Molecular cloning and functional characterization of theta class glutathione S-transferase fromApostichopus japonicus

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Glutathione S-transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play crucial roles in innate immunity. In the present study, a theta class GST homology was identified from A. japonicus (designated as AjGST-Θ) by RACE approaches. The full-length cDNA of AjGST-Θ was of 1013 bp encoded a cytosolic protein of 231 amino acids residues. Structural analysis revealed that AjGST-Θ processed the characteristic N-terminal GSH-binding site (G-site) and the C-terminal hydrophobic substrate binding site (H-site). Multiple sequence alignment and phylogenetic analysis together supported that AjGST-Θ belonged to a new member of theta class GST protein subfamily. Spatial expression analysis revealed that AjGST-Θ was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up-regulate the mRNA expression of AjGST-Θ when compared with control group. The recombinant protein was expressed in Escherichia coli and the purified AjGST-Θ showed high activity with GST substrate. Meantime, disc diffusion assay showed that recombinant AjGST-Θ protein could markedly improve bacterial growth under Cumene hydroperoxide exposure. More importantly, the recombinant AjGST-Θ could effectively prevent primary coelomocytes apoptosis after LPS exposure. Our present findings suggested that AjGST-Θ might play significantly roles in the modulation of immune response and protect cells from pathogens infection in A. japonicus.

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