Channel formation by the binding component of Clostridium botulinum C2 toxin:: Glutamate 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo
Channel formation by the binding component of Clostridium botulinum C2 toxin:: Glutamate 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo
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DOI：
10.1021/bi034199e
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发表时间：
2003-05-13
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：
Blöcker, D;Bachmeyer, C;Barth, H
肉毒梭菌C2毒素的结合组分(C2II)介导肌动蛋白ADP核糖化酶组分(C2I)进入靶细胞胞浆。C2II(80 KDa)被胰酶切割激活，蛋白水解性激活的C2II(60 KDa)在溶液中寡聚为七聚体。活化的C2II在具有高度阳离子选择性和电压门控的脂质双层膜上形成通道。讨论了该通道在C2I跨细胞膜进入胞浆转运中的作用。C2II的303-331氨基酸残基包含一种保守的疏水和亲水残基交替的模式，这可能通过创建两条反平行的β-链来促进膜插入和通道的形成。其中一些残基位于C2II通道的关键位置，特别是位于膜中央的谷氨酸307(E307)和位于膜反面的甘氨酸316(G316)。在体内和人工脂双层实验中，对这些氨基酸的单赖氨酸取代和C2II的双突变E307K/G316K进行了分析。C2I跨细胞膜转运的pH依赖性被改变，需要小于或等于5.2的pH才能将C2I转运到靶细胞；否则，没有观察到C2II促进C2I进入Vero细胞的变化。C2II的通道性质因突变而显著改变，阳离子选择性降低就是明证。有趣的是，野生型C2II的电压依赖性在E307K突变体中完全消失，这意味着e307负责电压门控。氯喹阻断了突变体C2II和C2I对Vero细胞的E307K突变通道和中毒作用，表明氯喹与E307的结合不涉及E307。总体而言，电压门控和C2II通道的阳离子选择性在C2I转运到胞浆中并不起重要作用。
The binding component (C2II) of the binary Clostridium botulinum C2 toxin mediates transport of the actin ADP-ribosylating enzyme component (C2I) into the cytosol of target cells. C2II (80 kDa) is activated by trypsin cleavage, and proteolytically activated C2II (60 kDa) oligomerizes to heptamers in solution. Activated C2II forms channels in lipid bilayer membranes which are highly cation selective and voltage-gated. A role for this channel in C2I translocation across the cell membrane into the cytosol is discussed. Amino acid residues 303-331 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which likely facilitates membrane insertion and channel formation by creating two antiparallel beta-strands. Some of the residues are in strategic positions within the putative C2II channel, in particular, glutamate 307 (E307) localized in its center and glycine 316 (G316) localized on the trans side of the membrane. Here, single-lysine substitutions of these amino acids and the double mutant E307K/ G316K of C2II were analyzed in vivo and in artificial lipid bilayer experiments. The pH dependence of C2I transport across cellular membranes was altered, and a pH of less than or equal to5.2 was needed for C2I translocation into target cells; otherwise, no change in C2II-promoted entry of C2I into Vero cells was observed. The channel properties of C2II were substantially changed by the mutations, as evidenced by reduced cation selectivity. Interestingly, the voltage dependence of wild-type C2II was completely lost for the E307K mutant, which means that E307 is responsible for voltage gating. Chloroquine blocked the E307K mutant channel and intoxication of Vero cells by mutant C2II and C2I, indicating that chloroquine binding does not involve E307. Overall, the voltage gating and cation selectivity of the C2II channel do not play an important role in translocation of C2I into the cytosol.