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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]

Clonorchiasis is a common zoonosis in southern and northeastern parts of China, especially in Guangdong, Guangxi and Jilin province. Anti- Clonorchis sinensis antibody detection by enzyme-linked immunosorbent assay (ELISA) has been used for epidemiological surveys of clonorchiasis for its convenience and celerity, but it is still a meaningful work to screen ideal diagnostic antigen or antibody subtype for improvement of diagnostic sensitivity and specificity and for judgement of curative effect. In the present study, recombinant CsCatL-propeptide (rCsCatL-propeptide) was highly expressed in form of inclusion body in Escherichia coli. Soluble rCsCatL-propeptide with high purity were obtained after purification in denatured condition by using His Bind Purification kit, and then renatured. The major antibody subtypes responding to rCsCatL-propeptide in sera from clonorchiasis patients were IgG1 and IgG4, but the level of IgG4 was more predominant ( P < 0.05). The sensitivity of specific IgG4 detection (91.7%) was statistically significantly higher than that of IgG1 (25.0%) with rCsCatL-propeptide ( P < 0.01). The specificities of IgG1 and IgG4 detection with rCsCatL-propeptide were 83.3% and 88.5%, respectively, and the difference between them was not statistically significant ( P > 0.05). Cross-reactions took place when we detected IgG1 of sera from patients infected with Schistosoma japonicum, Paragonimus westermani, hookworm, Trichuris trichiura and Ascaris lumbricoides with rCsCatL-propeptide, while cross-reactions only took place in sera from patients infected with S. japonicum and P. westermani when we detected specific IgG4. The positive rate of IgG4 detection in sera from clonorchiasis patients with <1,000, 1,000-4,999, 5,000-9,999, and ≥10,000 eggs per gram faeces (EPG) were 76.9%, 89.3%, 95.6%, and 100.0%, respectively. The positive rates of serodiagnosis correlated well with the EPG ( r = 0.93). Overall, rCsCatL-propeptide is a valuable candidate for specific IgG4 detection in sera from clonorchiasis patients by the method of ELISA for its few cross-reaction and acceptable sensitivity. In addition, specific IgG4 detection can be used to valuate infected degree and therapeutic effect of clonorchiasis patients. [ABSTRACT FROM AUTHOR]

Cathepsin L of parasite plays multiple roles in growth, food uptake, and invasion into host and pathogenesis, which makes it a valuable target for diagnosis, vaccine, and drug. In this study, we identified a cDNA encoding cathepsin L homolog (CsCPL) from the library of Clonorchis sinensis adult by bioinformatics analysis. Sequence encoding proenzyme of CsCPL (removal of signal peptide, CsproCPL) was highly expressed in form of inclusion body in Escherichia coli, and soluble rCsproCPL (about 1 mg/ml) in high purity were obtained after denaturation, purification, and renaturation. Western blot analysis indicated that CsCPL is a component of excretory-secretory products of adult, in mature form of protease. Reverse transcription polymerase chain reaction showed that CsCPL is also expressed in metacercaria and cercaria stage. Immunolocalization demonstrated that CsCPL is deposited at adult intestine, or tegument, and tegumentary cell of metacercaria and cercaria (especially at dorsal tegument of cercaria), indicating different secretory routine roles in adult and larva. The characteristics of CsCPL suggested that it may involve in invasion of cercaria into fish and development to metacercaria, excystment of metacercaria, and protein digestion of adult, which may render it a candidate antigen for fish vaccine and serodiagnosis of human clonorchiasis.