Institut Pasteur de Bruxelles, Laboratoire des Mycobactéries, Rue Engeland 642, B-1180 Bruxelles, Belgium1
Laboratoire de Chimie Biologique, Université Libre Bruxelles, Bruxelles, Belgium2
Faculté Universitaire de Gembloux, Centre de Biophysique Moléculaire Numérique, Belgium3
Laboratoire de Chimie Biologique, Université de Mons, Hainaut, Belgium4
Institut de Pharmacologie et Biologie Structurale du CNRS, 31077 Toulouse cedex, France5
Author for correspondence: J. De Bruyn. Tel: +32 2 3733357. Fax: +32 2 3733281. e-mail: jdebruyn{at}ben.ulb.ac.be
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ABSTRACT |
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Keywords: Mycobacterium bovis BCG, Hsp60 protein, GroEL
Abbreviations: CNBr, cyanogen bromide; Hsp, heat-shock protein; PB, phosphate buffer
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INTRODUCTION |
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The co-operating factor GroES (Hsp10) binds asymmetrically to GroEL, and stimulates ATP hydrolysis and substrate release (Hartl, 1996 ). Cell survival under stress conditions depends on the speed and efficiency of the activating mechanism responsible for the transcription of the hsp genes (Liberek & Georgopoulos, 1993
; Morimoto, 1993
) as well as on the ability of these Hsp proteins to prevent protein denaturation (Ziemienowicz et al., 1993
; Martin et al., 1988
). The Hsp proteins are highly conserved in nature from bacteria to humans (Harboe & Quayle, 1991
; Thole et al., 1988a
; Thole & Van Der Zee, 1990
). The immunological activities of Hsp60 in infectious diseases (Harboe & Quayle, 1991
; Kaufmann, 1990
; Welch & Winfield, 1992
), in autoimmune reactions (Van Eden et al., 1989
; Young, 1992
), in cancer (Jäättelä & Wissing, 1992
) as well as their use as potential antitumour vaccines (Ahsan & Sasaki, 1991
, 1993
) are active fields of investigation.
Large amounts of Hsp60-2 (P64; GroEL-2; 65 kDa) can be obtained from culture filtrates of Mycobacterium bovis BCG grown on zinc-deficient Sauton medium (De Bruyn et al., 1987a ). Further systematic analysis by gel electrophoresis of the different fractions obtained during stepwise elution with buffers of decreasing ionic strength from hydrophobic phenyl-Sepharose, performed as an initial phase of purification, showed the presence of proteins exhibiting an apparent molecular mass of 65 kDa in all the eluted fractions, suggesting the existence of several Hsp60s differing by their hydrophobicity. The new species, with respect to their order of elution, were named Hsp60-2a, Hsp60-2b and Hsp60-2d in reference to the protein we previously characterized, which we renamed Hsp60-2c.
To find an explanation for the different hydrophobic behaviour of Hsp60 proteins, the present study was undertaken by investigating the possible association of lipids with various Hsp60s, namely Hsp60-2 (65 kDa) from M. bovis BCG, the recombinant Hsp60-2 protein produced in E. coli and, as an external reference, E. coli Hsp60 (GroEL).
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METHODS |
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Construction of pRR3Hsp60-2.
A 3 kb SmaI fragment of the plasmid pRIB1000 containing the promoter and the coding regions of hsp60-2 was subcloned into a Bluescribe M13+ vector designated Bshsp. To construct the hsp expression vector under the control of its own promoter, Bshsp was digested by SmaI and the resulting 3 kb fragment was ligated to ScaI-digested pRR3, a kanamycin-resistant mycobacteria/E. coli shuttle vector.
Reagents.
Restriction enzymes, T4 DNA ligase and other DNA modifying enzymes were purchased from Boehringer Mannheim, Promega or USB.
Culture of bacteria.
M. bovis BCG 1173P2 (Pasteur Institute, Paris, France) was grown as a pellicle at 37·5 °C, on normal or zinc-deficient Sauton medium. The medium was prepared with Milli RO water (conductivity 20 S); zinc sulfate was added to a final concentration of 5 µM (normal Sauton medium) or 0·15 µM (zinc-deficient medium). E. coli was grown on LB medium containing 25 µg kanamycin ml-1 and harvested at an OD660 between 0·6 and 0·8.
Filtrate from zinc-deficient culture.
The culture medium was clarified by decantation. The remaining organisms were removed by filtration through a Pellicon filter unit of 0·22 porosity (Millipore). The culture filtrate was dialysed against 20 mM phosphate buffer (PB) pH 7·3 using a Pellicon filter unit equipped with membranes of 10 kDa cut off.
Preparation of cell extracts.
M. bovis BCG extracts from 7-d-old cultures were prepared as previously described (De Bruyn et al., 1987a ). Briefly, the bacterial pellets were suspended in 0·05 M PB, pH 7·3 (0·3 g wet weight cells per ml buffer). The suspension was homogenized with a potter homogenizer, disrupted at 83110 MPa in a French press and clarified by centrifugation at 4000 g. After a second centrifugation at 100000 g for 90 min, the resulting supernatant was analysed by hydrophobic chromatography.
E. coli was harvested at an OD660 between 0·6 and 0·8, centrifuged at 10000 g for 30 min and washed twice with 0·05 M PB pH 7·3. The bacterial pellet was resuspended in the same buffer using the same ratio of wet weight cells per ml buffer as described for M. bovis BCG. The bacterial suspension was disrupted at a maximum of 83 MPa in a French press and clarified by centrifugation under the same conditions as M. bovis BCG.
Protein determination.
Protein concentrations were determined with the Bio-Rad protein assay kit, according to the Bradford dye (Coomassie brilliant blue G)-binding procedure (Spector, 1978 ) with BSA as the standard. The amounts of non-covalently bound lipids on Hsp60 were estimated by dot blotting. Protein concentration was determined by the Lowry method.
Protein analysis by PAGE.
SDS-PAGE was conducted as described by Laemmli (1970) on 13% (w/v) acrylamide gels. Proteins from zinc-deficient culture medium and from fractions eluted from the first purification step on phenyl-Sepharose were precipitated by 10% (w/v) trichloroacetic acid in the presence of sodium deoxycholate (Sigma) at a concentration of 125 µg ml-1 and kept on ice for 2 h (Bensadoun & Weinstein, 1976
). The precipitates were centrifuged at 1200 g for 30 min, washed once with 1% (w/v) acetone/triethanolamine and twice with acetone, and then dissolved in the sample buffer (15 mM Tris/HCl, pH 6·8, 0·1% SDS, 1·25% ß-mercaptoethanol). Gels were stained with silver (Bio-Rad).
Immunoblotting.
After SDS-PAGE, proteins were transferred onto nitrocellulose sheets (Bio-Rad) by the method of Towbin et al. (1979) . mAbs XVIII G1 and IAI (anti-Hsp60-1 and anti-Hsp60-2; Thole et al., 1988b
), mAb 67-2 (anti-Hsp60-2; Anderson et al., 1988
), mAb 32TDS15 (anti-antigens 85), mAb 2F8-3 (anti-PstS-3; Braibant et al., 1996
), mAb F29-47 (anti-19 kDa lipoprotein; Young & Garbe, 1991
), mAb CS44 (anti-Hsp60-1) and mAb L7 (anti-Hsp70) were used. mAbs 67-2 and F29-47 were a gift from A. H. J. Kolk (Royal Tropical Institute, The Netherlands). mAbs L7 and CS44 were a gift from L. Walker (UNPD/World/WHO Special Programme for Research and Training in Tropical Disease). Rabbit anti-M. bovis BCG serum (Dako) was used at a dilution of 1/200. Alkaline phosphatase conjugated anti-mouse or anti-rabbit immunoglobulins (Promega) were used at a dilution of 1/7500 and 1/5000 respectively.
Purification of Hsp60-2b.
Except for hydrophobic chromatography on phenyl-Sepharose, all buffers contained glycerol (0·1%, w/v, final concentration), were adjusted to pH 7·3 and sterilized. All purification steps were performed at 4 °C. The zinc-deficient culture filtrate was dialysed against 20 mM PB and adjusted to 450 mM NaCl. This filtrate was applied to a phenyl-Sepharose column Cl-4B (10x30 cm; Pharmacia) (De Bruyn et al., 1987a ). The gel was first washed with 20 mM phosphate, 450 mM NaCl buffer (starting buffer) to remove unfixed materials, and then irrigated successively with 20 mM and 4 mM PB, and 10% ethanol. The purification scheme is shown diagrammatically in Fig. 1
. Fractions eluted with the 20 mM and 4 mM PB from the phenyl-Sepharose column were further purified by ion-exchange chromatography on DEAE Sephacel and eluted by a 20 to 160 mM phosphate gradient. Fractions showing one band on SDS-PAGE were pooled and concentrated using an Amicon stirred cell equipped with a 10 kDa cut-off membrane (Amicon). Purified recombinant 65 kDa protein (Batches MA-11A and MA-12B) was a gift from J. Van Embden (Bilthoven, The Netherlands) and M. Singh (Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany). E. coli GroEL (Batches 13676620 and 14439420) was purchased from Boehringer.
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Extraction of lipids.
The different purified Hsp60 proteins (about 100 µg protein ml-1 in 50 mM PB, pH 7·3) were successively extracted four times with chloroform/methanol (2:1, w/v) (Bligh & Dyer, 1959 ); the two first extractions were performed for 1 h at room temperature, and two other extractions were done for 1 h at 37 °C, with thorough mixing. In each extraction the volume of the organic solvent mixture used was equal to half that of the aqueous phase. The organic phases were pooled and washed once with 0·3% NaCl, twice with distilled water (using a volume of aqueous phase corresponding to 0·2 vol of the organic phase) and then evaporated to dryness under nitrogen. The samples were kept at -30 °C. Lipids from 1 mg purified protein were dissolved in 0·2 ml toluene/acetone (4:1, v/v) for subsequent analyses.
Lipid analysis.
Dot blotting was performed on Kieselgel 60W 254 DC Alufolien sheets (Merck). Equal volumes (10 µl) of serial twofold dilutions of the non-covalently bound lipid preparations from the Hsps and standards were used. Lipids were dissolved in toluene/acetone (4:1, v/v) and loaded using a Linomat (Camag) apparatus. Oleic acid (UCB) at an initial concentration of 0·5 µg ml-1 was used as the standard. Plates were sprayed with iodine and the highest dilution giving a positive result was determined in each case. TLC analysis of lipids was performed on the same support as that used for dot blotting. Toluene/acetone (4:1, v/v) was used as developing solvent. TLC plates were stained successively with iodine and -naphthol/sulfuric acid (5 min at 110 °C). Oleic acid (20 µl of a 200 µg ml-1 solution) was loaded on each chromatographic plate for estimation of RF variations from one TLC analysis to another. Galactosyl diglyceride from wheat flour (20 µl of a 20 µg ml-1 solution; Sigma) was also loaded on each chromatographic plate as a positive control for staining with
-naphthol/sulfuric acid. Scanning and quantification of the different TLC spots were performed with a GS-670 imaging densitometer (Bio-Rad). Densitometer pictures of the chromatograms were photographed.
To identify free carboxyl-group-containing lipid compounds, samples were treated with a diethylether solution of diazomethane for 1 h at room temperature. The diazomethane was evaporated under nitrogen and the samples were dissolved in toluene/acetone for TLC analysis as described above, and compared with the untreated samples.
Characterization of fatty acids and sugar components by GC.
Fatty acid and sugar constituents of lipids were determined by methanolysis. Erythritol (internal standard) was added to lipid extracts derived from about 2 mg Hsp60-2 and the mixtures were treated with 0·75 ml methanolic HCl (1·5 M) for 16 h at 80 °C. The solutions were dried under vacuum over phosphorus pentoxide and potassium hydroxide. For semi-quantitative determination of the relative percentages of fatty acid substituents in the lipid extracts, aliquots of the methanolysates were trimethylsilylated according to Sweeley et al. (1963) and analysed by GC, with erythritol as the reference. The remaining methanolysates were partitioned between water and diethylether.
The aqueous phases were dried and the methylglycosides were trimethylsilylated. Fatty methyl esters and sugar derivatives were both analysed by GC and compared to authentic standards, leading to the identification of fatty acid and sugar constituents.
GC.
GC was performed on a Girdel G30 apparatus equipped with a fused silica capillary column (25 m lengthx0·32 m internal diameter) coated with OV-1 (0·3 mm film thickness). A temperature gradient of 100280 °C (3 °C min-1) was used. The relative percentage of constituents was calculated by comparing the peak height of the various fatty acid methyl esters to that of glucose.
Metabolic labelling with [3H]palmitate.
M. bovis BCG was grown for 10 d in 25 ml Middlebrook 7H9/ADC medium (Difco) containing 25 µCi ml-1 of [9,10(n) 3H]palmitic acid (52 Ci mmol-1; Amersham). Bacteria were harvested by centrifugation and washed with either Tris-buffered saline or PB, depending on the experiment.
Preparation of soluble extracts.
Soluble extracts were prepared using a 375 W model Vibra Cell-Sonics Material (Analis) equipped with a 13 mm solid probe. The cooled bacterial suspension was sonicated for 1 min without interruption, followed by two 5 min pulses (30 s sonication min-1). After centrifugation for 10 min at 12000 g, the supernatant was used for further experimentation.
Analyses of soluble extract.
Triton X-114 phase separation was performed according to Bordier (1981) . Briefly, Triton X-114 was added to the soluble extract at a final concentration of 2% (v/v). After vigorous mixing, the preparation was first kept on ice and then incubated for 5 min in a 37 °C water bath; the mixture was centrifuged for 5 min at 5000 g and each phase was back-washed (Radolf et al., 1988
). The detergent washings of the aqueous phase and the aqueous washings of the detergent phase were added to the detergent phase and the aqueous phase, respectively, before analysis.
For immunoprecipitation mAb 67-2 (anti-Hsp60) or mAb L7 (mc0044; anti-Hsp70) was added to the soluble extract and the mixture was incubated overnight. Then, 100 µl ProteinASepharose CL-4B (Amersham Pharmacia Biotech) was added to the sample, which was incubated for 1 h at room temperature.
Visualization and identification of radiolabelled components.
To visualize radiolabelled components, SDS-PAGE gels were fixed and treated with Amplify according to the procedures recommended by the manufacturers (Amersham Life Science). Fluorographs were prepared by 3 d exposure to X-ray film (Kodak X-omat-AR) at -70 °C. About 80% of the material was used for autoradiography and 20% for immunoblotting.
Prediction of a tridimensional model of Hsp60-2 and comparison of the hydrophobic character with GroEL protein.
The Hsp60-2 sequence was compared with all sequences in the PDB database (release 79, January 1997) using FASTA (Pearson & Lipman, 1988 ). Modeller 4.0 program (Sali & Blundell, 1993
) was performed on PC586 microcomputers running under Linux. Visualization was performed using WinMGM software (Rahman & Brasseur, 1994
) from Ab Initio Technology.
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RESULTS |
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To check that the occurrence of Hsp60s in the different phenyl-Sepharose fractions was not due to the particular growth conditions, i.e. zinc-deficient culture filtrate, extracts from normal Sauton-grown bacteria harvested during the exponential phase of growth were analysed (Fig. 2, right). The corresponding electrophoretic pattern obtained for the first step of purification was similar to that from the zinc-deficient culture filtrate (Fig. 2a
, b
, c
). Importantly, an entirely different distribution pattern was observed when the P32 protein (antigen 85), the major secreted protein of M. bovis BCG, was analysed under the same conditions (Fig. 2d
). The P32 protein was present in large amounts in the last fraction only and, in agreement with our previous results (De Bruyn et al., 1987b
), the P32 protein was present in much lower amounts in the bacterial extract (23%) than in the culture filtrate (1520%) from which the antigen has been previously purified. Since this antigen family has a higher predicted hydrophobicity (hydrophobicity indexes of antigen 85A, B and C are 0·1382, 0·1437 and 0·1251, respectively) than Hsp60 (hydrophobicity index of Hsp60-2 is 0·0561), it was concluded that the observed distribution of Hsp60 fractions eluted from phenyl-Sepharose was not due to a non-specific adsorption of lipids on these proteins. Taken together, these data indicated that Hsp60 eluted in the different phenyl-Sepharose fractions correspond to various forms of the protein, differing from one another by their hydrophobicity.
The relative amounts of the Hsp60 present in the unfixed fraction and in the three fractions eluted from phenyl-Sepharose loaded with zinc-deficient culture filtrate or normal bacterial extract were estimated by ELISA using purified P64 as the standard and mAb IA1 (anti-Hsp60). This method gave highly reproducible results and showed that the unfixed Hsp60 represented about 5% of the total Hsp60 of the culture filtrate and less than 1% of that of the bacterial extract; each of the two peaks eluted with 20 mM and 4 mM phosphate buffer from the phenyl-Sepharose gel contained roughly 4045% of the Hsp60 from both sources. The remaining 10% of the Hsp60 was found in the last phenyl-Sepharose fraction. As Hsp60 was present in large amounts in zinc-deficient culture filtrate and was more easily purified from such culture medium, subsequent work was performed using Hsp60 derived from zinc-deficient culture filtrate.
Purification of Hsp60
Hsp60-2b was also easily purified from peak 5 of the phenyl-Sepharose column (Fig. 2a, left) by ion-exchange chromatography using the previously described procedure for Hsp60-2c (De Bruyn et al., 1987a
). As shown in Fig. 3
, only one major band was seen on SDS-PAGE after silver staining; the very faint bands of lower molecular mass revealed by Western blotting using mAb 67-2 probably correspond to degradation products often encountered with these Hsp60 proteins. The purification of Hsp60 from peak 7 (Fig. 2a
) was less easy than that of Hsp60-2b and c, because of the presence of several major contaminating proteins (see Fig. 2b
, lane 7). Nevertheless, after ion-exchange chromatography, Hsp60-2d was sufficiently pure for microsequence analysis after SDS-PAGE and electroblotting.
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Overexpression of the Hsp60-2 protein in E. coli
To determine whether the occurrence of various Hsp60-2 forms depended on the bacterial source, the protein was overexpressed in E. coli. Thus, the soluble extracts of E. coli containing pRR3Hsp60-2 or the empty pRR3 plasmid were brought in the starting buffer and applied on a phenyl-Sepharose column at a ratio of 0·51 mg protein (ml gel)-1 and submitted to the same stepwise elution as M. bovis BCG culture filtrate. In each case, the unfixed fraction and the three phenyl-Sepharose-eluted fractions were analysed by SDS-PAGE, followed by Western blotting using either mAb 67-2, which specifically recognizes the C-terminal part of the mycobacterial Hsp60-2, or mAb XVIIIG1, which recognizes both the E. coli GroEL and the mycobacterial Hsp60-2 (Fig. 4). As expected, a protein band corresponding to the mycobacterial Hsp60-2 was clearly seen in each lane, except in lane 1 since the E. coli GroEL doesnt react with the mAb 67-2 (Fig. 4a
). When the same experiment was performed using the control E. coli strain containing empty pRR3 (Fig. 4b
, c
), only M. bovis BCG Hsp60-2 control gave a band (Fig. 4b
, lane 3). Interestingly, Western blotting using mAb XVIIIG1, which recognizes both Hsp60 and GroEL, showed that the E. coli GroEL exhibits the same hydrophobic behaviour as M. bovis BCG Hsp60-2 (Fig. 4c
). It has to be noted, however, that the overexpression of Hsp60 in E. coli led to much degradation of this protein (Fig. 4a
, lanes 46), especially in Hsp60-2 of the unfixed fractions (Fig. 4a
, c
). As a control for the results presented in Fig. 4c
, the behaviour of commercially available GroEL on phenyl-Sepharose was analysed. Passage of GroEL in the starting buffer through a phenyl-Sepharose column resulted in extensive degradation (90%) of the protein (data not shown). The remaining 10% of the intact GroEL was recovered in the two most hydrophobic fractions eluted from the phenyl-Sepharose column, i.e. peaks 6 and 7; trace amounts of GroEL were found in peak 5 and it was just detectable in the unfixed phenyl-Sepharose fraction (peak 4). The observed instability of GroEL was not due to storage conditions, since incubation of the protein at 37 °C for 3 h at a concentration of 1 mg ml-1 did not affect the protein concentration and, more importantly, only one band of the same molecular mass as the non-incubated control was detected by SDS-PAGE analysis. It was thus concluded that the passage of GroEL through the phenyl-Sepharose column was responsible for the observed degradation of purified GroEL.
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Analysis of the non-covalently bound lipids associated with the purified Hsp60 proteins
Lipids extracted from the two major Hsp60-2s, recombinant Hsp60-2 and GroEL were analysed by TLC using toluene/acetone (80:20, v/v) as the developing solvent. The TLC profiles of the different samples were very similar (Fig. 5); at least ten lipid spots were visualized with iodine. Among these, four spots were stained with the
-naphthol/sulfuric acid reagent, suggesting that they corresponded to glycolipids. The migration of one of these spots (RF 0·43) was shifted following the methylation of the lipid extracts (RF around 0·50), suggesting the occurrence of some esterifiable groups in this compound. Furthermore, and most importantly, no spot was stained with the DittmerLester reagent (Dittmer & Lester, 1964
), a specific spray for the detection of phospholipids. The nature of the ester-linked fatty acid residues was determined by GC analysis of the methanolysis products of the lipids extracted from the different Hsp proteins. Fatty acids C16:0, C18:0 and C18:1 were the main fatty acyl substituents detected in all the methanolysates, C16:0 being the most abundant component (Table 1
). In addition, tuberculostearate (10-methyl octadecanoate) was present in the methanolysis products of lipids from Hsp60-2c. Glucose was the only abundant sugar constituent identified by GC in the methanolysis products of the four lipid samples. Interestingly, glucose represented (in relative percentage) 19±1 and 41±2% of the methanolysis product constituents from Hsp60-2c and Hsp60-2b, indicating that the various Hsp60-2s may also differ in their glycolipid content. Results presented in Table 1
are limited to the comparison of mycobacterial Hsp60-2b and Hsp60-2c fatty acid methyl esters contents as the recombinant Hsp60-2 and GroEL proteins were produced in a different host and their purification did not include a phenyl-Sepharose step.
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Molecular hydrophilic potentials (Brasseur, 1991 ) were displayed for Hsp60-2 and GroEL structures (Fig. 7
). This calculation allows visualization of the distribution of hydrophobic and hydrophilic domains of the molecules. The hydrophobicities of GroEL and Hsp60-2 assemblages are very slightly different. Considering the sequence conservation and hydrophobicity between GroEL and Hsp60-2, we can suggest that their macromolecular features are globally identical. However, their inside cavities are more hydrophobic than their respective outer parts. This feature of the GroEL binding surface cavity was previously reported by Frydman & Hartl (1996)
.
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DISCUSSION |
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Indeed, the occurrence of non-covalently bound lipids associated with Hsp60 was not due to an artefact since different sources and different purification schemes have been applied to the isolation of the natural BCG Hsp60-2, the BCG 65 kDa recombinant protein expressed in E. coli, and E. coli GroEL. The existence of the phenyl-Sepharose Hsp60 forms was dependent neither on the presence of zinc in the growth medium, nor on the bacterial compartment from which the proteins were isolated (culture filtrate or cell extract), nor on the bacterial source (M. bovis or E. coli). In addition, not all the proteins exhibit this behaviour on phenyl-Sepharose; for instance, the major secreted protein of M. bovis, P32 (antigen 85), which has a predictable hydrophobicity that is higher than that of Hsp60, was eluted from the phenyl-Sepharose column in a major fraction, proving that the hydrophobic behaviour of Hsp60 was not due to a non-specific adsorption of lipids on proteins. This conclusion was further supported by the absence of phospholipids in the lipids extracted from the purified Hsp proteins; these substances, which include the mannose-containing phosphatidyl inositol, are known to be present not only in the plasma membrane but also in the external envelope of mycobacteria (Ortalo-Magné et al., 1996 ) and thus represent sensitive controls for the absence of non-specific adsorption of lipids on proteins. The detection of glucose, but not mannose, as the unique sugar constituent of the methanolysis products of lipids extracted from Hsp60 was in agreement with the absence of phospholipids in Hsp lipids. Furthermore, no typical mycobacterial lipid could be detected in lipid extracts from Hsp60, another strong argument pointing towards the absence of non-specific adsorption of lipids. The very similar nature of the lipids extracted from the Hsp60 from the phylogenetically unrelated Gram-negative E. coli and the Gram-positive M. bovis suggests a common biological function of these lipids. The evidence of a selective labelling of Hsp60 with palmitate, but not Hsp70, reinforced the concept of a specific association of lipids to these Hsp60 proteins. Besides, the study brings an answer to an unexplainable result previously reported by Young & Garbe (1991)
. To identify lipoproteins of M. tuberculosis H37Rv, the authors performed two complementary experiments. On the one hand, they analysed the distribution of proteins from a Triton X-114-treated soluble extract of M. tuberculosis between the aqueous and detergent phases. In this experiment, the 65 kDa protein was found in the aqueous phase as expected for this water-soluble protein. On the other hand, they cultivated M. tuberculosis in the presence of [3H]palmitate and delipidated the labelled bacteria with chloroform/methanol prior to disruption of the bacterial cells. Proteins of the extract prepared from the delipidated bacteria were analysed by two-dimensional SDS-PAGE and fluorography. Following these analyses, the authors identified some well known lipoproteins and reported a strongly labelled signal which overlapped with the 65 kDa antigen spot; they could not explain the origin of this [3H]palmitate signal and have proposed that occurrence of a lipoprotein with similar electrophoretic mobility to the 65-kDa antigen remained an attractive possibility. Our data showed that the radioactive spot described in the experiment of Young & Garbe corresponds to the 65 kDa Hsp60 protein.
The existence of an hydrophobic channel, as visualized in the model, and consisting of hydrophobic amino acids (conserved in mycobacterial Hsp60) at the entrance of the cavity provides a suitable interface for interaction with lipids. The production of stable, purified lipid-associated Hsp60-2 protein complex is difficult. Moreover the stability of these complexes might be different in mycobacteria and E. coli, and vary with the nature of the Hsp60 (1 or 2). These facts could possibly be related to the very low yield of associated lipids found in E. coli GroEL. For these reasons, the role of these non-covalently bound lipids in protein folding can presently only be speculative and restricted to the in vivo situation. Distribution of the different Hsp60 proteins and the better stability of the most hydrophobic fractions has been reproducibly observed for years. This suggests to us that the less-lipidated Hsps with a higher glycolipid content (as based on the higher glucose content of lipids extracted from Hsp60-2b compared to Hsp60-2c) would bind more hydrophilic proteins and contribute to facilitating their folding; conversely, the more lipidated Hsp60-2 would participate in the folding of the more hydrophobic proteins. Possibly the involvement of lipids and glycolipids in the in vivo protein folding could somehow improve the yield of biologically active molecules produced.
The similar hydrophobic behaviour shown for mycobacterial Hsp60-2 and Hsp60-1 by using specific antibodies, and for E. coli GroEL in the overexpression control experiment may indicate that the presence of non-covalently associated lipids could be a general feature of cylindrical chaperones. An important input of work 20 years ago demonstrated the requirement of lipid for cytochrome oxidase activity (Vik & Capaldi, 1977 ), followed by the demonstration of the necessity of the presence of phospholipids for the activity of numerous membrane enzymes; why would lipids not play a role in such a complex function as protein folding?
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 June 1999;
revised 10 February 2000;
accepted 25 February 2000.