Merlen Clémence, Fayol-Messaoudi Domitille, Fabrega Sylvie, El Hage Tatiana, Servin Alain, Authier François
Institut National de la Santé et de la Recherche Médicale U510, Faculté de Pharmacie Paris XI, Châtenay-Malabry, France.
FEBS J. 2005 Sep;272(17):4385-97. doi: 10.1111/j.1742-4658.2005.04851.x.
We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.
我们已经确定了内化的霍乱毒素（CT）在大鼠肝脏内体装置中的体内和体外代谢命运。在体内，CT被内化并积聚在内体中，在那里它以pH依赖的方式进行降解。使用内体裂解物对CT进行体外蛋白水解需要酸性pH，并且对天冬氨酸蛋白酶抑制剂胃蛋白酶抑制剂A敏感。通过非变性免疫沉淀，酸性CT降解活性归因于内体组织蛋白酶D的腔内形式。发现使用内体裂解物或纯组织蛋白酶D对毒素的水解速率对于天然CT和游离CT-B亚基较高，而对于游离CT-A亚基较低。基于IC(50)值，竞争研究表明CT-A和CT-B亚基在组织蛋白酶D酶上共享一个共同的结合位点，天然CT和游离CT-B亚基对蛋白酶显示出最高的亲和力。通过免疫荧光，在肝癌HepG2和肠Caco-2细胞内吞作用的早期，证实了内化的CT与组织蛋白酶D的部分共定位。牛组织蛋白酶D在低pH下产生的CT水解产物对外源Gsα蛋白显示出ADP-核糖基转移酶活性，这表明CT细胞毒性至少部分可能与内吞小泡内的蛋白水解事件有关。总之，这些数据确定内吞装置是内吞CT积累和蛋白水解降解的关键亚细胞位点，并确定内体组织蛋白酶D是一种可能负责CT细胞毒性激活的酶。
