Mao Ming-Guang, Xu Jia, Liu Rui-Ting, Ye Lin, Wang Rui, Jiang Jie-Lan
Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture and Rural Affairs, College of Fisheries and Life Sciences, Dalian Ocean University, Dalian, 116023, China.
Dev Comp Immunol. 2021 Jun;119:104022. doi: 10.1016/j.dci.2021.104022. Epub 2021 Jan 19.
Fas and Fas ligand (FasL) pathway plays important roles in virus defense and cell apoptosis. In our previous work, nervous necrosis virus (NNV) was discovered in Pacific cod (Gadus macrocephalus), and the Fas ligand (PcFasL) was up-regulated when NNV outbreak, however, signal transmission of Fas/FasL in fish are still unclear. In the present study, Pacific cod Fas (PcFas), PcFasL and Fas-associating protein with a novel death domain (PcFADD) were characterized. The predicted protein of PcFas, PcFasL and PcFADD includes 333 aa, 90 aa and 93 aa, separately. 3-D models of PcFas, PcFasL and PcFADD were well constructed based on reported templates, respectively, even though the sequence homology with other fish is very low. The transcript levels of PcFas increased gradually from 15 day-post hatching (dph) to 75dph. PcFas was significantly up-regulated when cod larvae had NNV symptoms at 24dph, 37dph, 46dph, 69dph, and 77dph. Subcellular localization revealed that PcFasL was located in the cytoplasm, while PcFas was mainly located in the cell membrane. Exogenous expressed PcFasL of 900 μg/mL could kill the Epithelioma papulosum cyprinid (EPC) cells by MTT test, but low concentration has no effect on the cells. qPCR analysis showed that overexpression of PcFas could significantly up-regulate the expression of genes related to Fas/FasL signaling pathway, including bcl-2, bax, and RIP3, while overexpression of PcFasL significantly up-regulate the expression of caspase-3, caspase-9, and MLKL. Overexpression of PcFas or PcFasL could induce EPC apoptosis significantly by flow cytometry, which was consistent with the results of caspase-3 mRNA level increasing. The results indicated that NNV could induce apoptosis through Fas/FasL signal pathway.
Fas和Fas配体（FasL）通路在病毒防御和细胞凋亡中发挥着重要作用。在我们之前的工作中，在太平洋鳕鱼（大头鳕）中发现了神经坏死病毒（NNV），并且在NNV爆发时Fas配体（PcFasL）上调，然而，鱼类中Fas/FasL的信号传导仍不清楚。在本研究中，对太平洋鳕鱼Fas（PcFas）、PcFasL和具有新死亡结构域的Fas相关蛋白（PcFADD）进行了表征。PcFas、PcFasL和PcFADD的预测蛋白分别包含333个氨基酸、90个氨基酸和93个氨基酸。尽管与其他鱼类的序列同源性非常低，但分别基于已报道的模板成功构建了PcFas、PcFasL和PcFADD的三维模型。PcFas的转录水平从孵化后15天（dph）到75 dph逐渐增加。当鳕鱼幼体在24 dph、37 dph、46 dph、69 dph和77 dph出现NNV症状时，PcFas显著上调。亚细胞定位显示，PcFasL位于细胞质中，而PcFas主要位于细胞膜上。通过MTT试验，900 μg/mL的外源表达PcFasL可杀死鲤上皮瘤（EPC）细胞，但低浓度对细胞无影响。qPCR分析表明，PcFas的过表达可显著上调与Fas/FasL信号通路相关基因的表达，包括bcl-2、bax和RIP3，而PcFasL的过表达显著上调caspase-3、caspase-9和MLKL的表达。通过流式细胞术，PcFas或PcFasL的过表达可显著诱导EPC凋亡，这与caspase-3 mRNA水平升高的结果一致。结果表明，NNV可通过Fas/FasL信号通路诱导细胞凋亡。
