Proteolytic activation of cholera toxin and Escherichia coli labile toxin by entry into host epithelial cells - Signal transduction by a protease-resistant toxin variant
Proteolytic activation of cholera toxin and Escherichia coli labile toxin by entry into host epithelial cells - Signal transduction by a protease-resistant toxin variant
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DOI：
10.1074/jbc.272.24.15562
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发表时间：
1997-06-13
影响因子：
4.8
通讯作者：
Hirst, TR
中科院分区：
文献类型：
--
作者：
Lencer, WI;Constable, C;Hirst, TR
霍乱和大肠杆菌不耐热毒素（CT和LT）需要蛋白水解连接其酶A亚基的两个主要结构域的肽环以获得最大活性（称为“切口”）。为了测试宿主肠上皮细胞是否可以提供必需的蛋白酶，制备了重组rCT和rLT以及蛋白酶抗性突变体CTR 192 H。毒素作用被评估为从极化的人上皮T84细胞单层引起的Cl-分泌反应（Isc）。当施加到顶端细胞表面时，野生型毒素引起Isc的快速增加（80 μ A/cm（2））。伊势降低了1倍，然而，当毒素应用于基底外侧膜时，用胰蛋白酶体外预处理野生型毒素使“基底外侧"分泌反应恢复到”顶端"水平。毒素进入T84细胞通过顶端，但不是基底侧膜导致切口的丝氨酸型蛋白酶的A亚基，T84细胞，但是，没有切口CTR 192 H，和CTR 192 H引起的分泌反应仍然衰减，即使当应用于顶端膜。因此，T84细胞充分表达丝氨酸型蛋白酶，用于激活CT和LT的A亚基。然而，蛋白酶仅在毒素通过顶膜进入细胞时才可被激活。
Cholera and Escherichia coli heat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed ''nicking''). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared, Toxin action was assessed as a Cl- secretory response (Isc)elicited from monolayers of polarized human epithelial T84 cells, When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 mu A/cm(2)). Ise was reduced a-fold, however, when toxins were applied to basolateral membranes, Pretreatment of wild typo toxins with trypsin in vitro restored the ''basolateral'' secretory responses to ''apical'' levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease; T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient, for activating the A subunits of CT and LT, The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.