Multivalent sialyl-LeX: potent inhibitors of E-selectin-mediated cell adhesion; reagent for staining activated endothelial cells

Abstract

Free, monovalent SLeX (Neu5Acα2-3GalβI-4(Fucαl-3)-GlcNAc), SLn (Neu5Acα2-3Galβ1-4GlcNAc) and corresponding BSA-conjugated forms—displaying different ratios of SLeX and SLn to protein—were tested for their ability to inhibit binding of HL-60 cells to Immobilized E-selectin. Free SLeX and conjugated SLeX-BSA inhibited cell binding in a dose-dependent manner. SLn and SLn-BSA did not inhibit binding. SLeX₁₆BSA (16 mol tetrasaccharide/mol BSA) and monovalent SLeX inhibited cell binding with measured inhibitory concentrations (IC₅₀s) of 1 μM and 1 mM, respectively, demonstrating a three-order-of-magnitude enhancement of inhibitory activity with the multivalent form of SLeX. A SLex₇BSA conjugate was 10-fold less potent than those with 11 or 16 mol SLeX/mol BSA. An assay which measured neutrophil rolling on interleukin (IL)-1β-activated human umbilical vein endothelial cells (HUVECs) showed 50% reduction in the number of rolling neutrophils in the presence of 1 μM SLeX₁₆BSA, whereas the level of free, monovalent SLeX oligosaccharide required to produce the same effect was ˜0.3 mM. SLeX–BSA was found to be an excellent reagent for staining endothelial cells expressing E-selectin. Biotinylated SLeX–BSA in conjunction with Texas red avidin-stained lipopolysaccharide (LPS)-activated HUVECs, and co-incubation of activated cells with anti-E-selectin, specifically blocked staining. The distribution of E-selectin, as determined by binding of SLeX–BSA, was virtually identical with that obtained by binding of anti-E-selectin antibody. The pattern was punctate in nature, rather than being diffuse, suggesting that E-selectin may be organized as clusters within the plasma membrane. The results suggest that multivalent forms of SLeX bind to E-selectin with higher affinity than do monovalent glycans. Clustering of E-selectin in the membrane may be important for binding to counter-receptors on leukocyte cell surfaces.