First molluscan theta-class Glutathione S-Transferase: Identification, cloning, characterization and transcriptional analysis post immune challenges

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• 作者：Kasthuri Saranya Revathy^a ; Navaneethaiyer Umasuthan^a ; Youngdeuk Lee^a ; Cheol Young Choi^b ; Ilson Whang^a ; Jehee Lee^a ; ^c ; ^{jehee@jejunu.ac.kr}
• 关键词：Theta class Glutathione S-Transferase ; Detoxification enzyme in Ruditapes philippinarum ; Transcriptional expression ; Immune challenge ; Vibrio tapetis
• 刊名：Comparative Biochemistry and Physiology ; Part B
• 出版年：2012
• 期刊代码：50_10964959
• 类别：bio
• 出版时间：May, 2012
• 卷：162
• 期：1-3
• 页码：10-23
• 文件大小：2086 K

摘要

Glutathione S-Transferases (GSTs) are multifunctional cytosolic isoenzymes, distinctly known as phase II detoxification enzymes. GSTs play a significant role in cellular defense against toxicity and have been identified in nearly all organisms studied to date, from bacteria to mammals. In this study, we have identified a full-length cDNA of the theta class GST from Ruditapes philippinarum (RpGST胃), an important commercial edible molluscan species. RpGST胃 was cloned and the recombinant protein expressed, in order to study its biochemical characteristics and determine its physiological activities. The cDNA comprised an ORF of 693 bp, encoding 231 amino acids with a predicted molecular mass of 27 kDa and an isoelectric point of 8.2. Sequence analysis revealed that RpGST胃 possessed characteristic conserved domains of the GST_N family, Class Theta subfamily (PSSM: cd03050) and GST_C_family Super family (PSSM: cl02776). Phylogenetic analysis showed that RpGST胃 evolutionarily linked with other theta class homologues. The recombinant protein was expressed in Escherichia coli BL21(DE3) cells and the purified enzyme showed high activity with GST substrates like CDNB and 4-NBC. Glutathione dependent peroxidase activity of GST, investigated with cumene hydroperoxide as substrate affirmed the antioxidant property of rRpGST胃. By quantitative PCR, RpGST胃 was found to be ubiquitously expressed in all tissues examined, with the highest levels occurring in gills, mantle, and hemocytes. Since GSTs may act as detoxification enzymes to mediate immune defense, the effects of pathogen associated molecular pattern, lipopolysaccharide and intact Vibrio tapetis bacteria challenge on RpGST胃 gene transcription were studied. Furthermore, the RpGST胃 expression changes induced by immune challenges were similar to those of the antioxidant defense enzyme manganese superoxide dismutase (RpMnSOD). To our knowledge, RpGST胃 is the first molluscan theta class GST reported, and its immune-related role in Manila clam may provide insights into potential therapeutic targets for protecting this important aquaculture species.