Abstract
The issue of toxins produced by Helicobacter pylori (H. pylori) urgently requires clarification given that the bacterium causes gastric epithelial cell damage which may lead to precancerous and cancerous changes. During an investigation of the possibility of mono(adenosine 5′‐diphosphate (ADP)‐ribosyl)‐ation by H. pylori products, as observed for other bacterial toxins, we found that radioactivity of [adenylate‐32P]nicotinamide adenine dinucleotide (NAD) is incorporated into an H. pylori protein of 80 kDa after incubation with crude bacterial extract. In contrast, [carbonyl‐14C]NAD did not show any radioactivity incorporation. Unexpectedly, treatment of the modified protein with 0.1 N HC1, but not 0.1 JV NaOH, released the AMP moiety. Such chemical properties are characteristic of bacterial DNA ligase‐AMP complexes. We found that an antibody raised against Escherichia coli DNA ligase [EC 6.5.1.2] immunoprecipitated the modified 80 kDa protein. Our results indicate that incorporation of radioactivity derived from NAD into the 80 kDa protein was due to adenylation, but not mono‐(ADP‐ribosyl) ation, of the DNA ligase of H. pylori.
Keywords: Helicobacterpylori, DNA ligase, Mono(ADP‐ribosyl)ation, Adenylation, NAD
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REFERENCES
- 1. ) Sugiyama , T. and Yachi , A.Relationship of Helicobacter pylori to gastric carcinogenesis . Ann, Cancer Res. Ther. , 4 , 77 – 81 ( 1995. ). [Google Scholar]
- 2. ) Parsonnet , J. , Hansen , S. , Rodriguez , L. , Gelb , A. B. , Warnke , R. A. , Jellum , E. , Orentreich , N. , Vogelman , J. H. and Friedman , G. D.Helicobacter pylori infection and gastric lymphoma . N. Engl. J. Med. , 330 , 1267 – 1271 ( 1994. ). [DOI] [PubMed] [Google Scholar]
- 3. ) Seppala , K.H. pylori and gastric ulcer disease . In “ Helicobacter pylori, Basic Mechanisms to Clinical Cure ,” ed. Hunt R. H. and Tytgat G. N. J. , pp. 429 – 435 ( 1994. ). Kluwer Academic Publishers; , Netherlands . [Google Scholar]
- 4. ) Cover , T. L. and Blaser , M. J.Purification and characterization of the vacuolating toxin from Helicobacter pylori . J. Biol Chem. , 267 , 10570 – 10575 ( 1992. ). [PubMed] [Google Scholar]
- 5. ) Tummuru , M. K. , Sharma , S. A. and Blaser , M. J.Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL‐8 in gastric epithelial cells . Mol. Microbiol. , 18 , 867 – 876 ( 1995. ). [DOI] [PubMed] [Google Scholar]
- 6. ) Censini , S. , Lange , C. , Xiang , Z. , Crabtree , J. E. , Ghiara , P. , Borodovsky , M. , Rappuoli , R. and Covacci , A.Cag, a pathogenicity island of Helicobacter pylori, encodes type I‐specific and disease‐associated virulence factors . Proc. Natl Acad. Sci. USA , 93 , 14648 – 14653 ( 1996. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. ) Sekura , R. D. , Fish , K , Manclark , C. R. , Meade , B. , Zhang , Y. L.Pertussis toxin. Affinity purification of a new ADP‐ribosyltransferase . J. Biol. Chem. , 258 , 14647 – 14651 ( 1983. ). [PubMed] [Google Scholar]
- 8. ) Van Ness , B. G. , Howard , J. B. and Bodley , J. W.ADP‐ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl‐diph‐thamide and its hydrolysis products . J. Biol. Chem. , 255 , 10710 – 10716 ( 1980. ). [PubMed] [Google Scholar]
- 9. ) Van Ness , B. G. , Howard , J. B. and Bodley , J. W.ADP‐ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl‐amino acid and its hydrolysis products . J. Biol. Chem. , 255 , 10717 – 10720 ( 1980. ). [PubMed] [Google Scholar]
- 10. ) Little , J. W. , Zimmerman , S. B. , Oshinsky , C. K. and Gellert , M.Enzymatic joining of DNA strands, II. An enzyme‐adenylate intermediate in the DPN‐dependent DNA ligase reaction . Proc. Natl. Acad. Sci. USA , 58 , 2004 – 2011 ( 1967. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. ) Milligan , G. , Streaty , R. A. , Gierschik , P. , Spiegel , A. M. and Klee , W. A.Development of opiate receptors in neonatal rat brain . J. Biol. Chem. , 262 , 8626 – 8630 ( 1987. ). [PubMed] [Google Scholar]
- 12. ) Althaus , F. R. and Richter , C.ADP‐ribosylation of proteins: enzymology and biological significance . Mol. Biol. Biochem. Biophys. , 37 , 1 – 237 ( 1987. ). [PubMed] [Google Scholar]
- 13. ) Maehama , T. , Nishina , H. and Katada , T.ADP‐ribosylarginine glycohydrolase catalyzing the release of ADP‐ribose from the cholera toxin‐modified alpha‐sub‐units of GTP‐binding proteins. . Biochem. , 116 , 1134 – 1138 ( 1994. ). [DOI] [PubMed] [Google Scholar]
- 14. ) Modrich , P. , Anraku , Y. and Lehman , I. R.Deoxyribonucleic acid ligase. Isolation and physical characterization of the homogeneous enzyme from Escherichia coli . J. Biol. Chem. , 248 , 7495 – 7501 ( 1973. ). [PubMed] [Google Scholar]
- 15. ) Miyake , M. , Hirayama , T. , Wada , A. , Padilla , P. L , Moss , J. , Kato , I. and Noda , M.An arginine‐specific mono ADP‐ribosyltransferase activity in Helicobacter pylori cell lysate . The 12th International Symposium on ADP‐ribosylation Reactions: “Bacterial Pathogenesis to Cancer,” meeting abstracts , p. 30 ( 1997. ).
- 16. ) Tomkinson , A. E. , Totty , N. P. , Ginsburg , M. and Lindahl , T.Location of the active site for enzyme‐adenylate formation in DNA ligases . Proc. Natl Acad. Sci. USA , 88 , 400 – 404 ( 1991. ). [DOI] [PMC free article] [PubMed] [Google Scholar]