Lencer W I, Constable C, Moe S, Jobling M G, Webb H M, Ruston S, Madara J L, Hirst T R, Holmes R K
Combined Program in Pediatric Gastroenterology and Nutrition, Children's Hospital, Boston, Massachusetts 02115, USA.
J Cell Biol. 1995 Nov;131(4):951-62. doi: 10.1083/jcb.131.4.951.
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.
霍乱弧菌和大肠杆菌热不稳定毒素（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），引发肠道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进入该途径最初取决于毒素B亚基在细胞表面与GM1的结合。CT后续加工步骤发生的解剖学区域尚不清楚。然而，CT具有酶活性的A亚基含有内质网滞留信号KDEL（LT中为RDEL）。因此，如果KDEL基序是正常CT转运所必需的，那就意味着CT从高尔基体向内质网的移动。为了验证这一想法，制备了重组野生型（wt）和突变型CT及LT。CT的COOH末端KDEL序列被七个不相关的氨基酸取代：LEDERAS。在LT中，RDEL中的亮氨酸被缬氨酸取代产生了一个单点突变。野生型和突变型毒素表现出相似的酶活性以及与固定在塑料上的GM1的结合亲和力。使用标准电生理技术，将重组毒素的生物学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突变均延迟了毒素诱导的Cl-分泌的时间进程。在半衰期时，K(R)DEL突变毒素的剂量依赖性增加了≥10倍。KDEL突变体表现出不同程度的更高温度敏感性。与信号转导速率较慢直接一致，KDEL突变体转运至基底外侧膜的速度比野生型CT慢（通过选择性细胞表面生物素化评估为B亚基的转胞吞作用）。K(R)DEL突变对毒素内吞速率没有影响。这些数据提供了证据，表明CT和LT与内源性KDEL受体直接相互作用，并暗示两种毒素可能都需要通过高尔基体池和内质网逆行移动才能实现高效和最大的生物学活性。
