title: Characterization of rock bream (Oplegnathus fasciatus) cytosolic Cu/Zn superoxide dismutase in terms of molecular structure, genomic arrangement, stress-induced mRNA expression and antioxidant function.
abstract: Superoxide dismutases (SODs) are dedicated to scavenge and dismutate the superoxide anions in order to protect the cells from oxidative stress by establishing the redox homeostasis. In this study, we describe a cytosolic Cu/ZnSOD, the second SOD member from rock bream Oplegnathus fasciatus (Of-cCu/ZnSOD) at molecular, genomic structural-, transcriptional- and functional-levels. The determination of genomic arrangement of Of-cCu/ZnSOD by means of a BAC library revealed that its primary transcript is represented by five exons and encoded a peptide of 154 amino acids. In silico investigation of Of-cCu/ZnSOD indicated the presence of several family characteristics including two Cu/ZnSOD signatures, seven metal liganding residues and eight β-sheets forming a β-barrel topology. Alignment and modeling studies confirmed the conservation of Cu/ZnSOD at primary and tertiary levels. While invertebrate Cu/ZnSOD members mainly demonstrate a tetraexonic structure, the vertebrate members have acquired an additional intron in the third exon resulting in a quinquepartite arrangement with class-specific exon lengths. Although, teleost Cu/ZnSOD members resembled the mammalian orthologs in their genomic organization, they shared a proximal position with molluscan members in the phylogeny. The antioxidant (AO) activity of Of-cCu/ZnSOD was affirmed by a recombinant protein which was also used to examine the biophysical and biochemical properties. The pronounced activity was detected when the rOf-cCu/ZnSOD was expressed with the Cu(2+) and Zn(2+) supplementation. The optimum activities were observed at pH10 and 25°C, and KCN strongly inhibited the activity of the rOf-cCu/ZnSOD. Furthermore, a constitutive mRNA expression of Of-cCu/ZnSOD with higher levels in blood>liver>heart and brain was observed, which was consistent with the transcriptional profile of Of-mMnSOD, suggesting important physiological role(s). This idea was further strengthened by the temporal assessment of Of-cCu/ZnSOD transcripts in animals under pathological (bacteria- or viral-induced) and physiological (H2O2-induced oxidative) stress conditions using qPCR, in which it exhibited significantly up-regulated levels. Screening of Of-cCu/ZnSOD 5'-flanking region revealed the presence of several important transcription factor binding sites that potentially govern the Cu/ZnSOD expression. These findings conjointly contribute to expand our understanding regarding the piscine Cu/ZnSODs and; in particular, the AO enzyme network of rock bream.
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
authors.forename: Navaneethaiyer
authors.initials: N
authors.lastname: Umasuthan
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
authors.forename: S D N K
authors.initials: SD
authors.lastname: Bathige
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
authors.forename: William Shanthakumar
authors.initials: WS
authors.lastname: Thulasitha
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
authors.forename: Wan
authors.initials: W
authors.lastname: Qiang
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
authors.forename: Bong-Soo
authors.initials: BS
authors.lastname: Lim
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea. Electronic address: jehee@jejunu.ac.kr.
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D003198:Computer Simulation; D003300:Copper / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D009928:Organ Specificity; D010473:Perciformes / Q000378:metabolism*; D010802:Phylogeny; D011190:Potassium Cyanide / Q000378:metabolism; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D015032:Zinc / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D012333:RNA, Messenger; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc; D011190:Potassium Cyanide
keywords: Antioxidant activity; Cu/ZnSOD; Genomic arrangement; Immune/oxidative stress; Transcriptional expression