glycosciences status and perspectives (chapman&hall, 2002)

Hans-Joachim Gabius and Sigrun Gabius (Editors)

Glycosciences

Status and Perspectives

WILEY-VCH Verlag GmbH & Co. KGaA

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Hans-Joachim Gabius and Sigrun Gabius (Eds.)

Glycosciences

Hans-Joachim Gabius and Sigrun Gabius (Editors)

Glycosciences

Status and Perspectives

WILEY-VCH Verlag GmbH & Co. KGaA

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Foreword

Intuition has started to ascribe the potential of a versatile, but elusive code system to the at first sight puzzling array of sugar structures on proteins and lipids, as underscored by a quotation from the preface to an authoritative symposium nearly two decades ago: it states that many knowledge- able biologists would say, almost reflexly, that complex carbohydrates probably play a pivotal role in determining the specificity of many biological recognition pheno~nena (Marchesi et al., 1978). In the following years, the necessity to address comprehensively the enigmatic issue that the oligosaccharide chains of cellular glycoconjugates continued to be referred to as molecules in search of a function has been repetitively emphasized (Cook, 1986). The last decade has unmistakably witnessed marked upheavals in our understanding, with a dynamic development which can legitimately be called a permanent revolution. Technical advances in oligosaccharide synthesis, purification and structural analysis, together with the steadily growing knowledge about endogenous binding partners such as lectins, have enabled research activities in the different areas of glycosciences to exert an obvious influence on various disciplines in basic and applied sciences, as depicted in the figure. Due to the amazingly rapid flow of information, the impending danger that guiding concepts and ideas might easily be submerged should not be carelessly ignored. This line of reasoning pro- vided the impetus for us to contact colleagues from various branches of glycosciences and kindly confront them with our request to prepare a special chapter for this collection. First of all, it was intended to capture and transmit the spirit of jointly shared enthusiasm about our field, simul- taneously familiarizing the interested reader with

the status of each selected area based on an authoritative, though not necessarily encyclo- pedic appraisal of the evidence. Conceptual recognition is the essential step for one to be able to follow subsequently the inevitably subjective descriptions of the perspective with an appreci- ation of the inherently interdisciplinary character of glycosciences. Having been wholeheartedly delighted with the responses, we now have the pleasure of inviting the readers to wander with each author to the edges of the frontiers of our knowledge in glycosciences, to reflect on the cur- rent status and then to feel encouraged to go con- fidently beyond these frontiers. If turning perspective into status is stimulated by the con- tributions to this book, authors, publisher and editors will definitely have reached their goal.

References

Cook GMW (1986): Cell surface carbohydrates: molecules in search of a function? In J. Cell Sci. Suppl.

Marchesi VT, Ginsburg V, Robbins PW et al. (1978): Preface. In Cell Surface Carbohydrates and Bio- logical Recognition. Progr. Clin. Biol. Res. 23 (Mar- chesi VT, Ginsburg V, Robbins PW et al., eds) p 13, New York: Alan R. Liss.

4~45-70.

~~~

H.-J. and S. Gabius (Eds.), Glycosciences 0 Chapman & Hall, Weinheim, 1997 ISBN 3-8261-0073-5

Preface FRIEDRICH CRAMER: A decade of glycosciences

My first paper was published in 1948 and it was one in the field of glycosciences (Freudenberg and Cramer, 1948). Thus glycosciences opened for me the happy roads to science. It is therefore a great satisfaction for me that the editors have asked me to write this preface to their splendid book on GLYCOSCIENCES, in which so many highly important and relevant topics are treated by the most competent authors. That first paper of mine solved the structure and explained the properties of a-, p-, and y-cyclodextrin. From then on I was able to develop the story of inclusion compounds and supramolecular chemistry (Cramer, 1952, 1954, 1955; Cramer et al., 1967). Already in this first paper three facts had become apparent.

i: Carbohydrates are capable of forming hydrogen bonds. This fact is obvious, since there are so many hydroxyl groups, which normally make sugars hydrophilic molecules. However, this hydrogen bonding can also be used for internal stabilization of structure and for the interaction with other molecules, e.g. with proteins. This has now been shown in studies of sugar-binding proteins.

ii: Carbohydrates have a fairly rigid structure. For many years this was an assumption, but has now been proved by NMR studies in various ways. In oligosaccharides, as well as in complex carbohydrates, there are a number of internal hydrogen bonds which stabilize certain conformations to a surprising extent. There are no a- helices or p-pleated sheets as in proteins. Never- theless, complex carbohydrates can be recognized by sugar-binding molecules such as lectins in an unambiguous way. Complex carbohydrate structures on cell surfaces very often play an important role as antigens, whether in bacterial or eukaryotic cells.

iii: Complex carbohydrates may have hydro- phobic pockets. This became quite obvious in cyclodextrins (Freudenberg and Cramer, 1948), but it may also play an important role in a variety of carbohydrate interactions. It might well be that certain transport functions, lipid interactions and associated phenomena make use of this partial hydrophobicity of complex carbohydrates.

Chemistry and biochemistry of complex carbohydrates and glycosciences in general have been a neglected field until perhaps one decade ago. This does not mean that this part of science was not highly successful, but that it was a rather heroic area in many respects. Chemistry was just not sufficiently advanced to take up such complex problems. Without the modern techniques of natural product isolation, without NMR, without mass spectroscopy, today one can hardly imagine any success in this field. And yet the basic princi- ples and the basic structures had been determined a long time ago. I still remember when, during my early times in research, some colleagues next door worked on the characterization of blood group-specific substances in the lab of Karl Freudenberg. This was 45 years ago and it was indeed an heroic effort. Thus, it seems hardly surprising that only 12 years ago in a leading text- book one could read the following sentences: The function of the oligosaccharide side chains in membrane glycolipids and glycoproteins is unclear. It is possible that those in certain transmembrane proteins help to anchor and orient the proteins in the membrane by preventing them from slipping into the cytosol or from tumbling across the bilayer. The carbohydrate also may play a role in stabilizing the folded structure of a glycoprotein. In addition carbohydrate may play a role in guiding a membrane glycoprotein to its appro-


VIII I Preface priate destination in or on these cells, just as special carbohydrate chains on lysosomal enzymes direct these soluble glycoproteins to lysosomes. However, these cannot be the only function of carbohydrate in membrane glycoproteins.. . More- over, functions such as orienting, anchoring, stabilizing and targeting can not account for the carbohydrate in glycolipid molecules nor for the complexity of some of the carbohydrate chains in glycoproteins (Alberts et al., 1983).

Glycoproteins are an essential part of the cellular membrane, and I consider the cellular membrane as a multipurpose organelle. It is not simply a sacculus, which separates the inside from the outside. It has an enormous number of functions, it possesses gates, channels, receptors, it provides

specific contacts, it is important in cell aggrega- tions and dissociations, it exposes signals and can receive signals, it is responsible for the ordered and regular embryonic growth, and it enables the cell to make highly specific contacts such as syn- aptic connections in CNS. Most likely all these capacities of the cell reside in the structure and arrangement of the complex carbohydrates on the surface of the cell. Fig. 1 gives a schematic picture of the situation, in which lipids, proteins and complex carbohydrates cooperate through a three-dimensional interaction, the details of which are indeed still largely unknown (from Alberts et al., 1983).

Indeed, one must ask the question: what are these complex carbohydrates good for? The facts

Figure 1. A diagram of the cell code (Glycocalyx) which is made up of the oligosaccharide side chains of glycolipids and integral membrane glycoproteins, and polysaccharide chains of integral proteoglycans. In addition, glycoproteins and adsorbed proteoglycans contribute to the glycocalyx in some cells (Alberts et al . , 1983)

References 1 IX that these molecules are synthesized through the action of an enormous number of different and highly specific enzymes, that these enzymes are under strict genetic control, and that the complex carbohydrates are produced with very high precision, all this may well indicate that these glyco- structures play a very important and indispen- sable role in cellular events, and that they are not just there for solubilization purposes. Are they important for cell adhesion and cell recognition? If this were the case, there should exist specific receptors for complex carbohydrates, which would allow for such specific recognitions.

About 10 years ago we started the search for such sugar-recognizing molecules (Cramer and Gabius, 1985) and, indeed, found such carbohydrate receptors in many mammalian cells. Carbohydrate-recognizing molecules were known for a very long time in the form of plant lectins. However, the general opinion amongst the scientific community was that such lectins were restricted to the plant kingdom, and nobody really looked into the tissue of higher organisms. As several chapters in this book elegantly describe, tools for lectin detection and their application have enriched our view in this area profoundly. We are now even at a stage where functions can be understood at least in part, as will be discussed in the present volume.

I consider it a great merit of the editors to give - as far as I can see for the first time - a complete overview of modern glycosciences. The topics range from chemical analysis and chemical synthesis through structural determinations to the various kinds of carbohydrate interactions, which have become so important during the last years, and go right into biochemical topics like the glycobiology of host defense mechanisms, glycobiology of signal transduction, or glycobiology of development and fertilization. It is obvious that such a topic also touches problems of medical importance such as mechanisms of infection, tumor biology and histopathology. I believe that after a decade of intensive research in glycosciences, the effort of the editors will be greatly appreciated by the biochemical, biological and medical scientific community.

References

Alberts B, Brays D , Lewis J et al. (1983): Molecular Biology of the Cell. p 285, New York: Garland Publ. Inc. dto., p 284 (with kind permission).

Cramer F (1952): EinschluSverbindungen. In Angew. Chem. 64:437-47.

Cramer F (1954): EinschluSverbindungen. Heidelberg: Springer Verlag. Russian translation, Moskau 1957.

Cramer F (1955): Inclusion compounds. In Pure Appl. Chem. 5: 143-64.

Cramer F, Gabius H-J (1985): New carbohydrate- binding proteins (lectins) in human cancer cells and their possible role in cell differentiation and metasta- sis. In Interrelationship among Ageing, Cancer and Differentiation (Jerusalem Symp. on Quantum Chemistry and Biochemistry, Vol. 18) Pullmann B, TSO POP, Schneider EL (eds) pp 187-205, Dord- recht: Reidel.

Cramer F, Saenger W, Spatz HC (1967): Inclusion compounds. XIX.: The formation of inclusion compounds of a-cyclodextrins in aqueous solutions. Thermodynamics and kinetics. In J. Am. Chem. SOC.

Freudenberg K, Cramer F (1948): Die Konstitution der Schardinger-Dextrine a, p und y. In Z . Naturforsch.

89: 14-20.

3bA64-74.

Chapter 1 The Information-Storing Potential of the Sugar Code ROGERA.LAINE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2

2.1 3 3.1 3.2 4 5 6 7

8

9

Introduction . . . . . . . . . . . . . . . . . 1 10 Details of Calculation for Isomers of 11 a Trisaccharide . . . . . . . . . . . . . . . 2 Non-Reducing Oligosaccharides . . . . 3 12 Analysis . . . . . . . . . . . . . . . . . . . 3 13 NMR . . . . . . . . . . . . . . . . . . . . . 3 14 Mass Spectrometry . . . . . . . . . . . . 4 15 Synthesis . . . . . . . . . . . . . . . . . . . 4 16 Biologically Relevant Oligomer Size . Substitutions . . . . . . . . . . . . . . . . 4 16.1 Oligosaccharide Recognition by 16.2 Proteins . . . . . . . . . . . . . . . . . . . . 4 16.3 Evolution of Carbohydrate Code 17 Structures and Receptors . . . . . . . . 5 18 A High Level Biological Code . . .

4

6

Lectins in Biological Recognition . . . Biological Mechanisms of Lectins or other Carbohydrate-Binding Proteins Multivalent Effects . . . . . . . . . . . . BacteriaVEucaryotic Interactions . . .

G1 ycobiology . . . . . . . . . . . . . . . . Review of the Formal Calculation of

Pharmaceutical Development . . . . .

Oligosaccharide Isomers . . . . . . . . . Linear Oligosaccharides . . . . . . . . .

Oligosaccharide Building Blocks . . . . Summary . . . . . . . . . . . . . . . . . . .

Branched Oligosaccharides . . . . . . . Formulas . . . . . . . . . . . . . . . . . . .

Chapter 2 Methods of Glycoconjugate Analysis ELIZABETH F . HOUNSELL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 Introduction . . . . . . . . . . . . . . . . . 15 3 Biophysical Methods . . . . . . . . . . . 1.1 Monosaccharide Identification . . . . . 15 3.1 Mass Spectrometry . . . . . . . . . . . . 1.2 Chemical Reactions . . . . . . . . . . . . 18 3.2 NMR Spectroscopy . . . . . . . . . . . . 2 Biochemical Reagents . . . . . . . . . . 18 4 Glycoprotein Analysis . . . . . . . . . . 2.1 Lectin Affinity Interactions . . . . . . . 19 4.1 Proteases and Endoglycosidases . . . .

Digestion . . . . . . . . . . . . . . . . . . . 20 4.3 Capillary Electrophoresis . . . . . . . . 2.2 Sequential Exoglycosidase 4.2 HPLC Purification and Analysis . . . .

Chapter 3 Strategies for the Chemical Synthesis of Glycoconjugates RICHARD R . SCHMIDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

6

8 8 9

10 11 11

15

21 21 22 24 24 25 25

31

1 Introduction . . . . . . . . . . . . . . . . . 31 2.1 Typical Building Blocks for Glycoside 2 General Strategies . . . . . . . . . . . . . 31 Synthesis . . . . . . . . . . . . . . . . . . . 31

H.-J. andS . Gabius (Eds.), Glycosciences 0 Chapman & Hall, Weinheim. 1997 ISBN 3-8261-0073-5

XII 1 Contents 2.2 Methodologies in Enzymatic and in 3.4

Chemical Glycoside Bond Forma- 3.5 tion - a Comparison . . . . . . . . . . . . 32 4

2.3 General Aspects of Chemical Glycoside Bond Formation . . . . . . . 33

3 The Koenigs-Knorr Procedure and its Variations . . . . . . . . . . . . . . . . . . 34 4.1

3.1 The Koenigs-Knorr Procedure . . . . . 34 4.2 3.2 Fluoride as Leaving Group . . . . . . . 36 4.3 3.3 1, 2.Anhydro (1, 2.Epoxide) Sugars as

Glycosyl Donors . . . . . . . . . . . . . . 37 5

Sulfur as Leaving Group . . . . . . . . . Nitrogen as Leaving Group . . . . . . . The Trichloroacetimidate Method. the Phosphite Method. and Other Anomeric Oxygen Activation Procedures . . . . . . . . . . . . . . . . . . The Trichloroacetimidate Method . . . The Phosphite Method . . . . . . . . . . Other Anomeric Oxygen Activation Procedures . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . .

Chapter 4 Neoglycoconjugates REIKO T. LEE AND YUAN C . LEE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 2.1 2.2 2.3 2.4

2.5 3 3.1

3.2

3.3

3.4 3.5

3.6 4 4.1

Introduction . . . . . . . . . . . . . . . . . 55 4.2 Advantages of Neoglycoconjugates . . 55 Well-Defined Structure . . . . . . . . . . 55 5 Make as Much as Needed . . . . . . . . 56 Glycoside Cluster Effect . . . . . . . . . 56 6

Analogs . . . . . . . . . . . . . . . . . . . . 56 8 Changing Personalities . . . . . . . . . . 57 Neoglycoproteins . . . . . . . . . . . . . 58 8.1 Side Chains of Proteins Useful in 8.2 Modification . . . . . . . . . . . . . . . . . 58 8.3 Modification of Primary Amino Groups. . . . . . . . . . . . . . . . . . . . 58 9 Conjugation of Polysaccharides to 9.1 Proteins . . . . . . . . . . . . . . . . . . . . 60 Use of Enzymes . . . . . . . . . . . . . . 61 9.2 Glycoproteins of Non-Covalent Attachment . . . . . . . . . . . . . . . . . 63 9.3 Synthetic Glycopeptides . . . . . . . . . 63 9.4 Neoglycolipids . . . . . . . . . . . . . . . 64 9.5

Chromatographic Separation . . . . . . 64

Neoglycoconjugates Containing 7

Neoglycolipids for Thin Layer 10

Transglycosylation of Ceramide Glycanase . . . . . . . . . . . . . . . . . . Glycolipids into Neoglycoproteins and vice versa . . . . . . . . . . . . . . . . Neoproteoglycans . . . . . . . . . . . . . Neogl ycopolymers . . . . . . . . . . . . . Derivatives Useful for Preparation of Neoglycoconjugates . . . . . . . . . . . . G1 ycosides . . . . . . . . . . . . . . . . . . Glycosylamines and Glycamines . . . . Attachment of Glycopeptides with a Heterobifunctional Reagent . . . . . . . Application of Neoglycoconjugates . . Probing Carbohydrate-Protein Interactions . . . . . . . . . . . . . . . . . Use in Isolation of Carbohydrate- Binding Proteins . . . . . . . . . . . . . . Cytochemical Markers . . . . . . . . . . Neoglycoenzymes . . . . . . . . . . . . . Biomedical Applications . . . . . . . . . Conclusions and Perspectives . . . . . .

5 Glycosyltransferases Involved in N- and O-Glycan Biosynthesis INKA BROCKHAUSEN AND HARRY SCHACHTER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 Introduction . . . . . . . . . . . . . . . . . 79 3.1 Galactosyltransferases . . . . . . . . . 2 The Roads which Lead to Complex 3.2 Sialyltransferases . . . . . . . . . . . . .

N-Glycans . . . . . . . . . . . . . . . . . . 80 3.3 N-Acetylglucosaminyltransferases . . 3 Glycosyltransferases Involved in the 3.4 Fucosyltransferases . . . . . . . . . . .

Synthesis of Complex N-Glycan 4 Biosynthesis of 0.Glycans . . . . . . . Antennae . . . . . . . . . . . . . . . . . . 81

37 41

41 41 48

50 51

55

65

65 65 66

68 68 70

70 71

71

72 72 73 73 73

79

81 84 89 93 96

contents 1 XIII 4.1 Initiation of O-Glycan Biosynthesis. 4.2 Synthesis of O-Glycan Core

UDP-GalNAc: Polypeptide a1,3-N- Acet y lgalactosaminyltransferase (polypeptide GalNAc T; 4.4 Termination Reactions in the E.C. 2.4.1.41). . . . . . . . . . . . . . . . 97

Structures . . . . . . . . . . . . . . . . . . 97 Elongation of O-Glycans . . . . . . . . 98 Synthesis of O-Glycans . . . . . . . . . . 99 Conclusion. . . . . . . . . . . . . . . . . . 100

4.3

5

Chapter 6 Topology of Glycosylation - a Histochemists View MARGIT PAVELKA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . 115 1 2 3

Introduction. . . . . . . . . . . . . . . . . 115 Topology of N-Glycosylation . . . . . . 115 Topology of O-Glycosylation . . . . . . 118

4 Summarizing Remarks and Perspectives . . . . . . . . . . . . . . . . . 118

Chapter 7 Occurrence and Potential Functions of N-Glycanases TADASHI SUZUKI ETAL.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 1 2

3

4 4.1

4.2

Introduction. . . . . . . . . . . . . . . . . 121 N-GlycosylatiodDe-N-glycosylation. . . . . . . . . . . . . . . . . . . . 121 Occurrence of PNGases: The Discovery of Animal PNGases . . 122 PNGases inFish . . . . . . . . . . . . . . 123 Acid PNGase in Fish Oocyte is Responsible for the Detachment of

Glycophosphoproteins . . . . . . . . . . 123

PNGase during Embryogenesis . . . . 123

5

6

7

N-Glycan Chains from 8

N-Glycan Release by Alkaline 9

PNGase in Mouse-Derived Cultured Cells: L-929 PNGase . . . . . . . . . . . 124 Neutral, Soluble PNGases are Widely Distributed in Mouse Organs. . . . . . . . . . . . . . . . . . . . 125 Possible Biological Function of Neutral PNGase in Non-Lysosomal Degradation. . . . . . . . . . . . . . . . . 126 Plant PNGases: Possible Regulator Molecules in Cellular Processes . . . . 127 Concluding Remarks . . . . . . . . . . . 128

Chapter 8 Glycoproteins: Structure and Function NATHAN SHARON AND HALINA LIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 1 Modulation of Physicochemical 2 Properties . . . . . . . . . . . . . . . . . . 147 2.1 Modulation of Biological Activity . . . 149 2.2 Carbohydrate-Peptide Linking 3.4 Cellular Immune Functions . . . . . . . 153

Activities of Free Oligosaccharides . . 154 2.3 Carbohydrates as Recognition 3 Determinants . . . . . . . . . , . . . . , . 154 3.1

Introduction . . . . . . . . . . . . . . . . . 133 3.2 Structure.. . . . . . . . . . . . . . . . . . 134 Monosaccharide Constituents . . . . . 135 3.3

Groups . . . . . . . . . . . . , . . . . . . . 137 3.5 Oligo- and Polysaccharides . . . . . . . 139 3.6 Functions. . . . . . . . . . . . . . . . . . . 146 Methodology . . . . . . . . . . . . . . . . 146

XIV I Contents Chapter 9 Glycolipids: Structure and Function JORGEN KOPITZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

1 Structure. Classification and 5.2 Localization of Glycolipids . . . . . . . 163

1.1 Bacterial Glycolipids . . . . . . . . . . . 163 6.1 1.2 Plant Glycolipids . . . . . . . . . . . . . . 164 6.2 1.3 Animal Glycolipids . . . . . . . . . . . . 165 6.3 2 Elucidation of Glycolipid Structure . . 168 3 Physicochemical Properties and 6.4

Organization of Glycolipids . . . . . . . 169 4 Metabolism and Intracellular Traf- 6.5

ficking of Glycolipids . . . . . . . . . . . 169 5 Physiological Functions of

Glycolipids . . . . . . . . . . . . . . . . . . 172 7.1 5.1 Bacterial and Plant Glycolipids . . . . 172 7.2

6

7

Animal Glycolipids . . . . . . . . . . . . 173 Functions in Disease Mechanisms . . . 177 Oncogenic Transformation . . . . . . . . 177 Neurodegeneration . . . . . . . . . . . . 178 Disorders of Glycosphingolipid Catabolism . . . . . . . . . . . . . . . . . . 178 Attachment Sites for Bacteria or their Toxins . . . . . . . . . . . . . . . . . 179 Autoimmune Neuropathies . . . . . . . 179 Diagnostic and Therapeutic Functions . . . . . . . . . . . . . . . . . . . 180 Cancer . . . . . . . . . . . . . . . . . . . . 180 Neurological Disorders . . . . . . . . . . 180

Chapter 10 Lectins as Tools for Glycoconjugate Purification and Characterization RICHARD D . CUMMINGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

1 Introduction . . . . . . . . . . . . . . . . . 191 3.3 2 Structures of Plant

and Animal Lectins . . . . . . . . . . . . 191 3.4 3 Carbohydrate-Binding Specificities

of Lectins . . . . . . . . . . . . . . . . . . 192 4 3.1 Use of Immobilized Lectins in the

Oligosaccharides and Glycoproteins . 193

Affinity Chromatography . . . . . . . . 193

Chromatography-Based Isolation of 5

3.2 Immobilization of Lectins for 6

Elution of Glycoconjugates Bound to an Immobilized Lectin . . . . . . . . 196 Serial Lectin Affinity Chromatography (SLAC) . . . . . . . . 196 Uses of Lectins to Study Phenotypes ofcells . . . . . . . . . . . . . . . . . . . . 197 Uses of Lectins in Solid-Phase Assays for Glycosyltransferases . . . . . . . . . 197 Conclusion . . . . . . . . . . . . . . . . . . 198

Chapter 11 Proteoglycans . Structure and Functions HANS KRESSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

1 2 3 3.1

3.2 4 4.1

Introduction . . . . . . . . . . . . . . . . . 201 4.2 Glycosaminoglycan Structure . . . . . . 201 Hyaluronate . . . . . . . . . . . . . . . . . 202 4.3 Structure. Biosynthesis and Degradation . . . . . . . . . . . . . . 202 4.4 Selected Functions . . . . . . . . . . . . . 203 4.5 Proteoglycans . . . . . . . . . . . . . . . . 204 Glycosaminoglycan Chain 4.6 Assembly . . . . . . . . . . . . . . . . . . . 204 4.7

Large Hyaluronate-Binding Matrix Proteoglycans . . . . . . . . . . . . . . . . 208 Small. Leucine-Rich Extracellular

Basement Membrane Proteoglycans 211 Integral Membrane Heparan Sulfate

Intravesicular Proteoglycans . . . . . . 214

Matrix Proteoglycans . . . . . . . . . . . 209

Proteoglycans . . . . . . . . . . . . . . . . 212

Optional Proteoglycans . . . . . . . . . . 214

Contents I xv Chapter 12 GPI-Anchors: Structure and Functions VOLKER ECKERT ETAL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

1 1.1

1.2

1.3

1.4

1.5

2 2.1

Introduction. . . . . . . . . . . . . . . . . 223 2.2 The Discovery of GPI-Anchors: from an Exotic Way to Anchor a 2.3 Protozoal Surface Antigen to a General Principle among Eukaryotes 2.4 Discovery of Functions beyond the Mere Anchoring of Proteins to a Membrane : the GPI-Anchor as a Molecule with Multiple Functions 3.1 in Cell Biology . . . . . . . . . . . . . . . 224 Molecular Biology : from Function to Genes to Therapy?. . . . . . . . . . . 224 The Structure of GPI-Anchors : A Structure that has been Conserved

Species-Specific and Develop-

Additions to a GeneralTheme . . . . . 225 Structural Analysis. . . . . . . . . . . . . 225 4.1 An Exercise in Biochemistry and Logical Deduction: a Few 4.2

3

3.2 3.3

throughout Eukaryotic Evolution . . . 225

mentally Regulated Modification : 4

Representative Examples . . . . . . . . 225

Identification of GPI-Anchored Proteins. . . . . . . . . . . . . . . . . . . . 226 Structural Characterization of GPI-Anchors . . . . . . . . . . . . . . . . 228 The Biosynthesis of GPI-Anchors: a Conserved Pathway with Side- tracks and Backup Systems . . . . . . . 229 The Function of GPI-Anchors . . . . . 234 Anchoring of Membrane Proteins; Protozoa vs. Higher Eukaryotes; from a General Principle to Specialized Functions . . . . . . . . . . . 234 Transmembrane Signaling . . . . . . . . 236 GPI-Anchors in Parasite Patho- genicity: A Small Molecule with Dramatic Effects. . . . . . . . . . . . . . 236 The Molecular Biology of GPI-Anchor Biosynthesis . . . . . . . . 237 From Functions to Genes: Mutant Cells Lead the Way . . . . . . . 237 Cloning of Genes Involved in GPI- Anchoring by the Complementation of Defined Defects . . . . . . . . . . . . 238

Chapter 13 The Biology of Sialic Acids: Insights into their Structure, Metabolism and Function in particular during Viral Infection WERNER REUITER ET AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

1 Introduction. . . . . . . . . . . . . . . . . 245 4 Biological Functions of Sialic Acids. . 249

by Neuraminic Acid Analogs . . . . . . 251 2 Chemical Structure and Expression 5 Modification of Biological Functions

3 Metabolism of Sialic Acids . . . . . . . 247 6 Summary. . . . . . . . . . . . . . . . . . . 254 of Sialic Acids. . . . . . . . . . . . . . . . 246

Chapter 14 The Biology of Sulfated Oligosaccharides LORA V. HOOPER ET AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

1 Introduction . . . . . . . . . . . . . . . . . 261 4 Sulfated Oligosaccharides of 2 Sulfated Oligosaccharides of Unknown Function . . . . . . . . . . . . 271

Conclusion and Future Prospects . . . 272 Luteinizing Hormone. . . . . . . . . . . 263 5 3 Sulfated Carbohydrates in Leukocyte

Trafficking . . . . . . . . . . . . . . . . . . 269

XVI I Contents Chapter 15 Carbohydrate-Carbohydrate Interaction NICOLAI V. BOVIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

1 Introduction . . . . . . . . . . . . . . . . . 277 3.6 2 Historical Aspects . . . . . . . . . . . . . 277 3.7 3 Experimental Approaches . . . . . . . . 278 4 3.1 Cell Aggregation . . . . . . . . . . . . . . 279 3.2 Aggregation of Liposomes . . . . . . . . 279 5 3.3 Interaction of Liposomes with a

Plastic Surface Coated with Glyco- lipid or Another Glycoconjugate . . . . 280

3.4 Weak-Affinity Chromatography . . . . 281 Performed in the Following Way . . . . 281

6

3.5 Equilibrium Dialysis Method is 7

Enzyme-Linked Immunoassays . . . . 281 Langmuir-BlodgettTechnique . . . . . 282 Molecular Nature of Carbohydrate- Carbohydrate Interaction . . . . . . . . 282 Cell Interaction: Static and Dynamic Integration with Other Adhesion Mechanisms . . . . . . . . . . . . . . . . . 284 Polysaccharide-Carbo hydrate Interaction . . . . . . . . . . . . . . . . . . 286 Conclusions . . . . . . . . . . . . . . . . . 287

Chapter 16 Carbohydrate-Protein Interaction HANS-CHRISTIAN SIEBERT ET AL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

1 Theoretical Aspects of Carbohydrate- 2.3 Protein Interaction . . . . . . . . . . . . 291

1.1 Carbohydrate and Protein Flexibility . 291 2.4 1.2 Thermodynamic Parameters of

Carbohydrate-Protein Interaction . . . 292 2.5 Interactions . . . . . . . . . . . . . . . . . 295 3.1

2.1 X-ray Crystallography: Brookhaven 3.2 Protein Data Bank . . . . . . . . . . . . 295

2.2 Cambridge Structural Database . . . . 296 3.3

2 Modeling of Carbohydrate-Protein 3

Knowledge-Based Homology Modeling . . . . . . . . . . . . . . . . . . . 296 Basic Molecular Features in Carbohydrate-Protein Interactions . . 297 Estimation of Binding Constants . . . 299 NMR Spectroscopy . . . . . . . . . . . . 303 Free-State Conformation . . . . . . . . 303 Transferred NOE-Experiments of Oligosaccharide-Protein Complexes . 304 Perspectives . . . . . . . . . . . . . . . . . 306

Chapter 17 Antibody-Oligosaccharide Interactions Determined by Crystallography DAVID R . BUNDLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

1 1.1 1.2 1.3 1.4 1.5 1.6 2

3

3.1

3.2

Crystal Structure Determination . . . . 312 3.3 Sample Purification . . . . . . . . . . . . 312 Protein Crystals . . . . . . . . . . . . . . 312 3.4 Collecting X-ray Diffraction Data . . . 313 Electron Density . . . . . . . . . . . . . . 313 3.5 Map FittingModel Building . . . . . . 314 Precision of the Model . . . . . . . . . . 315 Recently Determined Crystal 3.6 Structures . . . . . . . . . . . . . . . . . . 315 The Structure of Mab Se 155.4 and 4 the Abequose-Based Epitope . . . . . . 319 5 Features of a Dodecasaccharide-Fab 6 Complex . . . . . . . . . . . . . . . . . . . . 320

Water Molecule . . . . . . . . . . . . . . . 322 Hydrogen-Bonding and the Structured 7

Attributing Structural Features to Binding Energy . . . . . . . . . . . . . 324 Oligosaccharide Conformational Change on Binding . . . . . . . . . . . . 324 A Different Bound Oligosaccharide Conformation in a Single Chain Fv-Trisaccharide Complex . . . . . . . . 325 Bound Conformation I1 in a Fab-Heptasaccharide Complex . . . . . 325 Role of Water Molecules . . . . . . . . . 328 Solvent-Exposed Hydrogen Bonds . . 329 Conformational Change in the Binding Site . . . . . . . . . . . . . . . . . 329 Summary . . . . . . . . . . . . . . . . . . . 329

Contents I XVII Chapter 18 Thermodynamic Analysis of Protein-Carbohydrate Interaction DIPTI GUPTA AND CURTIS F. BREWER. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

1 Introduction. . . . . . . . . . . . . . . . . 333 4 Thermodynamics of Binding of 1.1 Background Studies. . . . . . . . . . . . 334 Trimannoside 10 . . . . . . . . . . . . . . 338 2 Outline of Experimental Design. . . . 335 5 Thermodynamics of Binding of 2.1 Materials. . . . . . . . . . . . . . . . . . . 335 2.2 Methodology . . . . . . . . . . . . . . . . 336 6 Binding of Mono- and Dideoxy 3 Thermodynamics of Binding Derivatives of Trimannoside 10. . . . . 339

Summary and Perspectives . . . . . . . 341

Oligosaccharides 8, 11 and 12. . . . . . 338

of Mono- and Disaccharides . . . . . . 336 7

Chapter 19 Analysis of Protein-Carbohydrate Interaction Using Engineered Ligands DOLORES SOLIS AND TERESA DIAZ-MAURI~JO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

1 Overview. . . . . . . . . . . . . . . . . . . 345 4 Mapping of Subsites of Anti-Carbo- 2 Analysis of Hydrogen-Bonding and hydrate Antibodies . . . . . . . . . . . . 350

Steric Requirements for 5 Probing the Active Site Requirements Recognition . . . . . . . . . . . . . . . . . 346

Energetics and Protein Groups

of Carbohydrate-Binding Enzymes . . 350

for Recognition. . . . . . . . . . . . . . . 351 3 Analysis of Hydrogen-Bonding 6 Probing Conformational Requirements

Involved in Recognition . . . . . . . . . 348

Chapter 20 Application of Site-Directed Mutagenesis to Structure-Function Studies of Carbohydrate-Binding Proteins JUN HIRABAYASHI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

1 General Points on Site-Directed 4 Example of Mutagenesis: Human Galectin-1 . . . . . . . . . . . . . . . . . . 362

2 Special Points on Mutagenesis of 4.1 What is Galectin? . . . . . . . . . . . . . 362

3 Actual Procedures for Mutagenesis . . 358 Galectin Family . . . . . . . . . . . . . . 363 3.1 Conventional Procedures . . . . . . . . 359 4.3 Primer Design and Mutagenesis . . . . 363 3.2 New Procedures . . . . . . . . . . . . . . 360 4.4 Evaluation of the Results . . . . . . . . 365 3.3 PCR-Aided Mutagenesis . . . . . . . . . 361

Mutagenesis . . . . . . . . . . . . . . . . . 355

Carbohydrate-Binding Proteins . . . . 357 4.2 Conserved Amino Acids in the

Chapter 21 Bacterial Lectins: Properties, Structure, Effects, Function and Applications NECHAMA GILBOA-GARBER ET AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

1 Introduction. . . . . . . . . . . . . . . . . 369 2.2 Dependence of Lectin Production on

1.2. Bacterial Lectinology - Past and 2.3 Lectin Screening Technology . . . . . . 372 Present Status. . . . . . . . . . . . . . . . 369 3 Bacterial Lectin Properties . . . . . . . 373

2 Bacterial Lectin Prevalence, 3.1 Divalent Cation-Binding . . . . . . . . . 373 Expression and Detection . . . . . . . . 371 3.2 Sugar Specificities of the

2.1 State of the Art. . . . . . . . . . . . . . . 371 Bacterial Lectins . . . . . . . . . . . . . . 373

1.1. Prologue . . . . . . . . . . . . . . . . . . . 369 Growth Conditions . . . . . . . . . . . . 372

WIII I Contents 3.3

4 5 5.1

5.2

5.3

6

6.1 6.2 7 7.1 7.2 7.3

Physico-Chemical Properties of the 7.4 Lectins and their Purification . . . . . . 374 7.5 Bacterial Lectin Size and Structure . . 374 Bacterial Lectin Target Molecules . . . 376 Interactions of Bacterial Lectins

molecules . . . . . . . . . . . . . . . . . . 378 Interactions of Bacterial Lectind Adhesins with Cell Receptors 8.2 Including Blood Group Antigens . . . 378 The Receptor Specificity of Bacterial Lectins Determines Host Cell

Bacterial Lectin Effects on Macromolecules and Cells . . . . . . . . 381 Effects on Macromolecules . . . . . . . 381 9.1 Effects on Cells . . . . . . . . . . . . . . . 381 Bacterial Lectin Functions . . . . . . . . 383 9.2 Bacterial Self-Protection . . . . . . . . . 383 9.3 Bacterial Cell Organization . . . . . . . 384

10 Physiological Functions . . . . . . . . . . 384

8

with Free Glycosylated Macro- 8.1

Selectivity . . . . . . . . . . . . . . . . . . 379 9

Cell Contacts Supplying Nutrition and

Interactions for Proliferation . . . . . . 384 Cell Contacts Leading to Bacterial Death . . . . . . . . . . . . . . 384 Involvement of Bacterial Lectins in Pathogenesis . . . . . . . . . . . . . . . . 385 Enzyme/Toxin-Targeting and Trafficking to Host Macromolecules andcells . . . . . . . . . . . . . . . . . . . 385 The Role of Bacterial Lectins in Adherence of Individual and Coaggregated Bacteria to Host Cells . . . . . . . . . . . . . . . . . . 386 Preventive Strategies against Lectin- Mediated Adherence of Pathogenic Bacteria to Host Cells . . . . . . . . . . 388 Prevention of Lectin-Receptor Interactions . . . . . . . . . . . . . . . . . 388 Host Cell Receptor Elimination . . . . 388 Prevention of the Bacterial Lectin/ Adhesin Production . . . . . . . . . . . . 388 Applications of Bacterial Lectins . . . 389

Chapter 22 Glycobiology of Parasites: Role of Carbohydrate-Binding Proteins and their Ligands in the Host-Parasite Interaction HONORINE D . WARD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

1 Introduction . . . . . . . . . . . . . . . . . 399 4.1 2 Malaria . . . . . . . . . . . . . . . . . . . . 400 2.1 Sialic Acid and Sialic Acid- 4.2

Binding Proteins . . . . . . . . . . . . . . 401 4.3

Proteins . . . . . . . . . . . . . . . . . . . . 402 5.1 2.3 GlcNAc-Binding Proteins . . . . . . . . 402 5.2 3 Chagas Disease . . . . . . . . . . . . . . 402 6 3.1 Sialic Acid and Sialic Acid-Binding 6.1

Proteins . . . . . . . . . . . . . . . . . . . . 403 7 3.2 Heparin-Binding Protein . . . . . . . . . 403 7.1 4 Leishmaniasis . . . . . . . . . . . . . . . . 404 8

2.2 Glycosaminoglycan-Binding 5

Lipophosphoglycan (LPG) and LPG-Binding Proteins . . . . . . . 404 Heparin-Binding Proteins . . . . . . . . 405 GlcNAc-Binding Proteins . . . . . . . . 405 Amebiasis . . . . . . . . . . . . . . . . . . 406 GaYGalNAc-Binding Lectin . . . . . . 406 Chitotriose-Binding Proteins . . . . . . 407 Giardiasis . . . . . . . . . . . . . . . . . . 407 Mand-P-Binding Protein . . . . . . . . 407 Cryptosporidosis . . . . . . . . . . . . . . 408 GaYGalNAc-Binding Protein . . . . . . 408 Conclusion . . . . . . . . . . . . . . . . . . 409

Chapter 23 Structure and Function of Plant Lectins HAROLD RUDIGER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415

1 Introduction . . . . . . . . . . . . . . . . . 415 4 Functions . . . . . . . . . . . . . . . . . . . 423 2 Structure . . . . . . . . . . . . . . . . . . . 416 4.1 Internally Directed Activities . . . . . . 423

2.2 Non-Leguminous Plants . . . . . . . . . 419 5 Conclusion . . . . . . . . . . . . . . . . . . 429 3 Location . . . . . . . . . . . . . . . . . . . 421

2.1 Leguminosae . . . . . . . . . . . . . . . . 416 4.2 Externally Directed Activities . . . . . 425

Contents 1 XIX Chapter 24 Lectins and Carbohydrates in Animal Cell Adhesion and Control of Proliferation JEAN-PIERRE ZANEITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

1 2 3 3.1 3.2 3.3 4

4.1

Introduction . . . . . . . . . . . . . . . . . 439 4.2 The Galectins . . . . . . . . . . . . . . . . 439 4.3 The C-Type Lectins . . . . . . . . . . . . 440 5 The Selectins . . . . . . . . . . . . . . . . 440 6 The NK Sub-Family of C-Type Lectins 441 6.1 The Soluble C-Type Lectins . . . . . . . 441 6.2 Soluble Calcium-Independent Mannose-Binding Lectins . . . . . . . . 441 7 Glycoproteins Ligands of 8 Lectin CSL . . . . . . . . . . . . . . . . . 442

Lectin CSL as an Adhesion Molecule . 442 Lectin CSL as a Mitogen . . . . . . . . 444 Heparin-Binding Growth Factors . . . 446

Interleukin 2 (IL-2) . . . . . . . . . . . . 447 Interleukin 1 (IL-1) and Tumor

Conclusions and Perspectives . . . . . . 450 Appendix . . . . . . . . . . . . . . . . . . 452

Cytokines . . . . . . . . . . . . . . . . . . 447

Necrosis Factor (TNF) . . . . . . . . . . 449

Chapter 25 Galectins in Tumor Cells DAVID W . OHANNESIAN AND REUBEN LOTAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

1 2 2.1 2.2 3 4 5

Introduction . . . . . . . . . . . . . . . . . 459 6 Galectin Ligands . . . . . . . . . . . . . . 464 Galectins in Normal Cells . . . . . . . . 459 6.1 Laminin . . . . . . . . . . . . . . . . . . . 464 Galectin-1 . . . . . . . . . . . . . . . . . . 459 6.2 Lysosome-Associated Membrane Galectin-3 . . . . . . . . . . . . . . . . . . 460 Putative Functions of Galectins . . . . 461 6.3 Carcinoembryonic Antigen . . . . . . . 465 Galectins in Tumor Cells . . . . . . . . . 462 7 Conclusions . . . . . . . . . . . . . . . . . 466 Carbohydrate Specificity of Galectins . . . . . . . . . . . . . . . . . . . 463

Glycoproteins (LAMPS) . . . . . . . . . 465

Chapter 26 Glycoconjugate-Mediated Drug Targeting KEVING . RICE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

1 Introduction . . . . . . . . . . . . . . . . . 471 4.2 2 Mammalian Lectins that Mediate

Drug Delivery . . . . . . . . . . . . . . . 472 4.3 3 Design of Glycoconjugate Carriers 4.4

for Drug Delivery . . . . . . . . . . . . . 474 4 Applications of Carbohydrate- 4.5

Mediated Drug Targeting . . . . . . . . 475 4.1 Targeting Low Molecular 5

Weight Drug Molecules to Hepatocytes and Macrophages . . . . . 476

Targeting Antisense Oligonucleotides . . . . . . . . . . . . . . 476 Glyconjugate Targeting of Enzymes . . 477 Glycoconjugate-Mediated Targeting of DNA . . . . . . . . . . . . . . . . . . . . 477 Glycoconjugate Targeting of Liposomes and Lipoproteins . . . . . . 478 Conclusions . . . . . . . . . . . . . . . . . 479

Chapter 27 Glycobiology of Signal Transduction ANTONIO VILLALOBO ET AL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

1 Introduction . . . . . . . . . . . . . . . . . 485 4 Signaling by Membrane-Bound 2 Lectin-Induced Mitogenesis and Mammalian Lectins . . . . . . . . . . . . 492

Immunomodulation . . . . . . . . . . . . 489 5 Conclusions . . . . . . . . . . . . . . . . . 493 3 Lectin-Induced Apoptosis . . . . . . . . 492

xx 1 Contents Chapter 28 Glycobiology of Host Defense Mechanisms HANS-JOACHIM GABIUS ET AI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

1 Introduction . . . . . . . . . . . . . . . . . 497 2.3 Selectins . . . . . . . . . . . . . . . . . . . 500 2 The Family of Lectins in 2.4 NK Cell Lectins . . . . . . . . . . . . . . 500

Host Defense . . . . . . . . . . . . . . . . 497 2.5 Miscellaneous Lectins . . . . . . . . . . 501 2.1 Acute-Phase Reactants 3 Lectin-Dependent Activation

of Defense Mechanisms . . . . . . . . . 501 2.2 Collectins . . . . . . . . . . . . . . . . . . 498 4 Perspectives . . . . . . . . . . . . . . . . . 502

(Pentraxins) . . . . . . . . . . . . . . . . . . 497

Chapter 29 Transgenic Approaches to Glycobiology HELEN J . HATHAWAYAND BARRY D . SHUR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507

1 Introduction . . . . . . . . . . . . . . . . . 507 4.1 2 Protein Glycosylation . . . . . . . . . . . 508 4.2 3 Approaches to Genetic Manipulation

of Oligosaccharide Function . . . . . . 508 4.3 3.1 Overexpression of Gene Products . . . 508 5 3.2 Elimination of Genes . . . . . . . . . . . 508 5.1 3.3 Tissue- and Stage-Specific Alteration 5.2

of Gene Products . . . . . . . . . . . . . 509 5.3 4 Genetic Manipulation 6

of Glycosylation Pathways . . . . . . . . 510

Altering Terminal Glycosylation . . . . 510 Altering Complex N-Linked Glycosylation . . . . . . . . . . . . . . . . 511 Altering Glycosidases . . . . . . . . . . . 511 Altering Carbohydrate Receptors . . . 512 Selectins . . . . . . . . . . . . . . . . . . . 512 Galectin: L14 . . . . . . . . . . . . . . . . 512 fil,4.Galactosyltransferase . . . . . . . . 512 Future Directions . . . . . . . . . . . . . 515

Chapter 30 Biomodulation. the Development of a Process-Oriented Approach to Cancer Treatment PAUL L . MANN ETAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

1 Introduction . . . . . . . . . . . . . . . . . 519 4 The Biological Response Modifier

3 A Brief Summary of Other 5 Recent Uses of BRM/Biomodulator 2 Problem Definition . . . . . . . . . . . . 520 [BRM] Approach . . . . . . . . . . . . . 521

Terminology . . . . . . . . . . . . . . . . . 522 Approaches . . . . . . . . . . . . . . . . . 521

Chapter 31 Glycobiology in Xenotransplantation Research DAVID K.C. COOPER AND RAFAEL ORIOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531

1 Introduction . . . . . . . . . . . . . . . . . 531 7 2 Xenotransplantation . Basic

Immunobiology . . . . . . . . . . . . . . . 531 8 3 Allotransplantation across the

ABO Histo-Blood Group Barrier . . . 532 8.1 4 Identification of Human Anti-Pig

Antibodies as Anti-a-Galactosyl 8.2 Antibodies . . . . . . . . . . . . . . . . . . 533

Epitopes on Pig Vascular Endothelium 536 6 Experimental Studies in Baboons . . . 538 10

5 Identification of Oligosaccharide 9

Clinical Relevance of Anti-a-Gal Antibody in Xenotransplantation . . . 539 The Genetically Engineered a-Gal- Negative Pig . . . . . . . . . . . . . . . . . 540 Deletion of the Gene Encoding al.3.Galactosyltransferase . . . . . . . . 540 Increased Expression of Alternate Epitopes . . . . . . . . . . . . . . . . . . . 540 Other Therapeutic Approaches -

Comment . . . . . . . . . . . . . . . . . . 541 Gene Therapy . . . . . . . . . . . . . . . . 541

Contents I XXI Chapter 32 Modern Glycohistochemistry: A Major Contribution to Morphological Investigations ANDRE DANGUY ET AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547

1 2

3 3.1 3.2 3.3 4 4.1 4.2

Introduction. . . . . . . . . . . . . . . . . 547 4.3

are Attractive Reagents in Histology. . . . . . . . . . . . . . . . . . . 547 4.5 Methods . . . . . . . . . . . . . . . . . . . 550 Lectin Cytolabeling . . . . . . . . . . . . 550 4.6 Reverse Lectin Histochemistry . . . . . 550 Special Considerations . . . . . . . . . . 551 Functional Morphological Data . . . . 552 The Kidney . . . . . . . . . . . . . . . . . 552 Endocrine Status and the Glycohistochemical Expression . . . . 553

Why Lectins and Neoglycoproteins 4.4

5

The Integument . . . . . . . . . . . . . . 553 Glycan Expression in Skeletal Muscle . . . . . . . . . . . . . . . . . . . . 556 Lectin Histochemistry of the Teleost Intestine . . . . . . . . . . 557 Sugar-Binding Sites and Lectin Acceptors in Prokaryotes and Eukaryotic Parasites . . . . . . . . . . . 557 Conclusions and Perspectives. . . . . . 558

Chapter 33 Lectins and Neoglycoproteins in Histopathology S. KANNANAND M. KRISHNAN NAIR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563

1 1.1 1.2 2 2.1 2.2

2.3

3 3.1

Introduction. . . . . . . . . . . . . . . . . 563 3.2 Relevance of Histopathology . . . . . . 563 Glycoconjugates and Pathology . . . . 564 Lectins.. . . . . . . . . . . . . . . . . . . 564 Methods in Lectin Histochemistry. . . 564 Exogenous Lectins in 5.1 Histopathology . . . . . . . . . . . . . . . 565 Endogenous Lectins in 5.2 Histopathology . . . . . . . . . . . . . . . 568 Neoglycoproteins . . . . . . . . . . . . . 569 Neoglycoprotein Staining Protocols . . 570

4

5

Neoglycoproteins in Histopathology . . . . . . . . . . . . . . . 570 Precautions to be Taken in Lectin

Perspectives . . . . . . . . . . . . . . . . . 574 Modern Roles Played by Histopathology . . . . . . . . . . . . . . . 574 Perspectives of Lectins and Neoglycoproteins in Histo-

Histochemistry . . . . . . . . . . . . . . . 571

pathology . . . . . . . . . . . . . . . . . . 576

Chapter 34 Glycobiology of Development: Spinal Dysmorphogenesis in Rat Embryos Cultured in a Hyperglycemic Environment LORI KESZLER-MOLL ET AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585

1 Introduction. . . . . . . . . . . . . . . . . 585 2.4 The Kinetics of Neural Tube Defect 2 Problem Identification . . . . . . . . . . 586 Formation . . . . . . . . . . . . . . . . . . 589 2.1 The Model, Embryo Culture . . . . . . 587 3 Lectin Staining Patterns . . . . . . . . . 590 2.2 The Model, Serum Preparation . . . . 587 4 Surface Oligosaccharide and Fusion 2.3 Embryo Morphology . . . . . . . . . . . 587 Mechanism . . . . . . . . . . . . . . . . . 592

XXII I Contents Chapter 35 Glycobiology of Fertilization FRED SINOWATZ ET AL. . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

1 A Short Review of the Fertilization 3.1 3.2

2 3.3

2.1 4

2.2 Characterization of the Zona

Pathway . . . . . . . . . . . . . . . . . . . 595 Structure and Function of the Zona Pellucida . . . . . . . . . . . . . . . . . . . 596 The Glycoprotein Constituents of the ZonaPellucida. . . . . . . . . . . . . . . 596 Pellucida Oligosaccharides 4.1 by Lectins . . . . . . . . . . . . . . . . . . 597 4.2 Oligosaccharides of the Porcine Zona Pellucida . . , , . . . . . . . . . . . . . . . 597 4.3

Sperm-Zona Pellucida Interaction. . . 598

Carbohydrate-Binding Proteins . . . . 599

2.3

2.4 Carbohydrates Involved in

3 Sperm-Associated Zona Pellucida- and

Sperm Membrane C-Type Lectins . . . 600 Mouse Sperm fil,4-Galactosyl- transferase . . . . . . . . . . . . . . . . . . 601 Sperm Membrane-Associated Zona Pellucida-Binding Proteins . . . . . . . 602 Inhibition of Sperm-Egg Interactions by Saccharides and Glycosidases. . , . 605 Sperm Binding Assays . . . . . . . . . . 605 Inhibition of Sperm-Zona Binding by Saccharides . . . . . . . . . . . . . . . . . 605 Do Carbohydrates also Play a Role in the Fusion of the Oocyte Plasma Membrane with the Spermatozoon?. . 606

Chapter 36 Glycobiology of Consciousness RAYMONDE JOUBERT-CARON ET AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611

1 2

Introduction . . . . . . . . . . . . . . . . . 611 Brain Lectins . . . . . . . . . . . . . . . . 611

3 4

Neuroimmunomodulation . . . . . . . . 613 Brain Glycoproteins. . . . . . . . . . . . 615

List of Contributors

A N D R ~ , S. Institut fur Physiologische Chemie Tierarztliche Fakultat Ludwig-Maximilians-Universitat Veterinarstr. 13 80539 Munchen Germany

AVICHEZER, D . Department of Chemical Immunology The Weizmann Institute of Science Rehovot 76100 Israel

BAENZIGER, J. U. Department of Pathology Washington University School of Medicine 660 S. Euclid Ave. St. Louis, MO 63110-1093 USA

BAUM, 0. Freie Universitat Berlin, Institut fur Molekularbiologie und Biochemie Arnimallee 22 14195 Berlin-Dahlem Germany

BLADIER, D. UFR Leonard de Vinci Laboratoire de Biochimie et Technologie des ProtCines 74, rue Marcel Cachin 93012 Bobigny Cedex France

BOVIN, N. V. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences ul. Miklukho-Maklaya 16/10

Russian Federation 117871 GSP-7, V-437 MOSCOW

BRAUN, M. College of Pharmacy The University of New Mexico Albuquerque, NM 87131 USA BREWER, C. F. Departments of Molecular Pharmacology, Microbiology and Immunology Jack and Pearl Resnick Campus Albert Einstein College of Medicine 1300 Morris Park Ave. Bronx, NY 10461 USA BROCKHAUSEN, I. Biochemistry Department Research Institute Hospital for Sick Children 555 University Ave. Toronto M5G 1x8 Canada BUNDLE, D. R. Department of Chemistry Faculty of Science University of Alberta E3-52 Chemistry Bldg. Edmonton T6G 2G2 Canada CALVETE, J. J. Institut fur Reproduktionsmedizin Tierarztliche Hochschule Hannover Bunteweg 15 30559 Hannover Germany


XXlV 1 List of Contributors CAMBY, I. Laboratoire dHistologie FacultC de MCdecine UniversitC Libre de Bruxelles Route de Lennik 808 1070 Bruxelles Belgium

CARON, M. UFR LConard de Vinci Laboratoire de Biochimie et Technologie des ProtCines 74, rue Marcel Cachin 93012 Bobigny Cedex France

COOPER, D. K. C. Oklahoma Transplantation Institute Baptist Medical Center 3300 N.W. Expressway Oklahoma City, OK 73112-4481 USA

CRAMER, F. Max-Planck-Institut fur experimentelle Medizin Hermann-Rein-Str. 3 37075 Gottingen Germany

CUMMINGS, R. D. Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center 941 S. L. Young Blvd. Oklahoma City, OK 73190 USA

DANGUY, A. FacultC des Sciences Laboratoire de Biologie Animale et dHistologie ComparCe Universitt Libre de Bruxelles 50, avenue F. D. Roosevelt 1050 Bruxelles Belgium

DfAZ-MAuRIflO, T. Instituto de Quimica Fisica Rocasolano Consejo Superior de Investigaciones Cientificas Serrano 119,28006 Madrid Spain

ECKERT, V. Institut fiir Virologie AG Parasitologie Philipps-Universitat Robert-Koch-Str. 17 35037 Marburg Germany

GABIUS, H.-J. Institut fur Physiologische Chemie Tierarztliche Fakultat Ludwig-Maximilians-Universitat Veterinarstr. 13 80539Munchen Germany

GABIUS, S. Hamatologisch-Onkologische Schwerpunktpraxis Sternstr. 12 83022 Rosenheim Germany

GARBER, N. C. Department of Life Sciences Bar-Ilan University Ramat-Gan 52900 Israel

GARCIA, A. College of Pharmacy The University of New Mexico Health Sciences Center Albuquerque, NM 87131 USA

GEROLD, P. Institut fur Virologie AG Parasitologie Philipps-Universitat Robert-Koch-Str. 17 35037 Marburg Germany

GILBOA-GARBER, N. Department of Life Sciences Bar-Ilan University Ramat-Gan 52900 Israel

List of Contributors I xxv GILLERON, M. Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Department I11 GlycoconjuguCs et Biomembranes, CNRS 118, route de Narbonne 31062 Toulouse Cedex France

GUFTA, D. Departments of Molecular Pharmacology, Microbiology and Immunology Jack and Pearl Resnick Campus Albert Einstein College of Medicine 1300 Morris Park Ave. Bronx, NY 10461 USA

HANOSH, J . College of Pharmacy The University of New Mexico Albuquerque, NM 87131 USA

HATHAWAY, H. J. Department of Biochemistry and Molecular Biology The University of Texas MD Anderson Cancer Center 1515 Holcombe Blvd Cedex Houston, TX 77030 USA

HIRABAYASHI J. Department of Biological Chemistry Faculty of Pharmaceutical Sciences Teikyo University Sagamiko, Kanagawa 199-01 Japan

HOOPER, L. V. Department of Pathology Washington University School of Medicine 660 S . Euclid Ave. St. Louis, MO 63110-1093 USA

HORCAJADAS, J. A. Instituto de Investigaciones BiomCdicas Consejo Superior de Investigaciones Cientificas Arturo Duperier 4 28029 Madrid Spain

HOUNSELL, E. F. Department of Biochemistry and Molecular Biology University College of London Gower Street London WClE 6BT United Kingdom

INOUE, Y. Department of Biophysics and Biochemistry Graduate School of Science University of Tokyo Hongo-7 Tokyo 113 Japan

INOUE, S. School of Pharmaceutical Sciences Showa University Hatanodai-1 Tokyo 142 Japan

JOUBERT-CARON, R. UFR LConard de Vinci Laboratoire de Biochimie et Technologie des ProtCines 74, rue Marcel Cachin 93012 Bobigny Cedex France

KANNAN, S. Division of Cancer Research Regional Cancer Centre Thiruvananthapuram 695 011, Kerala State India

KAYSER, K. Abteilung Pathologie , Thoraxklinik Amalienstr. 5 , 69126 Heidelberg Germany

KELLEY, R. 0. College of Pharmacy The University of New Mexico Albuquerque, NM 87131 USA

KESZLER-MOLL, L. College of Pharmacy The University of New Mexico Albuquerque, NM 87131 USA

XXVl 1 List of Contributors KISS, R. Laboratoire dHistologie FacultC de Medecine UniversitC Libre de Bruxelles Route de Lennik 808 1050 Bruxelles Belgium

KITAJIMA, K. Department of Biophysics and Biochemistry Graduate School of Science University of Tokyo Hongo-7 Tokyo 113 Japan

KOPITZ , J . Institut fur Pathobiochemie und Allg. Neurochemie Universitat Heidelberg Im Neuenheimer Feld 220 69120 Heidelberg Germany

KRESSE, H. Institut fur Physiologische Chemie und Pathobiochemie Westfalische Wilhelms-Universitat Cedex Waldeyerstr. 15 48129 Miinster Germany

KRISHNAN NAIR, M. Division of Cancer Research Regional Cancer Centre Thiruvananthapuram 695 011, Kerala State India

LAINE, R. A. Deptartments of Biochemistry and Chemistry Louisiana State University and The Louisiana Agricultural Center Baton Rouge, LA 70803 USA

LEE, R. T. Department of Biology Johns Hopkins University 144 Mudd Hall 3400 N. Charles Street Baltimore, MD 21218-2685 USA

LEE,Y. C. Department of Biology Johns Hopkins University 144 Mudd Hall 3400 N. Charles Street Baltimore, MD 21218-2685 USA

LIS, H. Department of Membrane Research and Biophysics The Weizmann Institute of Science Rehovot 76100 Israel

LOTAN, R. Department of Tumor Biology MD Anderson Cancer Center The University of Texas 1515 Holcombe Blvd., Box 108 Houston, TX 77030 USA

LUTOMSKI, D. UFR LConard de Vinci Laboratoire de Biochimie et Technologie des Prottines 74, rue Marcel Cachin 93012 Bobigny Cedex France

MANN, P. L. College of Pharmacy The University of New Mexico Albuquerque, NM 87131 USA

MANZELLA, S. M. Department of Pathology Washington University School of Medicine 660 S. Euclid Ave. St. Louis, MO 63110-1093 USA

OHANNESIAN, D . W. Department of Tumor Biology MD Anderson Cancer Center The University of Texas 1515 Holcombe Blvd., Box 108 Houston, TX 77030 USA

List of Contributors I XXVll ORIOL, R. INSERM U.178 16, Paul Vaillant-Couturier 94807 Villejuif Cedex France

PAVELKA, M. Institut fur Histologie und Embryologie Universitat Innsbruck MullerstraBe 59 6020 Innsbruck Austria

RAYMOND-STINTZ, M. A. College of Pharmacy The University of New Mexico Albuquerque, NM 87131 USA

REUTER, G. Institut fur Physiologische Chemie Tierarztliche Fakultat Ludwig-Maximilians-Universitat Veterinarstr. 13 80539 Munchen Germany

REUITER, W. Institut fur Molekularbiologie und Biochemie Freie Universitat Berlin Arnimallee 22 14195 Berlin-Dahlem Germany

RICE, K. G. Division of Medicinal Chemistry and Pharmaceutics College of Pharmacy University of Michigan 428 Church Street Ann Arbor, MI 48109- 1065 USA

RUDIGER, H. Institut fur Pharmazie und Lebensmittelchemie Universitat Wurzburg Am Hubland 97074 Wurzburg Germany

SALMON, I. Service dAnatomie Pathologique HBpital Erasme UniversitC Libre de Bruxelles Route de Lennik 808 1050 Bruxelles Belgium

SCHACHTER, H. Biochemistry Department Research Institute Hospital for Sick Children 555 University Ave. Toronto M5G 1x8 Canada

SCHMIDT, R. R. Institut fur Organische Chemie Universitat Konstanz UniversitatsstraBe 10 78464 Konstanz Germany

SCHWARZ, R. Th. Institut fur Virologie AG Parasitologie Philipps-Universitat Robert-Koch-Str. 17 35037 Marburg Germany

SHARON, N. Department of Membrane Research and Biophysics The Weizmann Institute of Science Rehovot 76100 Israel

SHUR, B. D. Department of Biochemistry and Molecular Biology MD Anderson Cancer Center The University of Texas 1515 Holcombe Blvd, Houston, TX 77030 USA

SIEBERT, H.-C. Institut fur Physiologische Chemie Tierarztliche Fakultat Ludwig-Maximilians-Universit at Veterinarstr. 13 80539Munchen Germany

Xxvlll 1 List of Contributors SINOWATZ, F. Institut fiir Tieranatomie Tierarztliche Fakultat Ludwig-Maximilians-Universitat Veterinarstr. 13 80539 Munchen Germany SoLfs, D. Instituto de Quimica Fisica Rocasolano Consejo Superior de Investigaciones Cientificas Serrano 119 28006 Madrid Spain STASCHE, R. Institut fur Molekularbiologie und Biochemie Freie Universitat Berlin Arnimallee 22 14195 Berlin-Dahlem Germany STEHLING, P. Institut fur Molekularbiologie und Biochemie Freie Universitat Berlin Arnimallee 22 14195 Berlin-Dahlem Germany SUZUKI, T. Department of Biophysics and Biochemistry Graduate School of Science University of Tokyo, Hongo-7 Tokyo 113 Japan TOPFER-PETERSEN, E. Institut fur Reproduktionsmedizin Tierarztliche Hochschule Hannover Bunteweg 15 30559 Hannover Germ any VON DER LIETH, C.-W. Deutsches Krebsforschungszentrm Zentrale Spektroskopie Im Neuenheimer Feld 280 69120 Heidelberg Germany

VILLALOBO, A. Instituto de Investigaciones Biomedicas Consejo Superior de Investigaciones Cientificas Arturo Duperier 4 28029 Madrid Spain VLIEGENTHART, J . F. G. Department of Bio-Organic Chemistry Bijvoet Center for Biomolecular Research Utrecht University P.O. Box 80.075 3508 TB Utrecht The Netherlands WARD, H. D. Division of Geographic Medicine and Infectious Diseases Tufts University School of Medicine 750 Washington Street Boston, MA 02111 USA WENK, R. College of Pharmacy The University of New Mexico Albuquerque, NM 87131 USA WITTMANN , J . Institut fur Physiologische Chemie Tierarztliche Fakultat Ludwig-Maximilians-Universitat Veterinarstr. 13 80539 Munchen Germany ZANEITA, J.-P. Centre de Neurochimie du CNRS Laboratoire de Neurobiologie MolCculaire des Interactions Cellulaires 5 , rue Blaise Pascal 67084 Strasbourg Cedex France

The Information-Storing Potential of the Sugar Code ROGER A. LAINE

1 Introduction Carbohydrates, by their unique multi-linkage monomers and branching structure, contain an evolutionary potential of information content several orders of magnitude higher in a short sequence than any other biological oligomer. Therefore a high level of information potential is inherent in biological recognition systems com- prised of complex carbohydrate ligands on the one hand which are recognized for targeted activities on the other hand by hapten specific protein receptors, such as lectins.

The potential number of all possible linear and branched isomers of small oligosaccharides has recently been calculated to be much larger than previous estimations (Laine, 1994). Seven structural elements lead to the large number of isomers, including multiple ring sites as points of glycosidic attachment, a@ anomerity, pyranose/ furanose configuration and branching structure. For a trisaccharide composed from a set of 3 hexoses, these elements lead to isomer permutations exceeding 38,000. This can be compared with only 27 permutations for 3 amino acids or 3 nucleic acids.

Consider that the trisaccharide could be made up of any of our most commonly found sugars, glucose, mannose, galactose, fructose, N-acetyl- glucosamine, N-acetylgalactosamine, fucose, ara- binose, xylose, ribose, glucuronic acid, galactu- ronic acid, mannuronic acid, iduronic acid, and sialic acid, lets say 20 common sugars. The number of possible unsubstituted trisaccharides would be [permutations x anomerics x ring sizes X linkages] or [203 x 23 x 23 x 121 (linkage position potential ranges from 9 to 16, taking an aver- age around 12) or >6,000,000 linear struc-

tures, plus around 3,000,000 branched structures for a total of 9 x lo6 vs 8000 for 20 amino acids (calculation reviewed below).

Among all biological molecules, carbohydrates, in a short sequence, can potentially display the largest number of ligand structures to the binding sites of proteins in molecular recognition systems. The 3-dimensional presentation of carbohydrate structures to epitope-specific recognizing proteins comprises a high level language biochemical code. In this view, DNA can be looked upon as machine language, coding for the lectins and the sets of transferases that assem- ble the sugars. Antibodies are a prime example of binding proteins, being exquisitely sensitive to all of the carbohydrate structural elements. The isomer permutations of small (M,

2 1 The Information-Storing Potential of the Sugar Code mutations, the second, anomeric configuration, the third, ring size and the fourth, linkage position. Individual formulas and a master set of equations with graphs and tables of these results has been published for determination of all possible reducing end isomers for di- to octasaccha- rides (Laine, 1994). Oligomers higher than dp8, which contain the possibility for numerous branching isomers, generate astronomical numbers, larger than Avagadro's number. For 9-mers there is 1 mole of isomers! These numbers are artificially small, however due to the artifice of using the same size monomer library as the oligomer size. Using a common vocabulary of 20 different sugars to make up the hexasaccharide would give a number 1372 fold larger than either of the above (leading to 2.7X1Ol4 linear and 1.44 X 1015 total isomers including branched). In this case the first permutation term would be 206 instead of 66. A hexapeptide from 20 amino acids would yield 6 . 4 ~ 1 0 ~ structures, 8 orders of magnitude lower.

2 Details of Calculation for Isomers of a Trisaccharide

A calculation for the number of possible trisaccharides obtainable from a set of 3 hexoses, uses the formula: Structures = Enx 2: x 2; x 4"-'. The first term En represents the permutations from order of sequence including repetitions of the same sugar 33 = 27. In this term, E is the library of sugars (3 in this case), and n is the oligomer size (also 3 in this special case). The total is multiplied by another term for ring size, 2: or 23 = 8 since most sugars can occur in either pyranose or furanose forms. The total is again multiplied by 2;, a term for anomeric configuration: 23 = 8.

The linkage position term 4"-' is relevant for 2 of the 3 sugars, where 4 potential hydroxyls are available for linkage to the previous sugar (hence the n-1) and gives a number of 42 = 16. In furanose forms in a trisaccharide of sequence ABC, sugar A could have been connected through the 5 position of sugar B, for example. This factor is taken into account by the ring size term keeping the total possibilities of linkage positions at 16.

6 /OH

'OH

/AH

Pyranose ring form of Dgiucose

a Anorneric Configuration

CHPOH \ p -Anomeric Configuration

'OH 1 Furanose ring form of Dgiucose H

Figure 1

Thus, the correct number for permutations of linear trisaccharides made up from a set of 3 hexoses is 27 x 8 X 8 x 16 = 27,648.

In branched trisaccharides sugars A and B are both glycosides to sugar C by 2,3; 2,4; 2,6; 3,4; 3,6 or 4,6 branching presenting six possibilities. With sugar C as furanose, additional isomers include 2,3; 2,5; 2,6; 3 3 ; 3,6; or 5,6 for a total of 12 different branched structures. However, the ring size term 2:, takes into account the additional 6 structures engendered if C were furanose.

A(1->6) B(l->6) \ \ C(l->R)* or C(l->R)* / /

B(l->3) A(1->3)

*R = reducing end attachment site (aglycon)

Since each branch can occur in two different ways such as A6,B3 or B6,A3, there are again 12 different ways to branch these three sugars. The permutation term, En, however, takes care of the A6,B3 and B6,A3 branching duplex. Therefore, unique branched trisaccharides from a set of 3

3 Analysis I 3 hexoses are 27x8 X 8 x 6 = 10,368. The total number of structures from a trisaccharide com- prised of 3 hexoses, choosing among a set of only 3 different hexoses, is 27,648 (linear forms) plus 10,368 (branched forms) = 38,016. The formula for isomers of a trisaccharide having a reducing end is thus:

E x 2: x 2; x 4- (linear forms) + En x 2: x 2: * x 6n-2 (branched forms)

2.1 Non-Reducing Oligosaccharides

Trisaccharides can assume the trehalose-type di- saccharide aldose-1->1-aldose or the raffinose non-reducing aldose-1->2 ketose internal linkage structure, giving a larger number for possible trisaccharides. Longer oligosaccharides can form cyclodextrins. These kinds of permutations would add a large number of oligosaccharides to an isomers calculation. For cyclodextrin hexasaccharides, multiply by 4 the linear permutations number due to a term added by the extra head-to-tail linkage (to 5 possible hydroxyls), making the cy- clodextric versions of hexasaccharides alone close to 0.8 trillion. Some of the cyclic isomers might be identical, however, depending on the chosen cyclic starting position.

z 10

4 ::: 11 OLIGOSACCHARIDES

E 10

0 10

b PEPTIDES

3 Analysis

3.1 NMR

The use of NMR as a single spectroscopic method with the use of a chemical shift library is ques- tionable, even with saccharides as small as 3 sugars. Each trisaccharide from the conservative set using only 3 hexoses would contain 15 ring protons including the anomeric, thus the proton NMR spectrum library would require resolution of 38,016X 15 = 570,240 different proton envi- ronments within 0.5 ppm. This would require a resolution of ppm, (a terahertz instrument) if the line widths were also narrowed concomi- tantly. It is doubtful that a tenth of this number of lines could be resolved using multi-dimension proton NMR. In fact, the carbon-13 spectrum, thirty times more dispersed, to form a chemical shift library would need to resolve 38,016 X 18 car- bons = 684,288 lines if they all happened to be different. This would require a resolution of 2~ ppm, still in the terahertz range. NMR by itself, therefore cannot be used to establish a chemical shift library as a stand-alone identification system for trisaccharides and certainly not for larger oligomers to absolutely identify complete structure by virtue of chemical shift values. NOE and multi-dimension NMR can expand the available data to some degree, but not enough to overcome the chemical shift resolution problem, upon which all of the techniques must ultimately depend. However, much information is available from 1D proton NMR using 100 nanomoles of oligosaccharide.

On the other hand, having.1-5 micromoles of a pure trisaccharide with 15 lines to resolve in OSppm and with accidental overlaps mini- mized, the use of nuclear Overhauser effect (NOE) and 2-D NMR techniques can be used to completely identify the epimers, linkage positions and anomeric configurations. This is useful for confirmation of synthesis, but rarely useful in the case of identification of small amounts of biologically active saccharides where often only nanomole quantities are available.

0 1 2 3 4 5 6 7 DEGREE OF POLYMERIZATION

Figure 2

4 1 The Information-Storing Potential of the Sugar Code 3.2 Mass Spectrometry

v) 240-

z: 0 180.

180'

Mass spectrometry cannot be used by itself to identify oligosaccharides. All 10l2 isomers made of D-hexoses, for example, would have the same mass. There may be some fine structure in mass spectra due to linkage position and preferences in cleavage of the rings. Partial fragmentation in collisional activated mass spectrometry might provide the combination of partial degradation and spectral pattern to resolve such parameters as position of linkage (Laine et al., 1988, 1991; Laine, 1989; Yoon and Laine, 1992), but will not be sufficient without other sensitive chemical manipulations (see Hounsell, this volume). The advantage of mass spectrometry for partial structural analysis is its inherent nanomole to pico- mole sensitivity.

4 Synthesis

Chemical synthesis of a trisaccharide takes 20 man-weeks (Personal Communication: 0. Hinds- gaul, Edmonton, Alberta; K. Matta, Buffalo, NY; P. Garegg, Stockholm) compared with 3 hours for a tripeptide (automated solid state syn- thesizer). Part of the difficulty is the isomer problem. If the trisaccharide sought is one out of a possible 38,000 isomers, this is the crux of the synthetic problem and reason for the lack of automated systems.

5 Biologically Relevant Oligomer Size

With few exceptions in glycobiology, hexasaccharides are the upper limit for protein-recognized oligosaccharide sequences (Cisar et al., 1974, 1975; Takeo and Kabat, 1978; Smith-Gill et al., 1984), and repeating units in polysaccharides sel- dom exceed 6 sugars in size.

6 Substitutions

Often, carbohydrates are substituted with functional groups. Returning to the example of trisaccharides made up from a set of 3 hexoses, each

member of 38,000 isomeric structures could be substituted, for example, by one sulfate in any of 10 free positions. Therefore there are more than 380,000 possible singly sulfated trisaccharides composed from a set of 3 hexoses. Also there are 380,000 potential singly-0-methylated structures and a similar number of singly acetylated structures, to say nothing of phosphates, carbamoyla- tes, pyruvates, other kinds of derivatives and combinations. There are 44 ways to put 2 sulfates on one trisaccharide. Using 38,000 trisaccharides made of a 3 hexose vocabulary there would be 1.7 million possible structures with 2 sulfates. Using a more reasonable 20 sugar vocabulary for trisaccharides there would be 90,000,000 singly sulfated potential structures, and 4 x lo8 potential disulfaced trisaccharide isomers. Naturally, the numbers are much higher for oligomers such as hexasaccharides.

300 TWO DIFFERENT SUBSTlTUENTS 280 20

/ / 120 lm1 / A

MxlBLE 80 / // SUBSTITUTIONS 40 20 / NUMBER OF SINGLE SUBsmuTlONS

O O 1 2 3 4 6 6

OUGOMER

Figure 3

7 Oligosaccharide Recognition by Proteins

The cognate recognition partner for carbohydrate-based biological information is a protein with a specific sugar-binding site, such as a lectin, an antibody, a transferase or a glycosidase. A few investigators also believe that a specific carbo-

8 Evolution of Carbohydrate Code Structures and Receptors I 5 hydrate could be recognized by, and bind to, another carbohydrate (see Bovin, this volume). Lectins and antibodies are exquisitely sensitive to carbohydrate molecular structure, with precise recognition for the 7 major saccharide structural motifs:

0 epimers (including D and L) 0 sequence of sugars 0 anomeric configuration 0 ring size 0 linkage position 0 branching 0 charge (COOH, sulfate or NH3+, for exam-

ple)

Other structural motifs that make the biological recognition sites more complex include substitutions such as the following: (see also Sharon and Lis; Hooper et al., this volume)

0 phosphate 0 phosphonate 0 acyl groups (acetates, 0-1 fatty acids in nod

factors, mycolic acids in cord factor) 0 alkyl ether groups, most commonly

O-methyl 0 pyruvylation

sulfation 0 sulfonation 0 carbamoylation and others

The substitution of one methyl group anywhere on the 19 free hydroxyls of each of 1OI2 different hexasaccharides would give 19 x 1012new structures. For 2 methyl groups, this number would need to be multiplied by a factor of [(H-l)+(H- 2)+(H-3)+ ....+( H-(H-I))], where H is the number of free hydroxyls. For 2 different substituents, this factor would need to be multiplied by 2 to account for reciprocal locations.

8 Evolution of Carbohydrate Code Structures and Receptors

Evolution of receptorhigand pairs in carbohydrates is probably very slow. This is due to the necessity for development of specific sets of gly-

cosyltransferases to generate the carbohydrate ligand on one hand, and evolution of a binding site in a protein to recognize the new structure on the other. Cell-recognition factors and signal- ling systems would therefore be expected to be conserved across species and sometimes across genera. The selectins as leukocyte extravasation adhesins in mammals are a good cross-species example (Brandley et al., 1990; Polley et al., 1991; Yuen et al., 1992; Asa et al., 1995; Lasky, 1995). Single point mutations in glycosyltransferase proteins or lectins are not likely to alter target sugar structures or receptor binding sites. In a few known cases a minor amino acid change in a transferase or a lectin brings about recognition of a closely related sugar (Yamamoto et al., 1990; Jordan and Goldstein, 1995). For polypeptide-based carbohydrate recognition, such as in lectins, information is car- ried in one or more genes. It was an early hypo- thesis that adhesins could be related to binding sites in glycosyl transferases (Roth et al., 1971). There may be sets of motifs conserved among families of lectins such as the mannose-binding protein and selectins (Bajorath and Aruffo, 1995).

Evolution for biological recognition of just one additional or one altered sugar on an existing structure may re9uire a combination of the fol- lo wing: 1) On the carbohydrate side, mutation of the

peptide sequence of an existing glycosyltransferase, or evolution of an entirely novel transferase is needed to produce a new gene product. This new gene product could be a transferase that transfers another sugar, a different sugar, or the same sugar with a different linkage to the precursor structure. The complex carbohydrate ligand is coded into a set of glycosyltransferase genes coding sequentially act- ing enzymes. Each partially glycosylated precursor is recognized in the binding site of the subsequent glycosyltransferase. This could be called a sequential binding site pattern. Tri-antennary and tetra-antennary complex N-linked structures are among the largest non-template-driven precise structures in biology.

6 I The Information-Storing Potential of the Sugar Code 2) On the binding protein side, evolution of a

new or modified lectin is necessary to express a new bindinghecognition site.

9 A High Level Biological Code

In all natural polymers, the linear sequence of monomers comprises, in some fashion, a biological code. For carbohydrate polymers, as different from oligomers, this code may express itself as the chemical properties of the polymer (fi- brous, such as cellulose and chitin, soluble gel as starch or agar) which may include specific hydrogen bonds and other elements of self-association. Complex carbohydrate polymers may have subsets of internal oligosaccharides which possess biological activity based on specific proteins that bind small, specific, possibly rare internal sequences. Carbohydrate polymers themselves often contain a complex multifaceted sequence. Specific proteins can bind to relatively short subsets or haptens within longer saccharide sequences, such as in heparin (Riensenfeld et al., 1977; Atha et al., 1987; van Boeckel et al., 1993).

Heparin or heparan sulfate is an especially good example with the following biological activities being ascribed to specific sequences:

antithrombinII1 binding pentasaccharide (Lindahl et al., 1979,1980,1981; Rosenberg et al., 1981; Casu, 1981, 1994; Choay, 1983; Oscarsson, 1989), fibroblast growth factor binding hexasaccharide (Karlsson et al., 1988; Ornitz et al., 1992; Maccarana and Lindahl, 1993; Coltrini et al., 1994; Ishihara et al., 1993, 1994; Schmidt et al., 1995),

smooth muscle cell growth inhibition (Hoover et al., 1980),

interaction with platelet factors (Niewiarowski et al., 1979; Maccarana and Lindahl, 1993), virus receptors (Lycke et al., 1991), anti-neoplastic sequences and other binding activities such as superoxide dis- mutase (Karlsson et al., 1988).

The heparin cofactor I1 binding hexasaccharide sequence in dermatan sulfate is another example of a biologically active internal sequence in glyco- saminoglycans (Tollefsen et al., 1986; Tollefsen, 1992, 1994).

10 Lectins in Biological Recognition

Over the past 10 years, a dramatic number of new biological activities have been ascribed to recognition systems between carbohydrates and proteins.

The number of papers having the word lectin in the title or abstract has increased remarkably over the past 20 years. A Medline computer search today will show more than 7500 articles with lectin in the title or abstract. There are 6 chapters in this volume with lectin in the title, and many of the chapters describe biological recognition systems.

Lectins, enzymes and antibodies can exhibit dis- criminating binding specificities for the shape, charge, epimers, anomers, linkage positions, ring size, branching and monosaccharide sequence of carbohydrate ligand molecules where the maxi- mum recognized size is usually hexamer or smaller (Cisar et al., 1974, 1975; Takeo and Kabat, 1978; Smith-Gill et al., 1984). Carbohydrate sequences possess unique solution structures which, although dynamic, are shown by NOE NMR and molecular modeling to be populated mainly by minimum energy 3-dimensional conformations (Cumming and Carver, 1987; Poppe et al., 1990; French et al., 1993; Siebert et al., this volume). Oligosaccharide haptens, more rigid than short peptides because of steric crowding, must be envisioned in 3 dimensional space for specific recognition by proteins.

11 Biological Mechanisms of Lectins or other Carbohydrate Binding Proteins

Oligosaccharides can partake in diverse processes: signal transduction mechanisms, for example the well known activity of concanavalin A on certain leucocytes, and the activation of neu-

13 Bacterial/Eucatyotic Interactions I 7 galactose-binding protein (Rice et al., 1990). Spe- cific charge spacing on sulfated polymers may confer recognition by certain patterns of basic amino acids in cognate proteins. Possible higher complexity might occur where patterns in sets of carbohydrates form recognition systems with sets of binding proteins. Such systems may play a powerful role in intercellular sociology during development (Feizi, 1985, 1988), in the immune system (Brandley et al., 1990; Polley et al., 1991; Yuen et al., 1992; Asa et al., 1995; Lasky, 1995) and in parasitology (Friedman et al., 1985; Per- eira, this volume) and other microbial pathogenesis (Srnka et al., 1992; see Gilboa-Garber et al., this volume).

Numerous reviews and recent papers have been written regarding new discoveries in carbohydrate-based recognition systems as tumor markers (Hoff et al., 1989; Matsushita et al., 1990, 1991; Walz et al., 1990; Irimura et al., 1991; Miller et al., 1992; Chanrasekaran et al., 1995).

trophil integrins upon ligandation with P- selectin (Lasky, 1995) or in regulatory molecules (Villalobo et al. this volume; Zanetta, this volume), as signals for polypeptide location within the cell, such as lysosomal protein markers (Reit- man and Kornfeld, 1981; Hooper et al., this volume), as ligands for proper protein folding: Glyco- sylation is apparently important in recognition of proper folding for protein chaperones such as calnexin (Chen et al., 1995; Hebert et al., 1995; Ora and Helenius, 1995), in the metazoan, for specific cell surface recognition of one cell by another (Mann et al., this volume), in plants, the rhizobium recognition system utilizes a sulfated chitin oligosaccharyl glycolipid (nod factors) with specific acylation and sulfation for species specificity (Lerouge et al., 1990; Roche et al., 1991, 1992; Debelle et al., 1992; Denarie et al., 1992, 1994; Price et al., 1992; Demont et al., 1993, 1994; Denarie and Cullimore, 1993; Horvath et al., 1993; Mergaert et al., 1993; Ardourel et al., 1994; Journet et al., 1994; Lerouge, 1994; Relic et al., 1994; Schwedock et al., 1994; Spaink, 1994; Jabbouri et al., 1995; Price and Carlson, 1995; Stokkermans et al., 1995), in plants, pollen tube growth appears to be dependent on specific glycosylation gradients of a resident protein in the pistil (Balanzino et al., 1994), in plants, chitin oligosaccharides apparently can stimulate host responses to fungal infec- tions, and chitinases are utilized as plant defense systems (Scheel and Parker, 1990).

The multitude of known plant and animal lectins predicts a host of other very interesting biological activities, yet undiscovered.

12 Multivalent Effects

A collective of low avidity interactions may vastly strengthen intercellular binding (Lee et al., 1990). Specific spacing of carbohydrate moieties within a larger structure may confer several orders of magnitude tighter binding such as in the

13 BacteriaVEucaryotic Interactions

In another interesting area, microbes simulate mammalian complex carbohydrates to use as immune masks (E. coli K5 N-acetyl heparosan) (Casu et al., 1994)), E. coli K1 colominic acid, E. coli K4 fructosyl chondroitin analog, and Helico- bacter pylori using LeX (Noel et al., 1995). Some bacteria also produce hyaluronic acid. Microbes definitely use lectin-like interactions in pathogenic systems such as enterotoxic E. coli (Neeser et al., 1986), which are, interestingly, inhibited by mecomium oligosaccharides (Neeser et al., 1986), the well studied cholera toxidGM1 ganglioside system for which there are recent studies (Balan- zino et al., 1994).

In bacterial/eucaryotic interfaces, apparently there are general systems for recognition of certain microbial cell surface carbohydrate charac- teristics. For example, CD14 is apparently necessary for recognition of lipopolysaccharides by human endothelial cells (Noel et al., 1995). A long liter

glycosciences status and perspectives (chapman&hall, 2002)

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