Interaction between soluble P-selectin and soluble P-selectin glycoprotein ligand 1: equilibrium binding analysis.
Leukocyte rolling in the vasculature is mediated by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Since cell-cell interaction mediated by P-selectin and PSGL-1 is cooperative 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 PSGL-1, both lacking the transmembrane domain and both produced in Chinese hamster ovary (CHO) cells. Soluble PSGL-1 (sPSGL-1), which contains no tryptophan 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 was studied by monitoring changes in the intrinsic fluorescence of sP-selectin upon binding to sPSGL-1. Binding of sPSGL-1 to sP-selectin in the presence of calcium caused an increase in tryptophan fluorescence that could be reversed by the addition of ethylenediaminetetraacetic acid (EDTA). The fluorescence enhancement of sP-selectin by sPSGL-1 was used to generate binding isotherms, and these data were fitted to a bimolecular binding model. The binding constant, Kd, for the binding of sPSGL-1/Fuc-TIII and sPSGL-1/Fuc-TVII 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 PSGL-1. Furthermore, the fucosylation pattern of PSGL-1 can affect its affinity for P-selectin. These binding constants can be used to explore models of cell adhesion in flow systems.
Croce
K
K
Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
Freedman
S J
SJ
Furie
B C
BC
Furie
B
B
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D005454:Fluorescence Polarization; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019007:P-Selectin / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D012995:Solubility; D012996:Solutions; D013050:Spectrometry, Fluorescence
D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D012996:Solutions