EXPRESSION AND PARTIAL CHARACTERIZATION OF METALLOCARBOXYPEPTIDASES (MCPs) OF TRYPANOSOMA CRUZI.

GABRIELA NIEMIROWICZ, FABIOLA PARUSSINI, FERNÁN AGÜERO, AND JUAN J. CAZZULO

Iguazú

Congreso; XL Reunión Anual SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR; 2004

SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR

The genome of Trypanosoma cruzi, the causative agent of Chagas Disease, encodes two MCPs of the M32 family, with 64% of identity between them: TcCP-1 and TcCP-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 TcCP-1 and TcCP-2 were expressed as active recombinant enzymes in E. coli. TcCP-1 was purified to homogeneity in three steps, namely gel filtration and two ion exchange chromatography steps. TcCP-1 acted optimally at pH 6.2 on furylacryloyl(FA)-Ala-Lys with a Km of 0.166 mM. Activity against N-carbobenzoxy-Ala-X (ZAX) substrates revealed a P1´ preference for basic and some neutral C-terminal residues. Western blot analysis using a polyclonal antiserum raised against recombinant TcCP-1 showed that the enzyme is expressed in all life cycle stages of T. cruzi. Indirect immunofluorescence staining suggested that the protein is localized in the parasite cytosol. The polyHis-tagged TcCP-2 recombinant enzyme was purified to homogeneity by IMAC (Co+2). Preliminary characterization of this enzyme shows that it prefers FA-Phe-Phe at an optimum pH of 7.6-8.0. Therefore, the specificities of both MCPs are complementary.