Carbohydrate microarrays and the unravelling of ligands for effector proteins of the immune system

Abstract

Optimism (Feizi 1985) that the carbohydrate sequences of glycoproteins, glycolipids and proteoglycans are bearers of crucial biological information is being well borne out (Lindahl et al. 1994; Feizi 2000; Crocker 2001). Knowledge that the human genome encodes no more than 30 000–50 000 proteins has, in the mean time, served to emphasize the importance of oligosaccharide chains as modulators of the activities and functions of the proteins in health and disease, through recognition processes mediated by carbohydrate‐recognizing receptors. The challenge is to develop high throughput technologies to determine the roles of specific oligosaccharide sequences as ligands for carbohydrate‐recognizing proteins. The neoglycolipid (NGL) technology (Feizi et al. 1994) for generating lipid‐linked oligosaccharide probes has many features that render it adaptable for this challenge. This technology is now the basis of a carbohydrate array system that is applicable both to structurally defined oligosaccharides and to those derived from biological sources, glycoproteins, proteoglycans, cells and even whole organs (Fukui et al. 2002). We observe that such repertoires of NGLs are robust probes when presented on nitrocellulose membranes, thus permitting sensitive and high throughput detection of ligands for carbohydrate‐binding proteins. We show that carbohydrate‐recognizing proteins single‐out their ligands, not only in the arrays of homogenous oligosaccharides, but also in arrays of heterogeneous oligosaccharides. The unique feature is that deconvolution strategies are included with thin‐layer chromatography (TLC) and mass spectrometry for sequencing the ligand‐positive components. I shall discuss the applications in elucidating structures of carbohydrate differentiation antigens and the ligands of carbohydrate‐binding receptors of the immune system.