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，它们在免疫监测和免疫反应途径中起着非常重要的作用。 阐明与不同补体蛋白功能有关的分子特征需要进一步分析其结构。 初步实验强烈表明，可以通过比较来自不同物种的这些蛋白质的主要结构，“天然类似物”，并将这些信息与这些蛋白质与其他物种交叉反应的能力相关联，可以鉴定补体蛋白质的结构特征，这对其功能很重要。 除第三个补体成分外，C3对哺乳动物以外的其他物种中其他补体蛋白的存在和性质知之甚少。 该实验室具有克隆的鳟鱼（TR），xenopus（Xe）和Chicken（CH）C3，并提供了初步数据，鳟鱼和Xenopus具有带有I，B，H和D类似活动的蛋白质。 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 B.因子H和B也将从鳟鱼等离子体中分离出来，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质纯化程序，包括PEG降水，Mono Q阴离子交换，单声道和凝胶过滤色谱法用于净化XE，以及TR C3和TROUT C3和鳟鱼因子H和B。C3结合蛋白与鳟鱼C3的结合将通过直接结合ELISA来分析ELISA，并通过结合Insays Insays Insays进行分析。 将因子H的功能通过测试其抑制C3BBB转化酶的能力，B与C3b的抑制能力，以及通过因子I的C3b裂解中的辅助活性在C3b的裂解中发挥作用I的功能（s）的功能，将通过测试其结合C3B和COBRA VENOM因子（CVF）的能力来研究C3B的功能。 因子H和B的氨基酸序列信息将通过对其蛋白水解片段进行测序（纯化或电视为PVDF膜）来获得。 由获得的氨基酸序列，cDNA探针或亲和力纯化的抗C3抗体构建的寡核苷酸探针将用于筛选cDNA肝库，以获得C3的替代形式的完整主要结构，并为因子H和B. cDNA肝脏的替代形式提供了两种物种的cDNA肝脏库，并且已经准备好了XE和XE的部分c3和xe c3的部分结构。 所获得的序列将被比较并与功能数据相关。 特别感兴趣的是已知与其他补体蛋白质结合的蛋白质片段。 除了有关补体系统替代途径的系统发育数据外，所提出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。 %% %%补体系统是在脊椎动物的血液中发现的一组复杂的蛋白质。 这些蛋白质组在一起在免疫监测和免疫反应途径中起着非常重要的作用。 了解这些蛋白质的分子特征与它们的功能之间的关系需要进一步分析其结构。 鉴定补体蛋白质的结构特征的一种有效方法是对其功能很重要的是比较来自不同动物物种的这些蛋白质的主要结构，并将这些信息与这些蛋白质与其他物种的蛋白质作用的能力相关联。 除了对补体蛋白与其功能的关系的理解做出贡献，这项研究的结果将提供有关鱼类和两栖动物免疫防御系统的重要新信息。 ***