Consensus repeat domains of E-selectin enhance ligand binding.

To study the structural characteristics of E-selectin necessary for mediating cell adhesion, we examined the role of the consensus repeat (CR) domains in E-selectin function. Soluble constructs containing different numbers of CR domains were stably expressed in Chinese hamster ovary cells, purified to homogeneity, and characterized. The minimum functional unit of soluble E-selectin consisted of the lectin (Lec) and epidermal growth factor (EGF) domains alone (Lec-EGF) as indicated by its ability to mediate in vitro HL-60 cell adhesion. However, E-selectin 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 either immobilized E-selectin or cytokine-stimulated human umbilical vein endothelial cells. This increased potency of Lec-EGF-CR6 in blocking cell 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 ultrafiltration, and chemical cross-linking analysis. Therefore, although the lectin and EGF domains are necessary and sufficient for mediating cell adhesion, the additional six CR domains, present in native E-selectin, contribute to the enhanced binding of E-selectin to its ligand.