Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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Identification of the protease inhibitory domain of Trichinella spiralis novel cystatin (TsCstN)

  • Thassanee Yuthithum (Department of Helminthology, Faculty of Tropical Medicine, Mahidol University) ;
  • Orawan Phuphisut (Department of Helminthology, Faculty of Tropical Medicine, Mahidol University) ;
  • Onrapak Reamtong (Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University) ;
  • Nathamon Kosoltanapiwat (Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University) ;
  • Salisa Chaimon (Department of Preclinical Science, Faculty of Medicine, Thammasat University) ;
  • Porntida Kobpornchai (Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University) ;
  • Charin Thawornkuno (Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University) ;
  • Preeyarat Malaithong (Department of Helminthology, Faculty of Tropical Medicine, Mahidol University) ;
  • Orathai Sawatdichaikul (Institute of Food Research and Product Development, Kasetsart University) ;
  • Poom Adisakwattana (Department of Helminthology, Faculty of Tropical Medicine, Mahidol University)
  • Received : 2024.03.28
  • Accepted : 2024.07.03
  • Published : 2024.08.31
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Abstract

The Trichinella spiralis novel cystatin (TsCstN) inhibits cathepsin L (CatL) activity and inflammation of macrophages during lipopolysaccharide (LPS) induction. To identify the protease inhibitory region, this study applied an in silico modeling approach to simulate truncation sites of TsCstN (Ts01), which created four truncated forms, including TsCstN∆1-39 (Ts02), TsCstN∆1-71 (Ts03), TsCstN∆1-20, ∆73-117 (Ts04), and TsCstN∆1-20, ∆42-117 (Ts05). The superimposition of these truncates modeled with AlphaFold Colab indicated that their structures were more akin to Ts01 than those modeled with I-TASSER. Moreover, Ts04 exhibited the closest resemblance to the structure of Ts01. The recombinant Ts01 (rTs01) and truncated proteins (rTs02, rTs03, and rTs04) were successfully expressed in a prokaryotic expression system while Ts05 was synthesized, with sizes of approximately 14, 12, 8, 10, and 2.5 kDa, respectively. When determining the inhibition of CatL activity, both rTs01 and rTs04 effectively reduced CatL activity in vitro. Thus, the combination of the α1 and L1 regions may be sufficient to inhibit CatL. This study provides comprehensive insights into TsCstN, particularly regarding its protein function and inhibitory domains against CatL.

Keywords

Acknowledgement

This research project was supported by Mahidol University under the New Discovery and Frontier Research grant for Fiscal Year 2021 through Poom Adisakwattana (NDFR 03/2564).

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