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. 1989 May;86(9):3075–3079. doi: 10.1073/pnas.86.9.3075

Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.

C Locht 1, C Capiau 1, C Feron 1
PMCID: PMC287067  PMID: 2470088

Abstract

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barbieri J. T., Rappuoli R., Collier R. J. Expression of the S-1 catalytic subunit of pertussis toxin in Escherichia coli. Infect Immun. 1987 May;55(5):1321–1323. doi: 10.1128/iai.55.5.1321-1323.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Black W. J., Munoz J. J., Peacock M. G., Schad P. A., Cowell J. L., Burchall J. J., Lim M., Kent A., Steinman L., Falkow S. ADP-ribosyltransferase activity of pertussis toxin and immunomodulation by Bordetella pertussis. Science. 1988 Apr 29;240(4852):656–659. doi: 10.1126/science.2896387. [DOI] [PubMed] [Google Scholar]
  3. Capiau C., Petre J., Van Damme J., Puype M., Vandekerckhove J. Protein-chemical analysis of pertussis toxin reveals homology between the subunits S2 and S3, between S1 and the A chains of enterotoxins of Vibrio cholerae and Escherichia coli and identifies S2 as the haptoglobin-binding subunit. FEBS Lett. 1986 Aug 18;204(2):336–340. doi: 10.1016/0014-5793(86)80839-0. [DOI] [PubMed] [Google Scholar]
  4. Carroll S. F., Collier R. J. Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. J Biol Chem. 1987 Jun 25;262(18):8707–8711. [PubMed] [Google Scholar]
  5. Carroll S. F., Collier R. J. NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3307–3311. doi: 10.1073/pnas.81.11.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carroll S. F., McCloskey J. A., Crain P. F., Oppenheimer N. J., Marschner T. M., Collier R. J. Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7237–7241. doi: 10.1073/pnas.82.21.7237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Douglas C. M., Collier R. J. Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity. J Bacteriol. 1987 Nov;169(11):4967–4971. doi: 10.1128/jb.169.11.4967-4971.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gray G. L., Smith D. H., Baldridge J. S., Harkins R. N., Vasil M. L., Chen E. Y., Heyneker H. L. Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1984 May;81(9):2645–2649. doi: 10.1073/pnas.81.9.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hsia J. A., Moss J., Hewlett E. L., Vaughan M. ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding. J Biol Chem. 1984 Jan 25;259(2):1086–1090. [PubMed] [Google Scholar]
  10. Kaczorek M., Delpeyroux F., Chenciner N., Streeck R. E., Murphy J. R., Boquet P., Tiollais P. Nucleotide sequence and expression of the diphtheria tox228 gene in Escherichia coli. Science. 1983 Aug 26;221(4613):855–858. doi: 10.1126/science.6348945. [DOI] [PubMed] [Google Scholar]
  11. Kandel J., Collier R. J., Chung D. W. Interaction of fragment A from diphtheria toxin with nicotinamide adenine dinucleotide. J Biol Chem. 1974 Apr 10;249(7):2088–2097. [PubMed] [Google Scholar]
  12. Katada T., Tamura M., Ui M. The A protomer of islet-activating protein, pertussis toxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch Biochem Biophys. 1983 Jul 1;224(1):290–298. doi: 10.1016/0003-9861(83)90212-6. [DOI] [PubMed] [Google Scholar]
  13. Knowles J. R. Tinkering with enzymes: what are we learning? Science. 1987 Jun 5;236(4806):1252–1258. doi: 10.1126/science.3296192. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Locht C., Barstad P. A., Coligan J. E., Mayer L., Munoz J. J., Smith S. G., Keith J. M. Molecular cloning of pertussis toxin genes. Nucleic Acids Res. 1986 Apr 25;14(8):3251–3261. doi: 10.1093/nar/14.8.3251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Locht C., Cieplak W., Marchitto K. S., Sato H., Keith J. M. Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli. Infect Immun. 1987 Nov;55(11):2546–2553. doi: 10.1128/iai.55.11.2546-2553.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Locht C., Keith J. M. Pertussis toxin gene: nucleotide sequence and genetic organization. Science. 1986 Jun 6;232(4755):1258–1264. doi: 10.1126/science.3704651. [DOI] [PubMed] [Google Scholar]
  18. Marchitto K. S., Munoz J. J., Keith J. M. Detection of subunits of pertussis toxin in Tn5-induced Bordetella mutants deficient in toxin biological activity. Infect Immun. 1987 May;55(5):1309–1313. doi: 10.1128/iai.55.5.1309-1313.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  20. Michel A., Dirkx J. Occurrence of tryptophan in the enzymically active site of diphtheria toxin fragment A. Biochim Biophys Acta. 1977 Mar 28;491(1):286–295. doi: 10.1016/0005-2795(77)90064-2. [DOI] [PubMed] [Google Scholar]
  21. Munoz J. J., Arai H., Cole R. L. Mouse-protecting and histamine-sensitizing activities of pertussigen and fimbrial hemagglutinin from Bordetella pertussis. Infect Immun. 1981 Apr;32(1):243–250. doi: 10.1128/iai.32.1.243-250.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neer E. J., Lok J. M., Wolf L. G. Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase. J Biol Chem. 1984 Nov 25;259(22):14222–14229. [PubMed] [Google Scholar]
  23. Nicosia A., Bartoloni A., Perugini M., Rappuoli R. Expression and immunological properties of the five subunits of pertussis toxin. Infect Immun. 1987 Apr;55(4):963–967. doi: 10.1128/iai.55.4.963-967.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nicosia A., Perugini M., Franzini C., Casagli M. C., Borri M. G., Antoni G., Almoni M., Neri P., Ratti G., Rappuoli R. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4631–4635. doi: 10.1073/pnas.83.13.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pizza M., Bartoloni A., Prugnola A., Silvestri S., Rappuoli R. Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7521–7525. doi: 10.1073/pnas.85.20.7521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sato H., Sato Y. Bordetella pertussis infection in mice: correlation of specific antibodies against two antigens, pertussis toxin, and filamentous hemagglutinin with mouse protectivity in an intracerebral or aerosol challenge system. Infect Immun. 1984 Nov;46(2):415–421. doi: 10.1128/iai.46.2.415-421.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sato Y., Kimura M., Fukumi H. Development of a pertussis component vaccine in Japan. Lancet. 1984 Jan 21;1(8369):122–126. doi: 10.1016/s0140-6736(84)90061-8. [DOI] [PubMed] [Google Scholar]
  29. Tamura M., Nogimori K., Murai S., Yajima M., Ito K., Katada T., Ui M., Ishii S. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry. 1982 Oct 26;21(22):5516–5522. doi: 10.1021/bi00265a021. [DOI] [PubMed] [Google Scholar]
  30. Tamura M., Nogimori K., Yajima M., Ase K., Ui M. A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells. J Biol Chem. 1983 Jun 10;258(11):6756–6761. [PubMed] [Google Scholar]
  31. Tweten R. K., Barbieri J. T., Collier R. J. Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity. J Biol Chem. 1985 Sep 5;260(19):10392–10394. [PubMed] [Google Scholar]
  32. Watkins P. A., Burns D. L., Kanaho Y., Liu T. Y., Hewlett E. L., Moss J. ADP-ribosylation of transducin by pertussis toxin. J Biol Chem. 1985 Nov 5;260(25):13478–13482. [PubMed] [Google Scholar]
  33. Weiss A. A., Hewlett E. L. Virulence factors of Bordetella pertussis. Annu Rev Microbiol. 1986;40:661–686. doi: 10.1146/annurev.mi.40.100186.003305. [DOI] [PubMed] [Google Scholar]

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