Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D

Abstract

Cholera toxin (CT) produced by Vibrio cholerae is the major virulence factor responsible for the massive secretory diarrhea of infected humans [1]. CT interacts with intestinal epithelial cells and induces chloride secretion due to toxin-mediated activation of adenylate cyclase and elevation of intracellular cAMP. Activation of adenylate cyclase results from ADP-ribosylation of Arg201 of the a-subunit of the stimulatory GTP-binding regulatory protein, Gsa, catalyzed by the toxin [2].CT ( 84 kDa) is an oligomeric protein of the A-B type composed of one activating A subunit (CT-A, r.m.m. 27 400 Da) and five identical B subunits (CT-B, r.m.m. 11 600 Da) arranged in a ring-like configuration that bind ganglioside GM1 at the cell surface [3]. The CT-A subunit is comprised of two functional domains termed the A1 and A2 peptides linked by a single disulfide bond. The A1 peptide exhibits the toxin's ADP-ribosyltransferase activity which is necessary for CT cytotoxic action. The A2 peptide contains the endoplasmic reticulum-targeting motif KDEL at its C-terminus. For full ADP-ribosylation of the stimulatory heterotrimeric GTPase Gsa, enzymatic production of a degradative fragment generated from native CT and structurally related to the A1 peptide must occur followed by its targeting to the Gsa substrate. This process, which takes 30-40 min in most cell types, 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 Gsa 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.Abbreviations CT, cholera toxin; EN, endosomes; HI, human insulin; PA, pepstatin-A; PDI, protein...

Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

“…Arf6 also serves as an allosteric activator of the CT-catalyzed ADP-ribosylation of Gα s [35,36], with crystal structures of an Arf6-CT complex lending insight into how this occurs through changes in the active site loop structure that facilitates NAD binding [37]. Furthermore, CT has been shown to increase rates of intra-endosomal acidification [34] and while intra-endosomal acidification is not thought to be important for translocation of CT, the trafficking of CT through a low pH compartment has been demonstrated to be important for proteolytic activation of the catalytic subunit [38]. These observations suggest the possibility that PMT may use the same initial trafficking pathway as Tfn and/or CT, and may be trafficked to an acidified Arf6-positive endosome, from where it may translocate.…”

Omeprazole Antagonizes Virulence and Inflammation in Salmonella enterica -Infected RAW264.7 Cells

“…Many pathogens that target the endosomal compartment of host cells rely on vacuolar acidification for their ability to cause disease. For example, decreased vacuolar pH induces the activation of selected bacterial toxins such as cholera and tetanus toxins (27,43) and the intracellular replication of Leishmania (28) and Salmonella enterica (35). A chemical commonly used for inhibiting vacuolar acidification is the macrolide bafilomycin A 1 .…”

“…A chemical commonly used for inhibiting vacuolar acidification is the macrolide bafilomycin A 1 . It acts as a potent irreversible inhibitor of H ϩ -ATPases (40) and interferes with the activity of cholera toxin in liver cells and tetanus toxin in neuronal cells (27,43). Bafilomycin A 1 also has been reported to decrease the intracellular replication of L. monocytogenes and S. enterica serovar Typhimurium in both epithelial and monocytic cells (7,9,35).…”

Role of receptor‐mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity

“…Following CT binding to the plasma membrane of hepatocytes, CT accumulated in a low‐density endosomal compartment, with maximum accumulation observed by 15–30 min [4,7]. Following ATP‐dependent endosomal acidification, internalized CT was rapidly proteolyzed within hepatic endosomes by aspartic acid protease cathepsin D [7]. In vivo studies showed that the acidotropic agent chloroquine, as well as the carboxylic ionophore monensin, inhibited CT activation of adenylate cyclase and increased the lag period for this process [5,6].…”