LIPID INTERACTION OF DIPHTHERIA-TOXIN AND MUTANTS WITH ALTERED FRAGMENT-B .2. HYDROPHOBIC PHOTOLABELLING AND CELL INTOXICATION
LIPID INTERACTION OF DIPHTHERIA-TOXIN AND MUTANTS WITH ALTERED FRAGMENT-B .2. HYDROPHOBIC PHOTOLABELLING AND CELL INTOXICATION
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DOI：
10.1111/j.1432-1033.1987.tb13655.x
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发表时间：
1987-12-15
期刊：
影响因子：
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通讯作者：
MONTECUCCO, C
中科院分区：
文献类型：
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作者：
PAPINI, E;SCHIAVO, G;MONTECUCCO, C
白喉毒素及其B链突变体crm 45、crm 228和crm 1001的膜插入之后是疏水光标记和可光活化的磷脂酰胆碱类似物。结果表明，白喉毒素在中性pH时与磷脂双分子层表面结合，而在低pH时，其A链和B链也与磷脂的烃链相互作用。两种原体的光标记的pH依赖性不同：片段B的pKa约为5.9，而片段A的pKa约为5.2。后一个值与在酸性介质中与毒素一起孵育的细胞的半数最大中毒的pH值相关。这些结果表明，片段B首先进入双分子层并协助片段A的插入，片段B的脂质插入不是白喉毒素膜转位过程中的速率控制步骤。crm 45在光标记试验中表现为白喉毒素，但是，尽管如此，发现它对酸处理的细胞的毒性比白喉毒素低三个数量级，这表明白喉毒素的12-kDaCOOH-末端片段不仅对于它与细胞受体的结合而且对于毒素的膜转位都是重要的。这表明，crm 1001是无毒的，因为它的膜易位的缺陷，发生在一个较低的程度和在一个较低的pH值比天然毒素;因此，crm 1001可能无法逃脱的内涵体腔进入细胞质之前，融合的内涵体与溶酶体。
The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling and photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipid. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic medium. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.