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 Mg 2+ ; 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 × 10 5 M −1 s −1 and 2.98 × 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 μM in the absence (EGTA) and the presence of Ca 2+ (100 μM), respectively; in addition, K Mn was 2 μM in the presence of Ca 2+ (100 μM). Increasing Ca 2+ into the millimolar concentration range, however, resulted in a competitive displacement of Mg 2+ /Mn 2+ 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.