Fine mapping of inhibitory anti-α5 monoclonal antibody epitopes that differentially affect integrin‒ligand binding

Abstract

The high-affinity interaction of integrin α5β1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) 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 α5β1, identified by phage display, which blocks α5β1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-α5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-β1 mAbs, indicating that its epitope is close to the interface between the α and β subunits. Residues in human α5 were replaced with the corresponding residues in mouse α5 by site-directed mutagenesis; wild-type or mutant human α5 was expressed on the surface of α5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of α5. As expected, disruption of these epitopes had no effect on ligand recognition by α5β1. The locations of these epitopes are consistent with the β-propeller model for integrin α-subunit structure and allow us to propose a topological image of the integrin-ligand complex.