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CHAPTER 17 Preparation and Analysis of Glycoconjugates Ajit Varki,1 Hudson H. Freeze,2 and Adriana E. Manzi1 1University of California San Diego, La Jolla, California 2Sanford Children’s Health Research Center and Burnham Institute for Medical Research, La Jolla, California ABSTRACT Whereas DNA, RNA, and proteins are linear polymers that can usually be directly sequenced, glycans show substantially more complexity, having branching and anomeric configurations (α and β linkages). The biosynthesis of glycans, termed glycosylation, is extremely complex, is not template-driven, varies among different cell types, and cannot be easily predicted from simple rules. This overview discusses the stereochemistry of mono- and oligosaccharides and provides diagrammatic representations of monosaccharides (Fisher projections and Haworth representations) and formulas for representation of glycan chains. A glossary of terms used in glycobiology is also provided. Curr. Protoc. Mol. Biol. 88:17.0.1-17.0.12. C 2009 by John Wiley & Sons, Inc. Keywords: glycan r monosaccharide r glycan analysis r sugar symbols r glycoconjugate r glycobiology INTRODUCTION ing, solubility, stability, subcellular traffick- The modern revolution in molecular biol- ing, turnover, and half-life of the molecules ogy was driven to a large extent by advances to which they are attached. These are matters in methods for analysis and manipulation of of great importance to the cell biologist, pro- DNA, RNA, and proteins. Although oligosac- tein chemist, biotechnologist, and pharmacol- charides (sugar chains or glycans) are also ma- ogist. On the other hand, the successful growth jor macromolecules of the typical cell, they did of several glycosylation mutants as permanent not initially share in this molecular revolution. tissue culture cell lines indicates that the pre- The reasons for this were to a large extent cise structure of many glycans is not criti- technical. Whereas DNA, RNA, and proteins cal for the growth and viability of a single are linear polymers that can usually be di- cell in the protected environment of the cul- rectly sequenced, oligosaccharides show sub- ture dish. Thus, until recently, it was possible stantially more complexity, having branching for many researchers working with in vitro and anomeric configurations (α and β link- single-cell systems to ignore the existence of ages). Thus, whereas three amino acids or glycans. However, with the increasing empha- nucleotides can be combined into six possi- sis on studying cell-cell interactions in normal ble sequences, three hexose monosaccharides development, tissue morphogenesis, immune can theoretically generate 1056 possible gly- reactions, and pathological conditions such as cans. In addition, the syntheses of DNA, RNA, cancer and inflammation, the study of glycan and proteins are template-driven, and the se- structure and biosynthesis has become very quence of one can generally be predicted from important. that of another. In contrast, the biosynthe- The term “glycobiology” has found accep- sis of oligosaccharides, termed glycosylation, tance for denoting studies of the biology of is extremely complex, is not template-driven, glycoconjugates in both simple and complex varies among different cell types, and cannot systems. Many technical advances have oc- be easily predicted from simple rules. curred in the analysis of glycans, making it It is clear that glycans have important, al- now feasible to study them in detail. These beit varied, effects upon the biosynthesis, fold- advances include the development of sensitive Preparation and Analysis of Glycoconjugates Current Protocols in Molecular Biology 17.0.1-17.0.12, October 2009 17.0.1 Published online October 2009 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142727.mb1700s88 Supplement 88 Copyright C 2009 John Wiley & Sons, Inc. and specific assays and the availability of nu- quired to obtain final and definitive results. merous purified enzymes (glycosidases) with Nonetheless, armed with results obtained us- high degrees of specificity. ing the techniques described here, the typi- In spite of all these advances, many analyti- cal researcher can make intelligent decisions cal techniques in glycobiology remained in the about the need for such further analyses. domain of the few laboratories that specialized The appearance of many commercial kits in the study of glycans. Likewise, published for analysis of glycoconjugates is another sign compendia of carbohydrate methods were de- that the technology has arrived and that many signed mainly for use by experts. This chap- laboratories have developed an interest in gly- ter attempts to place some of this technology cobiology. It is worth noting that although within easy reach of any laboratory with basic some of these kits are designed to simplify capabilities in biochemistry and molecular bi- the use of well-established techniques, others ology. The techniques described here include employ methodologies that have been newly modern versions of time-honored methods and developed by the companies themselves. Ex- recently developed methods, both of which perience with the latter methodologies in aca- have widespread applications. However, it is demic scientific laboratories may be limited, important to emphasize that the protocols pre- and the techniques in question may therefore sented here are by no means comprehensive. not be represented in this chapter. However, Rather, they serve as a starting point for the although the admonition caveat emptor is ap- uninitiated scientist who wishes to explore the propriate, some kits may well become useful structure, biosynthesis, and biology of gly- adjuncts to the methods presented here. can chains. In most cases, further analysis us- Different types of glycosylation are ing more sophisticated techniques will be re- perhaps best defined by the nature of the Figure 17.0.1 Common glycan-linkage regions on animal cell glycoconjugates. The most common types of glycans found in animal glycoconjugates are shown, with an emphasis upon the linkage region between the glycan and the protein or lipid. Other rarer types of linkage regions and free glycans that can exist naturally are not shown (reproduced with permission from figure 1.6 in Varki et al., 2009). For the color version of this figuregotohttp://www.currentprotocols.com/protocol/mb1700. 17.0.2 Supplement 88 Current Protocols in Molecular Biology linkage region of the oligosaccharide to lated lactosamines, polylactosamines, O- a lipid or protein (Fig. 17.0.1). Although glycosaminoglycan chains, and blood group the linkage regions of these molecules are sequences), and does not deal with rarer unique, the sugar chains frequently tend to sequences (e.g., bisecting xylose residues, share common types of outer sequences. It N-linked glycosaminoglycans, and β-linked is important to note that this chapter deals GalNAc residues on N-linked glycans). For only with the major forms of glycosylation further information, the reader is directed to found in “higher” animal glycoconjugates— the Key References section at the end of this N-acetylglucosamine (GlcNAc)-N-Asn- chapter introduction. linked, N-acetylgalactosamine (GalNAc)- O-Ser/Thr-linked glycans on glycoproteins, CHOICE OF TECHNIQUES xylose-O-Ser-linked glycosaminoglycans For the novice experimenter in glycoconju- on proteoglycans, ceramide-linked gly- gate analysis, the greatest difficulty is in de- cosphingolipids, phosphatidylinositol-linked ciding which protocols are applicable to the glycophospholipid anchors, and O-linked question at hand, are sensitive enough to yield N-acetylglucosamine (GlcNAc-O-Ser). These results, and are most likely to give useful an- structures are depicted in Figure 17.0.1. Other swers. The following tables are therefore pro- less common forms of glycosylation may vided as a general guide to glycoconjugate need to be considered, especially if prior analysis. Suggestions are made for the pro- literature suggests their existence in a given tocols that are most likely to be useful based situation. Examples of rarer sugar chains upon the questions being asked (Table 17.0.1), include (1) O-linked glucose, mannose, and the amount of material that is available for fucose; (2) N-linked glucose; (3) glucosyl- analysis (Table 17.0.2), and the type of glyco- hydroxylysine; and (4) GlcNAc-P-Ser conjugate that is being studied (Table 17.0.3). (phosphoglycosylation). The user is advised to select protocols based Likewise, this chapter deals only with on the information in these tables and then the most common forms of shared outer to consult the commentary section of the sequences (e.g., sialylated and fucosy- selected protocols for further information Table 17.0.1 Protocol Choice for Glycoconjugate Analysis Based on Question Being Asked Question Suggested protocols (unit no.) Is there anything special about purifying my glycoconjugate? 17.1, 17.2, 17.3 Is my protein glycosylated? 17.1, 17.2, 17.4, 17.5, 17. 6, 17.7, 17.8, 17.9, 17.10, 17.12, 17.13, 17.17, 17.18 How much of my glycoconjugate consists of sugar chains? 17.9, 17.10, 17.12, 17.13, 17.17, 17.18 What monosaccharides are in my glycoconjugate, and in what ratio? 17.4, 17.9, 17.12, 17.16, 17.18, 17.19 How many glycosylation sites are there on my protein? 17.10, 17.13, 17.14B Can I specifically label the sugar chains on my glyconjugate? 17.4, 17.5, 17.6 Does my antibody recognize sugar chains on the glycoconjugate? 17.7, 17.8, 17.12, 17.13, 17.17 Can I selectively release the sugar chains
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