An upstream initiator caspase 10 of snakehead murrel Channa striatus, containing DED, p20 and p10 subunits: Molecular cloning, gene expression and proteolytic activity Jesu Arockiaraj a, * , Annie J. Gnanam b , Dhanaraj Muthukrishnan c , Mukesh Pasupuleti d , James Milton c , Arun Singh c a Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur, Chennai 603 203, 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 Centre for Aquaculture Research and Extension, St. Xavier’s College (Autonomous), Palayamkottai 627 002, Tamil Nadu, India d SRM Research Institute, SRM University, Kattankulathur, Chennai 603 203, Tamil Nadu, India article info Article history: Received 5 September 2012 Received in revised form 23 November 2012 Accepted 27 November 2012 Available online 16 December 2012 Keywords: Caspase Channa striatus Epizootic ulcerative syndrome (EUS) Gene expression Proteolytic activity abstract Caspase 10 (CsCasp10) was identified from a constructed cDNA library of freshwater murrel (otherwise called snakehead) Channa striatus. The CsCasp10 is 1838 base pairs (bp) in length and it is encoding 549 amino acid (aa) residues. CsCasp10 amino acid contains two death effector domains (DED) in the N-terminal at 2e77 and 87e154 and it contains caspase family p20 domain (large subunit) and caspase family p10 domain (small subunit) in the C-terminal at 299e425 and 449e536 respectively. Pairwise analysis of CsCasp10 showed the highest sequence similarity (79%) with caspase 10 of Paralichthys oli- vaceus. Moreover, the phylogenetic analysis showed that CsCasp10 is clustered together with other fish caspase 10, formed a sister group with caspase 10 from other lower vertebrates including amphibian, reptile and birds and finally clustered together with higher vertebrates such as mammals. Significantly (P < 0.05) highest CsCasp10 gene expression was noticed in gills and lowest in intestine. Furthermore, the CsCasp10 gene expression in C. striatus was up-regulated in gills by fungus Aphanomyces invadans and bacteria Aeromonas hydrophila induction. The proteolytic activity was analyzed using the purified recombinant CsCasp10 protein. The results showed the proteolytic activity of CsCasp10 for caspase 10 substrate was 2.5 units per mg protein. Moreover, the proteolytic activities of CsCasp10 in kidney and spleen induced by A. invadans and A. hydrophila stimulation were analyzed by caspase 10 activity assay kit. All these results showed that CsCasp10 are participated in immunity of C. striatus against A. invadans and A. hydrophila infection. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Caspases are intracellular cysteine proteinases that can cleave their substrates following an aspartic acid residue, and play crucial roles at different stages of the apoptotic mechanism [1,2]. It plays an important role in the action of programmed cell death (PCD), which is described by cell shrinkage, chromatin condensation, DNA frag- mentation, and formation of apoptotic bodies. PCD is a normal physiological function by which additional or repaired cells are terminated in order to regulate the homeostasis of various tissues and organs [3e5]. Apoptosis-influenced morphological variations are made by activation of enzymes in the caspase cascades [6]. Caspases are available in cytosol as a form of inactive proenzymes. Nevertheless, it gets activated when apoptosis is initiated. Caspases activate apoptosis over a highly combined and controlled biolog- ical, biochemical, and genetic mechanism [7]. Caspases are synthesized in the form of zymogene. The structural analysis of caspases revealed that all the procaspases consist a highly similar protease domain possessing two subunits, such as P20, a large subunit (mol. wt. 20 kDa) and P10, a small subunit (mol. wt. 10 kDa). A short peptide, approximately 10 amino acids is present between the P20 and P10 subunits in some procaspases [8]. Various amino acid residues and domains are highly homolo- gous and are known to be vital to maintain the catalytic function of casp 10 [9]. Specifically, the pentapeptide Gln-Ala-Cys-Gln-Gly, treated as a catalytic domain, is entirely similar between human and some lower vertebrates, inspite of their genetic variation [10]. * Corresponding author. Tel.: þ91 44 27452270; fax: þ91 44 27453903. E-mail address: jesuaraj@hotmail.com (J. Arockiaraj). Contents lists available at SciVerse ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2012.11.040 Fish & Shellfish Immunology 34 (2013) 505e513