The intercellular signaling activity of the Mycobacterium tuberculosis chaperonin 60.1 protein resides in the equatorial domain.
The major heat shock protein, chaperonin 60, has been established to have intercellular signaling activity in addition to its established protein-folding function. Mycobacterium tuberculosis is one of a small proportion of bacteria to encode two chaperonin 60 proteins. We have demonstrated that chaperonin 60.1 from this bacterium is a very active stimulator of human monocytes. To determine structure/function relationships of chaperonin 60.1 we have cloned and expressed the apical, equatorial, and intermediate domains of this protein. We have found that the signaling activity of M. tuberculosis chaperonin 60.1 resides in the equatorial domain. This activity of the recombinant equatorial domain was completely blocked by treating the protein with proteinase K, ruling out lipopolysaccharide contamination as the cause of the cell activation. Blockade of the activity of the equatorial domain by anti-CD14 monoclonal antibodies reveals that this domain activates monocytes by binding to CD14. Looking at the oligomeric state of the active proteins, using native gel electrophoresis and protein cross-linking we found that recombinant M. tuberculosis chaperonin 60.1 fails to form the prototypic tetradecameric structure of chaperonin 60 proteins under the conditions tested and only forms dimers. It is therefore concluded that the monocyte-stimulating activity of M. tuberculosis Cpn60.1 resides in the monomeric subunit and within this subunit the biological activity is due to the equatorial domain.
Tormay
Peter
P
Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, Cranmer Terrace, London. p.tormay@sghms.ac.uk
Coates
Anthony R M
AR
Henderson
Brian
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000276:immunology; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D001426:Bacterial Proteins; D018834:Chaperonin 60; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D021122:Protein Subunits; D011994:Recombinant Proteins; D019286:Endopeptidase K