Molecular regulation of the interaction between leukocyte function-associated antigen-1 and soluble ICAM-1 by divalent metal cations.
Surface plasmon resonance (SPR) was used to investigate and characterize the interaction between LFA-1 and sICAM-1 (a soluble form of ICAM-1). Full-length LFA-1 was immobilized on a hydrophobic surface, and sICAM-1 binding was monitored in a flow cell format. The binding of sICAM-1 to LFA-1 was specific and dependent upon Mg2+; Abs to both sICAM-1 and LFA-1 blocked the interaction, and EDTA abolished all binding. Association and dissociation rate constants (k(a) and k(d), respectively) for sICAM-1 were 2.24 x 10(5) M(-1) s(-1) and 2.98 x 10(-2) s(-1), respectively, giving a calculated K(ICAM) of 133 nM. Since the LFA-1/ICAM-1 interaction is highly sensitive to the presence of metal cations, SPR was also used to probe the affinity of the metal binding sites. The K(Mg) values were 160 and 12 microM in the absence (EGTA) and the presence of Ca2+ (100 microM), respectively; in addition, K(Mn) was 2 microM in the presence of Ca2+ (100 microM). Increasing Ca2+ into the millimolar concentration range, however, resulted in a competitive displacement of Mg2+/Mn2+ and decreased sICAM-1 binding. Based on these data, a synergistic model for the molecular regulation of LFA-1 by divalent metal cations is proposed, and implications to cellular adhesion are discussed.
Labadia
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Department of Biology, Boehringer Ingelheim Pharmaceuticals, Research and Development Center, Ridgefield, CT 06877-0368, USA.
Jeanfavre
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D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002413:Cations, Divalent; D004357:Drug Synergism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D007700:Kinetics; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D008956:Models, Chemical; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D012995:Solubility
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D016169:Lymphocyte Function-Associated Antigen-1; D008670:Metals; D018799:Intercellular Adhesion Molecule-1; D008345:Manganese; D008274:Magnesium; D002118:Calcium