A novel protein phosphatase 2A (MA) is involved in the transformation of human protozoan parasite Trypanosoma cruzi

• Autor(a) principal: Gonzalez, Jorge
• Data de Publicação: 2003
• Outros Autores: Cornejo, Alberto, Santos, Marcia Regina Machado [UNIFESP], Cordero, Esteban Mauricio [UNIFESP], Gutierrez, Bessy, Porcile, Patricio, Mortara, Renato Arruda [UNIFESP], Sagua, Hernan, Silveira, José F. da [UNIFESP], Araya, Jorge E.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: http://repositorio.unifesp.br/handle/11600/27414; http://dx.doi.org/10.1042/BJ20030215
• topic: enzyme purification; expression; gene cloning; genomic organization; phosphatase-specific inhibitor; trypanosome transformation

Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. in axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 muM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T cruzi PP2A. the enzyme displayed activity against 12 P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. the protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T cruzi PP2A enzyme by PCR. the isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.