'Glyco-epitope' assignments for the selectins: advances enabled by the neoglycolipid (NGL) technology in conjunction with synthetic carbohydrate chemistry.
The neoglycolipid (NGL) technology involving the preparation of lipid-linked oligosaccharide probes for binding experiments with carbohydrate-recognizing proteins, and their analysis by mass spectrometry, is a unique and powerful means of discovering oligosaccharide ligands for carbohydrate-binding proteins, and assigning details of their specificities. The key feature is that it enables the pinpointing and sequence determination of bioactive oligosaccharides within highly heterogeneous mixtures derived from natural glycoconjugates. A new generation of NGLs incorporating a fluorescent label now establishes the principles for a streamlined technology whereby oligosaccharide populations are carried through ligand detection and isolation steps, and sequence determination. Advances in selectin research made through applications of the NGL technology include (i) demonstration of the importance of density of selectin expression, and of oligosaccharide ligands, in the magnitude and the specificity of the binding signals; (ii) demonstration of the efficacy of lipid-linked oligosaccharides in supporting selectin-mediated cell interactions; (iii) the discovery of 3-sulphated Le(a)/Le(x) as selectin ligands; (iv) the isolation and sequencing of carbohydrate ligands for E-selectin on murine myeloid cells and kidney; (v) the finding that sulphation at position 6 of the penultimate N-acetylglucosamine confers superior L-selectin binding signals not only to 3-sialyl-Le(x) but also to 3'-sulpho-Le(x); and (vi) the finding that sialic acid de-N-acetylation, or further modification with formation of an intra-molecular amide bond in the carboxyl group, enhances or virtually abolishes, respectively, the potency of the 6'-sulfo-sialyl-Le(X) ligand. Working with biotinylated forms of the oligosaccharide ligands, we have observed that their presentation on a streptavidin matrix influences differentially the efficacy of interactions of the L- and P-selectins (but not E-selectin) with the sialylated and sulphated ligands.
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The Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Campus, Harrow HA1 3UJ, UK.
D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D019040:E-Selectin / Q000378:metabolism; D006017:Glycolipids / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D019041:L-Selectin / Q000378:metabolism; D008024:Ligands; D015335:Molecular Probes; D008969:Molecular Sequence Data; D019087:Selectins / Q000378:metabolism*; D015398:Signal Transduction
D019040:E-Selectin; D006017:Glycolipids; D008024:Ligands; D015335:Molecular Probes; D019087:Selectins; D019041:L-Selectin