Cystatin B homolog from rock bream Oplegnathus fasciatus: Genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein H.K.A. Premachandra a, 1 , Ilson Whang a, 1 , Young-deuk Lee a , Sukkyoung Lee a , Mahanama De Zoysa b , Myung-Joo Oh c , Sung-Ju Jung c , Bong-Soo Lim d , Jae Koo Noh e , Hae-Chul Park f, ⁎, Jehee Lee a, d, ⁎⁎ a Department of Marine Life Sciences, School of Marine Biomedical Science, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea b College of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon, 305-764, Republic of Korea c Department of Aqualife Medicine, Chonnam National University, Chonnam 550-749, Republic of Korea d Marine and Environmental Institute, Jeju National University, Jeju Special Self-Governing Province 690-814, Republic of Korea e Genetics & Breeding Research Center, National Fisheries Research & Development Institute, Geoje 656-842, Republic of Korea f Graduate School of Medicine, Korea University, Ansan, Gyeonggido 425-707, Republic of Korea abstract article info Article history: Received 10 April 2012 Received in revised form 15 May 2012 Accepted 15 May 2012 Available online 22 May 2012 Keywords: Rock bream Cystatin B Papain inhibition Bacterial challenge Transcriptional analysis 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 24 h post-infection (P b 0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demon- strated 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. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Cysteine proteases have been extensively studied and are known to play essential roles in the physiology of all living organisms. In humans, cysteine protease-mediated polypeptide degradation has been defined as a regulatory mechanism of diverse biological processes, such as apoptosis, major histocompatibility complex (MHC) class II immune responses, inflammation, pro-hormone processing, and extracellular matrix remodeling related to bone development (Chapman et al., 1997; Rzychon et al., 2004). In the pathogenic micro-organisms including bac- teria, fungi, viruses and parasites, cysteine proteases can act as virulence factors, which promote diseases in the host organisms (Takahashi et al., 1994; Mottram et al., 2004; Rudenskaya and Pupov, 2008). The cysteine proteases have been classified into 21 families (C1–C21) according to distinctive sequences and tertiary structures; however, the majority of cysteine proteases belong to the C1 family, which are largely known as papain-like proteases and include the lysosomal cysteine pro- teases, cathepsins. The C1 family members are regulated by a large group of reversible inhibitors, called cystatins (Anastasi et al., 1983; Barrett, 1987; Turk and Bode, 1991). Cystatins are recognized by the presence of at least one cystatin-like functional domain, and have been found in the genomes of diverse organisms. Studies implicated these protease in- hibitors in both biological and pathological processes, including immune Comparative Biochemistry and Physiology, Part B 163 (2012) 138–146 ⁎ Corresponding author. Tel.: + 82 31 412 6712; fax: + 82 31 412 6729. ⁎⁎ Correspondence to: J. Lee, Marine Molecular Genetics Lab, Department of Marine Life Sciences, College of Ocean Science, Jeju National University, 66 Jejudaehakno, Ara- Dong, Jeju, 690-756, Republic of Korea. Tel.: +82 64 754 3472; fax: +82 64 756 3493. E-mail addresses: hcpark67@korea.ac.kr (H.-C. Park), jehee@jejunu.ac.kr (J. Lee). 1 These authors contributed equally to this work. 1096-4959/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2012.05.012 Contents lists available at SciVerse ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb