Mapping of staphylococcal enterotoxin A functional binding sites and presentation by monoclonal antibodies and fusion proteins.

Bacterial superantigens (SAGs) are a group of structurally related proteins which have the ability to strongly activate the immune system. The staphylococcal enterotoxins (SE) are the best characterized among the bacterial SAGs, and they have been shown to bind as unprocessed proteins to a region on the major histocompatibility (MHC) class II molecule which is distinct from the peptide binding groove (1, 30, 34). T cells bearing certain T-cell receptor (TCR) Vβ families interact with the SAG-MHC class II complex, resulting in proliferation and the release of cytokines (1, 10, 30). A variety of techniques have been used to identify the regions of the SAGs involved in their various immunological activities (1, 5, 18, 24). However, the results are controversial. Some investigators localized the mitogenic activity of SEs to the N-terminal portion (5, 18, 24) while others blocked mitogenic function by deleting nine amino acid (aa) residues from the C-terminal region (22) or changed the Vβ specificity by mutation of one aa residue from this region (23). Several studies have also localized MHC class II binding to the amino terminus (13, 18, 25, 26), but others have attributed MHC class II binding to the C-terminal portion of SEs (15, 27). Recently, the structures of SE type B (SEB) and toxic shock syndrome toxin and their complexes with MHC class II molecules were elucidated by X-ray crystallography and the residues involved in these interactions identified (2, 17, 19, 32). SEA is still the most potent SAG, and considerable evidence from competition studies suggests that SEA and SEB do not bind in the same way to MHC class II molecules. SEA inhibits SEB binding to MHC class II molecules, but excess SEB does not prevent SEA binding (7, 11). Furthermore, SEA requires a single zinc atom for high-affinity binding to MHC class II molecules (12), and two distinct but cooperative binding sites have been reported (1, 16). More recently, the crystal structure of SEA was described. The metal binding site was identified, and based on the similarity between SEA and SEB, the sites of interaction of SEA with TCR and MHC class II molecule were suggested (29). In order to establish a functional map of SEA, I used a simple approach based on the biological interaction of a panel of 20 specific anti-SEA monoclonal antibodies (MAbs) with SEA and the consequences of this interaction on mitogenic and binding activities of SEA. Subsequently, I studied the interaction of this panel with two large fusion proteins overlapping the SEA molecule. The first SEA–glutathione S-transferase (GST) fusion protein corresponds to the N-terminal region (aa 1 to 110) and the second corresponds to the C-terminal (aa 93 to 233) region. Furthermore, I studied the ability of the MAbs to present SEA in vitro to human and murine cells. The results confirmed the presence of two functional binding sites of SEA on MHC class II molecules which differ by their requirement for T-cell activation. Antibodies recognizing MHC class II and TCR sites on SEA can present it to the immune cells and can start the cascade of cell activation and proliferation. All interaction sites of SEA depend only on the three-dimensional structure of SEA and not on its peptide sequence. Moreover, I confirmed the importance of the disulfide loop of SEA in TCR interaction and the presence of two different TCR-interacting sites.