Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL.
Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL.
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DOI：
10.1083/jcb.131.4.951
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发表时间：
1995-11
期刊：
影响因子：
--
通讯作者：
Holmes RK
中科院分区：
文献类型：
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作者：
Lencer WI;Constable C;Moe S;Jobling MG;Webb HM;Ruston S;Madara JL;Hirst TR;Holmes RK
霍乱弧菌和大肠杆菌不耐热毒素（CT 和 LT）通过结合顶端受体（神经节苷脂 GM1）并随后激活基底外侧效应器（腺苷酸环化酶），引发肠上皮细胞的分泌反应。我们最近提出，极化细胞中的信号转导可能需要含毒素膜的转胞吞作用（Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098）。 CT 靶向该途径最初取决于毒素 B 亚基与细胞表面 GM1 的结合。 CT 处理的后续步骤发生的解剖区室的定义不太明确。然而，CT 的酶活性 A 亚基包含 ER 保留信号 KDEL（LT 中的 RDEL）。因此，如果正常 CT 运输需要 KDEL 基序，则意味着 CT 从高尔基体到内质网的移动。为了测试这个想法，制备了重组野生型 (wt) 和突变体 CT 和 LT。 CT 中的 COOH 末端 KDEL 序列被七个不相关的氨基酸取代：LEDERAS。在 LT 中，进行了单点突变，将 RDEL 中的亮氨酸替换为缬氨酸。 Wt 和突变毒素表现出相似的酶活性和与固定在塑料上的 GM1 的结合亲和力。使用标准电生理学技术，将重组毒素的生物活性评估为从极化的人上皮细胞系T84引发的Cl-分泌反应。 CT 和 LT 的 K(R)DEL 突变延迟了毒素诱导的 Cl 分泌的时间过程。在 T1/2 时，K(R)DEL 突变毒素的剂量依赖性增加 > 或 = 10 倍。 KDEL 突变体表现出不同程度的更高的温度敏感性。与较慢的信号转导速率直接一致。 KDEL 突变体比 wt CT 更慢地运输到基底外侧膜（通过选择性细胞表面生物素化作为 B 亚基的转胞吞作用进行评估）。 K(R)DEL 突变对毒素内吞速率没有影响。这些数据提供了 CT 和 LT 与内源性 KDEL 受体直接相互作用的证据，并暗示这两种毒素可能需要通过高尔基池和 ER 逆行运动才能实现有效和最大的生物活性。
Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin- containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GM1 at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH- terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced Cl- secretion. At T1/2, dose dependencies for K(R)DEL-mutant toxins were increased > or = 10-fold. KDEL-mutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction. KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.