Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Centro de Ciências da Saúde (CCS), Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, RJ, Brazil.
Exp Parasitol. 2010 Jul;125(3):256-63. doi: 10.1016/j.exppara.2010.01.023. Epub 2010 Feb 6.
In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman's complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97kDa protein band in cellular extract and an 85kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.
在这项研究中，通过在含有明胶的酶谱中测量肽酶活性，分析了在改良罗伊特曼复合物（MRC）培养基中生长的克氏锥虫的细胞相关肽酶和细胞外释放的肽酶。我们的结果表明，细胞相关肽酶以及克氏锥虫细胞外释放的肽酶表现出两种不同的蛋白水解类：半胱氨酸和金属肽酶活性。主要的半胱氨酸肽酶，克鲁兹帕因，由 T. cruzi 细胞合成，在用抗克鲁兹帕因抗体探测后，在细胞寄生虫含量中被检测为 50kDa 的反应性多肽。此外，通过 Western blot 检测，金属型肽酶属于基质金属蛋白酶-9（MMP-9）家族，在细胞提取物中显示为 97kDa 的蛋白带，在细胞和分泌的寄生虫提取物中均显示为 85kDa 的多肽。存在于细胞和废弃培养物中的 MMP-9 样活性被抗 MMP-9 多克隆抗体免疫沉淀。通过流式细胞术分析也证明了 MMP-9 样蛋白在 T. cruzi 中的表面定位。此外，在体外具有直接 MMP-9 抑制特性的强力霉素抑制了凝胶酶谱、免疫沉淀和流式细胞术分析中的 MMP-9 样活性。这是首次报道 MMP-9 样分子存在于 T. cruzi 中。细胞外基质降解酶的存在可能在 T. cruzi-宿主细胞相互作用中发挥作用，使这种酶成为针对这种致病原生动物的未来药物开发的潜在靶标。
