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Molecular and immunological characterization of a Trypanosoma cruzi protein homologous to mammalian elongation factor 1 gamma

Abstract:

In previous studies, we reported the characterization of three Trypanosoma cruzi proteins with molecular masses of 45, 30 and 25 kDa eluted from a glutathione agarose column (these proteins were named TcGBP). Using antibodies against TcGBP native proteins we could isolate from a lambda ZAPII epimastigote cDNA library cDNA clones encoding the 30 and 25 kDa proteins. Comparison of the two sequences with amino acid sequences in several data banks revealed that both protein sequences were highly homologous to human and Artemia salina elongation factor 1β. Thus, the proteins were named TcEF-1 β25 and TcEF-1 β30. In the present study we used a double immunoscreening strategy that allowed us to isolate a cDNA clone corresponding to the 45 kDa protein. The protein sequence revealed 31% identity and 61% homology with human and Artemia salina EF1γ and therefore was named TcEF-1γ. Moreover, three putative phosphorylation sites at position 51 (CSPC), at position 90 (RTPL) and at position 265 (PSPF) were found in the TcEF-1γ sequence. These sites are compatible with the notion that TcEF-1γ could be the target of phosphorylation by protein kinase(s). Random primed cDNA hybridized with a single 1.4 kb mRNA found in epimastigote, trypomastigote and amastigote forms. In addition, Southern blot analysis of genomic DNA suggested that the protein is encoded by a single gene. The TcEF-1γ cDNA was subcloned into the pGEX-4T-3 vector for expression in Escherichia coli. Antibodies against the fusion peptide allowed us to identify the weight sizes of the native protein (48 kDa) and its major degradation product (24.4 kDa) which are in close agreement with those of EF1-γ from Artemia salina and Schizosaccharomyces pombe. These antibodies reacted against macrophage cell line J774 extracts which indicates that EF-1γ epitopes were conserved throughout evolution. © 1994.