Stenmark H, Olsnes S, Sandvig K
Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
J Biol Chem. 1988 Sep 15;263(26):13449-55.
The role of specific receptors in the translocation of diphtheria toxin A fragment to the cytosol and for the insertion of the B fragment into the cell membrane was studied. To induce nonspecific binding to cells, toxin was either added at low pH, or biotinylated toxin was added at neutral pH to cells that had been treated with avidin. In both cases large amounts of diphtheria toxin became associated with the cells, but there was no increase in the toxic effect. There was also no increase in the amount of A fragment that was translocated to the cytosol, as estimated from protection against externally added Pronase E. In cells where specific binding was abolished by treatment with 12-O-tetradecanoyl-phorbol 13-acetate, trypsin, or 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, unspecific binding did not induce intoxication or protection against protease. This was also the case in untreated L cells, which showed no specific binding of the toxin. When Vero cells with diphtheria toxin bound to specific receptors were exposed to low pH, the cells were permeabilized to K+, whereas this was not the case when the toxin was bound nonspecifically at low pH or via avidin-biotin. The data indicate that the cell-surface receptor for diphtheria toxin facilitates both insertion of the B fragment into the cell membrane and translocation of the A fragment to the cytosol.
研究了特定受体在白喉毒素A片段转运至胞质溶胶以及B片段插入细胞膜过程中的作用。为诱导毒素与细胞的非特异性结合，要么在低pH条件下添加毒素，要么将生物素化毒素在中性pH条件下添加至用抗生物素蛋白处理过的细胞。在这两种情况下，大量白喉毒素都与细胞结合，但毒性效应并未增强。根据对外部添加的链霉蛋白酶E的抗性估计，转运至胞质溶胶的A片段量也没有增加。在用12 - O - 十四烷酰佛波醇13 - 乙酸酯、胰蛋白酶或4,4'-二异硫氰酸 - 2,2'-二苯乙烯二磺酸处理以消除特异性结合的细胞中，非特异性结合并未诱导细胞中毒或产生对蛋白酶的抗性。未处理的L细胞也是如此，其未显示出毒素的特异性结合。当与白喉毒素结合于特异性受体的Vero细胞暴露于低pH时，细胞对K + 通透，而当毒素在低pH条件下非特异性结合或通过抗生物素蛋白 - 生物素结合时则并非如此。数据表明，白喉毒素的细胞表面受体促进了B片段插入细胞膜以及A片段转运至胞质溶胶。
