Skip to main content
SpringerLink
Log in

Identification, immunolocalization, and immunological characterization of nitric oxide synthase-interacting protein from Clonorchis sinensis

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Recently, accumulating evidences indicate that nitric oxide (NO) is a potent mediator with diverse roles in regulating cellular functions, signaling pathways, and variety of pathological processes. In the present study, using data from the published genomic for Clonorchis sinensis (C. sinensis), we investigated a gene encoding nitric oxide synthase-interacting protein (NOSIP) of C. sinensis. Recombinant CsNOSIP (rCsNOSIP) was expressed and purified from Escherichia coli BL21. The open reading frame of CsNOSIP comprises 867 bp which encodes 289 amino acids and shares 72.9, 45.2, 47, 46.4, and 45.8 % identity with NOSIP from Schistosoma mansoni, Xenopus laevis, Rattus norvegicus, Mus musculus, and Homo sapiens, respectively. Bioinformatics analysis suggested that the full-length sequence contains an eNOS-interacting domain and numerous B-cell epitopes. Quantitative RT-PCR indicated that CsNOSIP differentially transcribed throughout the adult worms, metacercariae, and egg stages of C. sinensis, and were highly expressed in the adult worms. Moreover, western blot analysis showed that the rCsNOSIP could be detected by the serum from BALB/c mice infected with C. sinensis and the serum from BALB/c mice immunized with excretory/secretory products (ESPs). Furthermore, immunolocalization assay showed that CsNOSIP was specifically localized in the intestine, vitellarium, and eggs of adult worm. Both immunoblot and immunolocalization results demonstrated that CsNOSIP was one component of ESPs of C. sinensis, which could be supported by SignalP analysis. Moreover, analysis of the antibody subclass and cytokine profile demonstrated that subcutaneously immunized BALB/c mice with rCsNOSIP could significantly enhance serum IgG1 level and up-regulate expression of IL-4 and IL-6 in the splenocytes. Our results suggested that CsNOSIP was an important antigen exposed to host immune system and probably involved in immune regulation of host by inducing Th2-polarized immune response.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Andrade MA, Siles-Lucas M, Espinoza E, Pérez Arellano JL, Gottstein B, Muro A (2004) Echinococcus multilocularis laminated layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro. Nitric Oxide 10(3):150–155

    Article  CAS  PubMed  Google Scholar 

  • Bredt DS, Snyder SH (1994) Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 63:175–195

    Article  CAS  PubMed  Google Scholar 

  • Cai GB, Bae YA, Kim SH, SohnWM LYS, Jiang MS, Kim TS, Kong Y (2008) Vitellocyte-specific expression of phospholipid hydroperoxide glutathione peroxidases in Clonorchis sinensis. Int J Parasitol 38(14):1613–1623

    Article  CAS  PubMed  Google Scholar 

  • Chen D, Chen J, Huang J, Chen X, Feng D, Liang B, Che Y, Liu X, Zhu C, Li X, Shen H (2010) Epidemiological investigation of Clonorchis sinensis infection in freshwater fishes in the Pearl River Delta. Parasitol Res 107(4):835–839

    Article  PubMed  Google Scholar 

  • Dedio J, König P, Wohlfart P, Schroeder C, Kummer W, Müller-Esterl W (2001) NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 15:79–89

    Article  CAS  PubMed  Google Scholar 

  • Duncan MR, Berman B (1991) Stimulation of collagen and glycosaminoglycan production in cultured human adult dermal fibroblasts by recombinant human interleukin 6. J Invest Dermatol 97:686–692

    CAS  PubMed  Google Scholar 

  • Erasmus DA, Popiel I, Shaw JR (1982) A comparative study of the vitelline cell in Schistosoma mansoni, S. haematobium, S. japonicum and S. mattheei. Parasitology 84(Pt 2):283–287

    Article  CAS  PubMed  Google Scholar 

  • Govers R, Oess S (2004) To NO or not to NO: where? is the question. Histol Histopathol 19:585–605

    CAS  PubMed  Google Scholar 

  • Hampl V, Herget J (2000) Role of nitric oxide in the pathogenesis of chronic pulmonary hypertension. Physiol Rev 80:1337–1372

    CAS  PubMed  Google Scholar 

  • Harinasuta T, Riganti M, Bunnag D (1984) Opisthorchis viverrini infection: pathogenesis and clinical features. Arzneimittelforschung 34:1167–1169

    CAS  PubMed  Google Scholar 

  • Hirata M, Hirata K, Kage M, Zhang M, Hara T, Fukuma T (2001) Effect of nitric oxide synthase inhibition on Schistosoma japonicum egg-induced granuloma formation in the mouse liver. Parasite Immunology 23:281–289

    Article  CAS  PubMed  Google Scholar 

  • Hu F, Yu X, Ma C, Zhou H, Zhou Z, Li Y, Lu F, Xu J, Wu Z, Hu X (2007) Clonorchis sinensis: expression, characterization, immunolocalization and serological reactivity of one excretory/secretory antigen-LPAP homologue. Exp Parasitol 117(2):157–164

    Article  CAS  PubMed  Google Scholar 

  • Huang L, Hu Y, Huang Y, Fang H, Li R, Hu D, Li W, Li X, Liang C, Yu X (2011) Gene/protein expression level, immunolocalization and binding characteristics of fatty acid binding protein from Clonorchis sinensis (CsFABP). Mol Cell Biochem 363:367–376

    Article  PubMed  Google Scholar 

  • Huang Y, Li W, Huang L, Hu F, Chen W, Wang X, Sun J, Liang C, Wu Z, Li X, Xu J, Yu X (2012) Identification and characterization of myophilin-like protein: a life stage and tissue-specific antigen of Clonorchis sinensis. Parasitol Res 111:1143–1150

    Article  PubMed  Google Scholar 

  • König P, Dedio J, Oess S, Papadakis T, Fischer A, Müller-Esterl W, Kummer W (2005) NOSIP and its interacting protein, eNOS, in the rat trachea and lung. Journal of Histochemistry & Cytochemistry 53(2):155–164

    Article  Google Scholar 

  • Liang P, Zhang F, Chen W, Hu X, Huang Y, Li S, Ren M, He L, Li R, Li X, Xu J, Wu Z, Lu G, Yu X (2013) Identification and biochemical characterization of adenylate kinase 1 from Clonorchis sinensis. Parasitol Res 112:1719–1727

    Article  PubMed  Google Scholar 

  • Long XC, Bahgat M, Chlichlia K, Ruppel A, Li YL (2004) Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni. J Helminthol 78:47–50

    Article  CAS  PubMed  Google Scholar 

  • Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, Fang YY (2005) Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis 5(1):31–41

    Article  PubMed  Google Scholar 

  • Lv X, Chen W, Wang X, Li X, Sun J, Deng C, Men J, Tian Y, Zhou C, Lei H, Liang C, Yu X (2011) Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis. Parasitol Res 110:2211–2219

    Article  PubMed  Google Scholar 

  • MacDonald AS, Straw AD, Dalton NM, Pearce EJ (2002) Cutting edge: Th2 response induction by dendritic cells: a role for CD40. J Immunol 168:537–540

    Article  CAS  PubMed  Google Scholar 

  • Maizels RM, Yazdanbakhsh M (2003) Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat Rev Immunol 3:733–744

    Article  CAS  PubMed  Google Scholar 

  • Mihara M, Moriya Y, Kishimoto T, Ohsugi Y (1995) Interleukin-6 (IL-6) induces the proliferation of synovial fibroblastic cells in the presence of soluble IL-6 receptor. Br J Rheumatol 34:321–325

    Article  CAS  PubMed  Google Scholar 

  • Nahrevanian H (2009) Involvement of nitric oxide and its up/down stream molecules in the immunity against parasitic infections. Braz J Infect Dis 13(6):440–448

    CAS  PubMed  Google Scholar 

  • Nam JH, Moon JH, Kim IK, Lee MR, Hong SJ, Ahn JH, Chung JW, Pak JH (2012) Free radicals enzymatically triggered by Clonorchis sinensis excretory-secretory products cause NF-kB-mediated inflammation in human cholangiocarcinoma cells. Int J Parasitol 42:103–113

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Prakobwong S, Pinlaor P, Charoensuk L, Khoontawad J, Yongvanit P, Hiraku Y, Pinlaor S (2012) The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases. Parasitol Int 61(1):112–117

    Article  CAS  PubMed  Google Scholar 

  • Rim HJ (2005) Clonorchiasis: an update. J Helminthol 79:269–281

    Article  PubMed  Google Scholar 

  • Rizzo V, McIntosh DP, Oh P, Schnitzer JE (1998) In situ flow activates endothelial nitric oxide synthase in luminal caveolae of endothelium with rapid caveolin dissociation and calmodulin association. J Biol Chem 273:34724–34729

    Article  CAS  PubMed  Google Scholar 

  • Sripa B, Kaewkes S (2000) Localisation of parasite antigens and inflammatory responses in experimental opisthorchiasis. Int J Parasitol 30:735–740

    Article  CAS  PubMed  Google Scholar 

  • Steudel W, Watanabe M, Dikranian K, Jacobson M, Jones RC (1999) Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension. Cell Tissue Res 295:317–329

    Article  CAS  PubMed  Google Scholar 

  • Wang L, Yu X, Yang Y, Chen J, Hu D, Deng C, Yang X, Hu X, Xu J (2010) Molecular characterization and expression of the MYND-ZF gene from Clonorchis sinensis. Parasitol Res 107(5):1249–1255

    Article  PubMed  Google Scholar 

  • Wang X, Chen W, Huang Y, Sun J, Men J, Liu H, Luo F, Guo L, Lv X, Deng C, Zhou C, Fan Y, Li X, Huang L, Hu Y, Liang C, Hu X, Xu J, Yu X (2011a) The draft genome of the carcinogenic human liver fluke Clonorchis sinensis. Genome Biol 12(10):R107

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang X, Chen W, Hu F, Deng C, Zhou C, Lv X, Fan Y, Men J, Huang Y, Sun J, Hu D, Chen J, Yang Y, Liang C, Zheng H, Hu X, Xu J, Wu Z, Yu X (2011b) Clonorchis sinensis enolase: identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products. Mol Biochem Parasitol 177(2):135–142

    Article  CAS  PubMed  Google Scholar 

  • Wang X, ChenW LX, Tian Y, Men J, Zhang X, Lei H, Zhou C, Lu F, Liang C, Hu X, Xu J, Wu Z, Li X, Yu X (2012) Identification and characterization of paramyosin from cyst wall of metacercariae implicated protective efficacy against Clonorchis sinensis infection. PLoS One 7(3):e33703

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Watanapa P, Watanapa WB (2002) Liver fluke-associated cholangiocarcinoma. Br J Surg 89:962–970

    Article  CAS  PubMed  Google Scholar 

  • Xue C, Botkin SJ, Johns RA (1996) Localization of endothelial NOS at the basal microtubule membrane in ciliated epithelium of rat lung. J Histochem Cytochem 44:463–471

    Article  CAS  PubMed  Google Scholar 

  • Yoo WG, Kim TI, Li S, Kwon OS, Cho PY, Kim TS, Kim K, Hong SJ (2009) Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR. Parasitol Res 104(2):321–328

    Article  PubMed  Google Scholar 

  • Young ND, Campbell BE, Hall RS, Jex AR, Cantacessi C, Laha T, Sohn WM, Sripa B, Loukas A, Brindley PJ, Gasser RB (2010) Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini. PLoS Negl Trop Dis 4(6):e719

    Article  PubMed Central  PubMed  Google Scholar 

  • Zahoor Z, Davies AJ, Kirk RS, Rollinson D, Walker AJ (2009) Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway. Parasites & Vectors 2:18

    Article  Google Scholar 

  • Zhan X, Li D, Johns RA (1999) Immunohistochemical evidence for the NO cGMP signaling pathway in respiratory ciliated epithelia of rat. J Histochem Cytochem 47:1369–1374

    Article  CAS  PubMed  Google Scholar 

  • Zhou C, Bian M, Liao H, Mao Q, Li R, Zhou J, Wang X, Li S, Liang C, Li X, Huang Y, Yu X (2013) Identification and immunological characterization of thioredoxin transmembrane-related protein from Clonorchis sinensis. Parasitol Res 112(4):1729–1736

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by National Basic Research and Development Project (973 programs) (No. 2010CB530000), National Natural Science Foundation of China (No. 81101270 and No.81171602.), National Important Sci-Tech Special Projects (No. 2012ZX10004220), and the Fundamental Research Funds for the Central Universities (No. 3164015), Guangdong Natural Science Foundation of China (No. S2012010008504).

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Department of Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China

    Meng Bian, Shan Li, Xiaoyun Wang, Yanquan Xu, Wenjun Chen, Chenhui Zhou, Xueqing Chen, Lei He, Jin Xu, Chi Liang, Zhongdao Wu, Yan Huang, Xuerong Li & Xinbing Yu

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

    Meng Bian, Shan Li, Xiaoyun Wang, Yanquan Xu, Wenjun Chen, Chenhui Zhou, Xueqing Chen, Lei He, Jin Xu, Chi Liang, Zhongdao Wu, Yan Huang, Xuerong Li & Xinbing Yu

  3. School of Nursing, Guangdong Medical College, Dongguan, 523808, People’s Republic of China

    Chenhui Zhou

Authors
  1. Meng Bian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaoyun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yanquan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wenjun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chenhui Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xueqing Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lei He
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jin Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Chi Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Zhongdao Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Xuerong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Xinbing Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xuerong Li or Xinbing Yu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bian, M., Li, S., Wang, X. et al. Identification, immunolocalization, and immunological characterization of nitric oxide synthase-interacting protein from Clonorchis sinensis . Parasitol Res 113, 1749–1757 (2014). https://doi.org/10.1007/s00436-014-3820-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-014-3820-1

Keywords

Search

Navigation