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 conser ved structure and are assumed to be key partners in the biogenesis of prote ins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60 from Mycobacterium bovis BCG zinc-deficient culture filtrate on phen yl-Sepharose followed by Western blotting revealed the existence of four Hs p60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 s pecies were further purified by ion-exchange chromatography and partial ami no acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 s pecies 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 g ene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coil us ing the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution p attern of the recombinant Hsp60-2, as well as that of Escherichia coil GroE L, was similar to that of the native Hsp60-2 from the culture filtrate of M . bovis BCC and entirely different from that of the mycobacterial antigen 8 5. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E , coli GroEL with organic solvents releases various amounts of non-covalent ly bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelli ng M. bovis BCC with radioactive palmitate. The radioactivity was specifica lly associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipopr oteins in the Triton X-114 phase. Analysis of the lipids extracted from pur ified 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 th e 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.