Comprehensive characterization of three glutathione S-transferase family proteins from black rockfish (Sebastes schlegelii)

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. GST omega, GST rho, and GST theta 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; RfGST omega, RfGST rho, and RfGST theta, respectively) were molecularly, biochemically, and functionally characterized to determine their antioxidant extent and protective aptitudes upon pathogenic stress. RfGST omega, RfGST rho, and RfGST theta, contained open reading frames of 717 bp, 678 bp, and 720 bp 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 RfGST omega (rRfGST omega) catalyzed the conjugation of GSH to dehydroascorbate (DHA), while rRfGST theta and rRfGST rho catalyzed to the model GST substrate 1-Chloro-2,4-dinitrobenzene (CDNB). Kinetic analysis revealed variation in K-m and V-max 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. RfGST omega and RfGST theta paralogs demonstrated their antioxidant potential towards H2O2 and heavy metals (Cd, Zn, and Cu) in vitro, while RfGST rho had an antioxidant potential only towards heavy metals (Zn and Cu). Though all the paralogs were ubiquitously expressed in different magnitudes, RfGST omega was highly expressed in blood, whereas RfGST rho and RfGST theta were highly expressed in liver. The mRNA expression of RfGST omega and RfGST theta, upon Streptococcus iniae and poly I:C stimulation, revealed a significantly up-regulated expression, whereas RfGST rho mRNA expression was significantly down-regulated. Collectively, our findings suggest that RfGST omega, RfGST rho, and RfGST theta 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. (C) 2016 Elsevier Inc All rights reserved.