Modulation of E-selectin structure/function by metal ions. Studies on limited proteolysis and metal ion regeneration.
E-selectin is a member of the selectin family of proteins that recognize carbohydrate ligands in a Ca(2+)-dependent manner. In order to better understand the role of Ca2+ in E-selectin-ligand interactions, we examined the E-selectin structure by limited proteolysis. Apo-Lec-EGF-CR6, a Ca(2+)-free form of soluble E-selectin containing the entire extracellular domain, was sensitive to limited proteolysis by Glu-C endoproteinase. Amino-terminal sequencing analysis of the proteolytic fragments revealed that the major cleavage site is at Glu98 which is in the loop (residues 94-103) adjacent to the Ca2+ binding region of the lectin domain. Upon Ca2+ binding, Lec-EGF-CR6 was protected from proteolysis. This Ca(2+)-dependent protection was further augmented upon sialyl Lewis x (sLex) ligand binding. These results implied that Ca2+ binding to E-selectin induces a conformational change and perhaps facilitates ligand binding. The sLex-bound complex in turn stabilizes Ca2+ binding. Lec-EGF-CR6 contains only one high-affinity Ca2+ site (Kd = approximately 3.5 microM) as determined by equilibrium dialysis. In addition, 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. Sr2+ also bound to apo-Lec-EGF-CR6 tighter than Ca2+. However, Sr(2+)-regenerated 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 any activity. Thus, E-selectin function can be modulated by different metal ions.
Anostario
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Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110, USA.
Huang
K S
KS
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D019040:E-Selectin; D006801:Humans; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D013329:Structure-Activity Relationship
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D008670:Metals; D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen; D002118:Calcium