METALLOCARBOXYPEPTIDASES OF TRYPANOSOMA CRUZI: CLONING, EXPRESSION AND CHARACTERIZATION.

GABRIELA NIEMIROWICZ; FABIOLA PARUSSINI; FERNÁN AGÜERO; JUAN JOSÉ CAZZULO

Glasgow

Congreso; 11th International Congress of Parasitology; 2006

British Society for Parasitology

The genome of Trypanosoma cruzi, the causative agent of Chagas Disease, encodes two metallocarboxypeptidases (MCPs) belonging to the M32 family, with 64% of identity between them: TcMCP-1 and TcMCP-2. These enzymes belong to a new family of peptidases whose members had been found so far exclusively in prokaryotes. This makes them possible candidates as targets for chemotherapy. Both full length TcMCP-1 and TcMCP-2 genes were cloned and expressed in E. coli as catalytically active polyHis-tagged recombinant enzymes. Despite their homology, purified TcMCPs displayed marked differences. TcMCP- 1 acted optimally at pH 6.2 on furylacryloyl(FA)-Ala-Lys with a Km of 166 mM. Activity against N-carbobenzoxy-Ala-X (ZAX) substrates reveled a P1´ preferencefor basic and, to a lesser extent, some neutral C-terminal residues. On the other hand, TcMCP-2 preferred aromatic and aliphatic residues at this position. The Km value for FA-Phe-Phe at pH 7.6 was 24 mM. Therefore, the specificity of both MCPs is complementary. Both TcMCPs were completely inactivated by dialysis for 16 hr at 4oC against 50 mM Tris-HCl, pH 7.6, 10 mM EDTA, and could be reactivated by 20 ? 27 % by 0.1 mM CoCl2 or MnCl2, in the case of TcMCP-1, and by 100 % by 0.1 mM CoCl2, for TcMCP-2.Western blot analysis revealed a different pattern of expression for both enzymes. Whereas TcCP-1 is present in all life cycle stages of T. cruzi, TcMCP-2 is mainly expressed in the insect vector ones. Indirect immunofluorescence staining suggested that both proteins are localized in the cytosol.