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Soluble Monomeric P-Selectin Containing Only the Lectin and Epidermal Growth Factor Domains Binds to P-Selectin Glycoprotein Ligand-1 on Leukocytes
Padmaja Mehta,
Kamala D. Patel,
Thomas M. Laue,
Harold P. Erickson, and
Rodger P. McEver
From the W. K. Warren Medical Research Institute, Departments of Medicine and Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK; the Department of Biochemistry, University of New Hampshire, Durham, NH; and the Department of Cell Biology, Duke University Medical Center, Durham, NC.
Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF than for sPS, whereas the IC50 for inhibiting binding of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether to or roll on Lec-EGF that was captured by an immobilized monoclonal antibody to the C-terminal epitope. These data show that P-selectin requires only the lectin and EGF domains to bind to PSGL-1.
Blood, Vol. 90 No. 6 (September 15), 1997:
pp. 2381-2389
© 1997 by The American Society of Hematology.
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