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Molecular and immunological characterization of cathepsin L-like cysteine protease of Paragonimus pseudoheterotremus

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Abstract

Cathepsin L is a cysteine protease belonging to the papain family. In parasitic trematodes, cathepsin L plays essential roles in parasite survival and host–parasite interactions. In this study, cathepsin L of the lung fluke Paragonimus pseudoheterotremus (PpsCatL) was identified and its molecular biological and immunological features characterized. A sequence analysis of PpsCatL showed that the gene encodes a 325-amino-acid protein that is most similar to P. westermani cathepsin L. The in silico three-dimensional structure suggests that PpsCatL is a pro-enzyme that becomes active when the propeptide is cleaved. A recombinant pro-PpsCatL lacking the signal peptide (rPpsCatL), with a molecular weight of 35 kDa, was expressed in E. coli and reacted with P. pseudoheterotremus-infected rat sera. The native protein was detected in crude worm antigens and excretory–secretory products and was localized in the cecum and in the lamellae along the intestinal tract of the adult parasite. Enzymatic activity of rPpsCatL showed that the protein could cleave the fluorogenic substrate Z-Phe-Arg-AMC after autocatalysis but was inhibited with E64. The immunodiagnostic potential of the recombinant protein was evaluated with an enzyme-linked immunosorbent assay (ELISA) and suggested that rPpsCatL can detect paragonimiasis with high sensitivity and specificity (100 and 95.6 %, respectively). This supports the further development of an rPpsCatL-ELISA as an immunodiagnostic tool.

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Acknowledgments

This study was supported by grant from Thailand Research Fund (TRF) in the program of initiative career development through Dr. Tippayarat Yoonuan (TRG5480011) and also partially supported by ICTM grant of the Faculty of Tropical Medicine. We thank Jaruchat Boonnachot from Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University for tissue processing in immunolocalization. Our gratitude also goes to the Faculty of Tropical Medicine for funding the proofreading, editing, and page charging of this manuscript.

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  1. Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, 10400, Thailand

    Tippayarat Yoonuan, Supaporn Nuamtanong, Paron Dekumyoy, Orawan Phuphisut & Poom Adisakwattana

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  1. Tippayarat Yoonuan
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  2. Supaporn Nuamtanong
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  3. Paron Dekumyoy
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  4. Orawan Phuphisut
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  5. Poom Adisakwattana
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Correspondence to Poom Adisakwattana.

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All animal works were conducted with the approval of the Faculty of Tropical Medicine Animal Care and Use Committee (no. FTM-ACUC 011/2012). Leftover helminth-infected human sera and healthy sera were used in this study with the permission of the Immunodiagnosis for Helminthiasis Unit, Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, and the approval of the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (no. MUTM 2011-056-01).

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Yoonuan, T., Nuamtanong, S., Dekumyoy, P. et al. Molecular and immunological characterization of cathepsin L-like cysteine protease of Paragonimus pseudoheterotremus . Parasitol Res 115, 4457–4470 (2016). https://doi.org/10.1007/s00436-016-5232-x

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