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Proteomic analysis of different period excretory secretory products from Clonorchis sinensis adult worms: molecular characterization, immunolocalization, and serological reactivity of two excretory secretory antigens—methionine aminopeptidase 2 and acid phosphatase

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Abstract

The excretory secretory products (ESP) of Clonorchis sinensis are the causative agents of clonorchiasis and biliary diseases. The parasites’ ESP play important roles in host–parasite interactions. The protein compositions of ESP at different secretory times are different and have not been systemically investigated so far. In this study, we collected ESP from six different periods (0–3 h, 3–6 h, 6–12 h, 12–24 h, 24–36 h, and 36–48 h) from C. sinensis adults. Using a shotgun LC–MS/MS analysis, we found 187, 80, 103, 58, 248, and 383 proteins, respectively. Among these proteins, we selected methionine aminopeptidase 2 (MAP-2, presented in 24–36 h and 36–48 h ESP) and acid phosphatase (AP, presented in 3–6 h, 12–24 h, 24–36 h, and 36–48 h ESP) for further study. Bioinformatics analysis showed that CsMAP-2 has metallopeptidase family M24, unique lysine residue-rich and acidic residue-rich domain, SGTS motif, and auto-cleavage point; and that CsAP has possible signal sequence cleavage site, acid phosphate domain, and two histidine acid phosphatases active regions. CsMAP-2 and CsAP’s cDNA have 1,425 bp and1,410 bp ORF, encoding 475 and 470 amino acid proteins and weighing 55.3840 kDa and 55.2875 kDa, respectively. MAP-2 and AP were identified as antigens present in the ESP and circulating antigens by immunoblot analysis, which were also found expressing in the eggs, metacercaria, and adult stages of C. sinensis. Immunofluorescence analysis showed that they were located in tegument and intestinal cecum of adult. MTT assay showed that they could inhibit hepatic stellate cell line (LX-2) proliferation. These findings presented the compositions of different period excretory secretary products from C. sinensis adults.

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Acknowledgments

This work was supported by the National Science and Technology Major Project of China (no. 2008ZX10004-011), National Natural Science Foundation of China (no. 30671831 and 81000936), and National High Technology Research and Development Program of China (973 program, no. 2010CB530000).

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Authors and Affiliations

  1. Department of Clinical Laboratory, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, 510120, People’s Republic of China

    Minghui Zheng & Hongyu Li

  2. Department of Parasitology, Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China

    Minghui Zheng, Jingfang Chen & Xinbing Yu

  3. Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China

    Kunhua Hu

  4. Guangdong Provincial Key Laboratory of Liver Disease, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China

    Wei Liu

  5. Changsha Center for Disease Control and Prevention, Changsha, 410001, People’s Republic of China

    Jingfang Chen

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  1. Minghui Zheng
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  2. Kunhua Hu
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  3. Wei Liu
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  4. Hongyu Li
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Correspondence to Xinbing Yu.

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Minghui Zheng, Kunhua Hu, and Wei Liu contributed equally to this work.

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Zheng, M., Hu, K., Liu, W. et al. Proteomic analysis of different period excretory secretory products from Clonorchis sinensis adult worms: molecular characterization, immunolocalization, and serological reactivity of two excretory secretory antigens—methionine aminopeptidase 2 and acid phosphatase. Parasitol Res 112, 1287–1297 (2013). https://doi.org/10.1007/s00436-012-3264-4

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