Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
권호 표지
DOI QR코드

DOI QR Code

Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis

  • Kang, Jung-Mi (Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine) ;
  • Yoo, Won-Gi (Department of Medical Environmental Biology, Chung-Ang University College of Medicine) ;
  • Le, Huong Giang (Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine) ;
  • Thai, Thi Lam (Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine) ;
  • Hong, Sung-Jong (Department of Medical Environmental Biology, Chung-Ang University College of Medicine) ;
  • Sohn, Woon-Mok (Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine) ;
  • Na, Byoung-Kuk (Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine)
  • Received : 2019.09.01
  • Accepted : 2019.11.12
  • Published : 2019.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

Keywords

References

  1. Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, Fang YY. Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis 2005; 5: 31-41. https://doi.org/10.1016/S1473-3099(04)01252-6
  2. Choi BI, Han JK, Hong ST, Lee KH. Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis. Clin Microbiol Rev 2004; 17: 540-552. https://doi.org/10.1128/CMR.17.3.540-552.2004
  3. Vennervald BJ, Polman K. Helminths and malignancy. Parasite Immunol 2009; 31: 686-696. https://doi.org/10.1111/j.1365-3024.2009.01163.x
  4. Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V. A review of human carcinogens-Part B: biological agents. Lancet Oncol 2009; 10: 321-322. https://doi.org/10.1016/S1470-2045(09)70096-8
  5. Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 2018; 46: 624-632.
  6. Hwang KP, Chang SH, Wang LC. Alterations in the expression level of a putative aspartic protease in the development of Angiostrongylus cantonensis. Acta Trop 2010; 113: 289-294. https://doi.org/10.1016/j.actatropica.2009.11.008
  7. Szecsi PB. The aspartic proteases. Scand J Clin Lab Invest Suppl 1992; 210: 5-22. https://doi.org/10.1080/00365519209104650
  8. Brinkworth RI, Prociv P, Loukas A, Brindley PJ. Hemoglobin-degrading, aspartic proteases of blood-feeding parasites: substrate specificity revealed by homology models. J Biol Chem 2001; 276: 38844-38851. https://doi.org/10.1074/jbc.M101934200
  9. Li J, Chi Z, Liu Z, Yue L, Peng Y, Wang L. Cloning and characterization of a novel aspartic protease gene from marine-derived Metschnikowia reukaufii and its expression in E. coli. Appl Biochem Biotechnol 2009; 159: 119-132. https://doi.org/10.1007/s12010-008-8400-3
  10. Williamson AL, Brindley PJ, Loukas A. Hookworm cathepsin D aspartic proteases: contributing roles in the host-specific degradation of serum proteins and skin macromolecules. Parasitology 2003; 126: 179-185. https://doi.org/10.1017/S0031182002002706
  11. Suttiprapa S, Mulvenna J, Huong NT, Pearson MS, Brindley PJ, Laha T, Wongkham S, Kaewkes S, Sripa B, Loukas A. Ov-APR-1, an aspartic protease from the carcinogenic liver fluke, Opisthorchis viverrini: functional expression, immunolocalization and subsite specificity. Int J Biochem Cell Biol 2009; 41: 1148-1156. https://doi.org/10.1016/j.biocel.2008.10.013
  12. Verity CK, McManus DP, Brindley PJ. Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum. Parasite Immunol 2001; 23: 153-162. https://doi.org/10.1046/j.1365-3024.2001.00369.x
  13. Na BK, Kang JM, Sohn WM. CsCF-6, a novel cathepsin F-like cysteine protease for nutrient uptake of Clonorchis sinensis. Int J Parasitol 2008; 38: 493-502. https://doi.org/10.1016/j.ijpara.2007.09.001
  14. Kang JM, Bahk YY, Cho PY, Hong SJ, Kim TS, Sohn WM, Na BK. A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite. Mol Biochem Parasitol 2010; 170: 7-16. https://doi.org/10.1016/j.molbiopara.2009.11.006
  15. Kang JM, Lee KH, Sohn WM, Na BK. Identification and functional characterization of CsStefin-1, a cysteine protease inhibitor of Clonorchis sinensis. Mol Biochem Parasitol 2011; 177: 126-134. https://doi.org/10.1016/j.molbiopara.2011.02.010
  16. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011; 8: 785-786. https://doi.org/10.1038/nmeth.1701
  17. Kang JM, Ju HL, Sohn WM, Na BK. Defining the regulatory and inhibitory elements within the prodomain of CsCF-6, a cathepsin F cysteine protease of Clonorchis sinensis. Mol Biochem Parasitol 2013; 190: 92-96. https://doi.org/10.1016/j.molbiopara.2013.07.001
  18. Krieger E, Nabuurs SB, Vriend G. Homology modeling. Methods Biochem Anal 2003; 44: 509-523.
  19. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25: 3389-3402. https://doi.org/10.1093/nar/25.17.3389
  20. Bernstein FC, Koetzle TF, Williams GJ, Meyer EF, Jr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M. The Protein Data Bank. A computer-based archival file for macromolecular structures. Eur J Biochem 1977; 80: 319-324. https://doi.org/10.1111/j.1432-1033.1977.tb11885.x
  21. Hanova I, Brynda J, Houstecka R, Alam N, Sojka D, Kopacek P, Maresova L, Vondrasek J, Horn M, Schueler-Furman O, Mares M. Novel structural mechanism of allosteric regulation of aspartic peptidases via an evolutionarily conserved exosite. Cell Chem Biol 2018; 25: 318-329. https://doi.org/10.1016/j.chembiol.2018.01.001
  22. Gradler U, Czodrowski P, Tsaklakidis C, Klein M, Werkmann D, Lindemann S, Maskos K, Leuthner B. Structure-based optimization of non-peptidic Cathepsin D inhibitors. Bioorg Med Chem Lett 2014; 24: 4141-4150. https://doi.org/10.1016/j.bmcl.2014.07.054
  23. Krieger E, Joo K, Lee J, Lee J, Raman S, Thompson J, Tyka M, Baker D, Karplus K. Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8. Proteins 2009; 77 (suppl): 114-122. https://doi.org/10.1002/prot.22570
  24. Lee H, Park H, Ko J, Seok C. GalaxyGemini: a web server for protein homo-oligomer structure prediction based on similarity. Bioinformatics 2013; 29: 1078-1080. https://doi.org/10.1093/bioinformatics/btt079
  25. Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol 2007; 372: 774-797. https://doi.org/10.1016/j.jmb.2007.05.022
  26. Huang CC, Meng EC, Morris JH, Pettersen EF, Ferrin TE. Enhancing UCSF Chimera through web services. Nucleic Acids Res 2014; 42: 478-484.
  27. Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 2014; 42: 320-324.
  28. Dalton JP, Neill SO, Stack C, Collins P, Walshe A, Sekiya M, Doyle S, Mulcahy G, Hoyle D, Khaznadji E, Moire N, Brennan G, Mousley A, Kreshchenko N, Maule AG, Donnelly SM. Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines. Int J Parasitol 2003; 33: 1173-1181. https://doi.org/10.1016/S0020-7519(03)00171-1
  29. Sajid M, McKerrow JH. Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 2002; 120: 1-21. https://doi.org/10.1016/S0166-6851(01)00438-8
  30. Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvorak J, Hsieh I, Bahgat M, Dissous C, McKerrow JH. A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. J Biol Chem 2006; 281: 39316-39329. https://doi.org/10.1074/jbc.M607128200
  31. Horn M, Fajtova P, Rojo Arreola L, Ulrychova L, Bartosova-Sojkova P, Franta Z, Protasio AV, Opavsky D, Vondrasek J, McKerrow JH, Mares M, Caffrey CR, Dvorak J. Trypsin- and Chymotrypsin-like serine proteases in schistosoma mansoni-- 'the undiscovered country'. PLoS Negl Trop Dis 2014; 8: e2766. https://doi.org/10.1371/journal.pntd.0002766
  32. Yoo WG, Kim DW, Ju JW, Cho PY, Kim TI, Cho SH, Choi SH, Park HS, Kim TS, Hong SJ. Developmental transcriptomic features of the carcinogenic liver fluke, Clonorchis sinensis. PLoS Negl Trop Dis 2011; 5: e1208. https://doi.org/10.1371/journal.pntd.0001208
  33. Young ND, Campbell BE, Hall RS, Jex AR, Cantacessi C, Laha T, Sohn WM, Sripa B, Loukas A, Brindley PJ, Gasser RB. Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini. PLoS Negl Trop Dis 2010; 4: e719. https://doi.org/10.1371/journal.pntd.0000719
  34. Cho PY, Lee MJ, Kim TI, Kang SY, Hong SJ. Expressed sequence tag analysis of adult Clonorchis sinensis, the Chinese liver fluke. Parasitol Res 2006; 99: 602-608. https://doi.org/10.1007/s00436-006-0204-1
  35. Li Y, Huang Y, Hu X, Liu X, Ma C, Zhao J, Wu Z, Xu J, Yu X. 41.5-kDa Cathepsin L protease from Clonorchis sinensis: expression, characterization, and serological reactivity of one excretory-secretory antigen. Parasitol Res 2012; 111: 673-680. https://doi.org/10.1007/s00436-012-2885-y
  36. Kang JM, Ju HL, Ju JW, Sohn WM, Kim TS, Bahk YY, Hong SJ, Na BK. Comparative biochemical and functional properties of two leucine aminopeptidases of Clonorchis sinensis. Mol Biochem Parasitol 2012; 182: 17-26. https://doi.org/10.1016/j.molbiopara.2011.11.009
  37. Kang JM, Lee J, Ju HL, Ju JW, Kim JH, Pak JH, Kim TS, Hong Y, Sohn WM, Na BK. Characterization of a gut-associated asparaginyl endopeptidase of Clonorchis sinensis. Exp Parasitol 2015; 153: 81-90. https://doi.org/10.1016/j.exppara.2015.03.015
  38. Metcalf P, Fusek M. Two crystal structures for cathepsin D: the lysosomal targeting signal and active site. EMBO J 1993; 12: 1293-1302. https://doi.org/10.1002/j.1460-2075.1993.tb05774.x
  39. Lapatto R, Blundell T, Hemmings A, Overington J, Wilderspin A, Wood S, Merson JR, Whittle PJ, Danley DE, Geoghegan KF, Hawrylik SJ, Lee SE, Scheld KG, Hobart PM. X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature 1989; 342: 299-302. https://doi.org/10.1038/342299a0

Cited by

  1. Identification and Analysis of the Tegument Protein and Excretory-Secretory Products of the Carcinogenic Liver Fluke Clonorchis sinensis vol.11, 2019, https://doi.org/10.3389/fmicb.2020.555730
  2. Dopaminergic antagonists inhibit bile chemotaxis of adult Clonorchis sinensis and its egg production vol.14, pp.3, 2019, https://doi.org/10.1371/journal.pntd.0008220
  3. pH-Dependent Structural Dynamics of Cathepsin D-Family Aspartic Peptidase of Clonorchis sinensis vol.10, pp.9, 2019, https://doi.org/10.3390/pathogens10091128