Caracterização bioquímica de uma Leucil-aminopeptidase de Trypanosoma cruzi (LAPTc)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

Pathogens depend on peptidase activities to accomplish many physiological processes, as well as to interact with their hosts, highlighting parasitic peptidases as virulence factors and, thus, potential drug targets. The kinetoplastid Trypanosoma cruzi genome sequencing, the aetiological agent of Chagas disease, has revealed 28 genes encoding putative aminopeptidases, among which there are three methionine, two aspartic, two puramycin-sensitive and three leucyl aminopeptidases of the M17 family. In this study, a major leucyl aminopeptidolytic activity was identified in the T. cruzi by using leu-AMC as substrate. It was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 320-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Interchain disulfide bonds do not take part in the oligomeric assembly of the active peptidase. Peptide mass fingerprinting revealed the molecular identity of the enzyme as the predicted T. cruzi aminopeptidase EAN97960, the product of the Tc00.1047053508799.240 gene. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80 C. Conversely, its recombinant form produced in E. coli, like other LAPs, is thermophilic and requires alkaline pH. The activity of this metalloaminopeptidase is inhibited by bestatin and metal chelants such as 1,10-phenanthroline, is restored by Zn2+, and potentiated by Mn2+ or Ca2+. The enzyme is expressed by all T. cruzi forms and localizes within vesicles in the cytoplasm of the parasite. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply. Furthermore, the peptidase activity could also play a role in peptide and protein processing. We postulate that the LAPTc might be a potential target for the development of new drugs to treat T. cruzi infection.

ASSUNTO(S)

chagas disease doença de chagas aminopeptidases leucina trypanosoma crúzi biologia molecular aminopeptidases leucine trypanosoma cruzi

ACESSO AO ARTIGO

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