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Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLeX) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLeX and sialyl Lewis-a (sLea) were ∼5−7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLeX-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galβ1,4(Fucα1,3)GlcNAcβ1,6(GalNAcβ1,3)GalNAcα-OMe was identified which blocked L- and P-selectin binding at 30−100-fold lower doses than sLeX. (iv) Surface plasmon resonance experiments determined that an sLeX analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K D of ∼111.4 μM. The measured on- and off-rates were high (k off > 3 s-1, k on > 27 000 M-1 s-1). Similar binding kinetics are expected for sLeX−selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.