Characterization of two 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 food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C-terminus and at selected sites in the N-terminal domain. Four amino acids in the C-terminus were 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 binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000-fold reduction in MHC 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, SEB, resulted in a 10-fold reduction in MHC class II affinity. The combination of these mutations in the N- and C-terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II-dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab-SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II-independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Abrahmsén
L
L
Pharmacia Biopharmaceuticals, Stockholm, Sweden.
Dohlsten
M
M
Segrén
S
S
Björk
P
P
Jonsson
E
E
Kalland
T
T
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D003601:Cytotoxicity Tests, Immunologic; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics; D000939:Epitopes; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000276:immunology*; D018089:Superantigens / Q000737:chemistry* / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000737:chemistry
D004768:Enterotoxins; D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D018089:Superantigens; C026066:enterotoxin A, Staphylococcal; D015032:Zinc; D000409:Alanine