title: Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1.
abstract: The selectin family of cell adhesion molecules mediates the tethering and rolling of leukocytes on blood vessel endothelium. It has been postulated that the molecular basis of this highly dynamic adhesion is the low affinity and rapid kinetics of selectin interactions. However, affinity and kinetic analyses of monomeric selectins binding their natural ligands have not previously been reported. Leukocyte selectin (L-selectin, CD62L) binds preferentially to O-linked carbohydrates present on a small number of mucin-like glycoproteins, such as glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), expressed in high endothelial venules. GlyCAM-1 is a soluble secreted protein which, following binding to CD62L, stimulates beta2-integrin-mediated adhesion of lymphocytes. Using surface plasmon resonance, we show that a soluble monomeric form of CD62L binds to purified immobilized GlyCAM-1 with a dissociation constant (Kd) of 108 microM. CD62L dissociates from GlyCAM-1 with a very fast dissociation rate constant (>/=10 s-1) which agrees well with the reported dissociation rate constant of CD62L-mediated leukocyte tethers. The calculated association rate constant is >/=10(5) M-1 s-1. At concentrations just above its mean serum level (approximately 1.5 microg/ml or approximately 30 nM), GlyCAM-1 binds multivalently to immobilized CD62L. It follows that soluble GlyCAM-1 may cross-link CD62L when it binds to cells, suggesting a mechanism for signal transduction.
authors.affiliation: Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: M W
authors.initials: MW
authors.lastname: Nicholson
authors.forename: A N
authors.initials: AN
authors.lastname: Barclay
authors.forename: M S
authors.initials: MS
authors.lastname: Singer
authors.forename: S D
authors.initials: SD
authors.lastname: Rosen
authors.forename: P A
authors.initials: PA
authors.lastname: van der Merwe
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015704:CD4 Antigens / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D019041:L-Selectin / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D009077:Mucins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D015398:Signal Transduction
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D009077:Mucins; D019041:L-Selectin; C075185:sulfated glycoprotein p50