对虾血蓝蛋白乙酰化修饰的分子机制与免疫学意义

Infectious disease and limited genetic characterization of shrimp are the main factors that have hindered sustainable shrimp aquaculture development in China. Delineating the immune defense mechanism of shrimp is therefore important for overcoming these problems. In recent years, shrimp hemocyanin has been shown to be a multifunctional protein with multiple immunological activities, such as antiviral and antibacterial activities. The molecular mechanism of hemocyanin’s functional diversity is probably related to its molecular polymorphism. Interestingly, our recent preliminary research revealed that hemocyanin undergoes acetylation and that the degree of this modification was closely related to shrimp defense against different pathogens. Thus, on the basis of these research findings，this proposed project seeks to further investigate the molecular mechanism of L. vannamei hemocyanin acetylation and immune significance by employing techniques such proteomics, molecular biology, interactomics, as well as molecular immunology techniques. We strongly believe that through this project we would be able to find the acetylation sites on hemocyanin, identify the acetylase/deacetylase involved, unravel the molecular mechanisms of the acetylation as well as uncover the crucial role this plays in immune defense against different pathogens. We anticipate that the results from this project will help deepen our understanding of the immune defense mechanism of shrimp, provide novel ideas for the search for new strategies for prevention/control of shrimp diseases, selection of improved varieties, and promote healthy and sustainable development of the shrimp aquaculture industry in China.

病害和种质退化被认为是影响目前我国对虾养殖业可持续发展的最关键因素，揭示对虾免疫防御机制是破解这些问题的重要基础。近年来研究发现血蓝蛋白是对虾中的一种具抗病毒、抑菌等多种免疫学活性的多功能蛋白，其功能多样性的分子基础可能与其分子多态性有关。有趣的是，最近本项目组研究发现对虾血蓝蛋白还存在乙酰化修饰，且其修饰程度与对虾抵御不同病原入侵密切相关。为此，本项目拟进一步通过联合运用蛋白质组学、分子生物学、相互作用组学和分子免疫学技术方法，对血蓝蛋白乙酰化修饰的分子机制与免疫学意义进行系统研究，阐明血蓝蛋白哪些乙酰化修饰位点在什么乙酰基转移酶/去乙酰化酶的作用下，通过何种方式参与对虾的免疫防御，揭示血蓝蛋白免疫防御的乙酰化修饰分子基础与作用机制，其成果将有助于深入揭示对虾的免疫防御机制，为寻找新的对虾病害防控、良种选育等策略提供新思路，促进我国对虾养殖业的健康可持续发展。

病害和种质退化严重制约我国对虾养殖业的可持续发展，解决此问题的关键环节之一就是揭示对虾免疫防御机制，为寻找有效的疾病防治、良品培育提供新的策略。有趣的是，近年来包括申请者在内的研究表明，血蓝蛋白是对虾中一种具有抗病毒、抑菌等多种免疫学活性和免疫调控活性的多功能蛋白，其功能多样性的分子基础可能与其分子多态性有关，其中，乙酰化修饰是近年来申请者课题组发现的一种新的血蓝蛋白翻译后修饰，其参与响应多种对虾病原胁迫。但迄今为止，血蓝蛋白乙酰化修饰的分子机制和免疫学意义尚不清楚。为此，本项目在此基础之上，联合蛋白质组学、分子生物学、分子免疫学、生物信息学等手段，揭示了血蓝蛋白乙酰化修饰形成的分子机制与免疫学意义，主要发现：1）血蓝蛋白大、小亚基分别存在K177、K188、K197、K239、K481、K484和K179、K188、K648、K653等10个乙酰化位点，其中，K481/484为血蓝蛋白抗革兰氏阴性菌相关性位点；2）p300/CPB和TIP60为血蓝蛋白乙酰基转移酶，HDAC3为血蓝蛋白去乙酰化酶；3）低乙酰化修饰尤其是K481/K484去乙酰化修饰不仅可以显著提高血蓝蛋白对革兰氏阴性菌的抗性与结合能力，还可以招募PP2a调控血蓝蛋白的去磷酸化修饰；4）血蓝蛋白乙酰化修饰可影响其泛素化修饰，其中，K197位点乙酰化修饰可调控PvMulan对血蓝蛋白的K63型泛素化修饰。所获研究结果深入揭示了对虾血蓝蛋白乙酰化修饰的免疫防御机制，为寻找新的对虾病害防控策略提供了新思路，有力的促进我国对虾养殖业的健康可持续发展。