CHARACTERIZATION OF 2 DISTINCT MHC CLASS-II BINDING-SITES IN THE SUPERANTIGEN STAPHYLOCOCCAL-ENTEROTOXIN-A

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram-positive septic shock and f ood poisoning, To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed ala nine substitution mutagenesis throughout the C-terminus and at selecte d sites in the N-terminal domain, Four amino acids in the C-terminus w ere shown to be involved in MHC class II binding, Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II bin ding and appeared to be involved in coordination of a Zn2+ ion, Alanin e substitution of H225 and D227 resulted in a 1000-fold reduction in M HC class II affinity, Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg SEE, resulted in a 10-fold reduction in MHC class LI affinity, Th e combination of these mutations in the N- and C-terminal sites result ed in a profound loss of activity, The perturbation of MHC class II bi nding in the various mutants was accompanied by a corresponding loss o f ability to induce MHC class II-dependent T cell proliferation and cy totoxicity, All of the SEA mutants were expressed as Fab-SEA fusion pr oteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II-independent T cell cytotoxi city assay, We propose a model in which the N- and C-terminal sites in SEA cooperate to form a high affinity interaction which involves bind ing to two separate MHC class II molecules, Considering the previously described SEB-HLA-DR complex, this study indicates that SAgs may bind monovalently or bivalently to MHC class II molecules and could be pre sented to the TCR as a dimeric or trimeric complex.