Oligomerization of a 45 kilodalton fragment of diphtheria toxin at pH 5.0 to a molecule of 20-24 subunits.
Oligomerization of a 45 kilodalton fragment of diphtheria toxin at pH 5.0 to a molecule of 20-24 subunits.
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在 pH 5.0 下，45 千道尔顿的白喉毒素片段寡聚成 20-24 个亚基的分子。
DOI：
10.1021/bi971301x
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Eisenberg,D
中科院分区：
文献类型：
--
作者：
Bell,CE;Poon,PH;Schumaker,VN;Eisenberg,D
白喉毒素（DT）是一种58 kDa的蛋白质，由白喉棒状杆菌溶原菌株分泌，导致人类白喉疾病。DT的催化(C)结构域通过进入细胞质和抑制蛋白质合成来杀死宿主细胞。C结构域的易位跨越内体膜进入宿主细胞的细胞质是由DT的易位(T)结构域介导的。这一过程是由核内体从pH ~ 7酸化到pH ~ 5触发的。在这里，我们展示了crm45 （45 kDa的交叉反应物质），一个45 kDa的DT缺失突变体，包含C和T结构域，但缺乏C末端受体结合(R)结构域，在从pH 7.0到pH 5.0的酸化过程中经历了从单体到大寡聚体的转变。在pH 5.0下，对crm45的动态光散射分析结果表明，该低聚物的多分散性值仅为8 ~ 17%，表明其大小均匀。使用分析性超离心，在pH 5.0下测量crm45的沉降速率和扩散系数，结果低聚物的分子质量测定为890±40 kDa（20±1亚基）。pH 5.0时，平衡沉降数据最适合于质量为1000±50 kDa（24±1亚单位）的单一物种。这些结果揭示了溶液中DT的C和T结构域的均匀大小，20−24亚基寡聚物的ph依赖性形成。由于crm45的低聚物在酸化的核内体的pH下形成，它可能与DT的C结构域跨核内体膜进入宿主细胞的细胞质易位有关。这种crm45寡聚物可能与DT C结构域的膜易位有关，这与早期DT中毒Vero细胞的动力学研究有关，该研究推断DT的~ 20 C结构域在一次事件中转移到宿主细胞的细胞质中。
Diphtheria toxin (DT) is a 58 kDa protein, secreted by lysogenic strains ofCorynebacterium diphtheriae, that causes the disease diphtheria in humans. The catalytic (C) domain of DT kills host cells by gaining entry into the cytoplasm and inhibiting protein synthesis. The translocation of the C domain across the endosomal membrane and into the cytoplasm of a host cell is mediated by the translocation (T) domain of DT. This process is triggered by acidification from pH ∼7 to pH ∼5 within the endosome. Here we show that crm45 (cross-reacting material of 45 kDa), a 45 kDa deletion mutant of DT which contains the C and T domains but lacks the C-terminal receptor-binding (R) domain, undergoes a transition from a monomer to a large oligomer upon acidification from pH 7.0 to pH 5.0. Dynamic light scattering analysis of crm45 at pH 5.0 results in a polydispersity value of only 8−17%, suggesting that the oligomer is uniformly sized. Using analytical ultracentrifugation, measurements of the sedimentation rate and diffusion coefficient of crm45 at pH 5.0 result in a molecular mass determination of 890 ± 40 kDa (20 ± 1 subunits) for the oligomer. Equilibrium sedimentation data on crm45 at pH 5.0 are best fit by a single species with a mass of 1000 ± 50 kDa (24 ± 1 subunits). These results reveal the pH-dependent formation of a uniformly sized, 20−24 subunit oligomer of the C and T domains of DT, in solution. Because the oligomer of crm45 forms at the pH of the acidified endosome, it could be relevant to the translocation of the C domain of DT across the endosomal membrane and into the cytoplasm of host cells. The possible relevance of this oligomer of crm45 to the membrane translocation of the C domain of DT correlates with earlier kinetic studies of DT intoxication of Vero cells, which inferred the transfer of ∼20 C domains of DT to the cytoplasm of host cells, in a single event.