Synergistic interactions of the two classes of ligand, sialyl-Lewis(a/x) fuco-oligosaccharides and short sulpho-motifs, with the P- and L-selectins: implications for therapeutic inhibitor designs.

Abstract

The E-, L- and P-selectins are carbohydrate-recognizing cell-adhesion molecules mediating selective leucocyte recruitment in inflammation. The 3'-sialyl- and 3'-sulpho-oligosaccharides of Lewis(x) (Le(x)) and Lewis(a) (Le(a)) series are bound by them, but for high-avidity binding of P- and L-selectins to the glycoprotein counter-receptor known as P-selectin glycoprotein ligand, PSGL-1, there is a requirement for sulpho-tyrosines neighbouring a sialyl-Le(x) glycan. The two selectins can also bind 3-O- or 6-O-sulphated galacto-lipids (sulphatides). Here we compare some features of the interactions of P- and L-selectins with a novel lipid-linked sulpho-tyrosine probe, and with the sulphatides and neoglycolipids of sialyl- and sulpho-Le(x)/Le(a) fuco-oligosaccharides. The sulpho-tyrosine probe is bound by both selectins. There are close similarities in the interactions of the two selectins with sulpho-tyrosine and the sulphatides; the binding is relatively resistant to chelation of calcium ions, in contrast to the absolute requirement of calcium ions with the long fuco-oligosaccharides, including 6-sulpho-sialyl-Le(x). With both selectins, there is striking synergy in binding signals elicited by the two ligand types when presented as equimolar mixtures on a matrix. Thus, there are two operationally distinct binding sites on both L- and P-selectin; and the binding sites for sulphate groups in the two ligand types are probably distinct. When sulpho-tyrosine and sialyl-Le(x) are presented on liposomes, a potent inhibitory activity is generated toward the binding of P-selectin to HL60 cells, with 50% inhibitory concentration (IC(50)) values in the nanomolar range. These features of the lipid-linked ligand analogues, and the simple approach for their display on liposomes, may have applications in designs and screening of selectin inhibitors as anti-inflammatory compounds.