Biochemical and mutational analysis of the histidine residues of staphylococcal enterotoxin A
Biochemical and mutational analysis of the histidine residues of staphylococcal enterotoxin A
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DOI：
10.1128/iai.64.3.885-890.1996
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发表时间：
1996-03-01
影响因子：
3.1
通讯作者：
Betley, M
中科院分区：
文献类型：
--
作者：
Hoffman, M;Tremaine, M;Betley, M
本研究的目的是研究组氨酸残基在葡萄球菌肠毒素A（SEA）生物活性中的作用。羧甲基化SEA不能刺激小鼠T细胞增殖，但对猴胃灌洗液降解具有抗性，表明天然构象是完整的。随后对SEA的组氨酸残基进行定点诱变，SEA-H44 A（组氨酸44被丙氨酸取代的SEA）、SEA-H44 D、SEA-H50 A、SEA-H50 D、SEA-H114 A、SEA-H114 D、SEA-H187 A和SEA-H187 D保留了超抗原和催吐活性，而SEA-H225 A和SEA-H225 D在刺激T细胞增殖的能力上是缺陷的。这些突变体不能与SEA竞争结合Raji细胞，表明SEA-H225 A和SEA-H225 D中的缺陷是由于受损的主要组织相容性复合物II类结合。SEA-H225 D只有在高剂量下才能引起猴子的呕吐反应，而SEA-H225 A在低剂量或高剂量下都不会引起呕吐反应。相比之下，SEA-H61 A和SEA-H61 D在催吐活性方面有缺陷，但在刺激鼠T细胞增殖的能力方面没有缺陷。总之，这些研究表明，羧基末端组氨酸在残基位置225 SEA是重要的超抗原和呕吐活动的肠毒素。组氨酸61似乎是重要的催吐活性，但不是超抗原活性，这与假设的两个活动是分离的葡萄球菌肠毒素一致。
The goal of this study was to examine the role of histidine residues in the biological activities of staphylococcal enterotoxin A (SEA), Carboxymethylated SEA was unable to stimulate murine T-cell proliferation but was resistant to monkey stomach lavage fluid degradation, suggesting that native conformation was intact. Site-directed mutagenesis of the histidine residues of SEA was subsequently performed, SEA-H44A (SEA with histidine 44 replaced with alanine), SEA-H44D, SEA-H50A, SEA-H50D, SEA-H114A, SEA-H114D, SEA-H187A, and SEA-H187D retained superantigen and emetic activities, whereas SEA-H225A and SEA-H225D were defective in the ability to stimulate T-cell proliferation. These mutants were unable to compete with SEA for binding to Raji cells, suggesting that the defect in SEA-H225A and SEA-H225D is due to impaired major histocompatibility complex class II binding. SEA-H225D provoked an emetic response in monkeys only if fed at high doses, while SEA-H225A did not provoke an emetic response at low or high doses. In comparison, SEA-H61A and SEA-H61D were defective in emetic activity but not in the ability to stimulate murine T-cell proliferation. Overall, these studies show that the carboxy-terminal histidine at residue position 225 of SEA is important for both the superantigen and emetic activities of this enterotoxin. Histidine 61 appears to be important for emetic activity but not for superantigen activity, consistent with the hypothesis that the two activities are separable in staphylococcal enterotoxins.