Molecular cloning and characterization of Aos1 and Uba2 from the orange-spotted grouper ( Epinephelus coioides )
Molecular cloning and characterization of Aos1 and Uba2 from the orange-spotted grouper ( Epinephelus coioides )
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斜带石斑鱼 (Epinephelus coioides) Aos1 和 Uba2 的分子克隆和表征
DOI：
10.1016/j.fsi.2018.07.043
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发表时间：
2018
影响因子：
4.7
通讯作者：
Qiwei Qin
中科院分区：
文献类型：
--
作者：
Jingguang Wei;Chen Li;Xin Zhang;Sheng Zhou;Shaoqing Zang;Shina Wei;Qiwei Qin
小泛素相关修饰物（SUMO）是一种与其他蛋白质发生后结合的蛋白质，是多种细胞过程的重要调节因子。SUMO的共价连接需要一个酶级联反应，包括一个单一的E1激活酶（Aos 1和Uba 2异源二聚体），一个单一的E2结合酶（Ubc 9），和几个E3连接酶，促进SUMO从Ubc 9转移到底物之一。本研究克隆了斜带石斑鱼（Epineoparus coioides）Aos 1和Uba 2同源基因（EcAos 1和EcUba 2），并分析了它们在鱼类免疫中的可能作用。EcAos 1的开放阅读框（ORF）包含1050个碱基对（bp），编码350个氨基酸的蛋白质，预测分子量为38.97 kDa，在193-203位残基处具有核定位信号（NLS）。EcUba 2的ORF全长1950 bp，编码650个氨基酸，预测分子量为71.3 kDa。实时定量聚合酶链反应分析表明，EcAos 1和EcUba 2分布在所有检查的组织。EcAos 1和EcUba 2在大肠杆菌脾脏和头肾中的表达水平。当用聚肌胞苷酸攻击时，类胡萝卜素差异上调。pEGFP-C1-EcAos 1和pEGFP-C1-EcUba 2均呈绿色荧光分布于GS细胞核内。当EcAos 1和EcUba 2的NLS被删除时，细胞定位都发生了变化。过量表达EcAos 1和EcUba 2可抑制赤点石斑鱼神经坏死病毒的感染和复制。这些结果对于更好地理解鱼类SUMO通路和深入了解病毒感染的调控机制具有重要意义。在养殖条件下。
Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are essential regulators of a wide range of cellular processes. Covalent attachment of SUMO requires an enzymatic cascade consisting of a single E1-activating enzyme (Aos1 and Uba2 heterodimer), a single E2-conjugating enzyme (Ubc9), and one of several E3 ligases that facilitate transfer of SUMO from Ubc9 to the substrate. In the present study, the Aos1 and Uba2 homologues (EcAos1 and EcUba2) from the orange-spotted grouper (Epinephelus coioides) were cloned and their possible roles in fish immunity were analyzed. The open reading frame (ORF) of EcAos1 contains 1050 base pairs (bp) encoding a 350 amino acid protein with a predicted molecular mass of 38.97 kDa EcAos1 has a nuclear localization signal (NLS) at residues 193–203. The ORF of EcUba2 contains 1950 bp encoding a 650 amino acid protein with a predicted molecular mass of 71.3 kDa EcUba2 has a NLS at residues 608–630. Quantitative real-time polymerase chain reaction analysis indicated that both EcAos1 and EcUba2 were distributed in all examined tissues. The expression levels of EcAos1 and EcUba2 in the spleen and head kidney ofE. coioideswere differentially up-regulated when challenged with polyinosine-polycytidylic acid. Green fluorescence of both pEGFP-C1-EcAos1 and pEGFP-C1-EcUba2 was distributed in the nucleus of GS cells. When the NLSs of EcAos1 and EcUba2 were deleted, the cellular localizations all changed. Over-expression of EcAos1 and EcUba2 inhibited red-spotted grouper nervous necrosis virus infection and replication. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection inE. coioidesunder farmed conditions.