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Molecular Biology Reports

, Volume 40, Issue 7, pp 4371–4382 | Cite as

Biochemical characterization and functional analysis of fructose-1,6-bisphosphatase from Clonorchis sinensis

  • Pei Liang
  • Jiufeng Sun
  • Yan Huang
  • Fan Zhang
  • Juanjuan Zhou
  • Yue Hu
  • Xiaoyun Wang
  • Chi Liang
  • Minghui Zheng
  • Yanquan Xu
  • Qiang Mao
  • Xuchu Hu
  • Xuerong Li
  • Jin Xu
  • Gang Lu
  • Xinbing Yu
Article

Abstract

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.

Keywords

Clonorchis sinensis CsFBPase Excretory/secretory products Liver fibrosis Hepatic stellate cell 

Notes

Acknowledgments

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).

References

  1. 1.
    Rim HJ (2005) Clonorchiasis: an update. J Helminthol 79(3):269–281PubMedCrossRefGoogle Scholar
  2. 2.
    Keiser J, Utzinger J (2005) Emerging foodborne trematodiasis. Emerg Infect Dis 11(10):1507–1514PubMedCrossRefGoogle Scholar
  3. 3.
    Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, Fang YY (2005) Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis 5(1):31–41PubMedCrossRefGoogle Scholar
  4. 4.
    Keiser J, Utzinger J (2009) Food-borne trematodiases. Clin Microbiol Rev 22(3):466–483PubMedCrossRefGoogle Scholar
  5. 5.
    Hong ST, Fang Y (2012) Clonorchis sinensis and clonorchiasis, an update. Parasitol Int 61(1):17–24PubMedCrossRefGoogle Scholar
  6. 6.
    Lim MK, Ju YH, Franceschi S, Oh JK, Kong HJ, Hwang SS, Park SK, Cho SI, Sohn WM, Kim DI, Yoo KY, Hong ST, Shin HR (2006) Clonorchis sinensis infection and increasing risk of cholangiocarcinoma in the Republic of Korea. Am J Trop Med Hyg 75(1):93–96PubMedGoogle Scholar
  7. 7.
    Shin HR, Oh JK, Lim MK, Shin A, Kong HJ, Jung KW, Won YJ, Park S, Park SJ, Hong ST (2010) Descriptive epidemiology of cholangiocarcinoma and clonorchiasis in Korea. J Korean Med Sci 25(7):1011–1016PubMedCrossRefGoogle Scholar
  8. 8.
    Kim TI, Na BK, Hong SJ (2009) Functional genes and proteins of Clonorchis sinensis. Korean J Parasitol 47(Suppl):59–68CrossRefGoogle Scholar
  9. 9.
    Yoon BI, Jung SY, Hur K, Lee JH, Joo KH, Lee YS, Kim DY (2000) Differentiation of hamster liver oval cell following Clonorchis sinensis infection. J Vet Med Sci 62(12):1303–1310PubMedCrossRefGoogle Scholar
  10. 10.
    Yoon BI, Choi YK, Kim DY (2004) Differentiation processes of oval cells into hepatocytes: proposals based on morphological and phenotypical traits in carcinogen-treated hamster liver. J Comp Pathol 131(1):1–9PubMedCrossRefGoogle Scholar
  11. 11.
    Ma C, Hu X, Hu F, Li Y, Chen X, Zhou Z, Lu F, Xu J, Wu Z, Yu X (2007) Molecular characterization and serodiagnosis analysis of a novel lysophospholipase from Clonorchis sinensis. Parasitol Res 101(2):419–425PubMedCrossRefGoogle Scholar
  12. 12.
    Hu F, Hu X, Ma C, Zhao J, Xu J, Yu X (2009) Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A(2) and investigation of its potential contribution to hepatic fibrosis. Mol Biochem Parasitol 167(2):127–134PubMedCrossRefGoogle Scholar
  13. 13.
    Zheng M, Hu K, Liu W, Hu X, Hu F, Huang L, Wang P, Hu Y, Huang Y, Li W, Liang C, Yin X, He Q, Yu X (2011) Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of a excretory-secretory antigen-fructose-1,6-bisphosphatase. Parasitol Res 109(3):737–744PubMedCrossRefGoogle Scholar
  14. 14.
    Tillmann H, Bernhard D, Eschrich K (2002) Fructose-1,6-bisphosphatase genes in animals. Gene 291(1–2):57–66PubMedCrossRefGoogle Scholar
  15. 15.
    Tielens AG, Van der Meer P, van den Heuvel JM, van den Bergh SG (1991) The enigmatic presence of all gluconeogenic enzymes in Schistosoma mansoni adults. Parasitology 102(Pt 2):267–276PubMedCrossRefGoogle Scholar
  16. 16.
    Naderer T, Ellis MA, Sernee MF, De Souza DP, Curtis J, Handman E, McConville MJ (2006) Virulence of Leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1,6-bisphosphatase. Proc Natl Acad Sci USA 103(14):5502–5507PubMedCrossRefGoogle Scholar
  17. 17.
    Mamczur P, Mazurek J, Rakus D (2010) Ubiquitous presence of gluconeogenic regulatory enzyme, fructose-1,6-bisphosphatase, within layers of rat retina. Cell Tissue Res 341(2):213–221PubMedCrossRefGoogle Scholar
  18. 18.
    Hu F, Yu X, Ma C, Zhou H, Zhou Z, Li Y, Lu F, Xu J, Wu Z, Hu X (2007) Clonorchis sinensis: expression, characterization, immunolocalization and serological reactivity of one excretory/secretory antigen-LPAP homologue. Exp Parasitol 117(2):157–164PubMedCrossRefGoogle Scholar
  19. 19.
    Stein S, Liehr T, Eschrich K (2001) Characterization of the mouse liver fructose-1,6-bisphosphatase gene. Gene 264(2):215–224PubMedCrossRefGoogle Scholar
  20. 20.
    Yoo WG, Kim TI, Li S, Kwon OS, Cho PY, Kim TS, Kim K, Hong SJ (2009) Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR. Parasitol Res 104(2):321–328PubMedCrossRefGoogle Scholar
  21. 21.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408PubMedCrossRefGoogle Scholar
  22. 22.
    Xu L, Hui AY, Albanis E, Arthur MJ, O’Byrne SM, Blaner WS, Mukherjee P, Friedman SL, Eng FJ (2005) Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 54(1):142–151PubMedCrossRefGoogle Scholar
  23. 23.
    Benkovic SJ, de Maine MM (1982) Mechanism of action of fructose 1,6-bisphosphatase. Adv Enzymol Relat Areas Mol Biol 53:45–82PubMedGoogle Scholar
  24. 24.
    Lehninger AL, Nelson DL, Cox MM (2000) Lehninger principles of biochemistry. Worth Publishers, New YorkGoogle Scholar
  25. 25.
    Wang X, Chen W, Huang Y, Sun J, Men J, Liu H, Luo F, Guo L, Lv X, Deng C, Zhou C, Fan Y, Li X, Huang L, Hu Y, Liang C, Hu X, Xu J, Yu X (2011) The draft genome of the carcinogenic human liver fluke Clonorchis sinensis. Genome Biol 12(10):R107PubMedCrossRefGoogle Scholar
  26. 26.
    Robinson MW, Menon R, Donnelly SM, Dalton JP, Ranganathan S (2009) An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host. Mol Cell Proteomics 8(8):1891–1907PubMedCrossRefGoogle Scholar
  27. 27.
    Hetsko ML, McCaffery JM, Svard SG, Meng TC, Que X, Gillin FD (1998) Cellular and transcriptional changes during excystation of Giardia lamblia in vitro. Exp Parasitol 88(3):172–183PubMedCrossRefGoogle Scholar
  28. 28.
    Pilkis SJ, Claus TH, Kurland IJ, Lange AJ (1995) 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme. Annu Rev Biochem 64:799–835PubMedCrossRefGoogle Scholar
  29. 29.
    Behrisch HW (1971) Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab. Biochem J 121(3):399–409PubMedGoogle Scholar
  30. 30.
    Ekdahl KN, Ekman P (1985) Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase. J Biol Chem 260(26):14173–14179PubMedGoogle Scholar
  31. 31.
    Kuznetsova E, Xu L, Singer A, Brown G, Dong A, Flick R, Cui H, Cuff M, Joachimiak A, Savchenko A, Yakunin AF (2010) Structure and activity of the metal-independent fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae. J Biol Chem 285(27):21049–21059PubMedCrossRefGoogle Scholar
  32. 32.
    Zhang R, Villeret V, Lipscomb WN, Fromm HJ (1996) Kinetics and mechanisms of activation and inhibition of porcine liver fructose-1,6-bisphosphatase by monovalent cations. Biochemistry 35(9):3038–3043PubMedCrossRefGoogle Scholar
  33. 33.
    Zhang Y, Liang JY, Huang S, Lipscomb WN (1994) Toward a mechanism for the allosteric transition of pig kidney fructose-1,6-bisphosphatase. J Mol Biol 244(5):609–624PubMedCrossRefGoogle Scholar
  34. 34.
    Ke HM, Liang JY, Zhang YP, Lipscomb WN (1991) Conformational transition of fructose-1,6-bisphosphatase: structure comparison between the AMP complex (T form) and the fructose 6-phosphate complex (R form). Biochemistry 30(18):4412–4420PubMedCrossRefGoogle Scholar
  35. 35.
    Nelson SW, Choe JY, Honzatko RB, Fromm HJ (2000) Mutations in the hinge of a dynamic loop broadly influence functional properties of fructose-1,6-bisphosphatase. J Biol Chem 275(39):29986–29992PubMedCrossRefGoogle Scholar
  36. 36.
    Nelson SW, Honzatko RB, Fromm HJ (2002) Hybrid tetramers of porcine liver fructose-1,6-bisphosphatase reveal multiple pathways of allosteric inhibition. J Biol Chem 277(18):15539–15545PubMedCrossRefGoogle Scholar
  37. 37.
    Skalecki K, Rakus D, Wisniewski JR, Kolodziej J, Dzugaj A (1999) cDNA sequence and kinetic properties of human lung fructose(1, 6)bisphosphatase. Arch Biochem Biophys 365(1):1–9PubMedCrossRefGoogle Scholar
  38. 38.
    Wang X, Chen W, Hu F, Deng C, Zhou C, Lv X, Fan Y, Men J, Huang Y, Sun J, Hu D, Chen J, Yang Y, Liang C, Zheng H, Hu X, Xu J, Wu Z, Yu X (2011) Clonorchis sinensis enolase: identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products. Mol Biochem Parasitol 177(2):135–142PubMedCrossRefGoogle Scholar
  39. 39.
    Saadat I, Higashi H, Obuse C, Umeda M, Murata-Kamiya N, Saito Y, Lu H, Ohnishi N, Azuma T, Suzuki A, Ohno S, Hatakeyama M (2007) Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature 447(7142):330–333PubMedCrossRefGoogle Scholar
  40. 40.
    Suttiprapa S, Loukas A, Laha T, Wongkham S, Kaewkes S, Gaze S, Brindley PJ, Sripa B (2008) Characterization of the antioxidant enzyme, thioredoxin peroxidase, from the carcinogenic human liver fluke, Opisthorchis viverrini. Mol Biochem Parasitol 160(2):116–122PubMedCrossRefGoogle Scholar
  41. 41.
    Smout MJ, Laha T, Mulvenna J, Sripa B, Suttiprapa S, Jones A, Brindley PJ, Loukas A (2009) A granulin-like growth factor secreted by the carcinogenic liver fluke, Opisthorchis viverrini, promotes proliferation of host cells. PLoS Pathog 5(10):e1000611PubMedCrossRefGoogle Scholar
  42. 42.
    Morris MJ, Craig SJ, Sutherland TM, Board PG, Casarotto MG (2009) Transport of glutathione transferase-fold structured proteins into living cells. Biochim Biophys Acta 1788(3):676–685PubMedCrossRefGoogle Scholar
  43. 43.
    Friedman SL (2008) Mechanisms of hepatic fibrogenesis. Gastroenterology 134(6):1655–1669PubMedCrossRefGoogle Scholar
  44. 44.
    Friedman SL (2004) Mechanisms of disease: mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol 1(2):98–105PubMedCrossRefGoogle Scholar
  45. 45.
    Marcos LA, Terashima A, Yi P, Andrade R, Cubero FJ, Albanis E, Gotuzzo E, Espinoza JR, Friedman SL (2011) Mechanisms of liver fibrosis associated with experimental Fasciola hepatica infection: roles of Fas2 proteinase and hepatic stellate cell activation. J Parasitol 97(1):82–87PubMedCrossRefGoogle Scholar
  46. 46.
    Lightowlers MW, Rickard MD (1988) Excretory-secretory products of helminth parasites: effects on host immune responses. Parasitology 96(Suppl):123–166CrossRefGoogle Scholar
  47. 47.
    Lv X, Chen W, Wang X, Li X, Sun J, Deng C, Men J, Tian Y, Zhou C, Lei H, Liang C, Yu X (2012) Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis. Parasitol Res 110(6):2211–2219PubMedCrossRefGoogle Scholar
  48. 48.
    Nam JH, Moon JH, Kim IK, Lee MR, Hong SJ, Ahn JH, Chung JW, Pak JH (2012) Free radicals enzymatically triggered by Clonorchis sinensis excretory-secretory products cause NF-kappaB-mediated inflammation in human cholangiocarcinoma cells. Int J Parasitol 42(1):103–113PubMedCrossRefGoogle Scholar
  49. 49.
    Hui AY, Friedman SL (2003) Molecular basis of hepatic fibrosis. Expert Rev Mol Med 5(5):1–23PubMedCrossRefGoogle Scholar
  50. 50.
    Gizak A, Pirog M, Rakus D (2012) Muscle FBPase binds to cardiomyocyte mitochondria under glycogen synthase kinase-3 inhibition or elevation of cellular Ca2+ level. FEBS Lett 586(1):13–19PubMedCrossRefGoogle Scholar
  51. 51.
    Gizak A, Majkowski M, Dus D, Dzugaj A (2004) Calcium inhibits muscle FBPase and affects its intracellular localization in cardiomyocytes. FEBS Lett 576(3):445–448PubMedCrossRefGoogle Scholar
  52. 52.
    Gizak A, Dzugaj A (2003) FBPase is in the nuclei of cardiomyocytes. FEBS Lett 539(1–3):51–55PubMedCrossRefGoogle Scholar
  53. 53.
    Gizak A, Zarzycki M, Rakus D (2009) Nuclear targeting of FBPase in HL-1 cells is controlled by beta-1 adrenergic receptor-activated Gs protein signaling cascade. Biochim Biophys Acta 1793(5):871–877PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Pei Liang
    • 1
    • 2
  • Jiufeng Sun
    • 3
  • Yan Huang
    • 1
    • 2
  • Fan Zhang
    • 1
    • 2
  • Juanjuan Zhou
    • 1
    • 2
  • Yue Hu
    • 1
    • 2
  • Xiaoyun Wang
    • 1
    • 2
  • Chi Liang
    • 1
    • 2
  • Minghui Zheng
    • 4
  • Yanquan Xu
    • 1
    • 2
  • Qiang Mao
    • 1
    • 2
  • Xuchu Hu
    • 1
    • 2
  • Xuerong Li
    • 1
    • 2
  • Jin Xu
    • 1
    • 2
  • Gang Lu
    • 5
  • Xinbing Yu
    • 1
    • 2
  1. 1.Department of Parasitology, Zhongshan School of MedicineSun Yat-sen UniversityGuangzhouChina
  2. 2.Key Laboratory for Tropical Diseases Control, Ministry of EducationSun Yat-sen UniversityGuangzhouChina
  3. 3.Guangdong Provincial Institute of Public HealthGuangdong Provincial Center for Disease Control and PreventionGuangzhouChina
  4. 4.Sun Yat-Sen Memorial HospitalSun Yat-Sen UniversityGuangzhouChina
  5. 5.Department of Pathogen BiologyHainan Medical CollegeHaikouChina

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