Molecular characterization of kappa class glutathione S‐transferase from the disk abalone (Haliotis discus discus) and changes in expression following immune and stress challenges
Molecular characterization of kappa class glutathione S‐transferase from the disk abalone (Haliotis discus discus) and changes in expression following immune and stress challenges
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DOI：
10.1016/j.fsi.2018.03.058
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发表时间：
2018-06
影响因子：
4.7
通讯作者：
Gayashani W.M. Sandamalika;Thanthrige Thiunuwan Priyathilaka;D. S. Liyanage;Sukkyoung Lee;H. Lim;Jehee Lee-Jehee
中科院分区：
文献类型：
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作者：
Gayashani W.M. Sandamalika;Thanthrige Thiunuwan Priyathilaka;D. S. Liyanage;Sukkyoung Lee;H. Lim;Jehee Lee-Jehee
谷胱甘肽S-转移酶（GST; EC 2.5.1.18）同工酶代表了一组复杂的蛋白质，它们参与了几种生物体的II期解毒。在这项研究中，GST κ（GSTκ）从盘鲍（Haliotis discus discus; AbGSTκ）的转录和功能水平的特点，以确定其潜在的能力，作为解毒剂在不同的压力条件下执行。预测的AbGSTκ蛋白由227个氨基酸组成，分子量为25.6kDa，理论等电点为7.78。计算机分析表明，AbGST κ是一个二硫键形成蛋白A（DsbA），由硫氧还蛋白结构域、谷胱甘肽结合位点（G-位点）和催化残基组成。结果表明，AbGSTκ基因与暗纹东方鲀同源性最高（60.0%）。在系统发育树中，AbGSTκ与其它鱼类GSTκ紧密聚在一起，与其它胞质GST在进化上距离较远。预测的三维结构清楚地表明，二聚体采用蝴蝶状的形状。组织分布分析表明，GST κ在消化道中高表达，具有解毒作用。根据不同的组织和时间，AbGSTκ显示不同的表达模式和表达水平，以下鲍鱼与免疫刺激剂的挑战。纯化的重组蛋白的酶动力学证明了其以1-氯-2，4-二硝基苯（CDNB）和谷胱甘肽（GSH）为底物的缀合能力，并表明其对两种底物具有低亲和力。rAbGSTκ GSH：CDNB结合活性的最适温度和pH分别为35 °C和8，表明鲍鱼对广泛的环境条件具有良好的适应性。Cibacron blue（100 μM）可完全抑制rAbGSTκ（100%），半数抑制浓度（IC 50）为0.05 μM。纸片扩散法显示rAbGSTκ对H2 O2、CdCl 2和ZnCl 2有明显的保护作用。总之，目前的研究表明，AbGST κ参与解毒和免疫宿主防御机制，并允许鲍鱼克服压力，以增加它们的生存机会。
Glutathione S-transferase (GST; EC 2.5.1.18) isoenzymes represent a complex group of proteins that are involved in phase II detoxification in several organisms. In this study, GST kappa (GSTκ) from the disk abalone (Haliotis discus discus; AbGSTκ) was characterized at both the transcriptional and functional levels to determine its potential capacity to perform as a detoxification agent under conditions of different stress. The predicted AbGSTκ protein consists of 227 amino acids, with a predicted molecular weight of 25.6 kDa and a theoretical isoelectric point (pI) of 7.78.In silicoanalysis reveals thatAbGSTκis a disulfide bond formation protein A (DsbA), consisting of a thioredoxin domain, GSH binding sites (G-sites), and a catalytic residue. In contrast, no hydrophobic ligand binding site (H-site), or signal peptides, were detected.AbGSTκshowed the highest sequence identity with the orthologue from pufferfish (Takifugu obscurus) (60.0%). In a phylogenetic tree, AbGSTκ clustered closely together with other fish GSTκs, and was evolutionarily distanced from other cytosolic GSTs. The predicted three-dimensional structure clearly demonstrates that the dimer adopts a butterfly-like shape. A tissue distribution analysis revealed thatGSTκwas highly expressed in the digestive tract, suggesting it has detoxification ability. Depending on the tissue and time,AbGSTκshowed different expression patterns, and levels of expression, following challenge of the abalone with immune stimulants. Enzyme kinetics of the purified recombinant proteins demonstrated its conjugating ability using 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates, and suggested it has a low affinity for both substrates. The optimum temperature and pH for the rAbGSTκ GSH: CDNB conjugating activity were found to be 35 °C and pH 8, respectively indicating that the abalone is well adapted to a wide range of environmental conditions. Cibacron blue (100 μM) was capable of completely inhibiting rAbGSTκ (100%) with an IC50(half maximal inhibitory concentration) of 0.05 μM. A disk diffusion assay revealed that rAbGSTκ could significantly protect cells from H2O2, CdCl2,and ZnCl2. Altogether, this current study suggests thatAbGSTκis involved in detoxification and immunological host defense mechanisms and allows abalones to overcome stresses in order for them to have an increased chance of survival.