Research paper Comparative analysis of CsCu/ZnSOD defense role by molecular characterization: Gene expression-enzyme activity-protein level Venkatesh Kumaresan a , Annie J. Gnanam b , Mukesh Pasupuleti c , Mariadhas Valan Arasu d , Naif Abdullah Al-Dhabi d , Ramasamy Harikrishnan e , Jesu Arockiaraj a, ⁎ a Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur, 603 203, Chennai, Tamil Nadu, India b Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USA c Lab PCN 206, Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India d Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia e Department of Zoology, Pachaiyappa's College for Men, Kanchipuram 631 501, Tamil Nadu, India abstract article info Article history: Received 5 December 2014 Received in revised form 2 March 2015 Accepted 18 March 2015 Available online 21 March 2015 Keywords: Murrel Epizootic ulcerative syndrome Superoxide dismutase Enzyme activity Gene expression Cu/ZnSOD (copper/zinc superoxide dismutase) primarily scavenges cytosolic reactive oxygen species (ROS) by converting ROS to hydrogen peroxide, which is then converted to water by the catalytic action of catalase, thus playing a pivotal role in the first line of defense mechanism against oxidative stress. In this study, we have report- ed a complete molecular characterization of cDNA sequence from striped murrel Channa striatus (Cs). Cellular lo- cation prediction reveals that CsCu/ZnSOD protein is cytosolic with an accuracy of 90%. Phylogenetic analysis showed that CsCu/ZnSOD belongs to SOD1 group and it shared a common clad with Asian seabass Lates calcarifer and then with other fishes. The highest CsCu/ZnSOD gene expression, SOD enzyme activity and total protein con- centration were observed in the liver and its regulation was studied upon fungus (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) challenges. Based on the results obtained from the above analysis, we conclud- ed a correlation of gene expression-enzyme activity-protein concentration. Overall, the findings demonstrated that the CsCu/ZnSOD plays a critical role in the antioxidant system especially in the liver during oxidative stress caused by fungus and bacteria. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Reactive oxygen species (ROS) are major antimicrobial substances which are generated by the host defense mechanism to neutralize the invading pathogens (Holmblad and Soderhall, 1999). However, the presence of these substances beyond the threshold limit may lead to cy- totoxic problem in the host (Munoz et al., 2000). ROS such as superox- ide anion, singlet oxygen, hydrogen peroxide and hydroxyl radicals damage the lipid per-oxidation, generating cross-linking and inactiva- tion of proteins and nucleic acids which results in cell death (Wiseman and Halliwell, 1996). Superoxide dismutases (SODs) are an- tioxidant enzymes which catalyze the dismutation of superoxide ion (O 2 - ) to hydrogen peroxide and oxygen molecule in the presence of water, thus reducing the destructive oxidative processes of ROS in cells. SODs are metalloenzymes which are broadly classified into four dis- tinct groups depending on the metal content. They are iron SOD (FeSOD), nickel SOD (NiSOD), manganese SOD (MnSOD) and copper/ zinc SOD (Cu/Zn SOD). FeSODs are mainly found in prokaryotes and plants (Cheng et al., 2010). NiSODs are found in bacteria especially in Streptomyces sp. (Wuerges et al., 2004). MnSODs and Cu/ZnSODs have been reported in various organisms including crustaceans, fish and mammals (Fridovich, 1975, 1995). Cu/ZnSODs are important members in the group due to their physiological and therapeutic importance which are believed to be evolved from a common ancestor. Further, Fridovich (1989) and Okado-Matsumoto and Fridovich (2001) stated that they are different from Mn and Fe SODs. On the basis of cellular lo- cation, Cu/ZnSODs are further divided into two types. They are cytoplas- mic isoforms which are non-glycosylated without signal sequence and extracellular isoforms which are glycosylated with N terminal signal se- quence. Thus, they are immunologically distinguishable with different amino acid sequences (Fujita et al., 2009). Extracellular Cu/ZnSOD (EC SOD) have been identified in two different structures based on the di- sulfide bridge formation (Petersen et al., 2003), whereas the cytosolic Cu/ZnSOD is a dimeric protein with two identical, non-covalently linked isoforms. Cu/ZnSOD is found in peroxisomes, nucleus and mitochondri- al inter-membrane space of eukaryotic cells (Valentine et al., 2005), Gene 564 (2015) 53–62 Abbreviations: Cu/ZnSOD,copper/zincsuperoxidedismutase;ROS,reactiveoxygenspe- cies; Cs, Channa striatus; FeSOD, iron SOD; NiSOD, nickel SOD; MnSOD, manganese SOD; EC SOD, extracellular Cu/ZnSOD; ALS, amyotrophic lateral sclerosis; EUS, epizootic ulcerative syndrome; UTR, untranslated regions; ORF, open reading frame; bp, base pair; aa, amino acid; PCR, polymerase chain reaction; PBS, phosphate buffer saline; ANOVA, analysis of variance. ⁎ Corresponding author. E-mail address: jesuaraj@hotmail.com (J. Arockiaraj). http://dx.doi.org/10.1016/j.gene.2015.03.042 0378-1119/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene