Cellular uptake of Clostridium botulinum C2 toxin:: Membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain
Cellular uptake of Clostridium botulinum C2 toxin:: Membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain
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DOI：
10.1021/bi0354278
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发表时间：
2003-12-30
期刊：
影响因子：
2.9
通讯作者：
Barth, H
中科院分区：
文献类型：
--
作者：
Haug, G;Wilde, C;Barth, H
肉毒梭菌C2毒素是二元肌动蛋白-腺苷三磷酸化毒素家族的原型。C2毒素由两个分离的非连接蛋白质组成。酶组分C2 I ADP-核糖基化靶细胞胞质溶胶中的肌动蛋白。结合/易位组分C2 II介导酶组分的细胞结合及其从酸性内体到胞质溶胶中的易位。在蛋白水解活化后，C2 II在内体膜中形成七聚体孔，并且最有可能的是，C2 I通过这些孔易位到胞质溶胶中。对于这一步，细胞热休克蛋白Hsp 90是必不可少的。我们分析了甲氨蝶呤对融合毒素细胞摄取的影响，其中二氢叶酸还原酶（DHFR）融合到C2 I的C-末端。在这里，我们报告说，展开的C2 I-DHFR是需要通过C2 IIa组件的毒素的细胞摄取。C2 I-DHFR融合毒素在体外催化肌动蛋白的ADP核糖基化，并且当与C2 IIa一起应用时能够毒害培养细胞。叶酸类似物甲氨蝶呤的结合有利于二氢叶酸还原酶结构域的稳定三维结构。用甲氨蝶呤预处理C2 I-DHFR防止胰蛋白酶切割C2 I-DHFR。在甲氨蝶呤的存在下，细胞与C2 I-DHFR/C2 II的中毒被抑制。甲氨蝶呤的存在减少了C2 I-DRFR融合毒素从内体区室到胞质溶胶的易位和C2 IIa介导的C2 I-DHFR跨细胞膜的直接易位。甲氨蝶呤对C2 I/C2 IIa的细胞中毒没有影响，也没有改变C2 IIa介导的C2 I-DHFR与细胞的结合。数据表明，甲氨蝶呤阻止了C2 I-DHFR融合毒素的解折叠，从而抑制了甲氨蝶呤结合的C2 I-DHFR从内体到靶细胞胞质溶胶的易位。
The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADPribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DRFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.