Structure and Functions of Complement Proteins from Different Species

不同物种补体蛋白的结构和功能

• 批准号: 9319111
• 负责人: John Lambris
• 金额: --
• 依托单位: University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-15 至 1997-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9319111&HistoricalAwards=false
• 关键词: Structure; Functions; Complement; Proteins; Different

9319111 Lambris The complement system is a complex group of proteins and glycoproteins which together play a very important role in immune surveillance and immune response pathways. The elucidation of the molecular features related to the functions of the different complement proteins require further analysis of their structure. Preliminary experiments strongly suggest that it is possible to identify the structural features of complement proteins that are important for their functions by comparing the primary structure of these proteins from different species, "natural analogs", and correlating this information with the ability of these proteins to cross react with proteins from other species. Except for the third complement component, C3, very little is known about the presence and nature of other complement proteins in species other than mammals. This laboratory has cloned trout (Tr), Xenopus (Xe), and chicken (Ch) C3 and provided preliminary data that trout and Xenopus have proteins with factor I, B, H, and D like activities. The goal of this project is to analyze the structure and functions of the alternative forms of C3 from trout and Xenopus and characterize the trout alternative pathway complement proteins, factor H and B. At the protein level, the objectives will be to purify the different forms of C3 from Xenopus and trout plasma and test their ability to react with human complement proteins CR1, CR2, H, B, I, C5 and P as well as with autologous H and B. Factor H and B will also be isolated from trout plasma and their function tested in the alternative complement pathway. A previously established purification procedure for mammalian complement proteins including PEG precipitation, Mono Q anion exchange, Mono S and gel filtration chromatography will be used to purify Xe, and Tr C3 and trout factors H and B. The binding of C3-binding proteins to trout C3 will be analyzed by direct binding ELISA, by binding inhibition assays. The functions of factor H will be analyzed by testing its ability to inhibit the C3bBb convertase, the binding of Factor B to C3b, and to exert cofactor activity in the cleavage of C3b by factor I. The function(s) of factor B will be investigated by testing its ability to bind C3b and cobra venom factor (CVF) and to form the C3bBb/CVFBb convertase. Amino acid sequence information for factors H and B will be obtained by sequencing their proteolytic fragments (HPLC purified or electroblotted to PVDF membranes). Oligonucleotide probes constructed from the obtained amino acid sequences, cDNA probes or affinity purified anti-C3 antibodies will be used to screen cDNA liver libraries to obtain complete primary structure for the alternative forms of C3 and that of factor H and B. cDNA liver libraries from both species have been prepared and partial cDNA clones for the alternative forms of Xe and Tr C3 have been isolated. The obtained sequences will be compared and correlated to functional data. Of special interest are the protein segments known to mediate the binding with other complement proteins. The proposed studies, in addition to the phylogenetic data on the alternative pathway of the complement system will provide basic information on the structural features of C3 and its binding proteins as they relate to their functions. %%% The complement system is a complex group of proteins found in the blood of vertebrate animals. These group of proteins together play a very important role in immune surveillance and immune response pathways. Understanding of how the molecular features of these proteins relate to their functions requires further analysis of their structure. One effective approach to identifying the structural features of complement proteins that are important for their functions is to compare the primary structure of these proteins from different species of animal and relating this information to the ability of these proteins to act in conjunction with proteins from other species. In add ition to contributing to the understanding of the relationship of complement proteins to their function, the results of this research will provide important new information on the immune defense systems of fish and amphibians. ***

9319111 Lambris 补体系统是一组复杂的蛋白质和糖蛋白，它们在免疫监视和免疫反应途径中共同发挥着非常重要的作用。 阐明与不同补体蛋白功能相关的分子特征需要进一步分析其结构。 初步实验强烈表明，通过比较来自不同物种的补体蛋白“天然类似物”的一级结构，并将这些信息与这些蛋白的能力相关联，可以鉴定对其功能很重要的补体蛋白的结构特征。与其他物种的蛋白质发生交叉反应。 除了第三个补体成分 C3 之外，人们对哺乳动物以外的物种中其他补体蛋白的存在和性质知之甚少。 该实验室克隆了鳟鱼（Tr）、非洲爪蟾（Xe）和鸡（Ch）C3，并提供了鳟鱼和非洲爪蟾具有具有因子I、B、H和D样活性的蛋白质的初步数据。 该项目的目标是分析来自鳟鱼和非洲爪蟾的 C3 替代形式的结构和功能，并表征鳟鱼替代途径补体蛋白、H 因子和 B 因子。在蛋白质水平上，目标是纯化不同形式的 C3从非洲爪蟾和鳟鱼血浆中提取 C3，并测试它们与人类补体蛋白 CR1、CR2、H、B、I、C5 和 P 以及自体 H 和 B 反应的能力。因子 H 和 B 也将从鳟鱼血浆中分离出来，并在替代补体途径中测试其功能。 先前建立的哺乳动物补体蛋白纯化程序（包括 PEG 沉淀、Mono Q 阴离子交换、Mono S 和凝胶过滤色谱）将用于纯化 Xe、Tr C3 以及鳟鱼因子 H 和 B。C3 结合蛋白与鳟鱼的结合C3 将通过直接结合 ELISA、结合抑制测定进行分析。 因子 H 的功能将通过测试其抑制 C3bBb 转化酶的能力、因子 B 与 C3b 的结合以及在因子 I 裂解 C3b 时发挥辅助因子活性的能力来分析。因子 B 的功能将是通过测试其结合 C3b 和眼镜蛇毒因子 (CVF) 并形成 C3bBb/CVFBb 转化酶的能力进行研究。 H 因子和 B 因子的氨基酸序列信息将通过对其蛋白水解片段进行测序（HPLC 纯化或电印迹至 PVDF 膜）来获得。 由获得的氨基酸序列构建的寡核苷酸探针、cDNA探针或亲和纯化的抗C3抗体将用于筛选cDNA肝脏文库，以获得C3替代形式以及因子H和B的完整一级结构。cDNA肝脏文库来自这两个物种均已制备完毕，并且 Xe 和 Tr C3 替代形式的部分 cDNA 克隆也已分离。 获得的序列将与功能数据进行比较和关联。 特别令人感兴趣的是已知介导与其他补体蛋白结合的蛋白质片段。 除了补体系统替代途径的系统发育数据之外，拟议的研究还将提供有关 C3 及其结合蛋白的结构特征的基本信息，因为它们与其功能相关。 %%% 补体系统是脊椎动物血液中发现的一组复杂的蛋白质。 这些蛋白质组一起在免疫监视和免疫反应途径中发挥着非常重要的作用。 要了解这些蛋白质的分子特征与其功能的关系，需要进一步分析其结构。 识别补体蛋白对其功能很重要的结构特征的一种有效方法是比较来自不同物种动物的这些蛋白质的一级结构，并将这些信息与这些蛋白质与来自其他物种的蛋白质联合作用的能力联系起来。 除了有助于理解补体蛋白与其功能的关系之外，这项研究的结果还将提供有关鱼类和两栖动物免疫防御系统的重要新信息。 ***