1887

Microbiology Society logo

Volume 150, Issue 7

Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16·3 and Hsp 70) of Free

Abstract

Several antigens of , identified by monoclonal antibodies, have been previously cloned and are being exploited in the development of improved vaccines and diagnostic reagents. In this study, the molecular characteristics of two of these antigens, the immunodominant proteins Hsp 16·3 and Hsp 70, were analysed in further detail by assessing their capacity to undergo protein phosphorylation, a chemical modification frequently used by organisms to adjust to environmental variations. Hsp 16·3 was overproduced in an expression system and purified to homogeneity. Upon incubation in the presence of radioactive ATP, it was shown to possess autophosphorylation activity. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at serine residues. In addition, cross-linking experiments demonstrated that it could tightly bind to ATP. Purified Hsp 70 was also shown to autophosphorylate but phosphorylation occurred exclusively at threonine residues. This reaction was found to be strongly stimulated by calcium ions. These data indicate that both structural and functional similarities exist between Hsp 16·3 (Acr) and -crystallin, a eukaryotic protein which plays an important role in maintaining the transparency of the vertebrate eye, and that the functional properties of Hsp 70 from are similar to those of other bacterial members of the Hsp 70 family, particularly the homologue DnaK.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26789-0
2004-07-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/7/mic1502135.html?itemId=/content/journal/micro/10.1099/mic.0.26789-0&mimeType=html&fmt=ahah

References

  1. Av-Gay Y., Jamil S., Drews S. J. 1999; Expression and characterization of the Mycobacterium tuberculosis serine/threonine protein kinase PknB. Infect Immun 67:5676–5682
     [Google Scholar]
  2. Chaba R., Raje M., Chakraborti P. K. 2002; Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division. Eur J Biochem 269:1078–1085 [CrossRef]
     [Google Scholar]
  3. Chang Z., Primm T. P., Jakana J., Lee I. H., Serysheva I., Chiu W., Gilbert H. F., Quiocho F. A. 1996; Mycobacterium tuberculosis 16-kDa antigen (Hsp16·3) functions as an oligomeric structure in vitro to suppress thermal aggregation. J Biol Chem 271:7218–7223 [CrossRef]
     [Google Scholar]
  4. Chow K., Ng D., Stokes R., Johnson P. 1994; Protein tyrosine phosphorylation in Mycobacterium tuberculosis. FEMS Microbiol Lett 124:203–207 [CrossRef]
     [Google Scholar]
  5. Cole S. T., Brosch R., Parkhill J. & 39 other authors; 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
     [Google Scholar]
  6. Courchesne P. L., Patterson S. D. 1999; Identification of proteins by matrix-assisted laser desorption/ionization mass spectrometry using peptide and fragment ion masses. Methods Mol Biol 112:487–511
     [Google Scholar]
  7. Cozzone A. J. 1993; ATP-dependent protein kinases in bacteria. J Cell Biochem 51:7–13 [CrossRef]
     [Google Scholar]
  8. Dannenberg A. M. Jr 1993; Immunopathogenesis of pulmonary tuberculosis. Hosp Pract (Off Ed) 28:51–58
     [Google Scholar]
  9. Drews S. J., Hung F., Av-Gay Y. 2001; A protein kinase inhibitor as an antimycobacterial agent. FEMS Microbiol Lett 205:369–374 [CrossRef]
     [Google Scholar]
  10. Duclos B., Marcandier S., Cozzone A. J. 1991; Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol 201:10–21
     [Google Scholar]
  11. Groenen P. J., Merck K. B., de Jong W. W., Bloemendal H. 1994; Structure and modifications of the junior chaperone alpha-crystallin. From lens transparency to molecular pathology. Eur J Biochem 225:1–19 [CrossRef]
     [Google Scholar]
  12. Haydel S. E., Dunlap N. E., Benjamin W. H. Jr 1999; In vitro evidence of two-component system phosphorylation between the Mycobacterium tuberculosis TrcR/TrcS proteins. Microb Pathog 26:195–206 [CrossRef]
     [Google Scholar]
  13. Hengge R., Bukau B. 2003; Proteolysis in prokaryotes: protein quality control and regulatory principles. Mol Microbiol 49:1451–1462 [CrossRef]
     [Google Scholar]
  14. Horwitz J. 1992; Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 89:10449–10453 [CrossRef]
     [Google Scholar]
  15. Hunter T. 1995; Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225–236 [CrossRef]
     [Google Scholar]
  16. Kato K., Hasegawa K., Goto S., Inaguma Y. 1994; Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. J Biol Chem 269:11274–11278
     [Google Scholar]
  17. Koul A., Choidas A., Treder M., Tyagi A. K., Drlica K., Singh Y., Ullrich A. 2000; Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis. J Bacteriol 182:5425–5432 [CrossRef]
     [Google Scholar]
  18. Koul A., Choidas A., Tyagi A. K., Drlica K., Singh Y., Ullrich A. 2001; Serine/threonine protein kinases PknF and PknG of Mycobacterium tuberculosis: characterization and localization. Microbiology 147:2307–2314
     [Google Scholar]
  19. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
     [Google Scholar]
  20. Matsuyama S., Kimura E., Mizushima S. 1990; Complementation of two overlapping fragments of SecA, a protein translocation ATPase of Escherichia coli, allows ATP binding to its amino-terminal region. J Biol Chem 265:8760–8765
     [Google Scholar]
  21. Mayuri Bagchi G., Das T. K., Tyagi J. S. 2002; Molecular analysis of the dormancy response in Mycobacterium smegmatis: expression analysis of genes encoding the DevR-DevS two-component system, Rv3134c and chaperone alpha-crystallin homologues. FEMS Microbiol Lett 211:231–237
     [Google Scholar]
  22. McCarty J. S., Walker G. C. 1991; DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc Natl Acad Sci U S A 88:9513–9517 [CrossRef]
     [Google Scholar]
  23. Moreno C., Mehlert A., Lamb J. 1988; The inhibitory effects of mycobacterial lipoarabinomannan and polysaccharides upon polyclonal and monoclonal human T cell proliferation. Clin Exp Immunol 74:206–210
     [Google Scholar]
  24. Nadeau K., Das A., Walsh C. T. 1993; Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases. J Biol Chem 268:1479–1487
     [Google Scholar]
  25. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. 1977; High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133–1141 [CrossRef]
     [Google Scholar]
  26. Olsen A. W., Andersen P. 2003; A novel TB vaccine; strategies to combat a complex pathogen. Immunol Lett 85:207–211 [CrossRef]
     [Google Scholar]
  27. O'Toole R., Smeulders M. J., Blokpoel M. C., Kay E. J., Lougheed K., Williams H. D. 2003; A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in Mycobacterium smegmatis. J Bacteriol 185:1543–1554 [CrossRef]
     [Google Scholar]
  28. Peake P., Winter N., Britton W. 1998; Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics. Biochim Biophys Acta 1387:387–394 [CrossRef]
     [Google Scholar]
  29. Peirs P., De Wit L., Braibant M., Huygen K., Content J. 1997; A serine/threonine protein kinase from Mycobacterium tuberculosis. Eur J Biochem 244:604–612 [CrossRef]
     [Google Scholar]
  30. Prabhakaran K., Harris E. B., Randhawa B. 2000; Regulation by protein kinase of phagocytosis of Mycobacterium leprae by macrophages. J Med Microbiol 49:339–342
     [Google Scholar]
  31. Roche P. W., Peake P. W., Davenport M. P., Britton W. J. 1994; Identification of a Mycobacterium leprae-specific T cell epitope on the 70 kDa heat shock protein. Immunol Cell Biol 72:215–221 [CrossRef]
     [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467 [CrossRef]
     [Google Scholar]
  33. Sharma S., Giri S., Khuller G. K. 1998; Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607. Mol Cell Biochem 183:183–191 [CrossRef]
     [Google Scholar]
  34. Shevchenko A., Wilm M., Vorm O., Mann M. 1996; Mass spectrometric sequencing of proteins on silver-stained polyacrylamide gels. Anal Chem 68:850–858 [CrossRef]
     [Google Scholar]
  35. Sudre P., ten Dam G., Kochi A. 1992; Tuberculosis: a global overview of the situation today. Bull World Health Organ 70:149–159
     [Google Scholar]
  36. Valdez M. M., Clark J. I., Wu G. J., Muchowski P. J. 2002; Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16·3 and human alphaB-crystallin. Eur J Biochem 269:1806–1813 [CrossRef]
     [Google Scholar]
  37. Verbon A., Hartskeerl R. A., Schuitema A., Kolk A. H., Young D. B., Lathigra R. 1992; The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins. J Bacteriol 174:1352–1359
     [Google Scholar]
  38. Wayne L. G. 1994; Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis 13:908–914 [CrossRef]
     [Google Scholar]
  39. Yuan Y., Crane D. D., Barry C. E. 3rd (1996; Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog. J Bacteriol 178:4484–4492
     [Google Scholar]
  40. Zylicz M., LeBowitz J. H., McMacken R., Georgopoulos C. 1983; 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 80:6431–6435 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26789-0
Loading
Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16·3 and Hsp 70) of Mycobacterium tuberculosis
Microbiology 150, 2135 (2004); https://doi.org/10.1099/mic.0.26789-0
/content/journal/micro/10.1099/mic.0.26789-0
/content/journal/micro/10.1099/mic.0.26789-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error