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A novel property of fWap65-2, the warm temperature acclimation-related 65-kDa protein from pufferfish Takifugu rubripes, as an antitrypsin

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  • Chemistry and Biochemistry
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Abstract

Protease inhibitors are widely distributed in the skin mucus of fish. The pufferfish, family Tetraodontidae, has high antitryptic activity among fish. In this study, we isolated and characterized a protease inhibitor from the skin mucus of the pufferfish Takifugu rubripes. The inhibitor termed T. rubripes trypsin inhibitor 2 (TRTI-2) is a serine protease inhibitor and specifically inhibits trypsin with the inhibitory constant (Ki) of 8.6 × 10–8 M. TRTI-2 is an acidic glycoprotein with a molecular mass of 63 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and deglycosylated to 46 kDa by treatment with glycopeptidase. Amino acid sequence and cDNA cloning analyses revealed that mature TRTI-2 comprises 421 amino acid residues with a theoretical molecular mass of 47,349.17 Da and isoelectric point (pI) of 5.47. A BLAST homology search revealed that TRTI-2 is homologous to hemopexin family protein and identical with fWap65-2, the warm temperature acclimation-related 65-kDa protein (Wap65) from T. rubripes. Wap65 shows functional diversification as a mediator of not only temperature acclimation but also inflammatory, antiinflammatory, and immune responses. The present study revealed a novel biochemical property of Wap65 as an antitrypsin, possibly to protect the functional molecule from damage by protease.

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References

  • Aliza D, Ismail I, Kuah M-K, Shu-Chien AC, Muhammad TST (2008) Identification of Wap65, a human homologue of hemopexin as a copper-inducible gene in swordtail fish. Xiphophorus Helleri Fish Physiol Biochem 34:129–138

    Article  CAS  Google Scholar 

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  Google Scholar 

  • Armenteros JJA, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, von Heijne G, Nielsen H (2019) SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol 37:420–423

    Article  Google Scholar 

  • Ascenzi P, Fasano M (2007) Heme-hemopexin: a ‘chronosteric’ heme-protein. IUBMB Life 59:700–708

    Article  CAS  Google Scholar 

  • Cho YS, Kim BS, Kim DS, Nam YK (2012) Modulation of warm-temperature-acclimation-related 65-kDa protein genes (Wap65-1 and Wap65-2) in mud loach (Misgurnus mizolepis, Cypriniformes) liver in response to different stimulatory treatments. Fish Shellfish Immunol 32:662–669

    Article  CAS  Google Scholar 

  • Choi CY, An KW, Choi YK, Jo PG, Min BH (2008) Expression of warm temperature acclimation-related protein 65-kDa (Wap65) mRNA, and physiological changes with increasing water temperature in black porgy, Acanthopagrus schelegeli. J Integr Biol 309A:206–214

    CAS  Google Scholar 

  • Delanghe JR, Langlois MR (2001) Hemopexin: a review of biological aspects and role in laboratory medicine. Clin Chim Acta 312:13–23

    Article  CAS  Google Scholar 

  • Diaz-Rosales P, Pereiro P, Figueras A, Novoa B, Dios S (2014) The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus). Fish Shellfish Immunol 41:80–92

    Article  CAS  Google Scholar 

  • Ellis AE (2001) Innate host defense mechanisms of fish against viruses and bacteria. Dev Comp Immunol 25:827–839

    Article  CAS  Google Scholar 

  • Gaur V, Chanana V, Jain A, Dinakar MS (2011) The structure of a haemopexin-fold protein from cow pea (Vigna unguiculate) suggests functional diversity of haemopexins in plants. Acta Cryst F 67:193–200

    Article  CAS  Google Scholar 

  • Hirayama M, Nakaniwa M, Ikeda D, Hirazawa N, Otaka T, Mitsuboshi T, Shirasu K, Watabe S (2003) Primary structures and gene organizations of two types of Wap65 from the pufferfish Takifugu rubripes. Fish Physiol Biochem 29:211–224

    Article  CAS  Google Scholar 

  • Hirayama M, Kobiyama A, Kinoshita S, Watabe S (2004) The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme. J Exp Biol 207:1387–1398

    Article  CAS  Google Scholar 

  • Jones SRM (2001) The occurrence and mechanisms of innate immunity against parasites in fish. Dev Comp Immunol 25:841–852

    Article  CAS  Google Scholar 

  • Kikuchi K, Watabe S, Suzuki Y, Aida K, Nakajima H (1993) The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish Carassius auratus. J Comp Physiol B 163:349–354

    Article  CAS  Google Scholar 

  • Kikuchi K, Watabe S, Aida K (1997) The Wap65 gene expression of goldfish (Carassius auratus) in association with warm temperature as well as bacterial lipopolysaccharide (LPS). Fish Physiol Biochem 17:423–432

    Article  CAS  Google Scholar 

  • Kim Y-O, Park E-M, Moon JY, Nam B-H, Kim D-G, Kong HJ, Kim W-J, Jee Y-J, Lee S-J (2013) Genetic organization of two types of flounder warm-temperature acclimation-associated 65-kDa protein and their gene expression profiles. Biosci Biotechnol Biochem 77:2065–2072

    Article  CAS  Google Scholar 

  • Kumaresan V, Harikrishman R, Arockiaraj J (2015) A potential Kazal-type serine protease inhibitor involved in kinetics of protease inhibition and bacteriostatic activity. Fish Shellfish Immunol 42:430–438

    Article  CAS  Google Scholar 

  • Kwon G, Ghil S (2018) Identification of warm temperature acclimation-associated 65-kDa protein-2 in Kumgang fat minnow Rhynchocypris kumagangensis. J Exp Zool 327:611–619

    Article  Google Scholar 

  • Machado JP, Vascondelos V, Antunes A (2014) Adaptive functional divergence of the warm temperature acclimation-related protein (WAP65) in fishes and the ortholog hemopexin (HPX) in mammals. J Hered 105:237–252

    Article  CAS  Google Scholar 

  • Matsumoto T, Ishizaki S, Nagashima Y (2011) Differential gene expression profile in the liver of the marine puffer fish Takifugu rubripes induced by intramuscular administration of tetrodotoxin. Toxicon 57:304–310

    Article  CAS  Google Scholar 

  • Nagashima Y, Takeda M, Ohta I, Shimakura K, Shiomi K (2004) Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis. Comp Biochem Physiol B 138:103–110

    Article  Google Scholar 

  • Ohashi H, Umeda N, Hirazawa N, Ozaki Y, Miura C, Miura T (2007) Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae). Intl J Parasitol 37:1483–1490

    Article  CAS  Google Scholar 

  • Paoli M, Anderson BF, Baker HM, Morgan WT, Smith A, Baker EN (1999) Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two beta-propeller domains. Nat Struct Biol 6:926–931

    Article  CAS  Google Scholar 

  • Patrick AK, Potts KE (1998) Protease inhibitors as antiviral agents. Clin Microbiol Rev 11:614–627

    Article  Google Scholar 

  • Pierre S, Coupe S, Prevot-d’Alvise N, Gaillard S, Richard S, Gouze E, Aubert J, Grillasca A (2010) Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues. Comp Biochem Physiol B 155:396–402

    Article  CAS  Google Scholar 

  • Rawlings ND, Tolle DP, Barrett AJ (2004) Evolutionary families of protease inhibitors. Biochem J 378:705–716

    Article  CAS  Google Scholar 

  • Sarropoulou E, Fernandes JMO, Mitter K, Magoulas A, Mulero V, Sepulcre MP, Figueras A, Novoa B, Kotoulas G (2010) Evolution of a multifunctional gene: the warm temperature acclimation protein Wap65 in the European seabass Dicentrarchus labrax. Mol Phylogen Evol 55:640–649

    Article  CAS  Google Scholar 

  • Sha A, Xu P, Takano T, Liu H, Terhune J, Liu Z (2008) The warm temperature acclimation protein Wap65 as an immune response gene: its duplicates are differentially regulated by temperature and bacterial infections. Mol Immunol 45:1458–1469

    Article  CAS  Google Scholar 

  • Shi YH, Chen J, Li CH, Li MY (2009) Molecular cloning of liver Wap65 in ayu (Plecoglossus altivelis) and mRNA expression changes following Listonella anguillarum infection. Mol Biol Rep. https://doi.org/10.1007/s11033-009-9551-1

    Article  PubMed  PubMed Central  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  CAS  Google Scholar 

  • Studer G, Rempfer C, Waterhouse AM, Gumienny G, Haas J, Schwede T (2020) QMEANDisCo-distance constraints applied on model quality estimation. Bioinformatics 36:1765–1771

    Article  CAS  Google Scholar 

  • Tahtinen V, Weber E, Gunther D, Ylonen A, Kalkkinen N, Olsen R, Jarvinen M, Soderstrom K-O, Rinne A, Bjorklund H, Bogwald J (2002) Immunolocalization of cysteine proteinases (cathepsins) and cysteine proteinase inhibitors (salarin and kininogen) in Atlantic salmon. Salmo Salar Cell Tissue Res 310:213–222

    Article  CAS  Google Scholar 

  • Tolosano E, Altruda F (2002) Hemopexin: structure, function, and regulation. DNA Cell Biol 21:297–306

    Article  CAS  Google Scholar 

  • Tort L, Balasch JC, Mackenzie S (2003) Fish immune system. A crossroads between innate and adaptive responses. Inmunologia 22:277–286

    Google Scholar 

  • Transue TR, Krahn JM, Gabel SA, DeRose EF, London RE (2004) X-ray and NMR characterization of covalent complexes of trypsin, borate, and alcohols. Biochemistry 43:2829–2839

    Article  CAS  Google Scholar 

  • Watabe S, Kikuchi K, Aida K (1993) Cold- and warm-temperature acclimation induces specific cytosolic proteins in goldfish and carp. Nippon Suisan Gakkaishi 59:151–156

    Article  CAS  Google Scholar 

  • Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: Homology modeling of protein structures and complexes. Nucleic Acids Res 46:W296–W303

    Article  CAS  Google Scholar 

  • Weng GQ, Wang EC, Wang Z, Liu H, Li D, Zhu F, Hou TJ (2019) HawkDock: a web server to predict and analyze the structures of protein-protein complexes based on computational docking and MM/GBSA. Nucleic Acids Res 47:W322–W330

    Article  CAS  Google Scholar 

  • Xu X, Liu J, Wang Y, Si Y, Wang X, Wang Z, Zhang Q, Yu H, Wang X (2018) Kunitz-type serine protease inhibitor is a novel participator in anti-bacterial and anti-inflammatory responses in Japanese flounder (Paralichthys olivaceus). Fish Shellfish Immunol 80:22–30

    Article  CAS  Google Scholar 

  • Ylonen A, Rinne A, Herttuainen J, Bogwald J, Jarvinen M, Kalkkinen M (1999) Atlantic salmon (Salmo salar L.) skin contains a novel kininogen and another cysteine proteinase inhibitor. Eur J Biochem 266:1066–1072

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thanks to Professor S. Watabe, M. Nakaya, PhD, and M. Hirayama, PhD, the University of Tokyo, for amino acid sequence analysis of the trypsin inhibitor TRTI-2.

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Correspondence to Yuji Nagashima.

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Nagashima, Y., Zhang, G.H., Sato, K. et al. A novel property of fWap65-2, the warm temperature acclimation-related 65-kDa protein from pufferfish Takifugu rubripes, as an antitrypsin. Fish Sci 87, 589–598 (2021). https://doi.org/10.1007/s12562-021-01533-6

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