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; Laboratory of Marine Fisheries Science and Food Production Process, 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.
Dev Comp Immunol. 2021 Sep;122:104137. doi: 10.1016/j.dci.2021.104137. Epub 2021 May 20.
The Methyltransf_FA domain is well-known as a key protein domain of enzyme synthesizing juvenile hormone, and Methyltransf_FA domain containing proteins (MFCPs) are widely existed in vertebrates and invertebrates. In the present study, a CgMFCP with a single Methyltransf_FA domain was screened from oyster Crassostrea gigas, and its open reading frame of CgMFCP was of 1128 bp, encoding a polypeptide of 376 amino acids with a signal peptide, a Methyltransf_FA domain and a transmembrane region. CgMFCP was clustered with FAMeTs from insecta and crustacea of arthropod. The mRNA transcripts of CgMFCP were detected in different tissues, with the extremely high expression level in haemocytes, which was 131.36-fold (p < 0.05) of that in gills. The expression level of CgMFCP protein was verified to be highly expressed in haemocytes. The expression level of CgMFCP mRNA in primarily cultured haemocytes significantly up-regulated at 3 h, 24 h and 48 h post LPS stimulation, which was 3.25-fold (p < 0.01), 2.04-fold (p < 0.05) and 3.59-fold (p < 0.01) compared to that in blank group. After the oysters were stimulated with Vibrio splendidus in vivo, the expression level of CgMFCP mRNA in haemocytes was also significantly up-regulated at 3 h, 12 h, and 24 h, which was 4.22-fold (p < 0.05), 4.39-fold (p < 0.05) and 6.35-fold (p < 0.01) of that in control group, respectively. By flow cytometry analysis, anti-rCgMFCP can label 95% of oyster haemocytes. And by fluorescence microscope analysis, CgMFCP was mainly distributed in cytomembrane of haemocytes. The recombinant CgMFCP (rCgMFCP) exhibited higher affinity towards MAN and LPS in a dose-dependent manner, while relatively lower affinity to PGN and poly (I:C). rCgMFCP also displayed binding activities towards Gram-negative bacteria (Vibrio anguillarum and V. splendidus), Gram-positive bacteria (Staphylococcu aureu) and fungi (Pichia pastoris). These results collectively indicated that CgMFCP specifically expressed in haemocytes and functioned as a pattern recognition receptor by binding to various microbes in oyster C. gigas, which provided insight into the function of Methyltransf_FA domain containing proteins.
甲基转移酶 FA 结构域是合成保幼激素的关键酶的蛋白质结构域，甲基转移酶 FA 结构域包含蛋白（MFCPs）广泛存在于脊椎动物和无脊椎动物中。本研究从牡蛎 Crassostrea gigas 中筛选到一个具有单个甲基转移酶 FA 结构域的 CgMFCP，其开放阅读框为 1128bp，编码一个含有信号肽、甲基转移酶 FA 结构域和跨膜区的 376 个氨基酸的多肽。CgMFCP 与昆虫和节肢动物甲壳类的 FAMeTs 聚类在一起。CgMFCP 的 mRNA 转录本在不同组织中均有检测到，在血细胞中表达水平极高，比鳃中高 131.36 倍（p<0.05）。CgMFCP 蛋白的表达水平在血细胞中被证实高度表达。在 LPS 刺激后 3h、24h 和 48h，原代培养血细胞中 CgMFCPmRNA 的表达水平显著上调，分别是空白组的 3.25 倍（p<0.01）、2.04 倍（p<0.05）和 3.59 倍（p<0.01）。在体内用灿烂弧菌刺激牡蛎后，血细胞中 CgMFCPmRNA 的表达水平也在 3h、12h 和 24h 时显著上调，分别是对照组的 4.22 倍（p<0.05）、4.39 倍（p<0.05）和 6.35 倍（p<0.01）。通过流式细胞术分析，抗 rCgMFCP 可以标记 95%的牡蛎血细胞。通过荧光显微镜分析，CgMFCP 主要分布在血细胞的细胞质膜上。重组 CgMFCP（rCgMFCP）以剂量依赖性方式对 MAN 和 LPS 表现出较高的亲和力，而对 PGN 和 poly（I:C）的亲和力较低。rCgMFCP 还与革兰氏阴性菌（鳗弧菌和灿烂弧菌）、革兰氏阳性菌（金黄色葡萄球菌）和真菌（毕赤酵母）具有结合活性。这些结果共同表明，CgMFCP 特异性表达于血细胞中，并通过与牡蛎 C. gigas 中的各种微生物结合作为一种模式识别受体发挥作用，为甲基转移酶 FA 结构域包含蛋白的功能提供了新的见解。
