Hemolin在柞蚕体液免疫中的作用及其分子机制

Hemolin, the remarkable insect defense protein, has been well studied on its functions of cellular responses. The roles of hemolin in humoral responses, however, remain to be discovered. Hence, with the support of crystal structure of Ap-hemolin we’ve acquired, this project investigates interactions between hemolin and various bacterial-/fungal-derived pathogen-associated molecular patterns (PAMPs) and further the rule of conformational flexibility and structural foundation of this binding. This project also evaluates the role of hemolin at humoral immune responses in aspects of microorganism agglutination, prophenoloxidase activation system (PPO-AS), Toll pathway and IMD pathway. Furthermore, the relationship between pattern recognition and the role mentioned above is also under concerned in structure-function aspect. Likewise, another destination of this project is to probe the direct targets which hemolin access to of humoral immune response through investigation on the in vitro activation mechanism and conditions as well as hemolin-interaction properties of HP14, the initial serine proteinase shared by both PPO-AS and Toll pathway..This study highlights the roles of hemolin in insect innate immunity, and will provide scientific basis for understanding the function of pattern recognition proteins as well as their structure-function relationship.

Hemolin作为昆虫体内重要的防御蛋白，在细胞免疫中作用的研究较为丰富，而在体液免疫的研究则有待于深入。基于此，本项目从考察Hemolin与细菌/真菌典型病原体相关分子模式(PAMP)的特异性结合入手，以前期获得的柞蚕Hemolin晶体结构为重要支撑，系统研究Hemolin与不同PAMP的结合特征、空间结构变化规律及其结合的结构基础；从微生物凝集、酚氧化酶原激活系统(PPO-AS)以及Toll途径和Imd途径等多方面评价Hemolin对柞蚕体液免疫应答的影响，从结构角度阐述该影响与其模式识别作用的关系；通过考察Hemolin与HP14这一PPO-AS和Toll途径共有的起始丝氨酸蛋白酶的相互作用及HP14体外激活机制与条件，探讨Hemolin激活体液免疫信号通路的直接靶点。上述研究为深入了解模式识别蛋白的功能及其构效关系，系统阐述Hemolin在昆虫先天性免疫防御中的作用提供科学依据。

Hemolin是迄今为止发现的唯一一个非脊椎动物免疫球蛋白家族成员。因其结构与哺乳动物的免疫球蛋白（Ig）类似而将其命名为类免疫球蛋白（Hemolin）。Hemolin本身无直接杀菌的能力但却参与清除病原微生物的免疫应答。截至目前，仅在鳞翅目昆虫体内发现Hemolin的存在并证实其在昆虫正常发育以及免疫防御中发挥重要作用。但关于Hemolin参与清除病原微生物的免疫应答的作用机制及其分子基础等方面的研究报道较少。本项目阐明Hemolin对微生物凝集、柞蚕幼虫血淋巴PPO-AS以及脂肪体中抗菌肽合成等体液免疫过程具有促进作用；明确其具有广谱模式识别特性并通过结构解析明确其结构中影响与不同类型病原菌结合相关的关键氨基酸残基；为阐述Hemolin参与柞蚕幼虫体液免疫的分子机制及其结构基础奠定基础。探索Hemolin与PPO-AS及Toll途径共有的核心起始丝氨酸蛋白酶HP14的相互作用的特点及其促进HP14酶原自水解的特性，并解析其结构中影响与HP14结合、自水解相关的关键氨基酸残基，其与模式识识别结构基础密切相关，这一结果为探索Hemolin激活PPO-AS以及Toll途径的具体分子机制奠定基础。此外，还获得柞蚕Hemolin-2的全序列并证实其具有广谱的模式识别作用并对不同病原体介导的PPO激活和诱导抗菌肽合成等体液免疫反应具有正向调控作用。综合上述实验结果，本研究这种针对两种Hemolin成员模式识别特性及其参与细菌介导的体液免疫信号通路调控的结构基础的研究，为制定有效的柞蚕病虫害防治策略提供理论依据；同时也为基于模式识别蛋白和Toll信号转导途径的激活及调控机制，研发具有自主知识产权和国际市场竞争力、适用微生物感染的快速诊断新技术、新试剂奠定基础。