Functional characterization of atherosclerosis-associated Ser128Arg and Leu554Phe E-selectin mutations

The cellular adhesion molecule E-selectin is expressed on activated endothe lial cells, and is involved in the process of adherence of blood cells to v essel endothelium in inflammatory events such as atherosclerosis. In a rece nt study we found a Ser128Arg mutation in the EGF domain as well as a Leu55 4Phe mutation in the membrane domain of E-selectin. We also established inc reased frequencies of both mutations among young patients with severe coron ary atherosclerosis. In the present study we investigated the influence of these mutations on cell adhesion and on the release of soluble E-selectin. Mutants were created by site-directed mutagenesis and COS cells were transf ected with E-selectin, either wild-type or mutant. Antibody-binding studies and cell-adhesion assays were performed on transfected COS cells and on in terleukin-1 beta-stimulated HUVECs. Soluble E-selectin in supernatants of w ild type and Leu554Phe mutant-transfected COS cells was measured by ELISA. We discovered significant differences in the strength of HL-60 cell adhesio n for the Ser128Arg mutant: in comparison with the wild type, the strength of adhesion to the mutant was reduced on transfected COS cells (P < 0.01) a s well as on stimulated HUVECs (P < 0.01). Significantly diminished release of soluble E-selectin was detected for the Leu554Phe membrane domain mutan t, in comparison with the wild type. In summary, the mutations studied here influence the E-selectin function in vitro and may be considered as one of the risk factors involved in the complex pathogenesis of atherosclerosis.