Fish Physiology and Biochemistry

, Volume 40, Issue 6, pp 1937–1955 | Cite as

A novel murrel Channa striatus mitochondrial manganese superoxide dismutase: gene silencing, SOD activity, superoxide anion production and expression

  • Jesu Arockiaraj
  • Rajesh Palanisamy
  • Prasanth Bhatt
  • Venkatesh Kumaresan
  • Annie J. Gnanam
  • Mukesh Pasupuleti
  • Marimuthu Kasi


We have reported the molecular characterization including gene silencing, superoxide activity, superoxide anion production, gene expression and molecular characterization of a mitochondrial manganese superoxide dismutase (mMnSOD) from striped murrel Channa striatus (named as CsmMnSOD). The CsmMnSOD polypeptide contains 225 amino acids with a molecular weight of 25 kDa and a theoretical isoelectric point of 8.3. In the N-terminal region, CsmMnSOD carries a mitochondrial targeting sequence and a superoxide dismutases (SOD) Fe domain (28–109), and in C-terminal region, it carries another SOD Fe domain (114–220). The CsmMnSOD protein sequence shared significant similarity with its homolog of MnSOD from rock bream Oplegnathus fasciatus (96 %). The phylogenetic analysis showed that the CsmMnSOD fell in the clade of fish mMnSOD group. The monomeric structure of CsmMnSOD possesses 9 α-helices (52.4 %), 3 β-sheets (8.8 %) and 38.8 % random coils. The highest gene expression was noticed in liver, and its expression was inducted with fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infections. The gene silencing results show that the fish that received dsRNA exhibited significant (P < 0.05) changes in expression when compared to their non-injected and fish physiological saline-injected controls. The SOD activity shows that the activity increases with the spread of infection and decreases once the molecule controls the pathogen. The capacity of superoxide anion production was determined by calculating the granular blood cell count during infection in murrel. It shows that the infection influenced the superoxide radical production which plays a major role in killing the pathogens. Overall, this study indicated the defense potentiality of CsmMnSOD; however, further research is necessary to explore its capability at protein level.


Channa striatus MnSOD Gene silencing SOD activity Superoxide production 



Channa striatus mitochondrial manganese superoxide dismutase


Reactive oxygen species


Hydroxyl radical


Hydrogen peroxide


Superoxide radical


Copper/zinc SOD


Iron SOD


Nickel SOD


Mitochondrial translocation sequence


Epizootic ulcerative syndrome


Mitochondrial targeting peptide


Fish physiological saline



This research is supported by DBT’s Prestigious Ramalingaswami Re-entry Fellowship (D.O.NO.BT/HRD/35/02/2006) funded by Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi.


  1. Abe R, Shimosegawa T, Moriizumi S, Kikuchi Y, Kimura K, Satoh A, Koizumi M, Toyota T (1995) Lipopolysaccharide induces manganese superoxide dismutase in the rat pancreas: its role in Cerulein Pancreatitis. Biochem Biophys Res Commun 217:1216–1222PubMedCrossRefGoogle Scholar
  2. Abirami A, Kumaresan V, Sathyamoorthi A, Palanisamy R, Prabha N, Bhatt P, Roy A, Thirumalai MK, Gnanam AJ, Pasupuleti M, Marimuthu K, Arockiaraj J (2013) Fish lily type lectin-1 contains β-prism architecture: immunological characterization. Mol Immunol 56:497–506CrossRefGoogle Scholar
  3. Abreu IA, Cabelli DE (2010) Superoxide dismutases-a review of the metal-associated mechanistic variations. Biochim Biophys Acta 1804:263–274PubMedCrossRefGoogle Scholar
  4. Amparyup P, Charoensapsri W, Tassanakajon A (2009) Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon. Dev Comp Immunol 33:247–256PubMedCrossRefGoogle Scholar
  5. Anderson R (1994) Hemocyte-derived reactive oxygen intermediate production in four bivalve mollusks. Dev Comp Immunol 18:89–96PubMedCrossRefGoogle Scholar
  6. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  7. Arockiaraj J, Haniffa MA, Perumalsamy PRR, Marimuthu K, Muruganandam M (2003) An effective treatment to the spotted murrel Channa punctatus for epizootic ulcerative syndrome (EUS). Fish Chimes 23:27–28Google Scholar
  8. Arockiaraj J, Sarasvathi E, Puganeshwaran V, Arunsingh SV, Rofina YO, Subha B (2011) Prophenoloxidase activating enzyme-III from giant freshwater prawn Macrobrachium rosenbergii: characterization, expression and specific enzyme activity. Mol Biol Rep 39:1377–1386PubMedCrossRefGoogle Scholar
  9. Arockiaraj J, Sarasvathi E, Puganeshwaran V, Arunsingh SV, Rofina YO, Subha B (2012) Immunological role of thiol-dependent peroxiredoxin gene in Macrobrachium rosenbergii. Fish Shellfish Immunol 33:121–129PubMedCrossRefGoogle Scholar
  10. Arockiaraj J, Annie JG, Dhanaraj M, Mukesh P, Milton J, Arun S (2013a) An upstream initiator caspase 10 of snakehead murrel Channa striatus, containing DED, p20 and p10 subunits: molecular cloning, gene expression and proteolytic activity. Fish Shellfish Immunol 34:505–513PubMedCrossRefGoogle Scholar
  11. Arockiaraj J, Gnanam AJ, Kumaresan V, Palanisamy R, Bhatt P, Thirumalai MK, Arpita R, Mukesh P, Marimuthu K (2013b) An unconventional antimicrobial protein histone from freshwater prawn Macrobrachium rosenbergii: analysis of immune properties. Fish Shellfish Immunol 35:1511–1522PubMedCrossRefGoogle Scholar
  12. Bannister JV, Bannister WH, Rotilio G, Fridovich I (1987) Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit Rev Biochem 22:111–180PubMedCrossRefGoogle Scholar
  13. Bao Y, Li L, Zhang G (2008) The manganese superoxide dismutase gene in bay scallop Argopecten irradians: cloning, 3D modelling and mRNA expression. Fish Shellfish Immunol 25:425–432PubMedCrossRefGoogle Scholar
  14. Bell KL, Smith VJ (1993) In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Dev Comp Immunol 17:211–219PubMedCrossRefGoogle Scholar
  15. Benard G, Faustin B, Passerieux E, Galinier A, Rocher C, Bellance N, Delage JP, Casteilla L, Letellier T, Rossignol R (2006) Physiological diversity of mitochondrial oxidative phosphorylation. Am J Physiol Cell Physiol 291:1172–1182CrossRefGoogle Scholar
  16. Borgstahl GEO, Parge HE, Hickey MJ, Beyer WF Jr, Hallewell RA, Tainer JA (1992) The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles. Cell 71:107–118PubMedCrossRefGoogle Scholar
  17. Boveris A, Chance B (1973) 0 Mitochondrial generation of hydrogen-peroxide-general properties and effect of hyperbaric-oxygen. Biochem J 134:707–716PubMedCentralPubMedGoogle Scholar
  18. Brouwer M, Brouwer TH, Grater W, Enghild JJ, Thogersen IB (1997) The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods. Biochemistry 36:13381–13388PubMedCrossRefGoogle Scholar
  19. Cadenas E, Davies KJ (2000) Mitochondrial free radical generation, oxidative stress and aging. Free Radical Biol Med 29:222–230CrossRefGoogle Scholar
  20. Cheng W, Tung YH, Chiou TT, Chen JC (2006) Cloning and characterisation of mitochondrial manganese superoxide dismutase (mtMnSOD) from the giant freshwater prawn Macrobrachium rosenbergii. Fish Shellfish Immunol 21:453–466PubMedCrossRefGoogle Scholar
  21. Cho YS, Choi BN, Ha EM, Kim KH, Kim SK, Kim DS, Nam YK (2005) Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis. Mar Biotechnol 7:350–362PubMedCrossRefGoogle Scholar
  22. Cho YS, Lee SY, Bang IC, Kim DS, Nam YK (2009) Genomic organization and mRNA expression of manganese superoxide dismutase (Mn-SOD) from Hemibarbus mylodon (Teleostei, Cypriniformes). Fish Shellfish Immunol 27:571–576PubMedCrossRefGoogle Scholar
  23. Clair DKS, Porntadavity S, Xu Y, Kiningham K (2002) Transcription regulation of human manganese superoxide dismutase gene. Method Enzymol 349:306–312CrossRefGoogle Scholar
  24. Dhanaraj M, Haniffa MA, Ramakrishna CM, Arunsingh SV (2008) Microbial flora from the epizootic ulcerative syndrome (EUS) infected murrel Channa striatus (Bloch, 1797) in Tirunelveli region. Turk J Vet Anim Sci 32:221–224Google Scholar
  25. Duttaroy A, Meidinger R, Kirby K, Carmichael S, Hilliker A, Phillips J (1994) A manganese superoxide dismutase-encoding cDNA from Drosophila melanogaster. Gene 143:223–225PubMedCrossRefGoogle Scholar
  26. Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem 64:97–112PubMedCrossRefGoogle Scholar
  27. Fukuhara R, Tezuka T, Kageyama T (2002) Structure, molecular evolution, and gene expression of primate superoxide dismutases. Gene 296:99–109PubMedCrossRefGoogle Scholar
  28. García-Triana A, Zenteno-Savín T, Peregrino-Uriarte AB, Yepiz-Plascencia G (2010) Hypoxia, reoxygenation and cytosolic manganese superoxide dismutase (cMnSOD) silencing in Litopenaeus vannamei: effects on cMnSOD transcripts, superoxide dismutase activity and superoxide anion production capacity. Dev Comp Immunol 34:1230–1235PubMedCrossRefGoogle Scholar
  29. Gomez-Anduro GA, Barillas-Mury CV, Peregrino-Uriarte AB, Gupta L, Gollas-Galvan T, Hernandez-Lopez J, Yepiz-Plascencia G (2006) The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei: molecular cloning and expression. Dev Comp Immunol 30:893–900PubMedCrossRefGoogle Scholar
  30. Greenberger JS, Epperly MW (2004) Radioprotective antioxidant gene therapy: potential mechanisms of action. Gene Ther Mol Biol 8:31–44Google Scholar
  31. Greenberger JS, Epperly MW, Gretton J, Jefferson M, Nie S, Bernarding M, Kagan V, Guo HL (2003) Radioprotective gene therapy. Curr Gene Ther 3:183–195PubMedCrossRefGoogle Scholar
  32. Hansen BH, Romma S, Garmo QA, Olsvik PA, Andersen RA (2006) Antioxidative stress proteins and their gene expression in brown trout (Salmo trutta) from three rivers with different heavy metal levels. Comp Biochem Physiol Part C 143:263–274Google Scholar
  33. Hensley K, Robinson KA, Gabbita SP, Salsman S, Floyd RA (2000) Reactive oxygen species, cell signaling, and cell injury. Free Radical Biol Med 28:1456–1462CrossRefGoogle Scholar
  34. Ho YS, Howard AJ, Crapo JD (1991) Molecular structure of a functional rat gene for manganese-containing superoxide dismutase. Am J Respir Cell Mol Biol 4:278–286PubMedCrossRefGoogle Scholar
  35. Holley AK, Bakthavatchalu V, Velez-Roman JM, St Clair DK (2011) Manganese superoxide dismutase: guardian of the powerhouse. Int J Mol Sci 12:7114–7162Google Scholar
  36. Jones PL, Kucera G, Gordon H, Boss JM (1995) Cloning and characterization of the murine manganous superoxide dismutase-encoding gene. Gene 153:155–161PubMedCrossRefGoogle Scholar
  37. Ken CF, Lee CC, Duan KJ, Lin CT (2005) Unusual stability of manganese superoxide dismutase from a new species, Tatumella ptyseos ct: its gene structure, expression and enzyme properties. Prot Expr Purif 40:42–50CrossRefGoogle Scholar
  38. Kim YI, Kim HJ, Kwon YM, Kang YJ, Lee IH, Jin BR, Han YS, Cheon HM, Ha NG, Seo SJ (2010) Modulation of MnSOD protein in response to different experimental stimulation in Hyphantria cunea. Comp Biochem Physiol Part B 157:343–350CrossRefGoogle Scholar
  39. Lee SJ, Lim KT (2007) UDN glycoprotein regulates activities of manganese-superoxide dismutase, activator protein-1, and nuclear factor-kB stimulated by reactive oxygen radicals in lipopolysaccharide-stimulated HCT-116 cells. Cancer Lett 254:274–287PubMedCrossRefGoogle Scholar
  40. Lesser MP (2006) Oxidative stress in marine environments: biochemistry and physiological ecology. Annu Rev Physiol 68:253–278PubMedCrossRefGoogle Scholar
  41. Lin CT, Tseng WC, Hsiao NW, Chang HH, Ken CF (2009) Characterization, molecular modelling and developmental expression of zebrafish manganese superoxide dismutase. Fish Shellfish Immunol 27:318–324PubMedCrossRefGoogle Scholar
  42. Lin YC, Lee FF, Wu CL, Chen JC (2010) Molecular cloning and characterization of a cytosolic manganese superoxide dismutase (cytMnSOD) and mitochondrial manganese superoxide dismutase (mtMnSOD) from the kuruma shrimp Marsupenaeus japonicus. Fish Shellfish Immunol 28:143–150PubMedCrossRefGoogle Scholar
  43. Livak KJ, Schmittgenm TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  44. Maningas MBB, Kondo H, Hirono I, Saito-Taki T, Aoki T (2008) Essential function of transglutaminase and clotting protein in shrimp immunity. Mol Immunol 45:1269–1275PubMedCrossRefGoogle Scholar
  45. Matityahu A, Hadar Y, Dosoretz CG, Belinky PA (2008) Gene silencing by RNA interference in the white rot fungus Phanerochaete chrysosporium [down-pointing small open triangle]. Appl Environ Microbiol 74:5359–5365PubMedCentralPubMedCrossRefGoogle Scholar
  46. Meyrick B, Magnuson MA (1994) Identification and functional characterization of the bovine manganous superoxide dismutase promoter. Am J Respir Cell Mol Biol 10:113–121PubMedCrossRefGoogle Scholar
  47. Mouyna I, Henry C, Doering TL, Latge JP (2004) Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigates. FEMS Microbiol Lett 237:317–324PubMedGoogle Scholar
  48. Mu C, Wang Q, Yuan Z, Wang C (2012) Identification of glutaredoxin 1 and glutaredoxin 2 genes from Venerupis philippinarum and their responses to benzo[a] pyrene and bacterial challenge. Fish Shellfish Immunol 32:482–488PubMedCrossRefGoogle Scholar
  49. Muller FL, Lustgarten MS, Jang Y, Richardson A, Van Remmen H (2007) Trends in oxidative aging theories. Free Radical Biol Med 43:477–503CrossRefGoogle Scholar
  50. Munoz M, Cedeno R, Rodriguez J, van der Knaap WPW, Mialhe E, Bachere E (2000) Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp Penaeus vannamei. Aquaculture 191:89–107CrossRefGoogle Scholar
  51. Olker C, Siese A, Stumpf S, Müller B, Gemsa D, Garn H (2004) Impaired superoxide radical production by bronchoalveolar lavage cells from NO(2)-exposed rats. Free Radical Biol Med 37:977–987CrossRefGoogle Scholar
  52. Parker MW, Blake CCF (1988) Crystal structure of manganese superoxide dismutase from Bacillus stearothermophilus at 2.4 Å resolution. J Mol Biol 199:649–661PubMedCrossRefGoogle Scholar
  53. Patterton HG, Graves S (2000) DNAssist: the integrated editing and analysis of molecular biology sequences in windows. Bioinform Appl Note 16:652–653CrossRefGoogle Scholar
  54. Porta J, Vahedi-Faridi A, Borgstahl GE (2010) Structural analysis of peroxide-soaked MnSOD crystals reveals side-on binding of peroxide to active-site manganese. J Mol Biol 399:377–384PubMedCrossRefGoogle Scholar
  55. Roch P (1999) Defense mechanisms and disease prevention in farmed invertebrates. Aquaculture 172:125–145CrossRefGoogle Scholar
  56. Rodrıguez I, Novoa B, Figueras A (2008) Immune response of zebrafish (Danio rerio) against a newly isolated bacterial pathogen Aeromonas hydrophila. Fish Shellfish Immunol 25:239–249PubMedCrossRefGoogle Scholar
  57. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  58. Schreck R, Rieber P, Baeuerle PA (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J 10:2247–2258PubMedCentralPubMedGoogle Scholar
  59. Sokolova IM, Ringwood AH, Johnson C (2005) Tissue-specific accumulation of cadmium in subcellular compartments of eastern oysters Crassostrea virginica Gmelin (Bivalvia: Ostreidae). Aquat Toxicol 74:218–228PubMedCrossRefGoogle Scholar
  60. Sun J, Folk D, Bradley Tower J (2002) Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics 161:661–672PubMedCentralPubMedGoogle Scholar
  61. Uinuk-Ool TS, Takezaki N, Kuroda N, Figueroa F, Sato A, Samonte IE, Mayer WE, Klein J (2003) Phylogeny of antigen-processing enzymes: cathepsins of a cephalochordate, an agnathan and a bony fish. Scand J Immunol 58:436–448PubMedCrossRefGoogle Scholar
  62. Wan XS, Devalaraja MN, St Clair DK (1994) Molecular structure and organization of the human manganese superoxide dismutase gene. DNA Cell Biol 13:1127–1136PubMedCrossRefGoogle Scholar
  63. Wang MQ, Su XR, Li Y, Jun Z, Li TW (2010) Cloning and expression of the MnSOD gene from Phascolosoma esculenta. Fish Shellfish Immunol 29:759–764PubMedCrossRefGoogle Scholar
  64. Winston GW, Di Giulio RT (1991) Prooxidant and antioxidant mechanisms in aquatic organisms. Aqua Toxicol 19:137–161CrossRefGoogle Scholar
  65. Wispe JR, Clark JC, Burhans MS, Kropp KE, Korfhagen TR, Whitsett JA (1989) Synthesis and processing of the precursor for human mangano-superoxide dismutase. Biochim Biophys Acta 994:30–36PubMedCrossRefGoogle Scholar
  66. Wool GD, Reardon CA, Getz GS (2008) Apolipoprotein A-I mimetic peptide helix number and helix linker influence potentially anti-atherogenic properties. J Lipid Res 49:1268–1283PubMedCentralPubMedCrossRefGoogle Scholar
  67. Xu Y, Porntadavity S, St Clair DK (2002) Transcriptional regulation of the human manganese superoxide dismutase gene: the role of specificity protein 1 (Sp1) and activating protein-2 (AP-2). Biochem J 362:401–412PubMedCentralPubMedCrossRefGoogle Scholar
  68. Yeh CC, Wan XS, St Clair DK (1998) Transcriptional regulation of the 5′ proximal promoter of the human manganese superoxide dismutase gene. DNA Cell Biol 17:921–930PubMedCrossRefGoogle Scholar
  69. Yu B, Zhao X, Lee LJ, Lee RJ (2009) Targeted delivery systems for oligonucleotide therapeutics. AAPS J 11:195–203PubMedCentralPubMedCrossRefGoogle Scholar
  70. Yu Z, He X, Fu D, Zhang Y (2011) Two superoxide dismutase (SOD) with different subcellular localizations involved in innate immunity in Crassostrea hongkongensis. Fish Shellfish Immunol 31:533–539PubMedCrossRefGoogle Scholar
  71. Zelko IN, Mariani TJ, Folz RJ (2002) Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radical Biol Med 33:337–349CrossRefGoogle Scholar
  72. Zhang Q, Li F, Wang B, Zhang J, Liu Y, Zhou Q, Xiang J (2007) The mitochondrial manganese superoxide dismutase gene in Chinese shrimp Fenneropenaeus chinensis: cloning, distribution and expression. Dev Comp Immunol 31:429–440PubMedCrossRefGoogle Scholar
  73. Zhang ZW, Li Z, Liang HW, Li L, Luo XZ, Zou GW (2011) Molecular cloning and differential expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Hypophthalmichthys molitrix. Fish Shellfish Immunol 30:73–79Google Scholar
  74. Zhang D, Cui S, Guo H, Jiang S (2013) Genomic structure, characterization and expression analysis of a manganese superoxide dismutase from pearl oyster Pinctada fucata. Dev Comp Immunol 41:484–490PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Jesu Arockiaraj
    • 1
  • Rajesh Palanisamy
    • 1
  • Prasanth Bhatt
    • 1
  • Venkatesh Kumaresan
    • 1
  • Annie J. Gnanam
    • 2
  • Mukesh Pasupuleti
    • 3
  • Marimuthu Kasi
    • 4
  1. 1.Division of Fisheries Biotechnology and Molecular Biology, Department of Biotechnology, Faculty of Science and HumanitiesSRM UniversityChennaiIndia
  2. 2.Institute for Cellular and Molecular BiologyThe University of Texas at AustinAustinUSA
  3. 3.Lab PCN 206, Microbiology DivisionCSIR-Central Drug Research InstituteLucknowIndia
  4. 4.Department of Biotechnology, Faculty of Applied SciencesAIMST UniversityBedongMalaysia

Personalised recommendations