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Olive Flounder (Paralichthys olivaceus) Cystatin C: Cloning, mRNA Expression, and Enzymatic Characterization of Olive Flounder Cystatin C

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Abstract

Cystatins are endogenous inhibitors of mammalian lysosomal cysteine proteinases, such as cathepsins B, L, H, and S. Cystatin C belongs to the type 2 cystatin family. In this study, the 751-bp cystatin C cDNA (PoCystatin C) of olive flounder (Paralichthys olivaceus) was cloned by screening from the olive flounder cDNA library. The mRNA expression of the PoCystatin C gene was examined in various tissues from normal and lipopolysaccharide (LPS)-stimulated olive flounder by RT-PCR and was compared with inflammatory cytokines IL-1β, IL-6, and IL-8. PoCystatin C transcripts ubiquitously existed in all normal and LPS-stimulated tissues that were tested. The recombinant PoCystatin C protein was expressed in Escherichia coli BL21(DE3) in pCold™ TF DNA expression vector as a 70-kDa fusion protein. The protease inhibitory activities of recombinant PoCystatin C toward papain cysteine protease, piscine cathepsins (L, S, K, F, and X), and bovine cathepsin B were measured with the synthetic fluorogenic peptide substrates. PoCystatin C tightly inhibited papain cysteine protease, whereas cathepsins L, S, K, F, X, and B were inhibited with lower affinities. Our results indicate that the P. olivaceus cystatin C is a homolog of mammalian cystatin C due to its sequence, structure, tissue expression, and biochemical activity.

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Acknowledgments

This work was supported by the National Research Foundation of Korea Grant funded by the Korean government (MEST; NFR-2010-0020475).

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Authors and Affiliations

  1. Department of Embryology, Carnegie Institution for Science, Baltimore, MD, 21218, USA

    Sang Jung Ahn

  2. Department of Biotechnology, Pukyong National University, Busan, 608-737, South Korea

    Hye Jin Bak, Ju Hyeon Park, Jin Young Lee, Jin Woo Han & Hyung Ho Lee

  3. Department of Aquatic Life Medicine, Pukyong National University, Busan, 608-737, South Korea

    Na Young Kim, Hyae In Jo & Joon Ki Chung

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  1. Sang Jung Ahn
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Correspondence to Joon Ki Chung or Hyung Ho Lee.

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Sang Jung Ahn and Hye Jin Bak contributed equally to this work.

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Ahn, S.J., Bak, H.J., Park, J.H. et al. Olive Flounder (Paralichthys olivaceus) Cystatin C: Cloning, mRNA Expression, and Enzymatic Characterization of Olive Flounder Cystatin C. Appl Biochem Biotechnol 170, 1216–1228 (2013). https://doi.org/10.1007/s12010-013-0248-5

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