Abstract
Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well-known E-selectin ligands, CD44/hematopoietic cell E-/L-selectin ligand(HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1), from hematopoietic cell extracts (KG1a, THP-1 and HL-60). A comprehensive characterization of their binding to E-selectin is presented here. We show that both ligands bind recombinant monomeric (m) E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric (d)E-selectin with remarkably slow on and off-rates. This binding requires the sialyl Lewis x (sLex) sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. This is the first documented binding kinetics of dE-selectin to PSGL-1 or CD44 purified from different cellular extracts determined by surface plasmon resonance.