de Melo Ana Cristina Nogueira, Dos Santos André Luis Souza, Leal Meirelles Maria Nazareth, Branquinha Marta Helena, Vermelho Alane Beatriz
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;
Int J Biomed Sci. 2008 Jun;4(2):97-102.
Cellular and extracellular peptidase profiles from non-infected and Trypanosoma cruzi-infected hepatocyte cell cultures were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing different copolymerized proteins as substrates. A 100 kDa metallopeptidase activity was detected in the cellular extracts and in the culture supernatant fluids of both systems, had the ability to exclusively degrade gelatin. However, non-infected hepatocytes produced an additional extracellular metallopeptidase of 85 kDa. In the non-infected and in the infected hepatocytes, a cysteine peptidase migrating in gelatin-SDS-PAGE at 60 kDa presented the broadest specificity, since it was also able to hydrolyze casein and hemoglobin. The 100 kDa component was only detected at alkaline pH and predominantly expressed in non-infected hepatocytes. Conversely, the 60 kDa cysteine peptidase was only observed in acidic condition and its production was robustly augmented in T. cruzi-infected cells, probably due to the cysteine peptidase synthesized by the parasites, as corroborated by immunoblotting assay using anti-cruzipain antibody. Collectively, these results suggest that peptidases may be involved in the interaction process between T. cruzi and hepatocytes in vitro.
通过含有不同共聚蛋白质作为底物的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），对未感染和克氏锥虫感染的肝细胞培养物中的细胞内和细胞外肽酶谱进行了表征。在两个系统的细胞提取物和培养上清液中均检测到一种100 kDa的金属肽酶活性，其能够特异性降解明胶。然而，未感染的肝细胞会产生一种额外的85 kDa细胞外金属肽酶。在未感染和感染的肝细胞中，一种在明胶-SDS-PAGE中迁移至60 kDa的半胱氨酸肽酶具有最广泛的特异性，因为它还能够水解酪蛋白和血红蛋白。100 kDa的组分仅在碱性pH下检测到，且主要在未感染的肝细胞中表达。相反，60 kDa的半胱氨酸肽酶仅在酸性条件下观察到，并且其产量在克氏锥虫感染的细胞中显著增加，这可能是由于寄生虫合成的半胱氨酸肽酶所致，使用抗克鲁兹蛋白酶抗体的免疫印迹分析证实了这一点。总体而言，这些结果表明肽酶可能参与了体外克氏锥虫与肝细胞之间的相互作用过程。
