The related staphylococcal toxins staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) are microbial superantigens. They require interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously identified a binding site on SEA, the N-terminal 45 amino acids, as well as its corresponding receptor on the MHC antigen, residues 65-85 of the fi chain. To further characterize the structural basis for SEA binding to class II MHC molecules we have examined its relationship to TSST-1 binding. Both toxins bound similarly to murine A20 cells, but blockage of binding was observed only with the homologous toxin, which suggests that the binding sites for the two toxins on A20 cells are distinct. In contrast, specific binding of SEA was greater than that of TSST-1 on human Raji Staphylococcal enterotoxin A (SEA) is the most potent T-cell mitogen known, stimulating DNA synthesis, interferon-y production, and interleukin 2 production at concentrations as low as 10-16 M (1-3). It has been described as a microbial superantigen because of its ability to stimulate all T cells bearing particular T-cell antigen receptor VB regions (4, 5). Antigen-presenting cells are required for SEA activity (6, 7), and recently it has been shown that class II major histocompatibility complex (MHC) molecules are the specific receptors on the antigen-presenting cell for SEA (8-10). Unlike classically presented antigens SEA is not processed prior to binding (6,7,11), nor is its presentation restricted by polymorphic portions of class II molecules (7).The N-terminal region of SEA has been identified previously as a site on SEA that is responsible for its interaction with MHC class II antigens (12). Moreover, the same region of SEA is capable ofbinding to class II antigens from different species-i.e., human leukocyte antigens (HLA) and murine immune-associated antigens (Ia) (12). SEA and staphylococcal enterotoxin B (SEB) are reported to compete for binding to HLA-DR, implying that they bind to the same region on the MHC molecule (10). In contrast, SEB and toxic shock syndrome toxin 1 (TSST-1) have been shown to interact at different sites (13). We have further examined the complex binding ofmicrobial superantigens to class II MHC molecules by using synthetic peptides, and we have detected multiple binding sites for SEA on human Raji cells, one of which overlaps with that for TSST-1. MATERIALS AND METHODSToxins. SEA and TSST-1 were obtained from Toxin Technology (Madison, WI). SEA was homogenous by SDS gel electrophoresis (1). For biotinylation, 1 mg ofSEA or TSST-1 was dissolved in 50 mM sodium bicarbonate buffer (pH = 9.6) with 0.02 mg of sulfosuccinimidyl 6-(biotinamide) hexanoate (NHS-LC-biotin; Pierce) and incubated on ice for 2 hr. 125The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Using the synthetic peptide approach, we have identified a part of the staphylococcal enterotoxin A (SEA) molecule that is responsible for stimulation of T cell proliferation and induction of the lymphokine IFN-gamma. Peptides were synthesized corresponding to amino acids 1 to 27, SEA(1-27), and 28 to 45, SEA(28-45). Both peptides were tested for direct competition with SEA for blockage of SEA induced proliferation and production of IFN-gamma by T cells. Further, antibodies were produced to the peptides and tested for their ability to bind to SEA and block SEA function. SEA (1-27), but not SEA (28-45), blocked proliferation of human peripheral T cells and induction of IFN-gamma by the T cell line, L12-R4. The inhibitory effects were specific, because SEA (1-27) did not inhibit the induction of T cell proliferation by the mitogen PHA. Consistent with the direct inhibition of function, antibodies to SEA (1-27), but not SEA (28-45), neutralized the mitogenic activity of SEA on human PBL. The data suggest that a functional site on SEA that is responsible for its modulation of T cell function involves the N-terminal 27 amino acids. Residues 1 to 27 of SEA could potentially interact at either the level of the TCR or may block the proposed binding of SEA to class II MHC Ag, based on recent data showing that these molecules are involved in SEA-induced proliferation.
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