文昌鱼 Apextrin 样蛋白家族的免疫功能研究

The innate immune system plays an essential role in the first line of host defense against pathogenic microbes. Microbes express signature molecules known as pathogen-associated molecular patterns (PAMPs), which are essential for survival and hence difficult for microbes to change via adaptive evolution. The innate immune system relies on germ-line-encoded pattern recognition proteins (PRPs) to detect and react with PAMPs. Apextrin-like proteins (ALPs) are extensively identified in invertebrates, especially in marine invertebrates，and 11 ALP were identified based on the whole genome sequences of amphioxus (Branchiostoma belcheri). Our previous study showed that two amphioxus ALPs (ALP1 and ALP2) function as PRPs and play important roles in the mucous immune system. Both ALP1 and ALP2 can bind and aggregate bacteria via their ApeC domain, a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). ALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram positive bacterium. Neutralization of secreted ALP1 by anti-ALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. ALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB signaling pathway. However, the molecular structure of ApeC domain remains unknown, and whether other members of amphioxus ALP family also function as PRPs need to be proved. The aims of this project are: Firstly, to reveal the crystal structure of ApeC domain using X-ray crystallography and nuclear magnetic resonance; Secondly, to clone the cDNA sequences of ApeC domain from other 9 ALPs and use the purified recombinant proteins to study the potential pattern recognition function of these peptides. On the base of previously mentioned studies, we plan to reveal the molecular structure of ApeC domain and the pattern recognition function of other ALPs, and pave the way for following applied research.

病原相关模式识别蛋白在天然免疫反应中发挥着至关重要的作用，它们通过识别病原微生物的特征性分子来感知其入侵并激活机体的免疫反应。Apextrin 样蛋白（ALP）广泛存在于各类无脊椎动物尤其是海洋无脊椎动物中，我们前期的研究首次阐明了文昌鱼的两个 ALP（ALP1 和 ALP2）作为模式识别蛋白在消化道的粘膜免疫系统中发挥着重要的作用，我们还鉴定出了 ALP 蛋白所特有的一种全新的模式识别结构域——ApeC 结构域。但 ApeC 结构域的分子结构特征以及 ALP 家族的其他成员是否也具有模式识别功能，还需要进一步研究。我们拟通过蛋白质晶体结构分析和基因突变技术来揭示 ApeC 结构域的分子特征，并进一步研究文昌鱼中其他 ALP 的模式识别功能。希望籍此来解析 ApeC 结构域的分子结构并揭示一个全新的模式识别蛋白家族——ALP 家族，为以后的应用研究提供理论基础。

病原相关模式识别蛋白在天然免疫反应中发挥着至关重要的作用，它们通过识别病原微生物的特征性分子来感知其入侵并激活机体的免疫反应。Apextrin 样蛋白（ALP）广泛存在于各类无脊椎动物尤其是海洋无脊椎动物中，我们前期的研究首次证明了文昌鱼的两个 ALP（ALP1 和 ALP2）作为模式识别蛋白在文昌鱼消化道的粘膜免疫中发挥着重要的作用，在此基础上我们还发现了一种全新的模式识别结构域—ApeC 结构域。但是 ApeC 结构域的分子结构特征以及 ALP 家族的其他成员是否也具有模式识别功能目前尚无研究。我们对已有序列信息的所有ALP蛋白进行了序列和进化分析，发现ApeC 结构域存在复制与缺失现象。我们对9个白氏文昌鱼ALP基因进行了表达谱分析，发现大多数ALP基因都在文昌鱼免疫器官高表达并且在感染后上调表达，表明这些基因可能具有重要的免疫功能。随后，我们对白氏文昌鱼中的ALP基因进行了全长cDNA克隆并通过荧光素酶报基因实验检测了这些蛋白对的NF-κB信号通路影响，发现它们都能够抑制NF-κB信号通路的活化。我们还在青岛文昌鱼中鉴定出了一个由于可变剪接而产生的的截短型bjALP2基因，该蛋白能够通过抑制NF-κB信号通路的活化来调节机体免疫反应。另外，我们还发现bjALP1蛋白也可以定位于细胞内并且以类似于bjALP2的方式调节NF-κB信号通路的活化。本研究证明了ALP蛋白是一类广泛存在于无脊椎动物（尤其是海洋无脊椎动物）中的免疫蛋白家族。