Concomitant loss of conformation and superantigenic activity of staphylococcal enterotoxin B deletion mutant proteins.
The T-cell-stimulating activity of staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases. To investigate the immunologically active domains of the SEB molecule, we have produced truncated fragments of recombinant SEB by C-terminal and N-terminal deletions. The fragments were expressed as fusion proteins with protein A, including a cleavage site to remove the protein A part. Mutant proteins were tested for the ability to stimulate human resting T cells and SEB-reactive T-cell clones. Deletion of only 9 amino acids from the C terminus leads to complete loss of T-cell-stimulating activity. Removing further amino acids from the SEB molecule did not lead to a reexpression of T-cell-mitogenic activity. A mutant protein, however, in which the 9 C-terminal amino acids were replaced with a tail of 68 amino acids encoded by the vector was fully active. Two mutant proteins with N-terminal deletions of 60 and 81 amino acids were inactive as well. A neutralizing monoclonal antibody against a conformational epitope lost binding with all the inactive mutant proteins only, whereas a monoclonal antibody recognizing an epitope involved in emetic activity reacted with all mutant proteins. These results suggest that even small deletions at the C terminus affect the three-dimensional conformation of the SEB molecule.
First Department of Medicine, University of Mainz, Germany.
B
B
Metzroth
T
T
Marx
M
M
Linnig
B
B
Fleischer
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004269:DNA, Bacterial; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017353:Gene Deletion; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology*; D013329:Structure-Activity Relationship
D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D004768:Enterotoxins; D011993:Recombinant Fusion Proteins; C031818:enterotoxin B, staphylococcal