Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus)
Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus)
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尼罗罗非鱼 (Oreochromis niloticus) NOD1、NOD2 和 NLRC3 的分子特征、表达和功能分析
DOI：
10.1016/j.fsi.2017.12.012
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发表时间：
2018-02-01
影响因子：
4.7
通讯作者：
Wang, Miao
中科院分区：
文献类型：
--
作者：
Gao, Feng-ying;Pang, Ji-cai;Wang, Miao
核苷酸结合寡聚化结构域蛋白NOD 1、NOD 2和NLRC 3是Nod样受体（NLR）家族的细胞质模式识别受体（PRR）。本研究克隆了尼罗罗非鱼（Oreochromis niloticus）NOD 1（ntWOD 1）、NOD 2（ntWOD 2）和NLRC 3（ntNLRC 3）基因，并对其进行了序列分析。ntWOD 1、ntNOD 2和ntNLRC 3基因全长分别为3924、3886和4574 bp，编码941、986和1130个氨基酸。三种Nod样受体具有NACHT结构域和C-末端富含亮氨酸重复序列（LRR）结构域。此外，ntNOD 1和ntNOD 2具有N-末端CARD结构域（ntWOD 2具有两个）。系统进化分析表明，这三个NLR高度保守。三种受体的组织表达分析表明，最高的mRNA和蛋白质水平的ntNOD 1，ntNOD 2和ntNLRC 3在脾脏。NLR在胚胎发育过程中的表达模式表明，ntNOD 2和ntNLRC 3的表达水平显着增加，从2至8天后受精（dpf）。ntWOD 1表达水平在2 ~ 6dpf显著升高，7 dpf降低，8dpf又升高。在腹腔注射无乳链球菌刺激后，血液、脾脏、肾脏、肠和鳃中ntWOD 1、ntNOD 2和ntNLRC 3 mRNA和蛋白的表达水平明显改变。此外，在与NF-κ B报告质粒共转染后，与对照细胞相比，在i-EDPA刺激之前或之后，ntNOD 1过表达的293 T细胞中的NF-κ B活化显著增加。相比之下，与对照相比，ntNOD 2和ntNLRC 3在293 T细胞中对NF-κ B活化没有影响，当它们的潜在配体不被刺激时。然而，在MDP刺激后，ntNOD 2和ntNLRC 3过表达增加了293 T细胞中NF-κ B的活化。NOD 1和NLRC 3在293 T细胞中均匀分布于胞浆中，而NOD 2则分布于胞浆和胞核中。我们的研究结果表明，这三个Nod样受体在功能上是保守的，可能在防御病原体如无乳链球菌中发挥关键作用。
The nucleotide-binding oligomerization domain proteins NOD1, NOD2 and NLRC3 are cytoplasmic pattern recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In the present study, the Nile tilapia (Oreochromis niloticus) NOD1 (ntWOD1), NOD2 (ntWOD2) and NLRC3 (ntNLRC3) genes were cloned and characterized. The full-length ntWOD1, ntNOD2 and ntNLRC3 genes were 3924, 3886 and 4574 bp, encoding 941, 986 and 1130 amino acids, respectively. The three Nod-like receptors have a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain. In addition, ntNOD1 and ntNOD2 have a N-terminal CARD domain (ntWOD2 has two). Phylogenetic analysis showed that the three NLRs are highly conserved. Tissue expression analysis of the three receptors revealed that the highest mRNA and protein levels of ntNOD1,ntNOD2 and ntNLRC3 were in the spleen. The expression patterns of NLRs during embryonic development showed that the expression levels of ntNOD2 and ntNLRC3 significantly increased from 2 to 8 days post-fertilization (dpf). The expression levels of ntWOD1 significantly increased from 2 to 6 dpf, decreased at 7 dpf and then increased at 8 dpf. Upon stimulation with an intraperitoneal injection of Streptococcus agalactiae, expression levels of the ntWOD1, ntNOD2 and ntNLRC3 mRNA and protein were clearly altered in the blood, spleen, kidney, intestine and gill. Furthermore, after cotransfection with an NF-kappa B reporter plasmid, NF-kappa B activation in ntNOD1-overexpressing 293T cells significantly increased compared with that in control cells, before or after i-EDPA-stimulation. By contrast, compared with control, ntNOD2 and ntNLRC3 had no effect on NF-kappa B activation in 293T cells, when their potential ligands were not stimulated. However, after MDP-stimulation, ntNOD2 and ntNLRC3 overexpression increased NF-kappa B activation in 293T cells. NOD1 and NLRC3 were uniformly distributed throughout the cytoplasm in 293T cells, whereas NOD2 was distributed throughout the cytoplasm and nucleus. Our results indicate that the three Nod-like receptors are functionally conserved and may play pivotal roles in defense against pathogens such as Streptococcus agalactiae.