title: Comprehensive characterization of three glutathione S-transferase family proteins from black rockfish (Sebastes schlegelii).
abstract: Glutathione S-transferases (GSTs, EC 2.5.1.18) are categorized as phase II enzymes, which form an important multifunctional family associated with a wide variety of catalytic activities. GSTω, GSTρ, and GSTθ are cytosolic GSTs which have been extensively studied in a variety of organisms; however, few studies have focused on teleosts. Those paralogs from black rockfish (Sebastes schlegelii; RfGSTω, RfGSTρ, and RfGSTθ, respectively) were molecularly, biochemically, and functionally characterized to determine their antioxidant extent and protective aptitudes upon pathogenic stress. RfGSTω, RfGSTρ, and RfGSTθ, contained open reading frames of 717bp, 678bp, and 720bp respectively, which encoded respective proteins of 239, 226, and 240 amino acids in length. In silico analysis revealed that all RfGSTs possessed characteristic N-terminal domains bearing glutathione (GSH)-binding sites, and C-terminal domains containing substrate-binding sites. Recombinant RfGSTω (rRfGSTω) catalyzed the conjugation of GSH to dehydroascorbate (DHA), while rRfGSTθ and rRfGSTρ catalyzed to the model GST substrate 1-Chloro-2,4-dinitrobenzene (CDNB). Kinetic analysis revealed variation in Km and Vmax values for each rRfGST, indicating their different conjugation rates. The optimum conditions (pH and temperature) and inhibition assays of each protein demonstrated different optimal ranges showing their wide range of activity as an assembly. RfGSTω and RfGSTθ paralogs demonstrated their antioxidant potential towards H2O2 and heavy metals (Cd, Zn, and Cu) in vitro, while RfGSTρ had an antioxidant potential only towards heavy metals (Zn and Cu). Though all the paralogs were ubiquitously expressed in different magnitudes, RfGSTω was highly expressed in blood, whereas RfGSTρ and RfGSTθ were highly expressed in liver. The mRNA expression of RfGSTω and RfGSTθ, upon Streptococcus iniae and poly I:C stimulation, revealed a significantly up-regulated expression, whereas RfGSTρ mRNA expression was significantly down-regulated. Collectively, our findings suggest that RfGSTω, RfGSTρ, and RfGSTθ paralogs are potent in detoxifying xenobiotic toxics, capable of protecting cells from oxidative stress generated by both H2O2 and heavy metals, and finally, yet importantly, stimulated under pathogenic stress signals.
authors.affiliation: Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea.
authors.forename: J D H E
authors.initials: JD
authors.lastname: Jayasinghe
authors.affiliation: Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea. Electronic address: sl_sanjaya@yahoo.com.
authors.forename: S D N K
authors.initials: SD
authors.lastname: Bathige
authors.affiliation: Biotechnology Research Division, National Institute of Fisheries Science, 408-1 Sirang-ri, Gijang-up, Gijang-gun, Busan 46083, Republic of Korea.
authors.forename: Bo-Hye
authors.initials: BH
authors.lastname: Nam
authors.affiliation: Genetics & Breeding Research Center, National Institute of Fisheries Science, Geoje 53334, Republic of Korea.
authors.forename: Jae Koo
authors.initials: JK
authors.lastname: Noh
authors.affiliation: Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, 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 / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005399:Fishes / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000633:toxicity; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000633:toxicity; D018384:Oxidative Stress; D010802:Phylogeny; D011070:Poly I-C / Q000633:toxicity; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000070058:Streptococcus iniae / Q000201:enzymology / Q000254:growth & development; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000975:Antioxidants; D004791:Enzyme Inhibitors; D029941:Fish Proteins; D019216:Metals, Heavy; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D011070:Poly I-C
keywords: Black rockfish; Catalytic activity; Glutathione S transferases; Oxidative stress; Pathogenic stress