Enhanced sensitivity to cholera toxin in ADP-ribosylarginine hydrolase-deficient mice

Cholera toxin (CT) produced by Vibrio cholerae causes the devastating diarrhea of cholera by catalyzing the ADP-ribosylation of the alpha subunit of the intestinal Gs protein (Gsalpha), leading to characteristic water and electrolyte losses. Mammalian cells contain ADP-ribosyltransferases similar to CT and an ADP-ribosyl(arginine)protein hydrolase (ADPRH), which cleaves the ADP-ribose-(arginine)protein bond, regenerating native protein and completing an ADP-ribosylation cycle. We hypothesized that ADPRH might counteract intoxication by reversing the ADP-ribosylation of Gsalpha. Effects of intoxication on murine ADPRH-/- cells were greater than those on wild-type cells and were significantly reduced by overexpression of wild-type ADPRH in ADPRH-/- cells, as evidenced by both ADP-ribose-arginine content and Gsalpha modification. Similarly, intestinal loops in the ADPRH-/- mouse were more sensitive than their wild-type counterparts to toxin effects on fluid accumulation, Gsalpha modification, and ADP-ribosylarginine content. Thus, CT-catalyzed ADP-ribosylation of cell proteins can be counteracted by ADPRH, which could function as a modifier gene in disease. Further, our study demonstrates that enzymatic cross talk exists between bacterial toxin ADP-ribosyltransferases and host ADP-ribosylation cycles. In disease, toxin-catalyzed ADP-ribosylation overwhelms this potential host defense system, resulting in persistence of ADP-ribosylation and intoxication of the cell.     

The sites on the regulatory component of adenylate cyclase which are ADP-ribosylated by cholera toxin

In rat liver membranes cholera toxin ADP-ribosylated two polypeptides (M_r 42000 and 47000) in the regulatory component of adenylate cyclase. L-arginine methyl ester specifically inhibited both the activation of adenylate cyclase and ADP-ribosylation by cholera toxin, suggesting that cholera toxin modified arginine, or arginine-like, residues. A hydrolysis-resistant analogue of GTP (β, γ-imidoguanosine 5′-triphosphate, p(NH)ppG) bound to the regulatory protein in an essentially irreversible manner. Pretreatment with the analogue failed to inhibit the labelling of polypeptides by cholera toxin showing that the sites for ADP-ribosylation were different from those at which guanyl nucleotides were bound.     

Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells

A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.     

Inhibition of ADP-ribosyltransferase activity of cholera toxin by MDL 12330A and chlorpromazine

ADP-ribosylation by cholera toxin of the guanine nucleotide binding regulatory protein (Gs) of rat liver membrane adenylate cyclase was inhibited by 0.1-1 mM MDL 12330A or 0.1-1 mM chlorpromazine. Basal as well as cholera toxin activated adenylate cyclase activity in liver membranes was also inhibited by the two drugs. NAD glycohydrolase activity and self-ADP-ribosylation of cholera toxin were also inhibited by MDL 12330A and chlorpromazine. These effects of MDL 12330A and chlorpromazine may be related to their effects on cholera toxin-induced fluid secretion in vivo.     

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.     

Use of substituted (benzylidineamino)guanidines in the study of guanidino group specific ADP-ribosyltransferase

A number of substituted (benzylidineamino)guanidines with different substitutents in the benzene nucleus are synthesized by coupling substituted benzaldehydes with aminoguanidine, and these compounds are tested as substrates for cholera toxin catalyzed ADP-ribosylation. A spectrophotometric assay method for the measurement of ADP-ribosyltransferase activity is developed, making use of the absorption characteristics of some of these compounds and the difference in the ionic character of the free compounds and the ADP-ribosylated products. The kinetic parameters for the ADP-ribosylation of these compounds are evaluated. A correlation between log kcat or log (kcat/Km) and the Hammett substituent constant sigma is observed. This correlation suggests the importance of substrate electronic effects on the enzymatic reaction. The reactivity of these compounds as acceptors of ADP-ribosyl groups in the reaction catalyzed by cholera toxin increases with increasing electron-donating power of the substituents in the benzene function. The effect is primarily on the catalytic rate constant, kcat, not on the binding constant, Km. The results are consistent with an SN2 reaction mechanism in which the deprotonated guanidino group makes a nucleophilic attack on the C-1 carbon of the ribose moiety.     

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

Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J272, 4385-4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsalpha proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha isoforms. Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H(+)-ATPase pump and in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H(+)-ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin. This cathepsin D-dependent action of cholera toxin under the control of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic response to cholera toxin. Thus, in hepatic cells, a unique endocytic pathway was revealed following cholera toxin administration, with regulation specificity most probably occurring at the locus of the endosome and implicating endosomal proteases, such as cathepsin D, as well as organelle acidification.     

Pertussis and cholera toxin ADP-ribosylation in Dictyostelium discoideum membranes

We report a 39 kDa substrate for cholera and pertussis toxins is present in D. discoideum membranes. This protein did not co-migrate with alpha subunits of either Gs (45 kDa and 52 kDa) or Gi (41 kDa) from control mammalian cells. The presence of GTP or its non-hydrolyzable analogs enhanced the ADP-ribosylation in response to cholera toxin, but did not significantly alter ADP-ribosylation by pertussis toxin. Divalent cations inhibited the ADP-ribosylation by both toxins. The possible association of this novel G-protein with D. discoideum adenylate cyclase may underlie some of the unique regulatory features of this enzyme. Alternatively, this G-protein may regulate one of several other cellular responses mediated by the cAMP receptor.     

Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.     

Differential activities of plant polyphenols on the binding and internalization of cholera toxin in vero cells

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.     

Differential effects of tumor promoters on cAMP production: inhibition of receptor-mediated and potentiation of cholera toxin-mediated stimulation

Tetradecanoylphorbol-acetate and other tumor promoters inhibit prostaglandin E2 and isoproterenol-induced cAMP accumulation in mouse thymocytes but markedly potentiate cAMP production induced by cholera toxin. Cholera toxin is known to stimulate cAMP production by inducing ADP-ribosylation of the alpha-subunit of a guanine nucleotide-binding regulatory (G) protein, resulting in activation of the catalytic unit of adenylate cyclase. G proteins have been implicated as plasma membrane transducers for a variety of additional signals. It is possible that the growth promoting and co-mitogenic properties of tumor promoters are related to their effects on G proteins.     

Effect of apple polyphenol extract on hepatoma proliferation and invasion in culture and on tumor growth, metastasis, and abnormal lipoprotein profiles in hepatoma-bearing rats

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 mug/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.     

Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein

Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.     

Inhibitory effect of ibuprofen on the cholera toxin-induced cyclic AMP response in Chinese hamster ovary cells

Abstract Ibuprofen, an inhibitor of prostaglandin synthesis in eukaryotic cells, was shown to inhibit the accumulation of 3',5'-cyclic adenosine monophosphate (cyclic AMP) in Chinese hamster ovary (CHO) cells exposed to cholera toxin. The inhibition was dose dependent, with a dose of 100 μg/ml reducing the cholera toxin response by approximately 50%, and maximal inhibition was observed when the drug was applied to the cells simulataneously with or 1 h before the toxin. Although ibuprofen also inhibited adenylate cyclase stimulation by forskolin, suggesting a nonspecific effect, the drug had no effect on cholera toxin-induced cyclic AMP accumulation when added to the culture medium 15 min or more after the toxin.     

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.     

ADP-ribosylation of a Mr 21,000 membrane protein by type D botulinum toxin

When crude membrane fraction from bovine adrenal gland was incubated with type D botulinum toxin in the presence of NAD, a membrane protein with a molecular weight of 21,000 was specifically ADP-ribosylated. This ADP-ribosylation occurred dependent on the dose of the toxin and was abolished by prior boiling ADP-ribose transfer to the membrane protein was significantly suppressed when agmatine and L-arginine methyl ester were included in the reaction mixture. Dithiothreitol stimulated this ADP-ribosylation about 3-fold. Incubation of membrane fractions from mouse brain and pancreas with this toxin also resulted in ADP-ribosylation of a protein of the same molecular weight. These results suggested that type D botulinum toxin catalyzed transfer of an ADP-ribose moiety of NAD to the specific membrane protein common to secretory cells.     

Mono(ADP-ribosyl)ation of poly(ADP-ribose)polymerase by cholera toxin.

Poly(ADP-ribose)polymerase (PADPRP) was found to be an efficient protein acceptor for the arginine-specific ADP-ribosylation reaction catalyzed by cholera toxin (CT). The covalent modification of PADPRP was carried out with [32P]2'-dNAD as a selective mono(ADP-ribosyl)ation substrate. Mono(2'-dADP-ribosyl)ated-PADPRP was identified by autoradiographic analysis of the CT reaction products following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Addition of recombinant ADP-ribosylation factor (rARF), a small GTP-binding protein that stimulates the enzymatic activity of CT, enhanced the mono(2'-dADP-ribosyl)ation of PADPRP in a time- and substrate-dependent manner. In contrast, rARF did not change the ADP-ribose polymerizing activity of PADPRP. Peptide mapping mapping of [32P] labeled (2'-dADP-ribose)-PADPRP, following partial proteolysis with papain, revealed that the DNA-binding domain of PADPRP contained the mono(2'-dADP-ribosyl)ated arginine residue(s). Our results are consistent with the conclusion that PADPRP is susceptible to arginine-specific mono(ADP-ribosyl)ation catalyzed by CT.     

The protective activity of tea catechins against experimental infection by Vibrio cholerae O1.

Tea catechins inhibited the fluid accumulation induced by cholera toxin in sealed adult mice. The catechins also reduced fluid accumulation by Vibrio cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea catechins may possess protective activity against V. cholerae O1.     

Studies on Hot Water-soluble and Dilute Alkali-soluble Polysaccharides from the Fruit Body of Mushroom (Psalliota campestris (Linn) Fr.)

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 μg/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.     

Mono(adenosine diphosphate ribosyl) transferase in Xenopus tissues. Direct demonstration by a zymographic localization in sodium dodecyl sulfate--polyacrylamide gels

A semiquantitative method to measure mono(adenosine diphosphate ribosyl) transferase activity [mADPRT] in tissue extracts is described. After electrophoretic separation in sodium dodecyl sulfate (SDS)--polyacrylamide gels, renatured enzymatic activity is demonstrated in situ by incubation of the slab gels with radiolabeled NAD+ and histones. Precipitation of the radiolabeled product in the gel allows localization of the enzyme by autoradiography. This method is suitable for two-dimensional gel electrophoresis, whereby proteins are electrofocused in the presence of 9 M urea and subsequently subjected to electrophoresis in SDS. A single major band showing mADPRT activity of Mr approximately 30 Kda was observed in all crude extracts of Xenopus tissues examined. Accumulation of acid-insoluble radiolabeled products was dependent on added histones and was specifically inhibited by agmatine. The ADPRT activity of cholera toxin A fragment could also be demonstrated by this technique. Reducing agents stimulated the activity of cholera toxin A fragment while depressing that of Xenopus mADPRT.