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. 1996 Mar;64(3):810–817. doi: 10.1128/iai.64.3.810-817.1996

Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen.

S Birkelund 1, P Mygind 1, A Holm 1, B Larsen 1, F Beck 1, G Christiansen 1
PMCID: PMC173841  PMID: 8641785

Abstract

Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized conformational epitopes of C. trachomatis DnaK.

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Selected References

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  1. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benjamin D. C., Berzofsky J. A., East I. J., Gurd F. R., Hannum C., Leach S. J., Margoliash E., Michael J. G., Miller A., Prager E. M. The antigenic structure of proteins: a reappraisal. Annu Rev Immunol. 1984;2:67–101. doi: 10.1146/annurev.iy.02.040184.000435. [DOI] [PubMed] [Google Scholar]
  3. Birkelund S., Larsen B., Holm A., Lundemose A. G., Christiansen G. Characterization of a linear epitope on Chlamydia trachomatis serovar L2 DnaK-like protein. Infect Immun. 1994 May;62(5):2051–2057. doi: 10.1128/iai.62.5.2051-2057.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birkelund S., Lundemose A. G., Christiansen G. Characterization of native and recombinant 75-kilodalton immunogens from Chlamydia trachomatis serovar L2. Infect Immun. 1989 Sep;57(9):2683–2690. doi: 10.1128/iai.57.9.2683-2690.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birkelund S., Lundemose A. G., Christiansen G. Chemical cross-linking of Chlamydia trachomatis. Infect Immun. 1988 Mar;56(3):654–659. doi: 10.1128/iai.56.3.654-659.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Birkelund S., Lundemose A. G., Christiansen G. The 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide is a DnaK-like protein. Infect Immun. 1990 Jul;58(7):2098–2104. doi: 10.1128/iai.58.7.2098-2104.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Birkelund S. The molecular biology and diagnostics of Chlamydia trachomatis. Dan Med Bull. 1992 Aug;39(4):304–320. [PubMed] [Google Scholar]
  8. Brunham R. C., Peeling R., Maclean I., McDowell J., Persson K., Osser S. Postabortal Chlamydia trachomatis salpingitis: correlating risk with antigen-specific serological responses and with neutralization. J Infect Dis. 1987 Apr;155(4):749–755. doi: 10.1093/infdis/155.4.749. [DOI] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flaherty K. M., DeLuca-Flaherty C., McKay D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990 Aug 16;346(6285):623–628. doi: 10.1038/346623a0. [DOI] [PubMed] [Google Scholar]
  11. Fukushi H., Hirai K. Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants. Int J Syst Bacteriol. 1992 Apr;42(2):306–308. doi: 10.1099/00207713-42-2-306. [DOI] [PubMed] [Google Scholar]
  12. Gragerov A., Zeng L., Zhao X., Burkholder W., Gottesman M. E. Specificity of DnaK-peptide binding. J Mol Biol. 1994 Jan 21;235(3):848–854. doi: 10.1006/jmbi.1994.1043. [DOI] [PubMed] [Google Scholar]
  13. Gupta R. S., Singh B. Cloning of the HSP70 gene from Halobacterium marismortui: relatedness of archaebacterial HSP70 to its eubacterial homologs and a model for the evolution of the HSP70 gene. J Bacteriol. 1992 Jul;174(14):4594–4605. doi: 10.1128/jb.174.14.4594-4605.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  15. Hearne C. M., Ellar D. J. Nucleotide sequence of a Bacillus subtilis gene homologous to the dnaK gene of Escherichia coli. Nucleic Acids Res. 1989 Oct 25;17(20):8373–8373. doi: 10.1093/nar/17.20.8373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heegaard P. M., Holm A., Hagerup M. Practical multipeptide synthesis: dedicated software for the definition of multiple, overlapping peptides covering polypeptide sequences. Pept Res. 1993 Jan-Feb;6(1):7–9. [PubMed] [Google Scholar]
  17. Kornak J. M., Kuo C. C., Campbell L. A. Sequence analysis of the gene encoding the Chlamydia pneumoniae DnaK protein homolog. Infect Immun. 1991 Feb;59(2):721–725. doi: 10.1128/iai.59.2.721-725.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krska J., Elthon T., Blum P. Monoclonal antibody recognition and function of a DnaK (HSP70) epitope found in gram-negative bacteria. J Bacteriol. 1993 Oct;175(20):6433–6440. doi: 10.1128/jb.175.20.6433-6440.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Larsen B., Birkelund S., Mordhorst C. H., Ejstrup L., Andersen L. S., Christiansen G. The humoral immune response to Chlamydia trachomatis in patients with acute reactive arthritis. Br J Rheumatol. 1994 Jun;33(6):534–540. doi: 10.1093/rheumatology/33.6.534. [DOI] [PubMed] [Google Scholar]
  20. Laver W. G., Air G. M., Webster R. G., Smith-Gill S. J. Epitopes on protein antigens: misconceptions and realities. Cell. 1990 May 18;61(4):553–556. doi: 10.1016/0092-8674(90)90464-p. [DOI] [PubMed] [Google Scholar]
  21. Lundemose A. G., Birkelund S., Larsen P. M., Fey S. J., Christiansen G. Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis. Infect Immun. 1990 Aug;58(8):2478–2486. doi: 10.1128/iai.58.8.2478-2486.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McCarty J. S., Walker G. C. DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9513–9517. doi: 10.1073/pnas.88.21.9513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McKenzie K. R., Adams E., Britton W. J., Garsia R. J., Basten A. Sequence and immunogenicity of the 70-kDa heat shock protein of Mycobacterium leprae. J Immunol. 1991 Jul 1;147(1):312–319. [PubMed] [Google Scholar]
  24. Mehra V., Sweetser D., Young R. A. Efficient mapping of protein antigenic determinants. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7013–7017. doi: 10.1073/pnas.83.18.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Meldal M., Holm C. B., Bojesen G., Jakobsen M. H., Holm A. Multiple column peptide synthesis, Part 2 (1, 2). Int J Pept Protein Res. 1993 Mar;41(3):250–260. doi: 10.1111/j.1399-3011.1993.tb00333.x. [DOI] [PubMed] [Google Scholar]
  26. Montgomery D., Jordan R., McMacken R., Freire E. Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK. J Mol Biol. 1993 Jul 20;232(2):680–692. doi: 10.1006/jmbi.1993.1418. [DOI] [PubMed] [Google Scholar]
  27. Narberhaus F., Giebeler K., Bahl H. Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J Bacteriol. 1992 May;174(10):3290–3299. doi: 10.1128/jb.174.10.3290-3299.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Newhall W. J., Batteiger B., Jones R. B. Analysis of the human serological response to proteins of Chlamydia trachomatis. Infect Immun. 1982 Dec;38(3):1181–1189. doi: 10.1128/iai.38.3.1181-1189.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Panagiotidis C. A., Burkholder W. F., Gaitanaris G. A., Gragerov A., Gottesman M. E., Silverstein S. J. Inhibition of DnaK autophosphorylation by heat shock proteins and polypeptide substrates. J Biol Chem. 1994 Jun 17;269(24):16643–16647. [PubMed] [Google Scholar]
  30. Peake P. W., Britton W. J., Davenport M. P., Roche P. W., McKenzie K. R. Analysis of B-cell epitopes in the variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein. Infect Immun. 1993 Jan;61(1):135–141. doi: 10.1128/iai.61.1.135-141.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rippmann F., Taylor W. R., Rothbard J. B., Green N. M. A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA. EMBO J. 1991 May;10(5):1053–1059. doi: 10.1002/j.1460-2075.1991.tb08044.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schröder H., Langer T., Hartl F. U., Bukau B. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 1993 Nov;12(11):4137–4144. doi: 10.1002/j.1460-2075.1993.tb06097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sherman M. Y., Goldberg A. L. Heat shock of Escherichia coli increases binding of dnaK (the hsp70 homolog) to polypeptides by promoting its phosphorylation. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8648–8652. doi: 10.1073/pnas.90.18.8648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  35. Stanley K. K., Luzio J. P. Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J. 1984 Jun;3(6):1429–1434. doi: 10.1002/j.1460-2075.1984.tb01988.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tilly K., Hauser R., Campbell J., Ostheimer G. J. Isolation of dnaJ, dnaK, and grpE homologues from Borrelia burgdorferi and complementation of Escherichia coli mutants. Mol Microbiol. 1993 Feb;7(3):359–369. doi: 10.1111/j.1365-2958.1993.tb01128.x. [DOI] [PubMed] [Google Scholar]
  37. Wagar E. A., Schachter J., Bavoil P., Stephens R. S. Differential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens. J Infect Dis. 1990 Oct;162(4):922–927. doi: 10.1093/infdis/162.4.922. [DOI] [PubMed] [Google Scholar]
  38. Young S. G., Koduri R. K., Austin R. K., Bonnet D. J., Smith R. S., Curtiss L. K. Definition of a nonlinear conformational epitope for the apolipoprotein B-100-specific monoclonal antibody, MB47. J Lipid Res. 1994 Mar;35(3):399–407. [PubMed] [Google Scholar]
  39. Zhong G., Brunham R. C. Antigenic analysis of the chlamydial 75-kilodalton protein. Infect Immun. 1992 Mar;60(3):1221–1224. doi: 10.1128/iai.60.3.1221-1224.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ziemienowicz A., Skowyra D., Zeilstra-Ryalls J., Fayet O., Georgopoulos C., Zylicz M. Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity. J Biol Chem. 1993 Dec 5;268(34):25425–25431. [PubMed] [Google Scholar]
  41. Zylicz M., LeBowitz J. H., McMacken R., Georgopoulos C. The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6431–6435. doi: 10.1073/pnas.80.21.6431. [DOI] [PMC free article] [PubMed] [Google Scholar]

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