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首页> 外文期刊>Molecular and Cellular Biology >Membrane Insertion by Anthrax Protective Antigen in Cultured Cells
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Membrane Insertion by Anthrax Protective Antigen in Cultured Cells

机译：炭疽保护抗原在培养细胞中的膜插入

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The enzymatic moieties of anthrax toxin enter the cytosol of mammalian cells via a pore in the endosomal membrane formed by the protective antigen (PA) moiety. Pore formation involves an acidic pH-induced conformational rearrangement of a heptameric precursor (the prepore), in which the seven 2β2-2β3 loops interact to generate a 14-strand transmembrane β-barrel. To investigate this model in vivo, we labeled PA with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) at cysteine residues introduced into the 2β2-2β3 loop. Each labeled PA was bound to CHO cells, and NBD fluorescence was monitored over time in stirred cell suspensions or by confocal microscopy. A strong increase was observed with NBD at positions 305, 307, 309, and 311, sites where side chains are predicted to face the bilayer, and little change was seen at residues 304, 306, 308, 310, and 312, sites where side chains are predicted to face the pore lumen. The increase at position 305 was inhibited by membrane-restricted quenchers, low temperature, or various reagents known to affect toxin action. Of the 24 NBD attachment sites examined, all but three gave results qualitatively consistent with the β-barrel model. Besides supporting the β-barrel model of membrane insertion, our results describe the time course of insertion and identify PA residues where NBD gives a strong signal upon membrane insertion in vivo.
机译：炭疽毒素的酶部分通过保护性抗原（PA）部分形成的内体膜中的孔进入哺乳动物细胞的细胞质。孔的形成涉及七聚体前体（前孔）的酸性pH诱导的构象重排，其中七个2β2-2β3环相互作用以生成14链跨膜β-桶。为了在体内研究该模型，我们在引入2β2-2β3环的半胱氨酸残基上用荧光团7-硝基苯-2-氧杂-1,3-二唑（NBD）标记了PA。每个标记的PA均与CHO细胞结合，并在搅拌的细胞悬浮液中或通过共聚焦显微镜随时间监测NBD荧光。观察到NBD在位置305、307、309和311处显着增加，这是预测侧链面向双层的位点，而在残基304、306、308、310和312处的侧位几乎看不到变化。预测链面对孔腔。膜限制淬灭剂，低温或已知影响毒素作用的各种试剂可抑制305位的增加。在检查的24个NBD附着位点中，除3个之外的所有位点在质量上均与β-桶模型一致。除了支持膜插入的β-桶模型外，我们的结果还描述了插入的时间过程，并鉴定了PA残基，其中NBD在体内插入膜后会发出强烈信号。

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