Clostridium botulinum C2 toxin -: Low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells
Clostridium botulinum C2 toxin -: Low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells
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DOI：
10.1074/jbc.m305849200
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发表时间：
2003-09-26
影响因子：
4.8
通讯作者：
Barth, H
中科院分区：
文献类型：
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作者：
Blöcker, D;Pohlmann, K;Barth, H
二元肉毒梭菌C2毒素由两种单独的蛋白质组成，即转运组分C2 II（80 kDa）和酶组分C2 I，其在细胞的胞质溶胶中使G-肌动蛋白ADP-核糖基化。胰蛋白酶激活的C2 II（C2 IIa）形成七聚体，其结合细胞受体并介导C2 I从酸性内体易位到靶细胞的胞质溶胶中。在这里，我们报告说，易位的C2 I跨细胞膜是伴随着孔形成的C2 IIa。我们使用放射性铷释放试验检测中国仓鼠卵巢细胞膜中的C2 IIa孔。C2 IIa的孔形成依赖于细胞C2毒素受体和酸性脉冲。当C2 IIa在中性pH下与细胞结合时以及当细胞随后转移到酸性培养基（pH小于或等于5.5）时形成孔，但当C2 IIa在酸性培养基中直接加入细胞时未检测到孔。最有可能的是，酸化诱导C2 IIa从“前孔”构象变为“孔”构象，并且在膜结合之前形成孔构象排除插入膜中。当在酸化步骤之前C2 IIa与细胞结合期间存在C2 I时，C2 IIa介导的铷释放减少，表明C2 I与C2 IIa孔的内腔相互作用。当C2 I在酸化步骤后加入到C2 IIa处理的细胞中时，也检测到铷流出的减少，这表明C2 I与C2 IIa在其孔构象中相互作用。此外，C2 I还与人工脂膜中的C2 IIa通道相互作用并部分阻断它们。C2 I只有易位穿过细胞膜时，C2 IIa加C2 I结合到细胞在中性pH值，随后转移到酸性pH值。当细胞结合的C2 IIa暴露于酸性pH值之前，C2 I此外，只有残留中毒的细胞观察到在高毒素浓度，并结合C2 I C2 IIa略有下降。总的来说，C2 IIa孔是必不可少的，但不足以易位的C2 I。用C2毒素中毒靶细胞需要严格协调的pH依赖性结合序列、C2 IIa孔形成和C2 I易位。
The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II ( 80 kDa) and the enzyme component C2I, which ADP-ribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium ( pH less than or equal to 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.