Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Liaoning Key Laboratory of Marine Animal Immunology and 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; Functional Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China; Liaoning Key Laboratory of Marine Animal Immunology and 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. 2022 Aug;127:129-139. doi: 10.1016/j.fsi.2022.05.057. Epub 2022 Jun 14.
The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p < 0.05) and haemocytes (3.62-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p < 0.001) to 24 h (0.20-fold of that in the control group, p < 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p < 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p < 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p < 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p < 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-β) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP.
RAC-alpha 丝氨酸/苏氨酸蛋白激酶（AKT）是真核生物中参与许多生物过程的最重要的蛋白激酶之一。在本研究中，从太平洋牡蛎（Crassostrea gigas）中鉴定出一种名为 CgAKT1 的新型 AKT 同源物。CgAKT1 cDNA 的开放阅读框（ORF）为 1482 bp，编码具有 493 个氨基酸残基的肽。预测的 CgAKT1 蛋白具有经典结构域，包括 N 端pleckstrin 同源结构域、中央催化结构域和 C 端疏水性结构域。在 C. gigas 的所有检查组织中均检测到 CgAKT1 的 mRNA 转录物，其中在鳃（比套膜高 8.24 倍，p<0.05）和血细胞（比套膜高 3.62 倍，p<0.05）中的水平较高。在用 poly（I:C）刺激后，血细胞中 CgAKT1 的 mRNA 表达在 3 h（对照组的 0.44 倍，p<0.001）至 24 h（对照组的 0.20 倍，p<0.001）时显著降低，然后在 48 h 时显著增加（对照组的 3.65 倍，p<0.05）。在用 rCgIFNLP 刺激后，CgAKT1 mRNA 的表达水平在 6 h 时显著增加，是对照组的 3.60 倍（p<0.001）。在血细胞中，CgAKT1 蛋白的 Alexa Fluor 488 阳性信号分布在细胞质和细胞膜中，而在用 poly（I:C）刺激后，细胞质中的信号变弱。在 CgAKT1-RNAi 牡蛎中，环状 GMP-AMP 合酶（CgcGAS）和 TANK 结合激酶 1（CgTBK1）的 mRNA 表达没有明显变化，但刺激干扰素基因（CgSTING）、干扰素调节因子-1（CgIRF-1）、干扰素调节因子-8（CgIRF-8）和 IFN 样蛋白（CgIFNLP）的 mRNA 表达水平显著增加，分别为 EGFP-RNAi 牡蛎的 1.40 倍、1.53 倍、1.72 倍和 1.99 倍（p<0.05）。在 CgIFNLP-RNAi 牡蛎中，与 EGFP-RNAi 牡蛎相比，CgAKT1 的转录物显著降低（0.16 倍，p<0.01）。此外，在用 poly（I:C）刺激后，用 p-AKT1 激活剂（SC-79）处理的牡蛎中 p-CgTBK1、CgSTING 和 CgIFNLP 的蛋白水平表达显著受到抑制。转染 CgAKT1 后，HEK293T 细胞中 p-cGAS 蛋白的表达显著增加，而细胞中环式 GMP-AMP 和细胞培养液中的干扰素（IFN-β）明显低于对照组。这些结果表明，CgAKT1 可能通过调节 CgIFNLP 的合成在牡蛎抗病毒免疫中发挥负调控作用。
