Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Liaoning of Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China.
Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China; Liaoning of Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China.
Fish Shellfish Immunol. 2020 Apr;99:442-451. doi: 10.1016/j.fsi.2020.02.042. Epub 2020 Feb 19.
The homeostasis of immune cells during immune response is vital for hosts to defend against invaders. Activating transcription factor 6 (ATF6) is an important transcription factor in the unfolded protein response (UPR) to maintaining cellular homeostasis. In the present study, one ATF6 homologue was identified from Pacific oyster Crassostrea gigas (designated as CgATF6β). The full length cDNA of CgATF6β was of 2645 bp with a 1596 bp open reading frame (ORF) encoding a polypeptide of 531 amino acids. The deduced amino acid sequence of CgATF6β was predicted to contain a transmembrane region, a conserved basic leucine zipper (bZIP) domain, a site 1 protease cleavage site, a site 2 protease cleavage site, and a Golgi localization signal. CgATF6β mRNA was constitutively expressed in hemocytes, gill, mantle, gonad, hepatopancreas and labial palp, with a slightly higher expression level in muscle (2.45-fold of that in gill, p < 0.05). After oysters were challenged with Vibrio splendidus, the mRNA expression levels of CgATF6β in hemocytes were significantly up-regulated at 3 h (2.68-fold of that in seawater group, p < 0.01) and peaked at 12 h (3.14-fold of that in seawater group, p < 0.01). The endogenic CgATF6β protein was mainly located in the cytoplasm of oyster hemocytes, and it was significantly transported into the nuclei of hemocytes at 1.5 h after the challenge with V. splendidus. After an injection with CgATF6β dsRNA, the mRNA expression of CgATF6β was knocked down to 0.26-fold of that in dsGFP group (p < 0.01). In CgATF6β dsRNA-injected oysters, the mRNA expressions of glucose-regulated protein 78 (GRP78), calnexin (CNX) and anti-apoptotic B-cell lymphoma-2 (Bcl-2) in hemocytes were significantly decreased at 12 h after V. splendidus challenge, which were 0.65-fold (p < 0.01), 0.54-fold (p < 0.01) and 0.17-fold (p < 0.01) of that in dsGFP-injected oysters, while the apoptotic rate of hemocytes was significantly up-regulated (1.97-fold of that in dsGFP group, p < 0.05). Collectively, these results suggested that CgATF6β was involved in apoptosis inhibition of oyster hemocytes upon V. splendidus challenge by regulating the expression of CgGRP78, CgCNX and CgBcl-2.
免疫细胞在免疫反应期间的动态平衡对于宿主抵御入侵至关重要。激活转录因子 6（ATF6）是未折叠蛋白反应（UPR）中维持细胞动态平衡的重要转录因子。在本研究中，从太平洋牡蛎（Crassostrea gigas）中鉴定出一种 ATF6 同源物（命名为 CgATF6β）。CgATF6β 的全长 cDNA 为 2645 bp，包含 1596 bp 的开放阅读框（ORF），编码 531 个氨基酸的多肽。CgATF6β 的推定氨基酸序列预测包含一个跨膜区、一个保守的碱性亮氨酸拉链（bZIP）结构域、一个位点 1 蛋白酶切割位点、一个位点 2 蛋白酶切割位点和一个高尔基定位信号。CgATF6β mRNA 在血细胞、鳃、套膜、性腺、肝胰腺和唇瓣中持续表达，在肌肉中的表达水平略高（比鳃高 2.45 倍，p<0.05）。在牡蛎受到灿烂弧菌挑战后，血细胞中 CgATF6β 的 mRNA 表达水平在 3 小时时显著上调（比海水组高 2.68 倍，p<0.01），并在 12 小时时达到峰值（比海水组高 3.14 倍，p<0.01）。内源性 CgATF6β 蛋白主要位于牡蛎血细胞的细胞质中，在受到灿烂弧菌刺激后 1.5 小时，其明显转运到血细胞的核内。在注射 CgATF6β dsRNA 后，CgATF6β 的 mRNA 表达被敲低至 dsGFP 组的 0.26 倍（p<0.01）。在 CgATF6β dsRNA 注射的牡蛎中，在受到灿烂弧菌刺激 12 小时后，血细胞中葡萄糖调节蛋白 78（GRP78）、钙连蛋白（CNX）和抗凋亡 B 细胞淋巴瘤-2（Bcl-2）的 mRNA 表达显著降低，分别为 dsGFP 注射牡蛎的 0.65 倍（p<0.01）、0.54 倍（p<0.01）和 0.17 倍（p<0.01），而血细胞的凋亡率显著升高（比 dsGFP 组高 1.97 倍，p<0.05）。综上所述，这些结果表明，CgATF6β 通过调节 CgGRP78、CgCNX 和 CgBcl-2 的表达，参与牡蛎血细胞在受到灿烂弧菌刺激时的凋亡抑制。
