Identification and characteristics of a cathepsin L-like cysteine protease from Clonorchis sinensis

  • Changling Ma
  • Kai Liang
  • Lili Tang
  • Shanshan He
  • Xiaoquan Liu
  • Mian He
  • Yanwen LiEmail author
Helminthology - Original Paper


Cathepsin L-like protease is an important member of the papain-like cysteine protease and plays numerous indispensable roles in the biology of parasitic organisms. In a previous study, we identified a gene encoding a cathepsin L-like protease of Clonorchis sinensis (CsCPL) that was detected in the cercaria, metacercaria, and adult worm stages by immunolocalization, suggesting that this cysteine protease may be important and involved in the development of C. sinensis. In this study, the mature domain of CsCPL (CsCPL-m) was cloned and expressed in the form of inclusion bodies in Escherichia coli. After refolding, the recombinant CsCPL-m displayed optimal protease activity towards Z-Phe-Arg-AMC substrates but not towards Z-Arg-Arg-AMC, and the activity of the protease was inhibited completely by the cysteine protease-specific inhibitors E-64 and IAA, which further demonstrated that CsCPL belongs to the cathepsin L-like cysteine protease family. Recombinant CsCPL-m exhibited considerable activity at temperatures ranging from 28 to 42 °C, with the highest activity observed at 42 °C. Furthermore, recombinant CsCPL-m exhibited activity across a broad range of pH values (pH 4.0–8.0), with an optimal pH of 5.5. The Km and Vmax of the recombinant CsCPL-m towards Z-Phe-Arg-AMC were determined to be 5.71 × 10−6 M and 0.6 μM/min, respectively, at 37 °C and pH 5.5. The recombinant CsCPL-m could degrade BSA and gelatine, but could not degrade human hemoglobin and human immunoglobulin G. These results implied that CsCPL might participate in the catabolism of host proteins for nutrition during the parasitic life cycle of C. sinensis; thus, CsCPL could be used as a potential vaccine antigen and drug target against C. sinensis infection.


Clonorchis sinensis Cathepsin L-like cysteine protease Enzymatic characteristics 



This work was supported by the Natural Science Foundation of Guangxi (No. 2013GXNSFAA019158) and the Science and Technology Research Project of Guangxi Educational Committee (No.2013YB046) China.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflicts of interest.


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© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Pathogen Biology & ImmunologyGuangzhou Medical UniversityGuangzhouPeople’s Republic of China
  2. 2.Department of ParasitologyGuangxi Medical UniversityNanningPeople’s Republic of China

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