Glycomics Hits the Big Time

Leading Edge Essay Glycomics Hits the Big Time Gerald W. Hart1,* and Ronald J. Copeland1 1Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA *Correspondence: [email protected] DOI 10.1016/j.cell.2010.11.008 Cells run on carbohydrates. Glycans, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules in nature. Recent advances in glycomics reveal the scope and scale of their functional roles and their impact on human disease. By analogy to the genome, transcriptome, O-GlcNAc (Hart et al., 2007). Even though Dynamic Structural Complexity or proteome, the ‘‘glycome’’ is the the generic term ‘‘glycosylation’’ is often Underlies Glycan Functions complete set of glycans and glycoconju- used to categorize and lump all glycan Glycoconjugates provide dynamic struc- gates that are made by a cell or organism modifications of proteins into one bin, tural diversity to proteins and lipids that under specific conditions. Therefore, side by side with other posttranslational is responsive to cellular phenotype, to ‘‘glycomics’’ refers to studies that attempt modifications such as phosphorylation, metabolic state, and to the developmental to define or quantify the glycome of a cell, acetylation, ubiquitination, or methylation, stage of cells. Complex glycans play crit- tissue, or organism (Bertozzi and Sasise- such a view is not only inaccurate, but ical roles in intercellular and intracellular kharan, 2009). In eukaryotes, protein also is completely misleading. If one only processes, which are fundamentally glycosylation generally involves the cova- considers the linkage of the first glycan important to the development of multicel- lent attachment of glycans to serine, to the polypeptide in both prokaryotic lularity (Figure 1). Unlike nucleic acids and threonine, or asparagine residues. Glyco- and eukaryotic organisms, there are at proteins, glycan structures are not hard- proteins occur in all cellular compart- least 13 different monosaccharides and wired into the genome, depending upon ments. Glycans are also attached to 8 different amino acids involved in glyco- a template for their synthesis. Rather, lipids, often ceramide, which is comprised protein linkages, with a total of at least the glycan structures that end up on of sphingosine, a hydrocarbon amino 41 different chemical bonds known to be a polypeptide or lipid result from the alcohol and a fatty acid. Complex glycans linking the glycan to the protein (Spiro, concerted actions of highly specific gly- are mainly attached to secreted or cell 2002). Importantly, each one of these cosyltransferases (Lairson et al., 2008), surface proteins, and they do not cycle unique glycan:protein linkages is surely which in turn are dependent upon the on and off of the polypeptide. In contrast, as different in both structure and function concentrations and localization of high- the monosaccharide O-linked N-acetyl- as protein methylation is from acetylation. energy nucleotide sugar donors, such as glucosamine (O-GlcNAc) cycles rapidly Of course, this modification is not only UDP-N-acetylglucosamine, the endpoint on serine or threonine residues of many about a single linkage. When structural of the hexosamine biosynthetic pathway. nuclear and cytoplasmic proteins. Identi- diversity of the additional oligosaccharide Therefore, the glycoforms of a glycopro- fying the number, structure, and function branches of glycans and the added diver- tein depend upon many factors directly of glycans in cellular biology is a daunting sity of complex terminal saccharides on tied to both gene expression and cellular task but one that has been made easier in glycans, such as fucose or sialic acids metabolism. recent years by advances in technology (about 50 different sialic acids are known There are at-least 250 glycosyltrans- and by our growing appreciation of how [Schauer, 2009]), are taken into account, ferases in the human genome, and it has integral glycans are to biology (Varki the molecular diversity and varied func- been estimated that about 2% of the et al., 2009). tions of protein-bound glycans rapidly human genome encodes proteins The scope of the glycomics challenge is increase exponentially. Just the ‘‘sia- involved in glycan biosynthesis, degrada- immense. The covalent addition of lome’’ (Cohen and Varki, 2010) rivals or tion, or transport (Schachter and Freeze, glycans to proteins and lipids represents exceeds many other posttranslational 2009). Biosynthesis of the nucleotide not only the most abundant posttransla- modifications in abundance and struc- sugar donors is directly regulated by nu- tional modification (PTM), but also by far tural/functional diversity. In addition, cleic acid, glucose, and energy metabo- the most structurally diverse. Although it chemical modifications, such as phos- lism, and the compartmentalization of is commonly stated that more than 50% phorylation, sulfation, and acetylation, these nucleotide sugar donors is highly of all polypeptides are covalently modified increase the glycan structural/functional regulated by specific transporters. Protein by glycans (Apweiler et al., 1999), even diversity even more. Thus, categorizing glycosylation is therefore controlled by this estimate is far too low because it fails glycosylation as a single type of post- rates of polypeptide translation and to include that myriad nuclear and translational modification is neither useful protein folding, localization of and compe- cytoplasmic proteins are modified by nor at all reflective of reality. tition between glycosyltransferases, 672 Cell 143, November 24, 2010 ª2010 Elsevier Inc. it is estimated that the binding sites of glycan-binding proteins (GBPs), such as antibodies, lectins, receptors, toxins, mi- crobial adhesions, or enzymes (Figure 1), can accommodate only up to two to six monosaccharides within a glycan structure (Cummings, 2009). Therefore, the number of specific glycan substructures that bind to biologically important GBPs in a cell may be fewer than 10,000, a number that is within the realm of current analytical and, if targeted, chemical or enzymatic synthetic capabilities. Until recently, the lack of tools and the inherent complexity of glycans have been major barriers preventing most biol- ogists from embracing the importance of glycans in biology. Recent technological advances have significantly lowered these barriers. Indeed, the tools of glycomics and the subfields of glycoproteomics, gly- colipidomics, and proteoglycomics have all progressed substantially in recent years (Krishnamoorthy and Mahal, 2009; Laremore et al., 2010). Major technological advances, many of which are shared with proteomics, have recently allowed Figure 1. Glycans Permeate Cellular Biology semiquantitative profiling of glycans and Complex glycans at the cell surface are targets of microbes and viruses, regulate cell adhesion and devel- glycoproteins (Krishnamoorthy and opment, influence metastasis of cancer cells, and regulate myriad receptor:ligand interactions. Glycans Mahal, 2009; Vanderschaeghe et al., within the secretory pathway regulate protein quality control, turnover, and trafficking of molecules to organelles. Nucleocytoplasmic O-linked N-acetylglucosamine (O-GlcNAc) has extensive crosstalk with 2010). Some of these advances are the phosphorylation to regulate signaling, cytoskeletal functions, and gene expression in response to nutrients result of the National Institute of General and stress. Medical Science’s (NIGMS) support of the Consortium for Functional Glycomics cellular concentration and localization of gene expression of glycan-processing (CFG), which has served to focus and nucleotide sugars, the localization of enzymes, by polypeptide structure at all assist more than 500 researchers on glycosidases, and membrane trafficking. levels, and by cellular metabolism. issues related to glycomics (Paulson Thus, individual glycosylation sites on the et al., 2006; Raman et al., 2006). same polypeptide can contain different Technology of Glycomics Kobata and colleagues were among the glycan structures that reflect both the A detailed understanding of cellular first to profile N-glycans, well before the type and status of the cell in which they processes will require a detailed appreci- current concepts of glycomics were are synthesized. For example, the glyco- ation of the glycans modulating proteins conceived. Despite the lack of many forms of the membrane protein Thy-1 are and pathways. Although this ultimate modern methods, their pioneering work very different in lymphocytes than they goal of glycomics is laudable, we are was characterized by a high level of rigor are in brain, despite having the same poly- a very long way from having the tech- in defining the arrays of N-glycan struc- peptide sequence (Rudd and Dwek, nology to completely characterize the gly- tures present in cells and tissues and on 1997). Conversely, even small changes in come of even a simple cell or tissue. Not specific proteins (Endo, 2010). Currently, polypeptide sequence or structure will only is the glycome much more complex a wide variety of high-resolution and alter the types of glycan structures than the genome, transcriptome, or pro- highly sensitive methods are available, attached to a polypeptide. For example, teome, as noted above, it is also much including capillary electrophoresis (CE), histocompatibility antigen polypeptides more dynamic, varying considerably

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