Nogueira de Melo Ana Cristina, Meirelles Maria Nazareth L., Porrozzi Renato, Costa Juliana Dias, Branquinha Marta Helena, Vermelho Alane Beatriz
Department of General Microbiology, Institute of Microbiology Prof Paulo de Góes, Federal University of Rio de Janeiro, CCS, Bl I, 21941-590, RJ, Rio de Janeiro, Brazil
Hepatol Res. 2004 Jan;28(1):49-56. doi: 10.1016/j.hepres.2003.09.002.
In this work, we are reporting differences in the proteolytic profile of Trypanosoma cruzi-infected and non-infected primary cultures of mouse embryo hepatocyte cells. In gelatin-SDS-PAGE, ours results showed the presence of a 100kDa metalloproteinase in the supernatant and in the cells of both systems and an 85kDa extracellular metalloproteinase found only in the non-infected hepatocyte cultures. An enzymatic assay using gelatin as substrate showed a decrease of 74 and 70% in metalloproteinase activity in the culture supernatant and in the cell hepatocyte system infected with T. cruzi, respectively. Western blotting analysis using anti-matrix metalloproteinase-9 (MMP-9) antibody recognized the 100 and 85kDa protein bands, indicating that hepatocyte metalloproteinases correspond to the latent and active forms of the gelatinase MMP-9, respectively. The localization of MMP-9 was established by immunocytochemistry analysis in the cytoplasm of the non-infected and infected hepatocyte cells. In normal and infected hepatocyte cells, cysteine-proteinases migrating in gelatin-SDS-PAGE at 60kDa were detected and should correspond to lysosomal cysteine-proteinases of T. cruzi (cruzipain) and hepatocytes. In T. cruzi-infected hepatocytes an increase of approximately 50% in this enzymatic activity was observed, possibly due to parasite's cruzipain.
在本研究中，我们报告了克氏锥虫感染和未感染的小鼠胚胎肝细胞原代培养物中蛋白水解谱的差异。在明胶-SDS-PAGE中，我们的结果显示，在两个系统的上清液和细胞中均存在一种100kDa的金属蛋白酶，而仅在未感染的肝细胞培养物中发现一种85kDa的细胞外金属蛋白酶。以明胶为底物的酶活性测定表明，克氏锥虫感染的培养上清液和肝细胞系统中金属蛋白酶活性分别降低了74%和70%。使用抗基质金属蛋白酶-9（MMP-9）抗体的蛋白质印迹分析识别出100kDa和85kDa的蛋白条带，表明肝细胞金属蛋白酶分别对应于明胶酶MMP-9的潜在形式和活性形式。通过免疫细胞化学分析确定了MMP-9在未感染和感染的肝细胞细胞质中的定位。在正常和感染的肝细胞中，检测到在明胶-SDS-PAGE中迁移至60kDa的半胱氨酸蛋白酶，它们应分别对应于克氏锥虫（克氏锥虫蛋白酶）和肝细胞的溶酶体半胱氨酸蛋白酶。在克氏锥虫感染的肝细胞中，观察到这种酶活性增加了约50%，这可能是由于寄生虫的克氏锥虫蛋白酶所致。
