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. 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. Cholera toxin (CT), 1The abbreviations used are: CT, cholera toxin; CTA, A subunit of cholera toxin; CTB, B subunit of cholera toxin; Stx-1, Shiga toxin 1; HBT, hop bract tannin; ACT, apple condensed tannin; HBSS, Hanks' balanced salt solution; HRP, horseradish peroxidase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GM1, Gal-Ga1NAc-[NeuAc]-Gal-Glc-Cer. a heterohexameric AB5 toxin produced by Vibrio cholerae, is responsible for the life-threatening diarrheal disease. The B subunits bind to cell surface ganglioside GM1 (1Cuatrecasas P. Biochemistry. 1973; 12: 3558-3566Crossref PubMed Scopus (327) Google Scholar, 2Holmgren J. Lonnroth I. Mansson J. Svennerholm L. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2520-2524Crossref PubMed Scopus (277) Google Scholar), following which the holotoxin is endocytosed by epithelial cells and delivered to the Golgi and endoplasmic reticulum (3Lencer W.I. Hirst T.R. Holmes R.K. Biochim. Biophys. Acta. 1999; 1450: 177-190Crossref PubMed Scopus (222) Google Scholar, 4Fujinaga Y. Wolf A.A. Rodighiero C. Wheeler H. Tsai B. Allen L. Jobling M.G. Rapoport T. Holmes R.K. Lencer W.I. Mol. Biol. Cell. 2003; 14: 4783-4793Crossref PubMed Scopus (192) Google Scholar), where reduction of a disulfide bond on CTA and proteolysis produce fragments A1 and A2. CTA1, which is enzymatically active, then enters the cytoplasm (5Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-215Crossref PubMed Scopus (124) Google Scholar, 6Schumitz A. Herrgen H. Winkeler A. Herzog V. J. Cell Biol. 2000; 148: 1203-1212Crossref PubMed Scopus (177) Google Scholar, 7Teter K. Allyn R.L. Jobling M.G. Holmes R.K. Infect. Immun. 2002; 70: 6166-6171Crossref PubMed Scopus (46) Google Scholar), where, in the presence of NAD, it catalyzes the ADP-ribosylation of Gαs (8Moss J. Vaughan M. J. Biol. Chem. 1977; 252: 2455-2457Abstract Full Text PDF PubMed Google Scholar, 9Cassel D. Selinger Z. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3307-3311Crossref PubMed Scopus (499) Google Scholar, 10Gill D.M. Meren R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3050-3054Crossref PubMed Scopus (478) Google Scholar), resulting in persistent activation of adenylyl cyclase (11Casey P.J. Gilman A.G. J. Biol. Chem. 1988; 263: 2577-2580Abstract Full Text PDF PubMed Google Scholar). The subsequent increase in cyclic AMP (12Taussig R. Gilman A.G. J. Biol. Chem. 1995; 270: 1-4Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar) induces the secretion of fluid and electrolytes into the lumen of the small intestine (13Field M. Fromm D. al-Awqati Q. Greenough III, W.B. J. Clin. Invest. 1972; 51: 796-804Crossref PubMed Scopus (218) Google Scholar). Natural products, like herbal medications, have long been used to treat diarrheal disease and are useful therapeutic agents. Recently, inhibition of the action of bacterial toxins by plant polyphenols, composed of highly condensed tannins, has been reported (14Saito T. Miyake M. Toba M. Okamatsu H. Shimizu S. Noda M. Microbiol. Immunol. 2002; 46: 249-255Crossref PubMed Scopus (47) Google Scholar, 15Oi H. Matsuura D. Miyake M. Ueno M. Takai I. Yamamoto T. Kubo M. Moss J. Noda M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3042-3046Crossref PubMed Scopus (60) Google Scholar, 16Tagashira, M., Yahiro, K., Morinaga, N., Moss, J., and Noda, M. (2003) US-Japan Cholera and Other Bacterial Enteric Infections, 38th Joint Panel Meeting, pp. 45-48, U. S. Department of Health and Human Disease, Public Health Service, National Institutes of Health, Bethesda, MDGoogle Scholar). These include applephenon, HBT, and RG-tannin, derived from apple, hop bract, and Daio (Rhei rhizoma), respectively. RG-tannin and applephenon suppress CT-catalyzed ADP-ribosylation and CT-induced fluid accumulation in rabbit or mouse ileal loops (14Saito T. Miyake M. Toba M. Okamatsu H. Shimizu S. Noda M. Microbiol. Immunol. 2002; 46: 249-255Crossref PubMed Scopus (47) Google Scholar, 15Oi H. Matsuura D. Miyake M. Ueno M. Takai I. Yamamoto T. Kubo M. Moss J. Noda M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3042-3046Crossref PubMed Scopus (60) Google Scholar). RG-tannin is most likely a procyanidine polymer (mainly 8-mer) containing galloyl groups. Applephenon is composed of chlorogenic acid, catechins, and condensed catechins and procyanidins. Its anti-CT activity was attributed to the highly condensed tannin fraction (14Saito T. Miyake M. Toba M. Okamatsu H. Shimizu S. Noda M. Microbiol. Immunol. 2002; 46: 249-255Crossref PubMed Scopus (47) Google Scholar), apple-condensed tannin (ACT), which contains procyanidine polymers (less than 15-mer and mostly 3–6-mer) (17Yanagida A. Kanda T. Takahashi T. Kamimura A. Hamazono T. Honda S. J. Chromatogr. A. 2000; 890: 251-259Crossref PubMed Scopus (67) Google Scholar). HBT was extracted from hop bract polyphenols, which contain procyanidine polymers (about 10–30-mers) with molecular weights around 6,000. HBT inhibited Shiga toxin 1 (Stx-1), which is produced by enterohemorrhagic Escherichia coli (16Tagashira, M., Yahiro, K., Morinaga, N., Moss, J., and Noda, M. (2003) US-Japan Cholera and Other Bacterial Enteric Infections, 38th Joint Panel Meeting, pp. 45-48, U. S. Department of Health and Human Disease, Public Health Service, National Institutes of Health, Bethesda, MDGoogle Scholar). Stx-1 is another AB5 toxin with B subunits that bind to Gb3 globotriaosyl ceramide on the cell surface (18Lindberg A.A. Brown J.E. Stromberg N. Westling-Ryd M. Schultz J.E. Karlsson K.A. J. Biol. Chem. 1987; 262: 1779-1785Abstract Full Text PDF PubMed Google Scholar, 19Lingwood C.A. Law H. Richardson S. Petric M. Brunton J.L. De Grandis S. Karmali M. J. Biol. Chem. 1987; 262: 8834-8839Abstract Full Text PDF PubMed Google Scholar), leading to its endocytosis. The A subunit of Stx-1 is a 33-kDa protein with RNA N-glycosidase activity that blocks protein synthesis in eukaryotic cells. HBT interfered with Stx-1-induced inhibition of protein synthesis and lethal toxicity in a mouse inoculated with enterohemorrhagic Escherichia coli producing Stx-1. HBT also suppressed CT ADP-ribosyltransferase activity. It is not clear, however, whether inhibition of fluid accumulation by RG-tannin or applephenon is, in fact, the result of suppression of CT enzymatic activity. Because polyphenols are generally believed to precipitate proteins (20Haslam E. J. Nat. Prod. 1996; 59: 205-215Crossref PubMed Scopus (1063) Google Scholar, 21Charlton A.J. Baxter N.J. Khan M.L. Moir A.J. Haslam E. Davies A.P. Williamson M.P. J. Agric. Food Chem. 2002; 50: 1593-1601Crossref PubMed Scopus (580) Google Scholar), we thought that RG-tannin, applephenon, and/or HBT might act by preventing CT binding to or internalization by cells. We report here that RG-tannin did not inhibit CT binding to cells but formed complexes with CT and thereby suppressed internalization from the cell surface. On the other hand, HBT or applephenon formed large aggregates with CT that interfered with its binding to and internalization by cells, indicating that the mechanism of RG-tannin action was different from those used by HBT or applephenon. Reagents—CT and CTA were purchased from List Biological Laboratories, and CTB was from Calbiochem. Applephenon 50 and ACT were kindly provided by Dr. Yanagida (Nikka Whisky Distilling Co., Ltd.). HBT was prepared by Asahi Beer Co., and RG-tannnin was prepared by Tsumura & Co. Applephenon, RG-tannin, and HBT were dissolved in PBS, and HBT was passed through a 0.22-μm membrane filter. 2-Mercaptoethanesulfonic acid, HRP-conjugated streptavidin, and BM blue substrate for peroxidase were purchased from Aldrich, Amersham Biosciences, and Roche Applied Science, respectively. Cell Viability—The number of viable cells was determined using a cell counting kit (Dojindo Laboratories, Kumamoto, Japan), and viability of cells was evaluated by trypan blue dye exclusion. The cell counting kit is based on the same principle as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (22Mosmann T. J. Immunol. Methods. 1983; 16: 55-63Crossref Scopus (46516) Google Scholar). Cells, grown on a 96-well plate, were incubated with 10 μl of cell counting reagent (company-supplied) at 37 °C for 1 h before color was quantified at 450 nm. Absorbance at 450 nm as a measure of cell number correlated well with the number counted directly using a hemacytometer. For Trypan blue dye exclusion assays, cells were detached from the plate by incubation with 0.25% of trypsin plus 0.02% EDTA. Equal volumes of cell suspension and 0.3% trypan blue in PBS were mixed before total and stained (dead) cells (at least 100 cells) were counted. Cell Enzyme-linked Immunosorbent Assay—Vero cells (2 × 104 cells/well) were cultured in Eagle's minimal essential medium with 10% fetal calf serum in 96-well plates overnight. Biotin-labeled CT was prepared according to the supplier's instructions using EZ-Link Sulfo-N-hydroxylsulfosuccinimide-SS-Biotin (Pierce). Approximately 84% of the label was incorporated into CTA. Cells were washed with cold Hanks' balanced salt solution with 0.1% of BSA (HBSS-BSA) three times before the addition of 100 μl of 20 nm CT in HBSS-BSA, incubated at 4 °C for 30 min, washed three times with cold HBSS, and fixed for 20 min with 0.25% in with BSA in PBS for 1 cells were incubated with HRP-conjugated for 1 and was with BM blue a the was with 1 and color was at 450 nm. was cells were washed with PBS three of toxin was quantified as binding of CT and to HBSS-BSA, to 37 was and the cells were incubated at 37 °C for the before with cold HBSS, and addition of acid, which not cells, to disulfide and from CT to the cell surface. incubation at for 30 min, cells were with for min and with HRP-conjugated as to GM1 Enzyme-linked Immunosorbent μl of GM1 ganglioside in were to well of a 96-well plate, which was incubated at 37 °C the had and the assay was at The plate was washed three times with 0.1% 20 in PBS and blocked for 1 h with BSA in 100 μl of CT or with polyphenols as were and incubation for 1 the plate was washed to toxin before the addition of and incubation for 1 was using BM the plate was washed three CT Binding and was with using a kit Cells (2 × were in plates containing and incubated at 37 °C overnight. cells were incubated at 4 °C for 30 min in containing Cy3-labeled CT with or polyphenols cells were washed to before or incubation at 37 °C for 10 min with containing Cells were fixed with and by fluorescence microscopy of as were incubated with 20 nm CT in PBS at for 30 min before centrifugation × was and was dissolved in μl of and for min, proteins were by in and to the same of CT and were in and used to was using HRP-conjugated by reagent was by and the density of was quantified using The of CT precipitated was as CTA, most of the label was in CTA. were as a of the total CT of CTA or CTB was determined using CTA or and CT in PBS were incubated at for 30 min before to the of a gradient of sucrose in PBS solution and × 4 were then the of were to in a and proteins were to Biotin-labeled CT was with HRP-conjugated and CT in fraction was quantified as CTA using and as of total of of of × were using CTA and of a and are molecular weight exclusion to a of a protein is in of and of a protein is in using a Biotin-labeled CT, CTA, or CTB was incubated with polyphenols in μl of PBS at for 30 The was then to and which were at × for min the was was to μl with PBS before were for of proteins by in and to Biotin-labeled toxin was and quantified as was quantified using a kit and were to Vero cells × cells/well) grown in plates and incubated at 4 °C for 30 Cells were then washed three and 20 nm CT was in the presence of by incubation at 37 °C for 30 was then and of in incubation for 20 min at cells were and and the was used for assay following the Applephenon, ACT, and HBT, but RG-tannin, CT Binding to Vero and internalization of CT by Vero cells was on CT a at nm not 20 nm CT and the of applephenon, ACT condensed tannin fraction of or HBT were incubated with cells at 4 °C for 30 min, toxin binding to cells was inhibited HBT and ACT were than HBT at 50 a at which applephenon was suppressed binding of CT by In contrast, RG-tannin, at to did not CT of Vero cells with polyphenols did not cell number or not a of in incubation of cells with polyphenols did not subsequent binding of CT or the to CT The of CT, to cells, did not following incubation with polyphenols, with the polyphenols These data that the of polyphenols did not result from binding to the cells with of CT binding or by polyphenols with CT, leading to its from the cells. of were CT was incubated with it before addition to the cells not The of HBT, applephenon, and ACT on CT binding to cells were confirmed by fluorescence microscopy using Cy3-labeled CT CT to Vero cells in the of polyphenols and in the presence of in the presence of applephenon or ACT, however, CT binding was CT to cells. incubation of cells at 37 °C for 10 min, toxin was and in the in and cells but not in or cells. whether cells were cells with polyphenols were detached from the plate with and total cell number and trypan blue exclusion were with polyphenols did not the cell number or cell viability by trypan blue dye exclusion with cells not to HBT and Applephenon CT into Vero whether HBT and applephenon suppress internalization of CT, cells were with CT at 4 °C for 30 min and then washed with to CT before incubation at 37 °C with CT internalization was quantified from the cell surface with CT internalization was suppressed by applephenon or HBT in a suppression was with RG-tannin were the not and were in viable cell number following incubation with RG-tannin of did not suppress binding of CT to Vero cells but suppressed CT internalization. We determined whether of CT and RG-tannin suppress internalization. CT and RG-tannin were incubated at 4 °C for 30 min before the addition to cells and incubation at 4 °C for 30 Binding of CT was not by with RG-tannin Cells were washed and incubated at 37 °C for the in RG-tannin suppressed internalization of CT in a HBT and Applephenon CT Binding to to surface ganglioside We whether polyphenols suppress CT binding to of polyphenols with GM1 did not CT binding CT however, was suppressed in a manner by applephenon and but not was with RG-tannin to the polyphenols suppress CT binding or we whether polyphenols CT. Biotin-labeled CT was incubated with the of polyphenols for 30 min at which the were and the resulting precipitate was on from polyphenols precipitated CT in a concentration-dependent 50 HBT, ACT, and applephenon a and precipitated the of CT. RG-tannin was were with CTA RG-tannin was and ACT was than applephenon. CTB was used of CTA RG-tannin precipitated CTB than other RG-tannin with with RG-tannin and CT was using sucrose density was mostly incorporated in CTA as and the CTA is The was in fraction 4 with CT and CT. the of fraction of CT and CT were a was that CT was in a fraction than CT. of the CT was in the fraction and CT were also in the fraction not These data that HBT, ACT, and applephenon large aggregates of than 250 with CT from the using molecular weight and RG-tannin with CT. RG-tannin did not inhibit CT binding to Vero cells, we that RG-tannin formed a with CTA but not to CTA incubated with RG-tannin was in a fraction than CTA of CTB to a fraction was also that RG-tannin might with CTA and CTB and complexes with CT. CTA or CTB incubated with HBT was in the with of a large of than 250 B and or CTA or CTB was also in the fraction not that RG-tannin complexes with CT, molecular weight exclusion were used and of CT was in with a molecular weight exclusion membrane not we used a membrane in which 10% of CT was in membrane molecular weight of CT incubated with RG-tannin was in and of the and complexes were in were using CTA or of the CTA or CTB was in the using a however, CTA or CTB was and in CTA or CTB was in the of a membrane with HBT or These data confirmed that RG-tannin with CTA and CTB and with of or using molecular weight exclusion in were quantified using and the of toxin in of a molecular weight exclusion is are of of three of toxin in of to to to to to to to to to to in a CTA with the by RG-tannin of CT that RG-tannin with CTA. CTA might interfere with the effect of RG-tannin on CT internalization. CT was incubated with of RG-tannin in the presence or of nm CTA before the addition to cells and incubation at 4 °C for 30 Cells were then incubated at 37 °C to CT internalization. in CTA the effect of RG-tannin on CT internalization. RG-tannin and condensed tannin fraction of apple ACT were reported to suppress CT-induced ADP-ribosylation of Gαs or and fluid accumulation in mouse or rabbit ileal loops (14Saito T. Miyake M. Toba M. Okamatsu H. Shimizu S. Noda M. Microbiol. Immunol. 2002; 46: 249-255Crossref PubMed Scopus (47) Google Scholar, 15Oi H. Matsuura D. Miyake M. Ueno M. Takai I. Yamamoto T. Kubo M. Moss J. Noda M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3042-3046Crossref PubMed Scopus (60) Google however, mechanism of action was not In we on the effect of RG-tannin, applephenon, and ACT, on the binding and internalization of CT. We also used HBT, which is from hop bract and contains similar to those in It suppressed ADP-ribosylation by CT and was to a of produced by Escherichia coli (16Tagashira, M., Yahiro, K., Morinaga, N., Moss, J., and Noda, M. (2003) US-Japan Cholera and Other Bacterial Enteric Infections, 38th Joint Panel Meeting, pp. 45-48, U. S. Department of Health and Human Disease, Public Health Service, National Institutes of Health, Bethesda, MDGoogle Scholar). HBT, ACT, and applephenon, but not RG-tannin, inhibited the binding of CT to Vero of the polyphenols inhibited CT internalization. The polyphenols used in report were not at the incubation of cells with polyphenols did not inhibit subsequent binding of and cell was not inhibition of internalization of CT was not to the of toxin by the These data that the of the polyphenols were not to binding to cells or to cell but from with CT in solution and on the cell thereby preventing CT binding and internalization. HBT, ACT, and applephenon CT, resulting in large which were by high These CT aggregates did not bind to GM1 or to Vero cells. RG-tannin, on the other hand, did not large aggregates with CT similar to applephenon or HBT, but it did with CT as demonstrated by sucrose density gradient centrifugation and through molecular weight exclusion RG-tannin did not inhibit CT binding to cells or to GM1 on It however, with CTB as well as CTA. It that the of CTB was not by with RG-tannin, did CT internalization. ACT was than applephenon for inhibition of CT binding and of CT, which ACT was than applephenon in of CT activity (14Saito T. Miyake M. Toba M. Okamatsu H. Shimizu S. Noda M. Microbiol. Immunol. 2002; 46: 249-255Crossref PubMed Scopus (47) Google Scholar). The different of RG-tannin and other polyphenols with CT the presence in RG-tannin, but not in applephenon and HBT, of galloyl H. Matsuura D. Miyake M. Ueno M. Takai I. Yamamoto T. Kubo M. Moss J. Noda M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3042-3046Crossref PubMed Scopus (60) Google Scholar) reported that RG-tannin the by CT of fluid In however, RG-tannin at 10 or 50 suppressed at 20 min CT internalization by Vero cells and in addition to internalization of the inhibition of might responsible for the suppression by RG-tannin of CT-induced fluid It is also not whether the is by cells and delivered from Golgi to endoplasmic reticulum and then or whether polyphenols like RG-tannin, applephenon, or HBT are and interfere with toxin activity. are to bind to however, the on of the and the protein surface (20Haslam E. J. Nat. Prod. 1996; 59: 205-215Crossref PubMed Scopus (1063) Google Scholar, 21Charlton A.J. Baxter N.J. Khan M.L. Moir A.J. Haslam E. Davies A.P. Williamson M.P. J. Agric. Food Chem. 2002; 50: 1593-1601Crossref PubMed Scopus (580) Google Scholar). RG-tannin was reported to interfere with CT activity but not with that of of Escherichia coli H. Matsuura D. Miyake M. Ueno M. Takai I. Yamamoto T. Kubo M. Moss J. Noda M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3042-3046Crossref PubMed Scopus (60) Google Scholar). We that RG-tannin from HBT and applephenon in its with CT. polyphenols in with bacterial is at the of the and the CT is thought to endocytosed binding to GM1 and to or A.A. Jobling M.G. S. M. J.L. Holmes R.K. Lencer W.I. J. Cell Biol. PubMed Scopus Google Scholar, M.L. B. K. J. Cell Sci. 2003; Scholar, J. Cell Sci. 2003; PubMed Scopus Google Scholar). These polyphenols interfere with in endocytosis. RG-tannin internalization of CT from the cell surface by or internalization of CT to the cell surface was suppressed by HBT and applephenon to HBT another therapeutic with applephenon and RG-tannin to disease. based on the it also on CT that had to cells. We Y. and T. for We Dr. Vaughan National Institutes of Health, Bethesda, for useful and of the

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