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

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... te constant of 7.4 ؋ 10 4 M ؊1 s ؊1 . We determined a dissociation constant (K d ) of 62 M, which resembles the affinity of L-selectin binding to glycosylationdependent cell adhesion molecule-1. The affinity of the E-selectin-ESL-1 interaction did not change significantly when the temperature was varied from 5°C to 37°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 ␣1,3-fucosyltransferase IV-expressing Chinese hamster ovary cells showed a very similar K d of 66 M, 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. Leukocyte extravasation into tissues is a multistep process involving different classes of adhesion molecules (1, 2). The selectins are a family of lectins, which mediate the initial step of tethering and rolling of leukocytes on vascular endothelium (3, 4). Whereas L-selectin is expressed on most leukocytes, Eand P-selectin are found on activated endothelial cells (5). Each of these type I transmembrane molecules displays an N-terminal C-type (i.e. Ca 2ϩ binding) lectin domain, followed by an epidermal growth factor domain as well as several consensus repeat domains (4). Although the selectins have been shown to bind to a variety of carbohydrates related to the tetrasaccharide sialyl Lewis x (sLe x ) 1 (6 -8), they preferentially bind to a limited number of glycoproteins, most of which are sialomucins (4, 8, 9) . It has been proposed that the ability of selectins to mediate the rapid and transient interactions typical of leukocyte tethering and rolling under shear is a consequence of the fact that they bind to their physiological ligands with fast kinetics and/or a high tensile strength (10, 11). Recently, the affinities and kinetics of two selectin/ligand interactions that can mediate rolling have been measured using SPR technology, namely L-selectin binding to glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) (12) and P-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) (13). GlyCAM-1 is a mucinlike glycoprotein that is secreted by endothelial cells and is found in serum (14 -16). Although the function of GlyCAM-1 as an adhesion molecule in vivo is not yet firmly established, it has been demonstrated that immobilized GlyCAM-1 can support L-selectin-dependent rolling of leukocytes in vitro (17). L-selectin binds to mouse GlyCAM-1 with an affinity or dissociation constant (K d ) of 108 M (12). The kinetics were too fast to measure precisely, but it was possible to show that the dissociation rate constant (k off ) was Ն10 s Ϫ1 , and the calculated association rate constant (k on ) was Ն10 5 M Ϫ1 s Ϫ1 (12). PSGL-1 is a homodimeric sialomucin and the major P-selectin ligand on leukocytes (18 -23). The human P-selectin-PSGL-1 interaction displays a much higher affinity (K d ϳ 0.3 M), a k off of 1.4 s Ϫ1 , and a very high k on of 4.4 ϫ 10 6 M Ϫ1 s Ϫ1 (13). The exceptionally high association rate constant of the P-selectin-PSGL-1 interaction is in good agreement with the well documented function of PSGL-1 as a selectin ligand that mediates leukocyte capturing under flow in vitro as well as in vivo (20, 24, 25) . Thus far, affinities and kinetics of E-selectin-ligand interactions have only been measured on a cellular level. The inhibition of leukocyte binding to immobilized E-selectin by soluble E-selectin molecules indicated a K d of Ͻ1 M (26). However, the soluble E-selectin that was used in that study was oligomeric, suggesting that the true affinity of monomeric E-selectin-ligand interactions may be considerably lower. When transient tethering of leukocytes to immobilized E-selectin was analyzed, a cellular k off of 0.7 s Ϫ1 was estimated (27). Here again, it was