title: Olive flounder (Paralichthys olivaceus) cystatin C: cloning, mRNA expression, and enzymatic characterization of olive flounder cystatin C.
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.
authors: Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218, USA.
authors: Sang Jung
authors: SJ
authors: Ahn
authors: Hye Jin
authors: HJ
authors: Bak
authors: Ju Hyeon
authors: JH
authors: Park
authors: Jin Young
authors: JY
authors: Lee
authors: Na Young
authors: NY
authors: Kim
authors: Jin Woo
authors: JW
authors: Han
authors: Hyae In
authors: HI
authors: Jo
authors: Joon Ki
authors: JK
authors: Chung
authors: Hyung Ho
authors: HH
authors: Lee
mesh_terms: D000818:Animals; D055316:Cystatin C / Q000032:analysis / Q000737:chemistry* / Q000502:physiology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005432:Flounder / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry*; D011480:Protease Inhibitors / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D055316:Cystatin C; D011480:Protease Inhibitors; D012333:RNA, Messenger; D010447:Peptide Hydrolases