Fas/FasL of pacific cod mediated apoptosis

• Published in: Developmental and comparative immunology 2021-06, Vol.119, p.104022-104022, Article 104022
• Main Authors: Mao, Ming-Guang, Xu, Jia, Liu, Rui-Ting, Ye, Lin, Wang, Rui, Jiang, Jie-lan
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Apoptosis; Apoptosis - genetics; Base Sequence; Bax protein; Bcl-2 protein; Caspase-3; Caspase-9; Cell membranes; Cloning, Molecular; Cod; Cytoplasm; FADD; Fas; Fas Ligand Protein - chemistry; Fas Ligand Protein - genetics; Fas Ligand Protein - metabolism; fas Receptor - chemistry; fas Receptor - genetics; fas Receptor - metabolism; Fas-Associated Death Domain Protein - chemistry; Fas-Associated Death Domain Protein - genetics; Fas-Associated Death Domain Protein - metabolism; FasL; FasL protein; Fish; Fish diseases; Fish Proteins - chemistry; Fish Proteins - genetics; Fish Proteins - metabolism; Flow cytometry; Gadiformes - genetics; Gadiformes - metabolism; Gadus macrocephalus; Gene expression; Gene Expression Profiling; Hatching; Homology; Larvae; Ligands; Localization; Models, Molecular; Necrosis; Nervous necrosis virus; Protein Binding; Protein Domains; Proteins; Sequence Analysis, DNA; Signal transduction; Signal Transduction - genetics; Signal transmission; Three dimensional models; Transcription; Viruses
• ISSN: 0145-305X; 1879-0089
• DOI: 10.1016/j.dci.2021.104022

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. [Display omitted] •Bioinformatic analysis of Fas, FasL and FADD in Pacific cod was conducted.•The relationship between NNV, Fas/FasL and apoptosis was examined.•Overexpression of PcFas and PcFasL in EPC cells will lead to cell apoptosis, respectively.