A mu class glutathione S-transferase from Manila clam Ruditapes philippinarum (RpGSTμ): Cloning, mRNA expression, and conjugation assays

Abstract

Glutathione S-transferases (GSTs) are enzymes that catalyze xenobiotic metabolism in the phase II detoxification process. GSTs have a potential for use as indicators or biomarkers to assess the presence of organic and inorganic contaminants in aquatic environments. In this study, a full-length cDNA of a mu (μ) class GST (RpGSTμ) was identified from Manila clam (Ruditapes philippinarum) and biochemically characterized. The 1356 bp of the cDNA included an open reading frame of 651 bp encoding a polypeptide of 217 amino acid residues with a molecular mass of 25.04kDa and an estimated pI of 6.34. Sequence analysis revealed that the RpGSTμ possessed several characteristic features of μ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione (GSH), a C-terminal domain containing substrate binding sites, and a μ loop. The recombinant RpGSTμ (rRpGSTμ) protein exhibited GSH-conjugating catalytic activity towards several substrates, and significantly strong activity was detected against 4-nitrophenethyl bromide (5.77±0.55) and 1-chloro-2,4-dinitrobenzene (CDNB, 3.19±0.05). Kinetic analysis as a function of GSH and CDNB concentrations revealed relatively low Km values of 1.03±0.46mM and 0.56±0.20mM, respectively, thereby indicating a GSH-conjugation attributed with high rates. The optimum pH and temperature for the catalytic activity of the rRpGSTμ protein were 7.7 and 37°C, respectively. The effect of two inhibitors, Cibacron blue and hematin, on the activity of rRpGSTμ was evaluated and the IC50 values of 0.65μM and 9μM, respectively, were obtained. While RpGSTμ transcripts were highly expressed in gills and hemocytes, a significant elevation in mRNA levels was detected in these tissues after lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C) and live bacterial (Vibrio tapetis) challenges. These findings collectively suggest that RpGSTμ functions as a potent detoxifier of xenobiotic toxicants present in the aquatic environment, and that its mRNA expression could be modulated by pathogenic stress signal(s).