Interaction between soluble P-selectin and soluble P-selectin glycoproteinligand 1: Equilibrium binding analysis

Leukocyte rolling in the vasculature is mediated by the interaction of endo thelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Since cell-cell interaction mediated by P-selectin and PSGL-1 is cooperati ve and complex, we have developed a model system to examine the binding of P-selectin to PSGL-1 in a soluble system. Equilibrium binding analyses were performed with truncated forms of soluble human P-selectin and dimeric PSG L-1, both lacking the transmembrane domain and both produced in Chinese ham ster ovary (CHO) cells. Soluble PSGL-1 (sPSGL-1), which contains no tryptop han residues and exhibits no intrinsic fluorescence, was harvested from CHO cells cotransfected with either fucosyltransferase III (sPSGL-1/Fuc-TIII) or fucosyltransferase VII (sPSGL-1/Fuc-TVII). Both fucosylation isoforms of sPSGL-1 bound to sP-selectin. The interaction of sP-selectin and sPSGL-1 w as studied by monitoring changes in the intrinsic fluorescence of sP-select in upon binding to sPSGL-1. Binding of sPSGL-1 to sP-selectin in the presen ce of calcium caused an increase in tryptophan fluorescence that could be r eversed by the addition of ethylenediaminetetraacetic acid (EDTA). The fluo rescence enhancement of sP-selectin by sPSGL-1 was used to generate binding isotherms, and these data were fined to a bimolecular binding model. The b inding constant, K-d, for the binding of sPSGL-1/Fuc-TIII and sPSGL-1/Fuc-T VII to sP-selectin was 3 +/- 2 nM and 80 +/- 44 nM, respectively. Monomeric sP-selectin bound to dimeric sPSGL-1 with a 2:1 stoichiometry. In a system in which both protein species are soluble and lack transmembrane domains, these results indicate high-affinity interaction between P-selectin and PSG L-1. Furthermore, the fucosylation pattern of PSGL-1 can affect its affinit y for P-selectin. These binding constants can be used to explore models of cell adhesion in flow systems.