**3.4 Proteolytic profile of** *T. cruzi* **isolates from Rio de Janeiro**

The differences in peptidase expression between TCI and TCII phylogenetic groups have recently been investigated. Since *T. cruzi* isolates from sylvatic triatomines were included in the third phylogenetic group, named Z3, our investigation contributes to investigate the expression of surface polypeptides and the major cysteine peptidase from the Z3 parasite population, thereby furthering understanding on the genetic variability in the pathogenesis of Chagas disease. In this context, we carried out an identification of the protein profile and peptidase from epimastigotes (replicative forms of this parasite) of sylvatic isolates of *T. cruzi* (classified as Z3) from triatomines captured in Santa Maria Madalena (SMM) in the State of Rio de Janeiro. The separation of soluble whole proteins revealed a different protein profile, with approximately 35 polypeptides presenting apparent molecular masses from 118 to 25 kDa in all the samples. The proteolytic activity was determined by zymograms analysis of all the samples, using SDS-polyacrylamide gel electrophoresis containing gelatin as substrate. Our main results demonstrate a major band of 45 kDa sensible to E-64, a powerful cysteine peptidase inhibitor, in all the samples. In order to confirm this data, western blotting was performed using the anti-cruzipain polyclonal antibody. These findings showed a strong polypeptide band with an apparent molecular mass between 40 and 50 kDa in all the sylvatic isolates: SMM10; SMM53; SMM88 and SMM98 respectively and also Dm28c (Figure 5).

Fig. 5. A – Gelatin-SDS-PAGE showing the proteolytic activity profiles of *T. cruzi* sylvatic isolates. Parasites (SMM10, SMM53, SMM88, SMM98, and Dm28c) grown for 7 days were harvested and lysed by SDS. The gel was incubated in 50 mM sodium phosphate buffer, pH 5.5, supplemented with 2 mM DTT for 40 h at 37°C; B- Western blotting showing the reactivity of cellular polypeptides of *T. cruzi* sylvatic isolates with the anti-cruzipain polyclonal antibody. Numbers on the left indicate the relative molecular mass markers, expressed in kilodaltons.

These results show the presence of a main cysteine peptidase, cruzipain, in the sylvatic isolates of *T. cruzi* from Santa Maria Madalena, in the State of Rio de Janeiro (Gomes et al., 2009). We also observed another gelatinolyti activity of 66 kDa that was recognized by the anti-cruzipain antibody, probably a cruzipain isoform; since cruzipain is a high mannosetype glycoprotein containing about 10% carbohydrate, its molecular mass can be estimated from the sequence, considering two high-mannose oligosaccharide chains, as about 40 kDa. However, this enzyme can present anomalous behavior in SDS-PAGE, yielding apparent molecular mass values of 35 to 60 kDa depending on the experimental conditions. The cysteine peptidases from parasites, including *T. cruzi*, have proven to be valuable targets for chemotherapy. Due to the biological importance of cruzipain in the life cycle of *T. cruzi*, many studies have sought to build specific inhibitors against the active core of this enzyme, in order to obtain a new drug capable of providing protection against human infection by *T. cruzi*.
