Garred O, Dubinina E, Holm P K, Olsnes S, van Deurs B, Kozlov J V, Sandvig K
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Exp Cell Res. 1995 May;218(1):39-49. doi: 10.1006/excr.1995.1128.
Cleavage of Shiga toxin A-fragment at a highly trypsin-sensitive site increases its enzymatic activity. To investigate the role of this cleavage site in intoxication of cells, we studied the routing, cleavage, and toxicity of mutant toxin where the trypsin-sensitive site had been eliminated. Ultrastructural analysis of toxin tagged with horseradish peroxidase demonstrated that wild-type and mutant toxins were transported from endosomes to the trans-Golgi network and further through the Golgi cisterns to the endoplasmic reticulum. Wild-type toxin was much more efficient than the mutants in provoking rapid intoxication, but after prolonged incubation time also mutants were highly toxic. The cells were able to cleave both wild-type Shiga toxin and the mutants, but the cellular location for cleavage appears to differ. Wild-type toxin was cleaved in the presence of brefeldin A, which disrupts the Golgi cisterns. This indicates that the cleavage occurs in the endosomes or in the trans-Golgi network. In contrast, the mutant Shiga-His (R248H/R251H) was not cleaved in the presence of brefeldin A, indicating that the cleavage can occur only after the toxin has left the trans-Golgi network. In vitro experiments showed that the cytosolic enzyme calpain is able to cleave Shiga-His, and results from in vivo experiments are consistent with the possibility that cleavage is carried out by calpain after the mutant A-fragment has reached the cytosol.
志贺毒素A片段在一个对胰蛋白酶高度敏感的位点发生切割会增加其酶活性。为了研究这个切割位点在细胞中毒过程中的作用，我们研究了消除了胰蛋白酶敏感位点的突变毒素的转运、切割及毒性。对用辣根过氧化物酶标记的毒素进行超微结构分析表明，野生型和突变型毒素均从内体转运至反式高尔基体网络，并进一步通过高尔基体潴泡转运至内质网。在引发快速中毒方面，野生型毒素比突变型毒素高效得多，但延长孵育时间后，突变型毒素也具有高毒性。细胞能够切割野生型志贺毒素和突变型毒素，但切割的细胞定位似乎有所不同。野生型毒素在布雷菲德菌素A存在的情况下被切割，布雷菲德菌素A会破坏高尔基体潴泡。这表明切割发生在内体或反式高尔基体网络中。相反，突变型志贺毒素-His（R248H/R251H）在布雷菲德菌素A存在的情况下未被切割，这表明切割仅在毒素离开反式高尔基体网络后才会发生。体外实验表明，胞质酶钙蛋白酶能够切割志贺毒素-His，体内实验结果与突变型A片段到达胞质溶胶后由钙蛋白酶进行切割的可能性一致。
