Affinity and kinetic analysis of L-selectin binding to their natural ligands and to synthetic oligosaccharides

We developed a flow cytometric methodology to measure the affinity and kinetics of L-selectin binding to their natural ligands on neutrophils (PMNs) and to synthetic selectin-ligand analogues under physiologically relevant conditions. The synthetic molecules tested include the sLe^x oligosaccharide, selected sLe^x analogues and core-2 based sialylated and sulfated sugars expressed on the natural selectin-ligands, including GlyCAM-1 and PSGL-1. Quantitative analysis was performed to determine the affinity constant (K_d) and binding rates of these molecules. We observed that: i) divalent soluble L-selectin bound their ligands on neutrophils (predominantly PSGL-1) with an on-rate of 9.5×10⁴/M/ss and off-rate of 2.3×10^-3/s. ii) The free acid forms of sialyl lewis-X (sLe^x-OH) and sialyl lewis-a (sLe^a-OH) were ∼2-4 fold poorer inhibitors of L-selectin mediated binding in comparison to sLe^x-OMe and sLe^a-OMe. iii) The binding affinity of selected core-2 di-O-sialylated and sulfated structures expressed on GlyCAM-1 and PSGL-1 were only 2-fold better than sLe^x-OH in spite of their homology to the natural selectin-ligands. In general, charged molecules tended to be poorer selectin inhibitors. iv) We compared L-selectin binding under static and shear conditions. In these studies, while we observed a direct correlation between the inhibition efficiency under static and shear conditions in most cases, a core-2 based non-fucosyalted molecule was observed to be a better inhibitor under shear rather than static conditions. Overall, we demonstrate that besides structural homology with the ligand, appropriate functionalization of the anomeric carbon and other physicochemical parameters, especially the solubility of the synthetic molecule, are important parameters influencing selectin-ligand affinities.