A delta-class glutathione transferase from the Chinese mitten crab Eriocheir sinensis: cDNA cloning, characterization and mRNA expression.
Glutathione-S-transferases (GSTs), a major superfamily of antioxidative enzymes, play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The delta-class GST cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) from the Chinese mitten crab, Eriocheir sinensis. The 938 bp E. sinensis GST gene, encoding a polypeptide of 216 amino acids, showed significant similarity to homologous genes in insects. The deduced amino acid sequence of the crab GST contains conserved features of this gene family including the G-site (1-82 aa, tripeptide glutathione binding site) in the N-terminal region and an H-site (88-213 aa, substrate binding site) in the C-terminus. Additionally, a kinase C phosphorylation site (ITI) and one putative N-linked glycosylation site (DNIT) for N-linked carbohydrate chains were also identified. Quantitative real-time RT-PCR (qRT-PCR) was employed to investigate the distribution of GST mRNA in different tissues and its temporal expression in haemocytes of crabs challenged with Aeromonas hydrophila. Different levels of GST mRNA expression were detected in haemocytes, hepatopancreas and muscle, while it was not detected in the gills, intestines and stomach. GST transcription was significantly induced in haemocytes at 6 h post-bacterial challenge (P < 0.05) and dropped to basal levels at 12 h, presumably down-regulated by increased bacteremia by that time. These findings suggest that GST could play a critical role in immunity, and this positive feedback mechanism can allow for efficient activation of the early innate immune response to infection.
Department of Biology, East China Normal University, Shanghai 200062, PR China.
Daxian
D
Zhao
Liqiao
L
Chen
Chuanjie
C
Qin
Hao
H
Zhang
Ping
P
Wu
Fengying
F
Zhang
D016980:Aeromonas hydrophila / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000382:microbiology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006434:Hemocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D012333:RNA, Messenger; D005982:Glutathione Transferase