Abstract
Previous studies have shown that lymphocyte function-associated antigen-1 (LFA-1) molecules containing the human alpha (CD11a) and human beta (CD18) subunits but not the murine alpha and human beta subunits can bind to human intercellular adhesion molecule 1 (ICAM-1). Using human/mouse LFA-1 alpha subunit chimeras, we mapped regions required for binding to ICAM-1 N-terminal to amino acid (aa) residue 350. Ligand binding sites were mapped in greater detail by scanning this region with murine sequences from 56 down to 17 aa in length and finally by introducing single or few murine aa residue replacements into the human sequence. Replacement of two non-contiguous regions of aa residues 119-153 and 218-248 in the me domain with the corresponding mouse sequences abolished most binding to human ICAM-1, without affecting alpha beta subunit association or expression on the surface of transfected COS cells. Specific residues within the I domain found to be important were Met-140, Glu-146, Thr-243, and Ser-245. Using the recently solved structure of the Mac-1 (CD11b) I domain as a model (Lee, J.-O., Rieu, P., Arnaout, M.A., and Liddington, R. (1995) Cell 80, 631-638), these residues are shown to be located on the surface of the I domain surrounding the site to which Mg2+ is chelated, and fine a ligand binding interface. Mapping of the epitopes of a panel of mouse anti-human and rat anti-mouse monoclonal antibodies gave concordant results. Epitopes were mapped to two different regions in the N-terminal domain, four regions within the I domain, and two regions between the I domain and the EF hand-like repeats. Monoclonal antibodies to epitopes within the mid- to C-terminal portion of the I domain and the N-terminal portion of the region between the I domain and the EF hand-like repeats gave good inhibition of LFA-1-dependent homotypic aggregation with cells that express either ICAM-1 or ICAM-3 as the major LFA-1 ligand.
Highlights
Mutation of residues that form the primary or secondary coordination shell with the M!f+ ion, including Asp-140 and Asp-242 in the I domain of Mac-l (Lee et al, 1995; Michishita et al, 1993), their homologues in the I domain of VLA-2 (Kamata et al, 1994) and VLA-l (Kern et al, 1994), and the homologue of Thr-209 in the I domain ofVLA-2 (Kamata and Takada, 1994), disrupt ligand binding. Such mutations abolish divalent cation binding in Mac-l (Michishitaet al., 1993) and are predicted to do the same in the o I and a2 subunits, suggesting that the divalent cation may indirectly stabilize a ligand binding site, or that the cation may directly coordinate with ligand
Construction and Expression of Human/Mouse CDlla Chimeras-To localize sites on the human LFA-1 a subunit cognate for human ICAM-1, a set of five human/mouse a subunit chimeras were constructed in the expression vector Ap'"M8 (Fig. lA)
The first approach relied on the observation that the human but not mouse 0: subunit enabled binding of the LFA-1 0:13 complex to human ICAM-1 (Johnstonet al., 1990)
Summary
Mutation of residues that form the primary or secondary coordination shell with the M!f+ ion, including Asp-140 and Asp-242 in the I domain of Mac-l (Lee et al, 1995; Michishita et al, 1993), their homologues in the I domain of VLA-2 (Kamata et al, 1994) and VLA-l (Kern et al, 1994), and the homologue of Thr-209 in the I domain ofVLA-2 (Kamata and Takada, 1994), disrupt ligand binding. Such mutations abolish divalent cation binding in Mac-l (Michishitaet al., 1993) and are predicted to do the same in the o I and a2 subunits, suggesting that the divalent cation may indirectly stabilize a ligand binding site, or that the cation may directly coordinate with ligand. Other residues required for ligand binding and that are likely to form direct contacts with ligand have yet to be identified in any integrin I domain
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