The Intercellular Signaling Activity of the Mycobacterium tuberculosis Chaperonin 60.1 Protein Resides in the Equatorial Domain
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m414158200
ISSN1083-351X
AutoresPeter Tormay, Anthony Coates, Brian E. Henderson,
Tópico(s)Protein Structure and Dynamics
ResumoThe 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. 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. The end of the 20th century saw the discovery of the cell stress response and the crucial role of molecular chaperones, such as the prototypic tetradecameric chaperonin (Cpn 1The abbreviations used are: Cpn, chaperonin; LPS, lipopolysaccharide; LAL, limulus amoebocyte lysate assay; Hsp, heat shock protein; TLR, Toll-like receptor; wt, wild type; IL, interleukin; PBS, phosphate-buffered saline; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. 1The abbreviations used are: Cpn, chaperonin; LPS, lipopolysaccharide; LAL, limulus amoebocyte lysate assay; Hsp, heat shock protein; TLR, Toll-like receptor; wt, wild type; IL, interleukin; PBS, phosphate-buffered saline; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.)60 protein (1.Saibil H. Curr. Opin. Struct. Biol. 2000; 10: 251-258Crossref PubMed Scopus (104) Google Scholar, 2.Thirumalai D. Lorimer G.H. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 245-269Crossref PubMed Scopus (322) Google Scholar), as protein-folding proteins vital for cell survival. In the past decade evidence has emerged that molecular chaperones are examples of moonlighting proteins with the capacity to act as intercellular signals of potential importance in controlling inflammation and immunity (3.Lewthwaite J. Skinner A. Henderson B. Trends Microbiol. 1998; 6: 426-428Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 4.Coates A.R. Henderson B. Mascagni P. Biotechnol. Genet. Eng. Rev. 1999; 16: 393-405Crossref PubMed Scopus (5) Google Scholar, 5.Wallin R.P. Lundqvist A. More S.H. von Bonin A. Kiessling R. Ljunggren H.G. Trends Immunol. 2002; 23: 130-135Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 6.Maguire M. Coates A.R. Henderson B. Cell Stress Chaperones. 2002; 7: 317-329Crossref PubMed Scopus (68) Google Scholar, 7.Panayi G.S. Corrigall V.M. Henderson B. Curr. Opin. Immunol. 2004; 16: 531-534Crossref PubMed Scopus (53) Google Scholar). It is now established that the Cpn60 proteins from a number of bacteria, and from the mitochondrion, can activate a range of mammalian cells, including myeloid cells and vascular endothelial cells (5.Wallin R.P. Lundqvist A. More S.H. von Bonin A. Kiessling R. Ljunggren H.G. Trends Immunol. 2002; 23: 130-135Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 6.Maguire M. Coates A.R. Henderson B. Cell Stress Chaperones. 2002; 7: 317-329Crossref PubMed Scopus (68) Google Scholar, 7.Panayi G.S. Corrigall V.M. Henderson B. Curr. Opin. Immunol. 2004; 16: 531-534Crossref PubMed Scopus (53) Google Scholar). In consequence, it is possible to hypothesize that bacterial Cpn60 proteins may be directly acting virulence factors (3.Lewthwaite J. Skinner A. Henderson B. Trends Microbiol. 1998; 6: 426-428Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The initial observation that Cpn60 proteins could activate myeloid cells (8.Friedland J.S. Shattock R. Remick D.G. Griffin G.E. Clin. Exp. Immunol. 1993; 91: 58-62Crossref PubMed Scopus (130) Google Scholar) used the Cpn60.2 protein from Mycobacterium tuberculosis, a protein more usually known as Hsp65 (9.Coates A.R. Shinnick T.M. Ellis R.J. Mol. Microbiol. 1993; 8: 787Crossref PubMed Scopus (26) Google Scholar). It has subsequently been discovered that this bacterium contains two genes coding for Cpn60 proteins and that these proteins have >70% sequence similarity (10.Kong T.H. Coates A.R. Butcher P.D. Hickman C.J. Shinnick T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2608-2612Crossref PubMed Scopus (98) Google Scholar). We have cloned both M. tuberculosis cpn60 genes and expressed and purified to homogeneity both recombinant Cpn60 proteins. Using these purified recombinant proteins we have shown that despite the substantial sequence identity there are major differences in their capacity to activate human monocytes (11.Lewthwaite J.C. Coates A.R. Tormay P. Singh M. Mascagni P. Poole S. Roberts M. Sharp L. Henderson B. Infect. Immun. 2001; 69: 7349-7355Crossref PubMed Scopus (99) Google Scholar). Thus the M. tuberculosis Cpn60.1 protein is 10–100 times more potent at stimulating human monocyte cytokine synthesis than is the Cpn60.2 protein and indeed may be the most potent cell signaling chaperonin 60 molecule. In addition, the monocyte-stimulating capacity of the Cpn60.1 protein is blocked by monoclonal antibodies binding to CD14. Such antibodies have no inhibitory effect with Cpn60.2. Such inhibition is not due to the blocking of contaminating lipopolysaccharide (LPS), because both recombinant proteins have equal, and biologically insignificant, levels of contaminating LPS. Nothing is known about the relationship between the structure of Cpn60 proteins and their intercellular signaling actions. These proteins are believed to exist as oligomers, although a recent study (12.Qamra R. Srinivas V. Mande S.C. J. Mol. Biol. 2004; 342: 605-617Crossref PubMed Scopus (69) Google Scholar) suggests that the mycobacterial proteins form dimers in vitro as well as in vivo. It is not known if their intercellular signaling capacity is due to the oligomer or the individual monomer. It is also not known if the cell-cell signaling activity requires the intact monomer or if activity resides in a smaller structural domain. To examine this we have cloned and expressed the M. tuberculosis cpn60.1 gene and examined the oligomeric status of the recombinant protein. We have also cloned, expressed, and purified the individual domains that constitute Cpn60.1 and have tested these individual domains to identify if one or other exhibit the signaling activity of the parent protein. Source of Proteins—The recombinant M. tuberculosis Cpn60.1 protein was produced and purified as described previously (11.Lewthwaite J.C. Coates A.R. Tormay P. Singh M. Mascagni P. Poole S. Roberts M. Sharp L. Henderson B. Infect. Immun. 2001; 69: 7349-7355Crossref PubMed Scopus (99) Google Scholar). To prepare the individual domains the complete coding sequences of the M. tuberculosis cpn60.1 gene was amplified by PCR using the Epicenter FailSafe PCR system (Cambio, Cambridge, United Kingdom) and cloned into the expression vector pBAD/Myc-HisB (Invitrogen). The resulting plasmid was designated pBTBc601. This construct expresses the full-length protein containing an additional 21 amino acids comprising the myc epitope and six histidine residues at the C terminus of the protein. The respective protein was designated HcCpn60.1. The individual protein domains were all based on this His-tagged version of the full-length protein. The equatorial domain as well as the equatorial domain in conjunction with the intermediate domain of Cpn60.1 was cloned by inverse PCR using the plasmid pBTBc601 as template. The resulting plasmids were called pBTBc601a and pBTBc601ab. The resulting proteins were designated HcCpn60.1a (equatorial domain of Cpn60.1) and HcCpn60.1ab (equatorial and intermediate domain of Cpn60.1). The apical domain of Cpn60.1 was obtained by amplifying the respective fragment by PCR and cloning the PCR product into the vector pBAD/Myc-HisB to produce the protein designated HcCpn60.1c. All recombinant proteins were expressed in the Escherichia coli strain, Top10. HcCpn60.1, HcCpn60.1a, and HcCpn60.1ab were all formed as inclusion bodies. To produce folded proteins the inclusion bodies were re-suspended in 6 m guanidine hydrochloride, and this was quickly diluted by adding a 10-fold volume of PBS. Refolding was continued overnight at 4°C, and the protein was clarified by centrifugation at 30,000 × g and subsequent filtration of the cleared protein through a 0.25-μm filter. The recombinant protein was then bound to Probond nickel-chelating resin (Invitrogen), and bound protein was washed with 5 column volumes of a 2 mg/ml solution of polymyxin B (Sigma) in PBS to remove any contaminating LPS. Protein was eluted from the column by application of a 10–300 mm imidazole gradient in 10 column volumes, and fractions containing the recombinant chaperonin protein/domain of interest were pooled and dialyzed against 20 mm sodium phosphate buffer, pH 6.8. Recombinant proteins were further purified by anion exchange chromatography using a Porous HQ column on a BioCad Sprint chromatography system (Applied Biosystems, Warrington, UK). The purification of HcCpn60.1c was essentially the same as described except for the fact that no renaturation was required, and soluble protein was directly bound to the nickel-nitrilotriacetic acid matrix. E. coli GroEL and GroES were purchased from Stressgen (Bioquote, York, UK). Analysis of Protein Purity—Protein purity was analyzed by SDS-PAGE. To do this 50 pmol of each purified recombinant protein (1.1–2.9 μg, dependent on protein) was run on a 4–12% Bis-Tris gradient gel (Invitrogen), and the gel was subsequently stained with Simply Blue Safe Stain (Invitrogen). Molecular Model Building—The sequence of the M. tuberculosis Cpn60.1 protein was first aligned to the E. coli homologue, GroEL, for which crystal structures are available. Subsequently, the alignments were submitted to the ExPASy modeling server (swissmodel.expasy.org/) (13.Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4376) Google Scholar). The resulting model was viewed using the Pdb viewer (www.expasy.org/spdbv/) (14.Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9399) Google Scholar). Oligomeric Structure Analysis—The oligomeric structure of the Cpn60 proteins was assessed by either cross-linking in conjunction with SDS-PAGE or by use of native polyacrylamide gel electrophoresis. For the cross-linking experiments, 8 μm of each recombinant protein was incubated with 400 μm disuccinimidyl suberate (Perbio, Tattenhall, UK) for 30 min at room temperature in PBS. The reaction was then quenched by adding an equal volume of 2 × LDS sample buffer (Invitrogen). After further incubation of the sample for 10 min the proteins were resolved on SDS-PAGE and visualized as described above. For the native polyacrylamide gel electrophoresis the proteins were incubated in 50 mm Tris/Cl, pH 7.5, 20 mm MgCl2, 150 mm KCl, 20% (v/v) glycerol before loading onto a 6% Tris/glycine gel. Proteins were resolved for 1.5 h at 130 V using Tris/glycine running buffer (25 mm Tris base, 192 mm glycine). Proteolysis of Recombinant Cpn60 Proteins—To analyze whether the cytokine-stimulating activity was directly associated with the recombinant proteins, 5 μm of each protein was digested with 1.3 μg/ml proteinase K at 37°C for 16 h. Proteins were used at a final concentration of 100 nm in each assay. Polymyxin B was subsequently added to these preparation where indicated. Measurement of LPS Contamination in Recombinant Proteins—To ascertain the levels of LPS in the various recombinant proteins used to activate human monocytes, use was made of a commercially available Limulus amebocyte lysate assay, which was used according to the manufacturer's instructions (Associates of Cape Cod, Liverpool, UK). Isolation of Human Monocytes—Human monocytes were prepared from buffy coat cells (obtained from the Blood Transfusion Centre, Colindale, London, UK) by density gradient centrifugation. Buffy coat cells were diluted 1:3 with PBS, and 30 ml of the diluted buffy coat cell suspension was carefully layered onto 15 ml of Lymphoprep (Axis-Shield Diagnostics Ltd., Dundee) and centrifuged at 400 × g for 30 min at room temperature. The mononuclear cell layer was collected, washed with PBS, and centrifuged at 400 × g for 15 min, the procedure being repeated. The final cell pellet was resuspended in Dulbecco's modified Eagle's medium containing 2% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine (all from Invitrogen). The cells were counted and diluted to 2 × 106 cells/ml, and 1-ml aliquots were dispensed into 24-well tissue culture plates. Cells were incubated for 1 h at 37°C and then washed with PBS to remove non-adherent cells. Finally, cells were re-suspended in RPMI medium containing 2% fetal calf serum 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine (all from Invitrogen). Induction and Measurement of Cytokines—Human monocytes were exposed to graded concentrations of the various recombinant Cpn60.1 proteins for 16 h, and the medium was removed and frozen to -70°C until analysis. The concentration of cytokines, namely IL-1β, IL-6, and tumor necrosis factor α, in the medium supporting the cells was assessed by enzyme-linked immunosorbent assay using paired antibodies to these cytokines from R&D Systems (Abingdon, UK). In most experiments the activating protein was incubated with cells in the presence of 20 μg/ml polymyxin B to ensure that activity recorded was not due to LPS. To ascertain the role of CD14 in cell activation, neutralizing antibodies to CD14 (R&D Systems) or the appropriate isotype antibody control were added to the activated cells at a concentration of 10 μg/ml. Results are means of three independent stimulation experiments using cells from the same buffy coat residue. In different experiments different buffy coat residue was used, resulting in some variation of the results. Physicochemical Characteristics of Recombinant Cpn60 Variants—All the recombinant proteins were analyzed by SDS-PAGE and stained with a sensitive commercially available Coomassie Blue stain. All proteins were of the expected protein mass. Even at high protein loadings there were no visible contaminating bands (Fig. 1). The predicted structure of the intact mycobacterial Cpn60.1 monomer and its constituent domains are shown in Fig. 2.Fig. 2Structural model of the M. tuberculosis heat shock protein Cpn60.1. The structural model was obtained by sequence alignment of M. tuberculosis Cpn60.1 with GroEL using the ExPASy modeling server. A, three-dimensional representation of the monomer structure of the Cpn60.1 protein. The different domains are indicated by different shading. B, the sequence of Cpn60.1 is provided with the domain structure outlined underneath. Arrows and boxes indicate β-sheets and helical regions, respectively. The domains are again indicated by different shading of the arrows and boxes (crosshatch, equatorial domain; 45° hatch, intermediate domain; vertical hatch, apical domain).View Large Image Figure ViewerDownload Hi-res image Download (PPT) LPS Contamination of Recombinant Proteins—Using polymyxin B to wash the recombinant proteins on the nickel affinity columns lowered the concentrations of LPS in the Cpn60 protein preparations to 70% sequence similarity between both proteins, it was found that Cpn60.1 was significantly more potent and efficacious as an inducer of monocyte cytokine synthesis than Cpn60.2. Indeed, this mycobacterial protein appears to be the most biologically active form of Cpn60 (6.Maguire M. Coates A.R. Henderson B. Cell Stress Chaperones. 2002; 7: 317-329Crossref PubMed Scopus (68) Google Scholar, 11.Lewthwaite J.C. Coates A.R. Tormay P. Singh M. Mascagni P. Poole S. Roberts M. Sharp L. Henderson B. Infect. Immun. 2001; 69: 7349-7355Crossref PubMed Scopus (99) Google Scholar). A major difference between these two proteins was that Cpn60.1 was blocked by neutralizing antibodies to CD14 implying that this protein activates monocytes via the CD14·TLR4 receptor complex. In contrast, Cpn60.2 was unaffected by antibodies to CD14 (11.Lewthwaite J.C. Coates A.R. Tormay P. Singh M. Mascagni P. Poole S. Roberts M. Sharp L. Henderson B. Infect. Immun. 2001; 69: 7349-7355Crossref PubMed Scopus (99) Google Scholar). This suggests that these two proteins bind to different receptors or to different parts of the CD14·TLR4·MD2 receptor complex. This has been confirmed by studies of competitive binding of Cpn60 proteins from different species to macrophages (24.Habich C. Kempe K. van der Zee R. Burkart V. Kolb H. FEBS Lett. 2003; 533: 105-109Crossref PubMed Scopus (40) Google Scholar). There is much that is not understood about the interaction of Cpn60 proteins and immune cells. To investigate the structure-function relationships of the M. tuberculosis Cpn60.1 protein we have developed a structural model based on the crystal structure of GroEL. This has allowed the development of a strategy for cloning and expressing the individual domains of this protein. Examination of the structure of GroEL reveals the presence of three domains in the monomer: (i) the equatorial domain, which makes contact with the corresponding equatorial domain in the oligomeric structure; (ii) the intermediate domain; and (iii) the apical domain (Fig. 1). To simplify the purification of the individual domains it was decided to produce all recombinant domains as C-terminal myc-6-His-tagged fusion proteins, and the parent protein was also recloned into the same vector. This allowed the production of: (i) the intact parent protein; (ii) the equatorial domain; (iii) the equatorial domain linked to the intermediate domain; and (iv) the apical domain. Some of these recombinant domain proteins formed inclusion bodies, but it proved possible to obtain them in soluble form. The first finding from this study was that addition of the myc epitope and 6-His residues to the C terminus of Cpn60.1 still allowed the generation of proteins with biological activity. Comparison of the fusion protein form of Cpn60.1 with the recombinant non-fusion protein form revealed that there had been some loss of potency. This may suggest that the C terminus of Cpn60.1 is also involved in the cell-cell signaling activity of this protein. With an active parent molecule it was now possible to determine which of the domains of Cpn60.1 contributed to the activity of this protein. This revealed that the equatorial domain and the equatorial domain plus the intermediate domain retained biological activity with the former being as active, or even more active, on a molar basis, as the parent molecule. The recombinant apical domain was inactive under the conditions tested. We have previously reported that the apical domain contained a peptide, residues 195–219, which had cytokine-stimulating activity, and it was surprising that this domain had no biological activity. It should be noted that this peptide was used at much higher molar concentration in the previous study (11.Lewthwaite J.C. Coates A.R. Tormay P. Singh M. Mascagni P. Poole S. Roberts M. Sharp L. Henderson B. Infect. Immun. 2001; 69: 7349-7355Crossref PubMed Scopus (99) Google Scholar) compared with the apical domain in this study. Moreover, the model we present here indicates that this particular sequence forms part of a β-sheet with a large loop in contrast to the helical structure prediction for the peptide. It also cannot be ruled out that the addition of the C-terminal His tag has a negative effect on any residual cytokine stimulating activity of the apical domain. Nevertheless, it is clear that the equatorial domain retains all the cytokine stimulating activity of the full-length protein. Adding the intermediate domain to this construct does not alter the activity. We therefore conclude that the sequence 195–219 is not responsible for the activity of the full-length protein and that the primary cytokine stimulating activity is associated with the equatorial domain. Recent studies with the human mitochondrial Cpn60 protein also implicate the equatorial domain as the primary domain for interaction with and activation of cells of myeloid origin. We have shown that deletion mutants from the C-terminal end of the human mitochondrial Cpn60 protein resulted in the loss of ability to induce bone resorption (25.Meghji S. Lillicrap M. Maguire M. Tabona P. Gaston J.S. Poole S. Henderson B. Bone. 2003; 33: 419-425Crossref PubMed Scopus (17) Google Scholar). Another study has identified that an epitope important for receptor binding on macrophages mapped to the C terminus of the equatorial domain (26.Habich C. Kempe K. Burkart V. Van Der Zee R. Lillicrap M. Gaston H. Kolb H. FEBS Lett. 2004; 568: 65-69Crossref PubMed Scopus (22) Google Scholar). The current paradigm is that the folding activity of chaperonin 60 proteins requires them to form higher oligomeric structures. GroEL, the prototypic member of this family, forms a tetradecamer consisting of two seven membered rings (1.Saibil H. Curr. Opin. Struct. Biol. 2000; 10: 251-258Crossref PubMed Scopus (104) Google Scholar, 2.Thirumalai D. Lorimer G.H. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 245-269Crossref PubMed Scopus (322) Google Scholar). It is not known if the cell-cell signaling activity of these proteins depends on the formation of such oligomeric structures. Studies with GroEL, in which trypsinization failed to block activity (27.Tabona P. Reddi K. Khan S. Nair S.P. Crean S.J. Meghji S. Wilson M. Preuss M. Miller A.D. Poole S. Carne S. Henderson B. J. Immunol. 1998; 161: 1414-1421PubMed Google Scholar), and the finding that boiling the mycobacterial Cpn60 proteins did not inhibit activity (11.Lewthwaite J.C. Coates A.R. Tormay P. Singh M. Mascagni P. Poole S. Roberts M. Sharp L. Henderson B. Infect. Immun. 2001; 69: 7349-7355Crossref PubMed Scopus (99) Google Scholar) suggest that the cell-cell signaling activity of Cpn60 proteins resides in the monomer and in submonomeric species. Here we show that even at high concentration neither the full-length M. tuberculosis Cpn60.1 protein nor its active subdomains form larger oligomers but exist as dimers. Thus under the conditions used for stimulating peripheral blood mononuclear cells the proteins are either in the dimeric or monomeric form. This is further evidence that the cell signaling activity is not associated with protein oligomerization. Indeed, it has recently been reported that the mycobacterial proteins reside as dimers in the cell and that the folding properties of M. tuberculosis Cpn60.1 do not require this protein forming a higher oligomeric structure (12.Qamra R. Srinivas V. Mande S.C. J. Mol. Biol. 2004; 342: 605-617Crossref PubMed Scopus (69) Google Scholar). There is still debate in the literature about the meaning of the cell-cell signaling activity of Cpn60 proteins, and one recent paper has attributed the bioactivity of human Cpn60 to contaminating LPS (28.Gao B. Tsan M.F. J. Biol. Chem. 2003; 278: 22523-22529Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). In another study the bioactivity of Hsp70 was also attributed to LPS contamination (29.Bausinger H. Lipsker D. Ziylan U. Manie S. Briand J.P. Cazenave J.P. Muller S. Haeuw J.F. Ravanat C. de la Salle H. Hanau D. Eur. J. Immunol. 2002; 32: 3708-3713Crossref PubMed Scopus (206) Google Scholar). We have developed methods for removing both LPS and contaminating proteins from recombinant chaperonin 60 proteins (27.Tabona P. Reddi K. Khan S. Nair S.P. Crean S.J. Meghji S. Wilson M. Preuss M. Miller A.D. Poole S. Carne S. Henderson B. J. Immunol. 1998; 161: 1414-1421PubMed Google Scholar, 30.Maguire M. Coates A.R. Henderson B. J. Chromatogr. B. 2003; 786: 117-125Crossref PubMed Scopus (7) Google Scholar), and all samples of proteins produced for this study have measurable, but biologically negligible (<2 pg of LPS per microgram of protein), concentrations of LPS. In a previous study we showed that the bioactivity of both recombinant mycobacterial Cpn60 proteins was blocked by exposure to proteinase K, which had no effect on LPS. In this study we have shown that the biological activity of the parent protein and the active recombinant proteins containing the equatorial domain are completely blocked by treatment with proteinase K. Because the LPS contamination of the proteins is below the level where LPS has an effect they could also be tested in the absence of polymyxin B, a small polypeptide used to neutralize the effect of LPS. Even in the absence of polymyxin B, proteolysis completely abrogates the biological activity. It has previously been argued that polymyxin B might not be able to neutralize LPS bound to heat shock proteins and that boiling or proteolysis releases the contaminating LPS, which in return can be neutralized by polymyxin B (5.Wallin R.P. Lundqvist A. More S.H. von Bonin A. Kiessling R. Ljunggren H.G. Trends Immunol. 2002; 23: 130-135Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). The fact that proteolysis in the presence as well as in the absence of polymyxin B completely blocks cell-cell signaling activity confirms that the activity of these proteins is not due to contaminating LPS or other nonproteinaceous contaminants. The activity of the non-fusion protein version of Cpn60.1 was completely inhibited by neutralizing antibodies to CD14 (11.Lewthwaite J.C. Coates A.R. Tormay P. Singh M. Mascagni P. Poole S. Roberts M. Sharp L. Henderson B. Infect. Immun. 2001; 69: 7349-7355Crossref PubMed Scopus (99) Google Scholar). The activity of the intact fusion protein version of Cpn60.1, the equatorial domain, and the equatorial and intermediate domain versions was also blocked by such antibodies implying that cell activation is CD14-dependent. The experimental data presented in this report allow the following conclusions to be reached. The biological activity of the M. tuberculosis Cpn60 proteins resides in the individual monomers. This does not rule out the possibility that oligomerization enhances activity. The equatorial domain contains the binding site for human myeloid cells. The recombinant Cpn60.1 equatorial domain is therefore an excellent starting point for defining the exact residues and structure conferring the potent cell signaling activity of this molecular chaperone. We thank Peter Lund and Ezster Kovac from Birmingham University for discussion on the oligomeric structure of the proteins.
Referência(s)