COMPREHENSIVE ANALYSIS OF THREE GLUTATHIONE S-TRANSFERASE PARALOGS FROM A TELEOSTEAN ORIGIN

Glutatione S-transferases (GSTs, EC 2.5.1.18) are coming under the phase II enzymes which are an important multifunctional family accompanied with a wide variety of catalytic activities. GSTs catalyze many endobiotic and electrophilic xenobiotics via the nucleophilic addition of the tripeptide glutathione (GSH) into their functional groups. In this study, three GST paralogs from black rockfish (Sebastes schlegeli); RfGSTθ, RfGSTω and RfGSTρ were molecularly and functionally characterized to determine the antioxidant extent.

Full length cDNA sequences were obtained from black rockfish cDNA database molecularly characterized. Using appropriate primers the complete ORF was replicated and cloned into pMAL-c5X vector and transformed into Escherichia coli ER2523 cells. Recombinant proteins were over expressed and purified. Spectrophotometric analysis was carried out to determine the specific activities towards various substrates with reduced Glutathione (GSH). The effect of temperature (10 °C - 60 °C), pH (3 - 12 pH), kinetic assays and the effect of inhibitors (cibacron blue (CB) and hematin) were assayed towards respective substrate. Disk diffusion assay was performed to determine the cell protective magnitude of three paralogs against oxidative stress developed by H₂O₂ and heavy metals (Cd, Cu and Zn).

All three paralogs were entailed their common domain architecture though they were slightly diverged from their corresponding orthologs in respect to activities, active moieties and GSH binding pocket architectures. Their enzymatic activities towards respective substrates, activities under different temperatures and pH values, kinetic parameters as well as inhibitory potential generated by known GST inhibitors reveled their functional variation and validated their existence as paralogs (Table 1). Additionally, three paralogs have revealed cell protective magnitudes against oxidative stress developed by H₂O₂ and heavy metals such as Cd, Cu and Zn in vitro. Collectively, these findings deliver a broadened scopes of GSTω, GSTρ, and GSTθ paralogs from a teleost species opening new dimensions for further studies with GST paralogs.