Molecular cloning and characterization of omega class glutathione S-transferase (GST-O) from the polychaete Neanthes succinea: biochemical comparison with theta class glutathione S-transferase (GST-T).
We cloned and sequenced a full-length cDNA of an omega class glutathione S-transferase (GST-O) from the polychaete Neanthes succinea (ns-GST-O). The full-length cDNA of ns-GST-O was 1562 bp in length, containing an open reading frame (OR) of 732 bp that encoded a 244 amino acid protein. The deduced amino acid sequence of ns-GST-O showed a low similarity with the theta class N. suucinea GST (ns-GST-T). As GSTs play a significant role in antioxidant defense, we checked the expression pattern of ns-GST-O in N. succinea after exposure to copper (CuCl(2) 12 to 72 mug/L), which is an oxidative stress-inducing agent. After exposure to CuCl(2), ns-GST-O gene was dramatically up-regulated and when compared with ns-GST-T the expression pattern was more pronounced at all the concentrations of copper. Even the basal transcription levels of ns-GST-O were higher than those of ns-GST-T. To further characterize the catalytic properties of ns-GST-O, we constructed a recombinant ns-GST-O plasmid with a 6x His-Tag at the N-terminal of the full-length ns-GST-O cDNA. Recombinant ns-GST-O protein was highly expressed in transformed Escherichia coli. The effect of pH, temperature and chemical inhibitors on the enzyme activity of ns-GST-O was also studied and compared with the reported effect of these factors on recombinant ns-GST-T protein. These results suggest that, like other types of GSTs, ns-GST-O protein plays a conserved antioxidant role in the polychaete N. succinea.
Rhee
Jae-Sung
JS
Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, South Korea.
Lee
Young-Mi
YM
Hwang
Dae-Sik
DS
Lee
Kyun-Woo
KW
Kim
Il-Chan
IC
Shin
Kyung-Hoon
KH
Raisuddin
Sheikh
S
Lee
Jae-Seong
JS
D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000502:physiology; D001483:Base Sequence; D015415:Biomarkers; D003001:Cloning, Molecular; D003300:Copper / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011077:Polychaeta / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D014874:Water Pollutants, Chemical / Q000633:toxicity
D000975:Antioxidants; D015415:Biomarkers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014874:Water Pollutants, Chemical; D003300:Copper; D005982:Glutathione Transferase; C029892:cupric chloride