Biochemical characterization and functional analysis of fructose-1,6-bisphosphatase from Clonorchis sinensis
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Fructose-1,6-bisphosphatase (FBPase), a key regulatory enzyme of gluconeogenesis, plays an essential role in metabolism and development of most organisms. To the wealth of available knowledge about FBPase from Clonorchis sinensis (CsFBPase), in this study, the characteristics of CsFBPase and its potential role in pathogenesis of clonorchiasis were investigated. The Km value of CsFBPase was calculated to be 41.9 uM. The optimal temperature and pH of CsFBPase were 37 °C and pH 7.5–8.0, respectively. In addition, Mg2+ or K+ played a regulatory role in enzyme activity of CsFBPase. Both transcriptional and translational level of CsFBPase were higher in metacercariae (one of larva stages) than those in adult worm (P < 0.05). CsFBPase were observed to extensively express in the intestine, vitellaria and tegument of adult worms and ubiquitously in metacercariae. Moreover, CsFBPase was confirmed as a component of excretory/secretory products. Consequently, the translocation of CsFBPase could be detected on epithelial cells of bile duct in liver of C. sinensis infected rat. Recombinant CsFBPase can specifically bind to the membrane of human hepatic stellate cell line LX-2 by immunofluorescence analysis and stimulated proliferation and activation of LX-2 which demonstrated by Cell Counting Kit-8 and upregulation of key fibrosis-related factors, such as α-smooth muscle actin, collagen I and collagen III using qRT-PCR. Thus, we predicated that CsFBPase might be a multifunctional enzyme which played as both regulatory enzyme and virulence factor in pathogenesis of C. sinensis infection.
KeywordsClonorchis sinensis CsFBPase Excretory/secretory products Liver fibrosis Hepatic stellate cell
This work was supported by National Key Basic Research and Development Project of China (973 project; No. 2010CB530000), The National S & T Major Program (No. 2012ZX10004-220), National Natural Science Foundation of China (No. 81101270 and No. 81171602), Fundamental Research Funds for the Central Universities (No. 3164015) and China Postdoctoral Science Foundation (No. 20110490952).
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