CELL-MEDIATED REDUCTION AND INCOMPLETE MEMBRANE TRANSLOCATION OF DIPHTHERIA-TOXIN MUTANTS WITH INTERNAL DISULFIDES IN THE A-FRAGMENT
CELL-MEDIATED REDUCTION AND INCOMPLETE MEMBRANE TRANSLOCATION OF DIPHTHERIA-TOXIN MUTANTS WITH INTERNAL DISULFIDES IN THE A-FRAGMENT
复制标题
DOI：
10.1074/jbc.270.35.20787
复制
发表时间：
1995-09-01
影响因子：
4.8
通讯作者：
OLSNES, S
中科院分区：
文献类型：
--
作者：
FALNES, PO;OLSNES, S
活性白喉毒素由通过二硫键连接的两个片段A和B组成。B片段与细胞表面受体结合，并有助于酶活性A片段易位至胞质溶胶。通常情况下，毒素A片段从酸性内体进入胞质溶胶，但易位也可以诱导在质膜的水平，通过暴露细胞与表面结合的毒素低pH值。最近，我们发现，二硫键引入到A片段的突变是抑制易位。在目前的工作中，我们发现，虽然完全易位的A片段被封锁，三个突变体毒素进行还原的interfragment二硫键后，低pH暴露，而内部的二硫键在A片段保持完整。在这些突变体中的两个的情况下，A片段被释放到细胞外介质后，细胞结合的毒素暴露于低pH值。释放的突变体A片段的pH值曲线是相同的野生型A片段的易位到胞质溶胶，和释放被抑制的条件，干扰A片段易位。在第三个突变体的情况下，它仍然是细胞相关的片段间二硫键还原后，易位中间检测。结果表明，片段间二硫键的还原可以发生在没有完全易位的A片段的胞质溶胶，他们表明，还原发生在易位过程的早期阶段。我们的研究结果表明，跨膜的A片段的易位是在C端启动。
Active diphtheria toxin consists of two fragments, A and B, joined by a disulfide bond. The B fragment binds to cell surface receptors and aids in the translocation of the enzymatically active A fragment to the cytosol. Normally, the toxin A fragment enters the cytosol from acidic endosomes, but translocation can also be induced at the level of the plasma membrane by exposing cells with surface-bound toxin to low pH. Recently, we showed that disulfide bonds introduced into the A fragment by mutation are inhibitory for translocation. In the present work, we found that although the complete translocation of the A fragment is blocked, three mutant toxins underwent reduction of the interfragment disulfide bond upon low pH exposure, whereas the internal disulfide in the A fragment remained intact. In the case of two of these mutants, the A fragment was released into the extracellular medium upon exposure of cell-bound toxin to low pH. The pH profile for the release of the mutant A fragments was the same as for translocation of wild-type A fragment to the cytosol, and the release was inhibited by conditions that interfere with A fragment translocation. In the case of the third mutant, which remained cell-associated upon reduction of the interfragment disulfide bond, a translocation intermediate was detected. The results show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction takes place at an early stage in the translocation process. Our findings suggest that the translocation of the A fragment across the membrane is initiated at the C terminus.