Structural/Functional Analysis of Chicken and Lamprey C3

鸡和七鳃鳗 C3 的结构/功能分析

• 批准号: 9018751
• 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: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1994-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9018751&HistoricalAwards=false
• 关键词: Structural; Functional; Analysis; Chicken; Lamprey

C3 plays a critical role in complement activation due to its ability to bind to other complement proteins, and interact with several cell surface receptors. The elucidation of the molecular features related to these C3 associated functions requires further analysis of its structure. The goal of this project is to analyze the structure and functions of C3 from two phylogenetically key species, the chicken and the lamprey, and to compare and correlate this information with what is known about mammalian C3 in order to further our understanding of this molecule. At the protein level, the objectives will be to purify C3 from the plasma of chickens and lampreys and test their ability to react with human complement proteins CR1, CR2, CR3, C5, H, B, I, and P as well as with autologous complement receptors. Previously established purification procedures will be used. The binding of C3-binding proteins to C3 from the two species will be analyzed by direct binding ELISA, by binding to C3 fragment-coated erythrocytes, and by inhibition assays. Amino acid sequences for the different C3s will be obtained by sequencing proteolytic fragments of C3 . In order to obtain N-terminal amino acid sequences of fragments generated by autologous C3 convertases and factor I (in the presence of H), serum of the two species will be activated by zymosan, the zymosan bound C3 fragments will be eluted, separated by SDS-PAGE, and then blotted to PVDF membranes. The different C3 fragments will be sequenced directly from the PVDF membranes. The presence of carbohydrates and thiolester bonds will also be determined. Oligonucleotide probes constructed from the determined amino acid sequences, cDNA probes or affinity purified anti-C3 antibodies will be used to screen lambda gt11 cDNA liver libraries to obtain complete primary structure for the various C3s. (Lambda gt11 cDNA liver libraries from the two species have been prepared already, and four candidate clones for chicken C3 have been isolated thus far.) The obtained sequences will be compared and correlated to functional data. Of special interest are the C3 segments known to mediate the binding of C3-binding proteins. The interaction of the protein, "C3", with other plasma proteins and with several cell surface receptors make it a key participant in phagocytic and immunoregulatory processes involved in animal defense mechanisms. Preliminary experiments suggest that it is possible to identify the structural features of C3 that are important for its functions by comparing the C3 amino acid sequences between species, and correlating this information with the ability of these C3s to bind to different C3-binding proteins. The proposed studies, in addition to providing new information on the phylogenetic history of C3, will provide basic information on how the structural features of C3 relate to its multiple functions.

C3 在补体激活中发挥着关键作用，因为它能够与其他补体蛋白结合，并与多种细胞表面受体相互作用。 阐明与这些 C3 相关功能相关的分子特征需要进一步分析其结构。 该项目的目标是分析两个系统发育关键物种（鸡和七鳃鳗）的 C3 结构和功能，并将这些信息与已知的哺乳动物 C3 进行比较和关联，以进一步了解该分子。 在蛋白质水平上，目标是从鸡和七鳃鳗的血浆中纯化 C3，并测试它们与人类补体蛋白 CR1、CR2、CR3、C5、H、B、I 和 P 以及自体补体蛋白反应的能力。补体受体。 将使用先前建立的纯化程序。 将通过直接结合 ELISA、通过与 C3 片段包被的红细胞结合以及通过抑制测定来分析 C3 结合蛋白与来自两个物种的 C3 的结合。 不同C3的氨基酸序列将通过对C3的蛋白水解片段进行测序来获得。 为了获得自体C3转化酶和因子I（在H存在下）产生的片段的N端氨基酸序列，用酵母聚糖激活两个物种的血清，将结合了酵母聚糖的C3片段洗脱，通过SDS-PAGE，然后印迹到 PVDF 膜上。 不同的 C3 片段将直接从 PVDF 膜上进行测序。 碳水化合物和硫醇酯键的存在也将被确定。 由确定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lambda gt11 cDNA肝文库以获得各种C3的完整一级结构。 （来自两个物种的 Lambda gt11 cDNA 肝脏文库已经制备完毕，并且迄今为止已分离出鸡 C3 的四个候选克隆。）所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 C3 结合蛋白结合的 C3 片段。 蛋白质“C3”与其他血浆蛋白和几种细胞表面受体的相互作用使其成为动物防御机制中吞噬和免疫调节过程的关键参与者。 初步实验表明，通过比较物种之间的 C3 氨基酸序列，并将这些信息与这些 C3 与不同 C3 结合蛋白结合的能力相关联，可以鉴定对其功能重要的 C3 结构特征。 拟议的研究除了提供有关 C3 系统发育史的新信息外，还将提供有关 C3 的结构特征与其多种功能如何相关的基本信息。