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Title: A novel protein phosphatase 2A (MA) is involved in the transformation of human protozoan parasite Trypanosoma cruzi
Authors: Gonzalez, Jorge
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.
Univ Antofagasta
Universidade Federal de São Paulo (UNIFESP)
Keywords: enzyme purification
gene cloning
genomic organization
phosphatase-specific inhibitor
trypanosome transformation
Issue Date: 14-Sep-2003
Publisher: Portland Press
Citation: Biochemical Journal. London: Portland Press, v. 374, p. 647-656, 2003.
Abstract: 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.
ISSN: 0264-6021
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