Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis Navaneethaiyer Umasuthan a,1 , Ilson Whang b, 1 , Youngdeuk Lee a , Sukkyoung Lee a , Yucheol Kim a , Hyowon Kim a , Sung-Ju Jung c , Myung-Joo Oh c , Cheol Young Choi d , Sang-Yeob Yeo e , Sang-Jun Lee f , Jehee Lee a, g, * a Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea b Department of Life Sciences, College of Natural Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea c Department of Aqualife Medicine, Chonnam National University, Chonnam 550-749, Republic of Korea d Department of Biotechnology, Division of Applied Chemistry & Biotechnology, Hanbat National University, Daejeon 305-719, Republic of Korea e Division of Marine Environment and Bioscience, Korea Maritime University, Busan 606-791, Republic of Korea f Biotechnology Research Division, National Fisheries Research and Development Institute, Busan 619-902, Republic of Korea g Marine and Environmental Institute, Jeju National University, Jeju 690-814, Republic of Korea article info Article history: Received 5 August 2010 Received in revised form 28 September 2010 Accepted 7 October 2010 Available online 15 October 2010 Keywords: Heparin cofactor II Serpin Rock bream Inhibitory activity Anti-coagulation abstract Heparin cofactor (HCII) is a serine protease inhibitor (SPI), and plays important physiological roles in various biological events including hemostasis. The gene encoding the HCII was isolated from GS-FLX Ô genomic data of rock bream (Oplegnathus fasciatus), designated as RbHCII. The RbHCII (1950 bp) consists of a 1512 bp open reading frame (ORF) encoding 504 amino acids (aa), with a signal peptide of 19 aa residues. The predicted molecular mass and the estimated isoelectric point of RbHCII were 58 kDa and 5.9, respectively. The deduced aa sequence of RbHCII displayed a characteristic serpin domain and a serpin signature motif (FTVDQPFLFLI). RbHCII demonstrated homology with vertebrate HCIIs and the greatest degree of similarity (90.1%) was observed with Gasterosteus aculeatus HCII. Various functional domains including the reactive center loop (RCL), glycosaminoglycan (GAG) and thrombin binding sites and acidic repeats of human and RbHCII were found to be orthologs through the molecular modeling studies. Phylogenetic analysis revealed that RbHCII belongs to the clade D serpins, and is closely related to the clade A members. Constitutive expression of RbHCII mRNA was detected at different levels in various tissues in a tissue-specific manner. Interestingly, RbHCII transcription was significantly down- regulated (p < 0.05) in liver after challenge with lipopolysaccharide (LPS), Edwardsiella tarda and rock bream iridovirus (RBIV). However, after the immune challenges, RbHCII showed a significant down- regulation in blood tissue only at the late-phase of investigation. The recombinant RbHCII (rRbHCII) was overexpressed in Rosetta-gami (DE3) cells and purified using the pMAL Ô system. The rRbHCII inhibited thrombin and chymotrypsin in a dose-dependent manner. Remarkably, heparin was found to be an enhancer of RbHCII’s thrombin-inhibitory activity. Correlating the heparin-dependent thrombin-inhibi- tion activity of RbHCII with its temporal downregulation against immune stimulants, it could be sug- gested that it is not only involved in the blood coagulation cascade, but also plays an incognito role in immune modulation. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Serine proteases (SPs) are a family of functionally related enzymes involved in a variety of important biological processes including digestion, blood clotting [1], immune activation [2,3] and cell differentiation [4]. The critical regulation of proteolytic activity of SPs is essential to maintain a balanced homeostasis and this is achieved by a functional pair of SPs known as serine protease inhibitors (Serpins). The serpins are a superfamily of ubiquitous ancient homologous proteins showing an extraordinary broad * Corresponding author. Marine Molecular Genetics Lab, Department of Marine Life Science, College of Ocean Sciences, Jeju National University, 66 Jejudaehakno, Ara-Dong, Jeju 690-756, Republic of Korea. Tel.: þ82 64 754 3472; fax: þ82 64 756 3493. E-mail address: jehee@jejunu.ac.kr (J. Lee). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.10.004 Fish & Shellfish Immunology 30 (2011) 194e208