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Carbohydrates and glycoconjugates
Carbohydrates and glycoconjugates Editorial overview Kurt Drickamer and Steve Homans Columbia University, New York, USA and University of Dundee, Dundee, UK Current Opinion in Structural Biology 1993, 3:667--668 Two themes dominate the reviews in this year's survey of structural glycobiology: flexibility and diversity. From the experimental point of view, these features can be frustrating. They are, of course, not unique to carbohydrates, but they seem to be more keenly felt: one three-dimensional structure does not describe an oligosaccharide as well as one structure can describe a DNA molecule, for example, and one approach to determining covalent structure does not apply to all oligosaccharides in the way that standardized techniques work for most proteins. Diversity and flexibility may however also endow oligosaccharides with some of the properties that define unique functional niches. The reviews in this section provide an overview of current efforts to deal with flexibility and diversity at the experimental level, and attempts to develop an understanding of how these features are biologically useful. From an experimental point of view, Rice et al. (pp669-674) note that important differences are observed between the structures of oligosaccharides studied as complexes with proteins analyzed by X-ray crystallography, and the corresponding preferred conformations found in solution using NMR. They note that the results are not incompatible, but that the more static X-ray view of the structures reflects the fact that interactions with proteins in the crystal lattice favor certain specific conformations selected from the many that are sampled by the free oligosaccharides. The importance of specific time scales for appropriate spectroscopic measurement of the oligosaccharide motions is not widely recognized; the value of optical methods as well as NMR is also pointed out. The energetic basis for the observed flexibility of oligosaccharides is discussed by Perez (pp 675-680). The available force fields are being improved, so that not only the existence of multiple secondary local energy minima but also the size of the energy barriers between them can be modeled with increased accuracy. In order to account for the observed data from optical and NMR data, appropriate averaging of the characteristics of all the conformations sampled must be undertaken. Important areas currently receiving attention are the application of available theory to larger structures, such as those found on natural glycoproteins and glycolipids, and the integration of these results with molecular information about carbohydrate-protein interactions in order to understand how particular conformations are stabilized by interaction with proteins.
Recent progress in the analysis of protein-carbohydrate interactions by X-ray crystallography is summarized by Bourne, Tilbeurgh and Cambillau ( p p 681-686). As with sugar structure, there is plenty of diversity. The most universal feature of these interactions is the importance of hydrogen bonds, often through water, that mediate sugar contacts with other portions of oligosaccharides as well as with proteins. On the other hand, direct sugar-bivalent metal ion interactions, which have not been seen in plant lectins, are evident in the calcium-dependent mannose-binding protein from mammalian serum, and hydrophobic interactions between sugar and protein are not seen in the mannose-binding protein but are comm o n features of many protein-sugar complexes, including the plant lectins. Interestingly, the enzyme xylose isomerase shows evidence of both of these latter types of interaction. The value of testing the implications of structural models by site-specific mutation of the proteins is emphasized. Our understanding of one protein-carbohydrate interaction, that between influenza virus neuraminidase and its substrate, has advanced to that point that it suggests the design of effective inhibitors. Clearly, these studies will serve as models for the design of ther apeutics that interfere with a variety of sugar-mediated recognition events. Two reviews emphasize the continuing need for technical advances in order to deal with the complexity of the carbohydrate universe just at the level of covalent structure. Dell and Khoo (pp687-693) remind us that, far from reaching a plateau, the number of new structures determined is increasing dramatically. The complexity defies structural analysis by any single method, but the power of advanced mass spectroscopic methods is clear. Examples cited come from bacteria and plants, as well as mammals. The continuing need for evolution in analytical technology is exemplified by recent studies of novel sulphated oligosaccharides found on an increasing number of glycoproteins. Analysis of the complex and selective nodulation signals produced by soil bacteria is an excellent example of how structure analysis is most revealing when correlated with specific biological activities. Just as no one method suffices for all structural analysis, no single approach to the synthetic preparation of oligosaccharides will provide all of the desired products. Halcomb and Wong (pp694-700) review recent progress in the use of both chemical and enzymatic methods. The chemical approaches have the potential advantage of not being limited to the constituent sug-
~) Current Biology Ltd ISSN 0959-440X
667
668
Carbohydrates and glycoconjugates ars used by glycosyl transferases and the linkages that they form. Novel approaches to achieving selective stereochemistry and to dealing with sialic acid are highlighted. Given the power of solid- phase methods in peptide and oligonucleotide synthesis, it is encouraging that progress using immobilized glycosyl donors is also reported. The use of enzymes to make novel oligosaccharides is proving to be more flexible than might have been anticipated. The activities of known enzymes towards new substrates are being explored, along with regeneration systems that eliminate the need for large quantities of expensive nucleotide donors. It is reasonable to hope that in the near future it will be possible to design and prepare modified sugars with enough facility to allow wide-scale analysis of the importance of individual sugar atoms in stabilizing particular conformations and in mediating specific interactions with protein receptors. In the absence of large libraries of synthetic oligosaccharides, much of our current understanding of sugar selectivity results from comparisons of how endogenous lectins such as the selectin cell-adhesion molecules discriminate between naturally occurring oligosaccharides. Ligands for the selectins are of particular interest because of the importance of these receptors in the initial phases of inflammation. The demonstration that specific saccharides form a dominant part of the target for the selectins provides convincing evidence that complex sugar structures at the cell surface serve as recognition elements. Yet there remains much that we do not understand: new oligosaccharides that bind to the selectins continue to be described, differences between the preferred ligands for the three selectins are becoming increasingly apparent, and glycoproteins (and glycolipids?) that present the saccharide ligands are still being defined. Current progress on all these fronts is reviewed by Feizi (pp 701-710). Of course, the enormous variety of natural glycoconjugate structures implies a corresponding diversity of enzymes that mediate their synthesis. In general, there has been a disappointing lack of similarity among glycosyl transferases. As pointed out by van den Eijnden and Joziasse (pp711-721), however, similarities within certain specific families, such as the sialyl, fucosyl, and a subset of the N-acetylglucosaminyl transferases, have started to emerge. In addition to describing novel transferases associated with the synthesis of new carbohydrate structures, a major emphasis in this field is determining how glycosyl transferase expression is temporally and spacially regulated. Regulatory signals that control transcription of these genes clearly represent one way in which control of oligosaccharide biosynthesis can be controlled during differentiation and development. In addition, studies on signals that lead to localization in various positions within the secretory pathway, and analysis of the behavior of
alternatively spliced forms of the transferases, provide an essential link between the genetic code that defines these proteins and the order in which the oligosaccharide chains are assembled. Recent experiments involving the selectin cell-adhesion molecules provide compelling evidence for the importance of carbohydrates as recognition markers and high light the importance of continuing structural work in this area. In a designed experiment, decreased efficiency of leukocyte extravasation observed in transgenic mice lacking P-selectin directly implicates this sugar-selective adhesion molecule in the biology of inflammation [1]. Conversely, in an experiment of nature, failure to produce an appropriate fucosylated ligand for the selectins has been linked to a clinical syndrome involving both susceptibility to infections and developmental abnormalities [2,3]. Exploitation of our knowledge of sugar-mediated adhesion to permit clinical control of inflammation is also moving closer to reality, with the demonstration that the sialyl Lewis-x tetrasaccharide can be used to inhibit experimentally induced alveolar hemorrhage [4]. Our understanding of biological processes mediated by carbohydrate-specific recognition is advancing rapidly. It can be hoped that elucidation of the structural basis for these and other carbohydrate-mediated processes will progress as rapidly.
References MAYADAS TN, JOHNSON RC, RAYI3URNH, HYNES RO, WAGNER DD: Leukocyte RoBing and Extravasation Are Severely C o m p r o m i s e d in P Selectin-Deficient ice. Cell 1993, 74:541-554. ETZIONI A, FRYI)MAN M, POLLACK S, AVIDOR I, PHILLIPS ML, PAULSON JC, CJEP~SHONI-BARUCH R: Brief Report: Recurrent Severe Infections Caused by a Novel Leucocyte Adhesion Deficiency. N Engl J Med 1992, 327:1789-1792. FRYDMAN M, ETZIONI A, EIDITZMARKUST, AVIDOR I, VARSANOl, SHECKTERY, ORLINJB, GERSHONI-BARUCHR: Ramban Hasharon Syndrome of ental Retardation, Short-Limbed Dwarfism, Defective Neutrophfl Chemotaxis and Bombay Phenotype. A m J Med Genet 1993, 44:297-302. MULIaGAN MS, PAULSONJC, FREES SD, ZHENG Z L, LOWE JB, WARD PA: Protective Effects of Oligosaccfiarides in PSelectin-Dependent Lung Injury. Nature 1993, 364:149-151.
K Drickamer, Department of Biochemistry, and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, New York 10032, USA. S Homans, Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, UK.
Recent progress in the analysis of protein-carbohydrate interactions by X-ray crystallography is summarized by Bourne, Tilbeurgh and Cambillau ( p p 681-686). As with sugar structure, there is plenty of diversity. The most universal feature of these interactions is the importance of hydrogen bonds, often through water, that mediate sugar contacts with other portions of oligosaccharides as well as with proteins. On the other hand, direct sugar-bivalent metal ion interactions, which have not been seen in plant lectins, are evident in the calcium-dependent mannose-binding protein from mammalian serum, and hydrophobic interactions between sugar and protein are not seen in the mannose-binding protein but are comm o n features of many protein-sugar complexes, including the plant lectins. Interestingly, the enzyme xylose isomerase shows evidence of both of these latter types of interaction. The value of testing the implications of structural models by site-specific mutation of the proteins is emphasized. Our understanding of one protein-carbohydrate interaction, that between influenza virus neuraminidase and its substrate, has advanced to that point that it suggests the design of effective inhibitors. Clearly, these studies will serve as models for the design of ther apeutics that interfere with a variety of sugar-mediated recognition events. Two reviews emphasize the continuing need for technical advances in order to deal with the complexity of the carbohydrate universe just at the level of covalent structure. Dell and Khoo (pp687-693) remind us that, far from reaching a plateau, the number of new structures determined is increasing dramatically. The complexity defies structural analysis by any single method, but the power of advanced mass spectroscopic methods is clear. Examples cited come from bacteria and plants, as well as mammals. The continuing need for evolution in analytical technology is exemplified by recent studies of novel sulphated oligosaccharides found on an increasing number of glycoproteins. Analysis of the complex and selective nodulation signals produced by soil bacteria is an excellent example of how structure analysis is most revealing when correlated with specific biological activities. Just as no one method suffices for all structural analysis, no single approach to the synthetic preparation of oligosaccharides will provide all of the desired products. Halcomb and Wong (pp694-700) review recent progress in the use of both chemical and enzymatic methods. The chemical approaches have the potential advantage of not being limited to the constituent sug-
~) Current Biology Ltd ISSN 0959-440X
667
668
Carbohydrates and glycoconjugates ars used by glycosyl transferases and the linkages that they form. Novel approaches to achieving selective stereochemistry and to dealing with sialic acid are highlighted. Given the power of solid- phase methods in peptide and oligonucleotide synthesis, it is encouraging that progress using immobilized glycosyl donors is also reported. The use of enzymes to make novel oligosaccharides is proving to be more flexible than might have been anticipated. The activities of known enzymes towards new substrates are being explored, along with regeneration systems that eliminate the need for large quantities of expensive nucleotide donors. It is reasonable to hope that in the near future it will be possible to design and prepare modified sugars with enough facility to allow wide-scale analysis of the importance of individual sugar atoms in stabilizing particular conformations and in mediating specific interactions with protein receptors. In the absence of large libraries of synthetic oligosaccharides, much of our current understanding of sugar selectivity results from comparisons of how endogenous lectins such as the selectin cell-adhesion molecules discriminate between naturally occurring oligosaccharides. Ligands for the selectins are of particular interest because of the importance of these receptors in the initial phases of inflammation. The demonstration that specific saccharides form a dominant part of the target for the selectins provides convincing evidence that complex sugar structures at the cell surface serve as recognition elements. Yet there remains much that we do not understand: new oligosaccharides that bind to the selectins continue to be described, differences between the preferred ligands for the three selectins are becoming increasingly apparent, and glycoproteins (and glycolipids?) that present the saccharide ligands are still being defined. Current progress on all these fronts is reviewed by Feizi (pp 701-710). Of course, the enormous variety of natural glycoconjugate structures implies a corresponding diversity of enzymes that mediate their synthesis. In general, there has been a disappointing lack of similarity among glycosyl transferases. As pointed out by van den Eijnden and Joziasse (pp711-721), however, similarities within certain specific families, such as the sialyl, fucosyl, and a subset of the N-acetylglucosaminyl transferases, have started to emerge. In addition to describing novel transferases associated with the synthesis of new carbohydrate structures, a major emphasis in this field is determining how glycosyl transferase expression is temporally and spacially regulated. Regulatory signals that control transcription of these genes clearly represent one way in which control of oligosaccharide biosynthesis can be controlled during differentiation and development. In addition, studies on signals that lead to localization in various positions within the secretory pathway, and analysis of the behavior of
alternatively spliced forms of the transferases, provide an essential link between the genetic code that defines these proteins and the order in which the oligosaccharide chains are assembled. Recent experiments involving the selectin cell-adhesion molecules provide compelling evidence for the importance of carbohydrates as recognition markers and high light the importance of continuing structural work in this area. In a designed experiment, decreased efficiency of leukocyte extravasation observed in transgenic mice lacking P-selectin directly implicates this sugar-selective adhesion molecule in the biology of inflammation [1]. Conversely, in an experiment of nature, failure to produce an appropriate fucosylated ligand for the selectins has been linked to a clinical syndrome involving both susceptibility to infections and developmental abnormalities [2,3]. Exploitation of our knowledge of sugar-mediated adhesion to permit clinical control of inflammation is also moving closer to reality, with the demonstration that the sialyl Lewis-x tetrasaccharide can be used to inhibit experimentally induced alveolar hemorrhage [4]. Our understanding of biological processes mediated by carbohydrate-specific recognition is advancing rapidly. It can be hoped that elucidation of the structural basis for these and other carbohydrate-mediated processes will progress as rapidly.
References MAYADAS TN, JOHNSON RC, RAYI3URNH, HYNES RO, WAGNER DD: Leukocyte RoBing and Extravasation Are Severely C o m p r o m i s e d in P Selectin-Deficient ice. Cell 1993, 74:541-554. ETZIONI A, FRYI)MAN M, POLLACK S, AVIDOR I, PHILLIPS ML, PAULSON JC, CJEP~SHONI-BARUCH R: Brief Report: Recurrent Severe Infections Caused by a Novel Leucocyte Adhesion Deficiency. N Engl J Med 1992, 327:1789-1792. FRYDMAN M, ETZIONI A, EIDITZMARKUST, AVIDOR I, VARSANOl, SHECKTERY, ORLINJB, GERSHONI-BARUCHR: Ramban Hasharon Syndrome of ental Retardation, Short-Limbed Dwarfism, Defective Neutrophfl Chemotaxis and Bombay Phenotype. A m J Med Genet 1993, 44:297-302. MULIaGAN MS, PAULSONJC, FREES SD, ZHENG Z L, LOWE JB, WARD PA: Protective Effects of Oligosaccfiarides in PSelectin-Dependent Lung Injury. Nature 1993, 364:149-151.
K Drickamer, Department of Biochemistry, and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, New York 10032, USA. S Homans, Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, UK.