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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. [ABSTRACT FROM AUTHOR]

Cysteine proteases (CPs) were associated with the pathogenicity and excystment of Clonorchis sinensis. Most of them were potential antigens for the immunodiagnosis of clonorchiasis. More researches on CPs will let us know more about their functions, and further employ them for the development of more efficient diagnostic reagent and prevention strategies. In the current study, a full-length sequence encoding cathepsin L from C. sinensis (CsCL41.5) was identified from our adult cDNA library. Bioinformatic analysis showed that CsCL41.5 included typical motifs of cathepsin L (ERFNIN and GNFD motifs) and conserved amino acid positions which constituted the active center of the enzyme. The identity of its amino acid sequence with the cathepsin L of Schistosoma japonicum was 49.6 %. Recombinant CsCL41.5 (rCsCL41.5) was highly expressed in the form of inclusion body in Escherichia coli, and soluble rCsCL41.5 was obtained after purification and renaturation. Western blotting analysis indicated that CsCL41.5 is an excretory-secretory antigen of C. sinensis adult. Immunolocalization demonstrated that CsCL41.5 is distributed in the intestine and eggs in the uterus of adult worm, tegument of metacercaria, oral suck, and tail of cercaria. ELISA assays showed that IgG4 was the predominant IgG isotype responding to rCsCL41.5 in sera from clonorchiasis patients. The sensitivity and specificity of specific IgG4 detection with rCsCL41.5 was 62.5 % (15/24) and 81.7 % (49/60), respectively. It was concluded that there were differences in biological function, efficiency of serodiagnosis, and characterization of immune reactivity between CsCL41.5 and other CPs of C. sinensis, combining with previous studies. [ABSTRACT FROM AUTHOR]