Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16.3 and Hsp 70) of Mycobacterium tuberculosis

Microbiology (Reading). 2004 Jul;150(Pt 7):2135-2141. doi: 10.1099/mic.0.26789-0.


Several antigens of Mycobacterium tuberculosis, 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 Escherichia coli 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 alpha-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 M. tuberculosis are similar to those of other bacterial members of the Hsp 70 family, particularly the E. coli homologue DnaK.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Chaperonins / genetics
  • Chaperonins / metabolism*
  • Crystallins / chemistry
  • Crystallins / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Escherichia coli / metabolism
  • HSP70 Heat-Shock Proteins / genetics
  • HSP70 Heat-Shock Proteins / metabolism*
  • Humans
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / growth & development
  • Mycobacterium tuberculosis / immunology
  • Mycobacterium tuberculosis / metabolism*
  • Phosphorylation


  • Antigens, Bacterial
  • Bacterial Proteins
  • Crystallins
  • HSP70 Heat-Shock Proteins
  • Hsp16.3 protein, Mycobacterium tuberculosis
  • Adenosine Triphosphate
  • Chaperonins