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 characteri ze the interaction between LFA-1 and sICAM-1 (a soluble form of ICAM-1 ). Full-length LFA-I was immobolized on a hydrophobic surface, and sIC AM-1 binding was monitored in a how cell format, The binding of sICAM- 1 to LFB-1 was specific and dependent upon Mg2+; Abs to both sICAM-1 a nd LFA-1 blocked the interaction, and EDTA abolished all binding. Asso ciation and dissociation rate constants (k(a) and k(d), respectively) for sICAM-1 mere 2.24 X 10(5) M-1 s(-1) and 2.98 x 10(-2) s(-1), respe ctively, 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 mas also used to probe the affinity of the metal binding sites. The K- Mg values mere 160 and 12 mu M in the absence (EGTA) and the presence of Ca2+ (100 mu M), respectively; in addition, K-Mn was 2 mu M in the presence of Ca2+ (100 mu M). increasing Ca2+ into the millimolar conce ntration range, however, resulted in a competitive displacement of Mg2 +/Mn2+ and decreased sICAM-1 binding. Based on these data, a synergist ic model for the molecular regulation of LFA-1 by divalent metal catio ns is proposed, and implications to cellular adhesion are discussed.