Abstract
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.
Highlights
Leukocyte extravasation into tissues is a multistep process involving different classes of adhesion molecules [1, 2]
The selectins have been shown to bind to a variety of carbohydrates related to the tetrasaccharide sialyl Lewisx1 (6 – 8), they preferentially bind to a limited number of glycoproteins, most of which are sialomucins [4, 8, 9]
The exceptionally high association rate constant of the P-selectin-P-selectin glycoprotein ligand-1 (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]
Summary
Antibodies—The following antibodies were used: 10E9.6, rat antimouse E-selectin, IgG2a [36, 37]; UZ4, rat anti-mouse E-selectin, IgM (38 – 40); R4 –22, rat IgM isotype control mAb (BD PharMingen, Heidelberg, Germany); Mel-14, rat anti-mouse L-selectin, IgG2a (BD PharMingen); 4RA10, rat anti-mouse PSGL-1, IgG1 [41]; 9E10, mouse anti-c-Myc, IgG1 (BD PharMingen); OX12, mouse anti-rat light chain, IgG2a [42]. Purification of Native Mouse ESL-1—Prior to purification of ESL-1 from mouse bone marrow cells, three batches of Protein A-Sepharose beads were prepared; 1.25 ml of drained Protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) was incubated with 2.5 mg of mouse P-selectin-Ig chimeric construct [47] at 4 °C for 16 h in 15 ml of wash buffer I (50 mM Tris-HCl, pH 8.4, 400 mM NaCl, 1 mM CaCl2, 0.1% Triton X-100, 6.2 mM sodium azide). The beads were sedimented and any remaining beads were removed by passing the supernatant over an empty PolyPrep column (Bio-Rad, Munchen, Germany) At this stage the lysate was free from PSGL-1 and could be used to precipitate ESL-1 with E-selectin-Ig. At this stage the lysate was free from PSGL-1 and could be used to precipitate ESL-1 with E-selectin-Ig To this end the lysate was added to 1.25 ml of drained Protein A-Sepharose coated with E-selectin-Ig and incubated at 4 °C for 16 h. Tis the temperature in Kelvin (K); To is an arbitrary reference temperature (e.g. 298.15 K); ⌬G° is the standard free energy of binding at T (kJ1⁄7molϪ1), and is calculated from the Kd using the equation ⌬G° ϭ R1⁄7To1⁄7lnKd, where R is the gas constant; ⌬HTo is the enthalpy change upon binding at To (kJmolϪ1); ⌬S°To is the standard state entropy change upon binding at To (kJ1⁄7molϪ1); and ⌬Cp is the specific heat capacity (kJ1⁄7molϪ11⁄7KϪ1), and is assumed to be temperature-independent
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