Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL.

Abstract

Abstract. 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 GMI 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 C1-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 C1-secretion. At T1/2, dose dependencies for K(R)DELmutant toxins were increased ~>10-fold. KDELmutants 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.ETROGRADE transport through Golgi cisternae has been shown to occur for soluble and membrane proteins of the ER (49) and for certain protein toxins (6, 55). Targeting of soluble ER and some type II membrane proteins in this pathway depends on the COOH-terminal sorting signal Lys-Asp-Glu-Leu (KDEL or HDEL

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

“…In contrast, when these same recombinant toxins were applied to apical membranes, there was little or no delay in the anticipated time course of the secretory response. In all past experiments (11,12,21,25,32), we consistently found that toxin preparations purified from V. cholerae supernatants elicited Cl Ϫ secretion with a shorter lag phase and more rapid rate of onset when applied to basolateral rather than apical surfaces of the same cells (Fig. 1, panel A).…”

“…Cell lysates were precleared again by a 30-min incubation with protein A-Sepharose (Pierce). This procedure solubilizes all cellular proteins and proteins associated with the cell monolayer including the entire fraction of toxin bound to the cell surface or internalized via endocytosis (11,12,25).…”

“…they did not elicit a secretory response or affect monolayer resistance). Periplasmic extracts of wt rCT and rLT were characterized in a previous publication (25).…”

Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

“…The time course and magnitude of electrogenic Cl Ϫ secretion in T84 cells demonstrated that Ctx (hereafter referred to as CtxAB) was significantly more active than Etx (hereafter referred to as EtxAB); CtxAB exhibited a shorter apparent lag period and generated a higher short circuit current (22). In this paper, we describe the structural basis for this difference in toxicity.…”

“…The observation that brefeldin A inhibits cholera toxin action (20,21,26), and the presence of a KDEL sequence at that the C terminus of CtxA (RDEL in EtxA) have suggested that the toxin is transported from the TGN to the endoplasmic reticulum (ER). Indeed, mutations in the K(R)DEL sequence of CtxA and EtxA reduce the efficiency of toxin-induced Cl Ϫ secretion in T84 cells (22). It has been speculated that the A-subunit may detach from the B-subunits in the TGN, since only the A-subunit (A1/A2-fragments) has been detected in the ER (23)(24)(25), although transport of the holotoxin from the Golgi to the ER, followed by rapid dissociation and anterograde transport of the B-subunit back to the Golgi, has not been excluded.…”