MULTIVALENT SIALYL-LEX - POTENT INHIBITORS OF E-SELECTIN-MEDIATED CELL-ADHESION REAGENT FOR STAINING ACTIVATED ENDOTHELIAL-CELLS

Free, monovalent SLeX (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)-Glc NAc), SLn (Neu5Ac alpha 2-3Gal beta 1-4GlcNAc) and corresponding BSA-c onjugated forms-displaying different ratios of SLeX and SLn to protein -were tested for their ability to inhibit binding of HL-60 cells to im mobilized E-selectin. Free SLeX and conjugated SLeX-BSA inhibited cell binding in a dose-dependent manner. SLn and SLn-BSA did not inhibit b inding. SLeX(16)BSA (16 mol tetrasaccharide/mol BSA) and monovalent SL eX inhibited cell binding with measured inhibitory concentrations (IC5 0S) of 1 mu M and 1 mM, respectively, demonstrating a three-order-of-m agnitude enhancement of inhibitory activity with the multivalent form of SLeX. A SLex(7)BSA conjugate was 10-fold less potent than those wit h 11 or 16 mol SLeX/mol BSA. An assay which measured neutrophil rollin g on interleukin (IL)-1 beta-activated human umbilical vein endothelia l cells (HUVECs) showed 50% reduction in the number of rolling neutrop hils in the presence of 1 mu M SLeX(16)BSA, whereas the level of free, monovalent SLeX oligosaccharide required to produce the same effect w as similar to 0.3 mM. SLeX-BSA was found to be an excellent reagent fo r staining endothelial cells expressing E-selectin. Biotinylated SLeX- BSA in conjunction with Texas red avidin-stained lipopolysaccharide (L PS)activated HUVECs, and co-incubation of activated cells with anti-E- selectin, specifically blocked staining. The distribution of E-selecti n, 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-selec tin may be organized as clusters within the plasma membrane. The resul ts suggest that multivalent forms of SLeX bind to E-selectin with high er affinity than do monovalent glycans. Clustering of E-selectin in th e membrane may be important for binding to counter-receptors on leukoc yte cell surfaces.