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Characterization of cysteine proteases from the carcinogenic liver fluke, Opisthorchis viverrini

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Abstract

Protease activities in extracts of Opisthorchis viverrini were investigated using gelatin zymography and fluorogenic peptide substrates. Using gelatin-impregnated X-ray film, 2 μg of O. viverrini excretory–secretory products (Ov-ES) and adult somatic extract (Ov-SE) showed proteolytic activity. Zymography of both O. viverrini extracts revealed bands at ~30 kDa. Using fluorogenic peptide substrates, the majority of O. viverrini activity was determined to be cathepsin L-like cysteine protease (cleaved Z–Phe–Arg–aminomethylcoumarin (AMC)) whereas little or no activity was ascribable to other classes of proteases. The O. viverrini cysteine protease activity was greatest at pH 6.0 and the activity was inhibited by the class-specific inhibitors, E-64 and Z–Ala–CHN2. Chromatographic purification of O. viverrini cysteine proteases on thiol-sepharose enriched for protein(s) of ~30 kDa from Ov-ES and Ov-SE. The activity profile of the purified enzyme was similar to that of the cathepsin L-like activity characterized in Ov-SE and Ov-ES. Furthermore, determination of cysteine protease activity in several developmental stages of the parasite revealed the highest protease activity in metacercariae soluble extract, followed by Ov-ES, egg soluble extract, and Ov-SE. These findings demonstrated that O. viverrini has a cathepsin L-like cysteine protease(s) and suggested that abundant cysteine protease activity was present in metacercariae where the hydrolase might be involved in cyst excystation during mammalian infection.

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Acknowledgements

This work was supported by the Thailand Tropical Diseases Research Programme (T-2, grant number ID 02-2-HEL-05-054), National Institute of Health–National Institute of Allergy and Infectious Diseases, International Collaboration in Infectious Disease Research award number AI065871 and Graduate School, Khon Kaen University.

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Authors and Affiliations

  1. Graduate School, Khon Kaen University, Khon Kaen, Thailand

    Natthawut Kaewpitoon

  2. College of Medicine and Public Health, Ubon Rajathanee University, Ubon Ratchathani, Thailand

    Natthawut Kaewpitoon

  3. Department of Parasitology, Khon Kaen University, Khon Kaen, Thailand

    Thewarach Laha & Sasithorn Kaewkes

  4. Department Biochemistry, Khon Kaen University, Khon Kaen, Thailand

    Puangrat Yongvanit

  5. Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand

    Banchob Sripa

  6. Department of Tropical Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA

    Paul J. Brindley

  7. Queensland Institute of Medical Research, Brisbane, Queensland, Australia

    Alex Loukas

  8. Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand

    Sasithorn Kaewkes, Puangrat Yongvanit & Banchob Sripa

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  1. Natthawut Kaewpitoon
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  2. Thewarach Laha
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Correspondence to Banchob Sripa.

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Kaewpitoon, N., Laha, T., Kaewkes, S. et al. Characterization of cysteine proteases from the carcinogenic liver fluke, Opisthorchis viverrini . Parasitol Res 102, 757–764 (2008). https://doi.org/10.1007/s00436-007-0831-1

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  • DOI: https://doi.org/10.1007/s00436-007-0831-1

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