Structure and Functions of Complement Proteins from Different Species

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应答途径中起着非常重要的作用。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的初级结构，“天然类似物”，并将这些信息与来自其他物种的蛋白质交叉反应的能力相关联，有可能确定补体蛋白的结构特征，这些结构特征对它们的功能很重要。除了第三种补体成分C3外，人们对哺乳动物以外的其他补体蛋白的存在和性质知之甚少。该实验室已经克隆了鳟鱼（Tr）、爪蟾（Xe）和鸡（Ch） C3，并提供了初步数据，表明鳟鱼和爪蟾具有因子I、B、H和D样活性的蛋白质。本项目的目的是分析鳟鱼和非洲爪鱼替代形式C3的结构和功能，并表征鳟鱼替代途径补体蛋白，因子H和B。在蛋白质水平上，目的是纯化非洲爪鱼和鳟鱼血浆中不同形式的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它们的功能。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和凝胶过滤层析，将用于纯化Xe、Tr 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和结合抑制试验进行分析。通过测试H因子对C3bBb转化酶的抑制能力、B因子与C3b的结合能力以及在i因子裂解C3b过程中发挥辅因子活性来分析H因子的功能。通过测试B因子与C3b和眼镜蛇毒因子（CVF）的结合能力以及形成C3bBb/CVFBb转化酶的能力来研究B因子的功能。因子H和B的氨基酸序列信息将通过对其蛋白水解片段（HPLC纯化或电印迹PVDF膜）进行测序获得。利用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，筛选cDNA肝文库，获得替代形式C3和因子H和因子b的完整一级结构，制备了两种物种的cDNA肝文库，并分离了替代形式Xe和Tr C3的部分cDNA克隆。得到的序列将与功能数据进行比较和关联。特别感兴趣的是已知介导与其他补体蛋白结合的蛋白质片段。拟议的研究，除了补体系统替代途径的系统发育数据外，将为C3及其结合蛋白的结构特征及其功能提供基本信息。补体系统是一组复杂的蛋白质，存在于脊椎动物的血液中。这些蛋白在免疫监视和免疫应答通路中起着重要的作用。要了解这些蛋白质的分子特征与其功能之间的关系，需要进一步分析它们的结构。鉴定补体蛋白结构特征的一种有效方法是比较来自不同动物物种的补体蛋白的初级结构，并将这些信息与这些蛋白质与来自其他物种的蛋白质协同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外，本研究结果还将为鱼类和两栖动物的免疫防御系统提供重要的新信息。＊＊＊

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. ***