Environmental stressors (salinity, heavy metals, H 2O 2) modulate expression of glutathione reductase (GR) gene from the intertidal copepod Tigriopus japonicus

Abstract

Glutathione reductase (GR) plays an essential role in cell defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant, glutathione. To address the effect of oxidative stresses on the intertidal copepod Tigriopus japonicus, we exposed specimens to hydrogen peroxide, heavy metals and different salinity levels, cloned and sequenced the oxidative stress-related GR gene. T. japonicus GR gene ( Tigriopus GR) cDNA contained 1526 bp including an open reading frame (ORF) encoding 458 amino acids with a theoretical p I of 6.58 and a calculated molecular weight of 49.6 kDa. Tigriopus GR showed a high similarity to frog Xenopus laevis GR (identity 57%) and the filarial parasite, Onchocerca volvulus GR (identity 57%). Specific motifs such as flavin adenine dinucleotide-binding site (LVLGGGSGGIASARRAAEF), pyridine nucleotide-disulphide oxidoreductases class-I active site (GGTCVNVGCVP), and NADPH binding motif (GxGYIAx18Rx5R) were highly conserved in the deduced amino acid sequence of Tigriopus GR. Interestingly, its expression and enzyme characteristics were different from GR homologue of filarial parasite O. volvulus. To investigate the biochemical and enzymatic characteristics of Tigriopus GR protein, we constructed the expression vector, pCRT7/TOPO NT containing Tigriopus GR. Tigriopus pCRT7/TOPO NT/GR was expressed in Escherichia coli, and the soluble protein was purified by 6× His-tag chromatography. The recombinant Tigriopus GR enzyme was found to make homodimer complexes of approximately 108 kDa on 12% native gel electrophoresis and showed enzymatic activity with NADPH and oxidized glutathione (GSSG) as substrates. To analyze the gene expression of Tigriopus GR against different environmental stresses (hydrogen peroxide, salinity, and heavy metals), we performed real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). Slight down-regulation in the expression of Tigriopus GR at the initial stage was observed upon exposure to hydrogen peroxide. The expression recovered in 2 h, while there were significant changes upon heavy metal (Cu and Mn) exposures in a time-dependent manner. Also, Tigriopus GR expression was significantly increased with moderately high salt stress (24 and 40 ppt). In the case of low salt stress (0 and 12 ppt) the expression was found to be down-regulated. These findings provide a better understanding of cellular protection mechanisms in the intertidal copepod T. japonicus against the environmental stressors caused by non-optimal salt levels.