Estudo das proteínas da família gp82 das formas metacíclicas do Trypanosoma cruzi

• Ano de defesa: 2008
• Autor(a) principal: Atayde, Vanessa [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• dARK ID: ark:/48912/00130000298w8
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Apoptose; Clone CL-14; Formas metacíclicas; Invasão celular; Melanoma; Proteínas da família gp82; Trypanosoma cruzi
• Link de acesso: http://repositorio.unifesp.br/handle/11600/9550

The main goal of this work was to characterize the proteins encoded by the gp82 gene fami ly, which is part of the large T. cruzi gp85/trans-sialidase multigenic family. Previous studies had focused on the surface gp82, which is engaged in the cell invasion by CL strain metacyclic forms and is identified by monoclonal antibody (MAb) 3F6. Firstly, we investigated the molecular basis of the non-virulence of T. cruzi clone CL-14 metacyclic forms in vivo and in vitro. We found that the low infectivity of these parasites is associated with reduced expression of surface gp82, reinforcing the role of this molecule in T. cruzi infection. In addition, our data suggested that instead of gp82, metacyclic forms of clone CL-14, like G strain, preferentially engage gp35/50 glycoproteins to interact with the host cell. Secondly, we analyzed the expression and cellular localization of molecules of the gp82 family in T. cruzi metacyclic forms of G and CL strains. We cloned a new member of this family, designated C03, which lacks the MAb 3F6 epitope, in contrast to the previously described gp82 cDNA clones J18 (G strain) and R31 (CL strain). The predicted amino acid sequence of the C03 clone displayed 59,1% identity to J18 clone and 60,2% to R31 clone. When the amino acid sequences of C03 and Tc-85 (tissue culture trypomastigote gp85) clones were aligned, the identity was 57,2%, indicating that this new clone belongs to the gp85/trans-sialidase family. Using anti-J18 and anti-C03 polyclonal antibodies, as well as MAb 3F6, we demonstrated that members of the gp82 family are localized on the cell surface and intracellularly in metacyclic forms, and some members have different cellular localization in parasites from T. cruzi I and II groups. As opposed to metacyclic stage-specific gp82 identified by MAb 3F6, other gp82 fami ly molecules were also detected in tissue culture trypomastigotes and amastigotes by polyclonal antibodies. Thirdly, we investigated the effect of gp82, as a recombinant protein (J18), on the murine melanoma lineage Tm5. We observed that J18 binds to these cells disrupting actin filaments similarly to cytochalasin-D, drug that induces apoptosis in mammalian cells. Based on these information, we comparatively analyzed alterations in actin cytoskeleton and apoptotic events in J18-treated Tm5 cells, and in the melanocyte lineage melan-a, from which Tm5 is derived. J18 selectively induced apoptosis in Tm5 melanoma cells. Our results show an activity of a protein from gp82 family that has not been described for any other T. cruzi molecule.