The Mycobacterium leprae hsp65 Displays Proteolytic Activity. Mutagenesis Studies Indicate That the M. leprae hsp65 Proteolytic Activity Is Catalytically Related to the HslVU Protease

Abstract

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P₁, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T¹³⁶, K¹⁶⁸, and Y²⁶⁴) and the other in the C-domain (T³⁷⁵, K⁴⁰⁹, and S⁵⁰²). Mutagenesis studies showed that the replacement of K⁴⁰⁹ by A caused a complete loss of the proteolytic activity, whereas the mutation of K¹⁶⁸ to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T³⁷⁵, K⁴⁰⁹, and S⁵⁰² at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.