Fine mapping of inhibitory anti-alpha 5 monoclonal antibody epitopes that differentially affect integrin-ligand binding

The high-affinity interaction of integrin alpha 5 beta 1 with the central c ell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) seque nce (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (P HSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg -Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for alpha 5 b eta 1, identified by phage display, which blocks alpha 5 beta 1-mediated ce ll adhesion to fibronectin. A key question is the location of the binding s ites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti -alpha 5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs hav e distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding o f SNAKA52 is inhibited by anti-beta 1 mAbs, indicating that its epitope is close to the interface between the alpha and beta subunits. Residues in hum an alpha 5 were replaced with the corresponding residues in mouse alpha 5 b y site-directed mutagenesis; wild-type or mutant human alpha 5 was expresse d on the surface of alpha 5-deficient Chinese hamster ovary cells. mAb bind ing was assessed by flow cytometry and by adhesion to the central cell-bind ing domain of fibronectin or RRETAWA by cell attachment assay. All three ep itopes were located to different putative loops in the N-terminal domain of alpha 5. As expected, disruption of these epitopes had no effect on ligand recognition by alpha 5 beta 1. The locations of these epitopes are consist ent with the beta-propeller model for integrin alpha-subunit structure and allow us to propose a topological image of the integrin-ligand complex.