Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG hsp60 proteins, and to the Escherichia coli homologue GroEL.
Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.
De Bruyn
J
J
Institut Pasteur de Bruxelles, Laboratoire des Mycobactéries, Rue Engeland 642, B-1180 Bruxelles, Belgium1.
Soetaert
K
K
Institut Pasteur de Bruxelles, Laboratoire des Mycobactéries, Rue Engeland 642, B-1180 Bruxelles, Belgium1.
Buyssens
P
P
Institut Pasteur de Bruxelles, Laboratoire des Mycobactéries, Rue Engeland 642, B-1180 Bruxelles, Belgium1.
Calonne
I
I
Institut Pasteur de Bruxelles, Laboratoire des Mycobactéries, Rue Engeland 642, B-1180 Bruxelles, Belgium1.
De Coene
J L
JL
Laboratoire de Chimie Biologique, Université Libre Bruxelles, Bruxelles, Belgium2.
Gallet
X
X
Faculté Universitaire de Gembloux, Centre de Biophysique Moléculaire Numérique, Belgium3.
Brasseur
R
R
Faculté Universitaire de Gembloux, Centre de Biophysique Moléculaire Numérique, Belgium3.
Wattiez
R
R
Laboratoire de Chimie Biologique, Université de Mons, Hainaut, Belgium4.
Falmagne
P
P
Laboratoire de Chimie Biologique, Université de Mons, Hainaut, Belgium4.
Montrozier
H
H
Institut de Pharmacologie et Biologie Structurale du CNRS, 31077 Toulouse cedex, France5.
Lanéelle
M A
MA
Institut de Pharmacologie et Biologie Structurale du CNRS, 31077 Toulouse cedex, France5.
Daffé
M
M
Institut de Pharmacologie et Biologie Structurale du CNRS, 31077 Toulouse cedex, France5.
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D005227:Fatty Acids / Q000032:analysis; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008759:Methylglycosides / Q000032:analysis; D008958:Models, Molecular; D009163:Mycobacterium bovis / Q000737:chemistry*; D010168:Palmitates / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D016622:Silver Staining; D014316:Tritium
D001426:Bacterial Proteins; D018834:Chaperonin 60; D005227:Fatty Acids; D008055:Lipids; D008759:Methylglycosides; D010168:Palmitates; D011994:Recombinant Proteins; D014316:Tritium