A mu class glutathione S-transferase from Manila clam Ruditapes philippinarum (RpGSTμ): cloning, mRNA expression, and conjugation assays.
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 25.04 kDa 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 ± 0.46 mM and 0.56 ± 0.20 mM, 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 0.65 μM and 9 μM, 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).
Department of Marine Life Sciences, , Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
S D N K
SD
Bathige
Department of Marine Life Sciences, , Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
Navaneethaiyer
N
Umasuthan
Department of Marine Life Sciences, , Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
Kasthuri
K
Saranya Revathy
Department of Marine Life Sciences, , Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
Youngdeuk
Y
Lee
Aquatic Life Disease Control Division, National Fisheries and Research & Developmental Institute, Busan 619-705, Republic of Korea.
Seokryel
S
Kim
Aquatic Life Disease Control Division, National Fisheries and Research & Developmental Institute, Busan 619-705, Republic of Korea.
Mi Young
MY
Cho
Aquatic Life Disease Control Division, National Fisheries and Research & Developmental Institute, Busan 619-705, Republic of Korea.
Myoung-Ae
MA
Park
Department of Marine Life Sciences, , Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea. Electronic address: ilsonwhang@hanmail.net.
Ilson
I
Whang
Department of Marine Life Sciences, , Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea. Electronic address: jehee@jejunu.ac.kr.
Jehee
J
Lee
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D049892:Mya / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014735:Vibrio Infections / Q000378:metabolism
D000276:Adjuvants, Immunologic; D012333:RNA, Messenger; D005982:Glutathione Transferase
Catalytic activity; Immune response; Inhibitory activity; Mu class glutathione S-transferase; Ruditapes philippinarum