Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1.
Leukocytes use the cell-surface mucin P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin on activated endothelial cells and platelets. By using surface plasmon resonance, we measured the affinity and kinetics of binding of soluble monomeric human P-selectin to immobilized PSGL-1 from human neutrophils. Binding was specific, as documented by its Ca2+-dependence, its inhibition by specific monoclonal antibodies to P-selectin and PSGL-1, and its abrogation by treating PSGL-1 with sialidase. Similar binding was observed for soluble P-selectin that contained the lectin and epidermal growth factor domains plus all nine consensus repeats, and for a soluble construct that contained only the lectin and epidermal growth factor domains. Soluble P-selectin bound saturably to a single class of sites on PSGL-1 with a dissociation constant (Kd) of 320 +/- 20 nM. The measured koff was 1.4 +/- 0.1 s-1, and the calculated kon was 4.4 x 10(6) M-1 s-1. We conclude that monomeric P-selectin binds to PSGL-1 with fast association and dissociation rates and relatively high affinity. These features may be important for efficient tethering and rolling of leukocytes at physiologic densities of PSGL-1 and P-selectin.
Mehta
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W. K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Cummings
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McEver
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009439:Neuraminidase / Q000737:chemistry; D019007:P-Selectin / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D004815:Epidermal Growth Factor; D009439:Neuraminidase