A monoclonal antibody enhances ligand binding of fibronectin MSCRAMM (adhesin) from Streptococcus dysgalactiae

Abstract

A monoclonal antibody 3A10, generated from a mouse immunized with the Streptococcus dysgalactiae fibronectin (Fn) binding protein FnbA, was isolated, and its effect on ligand binding by the antigen was examined. The epitope for 3A10 was localized to a previously unidentified Fn binding motif (designated Au) just N-terminal of the repeat domain which represents the primary ligand binding site on FnbA. Fn binding to Au was enhanced by 3A10 rather than inhibited. This effect was demonstrated in two different assays. First, in the presence of 3A10 the Au-containing proteins and synthetic peptide more effectively competed with bacterial cells for binding to Fn. Second, 3A10 dramatically increased the binding of biotin-labeled forms of the Au-containing proteins to Fn immobilized on a blotting membrane. Pure 3A10 IgG did not recognize the antigen by itself, and Fn was required for the immunological interaction between the antibody and the epitope. This induction effect of Fn was shown in both Western blot and enzyme-linked immunosorbent assay in which immobilized Au-containing molecules were probed with 3A10 in the presence of varying concentrations of Fn. Specificity analyses of 3A10 revealed that the monoclonal also recognized a ligand binding motif in a Streptococcus pyogenes Fn binding MSCRAMM but not the corresponding motifs in two related adhesins from Staphylococcus aureus and S. dysgalactiae. Furthermore, 3A10 stimulated Fn binding by S. pyogenes cells. These results together with subsequent biophysical studies presented in the accompanying paper (House-Pomepeo, K., Xu, Y., Joh, D., Speziale, P., and Hook, M. (1996) J. Biol. Chem. 271, 1379-1384) indicate that the ligand binding sites of Fn binding MSCRAMMs have little or no secondary structure. However, on binding to Fn, they appear to undergo a structural rearrangement resulting in a defined structure rich in β sheet and expressing a ligand- induced binding site for antibodies such as 3A10.