The challenge and promise of glycomics

Abstract

Glycomics is a broad and emerging scientific discipline focused on defining the structures and functional roles of glycans in biological systems. The staggering complexity of the glycome, minimally defined as the repertoire of glycans expressed in a cell or organism, has resulted in many challenges that must be overcome; these are being addressed by new advances in mass spectrometry as well as by the expansion of genetic and cell biology studies. Conversely, identifying the specific glycan recognition determinants of glycan-binding proteins by employing the new technology of glycan microarrays is providing insights into how glycans function in recognition and signaling within an organism and with microbes and pathogens. The promises of a more complete knowledge of glycomes are immense in that glycan modifications of intracellular and extracellular proteins have critical functions in almost all biological pathways.

Figure 1

The general roles of glycans in glycoproteins involve both their direct recognition by glycan-binding proteins (GBPs), and indirect effects of glycans on glycoprotein interactions, which may be dependent on protein-protein, protein-lipid, or even carbohydrate-to-carbohydrate interactions. This includes both glycoprotein on the cell surface, cellular organelles, and in secretions, as well as intracellular glycoproteins, e.g. O-GlcNAc glycoprotein. The bottom depicts many of the major classes of glycan linkages to proteins and lipids, along with the symbol key for representing glycan structures with abbreviations of monosaccharides and other substituents.

Figure 2

Glycan-Related Genes. Compilation of data from several databases identifies glycosylation-related human and mouse genes (glycogenes). Modified and updated from Nairn et al., (Nairn et al., 2008).

Figure 3

To explore protein-glycan interactions one major technology in the field is glycan microarrays. In such an approach, individual glycans from natural sources or chemo/enzymatic syntheses, are modified to allow their automated printing and attachment, either covalently or non-covalently, to a slide-type surface in defined positions, akin to a gene array. The glycan microarray can be interrogated with a GBPs or other reagents or even cells and viruses, to identify those glycan “spots” that are recognized, which can be visualized by either direct or indirect fluorescent tagging. The spot pattern on the image, which should also incorporate replicates of each glycans, can be averaged to generate a histogram. In the example shown, the results indicate that that the GBPs in question bound strongly to one glycan, less strongly to another glycan, and did not bind appreciably to any other glycan. Such microarrays can also be prepared from glycolipids, glycopeptides, whole glycoproteins, or polysaccharides of animal, plant, or microbial origins.

Figure 4

GBPs often recognize 2–6 linear or branched monosaccharides with additional modifications, such as phosphorylation, sulfation, O-acetylation, etc. Such recognition can be termed a glycan determinant, which is the minimal glycan structure that confers maximum binding. These glycans determinants can be thought of as being assembled from partial determinants, that in the example shown are modified galactose, modified Gal-GlcNAc, and modified GlcNAc-Man. With the partial determinants shown, which are respectively, 6, 9, and 3 in number, it is possible to assemble these into 162 different glycan determinants. Shown also are two of these as Determinant 1 and 2, which are differently recognized by a GBPs or GRM. If one considers all such known partial determinants in human glycans defined to date, it is possible to predict that there are over 5,000 glycan determinants; if the GAG sequences are also included up to pentasaccharides, then there are an additional 10,000 or so. It is likely that this is an underestimate for the total theoretical glycan determinants, since it is likely that other partial determinants will be identified in the future. In addition, it is possible that in a single branched or linear glycan, the one set of glycan determinants may attenuate the recognition of the same or a different glycan determinant on the same molecule.

Figure 5

The different classes of glycans, from the GPI glycans within GPI-anchored glycoproteins, to the GAGs, represent different levels of complexity and numbers and diversity of glycan determinants. Of course with complexity and increased numbers of determinants, the analytical difficulties expand tremendously. Thus, a great challenge of glycomic technologies is to difficult better methods of preparing and analyzing glycans to overcome the challenges of their complexities.

Figure 6

The general strategies for glycomic analyses typically involves isolating or generating free glycans from glycoproteins/proteoglycans and glycolipids. The obtained mixture of glycans can then be derivatized and directly analyzed by MS, or derivatized, separated by HPLC and other approaches, and further analyzed by MS or NMR. In shotgun glycomics for functional studies, the released glycans after separation can be printed to generate glycan microarrays. For site-specific glycosylation and identification of protein carriers, glycopeptides can be generated by proteolysis and then analyzed directly before or after glycan removal.

Figure 7

The higher order functions of glycans are typically revealed in physiological studies of organisms, and to a lesser extent perhaps in studies based on isolated tissues and cells. Thus, mutations of genes involved in specific glycosylation pathways of anabolism or catabolism might have little effects on cultured cells, but are deleterious to organismal development; increasing biological complexity is denoted by the green arrow. Of course, the analytical ease of defining the glycome itself is simplified by considering free or released glycans, compared to individual glycoconjugates, such as a glycoprotein, and becomes increasingly difficult with larger biological complexity to the organism itself, denoted by the blue arrow.