MODULATION OF E-SELECTIN STRUCTURE-FUNCTION BY METAL-IONS - STUDIES ON LIMITED PROTEOLYSIS AND METAL-ION REGENERATION

E-selection is a member of the selectin family of proteins that recogn ize carbohydrate ligands in a Ca2+-dependent manner. In order to bette r understand the role of Ca2+ in E-selectin-ligand interactions, we ex amined the E-selectin structure by limited proteolysis. Apo-Lec-EGF-CR 6, a Ca2+-free form of soluble E-selectin containing the entire extrac ellular domain, was sensitive to limited proteolysis by Glu-C endoprot einase. Amino-terminal sequencing analysis of the proteolytic fragment s revealed that the major cleavage site is at Glu(98) which is in the loop (residues 94-103) adjacent to the Ca2+ binding region of the lect in domain. Upon Ca2+ binding, Lec-EGF-CR6 was protected from proteolys is. This Ca2+-dependent protection was further augmented upon sialyl L ewis x (sLe(x)) ligand binding. These results implied that Ca2+ bindin g to E-selectin induces a conformational change and perhaps facilitate s ligand binding. The sLe(x)-bound complex in turn stabilized Ca2+ bin ding. Lec-EGF-CR6 contains only one high-affinity Ca2+ site (K-d = app roximate to 3.5 mu M) as determined by equilibrium dialysis. In additi on, we found that Ba2+ was a potent antagonist in blocking Lec-EGF-CR6 -mediated HL-60 cell adhesion. By competitive equilibrium dialysis and proteolysis analysis, we demonstrated that Ba2+ bound to apo-Lec-EGF- CR6 5-fold tighter than Ca2+ and abolished ligand binding activity. Sr 2+ also bound to apo-Lec-EGF-CR6 tighter than Ca2+. However, Sr2+-rege nerated Lec-EGF-CR6 showed 50% ligand binding activity. Mg2+ bound to apo-Lec-EGF-CR6 with much weaker affinity than Ca2+ and did not show a ny activity. Thus, E-selectin function can be modulated by different m etal ions.