Affinity, kinetics, and thermodynamics of E-selectin binding to E-selectin ligand-1.
E-selectin is an endothelial adhesion molecule, which mediates the tethering and rolling of leukocytes on vascular endothelium. It recognizes the glycoprotein E-selectin ligand-1 (ESL-1) as a major binding partner on mouse myeloid cells. Using surface plasmon resonance, we measured the kinetics and affinity of binding of monomeric E-selectin to ESL-1 isolated from mouse bone marrow cells. E-selectin bound to ESL-1 with a fast dissociation rate constant of 4.6 s(-1) and a calculated association rate constant of 7.4 x 10(4) m(-1) s(-1). We determined a dissociation constant (K(d)) of 62 microm, which resembles the affinity of L-selectin binding to glycosylation-dependent cell adhesion molecule-1. The affinity of the E-selectin-ESL-1 interaction did not change significantly when the temperature was varied from 5 degrees C to 37 degrees C, indicating that the enthalpic contribution to the binding is small at physiological temperatures, and that, in contrast to typical protein-carbohydrate interactions, binding is driven primarily by favorable entropic changes. Interestingly, surface plasmon resonance experiments with recombinant ESL-1 from alpha 1,3-fucosyltransferase IV-expressing Chinese hamster ovary cells showed a very similar K(d) of 66 microm, suggesting that this fucosyltransferase is sufficient to produce fully functional recombinant ESL-1. Following the recent description of the affinity and kinetics of the selectin-ligand pairs L-selectin-glycosylation-dependent cell adhesion molecule-1 and P-selectin-P-selectin glycoprotein ligand-1, this is the first determination of the parameters of E-selectin binding to one of its naturally occurring ligands.
Wild
M K
MK
Institute of Cell Biology, Zentrum für Molekularbiologie der Entzündung, University of Muenster, 48149 Muenster, Germany.
Huang
M C
MC
Schulze-Horsel
U
U
van der Merwe
P A
PA
Vestweber
D
D
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D019040:E-Selectin / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012795:Sialoglycoproteins; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D017931:DNA Primers; D019040:E-Selectin; D008562:Membrane Glycoproteins; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C086711:cysteine-rich fibroblast growth factor receptor