Modulation of E-selectin structure/function by metal ions. Studies on limited proteolysis and metal ion regeneration.

The Journal of Biological Chemistry
M Anostario, K S Huang

Abstract

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 ...Continue Reading

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Citations

Oct 1, 1995·Current Opinion in Structural Biology·K Drickamer
Dec 14, 2002·Biochimica Et Biophysica Acta·Tae Jun ParkIn Kyoung Lim
Aug 13, 1998·Immunological Reviews·W I WeisK Drickamer
Nov 12, 2016·Biosensors & Bioelectronics·Karuppuchamy Selvaprakash, Yu-Chie Chen
Jul 12, 1996·The Journal of Biological Chemistry·K V EwartC L Hew
Mar 29, 1996·The Journal of Biological Chemistry·O BlanckK Drickamer

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