of the oligosaccharide structures could contain blood group A (terminal a-Fuc and aGalNAc) or H (terminal a-Fuc) antigens. B. Sialic Acids

The sialic acids are a family of nine carbon carboxylated sugars which confer a formal negative charge on certain classes— hybrid and complex—of A^-linked chains through terminating the branch antennae (see Table 1). This results in sialic acids, or any oligosaccharide chain containing them, binding more strongly to anion-exchange resins. Thus, in comparison to neutral and amino sugars, stronger elution conditions are required to elute the retained charged molecules from the column. This is achieved by the application of an increasing linear gradient of sodium acetate (50-250 mM sodium acetate over 30 min) in addition to the constant alkaline pH conferred by 100 mM


79

GalNAc

100-lA

>

I 50C

o

Q. tf) Q» OH

a

IXJ Q-

10

I

15

100-

> E

en a lU CL

50 H

Fuc

Gal GalNAcyv

Man

10

15

Figure 2. The HPAEC separation of (A) 50 ng each of fucose (Fuc), N-acetylgalactosamine (GalNAc), galactose (Gal), N-acetylglucosamine (GlcNAc) and mannose (Man); (B) the monosaccharides hydrolyzed from 2 jj,g of rEGFR. The chromatography was carried out as described in Smith et al. [75].

80

SMITH ETAL

Table 3. The Monosaccharide Composition of rEGFR as Determined by HPAEC (Figure 2) Monosaccharide

Amount per 2 [ig rEGFR

Fucose A^-Acetylgalactosamine Galactose A^-Acetylglucosamine Maimose

14.4 ng 12.1 ng 67.8 ng 236.6 ng 131.7 ng

Amount per mol rEGFR 4.8 mol 3.0 mol 20.7 mol 58.8 mol 40.2 mol

sodium hydroxide. The sialic acid content of a glycoprotein can be determined after hydrolysis either by enzyme (neuraminidase, 10 mU, 37 °C, 1 h) or acid (0.1% hydrochloric acid, 70 °C, 1 h). HPAEC is capable of separating the two major sialic acids, A^-glycolyl- and A/'-acetylneuraminic acid, by more than 10 minutes on the sodium acetate gradient despite only differing in structure by one extra hydroxyl group (Figure 3A). Unfortunately, this gradient is unable to separate the individual O-acetylated forms (reviewed in ref. 74) of each sialic acid. Aliquots (45 pmol) of the recombinant form of EGFR was desialylated using both enzyme digestion (Figure 3B) and acid hydrolysis (Figure 3C). The protein was found by resorcinol staining to contain the equivalent amount of sialic acid to 485 pmole of standard iV-acetylneuraminic acid, which is consistent with the presence of bi- or trisialylated chains on each of the 11 potential A^-glycosylation sites. However the HPAEC profiles show that the time of the released product did not correlate with the retention of the standards. This may represent a five-deaminated derivative of sialic acid (H rather than NHCOCH3 at C5 of Neu5Ac) resulting from the cell type used in the recombinant production. However this type of modification has not been previously reported. C.

Oligosaccharides

So far, HPAEC has been discussed in the context of determining the monosaccharide and sialic acid compositional analysis of glycoproteins and/or individual oligosaccharide chains.

NeuSGc

mm

100 n (/) > E

Si

c o

Q. V)

50H

Q UJ Q.

:L 10

20 min

30

40

Figures. HPAECprofilesof(A)N-acetylneuraminicacid (NeuSAc)and N-glycolylneuraminic acid (NeuSGc); (B) sialic acid from rEGFR released by neuraminidase; (C) sialic acid from rEGFR released by HCI hydrolysis. The chromatography was carried out as described in Smith etal. [75]. 81

82

SMITH ETAL.

However the diversity of the technique extends to the qualitative determination of the iV-linked oligosaccharides present on a glycoprotein as a whole or at a particular glycosylation site. It is particularly useful in the resolution of complex sialylated structures. The TV-linked structures are released enzymically using peptide: A^-glycosidase F [PNGase F, peptide-N'^-Cacetyl-P-glucosaminyl)asparagine amidase] which catalyses the hydrolysis of the A^,jV'-diacetylchitobiose bond adjacent to the asparagine residues of all A^-linked oligosaccharides [71]. The reaction mechanism differs from that of endo-glycosidasQS D, H, and F. These enzymes cleave the glycosidic linkage between the two GlcNAc residues. The released oligosaccharides, each bearing a reducing terminal p-GlcNAc residue, are resolved by HPAEC using an acetate gradient (50-200 mM IM sodium acetate in 40 min in the presence of 100 mM sodium hydroxide) similar to that used for sialic acids [72,75]. An HPAEC profile for the A^-linked oligosaccharides released from bovine fetuin by PNGase F gives a complex pattern (Figure 4). The initial separation is in terms of the number of sialic 100

> E g o

triantcmary 2|

50-

Q.

Q UJ £L

10

20

30 min

1^ AO

50

60

1 70

Figure 4. HPAEC profile of 200 pmol of the N-linked oligosaccharides released from bovine fetuin using PNGase F. The chromatography was carried out as described in Smith et al. [75].

N-L inked Oligosaccharides

83

acid residues per chain and thus the degree to which an individual structure is retained on the column. Thus fetuin can be shown to contain bisialylated, trisialylated, and tetrasialylated structures. The presence of fucosylation causes an oligosaccharide to elute earlier than its non-fucosylated counterpart [67,68,76]. Thereafter, separation occurs within each charge band on the basis of size (a bisialylated, biantennary chain will be retained to a lesser extent than a bisialylated, triantennary chain) and, for similarly sized structures, in terms of isomeric differences normally differing intrachain linkages. The involvement of one or more hydroxyl groups of a monosaccharide in glycosidic bonds to other monosaccharides will alter its ionization. Thus two oligosaccharides which have identical monosaccharide sequences but different glycosidic linkage positions can be separated. This effect can be illustrated by reference to Figure 4 and structures II-IV. Structures II and IV are separated due to differing linkages between the Gal and GlcNAc in the lactosamine branches [63], i.e. -P-Gal-(l->3)-p-GlcNAc-(l-^4) (structure IV) elutes after -P-Gal-(l-^4)-P-GlcNAc-(l->4) (structure II). A similar effect occurs with the linkages between sialic acids [65]; the greater the proportion of (2-^3) to (2->6) linkages the longer the elution time; structure II is a trisialylated chain which contains two a-Neu5Ac-(2->6) and one a-Neu5Ac-(2-^3) linkages while

a-Neu5Ac-(2->6)-p-Gal-(l-»4)-p-GlcNAc-(l->2)-a-Man-l

i 6 p-Man-(l->4)-R 3

t

a-Neu5Ac-(2->6)-p-Gal-(1^4)-P-GlcNAc-(l->6)-a-Man-l 4

t

a-Neu5Ac-(2->3)-P-Gal-(l-^4).p-GlcNAc-l II

84

SMITH ETAL.

a-Neu5Ac-(2->3)-P-Gal-(l->4)-P-GlcNAc-(l->2)-a-Man-l

i 6 P-Man-(l-»4)-R 3

t a-Neu5 Ac-(2-»6)-p-Gal-( 1 ■^4)-p-GlcNAc-( 1 -»6)-a-Man-1 4

t

a-Neu5 Ac-(2-^3)-P-Gal-( 1 -M)-p-GlcNAc-1 III a-Neu5Ac-(2->3)-p-Gal-(l-»4)-p-GlcNAc-(l-^2)-a-Man-l

i 6 P-Man-(l->4)-R 3

t

a-Neu5Ac-(2-^6)-p-Gal-(l->4)-P-GlcNAc-(1^6)-a-Man-l 4

t

a-Neu5Ac-(2->3)-p-Gal-(l-^3)-p-GlcNAc-l IV R = -P-GlcNAc-(1^4)-p-GlcNAc-(l-).Asn

Structure III contains the reverse. Reduction of the released iV-linked oligosaccharides by alkaline borohydride treatment [71] decreases the retention times in the profile (Figure 5) without any major decrease in response or resolution [77]. PNGase F treatment of rEGFR and HPAEC analysis of the released oligosaccharides (Figure 6) gave one major peak at approximately 35 minutes in the region corresponding to a relatively slow eluting biantennary chain, perhaps as result of trisialyla-


85 Reduced triantennary

Figure 5. HPAEC profile of 1 nmol of the N-linked oligosaccharides released from bovine fetuin and then reduced to alditols by alkaline borohydride treatment [71 ]. The chromatography was carried out on a Dionex-500 HPAEC system using the gradient of McGuire et al. [77].

100-1

Figure 6. HPAEC profile of the N-linked oligosaccharides released from 45 pmol of rEGFR The chromatography was carried out as described in Smith et al. [75].

86

SMITH ETAL

loo-

se-

1

1

10

20

1 30 min

1

40

1 50

1

60

1 70

Figure 7. HPAEC profile of the N-linked oligosaccharides released from rEGFR after fucosidase treatment. The chromatography was carried out as described in Smith et al. [75].

tion, or a fast eluting trisialylated chain. Treatment with a fucosidase which removes inner core a-Fuc-(l-^6) linked residues (Structure V), prior to PNGase F treatment, significantly increased the elution time of the peak (Figure 7) to a time compatible with a non-fucosylated triantennary structure.

a-Man-(l->3)-P-Man-(l->4)-p-GlcNAc-(l->4)-|3-GlcNAc-(l-Âsn 6 6

t

1 p-GlcNAc-( 1 ->6)-a-Man 4

t

1 p-GlcNAc

t

1 a-Fuc


87

Vni. APPLICATIONS OF HPAEC The ability to generate an oligosaccharide profile for a specific glycoprotein provides a sensitive diagnostic mechanism to monitor changes in glycosylation between the normal molecule and those generated under abnormal (disease) conditions or in a different system, e.g. recombinant production. As mentioned earlier, the glycosylation pattern at each of the five glycosylation sites of a-1-acid glycoprotein (AGP) is known to alter. In certain diseases abnormally glycosylated variants (glycoforms) which may be functionally significant, are expressed. We have compared the glycosylation patterns of AGP from patients with contrasting degrees of rheumatoid arthritis using high pH anion exchange chromatography (Figure 8) after deglycosylation of the glycoprotein (Table 4). The conclusion from a limited study is that the glycosylation pattern of AGP in rheumatoid arthritis can be directly correlated with the disease activity. Results indicate that a stepwise increase in the mean grade of disease activity (II->III->IV) is correlated with an increase in the biantennary portion of total AGP and a significant reduction in the content of tri- and tetraantennary chains. This type of pattern has previously only been reported in acute inflammatory conditions [79]; normally patients with chronic symptoms have dis-

Table 4. The Classification of the Patients Used to Study Alterations in the Glycosylation Patterns of AGP during Rheumatoid Arthritis (Figure 8)

Patient 1 2 3 Abbreviations: Hb ESR MGDA

Disease Duration (years)

ESR (mm/h)

Hb (g/dl)

MGDA [78]

16 14 2

20 50 106

12.1 13.1 10.1

II III IV

Hemoglobin level Erythrocyte sedimentation rate Mean grade of disease activity

88

SMITH ETAL

PATIENT 1

100 H

triantennary

> E

I

tetraantennary

Q UJ

a.

OH 20

25

30

n

35

40

\

1

1

45

50

55

45

50

55

45

50

55

100 H PATIENT 2

>

Q

ID

OH 15

20

1^

25

I

30

35

"1— 40

1 ^

I

PATIENT 3

100-

20

25

-T 30

r. 35 mm

40

Figure 8. HPAEC profile of the N-linked oligosaccharides released from AGP isolated from rheumatoid human plasma of patients with varying disease activity. The chromatography was carried out on a Dionex-300 HPAEC system using the gradient of Smith et al. [75].


89

played an increased expression of tri- and tetraantennary chains to the detriment of biantennary structures. REFERENCES [1] [2] [3] [4] [5]

Varki, A., Glycobiology, 3 (1993) 97-130. Schauer, R., Adv. Carbohydr. Chem. Biochem., 40 (1982) 131-234. Gavel, Y. and von Heijne, G., Prot. Eng., 3 (1990) 433-443. Marshall, R.D., Ann. Rev. Biochem., 41 (1971) 673-702. Titani, K., Kumar, S., Takio, K., Ericsson, L.H., Wade, R.D., Ashida, K., Walsh, K.A., Chopek, M.W., Sadier, J.E. and Fujikawa, K., Biochemistry, 25 (1986) 3171-3184. [6] Stenflo, J. and Femlund, J., J. Biol. Chem., 257 (1982) 12180-12190. [7] Kronquist, K.E. and Lennarz, W.J., J. Supramol. Struct, 8 (1978) 51-55. [8] Komfeld, R. and Komfeld, S., Ann. Rev. Biochem., 54 (1985) 631-664. [9] Marshall, R.D., In Smellie, R.M.S. and Beeley, J.G. (eds.). The Metabolism and Function of Glycoproteins, Biochemical Symposia No. 40, The Biochem. Soc, London (1974) 17-26. [10] Avanov, A. Ya., Mol. Biol., 25 (1991) 237-249. [11] Bause, E., Biochem. J., 209 (1983) 331-336. [12] Yet, M.G. and Wold, R, Arch. Biochem. Biophys., 278 (1990) 356-364. [13] Montreuil, J., Adv. Carbohdr. Chem. Biochem., 37 (1980) 157-223. [14] Yamashita, K., Tachibana, Y. and Kobata, A., J. Biol. Chem., 253 (1978) 3862-3869. [15] Miller, R, Immunochemistry, 9 (1972) 217-228. [ 16] Rademacher, T.W, Parekh, R.B. andDwek, R.A., Ann. Rev. Biochem 57 (1988) 785-838. [17] van Dijk, W., Turner, G.A. and Mackiewicz, A., Glycosylation and Disease, 1 (1994)5-14. [18] Lis, L. and Sharon, N., Eur. J. Biochem., 218 (1993) 1-27. [19] Begbie, R., Biochim. Biophys. Acta., 371 (1974) 549-567. [20] Spiro, R.G. and Bhoyroo, V.D., J. Biol. Chem., 249 (1974) 5704^5717. [21] Spiro, R.G., Adv. Protein Eng., 27 (1973) 349-367. [22] Takasaki, S. and Kobata, A., Biochemistry, 25 (1986) 5709-5715. [23] Baenziger, J.U. and Fiete, D., J. Biol. Chem., 254 (1979) 789-795. [24] Baumann, H. and Gauldie, J., Mol. Biol. Med., 7 (1990) 147-159. [25] Schmid, K., Kaufmann, H., Isemura, S., Bauer, R, Emura, J., Motoyama, Ishiguro M.T. andNanno, S., Biochemistry, 12 (1973) 2711-2722. [26] Turner, G.A., Clin. Chim. Acta., 208 (1992) 149-171. [27] De Graaf, T.W, Van der Stelt, M.E., Anbergen, M.G. and van Dijk, W, J. Exp. Med., 177(1993)657-666. [28] Mayes, E.L.V. and Waterfield, M.D., EMBO. J., 3 (1984) 531-537. [29] Soderquist, A.M., Stoscheck, C. and Carpenter, G., J. Cell. Physiol. 136 (1988) 447-454.

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SMITH ETAL

[30] Slieker, L.J. and Lane, M.D., J. Biol. Chem., 260 (1985) 687-690. [31] Defize, L.H.K., Amdt-Javin, D.J., Jovin, T.M., Boonstra, J., Meisenhelder, J., Hunter, T., de Hey, H.T. and de Laat, S.W., J. Cell. Biol., 107 (1988) 939-949. [32] Honda, S., Anal. Biochem., 140 (1984) 1^7. [33] Hicks, K.S., Adv. Carbohydr. Chem. Biochem., 46 (1988) 17-72. [34] Hounsell, E.F., In Lim, C.K., (ed.), HPLC of Small Molecules, IRL Press, Oxford, (1986) 49-^8. [35] Yamashita, K., Mizouchi, T. and Kobata, A., Meth. EnzymoL, 83 (1982) 105-126. [36] Sutton, C.W., O'Neill, J.A. and Cottrell, J.S., Anal. Biochem., 218 (1994) 3^M6. [37] Pfeiffer, G., Schmidt, M., Strobe, K-H. and Geyer, R., Eur. J. Biochem., 186 (1989)273-286. [38] Edge, C.J., Rademacher, T.W., Wormald M.R., Parekh, R.B., Butters, T.D., Wing, D.R. andDwek, R.A., Proc. Natl. Acad. Sci. USA, 89 (1992) 6338-6342. [39] Hounsell, E.F., Jones, N.J. and Stoll, M.S., Biochem. Soc. Trans., 15 (1985) 1061-1064. [40] Hounsell, E.F., Lawson, A.M., Feeney, J., Gooi, H.C., Pickering, N.J., Stoll, M.S., Lui, S.C. and Feizi, T., Eur, J. Biochem., 148 (1985) 367-377. [41] Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K. andNakamura, J., Anal. Biochem., 180 (1989) 351-357. [42] Daniel, RF., de Feudis, D.F., Lott, LT. and McCleu, R.H., Carbohydr. Res. 97 (1981)161-189. [43] Webb, J.W, Jiang, K., Gillece-Castro, B.L., Tarentino, A.L., Plummer, T.H., Byrd, J.C, Fisher, S.J. and Burlingame, A.L., Anal. Biochem., 163 (1988) 337-349. [44] Bendiak, B. and Gumming, D.A., Carbohydr. Res., 151 (1986) 89-103. [45] Arbatsky, N., Martynova, M.D., Zhelrova, A.O., Derevitskaya, V.A. and Kochetkov, N.K., Carbohydr. Res., 187 (1989) 165-171. [46] Bendiak, B., Harris-Brandts, M., Michnik, S.W., Carver, J.P. and Gumming, D.A., Biochemistry, 28 (1989) 6491-6499. [47] Kakehi, K. and Honda, S., J. Chromatog., 379 (1986) 27-55. [48] Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y. and Takahashi, N., Anal. Biochem., (1988) 171, 73-90. [49] Oku, H., Hase, S. and Ikenaka, T., Anal. Biochem., 185 (1990) 331-334. [50] Hase, S. and Ikenaka, T., Anal. Biochem., 184 (1990) 135-138. [51] Tomiya, N., Lee, YC., Yoshida, T., Wada, Y, Awaya, J., Kurono, M. and Takahashi, N., Anal. Biochem., 193 (1991) 90-100. [52] Kakehi, K., Suzuki, S., Honda, S. and Lee, Y C , Anal. Biochem., 199 (1991) 256-268. [53] Davies, M., Smith, K.D., Harbin, A-M. and Hounsell, E.F., J. Chromatog., 609 (1992)125-131. [54] Davies, M., Smith, K.D., Harbin, A-M., Carruthers, R.A., Chai, W., Lawson and Hounsell, E.F., J. Chromatog., 646 (1993) 317-326. [55] Baenziger, J.U. and Notowicz, M., Anal. Biochem., 112 (1981) 357-361.


91

[56] Green, E.D. and Baenziger, J.U., Anal. Biochem., 158 (1986) 42-^9. [57] Green, E.D., Adelt, G., Baenziger, J.U., Wilson, S. and van Halbeek, H., J. Biol. Chem., 263 (1988) 18253-18268. [58] Green, E.D., Baenziger, J.U. and Boime, I., J. Biol. Chem., 260 (1985) 1563115638. [59] Spellman, M.W., Anal. Chem., 62 (1990) 1714-1722. [60] Lee, Y.C., Anal. Biochem., 189 (1990) 151-162. [61] Hardy, M.R., Meth. Enzymol., 179 (1989) 76-82. [62] Hardy, M.R. and Townsend, R.R., Meth. Enzymol, 230 (1994) 208-225. [63] Hardy, M.R. and Townsend, R.R., Proc. Natl. Acad. Sci. USA, 85 (1988) 3289-3293. [64] Hardy, M.R. and Townsend, R.R., Carbohydr. Res., 188 (1989) 1-7. [65] Townsend, R.R., Hardy, M.R., Gumming, D.A., Carver, J.R and Bendiak, B., Anal. Biochem., 182 (1989) 1-8. [66] Townsend, R.R., Hardy, M.R. and Lee, Y.C., Meth. Enzymol., 179 (1989) 65-76. [67] Pfeiffer, G., Geyer, H., Kalsner, L, Wendorf, R and Geyer, H., Biomed. Chromatog., 4, (1990) 193-199. [68] Basa, L.J. and Spelhnan, M.W., J. Chromatog., 499 (1990) 205-220. [69] McGuire, J.M., Douglas, M. and Smith, K.D., Carbohydr. Res., 292 (1996) 1-9. [70] Hermentin, R, Witzel, R., Doenger, R., Bauer, R., Haupt, H., Patel, T, Parekh, R.B and Brazel, D., Anal. Biochem., 206 (1992) 419-429. [71] Davies, M.J., Smith, K.D. and Hounsell, E.F., Meth. Molec. Biol., 32 (1993) 129-141. [72] Smith, K.D., Davies, M.J., and Hounsell, E.R,: Meth. Molec. Biol. 32 (1993) 143-155. [73] Hardy, M.R. and Townsend, R.R., Glycobiology, 1 (1991) 139-147. [74] Varki, A., Glycobiology, 2 (1992) 25-40. [75] Smith, K.D., EUiott, M.A., Elliott, H.G., McLaugWin, CM., Wightman, P and Wood, G.C., J. Chromatog. Biomed. AppL, 661 (1994) 7-14. [76] Anumula, K.R. and Taylor, PB., Eur. J. Biochem., 195 (1989) 269-280. [77] McGuire, J.M., Elliott, M.A., Elliott, H.G. and Smith K.D., Carbohydr. Res., 270(1995)63-69. [78] Mallya, R.K. and Mace, B.E.W., Rheumatol, and Rehabil, 20 (1981) 14-17. [79] Raynes, J., Biomedicine, 36 (1982) 77-86.


NEOGLYCOCONJUGATES AS ARTIFICIAL ANTIGENS: CHEMICAL ASPECTS

Nikolay K. Kochetkov and Anatoly Ya. Chernyak

I. Introduction 94 II. The Principles of Design and Architecture of Glycoconjugates 95 III. Glycoconjugates with Oligosaccharide as a Carbohydrate Moiety 98 A. Carbohydrate Carriers of Specificity: Their Preparation . . 98 B. Conjugation to a Polymer Carrier 106 C. Preparation of Oligosaccharide Derivatives Suitable for Conjugation 116 D. Discussion 130 E. Polymerization as a Method for Preparing Glycoconjugates with a Synthetic Polymer Carrier . . . . 132


93

94

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

IV. Neoglycoconjugates with Polysaccharide as a Carbohydrate Moiety A. Utilization of Natural Polysaccharides B. Utilization of Synthetic Polysaccharides V. Conclusion Acknowledgments Notes References

152 153 161 164 166 166 166

I. INTRODUCTION Novel aspects have appeared in the chemistry and biochemistry of carbohydrates in the last 10 to 20 years. It became clear that their role in the functioning of living cells is much wider and more important than was long believed. This is primarily because in many carbohydrate structures information is encoded which determines, in particular, cell-to-cell and cell-to-cytoplasm interactions [1,2]. This can be best exemplified by the well-known role of surface or capsular polysaccharides of microbial cells which determine their antigenic properties [3] or of group-specific animal and human glycoproteins, the carbohydrate chains of which are responsible for group specificity [4]. Studies on the essence and mechanisms of these finest biological events require, naturally, not only the knowledge of the structures of the carbohydrate-containing compounds involved but also of the very compounds under study in sufficient amounts and, even more critical, of an appropriate purity. If additionally it is taken into account that in many cases their concentration in nature is extremely low and they are the components of very intricate complexes or mixtures, it becomes evident that their isolation for subsequent experimental studies may require processing of a vast amount of a natural material and the application of skilled labor. In this connection semisynthetic compounds became of definite importance in the very beginning of studies on biological specificity of carbohydrate structures. They were prepared by

Neoglycoconjugates Synthesis

95

covalent attachment of a carbohydrate molecule under investigation, or its fragment, to a high molecular weight carrier, frequently a protein. This approach, which was initiated by the classical works of Landsteiner [5] on the synthesis of artificial antigens and further developed by Goebel et al. [6], is currently very popular. Compounds of this type with a carbohydrate fragment immobilized on a high molecular weight carrier are named neoglycoconjugates^ and constitute an independent and rapidly expanding class of polymers. Neoglycoconjugates, which in a broad sense involve both synthetic analogs and modified carbohydrates with complex substituents, in particular, have found not only biomedical and pharmacological applications [7] but also some technical uses. The latter were usually developed quite independently although some approaches that resulted were also used for the preparation of neoglycoconjugates of biological significance. This review deals mainly with the latter group which are employed as artificial antigens. Other studies not related directly to this topic will be briefly discussed. Methods for preparation of neoglycoconjugates, mostly neoglycoproteins, and the diversity of their uses have been covered in several excellent reviews published a decade ago [8-10]. The rapidly increasing use of compounds of this kind for the solution of many problems in life sciences has resulted in the equally rapid development of synthetic approaches to obtain them. Relevant novel data appearing in the last few years are essential for practical work in this field and are also reviewed here.

II. THE PRINCIPLES OF DESIGN AND ARCHITECTURE OF GLYCOCONjUGATES Generally, the problem of synthesis of glycoconjugates can be considered as covalent incorporation of carbohydrate fragments into a macromolecule. This was traditionally performed by conjugation of a carbohydrate moiety of a given specificity, e.g. a hapten in the synthesis of artificial antigens to an existing polymeric support (route A, Scheme 1). The support which is most


96 SUGAR

SUGAR (carbohydrate monomer)

(polymer carrier )

( noncar bo hydrate monomer)

SUGAR

SUGAR

Scheme 1.

often used is a protein [8], although sometimes it is a synthetic polypeptide [11] or some other macromolecule. A novel alternative approach for the preparation of neoglycoconjugates consists of the copolymerization of carbohydrate fragments (haptens in the case of antigens) bearing functional groups suitable for polymerization with non-carbohydrate monomers (route B, Scheme 1) [12,13]. In this case the polymeric carrier is formed in the very process of synthesis of the neoglycoconjugate. When using neoglycoconjugates as models of the natural carbohydrate-containing biopolymers it should be kept in mind that quite often their overall design differs considerably from that of natural compounds and results in the alteration of biological properties. This should be taken into account when planning glycoconjugate synthesis. Thus, artificial antigens, which mimic bacterial ones, are usually built on to a central polymer chain to which oligosaccharides are laterally attached which, in turn, correspond to a


97

repeating unit of a natural polysaccharide. This is essentially in contrast to the design of the natural prototypes: for instance, somatic antigens of a Gram-negative microbial cell surface (the so-called 0-antigens) contain long polysaccharide chains comprising 10 or more repeating units [14], while capsular antigens are polysaccharides of extremely high molecular mass also built of repeating oligosaccharide units [3]. These differences in design may result in disappearance from the neoglycoconjugate structure of certain elements typical of the natural prototype and the appearance of new ones. Thus, the fragment A-^C inherent to the prototype is modified when present as a component of a neoglycoconjugate shown in Scheme 2: the monosaccharide A is not linked to the residue C of the neighboring repeating unit but is coupled to a carrier to leave C unsubstituted. The relative proportion of these Cterminal residues can be much higher than in the natural structure. This, in turn, can alter to a certain extent the serological specificity of neoglycoconjugates, especially if it is taken into

RU (repeating unit)

RU

«~

spacer

98


account that monosaccharide residues at the nonreducing end of oligo- and polysaccharides are, as a rule, immunodominant [3]. It is thus of importance to identify the fragment of the carbohydrate polymer which is introduced into the neoglycoconjugate, with the artificial antigen in particular. Two fundamentally different types of artificial antigens should be distinguished: one contains oligosaccharide or in extreme cases even monosaccharide fragments, while the other less frequently comprises polysaccharide chains attached to a macromolecular support. Distinction between these two types of artificial antigens is associated not so much with different conjugation procedures (although this is also the case, cf. Section IV) but rather with the aims of their preparation. In fact, based on its general character of the principle of regularity, repetition for the majority of the natural polysaccharide antigens leads to the conclusion that the information on the antigenic specificity is encoded, for the most part, in the structure of one-two repeating oligosaccharide units. Therefore, artificial antigens, with short oligosaccharide fragments, simulate partial antigenic specificities and serve as an indispensable tool in studies of immunochemistry of natural carbohydrate-containing antigens. Conjugation of purified polysaccharide preparations to a polymeric carrier pursues another goal; namely the preparation of an immunogenic material which can be used for eliciting anti-polysaccharide antisera and serve as a model of polysaccharide vaccines for prophylaxis of infectious diseases. These two kinds of glycoconjugates differ considerably in the nature of the starting carbohydrates and in techniques of their immobilization on a high molecular weight carrier. Therefore their preparation will be discussed here separately. III. GLYCOCONJUGATES WITH OLIGOSACCHARIDE AS A CARBOHYDRATE MOIETY A. Carbohydrate Carriers of Specificity: Their Preparation

The preparation of an artificial carbohydrate antigen requires the oligosaccharide fragment to be present in a pure state and


99

in a sufficient amount. Natural glycoconjugates themselves are sources for the oligosaccharides from which they can be isolated by means of chemical or enzymic degradation. However, the oligosaccharide fragments obtainable by these methods are few compared to those which exist in natural glycoconjugates since it is not always possible to split linkages specifically by chemical methods and substrate specificity of enzymes sets its limits for enzymatic degradation. Thus the series of oligosaccharides which may actually be prepared from the natural sources can be rather limited. Chemical Degradation

Solvolytic splitting of glycosidic linkages is the most widespread method for isolation of oligosaccharides from natural polysaccharides. Hydrolysis with mineral or organic acids [15] under controlled conditions quite often splits the most labile glycosidic bonds present in a given structure rather selectively. Thus, for instance, furanosidic linkages and glycosidic linkages of deoxy- and dideoxysugars can be split with sufficient selectivity. On the other hand, glycosidic linkages of hexuronic acids are more resistant to hydrolysis than those of neutral sugars. Thus, partial acid hydrolysis of the capsular polysaccharide of Streptococcus pneumoniae type 3 (or type 8) afforded cellobiouronic acid [16] which was used to prepare one of the first artificial antigens for modeling bacterial polysaccharides [17]: ^3)-P-D-GlcA-(l->4)-p-D-Glc-(1^3)-|3-D-GlcA-(l->4)-p.D-Glc-(l->

P-D-GlcA-(l->4)-D-Glc

The resistance of glycosidic linkages towards acetolysis can sometimes differ from that towards acid hydrolysis, as is the case with (l->6)-linkages [18]. Splitting of natural polysaccharides by this method gives rise to peracetylated fragments. A new and promising method for selective scission of glycosidic linkages is the solvolysis with anhydrous hydrogen fluoride (Scheme 3) [19]. Variation of conditions, mainly reaction

100


"'»(A)

rVo„

R'OH

Scheme 3.

temperature, affects the specificity of splitting and produces different oligosaccharides from the same starting material. The regularities of the solvolysis with hydrogen fluoride have been reviewed [19]. According to the accepted mechanism (Scheme 3), reaction with hydrogen fluoride results in the formation of glycosyl fluorides. Their subsequent fate is determined by the mode of treatment of the reaction mixture. Addition of water (pathway A) gives reducing oligosaccharides, while treatment with methanol (pathway B) affords methyl glycosides. If treatment of glycosyl fluorides with other alcohols suitable for subsequent conjugation (e.g. allyl alcohol) also gives rise to glycosides, the solvolysis with hydrogen fluoride can become a valuable method for production of oligosaccharides which can be directly employed in designing neoantigens (see below. Section III.E). Oligosaccharides can also be released by other methods specific for the degradation of carbohydrate-containing biopolymers, e.g. Smith degradation [20]. The presence in the starting structure of a sequence of several monosaccharides which are not oxidized with periodate is the prerequisite for preparation of an oligosaccharide fragment (Scheme 4) [21].

Neoglycoconjagates

Synthesis

101

OOH

HO

OH

CH3

NHAc

CH2OH

NHAc

Scheme 4.

The elimination of oligosaccharide fragments, which are the components of glycoproteins, can also be effected by solvolytic methods. This usually results in a complex mixture of oligosaccharides due to heterogeneity of oligosaccharide chains in the starting glycoprotein and nonspecific degradation of carbohydrate chains. Whenever intact oligosaccharide fragments of glycoproteins are required, specific methods are employed. To split oligosaccharide chains of glycoproteins attached to serine or threonine residues through 0-glycosidic linkage, Pelimination is most often used. It is performed by the action

102


of an alkali in the presence of a reducing agent which blocks chain degradation from the reducing end. Conventionally, the reagent used is 1 M NaBH^ + 50 mM NaOH (50 °C, 16 h) [22]. Treatment with this reagent, however, results in release of not only 0-linked oligosaccharide chains but also 10-13% of A^-linked chains due to reductive splitting of A^-glycosylamide bonds. This side reaction can be suppressed if degradation is carried out in the presence of Cd^"^, resulting in the selective splitting of 0-linked carbohydrate chains [23]. Alkaline acetolysis may also be used for the same purpose [24]. Under conditions specified (1:1 acetic acid—acetic anhydride, saturated with sodium acetate, 80-85 °C, 100-200 h) Olinked chains are split selectively and in high yield (75-90%), with A^-linked chains being unaffected. This degradation method is based on P-elimination and the oligosaccharides liberated are simultaneously acetylated. A mild method for chemical splitting of iV-glycosylamide bonds in glycoproteins was recently developed. This involves treatment with lithium borohydride in 70% aqueous tert-hutyl alcohol followed by hydrolysis of glycosylamines produced with aqueous acetic acid (Scheme 5). This allows oligosaccharide chains of

-OH p NH-■OvNHCCHjCH .OH C-NH^HCO... RON—{ § \f NHAc

LIBHt, 45, 5 h

NH4HCO3

||p„3.,

30* 6-7 days pOH

Scheme 5.

NHAc


103

A^-glycoproteins to be obtained in yields of 60-80% [25]. A combination of these two methods [23,25] allows sequential and selective splitting of O- and then A^-linked carbohydrate chains of A^,0-glycoproteins to be performed [26]. Enzymic Degradation The advantage of enzymic hydrolysis is its regio- and stereospecificity, which allows splitting of one particular type of glycosidic linkage in a natural carbohydrate-containing biopolymer, albeit the very isolation of an enzyme, estimation of its substrate specificity, and optimization of the degradation procedure requires considerable preliminary work. An interesting and rather widespread access to oligosaccharides from natural sources is based on enzymic cleavage of bacterial polysaccharides by bacteriophages [27]. The tail region of a phage is a natural compartment for the glycosidases responsible for cleavage. If phage-induced hydrolysis is combined with simultaneous dialysis, a set of large fragments (di-, tri-, tetra-, and higher oligomers of a repeating unit) can be prepared [28]. These fragments are hardly accessible by partial solvolysis or specific chemical degradation. This hydrolytic procedure does not imply isolation of an enzyme in a pure state; it simply consists of incubation, at 37 °C, of abacterial lipopolysaccharide preparation with a purified inactivated bacteriophage in a dialysis bag. The oligosaccharides thus released are separated by gel-chromatography. However, practical application of this approach also requires serious preliminary work involving the search for an appropriate phage, specific to a given polysaccharide chain, and characterization of substrate specificity of glycanases of the phage. This can be exemplified by degradation of Salmonella 0-polysaccharides. Studies of 18 bacteriophages specific to LPS of serological groups A, B, and Dj revealed all of them to contain eM(io-a-rhamnosidases which catalyze splitting of the a-L-Rhap(1—>3)-D-Galp bond [29]. They differ from each other in subtle details of substrate specificity (Scheme 6). The presence of an a-glucose substituent at position 4 of a galactose residue pre-

104


R 1

a-D-Glc 1

4

i

3 4 l-a-D-Man-(l-^)- a-L -Rha-(1 -»3)-a-D-Gal-(l^ ft phages P22, P27 ® phages 9N A, KB 1© R 1

a-D-Glc 1

i

i

6 3 -^ 2)-a-D-Man-( 1 ->4)-a-L-Rha-( 1 ->3)-a-D-Gal-( 1 -> i\

phages P22, P27 e phages 9NA, KB 1®

R = 3,6-dideoxy-a-hexosyl (D-arabino, D-xylo, D-ribo) Scheme 6,

vents hydrolysis of a rhamnosyl—galactose linkage under the action of phage 9NA and KBl glycanases, whereas phages P22 and P27 are active. Quite an opposite situation is observed if galactose bears an a-glucose substituent at position 6. The presence of a 3,6-dideoxyhexose residue attached at position 3 of mannose is the prerequisite for the phage P22 to manifest its rhamnosidase activity irrespective of the nature of the 3,6-dideoxyhexose. Thus, enzymic hydrolysis allows the isolation of oligosaccharide fragments containing 3,6-dideoxyhexoses, the latter being immunodominant sugars. These fragments cannot be achieved if conventional hydrolytic procedures are applied. This was taken into account when conditions for preparativescale degradation of Salmonella lipopolysaccharides had to be chosen—^the most frequently used was phage P22 [30]. Enzymatic hydrolysis has not apparently been applied to the preparative-scale isolation of oligosaccharide fragments of glycoproteins despite the existence of specific enzymes which en-


105

sure highly selective splitting of nondegraded carbohydrate chains. Flavobacterium meningosepticum is the source for peptide A^-glycosidase F which cleaves the 7V-glycosylamide bond between asparagine residues and carbohydrate chains [31]. OLinked carbohydrate chains can be split off by using a 0-glycan-peptide hydrolase (0-glycopeptide e«ô-D-galactosylN-acetyl-a-D-galactosaminyl hydrolase, EC 3.2.1.97) from Diplococcus pneumoniae [32a]. Substrate specificity of this enzyme is confined to asialo-oligosaccharide chains; moreover, it is only disaccharide p-D-Gal-(l-^3)-D-GalNAc that is the major product."^ Synthesis of Oligosaccharides

A versatile method for preparation of oligosaccharide fragments representing biological determinants is by chemical synthesis. In principle, this route allows access to any oligosaccharide, including structural analogs of the natural determinant fragments. Analogs of this kind are often very useful in elucidation of functionally important structural details of carbohydrate antigens [33,34]. Preparation of the first artificial carbohydrate antigens made use of available mono- and disaccharides [17,35]. However, the progress in this field necessitated the synthesis of more complex oligosaccharides and this was one of the most powerful stimuli for development of oligosaccharide synthesis. On the basis of better comprehension and by developing synthetic methods the solution to more and more complex problems is becoming possible. This can be exemplified by syntheses of oligosaccharides which are repeating units of microbial polysaccharides (cf. Table III in ref. 36) and carbohydrate fragments of several glycoproteins (ref. 37 and references cited therein; ref. 38). Today the number of reports on the synthesis of oligosaccharides is literally compared with an avalanche that has swept over scientific journals. A chemical synthesis of oligosaccharides is a more complicated problem than that of polypeptides or polynucleotides. It is practically devoid of general solutions, and the synthesis of any particular oligosaccharide is a rather specific challenge [39].

106


Nevertheless, synthesis of oligosaccharide structures comprising up to 10 monosaccharide residues is currently quite a realistic task [38,40]. The modern methodology of oligosaccharide synthesis is covered in several excellent reviews [39,41,42] and will not be dealt with here. What should be emphasized is that if synthetic oligosaccharides are to be used for the preparation of neoglycoconjugates, their synthesis should be designed and accomplished in such a way as to produce the target oligosaccharides directly in a form suitable for subsequent conjugation. As a rule, the synthetic oligosaccharides are obtained as O- or S-glycosides with a fixed configuration of a glycosidic bond and an aglycone with a reactive functionality. This circumstance may seriously affect the general strategy of the synthesis, especially in the case of complex structures because problems of the oligosaccharide synthesis, as such, are supplemented by those associated with the introduction and transformation of a more or less complex aglycone. B. Conjugation to a Polymer Carrier

Covalent attachment of carbohydrate determinants to a polymer carrier (conjugation) is the key stage in preparation of neoglycoconjugates. Several versions of conjugation have been developed, each requiring preliminary derivatization of an oligosaccharide. The generally accepted methods for conjugation will be discussed first followed by ways to prepare the corresponding oligosaccharide derivatives. Polymer Carriers

Most often neoglycoconjugates, especially artificial antigens, are prepared as neoglycoproteins [8-10] by attachment of a carbohydrate moiety to a protein. In the majority of cases, easily accessible serum proteins such as bovine, rabbit, or human albumin are employed as a protein carrier. These proteins, molecular weight of about 67,000-69,000, can be purified and obtained in a crystalline state. They are readily soluble in water.


10 7

and are convenient for conjugation. Hemocyanin (M^9,000,000) of different origins [43] or the hempseed protein, edestin (M^ 310,000) [44,45], are less commonly used. The latter exhibits higher immunogenicity than albumins but its utilization is hampered by its solubility only at high pH values. This creates difficulties both at the conjugation step and at isolation of the carbohydrate-protein conjugate. The use of other protein carriers is also documented. They include diphtheria toxin [46], bacterial protein, porin (Mj. 34,000-36,000), isolated from the outer membrane of Salmonella typhimurium [47], cytochrome c (Mj. 12,300), and some others. Of especial interest is the utilization of porin for conjugation with oligosaccharide fragments of 0-antigen from the same bacteria since it affords a neoglycoconjugate of enhanced protective effect. This is believed to be associated with the ability to elicit antibodies against two independent targets on a bacterial cell at the same time [47]. The use of synthetic polypeptides, such as poly-L-lysine (M^ 70,000) [48] or poly-(L-aspartic acid) [49] as the protein carriers are less common. Methods for Conjugation of Oligosaccharides with Protein Carriers As a rule, covalent attachment of oligosaccharide determinants to a protein carrier does not require any preliminary modification of the latter. Free 8-amino groups of L-lysine residues (e.g. in the case of BSA they amount to 57) or free carboxylic groups that are involved in coupling, by means of conventional organic reactions with carbohydrate residues, may result in the formation of a C-N bond. Formation of a N=N-X Bond (X = NH, C). Diazo coupling is historically the first conjugation method and was the most widespread for a long time. It dates back to the work of K. Landsteiner [5] and it is the modified procedure by Westphal and Feier [50] that is usually employed (Scheme 7). This procedure involves the introduction of the 4-aminophenyl moiety into the oligosaccharide; diazotization leads to an

108


NaN02, HCl,

Sug-O^NH^

„ . , ; ^ / ^

Sug-0-*~0.

S H2N-C-NH2

♦

c^N5C(NH2)2f Br" OAc

P

C

pOH

Vj^H2CN

K2CO3. NQHSO3

\

/

H2N-protcin, pH 7-10. 4-20* 2 h*^

001M MeONa,

J ~ 0 SCH2COCH3

30-3?: 24-48 h

rOH NH / — 0 . scH2CNH-protein C^_/

Scheme 14.

yn

114


thiosugar and then, by S'-alkylation with chloroacetonitrile, into the S-cyanomethyl 1-thioglycoside. Removal of 0-acetyl groups and simultaneous conversion of the cyanomethyl group into imido ester is performed by treatment with sodium methoxide to produce 2-imino-2-methoxyethyl 1-thioglycosides which react readily with the free amino groups of proteins. Under alkaline conditions and with a 5- to 10-fold excess of the imido ester, about 50-60% of amino groups in BSA and other proteins are modified by carbohydrates. This method has found application mainly in the conjugation of mono- and disaccharides. Quantitation of the degree of amidination in this case can easily be accomplished because the thioglycoside linkage undergoes selective mercuric salt-catalyzed hydrolysis [70]. Formation of C-NH Bonds. Conjugation methods involving reductive amination result in C—NH bond formation (Scheme 15). The method is based on the shift of equilibrium between an aldehyde and the respective Schiff base, formed with the amino group of the protein, by means of selective reduction of the aldimine into secondary amine [71]. The aldehyde group may be specially introduced in an aglycone portion of the carbohydrate determinant [72,73]. On the other hand, the reducing oligosaccharide itself possesses an aldehyde group masked as a hemiacetal. In the latter case the concentration of the free aldehyde group in solution is low and therefore conjugation of unprotected oligosaccharides proceeds very slowly, often taking several days [71]. The use of sodium cyanoborohydride as a reducing agent is advantageous since, unlike sodium borohydride, it reduces selectively aldimines and not aldehyde groups. In order to achieve a reasonable extent of modification of proteins, large excesses of the aldehyde components are required that, in turn, result in the formation of tertiary as well as secondary amines [74]. Application of this conjugation procedure to reducing oUgosaccharides results in transformation of the cyclic structure of the monosaccharide at the reducing HgN-protein Sug~CH=0 ^=^

NaBHaCN

^ Sug-CH=N-proteln pH T~9^ Sug-CHgNH-proteln Scheme 15.

Neoglycoconjugates

Synthesis

115

terminus into acyclic 1-amino-1-deoxyalditol. With this unit, a new specificity arises in the neoglycoconjugate that, provided small molecular weight oligosaccharides are immobilized, lowers the merits of this method. Conjugation methods wherein the C-NH bond is produced include the stepwise substitution of chlorine atoms in the bifunctional reagent cyanuric chloride (2,4,6-trichloro-5-triazine) [75,76] with a carbohydrate component and a protein (Scheme 16). Nucleophilic substitution of one chlorine atom in cyanuric chloride with an amino derivative of an oligosaccharide essentially deactivates the remaining chlorine atoms so that no disubstitution takes place (Scheme 16). This first step is usually carried out at Q-5 °C. Increase in the reaction temperature to 45 "^C enables nucleophilic substitution of the second chlorine atom. Provided the protein amino group acts as a nucleophile at the second step, a neoglycoprotein is obtained with an asymmetric disubstituted chlorotriazine moiety at the junction site. Besides introducing this hydrophobic and possibly antigenic fragment, solubility problems may also be a disadvantage of this method. The use of 0-acetylated sugar derivatives may be necessary in the first step. Deacetylation of the conjugates proCi

R-NH2

♦

ci

IOJL

R « Sug. Sug(OAc)„

-^r-^

^

'lOj ^

CI Ur\H

H2N-proteln.

CI Sug-HN^ ^ N ^ N H --protein p

Scheme 16.

^"^* " * ° " *

116


duced thereafter, may not completely overcome the solubility problem [54]. The use of cyanuric chloride in the preparation of glycoconjugates based on polysaccharides will be dealt with in Section IV.A. C. Preparation of Oligosaccharide Derivatives Suitable for Conjugation To effect conjugation of an oligosaccharide with a protein the former should possess a functional group which enables its successful condensation to amino groups of the protein by one of the aforementioned methods. The existence of such a group should be envisaged for a synthetic oligosaccharide. But provided an oligosaccharide is prepared by degradation of a natural product and produces a reducing species, this should be properly functionalized. Derivatization of Reducing Oligosaccharides The transformation of oligosaccharides into derivatives, which can be conjugated, does not require, as a rule, preliminary hydroxyl protection and subsequent deprotection. This is very advantageous, albeit the cost is the "loss" of a monosaccharide residue at the reducing end of the oligosaccharide. This is due to the fact that almost all the transformations, with the exception of formation of glycosylamines (cf. Scheme 5) [77,78], results in elimination of the natural cyclic form and configuration of the glycosidic linkage of this monosaccharide unit. The most frequent are the following transformations: 1. The reducing (terminal) monosaccharide residue is oxidized into an aldonic acid (Scheme 17) in order to activate the resulting carboxyl group with carbodiimide (Scheme 8) or via mixed anhydride (Scheme 9) prior to conjugation. 2. The monosaccharide unit is converted into 1-amino-1-deoxyalditol by reductive amination. If amino groups of a protein are involved in this slow transformation, i.e. the reductive amina-


11 7

IH

pOH \2. N Q O H

\

SugC^N

/ AH

)-0H

►

OH

SugO

V -

COOH 6H

pOH

J—OH

H2N—protein

SugO

^

yCONH-protein

Scheme 17,

tion is carried out in the presence of the protein, the product is directly a neoglycoprotein [74,79] (cf. Scheme 15). Alternatively, the reaction can be performed with 2-(4-aminophenyl)ethylamine (Scheme 18) [80]. The oligosaccharide is dissolved in the reagent [80] or in anhydrous dimethyl sulfoxide as a solvent. Anhydrous conditions in this case favor dehydration of a carbinolamine intermediate [81]. If reductive amination with sodium cyanoborohydride is performed in aqueous solution at pH 8.0 [82], it is the aromatic amino group that is involved in the reaction [83] so that another 1-amino-1-deoxyalditol is formed (cf Scheme 18):

CH2CH2NH2

The aromatic amino group, introduced by reductive amination, can be used effectively in the conjugation with a protein (Scheme 18) by means of the diazo coupling procedure [84] or the isothiocyanate method [82]. Reductive amination of oligosaccharides with 4-trifluoroacetamidoaniline (TFAN) (pH 6, 15-48 h) also affords

A A

03

-

SugO ~ l i = N C H 2 C H 2 e N ~

I

ugo~~~HC~CH2eN=-NH-profein

NaBHG

-

C H ~ N H C H ~ ( H ~ ~ N H ~ sugo

sugo

Scheme 18.

C H ~ N H C H ~ C H ~ ~ N H \ N rHi n- P ~ O ~ 5


119

r-OH /—OH

TFAN. NaBH^CN, 3)-a.D-Man-(l-OCH2CH=CH2 a-Abe-(1^3)-a-D-Man.(l-OCH2CH2NHCOCH=CH2 a-Tyv-( 1 ^3)-a-D-Man-( 1 -OCH2CH=CH2 a-Par-( 1 ->3)-a-D-Man-( 1 -OCH2CH2NHCOCH=CH2 a-Abe-(l-^3)-p-L.Rha-(l-0(CH2)„NHCOCH=CH2 (n = 2,6) a-Abe-(l->3)-p-L-Rha-(1^2)-a-D-Man-(l-OCH2CH2NHCOCH=CH2 P-D-Man-(l->4)-a-L-Rha-(1^3)-p-D-Gal-(l-OCH2CH=CH2)

13,142 161 161 161 151 152 12,13, 122,142 P-D-Man-( 1 ->4)-a-L-Rha-( 1 -^3)-p-D-Gal-( 1 -OC6H4NHCOCH=CH2 154 a-D-Glc-(l->3)-a-D-Man-(l-OCH2CH2NHCOCH=CH2 162 2-0-Ac-a-D-Glc-( 1 ->3)-a-D-Man-( 1 -OCH2CH2NHCOCH=CH2 162 [-^3)-a-D-Gal-( 1 -^2)-a-D.Man-( 1 ->4)-a-L-Rha-( l-]-„ {n = 1,2) tl,3 a-Abe ^3)-a-D-Gal-(l-^2)-a-D-Man-(l-^)-a-L-Rhaol-(l-N-C6H4NHCOCH=CH2 ^

Ac

1 a-Abe

159

[->3)-a-D-Gal-(l-^2)-a-D-Man-(1^4)-a-L-Rha-(l-]-„ 3

(n = 1,2)

t

1 a-Tyv -^3)-a-D-Gal-(l->2)-a-D-Man-(l-^4)-a-L-Rhaol-(l-N-€6H4NHCOCH=CH2

f

Ac

1 a-Tyv Salmonella minnesota R^ 595 LPS (Kdo-Region) *a-Kdo-(2-OCH2CH=CH2 *a-Kdo-(2-4)-a-KDO-(2-OCH2CH=CH2

159

163,164 163, 164 {continued)


145

Table 3. (Continued) Carbohydrate Moieties in Form Suitable for Copolymerization

References

Escherichia coli *P-Kdo-(2-OCH2CH=CH2 p.D-Rib/-(l->7)-p-KDO-(2-OCH2CH=CH2 p-D-GlcA-(l-^3)-a-L-Rha-(l-OCH2CH2NHCOCH=CH2 a-L-Rha-( 1 ^3)-p-D-GlcA-( 1 -OCH2CH2NHCOCH=CH2

163 163 153 153

Chlamydia *a-Kdo-(2->8).a-KDO-(2-OCH2CH=CH2 *p-Kdo-(2^8)-a-KDO-(2-OCH2CH=CH2 *a-Kdo-(2->8)-a-KDO-(2^4)-a-KDO-(2-OCH2CH=CH2

167 167 167

Streptococcus pneumoniae, type 3 p-D-GlcA-(1^4)-p-D-Glc-(l-OCH2CH2=CH2 p-D-Glc-( 1 ^3)-p-D-GlcA-( 1 -OCH2CH=CH2 p-D-Glc-( 1 ->3)-p-D-GlcA-( 1 ^4)-p-D-Glc-( 1 -OCH2CH=CH2 p-D-GlcA-( 1 -^4)-p-D-Glc-( 1 ^3)-P-D-GlcA-( 1 -OCH2CH=CH2 p-D-GlcA-(l->4)-p.D-Glc-(l->3)-p-D-GlcA-(l-^4)-p-D-Glc(l-OCH2CH=CH2

143, 168 143, 168 169 169 169

Mycobacterium leprae Glycolipid p-3,6Me2-D-Glc-(l-OCH2CH=CH2 p-3,6Me2-D-Glc-(l->4)-a-2,3Me2-L-Rha-(l-OCH2CH=CH2 p-3,6Me2-D-Glc-( 1-> 4)-a-2,3Me2-L-Rha-( 1-0(CH2)20(CH2)20CH2CH=CH2 p-3,6Me2-D-Glc-( 1 ->4)-a-2,3Me2-L-Rha-( 1 ^2)-a-3Me-L-Rha(l-OCH2CH=CH2

170 170 171 170

Group Specific Glycoproteins (Le*, H, A) (« = 3,4) p-D-Gal-(l-3)-p-D-GlcNAc-(l-0(CH2)„OCH2CH=CH2 4

141

t

1 a-L-Fuc p-Gal-( 1 -3)-p-D-GlcNAc-( 1 -0(CH2)3NHCOCH=CH2 4

111,160, 172

T 1 a-L-Fuc

{continued)

146

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK Table 3. (Continued)

Carbohydrate Moieties in Form Suitable for Copolymerization a-L-Fuc-( 1 ->2)-p-D-Gal-( 1 ^3)-p-D.GlcNAc-( 1 0(CH2)„OCH2CH=CH2 a-D-GalNAc-(1^3)-p-i>Gal-(l->3)-p-D-GlcNAc-(l-0(CH2)„OCH2CH=CH2 a-D-GalNAc-(l-^3)-p-i>Gal-(l->3)-p-D-GlcNAc-(l-0(CH2)„OCH2CH=CH, 2

References 141 141 141

t

1 a-L-Fuc Human Milk Oligosaccharides Lacto-N-tetraose p-D-Gal-( 1 ->3)-P-D-GlcNAc-( 1 ^3)-p-D-Gal-( 1 ->4)-D-G1CO1-

159

(1 -NAc-C6H4NHCOCH=CH2 Lacto-N-tetraose p-D-Gal-(l->3)-p.D-GlcNAc-(l->3)-p-D.Gal-(l->4)-D-Glc-(l-

78

NHC0CH=CH2 Lacto-N-fucopentaose I a-L-Fuc-( 1 ^2)-p-D-Gal-( 1 ^3)-p-D-GlcNAc-( 1 -^3)-p-D-Gal-( 1 ^ 4 ) P-D-Gal-(1-NHC0CH=CH2 A-Tetrasaccharide a-D-GalNAc-( 1 ->3)-p-D-Gal-( 1 -^4)-p-D-Glc-( 1 -NHC0CH=CH2 2

78

78

T 1 a-L-Fuc 2'-Fucosyllactose a-L-Fuc-(1^2)-p-D-Gal-(1^4).p-D-Glc-(l-NHCOCH=CH2

78

Other Structures a-Neu5Ac-(2-0(CH2(3S(CH2)2NHCOCH=CH2 a-Neu5Ac-(2-OCH2CH=CH2 a-L-Rha-(l-0(CH2)3S(CH2)2NHCOCH=CH2 a-D-GlcNAc-(l-OCH2CH=CH2 p-D-GlcNAc-(l-OCH2CH=CH2 a-D-GlcNAc-(l-0(CH2)3S(CH2)2NHCOCH=CH2 p-D-GlcNAc-(l-0(CH2)3S(CH2)2NHCOCH=CH2

156 146 155 157 157 157 157

Note: "Hapten analogs marked (*) with altered structures [other combinations of configuration and linkage type, (2->7), (2->4)-(2^4), and with 5d-Kdo residues] as neoantigens of copolymer type were used to characterize epitope specificity of monoclonal antibodies against inner core region of enterobacterial LPS [165,166].

Neoglycoconjagates Synthesis

147

If a copolymerization method is applied for the preparation of neoantigens, the determinant enters the neoglycoconjugate in virtually quantitative yield and its percentage can easily be regulated. Thus, the route is open to prepare well-standardized neoantigens whose compositions can be preset and carefully monitored, e.g. by ^^C NMR spectroscopy. ^^C NMR spectra of glycoconjugates of this type are very similar to those of the starting carbohydrate monomers, with regular polyacrylamide carriers exhibiting a limited number of signals which do not overlap, as a rule, with those of a carbohydrate moiety. Spectroscopic confirmation of the structure of the neoglycoconjugate at every stage of its synthesis substantiates any conclusion on the relationship between immunochemical properties of neoantigens and their structure. Copolymerization is a very flexible approach for designing neoantigens. It allows several determinants to be incorporated at one time as well as other components as required. Thus, copolymerization of several synthetic Salmonella determinants gave polyvalent (complex) neoantigens of a wide diagnostic value in performing screening of sera from patients and infected animals (Scheme 44) [154].

CONH2

CH2

CONH2

CH2

CONH2

6R

Serological Specificity AM,TM PM, AM, TM MRG, AM, TM

0:4 + 0:9 (B + D) 0:2 + 0:4 + 0:9(A + B + D) 0:3 + 0:4 + 0:9 (E + B + D)

AM = a-Abe-( 1 ->3)-a-D.Man-( 1 PM = a-Par-( 1 ^3)-a-D-Man-( 1 TM = a-Tyv-( 1 ->3)-a-D-Man-( 1 MRG = p-Man.(1^4)-a-L-Rha-(l->3)-p-D-Gal-(l-^ Scheme 44.

(0:4) (0:2) (0:9) (0:3)


148

The copolymerization concept enabled incorporation into neoantigen of a synthetic A^-acetylmuramoyl-L-alanyl-D-isoglutamine (muramoyldipeptide, MDP) [173]. This is the minimal immunoactive fragment of cell-wall peptidoglycan of bacteria which are used in Freund's complete adjuvant. A spacer was introduced into MDP by condensation of C-terminal amino acid carboxyl group with mono-A^-acryloylhexamethylenediamine [173]. Copolymerization of a mixture of three components—^viz.

CONHg

CONH

CONHg

(CH^), NH i

CONH I (CH2)3

CONHg

hapten Le

MDP .a - PAA - MDP, Le^ 1:58:1

CHgOH

ÔH NHAc (CHg )2C0-NH (CHg )gNHC0CH=CH2 CONHCHCONHCH I I CH3 CONHg

HDP

'

a-Fuc-(l-^4) v^

hapten Le"^ Scheme 45.


149

acrylamide and two modified acrylamides—one modified with a trisaccharide component of Le^ glycoprotein and the second with MDP, gave rise to a neoantigen with the minimal adjuvant build-in (Scheme 45) [160]. The polyacrylamide backbone of neoantigens can be modified chemically if needed. Thus, to impart to these antigens the ability to adsorb onto erythrocyte surfaces facilitating their use as erythrocyte diagnostic tools, the neoglycoconjugates were modified with fatty acid residues (Scheme 46) [174]. Treatment of a polymer with anhydrous 1,2-diaminoethane results in partial transamidation of acrylamide units; at the same time the level of carbohydrate determinants remains unaltered due to the use of allyl glycoside for preparation of the copolymer. Acyl- ation of the introduced aminoethyl groups with palmitoyl chloride gives a neoantigen with lipophilic fragments [174]. It should be stressed that this method allows the preparation of oligosaccharide-polyacrylamide gels which are promising as immunoadsorbants [149,150]. An access to gels is readily realized by incorporation of a cross-linking agent, 7V,iV-methylenebisacrylamide into a mixture of monomers to be polymerized.

)H2NCH2CH2NH2.

°°^

IH2

90° 4 h

2)CH3(CH2)i4C0Cl,

CONHR'

R = a-Abe-(l-^3)-a-D-Man(l-» R' = H, or CH3(CH2)i4CONHCH2CH2- (2-3% of fatty acid residues) Scheme 46.

OH"

150


a-Abe-(l->3)-a-L-Rha-(l-0(CH2)2NHCOCH=CH2 + CH2=CHCONH2 + CH2=CHC00H

a-Abe-( 1 -^3)-a-L-Rha-( 1 -O a-Abe-( 1 ->3)-a-L-Rha-( 1 -O R = NH2,orOH(~l:l) ~20% of carbohydrates

Scheme 47. Obviously, a wide range of unsaturated compounds, other than acrylamide, can be used as non-carbohydrate monomers with the aim of preparation of neoglycoconjugates with novel, interesting properties, and this is one of the perspective directions of development in synthesis of neoglycoconjugates. For instance, data on the immunostimulating effect of polyanions (in particular, polyacrylic acid [175,176]) on eliciting humoral antibodies upon immunization, suggest that the preparation of neoantigens with polyacrylic acid as a carrier is feasible. However, polyacrylic acid itself exhibits pronounced toxicity and to reduce it, without affecting the adjuvant activity, it is worthwhile to use copolymers of acrylic acid with incorporated units of an uncharged monomer [177]. The copolymerization principle has enabled the preparation of a neoantigen with a carrier of this mixed type (Scheme 47) [154], the ratio of charged and neutral units therein being variable.


151

Immunobiological Properties of Neoantigens of Copolymer Type. The only serologically active component in neoglycoconjugates of the copolymer type is represented by its sugar moiety which is the carbohydrate fragment of a natural antigen. This represents a sharp distinction from the neoglycoproteins with two serologically active components derived from carbohydrate and proteins. Thus neoglycoconjugates of this type with strictly defined chemical structures are especially suitable for serological studies when used as antigens. Neoantigens of this type can be used successfully in Ouchterlony double radial immunodiffusion tests [140,142,155,178], with the sensitivity of assay being in some cases 10-100 times as high as that with homologous natural antigens [140,142,178]. They are also efficient inhibitors of passive hemagglutination tests [140,178]. The oligosaccharide-polyacrylamide conjugates display an excellent adsorption capacity onto polystyrene surfaces allowing them to be utilized as coating antigens in enzyme-linked immunosorbent assays (ELISA) [154]. It should be emphasized that polyacrylamide conjugates are extremely effective as coating antigens, with optimal doses being 0.01-0.1 |ig/ml as comp a r e d with 1 — 10 |Lig/ml for lipopolysaccharides and neoglycoproteins [159]. Determination of antibodies of three classes (IgG, IgM, and IgA) in antisera from patients with salmonellosis [179] and leprosy [170], has successfully been carried out by ELISA using oligosaccharide-polyacrylamide conjugates of respective specificity. Neoantigens of this type were very helpful in immunochemical studies for characterization of the epitope specificity of monoclonal antibodies [164^166]. Much less studied are other immunobiological properties, in particular immunogenicity of the copolymer-type neoantigens. However, the very possibility of using the polyacrylamide carrier for creation of nontoxic immunogens on the basis of oligosaccharide haptens has been demonstrated in experiments on immunization of rabbits and mice without recourse to adjuvants [140,180]. In this way, antisera were prepared against the Salmonella 0:3 antigenic determinant [140] and Le^-group specific

152


glycoproteins [180]. Protective activity of neoantigens with polyacrylamide carrier and bacterial oligosaccharide fragments has also been documented [181].

IV. NEOGLYCOCONjUGATES WITH POLYSACCHARIDE AS A CARBOHYDRATE MOIETY Polysaccharide antigens possess a whole set of specificities encoded in a structure of the constituent oligosaccharide fragments overlapped in the chain. Therefore neoantigens based on polysaccharides are less informative for studying the relationship between structure and immunochemical properties. On the other hand, synthesis of neoglycoconjugates with polysaccharides as a carbohydrate moiety is associated primarily with attempts to obtain immunogenic polysaccharide vaccines for prophylaxis of infectious diseases [182]. Covalent attachment of polysaccharides to "T-dependent" protein-carriers is thought to be the way to overcome "T-independence" of polysaccharide antigens^ and thereby to obtain neoantigens with enhanced immunogenicity which are capable of eliciting protective antibodies. In this context, of essence is the formation of a sufficiently strong covalent bond between the polysaccharide and carrier protein upon conversion of the former to neoglycoproteins, whereas the structure of every linkage unit and, hence, the character of modification of the polysaccharide molecule associated therewith is not as important as in the case of oligosaccharides. Partial modification of a polysaccharide in the course of conjugation, as a rule, does not result in essential changes of its structure because the majority of natural polysaccharide antigens are built of repeating units, ensuring retention of sufficient amounts of intact fragments with inherent immunochemical specificity along with modified ones. Modification of polysaccharides differs from that of oligosaccharide haptens in the course of preparation for conjugation (cf pp. 116-121) in the following two aspects. First, it often affects several monosaccharide units of the polysaccharide chains. This may result, of course, in formation of cross-linked polysaccharide—protein complexes in the process of conjugation. Secondly, partial modi-


153

fication of a polysaccharide is, as a rule, chemically ambiguous since the modification can affect different monosaccharide units and/or elements of their structure. This is to be kept in mind when methods are discussed for the conjugation of polysaccharides with polymer carriers, although they are usually the same as for oligosaccharides (see Section III.B). A. Utilization of Natural Polysaccharides Preparation of neoglycoconjugates with polysaccharide carbohydrate moieties preferentially makes use of the natural polysaccharides. Isolation of polysaccharides from natural sources can be a rather complicated problem by itself [184]. It may also involve degradation of a natural carbohydrate-containing biopolymer, thus making the preparation of a pure polysaccharide more complex. This can be exemplified by isolation of 0-specific polysaccharides from lipopolysaccharides of Gram-negative bacteria by acid hydrolysis [185] or splitting of carbohydrate chains of glycoproteins by reductive degradation [22,23]. The critical point in isolation of a natural polysaccharide preparation is its purification. It should be kept in mind that application of even the most sophisticated methods does not guarantee isolation of a homogeneous polysaccharide specimen, free from antigenic impurities of other specificities. The extent of purity of the polysaccharide preparation can, in turn, influence the immunogenic properties of the polysaccharide-protein conjugate made therefrom. The first experiments aimed at preparation of polysaccharideprotein conjugates for their subsequent use as immunogens with a desirable specificity were undertaken by Goebel and Avery [186]. Their procedure involved partial 4-nitrobenzylation of a natural polysaccharide, reduction of the 4-nitrobenzyl ether to the 4-aminobenzyl ether (e.g. by the action of sodium dithionite [187]) followed by conversion into the diazonium salt, and diazo coupling with a protein carrier at a final stage (Scheme 48). When the capsular polysaccharide of Streptococcus pneumoniae type 3 (S3) was subjected to the modification the degree of

154


p-NO^-CgH^-CH^Br, S3-0H

Na2S205

► S3-0CHp-C^H,-N0p ^ ^ ^ ^ «N02 + - HSA. S3-0CHo-C^H,-NH•2-C6H4-NH2 - j j ^ S 3 - 0 C H 2 - C ^ H , - N ^ C l H a ^ ^ D ^ NaOH

— ► S3-0CH2-CgH^-N=N-HSA ('-13% of sugars)

S3 = capsular polysaccharide (soluble substance) ot S.pnetmonioe type 3. HSA = horse serum albumin Scheme 48.

substitution was very high: not less than one 4-aminobenzyl substituent per disaccharide repeating unit, as judged from the nitrogen content [186]. This could result in considerable crosslinking upon subsequent coupling to the horse serum albumin. Nevertheless, the polysaccharide moiety of the conjugate (Scheme 48) retained its original serological specificity when tested with type 3 antipneumococcal serum, and the conjugate itself exhibited immunogenicity [186]. Polysaccharides, which are stable to treatment with alkali (pH 12—13, 20 °C) for short periods, can be modified, for subsequent preparation of polysaccharide—azo—protein conjugates, with 2[(4-aminophenyl)-sulfonyl-ethyl] hydrogen sulfate (APSE hydrogen sulfate) [188]. On treatment of a dextran with this reagent (1:1, w/w) an average degree of substitution amounted to 0.05, i.e. on average every twentieth monosaccharide residue underwent modification (Scheme 49). Following diazotization and diazo coupling with a protein under standard conditions (see pp. 107-108) immunogenic polysaccharide—ethyl-sulfonylphenyl-azo-protein conjugates could be prepared. Activation of the 0-specific polysaccharide of Salmonella typhimurium was performed by partial acylation with bromoacetyl


155

p-H2N-CgH^-S02CH2CH20S03H, PS

► 0.5 N NaHC03/Na2C03, NaOH, pH 11-13. 20°, 1 h

1)NaN02-HCl. 0°, 20 mln -»- PS-0CH2CH2S02-CgH^-NH2 ► PS-0CH2CH2S02-CgH^-N=N-edestln c.) 60.6Suin,

0.2 N NaOH. 0°, 2 h PS = dextran

Scheme 49. bromide [189]. The 0-bromoacetyl groups introduced reacted further with primary amino groups of side chains of a synthetic macromolecular polyelectrolyte carrier (Scheme 50) [190], the latter being the copolymer of acrylic acid and A^-vinylpyrrolidone containing equimolar proportion [177]. Its molecular mass was 10,000 and it contained about 20% of primary amino groups in side chains. This is one of rather few examples of the conjugation of polysaccharides with a carrier other than a protein. In development of a concept by Diamanstein, et al. on the immunoadjuvant effect of polyelectrolytes [175], a synthetic polysaccharide—polyelectrolyte conjugate retained its inherent T-independence to the polysaccharide antigen and exhibited enhanced immunogenicity [190]. From the practical point of view methods for conjugation of polysaccharides with proteins based on the use of cyanuric chloride as a bifunctional reagent [191] or cyanogen halides are more convenient [192]. The use of cyanuric chloride as a coupling reagent has already been discussed (see pp. 115-116). In this context two examples can be mentioned. Activation of a galactomannan from a dermatophyte Epidermophyton floccosum with cyanuric chloride at 4 °C for 1 h, followed by incubation with bovine y-globulin (25 "^C, 1 h) afforded an immunogenic polysaccharide—protein conjugate [191]. Similarly S. pneumoniae type 3 capsular poly-

156

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK - CH2CH—CH2CH—CH2CH CONH

(PS)-0H

T

BrCH2C0Br ► (ps)-(0C0CH2Br

CH2CH Oy-N^

I

I

COOH

t^»2)n I NH2

CH2CH—CH2CH— CONH COOH I (CH2)n NH I CH2 0-C OH

Scheme 50.

saccharide [193] produced an immunogenic conjugate with bovine y-globulin. Activation of polysaccharides with cyanogen halides, of which the commercially available cyanogen bromide is the most popular, is based on a reaction of hydroxyl groups with this reagent at alkaline pH [192]. The second stage involves interaction of the activated polysaccharide, which supposedly contains cyanate groups, - O C = N , with amino groups of a protein at pH 7.5—9.0 to produce isoureido linkages, -OC(=NH)NH— and, in part, some other linkages [192]. Conjugation of CNBr-activated polysaccharide antigen of Hemophilus influenza type b (Hib Ps) was carried out with proteins modified with a C^-spacer [194]. This is a rather rare example of the use of premodified proteins in preparation of neoglycoproteins. Activation of proteins (hemocyanin, diphtheria toxin, serum albumins) involved their treatment with adipic dihydrazide (ADH) in the presence of water-soluble carbodiimide at pH 4.7 ± 0.2 (20 °C, 3 h). Subsequent interaction of the modified protein containing about 20 mol ADH per mol protein with a CNBr-activated polysaccharide afforded immu-

Neoglycoconjagates Synthesis

157

nogenic Hib Ps—protein conjugate which was used as a model of a protective polysaccharide vaccine [182]. Activation of polysaccharides can also be effected by chloroacetaldehyde dimethyl acetal [195]. Partial alkylation with this reagent in methyl sulfoxide in the presence of dimsyl sodium (50 °C, 1 h) results in introduction of 0-(2,2-dimethoxyethyl) groups. Mild acid treatment (50 mM HCl, 100 °C, 20 min) deprotects aldehyde groups, enabling subsequent coupling of this modified polysaccharide to proteins which are stabilized by reductive amination (Scheme 51). Partial periodate oxidation of polysaccharides can result in transformation of some monosaccharide residues into dialdehyde units and their reductive amination can be used to effect coupling to proteins. Conjugation of this type with periodateoxidized dextrans was performed under the action of sodium borohydride at pH 7.8-9.05 (25 °C, 6-24 h) [196]. Carbonyl groups in polysaccharides can also be generated by treatment with bromine in aqueous solution at pH 7.0 [197]. The oxidized polysaccharides can be coupled to proteins by the action of sodium cyanoborohydride at pH 6.5 (20 °C, 3 days); an increase in pH favors degradation of the oxidized polysaccharides [197]. These conjugation procedures are based on activation of several functional groups within a polysaccharide molecule (the

PS-OH

ClCH2CH(0Me)2, dimsyl sodlijm, ►PS-0CH2CH(0Me)2 DMSO. 50^, 1 h

50 mM HCl, H2N-Droteln, ^^^5--^^^PS.0CH2CH=0 NaBHsCN pH 7.0. ^^ ^S-0CH2CH2NH-proteln

20^, Bh PS = curdlan C (i-^3)-p-D-gl.ucan protein = HSA, IgG Scheme 51.

]

158


so-called "polyfunctional approach" for designing polysaccharide—protein conjugates). When performing conjugation of a meningococcal group C polysaccharide with BSA under the action of cyanogen bromide or water-soluble carbodiimide (EDC), Jennings and Lugowski detected the formation of water-soluble complexes due to cross-linking of both macromolecules [198]. To overcome this undesirable effect, which is produced by activation of several functional groups of the polysaccharide molecule, and to localize more precisely the polysaccharide-toprotein linkage region, a directed introduction of an aldehyde group into the polysaccharide was attempted (Scheme 52). Timecontrolled periodate oxidation of meningococcal group A and C polysaccharides introduced an aldehyde group selectively at the nonreducing terminus of polysaccharides, viz. at C7 of the terminal sialic acid unit (Scheme 52) [198]. Subsequent conjugation by means of reductive amination with a protein gave immunogenic conjugates of a more definite structure. Another example of such a monofunctional approach is the selective introduction of an aldehyde group by means of rapid oxidation of a terminal nonreducing residue of L-glycero-Dmanno-heptose which forms a branching of the inner part of the core of a partially 0-deacylated lipopolysaccharide from Vibrio anguillarum [66]. This oxidation converts the terminal heptose residue into 6-aldo-mannose (Scheme 53). The aldehyde formed was conjugated to a bifunctional spacer 1,6-diaminohexane, using a reductive amination procedure. Then the A^-(6aminohexyl)amino derivative of the polysaccharide was converted into the corresponding isothiocyanate as described

COONa

R.R' -

H or/and COCH3

Scheme 52.

159


1)H2N{CH2)6NH2, NaBHgCN

\0l

2) CSCI2

CH2NH(CH2)6N=C=S

(:H2NH{CH2)6NHCNH-BSA

H2N—BSA I

Scheme 53.

above (cf. pp. 111-112) which, in turn, was coupled to BSA. The conjugate obtained with about 14 mol of the modified LPS per mol BSA seems to be the first example of LPS covalently attached to a protein carrier. Provided the reducing end of a polysaccharide is represented by a ketose residue, e.g. 3-deoxy-D-mannooctulosonic acid (Kdo), direct conjugation with proteins by means of reductive amination becomes impossible due to low reactivity of this carbonyl group [199]. If, however, the bifunctional spacer 1,6diaminohexane is used in 100-fold excess, efficient reductive amination of the carbonyl group of dOclA takes place. The remaining, free amino group of the spacer enables subsequent covalent coupling to a protein carrier (Scheme 54). With this approach a directed conjugation of 0-specific polysaccharides from Aeromonas hydrophila and Aeromonas salmonicida, which involved the terminal-reducing Kdo residue of the core oligosaccharide as an anchor to BSA, was achieved (Scheme 54) [66]. Thus it demonstrates that the "monofunctional approach" makes possible the preparation, starting from polysaccharides, of neoglycoconjugates possessing properties similar to those of oligosaccharide—protein conjugates, i.e. noncross-linked conjugates, wherein covalent linkage to a carrier involves one of the

160

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK n^'"*

U ^^, A |

COONa

X

NaBHjCN pH 8.0

^

/ OH

?% OCH HOCH CHOH CHOH CH2OH

COONa CHNH(CH2)eN=C=S

CSCI2. pH T . O ,

^ 80% aq. ethanol

CHo

^^N-BSA, pH 9 . 0

- -OCH I HOCH I

CHOH I

CHOH I

CH2OH COONa CHNH(CH2)5NHCNH-BSA CH2

I

-OCH I

HOCH CHOH CHOH I CH2OH

( 1 0 m o l P S / m o l BSA)

Scheme 54.

terminal monosaccharides, reducing or nonreducing. The latter statement in its strict sense is applicable to conjugates based on oligosaccharides. Involvement in covalent bond formation of structural elements proximal to one of the termini of a polysaccharide molecule (cf. pp. 97-98) can be regarded as a conjugation via the "reducing" or "nonreducing" end.

^


161

B. Utilization of Synthetic Polysaccharides

Until recently only naturally occurring polysaccharides were utilized for preparation of polysaccharide-protein conjugates. Now samples of pure polysaccharide can be reliably obtained by chemical synthesis. However, approaches to synthesize regular polysaccharides did not exist for a long time. In the authors' laboratory a general approach to the synthesis of homo- and heteropolysaccharides with l,2-/ra«5-glycosidic bonds between repeating units was developed [200]. This is based on stereospecific glycosylation of trityl ethers with sugar l,2-0-(l-cyano)ethylidene derivatives catalyzed by triphenylmethylium perchlorate (Scheme 55) [201]. With both the trityl ether and cyanoethylidene functions combined in one carbohydrate molecule, polycondensation of this monomer is possible under glycosylation conditions giving rise to a regular polysaccharide, which is a homo- or heteropolysaccharide depending on the structure of the starting bifunctional monomer [Scheme 56, Eq. (1)] [200]. On the basis of this principle, the first synthesis of complex natural heteropolysaccharides from the respective functionalized oligosaccharides was accomplished. The polysaccharides synthesized involved 0-antigenic polysaccharides of Salmonella TrO-^ TrClO^ TrO^

—————^^

ofcN CH3

^

^°{^\ 1

CH3

Scheme 55.

162

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK / ~ \

TrClO. (1) Wc

CH3

^ ^ \

TrO^ „ A'°\0(CH2)nNPhf

^q^.. • CT

TrCl04

TrO'

CH3

(7)

.iQT-cr'

»NPhf

Scheme 56.

newington (8-12 trisaccharide repeating units) [202], Shigella flexneri (10 tetrasaccharide repeating units) [203], and capsular polysaccharide of Streptococcus pneumoniae type 14 (10 tetrasaccharide repeating units) [204]. These chemically pure synthetic polysaccharide antigens, which were spectroscopically and serologically identical with their natural counterparts, can in principle be utilized for the preparation of neoglycoconjugates by one of the aforementioned procedures. At the same time, the potential of the trityl-cyanoethylidene condensation is far from being exhausted and it has recently been modified so as to enable the introduction into a polysaccharide molecule, in the course of its synthesis, of an aglycone spacer suitable for subsequent conjugation with polymeric carriers [205]. For this purpose the trityl-cyanoethylidene condensation is carried out in the presence of co-phthalimidoalkyl glycoside of a tritylated monosaccharide which serves as a "terminator" for the growing polysaccharide chains [Scheme 56, Eq. (2)] [205] and occupies the "reducing" position of the synthetic polysaccharide.


163

On removal of all the protective groups from the products of polycondensation, liberated free amino groups in the aglycone spacer allow the separation, by cation-exchange chromatography, of polysaccharide chains with co-aminoalkyl aglycone termini from neutral p o l y s a c c h a r i d e s p r o d u c e d upon "self-condensation" of the bifunctional monomer. Several homopolysaccharides and block-type heteropolysaccharides with a reactive aglycone spacer suitable for immobilization on a macromolecular carrier were synthesized using this approach [205]. Among them were 6-aminohexyl glycosides of the polysaccharide of Streptococcus A-variant [206], -[->2)-a-L-Rha-( 1 -^3)-a-L-Rha-( 1 -]„->2)-a-L-Rha-( 1 -0(CH2)6NH2 « = 6-7

and the common polysaccharide antigen of Pseudomonas aeruginosa [207]: [->3)-a-D-Rha-( 1 ->2)-a-D-Rha-( 1 ->3)-a-D-Rha-( 1 -]„->3)a-D-Rha-l-0(CH2)6NH2

«= 5 The presence, in these synthetic polysaccharides of an amino group-containing an aglycone spacer allows their conjugation to protein and synthetic polymeric carriers, by using standard methods, similar to those described for analog derivatives of oligosaccharides (see Table 1). Thus, a synthetic (1^6)-a-Dmannan with 6-aminohexyl glycopyranoside as its "reducing" end [208] was converted, upon action of thiophosgene, into the respective isothiocyanate (cf. pp. 111-112) and then conjugated to BSA. This conjugate, with about 11 mol polysaccharide per mol BSA, elicited (anti-polysaccharide)-specific antibodies upon immunization of rabbits, as shown by ELISA [209]. Analogously, the 6-aminohexyl glycoside of the synthetic polysaccharide antigen of Pseudomonas aeruginosa [207] was converted into neoglycoprotein (--5 mol polysaccharide/mol BSA) (Scheme 57) [210]. Immunobiological studies of an immunogen produced gave a reliable proof of the identity of im-

164


CSCI2, PS-0(CH2)6NH2 70% aq.ethanol. pH 7.0 ' PS-0(CH2)6N=C=S

HgN-BSA, pH 9 . 0 , —p— ► PS-0(CH9)ftNHCNH-BSA 20^, 72 h

I

PS = common polysaccharide antigen of

P.Qervginoaa

Scheme 57.

munological properties of the synthetic and natural samples of the common polysaccharide antigen from P. aeruginosa [210]. With further developments in the methods for synthesis of polysaccharides, in combination with these approaches, the preparation of polysaccharides in a form directly suitable for conjugation with macromolecular carriers is possible. Thus prospects for application of a unified strategy in designing neoglycoconjugates of oligo- and polysaccharide character will be possible. This may favor elucidation of many complicated problems of immunochemistry, in particular, of the role of repeats of oligosaccharide blocks for immunochemistry of bacterial antigens. V. CONCLUSION Neoglycoconjugates as artificial antigens are extensively used in studies of various carbohydrate-mediated biological processes. In conjugates of this kind the carbohydrate moiety is covalently attached to a polymeric carrier. Two general approaches for designing neoglycoconjugates have been developed: (1) modification with carbohydrates of a preexisting polymeric carrier, and (2) formation of a polymeric carrier in the course of copolymerization of carbohydrate and non-carbohydrate monomers. The role of a preexisting carrier in the majority of cases is played by a protein and therefore the first approach can be regarded as a route to neoglycoproteins. The


165

second approach affords conjugates with a synthetic polymeric carrier, which is as a rule currently a polyacrylamide. Recently, the first general approach has also been applied to the synthesis of polyacrylamide conjugates [211,212]. Used as a precursor to the polyacrylamide backbone is an activated form of polyacrylic acid, poly(4-nitrophenylacrylate), accessible by azo-bisisobutyronitrile (AIBN)-induced polymerization of 4-nitrophenylacrylate. Displacement of nitrophenyloxy groups with synthetic saccharides in the form of glycosides with an aminocontaining spacer leads to formation of amide linkages. Treatment with ammonia or 2-aminoethanol is used to complete the formation of the polyacrylamide backbone in the latter case modified with A^-(2-hydroxyethyl)amide groups [213]. Analysis of conjugation procedures reveals the most versatile strategy for preparation of neoglycoconjugates associated with the use of spacers with a terminal amino group, ensuring both effective coupling to proteins (e.g. by the isothiocyanate method [62-64]) and an access to neoantigens of the copolymer type via preliminary A^-aeryloylation [111,151-153,155-157]. Recent findings that the A^-acryloyl group is able to act as an acceptor of amino functions of proteins in Michael-type addition opens more prospects for preparation of both neoglycoproteins and polyacrylamide conjugates from the same saccharide precursor [214,215]. This strategy is applicable to oligosaccharides and, apparently, to polysaccharides. Conjugates of the copolymer type derived from polysaccharides have not been documented yet. This versatile strategy allows for the preparation, from the same carbohydrate fragment, of glycoconjugates with carriers having various immunobiological properties. Conversion of a polysaccharide into a neoglycoprotein enhances, as a rule, immunogenicity of the former. This, however, occurs due to coupling of a carbohydrate moiety with the protein molecule which possesses its own antigenic and immunogenic properties. Protein carriers being T-dependent antigens, modulate an immune response to neoglycoproteins so as to direct it along the T-dependent route whereas many polysaccharide antigens are T-independent [216]. Therefore, from this point of view, neo-

166


glycoproteins cannot be regarded as completely adequate models for the natural polysaccharide antigens. Neoglycoconjugates of the copolymer type with inherent multiple repeats of the same monomer along the polymeric chain of the carrier are close in structural design to the T-independent antigens (cf. [217]), although the nature of their immunogenicity requires serious study. To date few results are available on immunogenic properties of neoglycoconjugates of this type [140,180]. Their distinction from neoglycoproteins is that they contain only a single serologically active component in a macromolecule, related to natural glycoconjugates, i.e. the carbohydrate moiety. It is this circumstance that makes them especially promising as antigens for serological studies. ACKNOWLEDGMENTS The authors wish to thank Dr. L.V. Backinowsky and Mrs. E.E. Trusikhina for their assistance in preparation of the manuscript.

NOTES 1. The term "neoglycoconjugates" is also applicable to conjugates of carbohydrates with low-molecular-weight compounds, e.g. with higher alcohols (neoglycolipids), or even with particles, e.g. latexes or macroporous sorbents. Neoglycoconjugates of macromolecular character are mainly considered in the present review. 2. This enzyme was shown recently to have a much broader substrate specificity [32b]. In addition to the aforementioned disaccharide, p-D-GlcNAc-(l->3)-GalNAc and P-D-GalNAc-(l->3)-D-GalNAc and even trisaccharide a-L-Fuc-(l-^2)-P-DGal-(l->3)-D-GalNAc were identified in the mixture of oligosaccharides released from desialylated bovine submaxillary mucin by the enzyme. 3. The difference in the character of immune response to T-dependent (T-D) and T-independent (T-I) antigens is discussed in [183].

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GLYCOSYLATION PATTERNS IN MUCUS GLYCOPROTEINS

Amalia Slomiany, Chinnaswamy Kasinathan, and Bronislaw L. Slomiany

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction 178 Apomucin Synthesis-Mucin Genes 179 Rough Endoplasmic Reticulum (RER) Processing 184 Transport and Topography of Glycosylation in the Golgi Apparatus 189 Peptide-Specific Initial Glycosylation 190 Localization of Glycosyltransferases 192 Role of Nucleotide Transport System in Synthesis of Oligosaccharide Chains 193 Other Factors Regulating Glycosylation 194 Role of Expression of Glycosyltransferases . . . . . . . . 195 Role of Glycosyltransferase Structure 196 Carbohydrate Chains 198


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A. SLOMIANY, C. KASINATHAN, and B. L. SLOMIANY

XII. 0-Glycosylation of Nucleus and Cytoplasm Protein . . .201 XIII. Concluding Remarks 203 Acknowledgments 204 References 204

I. INTRODUCTION For decades mucus glycoproteins (mucins) have preoccupied and challenged the skills and imagination of many investigators interested in resolving the pathological abnormalities of the mucus of gastrointestinal and respiratory diseases, pathologies of salivary glands, and reproductive tract secretions in infertility [1-6]. In recent years, the interest in the biological role of mucus glycoproteins intensified even further when examples of their importance were found in recognition determinants in hostpathogen interactions, protein targeting, cell-cell interactions, and cell surface receptors [7-11]. However, in spite of renewed attention and efforts to understand the functional roles of carbohydrate chains of glycoproteins, and how they relate to the cellular glycosylation processes and the primary sequence of the apomucin core, the glycoprotein, and particularly the Oglycosidic mucus glycoprotein, such as mucin, still remains largely unknown. In the past, the glycosylation patterns of mucus glycoproteins were usually taken literally and overviewed as an impressive array of oligosaccharides varying in composition, anomeric linkages, branching, acidity, and length from a single A^-acetylgalactosamine (GalNAc) residue to long multiple-branched structures containing 20 or more sugar units [1]. This approach was possible in previous reviews of the topic when a number of known structures was manageable and helpful in establishing the structural elements of the oligosaccharides derived from divergent sources. Recent advances in the structural studies of mucins, however, have revealed almost limitless combinations of carbohydrate chains which may be attached to single apopeptide with remarkable diversity of biologically active determinants. However the information regarding the potential importance of

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the particular order of carbohydrate substitution in the oligosaccharide chains, apomucin sequence, and its early intracellular processing still remain poorly understood and unexplored. Therefore, the present review, which will be limited to glycoproteins containing O-linked sugar chains, addresses the area of research relating to apomucin synthesis and co-translational modifications. The issue of the factors which influence glycosylation pattern and cause structural diversity of the sugar chains will be discussed, and evidence that the so-called "link protein" is an extraneous protein derived from fibronectin will be provided. IL APOMUCIN SYNTHESIS-MUCIN GENES Three independent studies suggest that several genes are involved in mucin peptide synthesis and propose that the molecules consist of tandemly repeated identical peptide fragments [12-14] with unique amino-N- and carboxyl-C-terminal sequences [12]. As suggested, either the number of internal repeats or the N- and C-terminal fragment length and composition reflect on mucin derivation and the differences in the apopeptides molecular weight. For ovine and bovine submaxillary mucins [12-15] and for human and rat gastric and intestinal mucins (Figure 1), the translation products which were immunoprecipitated with apomucin polyclonal and monoclonal antibodies are 60-64-kDa proteins. While the in vitro translation studies and analyses of the enzymatic deglycosylation products from several sources indicate that the apomucin is a 60-kDa protein, a 200400-kDa protein core has been postulated for bronchial mucus glycoprotein [16]. In addition, in studies with intestinal mucins by Mantle et al. [17-20] and Fahim et al. [21,22], the existence of a linking peptide enriched in cysteine and A^-glycosidically linked mannose-rich oligosaccharides has been strongly emphasized and its contribution to mucin properties in cystic fibrosis has been advocated [20]. Just as estimates of the size of mucin core have varied, the earlier concept of a linking protein has been modified over the years. The earlier findings of Allen et al. [23] suggesting that

180


Figure 1. Cell-free synthesis of gastric apomucin. The rat gastric mucous cell mRNA was translated in presence of microsomal membranes. (1) Aliquots of total translation products, and immunoprecipitates obtained with antimucin monoclonal antibody (3G12MAb) from (2) translation mixture without mRNA. (3) Translation mixture with mRNA at 0 time incubation, and (4) translation mixture with mRNA after 60 min at 30 °C. The samples were analyzed on 7.5% SDS-PAGE.

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a 70-kDa protein is involved in holding four glycosylated subunits, and that the link protein may be a 118-kDa glycosylated peptide incorporated between the glycosylated mucin subunits as in human intestinal mucin [24]. Alternatively, it has been considered that it is a 60-kDa component as in the case of canine tracheobronchial mucin [25], or that neither link protein is present in rectal mucin preparations [2]. Several interpretations were offered to explain the variations in size, quantity, and appearance of a linking protein, but none of these has provided sufficient evidence indicating that the "link protein" represents an integral portion of the mucin protein core. Our studies revealed that a mucin preparation obtained according to a procedure utilized by Pearson and Allen [23], Mantle et al. [17-20], Fahim et al. [22], Carlstedt et al. [26], Honsson et al. [27], Roussel et al. [29] contains fibronectin fragments which apparently were falsely identified as an integral part of mucin protein. Western immunoblotting with fibronectin antibodies, the production of proteolytic fragments of fibronectin obtained with pepsin, trypsin, chymotrypsin and elastase, and the compositional analysis of the fragments has provided overwhelming evidence that the mucin-associated peptides are derived from fibronectin molecules (Figure 2). Furthermore, analysis of the mucin obtained according to the procedure of Carlstedt [26—28], revealed that such a preparation consists of mucin-fibronectinDNA complexes which were assumed by this method to represent the undegraded native mucin only. This fallacy led to a series of misinterpretations and miscalculations displayed in many papers that followed [4,27,28,30,31]. Therefore, many findings and ideas derived from the studies of Mantle et al. [17-20], Carlstadt et al. [4,26,28], and the others [21-25,27,2931] utilizing similar criteria of mucin preparations require thorough reevaluation. Particular priority should be assigned to the reassessment of the so-called "link protein" antibodies [19—24] which appear to recognize the iV-glycosylated fragment of the fibronectin or fibronectin-like protein. The general picture that emerges from the evidence which refers to mucin and not to its complexes with extraneous proteins, or other components released by the destruction of the

1 2345

Figure 2. SDS-PAGE and Western blots with antifibronectin antibodies of the native-nonreduced (A) and reduced (B) mucins. Panel A, intestinal mucin prepared according to procedures described in 1, Slomiany et al. [32]; 2, Mantle etal. [19]; tracheobronchial mucin prepared according to procedure described in 3, Carlstedt et al. [26]; 4, Slomiany et al. [32]; 182

1

2

118H

92-1 70-

^1 Figure 2. (Continued) 5, intestinal mucin preparation after DNase treatment prepared according to procedure of Fahim et al. [21]. Panel B-1, components of the reduced human intestinal mucin reacting with antifibronectin antibodies and 2, with affinity to Concanavalin A. 183

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cells, is that mucus glycoprotein does not contain any linking protein, and that in the majority of tissues, its protein core constitutes a 58-62-kDa peptide. The carboxyl terminal of this peptide is not glycosylated (naked) and is responsible for mucin self-association and also for the interaction with fibronectin and fibronectin-like molecules. Hence, it is important to determine whether the physicochemical properties of mucins are directly related to the size, composition, sequence, and the processing (fatty acylation) of the C-terminal end of their peptide core [12,32]. III. ROUGH ENDOPLASMIC RETICULUM (RER) PROCESSING Recent work from our laboratory has begun to uncover the initial stages of 0-glycosidic glycoprotein synthesis in the RER [34— 37]. The data indicate that the apopeptide precursor, after initial N-terminal palmitoylation and the release of signal sequence, is transferred to the luminal site of RER, and while the peptide is being translated, the luminal portion of the apomucin is subjected to systematic 0-glycosylation with A^-acetylgalactosamine [36]. Considering the events involving the immediate peptide folding in RER which closely follow the nascent peptide translocation [38], this stage appears to be the only feasible moment for complete glycosylation of the peptide chain. Using mucus glycoprotein synthesizing polysomes, and monoclonal antibodies recognizing GalNAc, the fraction of the peptides ranging from 6-60 kDa were isolated [34—37]. These peptides were also retarded on an affinity column of Helix pomatia lectin [35-37]. The results suggest that once the initiated mucin peptides reach the luminal surface they undergo immediate glycosylation. Considering the length of the signal sequence and the size of the shortest glycosylated nascent chains, it may be concluded that glycosylation cannot occur until there is a segment of 50-54 amino acid residues between the acceptor site and the ribosomes [39]. This segment would be long enough to undergo cleavage of 20-25 residues of signal sequence and to extend across the membrane of the ER into the lumen.

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By examining the protease sensitivity of the luminal peptides and monitoring for palmitate label, it was demonstrated that the protease cannot digest the A^-terminal amino acid sequences beyond the fatty acyl residues of the peptides. This resistance to proteolytic degradation could be interpreted as an indication of an immediate, complete, and systematic 0-glycosylation of the nascent peptide, and that the first GalNAc residues are in close proximity to the palmitoylated amino acids since the protease-treated peptides also retained the fatty acyl substituent [36,37,40]. Additional information on the processing of (9-glycosylated peptides in RER was obtained from a study with isolated precursors of mucus glycoprotein since the treatment of the completed mucin precursors with trypsin released a 8-12-kDa peptide which was acylated but lacked GalNAc residues [41]. This result is consistent with the cytoplasmic orientation of the fatty acyltransferase enzyme(s) which at first acylates the Nterminal end of the peptide. Once the translation is completed and the ribosomal subunit has dissociated, the C-terminal unprotected segment is acylated again [41]. The susceptibility of the C-terminal end of the peptide to proteases suggests that the 0-glycosylation is terminated as soon as translation is completed since the 8-12-kDa fragment corresponding to the peptide spans across the membrane and the ribosomal subunit is not substituted with 7V-acetylgalactosamine. Taken together, these results suggest that 0-glycosidic glycosylation is initiated in rough endoplasmic reticulum [6,32—37,41,42]. Although sufficient evidence on the role of N-terminal and C-terminal acylation is not available, it is tempting to speculate that the acyl residue directs the peptide to the lumen of endoplasmic reticulum and serves as the recognition signal for the initiation of the peptide 0-glycosylation. The data reviewed above, thus, support the model for the co- and early posttranslational acylation and 0-glycosylation in the RER as shown in Figure 3. It should be noted that some features of this model at this time are supported only by the experiments performed in this laboratory on gastric and salivary mucin. Thus, it is

Cytosol ER Membrane Protein Fatty Acyl Transferase

ER Lumen protease Figure 3. Mucin peptide synthesis and oligosaccharide chain initiation.

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likely that some aspects of this process may be modified in the future as more information becomes available. It has been speculated that protein acylation would only retain the peptide in the endoplasmic membrane and could not help in its translocation [43]. If, indeed, this was the event, then one should expect the growing peptide by sheer force of the enlargement finding its way to the ER lumen and to appear there in the form of a growing loop until translation is completed. Another possibility is that the process is similar to the synthesis of Man-P-Dol which supposedly flips from the cytoplasmic to luminal face of the ER [44]. Such a flip or drag is probably spontaneous, since fatty acids alone have been shown to flip freely within the membranes [45]. This path of events would be consistent with the findings that the N-terminal portion of the apomucin is glycosylated in an immediate vicinity of acyl residue and thus not susceptible to proteolytic cleavage. However, if the first situation prevails, then the 25 amino acid residues buried in the membrane would remain unsubstituted and, most likely, susceptible to proteolytic degradation. Depending on the topography of these processes, the fatty acyl residue may be dragged or pooled to the inside of the ER lumen by the force of peptide elongation or may flip across the RER membrane, and the peptide remains anchored to the membrane throughout the process of translation (Figure 4). This model has several interesting and possibly significant features. It suggests that the peptides entering the RER-Golgi secretory pathway are first lipid-linked, a modification occurring on the cytosolic face of ER membrane. Then the fatty acyllinked peptides flip or are forced to the luminal face of the membrane. The demonstration that the protein fatty acyltransferase, which attaches the fatty acyl group, has a cytosolic protease-sensitive site, tends to support this view. The site of the initial 0-glycosylation is still controversial. Nevertheless, the evidence obtained by Carraway et al. [6,42] and others [46] and the successful isolation in our laboratory of A^-acetylgalactosamine containing peptides, but not completely translated mucin nascent peptides [35-37,40], suggest that the apomucin— a-A^-acetylgalactosaminyltransferase—is the luminal ER en-

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Apical Plasma Membrane

Figure 4. Mucin synthesis; vesicles transfer between ER-Golgi-Plasma membranes.

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zyme which is expressed in the tissues synthesizing 0-glycosidic glycoproteins regardless of ABH blood group status of the analyzed tissue.

IV. TRANSPORT AND TOPOGRAPHY OF GLYCOSYLATION IN THE GOLGI APPARATUS The secretory pathway of eukaryotic cells consists of distinct membrane-bound organelles and transport vesicles that collectively function to transport proteins to the cell surface [47]. While all proteins enroute to the cell surface enter and traverse the endoplasmic reticulum and Golgi—^the fundamental processes to interorganelle transport—^the specific intracellular signals for processing and secretion are not very well known. At least four factors are found to be essential for the transport of a protein between more than one stage of the pathway [48]. They are required in a sequence to facilitate vesicle budding and fusion and include ATP, GTP, Ca^"^, and protein involved in intracellular fusion [49]. Reconstitution of the export of protein from ER to Golgi requires ATP and cytosol, while GTP is now recognized as an essential step for transport of proteins between all secretory compartments [49—53]. An addition of guanosine-5'-0-(3-thiotriphosphate), GTPyS, to the system inhibits transport between the ER and Golgi compartment [52]. A similar effect is achieved when the A^-ethylmaleimide sensitive factor (NSF) of the cytosol is inactivated with either N-Qthylmaleimide (NEM) or a monoclonal antibody to NSF [50,52,54]. At present, the NSF protein is believed to be one of the components of the protein complex that is assembled and disassembled to ensure fusion between transport vesicle and its target membrane. Ca^"^ is also essential for the fusion of transport vesicles and for the transport between the ER and Golgi [49]. The results of studies with semi-intact cells suggest that the delivery and/or fusion of the carrier vesicle to the Golgi compartment, but not its formation requires Ca^"^ [48]. What is the structural basis for efficient export of protein from the ER? It is conceivable that the glycosylated proteins are not recruited for matrix pro-

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teins of ER [56,57]. A second possibility is that similarly to the transport of the transmembrane vesicular stomatitis virus protein (VSV G protein), the secretory protein signals for translocation from ER to Golgi are located in their cytoplasmic domains [57-59]. While there is no direct evidence that mucin C-terminal cytoplasmic acylated domain is necessary for the efficient export from the ER, the mucin assembled in a system treated with physiologically tolerated dose of alcohol, which is known to inhibit the protein acylating enzyme, is retained or degraded in the ER [60-61]. Similarly, the retention of apomucin is observed in tissues derived from patients with Sjogren syndrome [62]. However, in the latter case, the accumulating peptides not only remain in the ER, but also evoke an autoimmune response [62,63]. These results and intriguing effects of Brefeldin A (prostaglandin analog) on protein transport [64,65] suggest the possibility that a common biochemical mechanism disrupted by these agents (Brefeldnin A, alcohol) is fundamental for transport and segregation of the proteins, which are distributed in some unknown but orderly way [59]. The delineation of the mechanism underlying the effect of ethanol, Sjogren syndrome pathology, and the Brefeldin A on the normal vectorial flow of mucin precursors from ER to Golgi compartment may be expected to provide a new insight into the possible function of (9-glycosylation, protein C-terminal sequence, and its early post-translational acylation on the secretory protein assembly.

V. PEPTIDE-SPECIFIC INITIAL GLYCOSYLATION Examples of a peptide-directed attachment of glycosyl residues are so far limited to A/^-linked sugar chains [66]. Substitution of mannose 6-phosphate on A^-linked sugar chains of lysosomal enzymes [66], where the enzyme recognizes a specific peptide determinant or terminal sequence on A^-linked oligosaccharides of pituitary hormones (LH, TSH), apparently results from recognition of a peptide by specific glycosyltransferase [67]. In these and other examples [68], the peptide-specific glycosylation of A^- glycosidically linked oligosaccharide chains is limited to trans-Golgi enzymes. However, there is no evidence as to what

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sequence on the polypeptide direct the attachment of the Olinked sugar chains to the hydroxyl group of threonine and serine. A recent idea is that the initial glycosylation of 0-glycosylated and apomucin type peptides is attributed to the recognition of the growing peptide by luminal apopeptide: GalNActransferase [35-37]. In spite of numerous claims that the (9-glycosidic chain initiation is not directed by peptide structure [2,3,5,69,70], the fact that the majority of serine and threonine residues in A^-glycosidic glycoproteins are not glycosylated, while apomucin peptides are consistently 0-glycosylated, suggest that the apomucin-aGalNActransferase recognizes some specific peptide determinant on the protein undergoing 0-substitution. This recognition appears to be an early cotranslational event since the partially translated apopeptides are already Oglycosylated [36,37,40]. Since the initial glycosylation represents the RER-luminal modification, the ultimate signal for (9-glycosylation must be determined early, perhaps by cotranslational N-terminal fatty acylation [35,36]. This is in accord with the evidence that the 0-glycosylated peptides are also fatty acylated [34,36,37]. Examination of the in vitro translated mRNA in the presence of microsomes indicates that acylation and O-glycosylation does not occur if microsomes are first subjected to incubation with trypsin (A. Slomiany, unpublished). Since trypsin inactivates protein fatty acyltransferase but not GalNActransferase [71], it is possible that initial 0-glycosylation is signaled by N-terminal acylation of the translocated peptide. Most likely, however, these observations imply that the processing of 0-glycosidic glycoproteins is initiated in the rough endoplasmic reticulum and that this event may be controlled by at least two enzymes: protein acyltransferase, which is responsible for translocation of the peptide to the luminal site of the ER, and GalNActransferase which recognizes the specific 0-glycosylation sequences unique to mucin protein or a segment of proteins which become Oglycosylated. Such a schedule of events in processing of apoproteins is also reflected in the common structural element in 0-linked carbohydrate chains; i.e., it is restricted to a-GalNAc residues only. If the initial processing events were limited to

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Golgi, the peptide folding which is known to take place in the RER [38], would prevent systematic substitution of the 0-galactosaminyl residues and most likely would result in an irregular processing as the one observed for the terminal glycosylation of the chains. VI. LOCALIZATION OF GLYCOSYLTRANSFERASES Localization of the enzymes involved in O- and A^-linked glycosylation has been studied extensively, and it is firmly established that the terminal glycosyltransferases are present in Golgi apparatus [72—77]. Precise localization of these enzymes is still, however, uncertain, and recent studies suggest that in some cells the terminal glycosyltransferases may have a dispersed distribution throughout the Golgi stocks [78-80]. It is believed that the common trade of the Golgi glycosyltransferases should be a specific retention signal that is absent in plasma proteins, secreted proteins, or ER integral proteins [58,81—83]. The available data suggest that the Golgi enzyme retention signal must reside in the N-terminal portion of the protein, but whether it is within the cytoplasmic tail, membrane anchor, or stem region is not precisely known [80,84]. Demonstration of such a signal sequence for Golgi glycosyltransferases would help to further evaluate the properties of the apomucin—GalNAc transferase which certainly should exhibit the RER integral protein qualities. The presence of a DKEL sequence, a common tetrapeptide signal sequence in proteins in the lumen of the ER [85], would provide unquestionable evidence for the concept of RER localization of the initial processing of 0-glycosylated secretory proteins. There is, however, ample evidence supporting the luminal orientation of the active sites of Golgi glycosyltransferases [80,86,87]. Membrane and secretory proteins containing carbohydrate chains are detected within the Golgi vesicle complex, and glycosyltransferases inactive in vitro and protease resistant in sealed vesicles [80], are glycosylated, and the soluble glycosyltransferases are secreted from the cell in active form [88]. Thus, it appears that the specificity of the enzymes for their

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donor and acceptor substrates constitutes the primary basis for determining the structure of the sugar chains produced by Golgi enzymes. However, the carbohydrate residues attached in Golgi compartments (farther away from the core peptide chain) are differentially expressed and seem to also be regulated in the cell by the development, differentiation, oncogenic transformation [80], and other as yet unknown factors.

Vll. ROLE OF NUCLEOTIDE TRANSPORT SYSTEM IN SYNTHESIS OF OLIGOSACCHARIDE CHAINS The luminal exposure of Golgi glycosyltransferases suggests that there should be a pool of nucleotide sugars within the lumen to serve as substrates in glycosylation reactions. However, nucleotide sugars are synthesized in the cytosol, with the sole exception of CMP-Neu5Ac which appears to be synthesized in the nucleus [89]. It is possible that the ability of the sugar nucleotides to cross membranes is important in determining the structure of oligosaccharides in glycoproteins, glycolipids, and proteoglycans. This has led to the hypothesis that glycosylation (in ER and Golgi) involve the transport of sugar nucleotides from the cytosol to the ER and Golgi lumen. Evidence strongly suggesting that nucleotide sugar translocation is of physiological importance and that it is a determining factor of oligosaccharide structure has been obtained through the analyses of mutant cultured cells [90-93] which were unable to translocate individual nucleotide sugars. Cells of these mutants had an 80-90% decrease in galactose and sialic acid in their glycoproteins and glycolipids although the levels of UDPGal and CMP-Neu5Ac, as well as activities of galactosyl- and sialyltransferases, appeared to be normal, with the endogenous acceptor for galactose also showing the characteristics similar to the parental cell lines. However, the rate of UDP-galactose translocation into the Golgi vesicles from the mutant cell lines was only 3% of the rate of transport into the vesicles derived from parental cell lines, whereas the other nucleotide derivatives, such as UDP-GlcNAc, UDP-GalNAc, and 3'-phosphoade-

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nosine 5'-phosphosulfate (PAPS) were transported at the rates comparable with that of wild-type cells [93]. These results provide strong evidence as to the importance of nucleotide sugar translocation for glycosylation in ER and Golgi. The results also suggest that there are separate translocator proteins for each nucleotide sugar and PAPS, particularly for uridine nucleotide sugars which have been thought to use a single translocator [89,94].

VIII. OTHER FACTORS REGULATING GLYCOSYLATION All stages of glycosylation are regulated by various agents which may produce qualitative changes in the level of cell surface terminal glycosylation sequences or quantitative changes specifically noticeable in cells producing large quantities of mucins [1,2,69,95,96]. Exposure of the cells to agents such as alcohol produces quantitative changes in the levels of initial synthesis of the glycoprotein without appreciable effect on the Golgi glycosyltransferases [97-100]. Differentiation agents such as butyrate [101], phorbol esters [102], and retinoic acid [103,104] have been reported to produce qualitative changes in glycosylation by affecting specific glycosyltransferases, by modifying expression of glycosyltransferase genes, and their regulation at the transcription level. In accord with these observations, the expression of a new mucin and the sequence of glycosylation correlate with the enzyme activity and the mRNA levels for their molecules [80]. Support for this idea comes from studies with altered cellular glycosylation in cells transfected with DNA fragments or expression vectors containing cDNA coding for glycosyltransferases which synthesize terminal sequences of apomucins. At present, the idea is entertained that different mucosal cells are responsible for different mucins [2,4,5,105,106]. The supporting results for this notion have been obtained using various monoclonal antibodies and lectins. Although many papers present evidence to support this view [17-22,105-107], caution should be exercised in further propagation of the idea as the

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poly- and monoclonal antibodies, which were believed to be highly discriminant and specific for mucus glycoprotein, appear to be antifibronectin antibodies. This error alone may disqualify many statements which have been made with reference to the heterogeneity of the intestinal mucins [20,105-107]. Thus, it is far more appealing to implicate the function of glycosyltransferase genes in the final expression and complexity of carbohydrate chains [78,80,108,109]. Whether glycosyltransferase genes are differently expressed and differently affected at transcriptional level in the individual mucous cells remains to be determined. It is known, however, that the cell transformation results in the activation of specific glycosyltransferase genes, whereas hormones provoke increased activity of the enzymes in the entire group of cells which has been subjected to a particular treatment. Thus, the diversity of mucin carbohydrate chains is dictated by the cellular make up and the physiological stimuli [80,101-104,108,110]. IX. ROLE OF EXPRESSION OF GLYCOSYLTRANSFERASES

To date, no evidence has been presented that apomucin-A/'-acetylgalactosaminyltransferase is affected and initiation of the Olinked oligosaccharides is differentially expressed in cells undergoing differentiation during development and oncogenic transformation [108,111—113]. In contrast, the terminal glycosylation and the expression of the corresponding glycosyltransferases were observed to change. Direct dependence of glycosylation on specific glycosyltransferases was shown in the experiments with cells transfected with DNA fragments or expression vectors containing cDNA coding for glycosyltransferase [78,109,114]. Transfection of the cells with cDNA for Gala(l-^3)-glycosyltransferase resulted in the production of Gala(l->3)-Gal-R carbohydrate chains which were absent prior to this manipulation. Similar examples of the induced expression of the carbohydrate sequences are described in the case of A^-glycosidically linked carbohydrates [72,80]. In all instances, the terminal sac-

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charides were affected. Whether this indicates that the enzymes residing in the Golgi are regulated differently than those of the ER membrane is too speculative to advance. First, the functional cloning of the a-A^-acetylgalactosaminyltransferase must be performed to verify the synthesis of the specific oligosaccharides with expression of the enzymes responsible for their initiation. In support of this s u g g e s t i o n , several reports have [80,108,111,115] demonstrated that the changes in glycoconjugate glycosylation correspond, not only to quantitative, but also qualitative changes. Such results are particularly interesting in conjunction with the fact that glycosylation may play a role in both cell differentiation pathways and in expression of the specific glycosyltransferases, which are indispensable in mucin synthesis. Developmentally regulated synthesis and processing of the apomucin may be reflected in oncogenic transformation of mucus secreting cells into nonsecreting cells, while the quantitative changes are observed in aged cells. Although large number of oligosaccharide structures have been studied and their functional significance determined, it appears that initial specific sequences are important in cell differentiation pathways [116-119]. On the other hand, the terminal sequences, which are relevant in biological recognition; cellular, microbial [120-123], and virus specific interaction [120-123]; maturation of thymocytes; compaction of embryo cells; or cell adhesion, implicate the direct involvement of carbohydrate interaction [124—129]. Oligosaccharide chains of glycoproteins and glycosphingolipids have been implicated in numerous cell functions, but evidence depicting precisely the role of cell sequential addition of carbohydrate residues on the developmental status of the cell is not available. The elucidation of the biological role of each individual enzyme and its expression in glycoconjugate is unknown and certainly requires further attention. X. ROLE OF GLYCOSYLTRANSFERASE STRUCTURE It is estimated that the carbohydrate sequences of glycoprotein are elaborated with the aid of 100 or more highly specific gly-

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cosyltransferases, which determine the highly diverse sequences of the sugar chains produced by the cell. Recently, the structures of six glycosyltransferases have been deduced [80,130-133]. The enzymes have no sequence homology, but each has a short N-terminal cytoplasmic tail, 16-20 amino acid membrane anchor domain, a flexible stem region, and a large catalytic domain. The membrane spanning domain retains and orients the enzymes within the lumen of the Golgi, whereas the stem region, which probably differs in length and flexibility, allows the catalytic C-terminal domain to glycosylate the arriving vesicular peptides, which fuse with the Golgi segment that contains one or the other particular glycosyltransferase. Depending on the length of the stem region and the size of the catalytic domain, various sites on the apomucin molecule have a chance to undergo glycosylation. The flexible stem of GlcNAcpl,4-galactosyltransferase consists of 62 amino acids, and that of Gala,2,6-sialytransferase is 35 amino acid residues long [133,134]. Although direct evidence for the function of this "reaching arm" is not available, one can only speculate that the variations in the flexible tether of glycosyltransferases are tissue specific and that they may dictate the oligosaccharide make up. The fact that sialytransferase has a shorter arm than galactosyltransferase may be expressed in structures directly related to oligosaccharide size, showing that sialic acid is either terminating very short oligosaccharides or is present in terminal locations. On the other end, the galactosyltransferase flexibility, the length of the arm, and the species variation in the enzyme stem region are the attributes for easy access to more complex and branched oligosaccharide chains with a high degree of heterogeneity. Further studies on the structural assets of various glycosyltransferases should provide more evidence in this area and give broader bases for such speculation. Obviously, this type of restriction on the enzymatic activity would not apply to the soluble forms of glycosyltransferase enzymes. Their activity and expression in the oligosaccharide structure would be limited by the enzyme—substrate specificity and by the size and structure of the catalytic domain. This would, perhaps, explain why the

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oligosaccharides isolated from sources containing soluble glycosyltransferases are not identical with those assembled in more structured and compartmentalized path in Golgi apparatus. The oligosaccharide make up of the body fluids probably reflects on health status of the individual, since it has been documented that inflammation or some other disease states increase cathepsin D-like protease activity within Golgi compartments [87,88,135] and that the elevated levels of soluble glycosyltransferases in serum, milk, and other body fluids may originate from the release of membrane-bound enzymes by endogenous proteases [135]. XI. CARBOHYDRATE CHAINS The 0-glycosidic linkage of GalNAc to serine or threonine of apomucin constitutes the first step in the synthesis of mucus glycoprotein oligosaccharides and is identical for every chain initiated. Following stages referred to as core formation, elongation or backbone assembly and termination display almost infinite structural variability. Nevertheless, some rules that govern the biosynthetic processes can be delineated. A most rational and comprehensive presentation of the biosynthetic pathways of 0-glycans has been provided in a review by Schachter and Brockhausen [69]. At a very early stage of synthesis, the factors controlling assembly of carbohydrate chains appear to be the substrate specificity and competition between 0-glycan glycosyltransferases. Three or more different enzymes compete for GalNAc-R depending on the tissue; the 0-3 of GalNAc undergoes substitution with galactose, or A^-acetylglucosamine in presence of a highly specific p3-galactosyltransferase or p3A^-acetylglucosaminyltransferase, respectively [136-139,143,144]. The C-6 of GalNAc in ovine submaxillary glands is substituted with sialic acid in presence of a6-sialytransferase [140-142]. Gaip(l->3)GalNAcR and GlcNAcP(l->3)GalNAc-R serve to form branched structures shown in Figure 5. Five enzymes compete for Gaip(l->3)GalNAc-R (core 1) [69,136,14^148] and any substitution of core 1 prevents synthesis of core 2. The key enzyme

-

Fucal

Fucal Termination

lnitiarion

Elongation

Figure 5. Assembly of the 0-linked oligosaccharides in mucins.

Termination

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responsible for switching from core 1 to core 2 0-glycans is p6-GlcNActransferase [146,147]. Formation of the larger, linear or branched oligosaccharide structures is accomplished by the addition of Gaip(l->3)GlcNAc- or Gaip(l->4)GlcNAc-disaccharide sequences in p(l-^3), p(l->4), and p(l-^6) linkages, which considerably amplify the potential for structural variability. The number of crossroads in the synthesis of these structures are shown in schemes presented by Schachter and Brockhausan [69]. The path taken by the synthetic pathway is primarily dictated by the relative activity of the competing transferases, since at each point more than one enzyme competes for a common substrate. A common feature of many elongated oligosaccharide structures is the presence of a-linked chain-terminating carbohydrate residues which confer various antigenic properties. The wide diversity of a-linked terminal structures is shown in Figure 5. Their synthesis has been reviewed previously [69] and the enzymes involved in the assembly of these complex structures have also been described [148—151]. In addition to commonly recognized blood group antigens, a diverse group of carbohydrate determinants is involved in agglutination, elimination, and the bacterial attachment to cellular surfaces [5]. Indeed, an impressive list of carbohydrate determinants recognized by microorganisms has been compiled in the recent review of glycosphingolipids [152]. Although the review does not deal with carbohydrate structures of mucins, it is more than likely that glycoprotein-bound carbohydrate determinants are also involved in such processes in a manner similar to blood group antigens identified on glycosphingolipids and 0-glycosidic glycoproteins [153-157]. In conclusion, there is a sufficient amount of evidence to support the idea that the diversity of the synthesized carbohydrate chains depends on many factors, which are inherent to a particular cell or organ function, along with many physiological correlations. The composition and sequence of the mucin backbone, the structure, the amount and the activity of the glycosylatransferases involved in the assembly, the availability and transport of substrates and cofactors, and the status of the in-

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tegral proteins of the secretory pathway are all involved in the moulding and structuring of the carbohydrate chains that are being assembled in the postranslational non-template pathway. Variations in the oligosaccharide structures may be the most important function of the mucin's carbohydrate moieties arising in response to the demand of the changing environment, physiological stimuli, or pathological transformation.

XII. O-GLYCOSYLATION OF NUCLEUS AND CYTOPLASM PROTEIN While it is widely accepted that the glycosyl moieties on glycoprotein are localized on the outer surface of the cell membrane and the luminal compartments of the intracellular organelles, the existence of cytoplasmic or nucleoplasmic glycoproteins has been largely ignored since the contamination from plasma membranes and broken endoplasmic reticulum and Golgi is usually difficult to rule out [158—161]. Furthermore, none of the accepted models of glycoprotein synthesis and transport provide an explanation for the transport of some glycoconjugates to nucleoplasmic or cytoplasmic compartments of the cell. Recently, however, O-glycosidically linked residues of A^-acetylglucosamine were detected in purified proteins of nuclear pores [162-168], cytoskeletal proteins [169], and transcription factor [170,171]. 0-Linked mannosyl residues on cytoplasmic proteins [172,175], and glucosyl residues attached to hydroxyl moiety of tyrosine were found on cytoplasmic "primer" for glycogen synthesis [173,174]. There are also studies providing evidence for the existence of nucleoplasmic and cytoplasmic glycosidases and glycosyltransferases [170,176,177]. On incubating UDP-GlcNAc with isolated nuclei, heavy labeling was detected in autoradiographed nuclei and the chemical analysis indicated the acid and alkali susceptibility of the incorporated sugar [168,178]. 0-linked Nacetylglucosamine was found on the intracellular proteins of B lymphocytes where it accounted for 5-12% of the total metabolic incorporation of [^H]glucosamine [179]. Other studies indicated that proteins containing O-linked GlcNAc residues are

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particularly concentrated in the nuclear envelope, but substantial amounts were also present in the integral endoplasmic reticulum and Golgi membrane protein facing the cytosolic environment [167,168]. In contrast, proteins containing 0-linked GlcNAc residues were not detected in the serum. The 62-kDa GlcNAc(9-nuclear pore glycoprotein and several different subsets of the proteins associated with the nuclear pore complex were isolated and characterized [165]. It has been demonstrated that the 61kDa precursor of the 62-kDa protein is synthesized in the cytoplasm, and antibody staining demonstrated that the nuclear pore glycoprotein undergoes dispersion in the cytoplasm during mitosis. Recently, Schindler et al. [163] suggested that 0-GlcNAc moieties are to protect the proteins from proteolysis, or serve as targeting signals for nuclear proteins, while other studies suggested their involvement in nuclear transport processes [180,181], such as ATP-dependent RNA transport. One potentially significant finding is that the group of proteins which constitute a particular RNase polymerase II transcription factor appear to contain 0-GlcNAc residues. For example, the proteins labeled by galactosylation of AP-1 family appeared to be c-Jun, c-Fos, and Fos-related antigen proteins [171]. From available data, it may be concluded with certainty that there are several types of 0-glycosidic glycoproteins present in the cytoplasm and nucleoplasm. However, the site of their synthesis and their transport are unknown. They could be synthesized in the luminal spaces of the ER and Golgi, similar to conventional (9-linked mucus glycoproteins, or are synthesized on free ribosomes and then glycosylated in the cytoplasm or nucleoplasm [170]. Demonstration of several glycosyltransferase activities in these subcellular spaces indicates that such a biosynthetic pathway is possible for these glycoproteins [170]. As with synthesis and processing, the role of glycosyl residues in these proteins has yet to be determined. Targeting of the proteins to specific intracellular spaces, nuclear transport or role in transcription are the possible functions for these 0-glycosidic glycoproteins. Further, careful characterization of these protein— oligosaccharide structures and patterns should be the first step

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necessary to address the issue of the synthesis, transport, and function of these novel glycoconjugates. Xm. CONCLUDING REMARKS The challenge towards understanding the role of carbohydrate groups in biological recognition is in identifying the potential information coded in the glycosylation patterns and in the recognition of the cellular factors which dictate the particular sequence of events. The A^-linked carbohydrate groups in most of the glycoproteins seem to have no apparent effect on the biological activity since the mutant cells deficient in A^-linked sugar chains grow normally. However, the cell lines deficient of UDPglucose 4-epimerase, express glycoprotein which is rapidly degraded, demonstrating that 0-linked carbohydrate chains are of crucial importance for the membrane glycoprotein stability. The consequences of such deficiency in epithelial cells producing mucus glycoprotein have not been investigated extensively and, thus, it remains to be seen whether the initial (9-glycosylation with A^-acetylgalactosamine is the ultimate signal for the endoplasmic transit and delivery for further processing in the Golgi. Another uncharted area of mucus glycoprotein research lies in unravelling the potential information stored in the apopeptide sequence and its relevance to the mucus glycoprotein glycosylation pattern. Obviously, further developments in this area depend on the successful detailing of structural features of apomucin itself, which, at this point, is immensely complicated by researchers promoting the existence of the enigmatic "link protein" or perhaps proteins which recently were characterized as segments of apomucin undergoing extensive A^-glycosylation. The sequence analyses of the fibronectin-free mucin are as desirable as reappraisal of the specificity of the antibodies which were intended to recognize pure mucin but instead actually recognized the fibronectin. Finally, the task of understanding the glycan organization along the polypeptide core remains to be addressed. Despite extensive information on the oligosaccharide sequences, the

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methods available for oligosaccharide release are indiscriminate. If only this could be overcome we could learn whether organization of the glycans is controlled, or reflects dynamic status of the cell and availability of the substrate. If, indeed, these factors and extracellular stimuli are pertinent, it might be that these glycosylated patterns reflect an essential response to challenges of the environment. Perhaps such responsiveness is necessary to accommodate, serve, and create the cell ligands for blood group and tumor-associated antibodies, toxins, bacterial aggregation and attachment, mycoplasma, animal viruses, lectins, and other still unknown agents. ACKNOWLEDGMENTS This work was supported by USPHS Grants from National Institute on Alcohol Abuse and Alcoholism #AA05858, National Heart, Lung and Blood Institute #HL32553, Institute of Diabetes and Digestive and Kidney Diseases #DK21684, and from the National Institute of Dental Research #DE05666.

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[167] Holt, G.D., Snow, CM., Senior, A., Haltiwanger, R.C., Gerace, L. and Hart, G.W, J. Cell Biol., 104 (1987) 1157-1164. [168] Holt, G.D. and Hart, G.W., J. Biol. Chem., 261 (1986) 8049-8057. [169] Holt, G.D., Haltiwanger, R.S., Torres, C.R. and Hart, G.W., J. Biol. Chem., 262 (1987) 14847-14850. [170] Hart, G.W., Haltiwanger, R.S., Holt, G.D. and Kelly, W.G., Ann. Rev. Biochem., 58 (1989)841-874. [171] Jackson, S.R and Tjian, R., Cell, 55 (1988) 125-133. [172] Ripellino, J.A., Bailo, M., Margolis, R.V. and Margolis, R.K., J. Cell Biol, 106 (1988) 845-855. [173] Rodriguez, I. and Fleisler, S., Arch. Biochem. Biophys., 260, (1988) 628-637. [174] Pitcher, J., Smythe, C, Campbell, D.G. and Cohen, R, Eur. J. Biochem., 169 (1987)497-502. [175] Srisomsap, C, Richardson, K.L., Jap, J.C. and Marchase, R.B., J. Biol. Chem., 263(1988)17792-17797. [176] Rogge, H., Neises, M., Passow, H., Grunz, H. and Rise, H.J., In Neubet, D. and Meeker, H.J. (eds.). New Approaches to the Evaluation of Abnormal Embryonic Development, Stuttgart, Thieme, 1975, pp. 772-791. [177] Shaper, N.L., Hollis, G.F., Douglas, J.G., Kirsch, I.R., Shaper, J.H., J. Biol. Chem., 263 (1988) 10420-10428. [178] Davis, L.I. and Blobel, G., Proc. Natl. Acad. Sci. USA, 84 (1987) 7552-7556. [179] Torres, C.R. and Hart, G.W., J. Biol. Chem., 259 (1984) 3308-3317. [180] Dingwall, C. and Laskey, R.A., Ann. Rev. Cell Biol., 2 (1986) 367-390. [181] Newport, J.W. and Forbes, D.J., Ann. Rev. Biochem., 56 (1987) 535-565.


INDEX

Aeromonas hydrophila, 0-antigen, 159 Aeromonas salmonicida, 0-antigen, 159 AGP, see a i-acid glycoprotein AIR, see Alcohol insoluble residues Alcohol insoluble residues, 8 Antibodies anti-polysaccharide, 98, 120, 131, 151, 154, 163 apomucin, 179 fibronectin, 181 Antigen artificial, 96, 105, 144 blood group A, 72, 78, 144, 149, 151 blood group B, 72 blood group H, 72, 78, 144, 151 Lewis A, 72, 144 Lewis X, 72 sialyl Lewis A, 72 sialyl Lewis X, 72, 73 Antigens, blood group specific, 145 Apomucin synthesis, 179—184 Arabinanases

classification, 20-33, 42-52, 53 ^«(i(9-(l—>5)-a-L-, 21 action on branched arabinan, 33 amino acid sequences, 52 assays, 36-40, 54-55 catalytic mechanisms, 52, 54 cooperative action with galactanase, 36 isolation, 31 physicochemical properties, 47, 48 properties, 32 reaction products, 39, 40 purification, assay methods, 36-40, 54^55 Arabinanase, ejco-a-L-, 28 Arabinans characterization, 6, 11—20 chemical synthesis, 10 composition, 3, 11 computer modeling, 42 cooperative effects during enzymic degradation, 33-36 debranched, 41 213

214

enzymic degradation, 20-36 hydrolysis by a-L-arabinofuranosidase, 20 isolation, 5, 8 methylation analysis, 12, 19 NMR spectroscopy, 17, 42, 50 pectic, 10, 15 periodate oxidation, 11 purification, 9 X-ray crystallographic analysis, 18, 4 1 ^ 2 Arabinofuranohydrolase, a-L-, (l->4)-P-D-arabinoxylan, isolation, 27 Arabinofuranohydrolase, properties, 28, 41-49 Arabinofuranosidase A, a-Laction on arabinogalactan, 35 action on arabinoxylan, 35 action on branched arabinan, 33 assay, 37 definition, 22 physicochemical properties, 43 properties, 23 purification, 37 Arabinofuranosidase, a-L-, 21 Arabinofuranosidase, B, a-Laction on arabinogalactan, 35, 45 action on arabinoxylan, 35 a-L-, action on branched arabinan, 33 a-L-, assay, 37 a-L-, definition, 22 identification of hydrolysis products, 38 physicochemical properties, 45

INDEX

properties, 25 purification, 37 Arabinogalactan structural types, 5 substrate for a-L-arabinofuranosidases, 20, 21, 26, 35, 47 Arabinopyranosidase, P-L-, substrate specificity, 30 Arabinoxylan structure, 5 substrate for arabinofuranosidases, 21, 26, 35 Arafur A, see Arabinofuranoside A, a-LArafur B, see Arabinofuranosidase B, a-LArthritis, rheumatoid, 73, 87 AXH, see Arabinofuranohydrolase, a-L-, (l-»4)-P-D-Arabinoxylan B lymphocytes, 201 Carbohydrate determinants, 143 Chlamydia, capsular polysaccharide, 145 Chromatography glycopeptide, 74 high performance liquid, 38, 73-89 high pH anion-exchange, 75-89 applications, 87-89 separation of monosaccharides, 75-78

Index

separation of oligosaccharides, 80-86 separation of sialic acids, 78-80 ion-suppression amine absorption, 75 applications, 87 separation of oligosaccharides, 80, 82, 84, 86, 88 pulsed amperometric detection, monosaccharide analysis, 76 A^-linked oligosaccharides, 73, 75 CMP-Neu 5Ac, see Cytidine monophosphate-neuramin ic acid Conjugates, polysaccharide-protein, 153 Cytidine monophosphateneuraminic acid, 193

3-Deoxy-a-D-mannooctulosonic acid, 159 Detection, pulsed amperometric, 76

EGFR, see Epidermal growth factor receptor Endoplasmic reticulum apomucin-a-A^-acetylgalactosam inyltransferase, 187 DKEL sequence, 192 Golgi-plasma membranes, 188 lumen, 184, 187, 191, 192, 202

215

A^-acetylgalactosaminyltransferase, 191 protein transport, 189 transport of sugar nucleotides, 193 Enzymes, multifunctional, 50 Epidermal growth factor receptor, 71, 73, 77, 80, 85, 86 Epidermaphyton floccosum, galactomannan, 155 ER, see, Endoplasmic reticulum Escherichia coli, Kdo region, 144

Fetuin, 71, 72, 82, 83 Fibronectin, 181 Fucose, L-, 69, 77, 78, 80, 83

Galactanase, endo-, 36 Galactomannan, 155 Glycan-peptide hydrolase, 105 Glycanases, phage, 103, 131 Glycoconjugates with oligosaccharide moieties, 98—152 principles of design, 95—98 Glycoforms, 71—73 Glycolipids, 129, 144, 200 Glycoprotein P-elimination of 0-glycosidic linkage, 101 a i - acid, 71, 72, 73, 87 alkaline acetolysis, 102 enzymic degradation, 103 hydrolysis of A^-glycosylamide bond, 102

216

monosaccharide composition, 75, 77 Glycoproteins, exoglycosidase digestion, 74 Glycosidase, cytoplasmic, 201 Glycosidase D, endo-, 82 Glycosidase F, endo-, 82 Glycosidase F, N-, 82, 86, 105 Glycosidase H, endo-, 82 Glycosidase, nucleoplasmic, 201 Glycosphingolipids, 129, 200 Glycosylated variants, see Glycoforms Glycosylation, A^-linked antennae, 68, 78 biantennary chains, 69, 72, 73, 77, 83, 86 biosynthesis, 69, 190 chromatography, 73 classes, 69 complex A^-glycan, 69, 73 high mannose A^-glycan, 69 hybrid A^-glycan, 69 complex A^-glycans, 69, 73 consensus sequence, 67 heterogeneity, 69, 71 high mannose glycans, 69 hybrid A^-glycans, 69 oligosaccharide sequence, 68-71 pentaantennary chains, 69 pentasaccharide core, 68 sialylated chains, 70, 75, 82 sites, 67 tetraantennary chains, 69 triantennary chains, 69, 72, 77, 83 Glycosylation, 0-, apopeptide: GalNac transferase, 191, 192

INDEX

assembly of chains, 198-201 cytoplasmic proteins, 201-203 linkage to serine and threonine, 191, 198 nucleoplasmic, 201-203 processing, 185 with 7V-acetylgalactosamine, 184, 185, 198, 203 with A^-acetylglucosamine, 201, 202 Glycosylation, peptide-directed attachment, 190-192 Glycosylation, regulation, 194 Glycosyltransferase P-3-A^-acetylglucosaminyltransferase, 198 P-6-7V-acetylglucosaminyltransferase, 200 apomucin-A^-acetylgalactosaminyltransferase, 187, 191, 192, 195 cytoplasmic, 201 expression, 195 P-3-galactosyltransferase, 198 P-4-galactosyltransferase, 197 genes, 194, 195 levels of soluble enzyme, 198 localization, 192, 202 nucleoplasmic, 201 role of structure, 195-198 a-6-sialyltransferase, 197, 198 Golgi, glycosyltransferases, 192, 193, 194, 197 lumen, 193, 197, 202 protein transport, 189 transport of sugar nucleotides, 193

Index

Hapten, see Carbohydrate determinants Hemophilus influenza, 156 Hormones, pituitary, 190 HPAEC, see Chromatography, high pH anion-exchange Hydrolases, lysosomal, 70, 190 ISAA-HPLC, see Chromatography, ion-suppression amine absorption Kdo, see 3-Deoxy-a-D-mannooctulosonic acid Lactosamine, 72, 83 Lipopolysaccharide, 103, 104, 144, 158, 159 LPS see Lipopolysaccharide Lyase, pectate, endo-, 4 pectin, endo-, 4 Mannan, D-, synthetic, 163 Mucin, bovine submaxillary, 179 canine tracheobronchial, 181 fibronectin-DNA complexes, 181 genes, 179 human gastric, 179 human intestinal, 179, 181 oligosaccharide chain assembly, 198-201 oligosaccharide chain initiation, 186 ovine submaxillary, 179, 198

217

rat gastric, 179 rat intestinal, 179 rectal, 181 synthesis, 186, 188 Mycobacterium leprae, glycolipid, 144 Neoglycoconjugates, artificial antigen, 96, 105, 110, 129, 130, 143, 147, 164 conjugation of oligosaccharides, 107, 112-115 containing fatty acid residues, 149 containing natural polysaccharides, 153-160 containing synthetic polysaccharides, 161-164 copolymer type, 143 immunological properties, 102, 130, 151 preparation of carbohydrate carriers, 98 preparation of protein carriers, 106 preparation with synthetic polymer carriers, 132 synthesis from allyl glycosides, 137 synthesis from allyloxyalkyl glycosides, 137 synthesis of oligosaccharides, 105 with polysaccharide moieties, 152—164 Neoglycolipid, 129 Neoglycoprotein, 156-163

218

Neuraminic acid, TV-acetyl, see sialic acid Nucleotide transport system, 193 Oligosaccharide, conversion to glycosylamines, 121 derivitization, 116-121, 141 from alkaline acetolysis, 102 from chemical degradation of polysaccharides, 99 from P-elimination of 0-glycan chains, 101 from enzymic degradation, 103-105 from hydrolysis of A^-glycosylamide bonds, 102 from Smith degradation of polysaccharides, 100 synthesis, 105, 121 as glycosides with aglycone spacer, 121 role of nucleotide transport system, 193 Oligosaccharides conjugation with protein carriers, 107—115 human milk, 144 isomeric, 75 A^-linked, antennae, 68, 69 biosynthesis, 69, 190 chromatography, 73 classes, 69 complex A^-glycan, 69, 73 high mannose A^-glycan, 69 hybrid A^-glycan, 69 heterogeneity, 69, 71 pentasaccharide core, 68 sialylated, 70, 74, 75

INDEX

Ovalbumin, 70 Ovotransferrin, 70 PAPS, see 3' Phosphoadenosine 5' phosphosulphate Pectin lyase, endo-, 4 Pectin, attached arabinan, 4 degradation of, 4 rhamnogalacturonan backbone, 3, 4 3 '-Phosphoadenosine 5'-phosphosulphate, 193 PNGase F see Glycosidase F, Polyacrylamide, immunological properties, 151 modification of conjugates, 149 oligosaccharide conjugates, 136, 138 Polygalacturonase, endo-, 4 Polysaccharide, vaccines, 98 Polysaccharides, activation with bromoacetyl bromide, 154 activation with chloroacetaldehyde dimethylacetyl, 157 activation with cyanuric chloride, 155 activation with sodium periodate, 157 capsular, 99, 124, 144, 153, 155, 161, 162 chemical degradation, 99 enzymic degradation, 103-105

Index

for synthesis of neoglycoconjugates, 153 immunodominant sugars, 98 meningococcal, 158 0-antigens, 97, 103, 153, 154, 159, 161 repeating units, 97 synthesis of homo- and heteropolysaccharides, 161-163 Pseudomonas aeruginosa, synthetic antigen, 163 Pseudopolysaccharides, 133

RER, see Rough endoplasmic reticulum Rhamnogalacturonan, 3, 4 Rhamnosidase, endo-a-, 103, 131 Rough endoplasmic reticulum, 184-189 luminal site, 184-191 membranes, 187 O-glycosylation, 184, 185, 187 peptide folding, 192 processing, 184-189

Salmonella illinois, 0-antigen, 119 Salmonella minnesota, Kdo region, 144 Salmonella newington, Oantigen, 162 Salmonella typhimurium, Oantigen, 131, 154 Salmonella lipopolysaccharide, 104, 144

219 Salmonella, 0-antigen, 103, 130, 136, 143, 151 Salmonella, serogroups, 144 Shigella flexneri, 0-antigen, 162 Sialic acid, 66, 74, 77, 78-80, 138, 158 Sialyllactosamine, 70 Spectroscopy, ^^C NMR, 11, 17, 42, 50, 138, 139, 147 Streptococcus pneumoniae, capsular polysaccharides, 99, 124, 143, 145, 162 Streptococcus, A variant, 163 UDP-Gal, see Uridine diphosphate-galactose UDP-GalNAc, see Uridine diphosphate-A^-acetylgalactosamine UDP-GlcNAc, see Uridine diphosphate-A/^-acetylglucosamine Uridine diphosphate-A^-acetylgalactosamine, 193 Uridine diphosphate-Aâcetylglucosamine, 193 Uridine diphosphate-galactose, 193 Uridine diphosphate-glucose 4-epimerase, 203 Vibrio anguillarium, lipopolysaccharide, 158 Xylanase arabinose-releasing, 50 endo-, 35

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