| This study examined the N-terminal third of staphylococcal enterotoxin type A (SEA) for involvement in emetic and superantigen activities. Mutations resulting in either in-frame deletions or in single amino acid residue substitutions in SEA were constructed by site-directed mutagenesis. The mutant SEAs were tested in murine splenocyte prolifection and monkey emetic assays to determine their superantigen and emetic activities, respectively. Mutant SEAs with deletions of residues 3 to 17 or 56 to 59) retained significant superantigen activity but all others were inactive. The deletion mutants were not tested for emetic activity as they were degraded in vitro by monkey stomach lavage fluid, in contrast to wild-type SEA. Substitution of L12, K14, S16, K27, D45, Q46, T51, K81, or D86 with glycine or alanine did not significantly affect the superantigen or emetic activities of SEA. Substitution of V85 with glycine (designated SEA-V85G) did not affect superantigen activity; emetic activity of SEA-V85G was not determined as it was degraded by monkey stomach lavage fluid. Substitution of N25 and L48 with glycine or alanine, or F47 with glycine or serine decreased superantigen activity, with the mutants at position 47 being the most defective. Results of a cell binding assay suggested that F47 and L48 of SEA are important for interactions with major histocompatibility complex class II molecules, whereas N25 of SEA is important for interactions with T-cell receptors. With respect to emetic activity, the mutant SEAs at position 25 or 48 exhibited decreased, but significant activity. Interestingly, the two mutant SEAs at position 47 had different emetic activities; SEA-F47G was non-emetic when administered intragastrically at 500 {dollar}mu{dollar}g per animal whereas SEA-F47S was emetic at this dosage. The difference in the emetic activity of the position 47 mutants did not appear to be due to differences in superantigen activity. The corresponding substitution mutant of SEB, SEB-F44S, also retained significant emetic activity, despite its previously reported defect in murine T-cell activation. Taken together, these findings support the hypothesis that there is not an absolute correlation between SE emetic and superantigen activities. |
