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. 1990 Mar;58(3):746–752. doi: 10.1128/iai.58.3.746-752.1990

Monoclonal antibodies that inhibit ADP-ribosyltransferase but not NAD-glycohydrolase activity of pertussis toxin.

H R Kaslow 1, J D Schlotterbeck 1, J G Kenimer 1
PMCID: PMC258528  PMID: 2155182

Abstract

Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identified three classes of monoclonal antibodies, termed A, B, and C, that recognize the S1 subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the S1 subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or S1 subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native S1 proteins and fails to distinguish between neutralizing and nonneutralizing antibodies.

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Selected References

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  1. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [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. Burnette W. N., Cieplak W., Mar V. L., Kaljot K. T., Sato H., Keith J. M. Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope. Science. 1988 Oct 7;242(4875):72–74. doi: 10.1126/science.2459776. [DOI] [PubMed] [Google Scholar]
  4. Burns D. L., Hausman S. Z., Lindner W., Robey F. A., Manclark C. R. Structural characterization of pertussis toxin A subunit. J Biol Chem. 1987 Dec 25;262(36):17677–17682. [PubMed] [Google Scholar]
  5. Burns D. L., Kenimer J. G., Manclark C. R. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect Immun. 1987 Jan;55(1):24–28. doi: 10.1128/iai.55.1.24-28.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burns D. L., Manclark C. R. Adenine nucleotides promote dissociation of pertussis toxin subunits. J Biol Chem. 1986 Mar 25;261(9):4324–4327. [PubMed] [Google Scholar]
  7. Burns D. L., Manclark C. R. Role of cysteine 41 of the A subunit of pertussis toxin. J Biol Chem. 1989 Jan 5;264(1):564–568. [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Cieplak W., Burnette W. N., Mar V. L., Kaljot K. T., Morris C. F., Chen K. K., Sato H., Keith J. M. Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4667–4671. doi: 10.1073/pnas.85.13.4667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cockle S. A. Identification of an active-site residue in subunit S1 of pertussis toxin by photocrosslinking to NAD. FEBS Lett. 1989 Jun 5;249(2):329–332. doi: 10.1016/0014-5793(89)80652-0. [DOI] [PubMed] [Google Scholar]
  12. Cody C. L., Baraff L. J., Cherry J. D., Marcy S. M., Manclark C. R. Nature and rates of adverse reactions associated with DTP and DT immunizations in infants and children. Pediatrics. 1981 Nov;68(5):650–660. [PubMed] [Google Scholar]
  13. Fling S. P., Gregerson D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem. 1986 May 15;155(1):83–88. doi: 10.1016/0003-2697(86)90228-9. [DOI] [PubMed] [Google Scholar]
  14. Kaslow H. R., Lesikar D. D. Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin. Biochemistry. 1987 Jul 14;26(14):4397–4402. doi: 10.1021/bi00388a031. [DOI] [PubMed] [Google Scholar]
  15. Kaslow H. R., Lim L. K., Moss J., Lesikar D. D. Structure-activity analysis of the activation of pertussis toxin. Biochemistry. 1987 Jan 13;26(1):123–127. doi: 10.1021/bi00375a018. [DOI] [PubMed] [Google Scholar]
  16. Kaslow H. R., Schlotterbeck J. D., Mar V. L., Burnette W. N. Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin. J Biol Chem. 1989 Apr 15;264(11):6386–6390. [PubMed] [Google Scholar]
  17. Katada T., Ui M. ADP ribosylation of the specific membrane protein of C6 cells by islet-activating protein associated with modification of adenylate cyclase activity. J Biol Chem. 1982 Jun 25;257(12):7210–7216. [PubMed] [Google Scholar]
  18. Katada T., Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci U S A. 1982 May;79(10):3129–3133. doi: 10.1073/pnas.79.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim K. J., Burnette W. N., Sublett R. D., Manclark C. R., Kenimer J. G. Epitopes on the S1 subunit of pertussis toxin recognized by monoclonal antibodies. Infect Immun. 1989 Mar;57(3):944–950. doi: 10.1128/iai.57.3.944-950.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. 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]
  22. Lim L. K., Sekura R. D., Kaslow H. R. Adenine nucleotides directly stimulate pertussis toxin. J Biol Chem. 1985 Mar 10;260(5):2585–2588. [PubMed] [Google Scholar]
  23. Locht C., Capiau C., Feron C. Identification of amino acid residues essential for the enzymatic activities of pertussis toxin. Proc Natl Acad Sci U S A. 1989 May;86(9):3075–3079. doi: 10.1073/pnas.86.9.3075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Miller D. L., Ross E. M., Alderslade R., Bellman M. H., Rawson N. S. Pertussis immunisation and serious acute neurological illness in children. Br Med J (Clin Res Ed) 1981 May 16;282(6276):1595–1599. doi: 10.1136/bmj.282.6276.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moss J., Stanley S. J., Burns D. L., Hsia J. A., Yost D. A., Myers G. A., Hewlett E. L. Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). J Biol Chem. 1983 Oct 10;258(19):11879–11882. [PubMed] [Google Scholar]
  27. Moss J., Stanley S. J., Watkins P. A., Burns D. L., Manclark C. R., Kaslow H. R., Hewlett E. L. Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents. Biochemistry. 1986 May 6;25(9):2720–2725. doi: 10.1021/bi00357a066. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Noble G. R., Bernier R. H., Esber E. C., Hardegree M. C., Hinman A. R., Klein D., Saah A. J. Acellular and whole-cell pertussis vaccines in Japan. Report of a visit by US scientists. JAMA. 1987 Mar 13;257(10):1351–1356. [PubMed] [Google Scholar]
  30. 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]
  31. Sato H., Ito A., Chiba J., Sato Y. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections. Infect Immun. 1984 Nov;46(2):422–428. doi: 10.1128/iai.46.2.422-428.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sato H., Sato Y., Ito A., Ohishi I. Effect of monoclonal antibody to pertussis toxin on toxin activity. Infect Immun. 1987 Apr;55(4):909–915. doi: 10.1128/iai.55.4.909-915.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sato Y., Arai H., Suzuki K. Leukocytosis-promoting factor of Bordetella pertussis. 3. Its identity with protective antigen. Infect Immun. 1974 May;9(5):801–810. doi: 10.1128/iai.9.5.801-810.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sekura R. D., Fish F., Manclark C. R., Meade B., Zhang Y. L. Pertussis toxin. Affinity purification of a new ADP-ribosyltransferase. J Biol Chem. 1983 Dec 10;258(23):14647–14651. [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Wessling-Resnick M., Johnson G. L. Kinetic and hydrodynamic properties of transducin: comparison of physical and structural parameters for GTP-binding regulatory proteins. Biochemistry. 1987 Jul 14;26(14):4316–4323. doi: 10.1021/bi00388a020. [DOI] [PubMed] [Google Scholar]

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