Abstract
Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. They are composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of consensus repeats CR (also called "sushi" domains), a transmembrane domain and a C-terminal cytoplasmic tail. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e., E-selectin) occurs through the interaction between the lectin domain of selectins and sLeX presenting ligands. Investigating the mechanism by which structural domains of selectins are involved in the binding functionality and kinetic behavior of selectin-ligand interaction primarily
focused on lectin domain as the main binding domain while other domains received less attention. We prepared a number of different recombinant E-selecin proteins where various domains were removed, the E-selectin proteins were created as a monomer or dimer, and/or strategic residues were mutated to change the conformation of the selectin molecule.
Using a novel real time immunoprecipitation surface plasmon resonance (SPR)-based in vitro binding study developed in our
lab, the interaction of recombinant E-selectin proteins with immunoprecipitated endogenous ligands (i.e. CD44 and PSGL-1) captured on a CM-5 chip was assessed. These studies provided quantitative binding kinetics of on and off rates of selectin-ligand interactions and suggested that robust binding is dependent on the presence of the sushi domain(s) and oligomerization. These results provide significant implications on the mechanism of E-selectin binding to its ligands.