CONSENSUS REPEAT DOMAINS OF E-SELECTIN ENHANCE LIGAND-BINDING

To study the structural characteristics of E-selectin necessary for me diating cell adhesion, we examined the role of the consensus repeat (C R) domains in E-selectin function. Soluble constructs containing diffe rent numbers of CR domains were stably expressed in Chinese hamster ov ary cells, purified to homogeneity, and characterized. The minimum fun ctional unit of soluble E-selectin consisted of the lectin (Lee) and e pidermal growth factor (EGF) domains alone (Lec-EGF) as indicated by i ts ability to mediate in vitro HL-60 cell adhesion. However, E-selecti n containing all six CR domains (Lec-EGF-CR6) at its COOH terminus was the most potent in blocking neutrophil or HL-60 cell adhesion to eith er immobilized E-selectin or cytokine-stimulated human umbilical vein endothelial cells. This increased potency of Lec-EGF-CR6 in blocking c ell adhesion was not due to CR-mediated oligomerization of the protein . Lec-EGF-CR6 was most likely monomeric in solution, as judged by gel filtration fast protein liquid chromatography, membrane ultrafiltratio n, and chemical cross-linking analysis. Therefore, although the lectin and EGF do mains are necessary and sufficient for mediating cell adhe sion, the additional six CR domains, present in native E-selectin, con tribute to the enhanced binding of E-selectin to its ligand.