Affinity and kinetics of selectin‐carbohydrate interaction

Abstract

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion during 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 static and fluid flow conditions. Surface plasmon resonance (SPR) studies quantified binding kinetics. We observed that: i) The functional group at the anomeric position of carbohydrates play an important role during selectin recognition, since sLeX and sialyl Lewis-a were ~5–7 fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. ii) Despite their homology to physiological glycans, carbohydrates of GlyCAM-1 and PSGL-1 bound selectins with low-affinity. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of sugars in clusters on this glycoprotein may contribute to selectin recognition. iii) A novel, neutrally-charged small molecule was identified which blocked L- and P-selectin binding at 30–100 fold lower doses than sLeX. iv) SPR experiments determined that a sLeX-analog (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 KD of ~0.11mM and high off-rates (>3/s). Overall, our study provides new insight into the kinetics and mechanism of carbohydrate interaction with selectins. Supported by NIH HL63014, HL77258