Glutathione S-transferase (GST) is a multifunctional protein. It is involved in phase II detoxification of chemical substances through conjugation to reduced glutathione. Further it is involved in various biological functions such as, prostaglandin D2 synthesis, fatty acid β-oxidation and signal transduction pathway. The GSTs are divided into three major families according to their localization in cell. Such as; as cytosolic, mitochondrial, and microsomal. The GSTs are further characterized into several subclasses, namely alpha (α), beta (β), delta (δ), epsilon (ε), zeta (ζ), and theta (θ) based on their substrate specificity, N-terminal amino acid sequence, antibody cross-reactivity and sensitivity to inhibitors. In this current study, we are analyzing the molecular, transcriptional roles, and substrate specificity of GST Alpha4 in Hippocampus abdominalis (HaGSTA4).
The cDNA sequence of HaGSTA4 was obtained from seahorse database. In-silico analysis of HaGSTA4 was conducted using various bioinformatics tools. The abiotic and biotic challenges were conducted with Poly I.C, LPS, gram-negative Edwardsiella tarda and gram-positive Streptococcus iniae. The blood was collected from immune challenged seahorses at different time points. The qPCR was carried out to determine the spatial and temporal expression of HaTRx-2. Further, the GSTA4 was recombinantly expressed in Escherichia coli BL21(DE3) and the substrate specificity check with various substrates.
The GST Alpha4 has 223 amino acids which encoded by 690 bp ORF with molecular weight of 25.7kDa. Its calculated iso electric pI is 8.47. It comprised GST_C_ super family and Thioredoxin _like superfamily. The HaGSTA4 showed highest sequence identity (78.5%) and similarity (85.7%) with the orthologue Parambassis ranga. The phylogenetic analysis revealed that HaGSTA4 is closely clustered together with other fishes. According to the qPCR results, tissue distribution profile revealed that HaGSTA4 highly expressed in kidney compared to other examined tissues (Fig.1.). In blood, HaGSTA4 showed down regulation of most of the time points other than 6 h post injection(p.i.) when challenged with LPS and S.iniae. The significant upregulation of HaGSTA4 was observed with poly I.C at 3 h, 12 h, and 24 h p.i. Further, the immune challenged by E.tarda showed significant up regulation at 24 h p.i. The recombinant HaGSTA4 was exhibited higher activity toward 1-chloro-2,4-dinitrobenzene (CDNB) than other substrates (18.69 μmol.min-1mg-1). Together, the current study suggested that HaGSTA4 has indispensable role in immune defense regulation when pathogen attacks and xenobiotics detoxification.