Molecular characterization of kappa class glutathione S-transferase from the disk abalone (Haliotis discus discus) and changes in expression following immune and stress challenges

Glutathione S-transferase (GST; EC 2.5.1.18) isoenzymes represent a complex group of proteins that are involved in phase II detoxification in several organisms. In this study, GST kappa (GST kappa) from the disk abalone (Haliotis discus discus; AbGST kappa) was characterized at both the transcriptional and functional levels to determine its potential capacity to perform as a detoxification agent under conditions of different stress. The predicted AbGST kappa protein consists of 227 amino acids, with a predicted molecular weight of 25.6 kDa and a theoretical isoelectric point (pI) of 7.78. In silico analysis reveals that AbGST kappa is a disulfide bond formation protein A (DsbA), consisting of a thioredoxin domain, GSH binding sites (G-sites), and a catalytic residue. In contrast, no hydrophobic ligand binding site (H-site), or signal peptides, were detected. AbGST kappa showed the highest sequence identity with the orthologue from pufferfish (Takifugu obscurus) (60.0%). In a phylogenetic tree, AbGST kappa clustered closely together with other fish GST kappa s, and was evolutionarily distanced from other cytosolic GSTs. The predicted three-dimensional structure clearly demonstrates that the dimer adopts a butterfly-like shape. A tissue distribution analysis revealed that GST kappa was highly expressed in the digestive tract, suggesting it has detoxification ability. Depending on the tissue and time, AbGST kappa showed different expression patterns, and levels of expression, following challenge of the abalone with immune stimulants. Enzyme kinetics of the purified recombinant proteins demonstrated its conjugating ability using 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates, and suggested it has a low affinity for both substrates. The optimum temperature and pH for the rAbGST kappa GSH: CDNB conjugating activity were found to be 35 degrees C and pH 8, respectively indicating that the abalone is well adapted to a wide range of environmental conditions. Cibacron blue (100 mu M) was capable of completely inhibiting rAbGST kappa (100%) with an IC50 (half maximal inhibitory concentration) of 0.05 mu M. A disk diffusion assay revealed that rAbGST kappa could significantly protect cells from H2O2 , CdCl2, and ZnCl2. Altogether, this current study suggests that AbGST kappa is involved in detoxification and immunological host defense mechanisms and allows abalones to overcome stresses in order for them to have an increased chance of survival.