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Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis

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Abstract

Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560 bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0 %), Trypanosoma cruzi (18.0 %), Mus musculus (19.3 %), and relatively high homology with Schistosoma mansoni (65.6 %). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37 °C and pH 7.5. The K m and V max values of rCsLAP2 were 18.2 μM and 10.7 μM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.

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Acknowledgments

This work is supported by grants from the National Basic Research Program of China (973 program, No. 2010CB530000), and science and technology projects of Guangzhou (10A31021473).

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Authors and Affiliations

  1. Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, Guangdong, 510080, China

    Chuanhuan Deng, Jiufeng Sun, Xuerong Li, Lexun Wang, Xuchu Hu, Xiaoyun Wang, Wenjun Chen, Xiaoli Lv, Chi Liang, Wenfang Li, Yan Huang, Ran Li, Zhongdao Wu, Xinbing Yu & Jin Xu

  2. Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, 510080, China

    Chuanhuan Deng, Jiufeng Sun, Xuerong Li, Lexun Wang, Xuchu Hu, Xiaoyun Wang, Wenjun Chen, Xiaoli Lv, Chi Liang, Wenfang Li, Yan Huang, Ran Li, Zhongdao Wu, Xinbing Yu & Jin Xu

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  1. Chuanhuan Deng
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  2. Jiufeng Sun
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Correspondence to Jin Xu.

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Deng, C., Sun, J., Li, X. et al. Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis . Mol Biol Rep 39, 9817–9826 (2012). https://doi.org/10.1007/s11033-012-1848-9

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