IDENTIFICATION OF CRITICAL RESIDUES OF STAPHYLOCOCCAL-ENTEROTOXIN-B FOR LYMPHOMONOCYTE PROLIFERATION AND CYTOKINE PRODUCTION

Superantigens bind to major histocompatibility complex class II molecu les and stimulate large numbers of T cells expressing particular v bet a elements of the T-cell receptor. Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that causes food poisoning and toxic-shock syndrome. The toxicity of SEB is thought to be mediated by T-cell sti mulation and cytokine production. Different regions of the SEB molecul e are important for mitogenic activity. To identify critical residues of SEB in the region 124-154, which competitively inhibits the mitogen ic activity of the toxin, we used the synthetic peptide approach and a lanine scanning mutagenesis as a probe. We synthesized eight peptides with alanine replacement of all residues in the SEB sequence 131-138 a nd tested them for the capacity to inhibit both SEB-induced proliferat ion of human lymphomonocytes and the production of tumor necrosis fact or alpha and interferon gamma. Mutation to alanine of the residue Thr 133 improved the inhibition of SEB-induced proliferation and cytokine production, whereas the substitution of Ser 131 also increased the inh ibition, albeit to a lesser degree. The peptide obtained by substituti on of Val 136 with alanine was unable to inhibit SEB-induced prolifera tion and cytokine production, suggesting that Val 136 is essential for mitogenic activity. Thus hydrophobic interactions apparently are very important for mitogenic activity. The identification of critical resi dues in this active site in the SEB and the computer modeling based on crystal X-ray data contribute to a better understanding of the molecu lar mechanism of the superantigen and may be useful for therapeutical applications.