title: Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein.
abstract: Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) μg μL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Science, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
authors.forename: H K A
authors.initials: HK
authors.lastname: Premachandra
authors.forename: Ilson
authors.initials: I
authors.lastname: Whang
authors.forename: Young-deuk
authors.initials: YD
authors.lastname: Lee
authors.forename: Sukkyoung
authors.initials: S
authors.lastname: Lee
authors.forename: Mahanama
authors.initials: M
authors.lastname: De Zoysa
authors.forename: Myung-Joo
authors.initials: MJ
authors.lastname: Oh
authors.forename: Sung-Ju
authors.initials: SJ
authors.lastname: Jung
authors.forename: Bong-Soo
authors.initials: BS
authors.lastname: Lim
authors.forename: Jae Koo
authors.initials: JK
authors.lastname: Noh
authors.forename: Hae-Chul
authors.initials: HC
authors.lastname: Park
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055313:Cystatin B / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D010473:Perciformes / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D017433:Protein Structure, Secondary; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; D055313:Cystatin B; D010206:Papain