Structural/Functional Analysis of Chicken and Lamprey C3

由于C3能够与其他补体蛋白结合，并与几种细胞表面受体相互作用，因此它在补体活化中起着关键作用。阐明与这些C3相关功能的分子特征需要进一步分析其结构。该项目的目的是分析鸡和七鳃鳗这两个系统发育关键物种的C3的结构和功能，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，目的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以及与自身补体受体的反应能力。将使用先前建立的净化程序。将通过直接结合ELISA、结合C3片段包被红细胞和抑制试验来分析这两个物种的C3结合蛋白与C3的结合。通过对C3蛋白水解片段进行测序，可以得到不同C3的氨基酸序列。为了获得由自体C3转化酶和因子I（在H存在的情况下）产生的片段的n端氨基酸序列，将两种动物的血清用酶酶酶激活，酶酶酶结合的C3片段被洗脱，用SDS-PAGE分离，然后印迹到PVDF膜上。不同的C3片段将直接从PVDF膜上测序。碳水化合物和硫酯键的存在也将被确定。利用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抗体筛选lambda gt11 cDNA肝文库，获得各种c3的完整一级结构。（两个物种的Lambda gt11 cDNA肝脏文库已经制备完成，目前已分离出4个鸡C3候选克隆。）得到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。C3蛋白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参与动物防御机制的吞噬和免疫调节过程的关键参与者。初步实验表明，通过比较不同物种之间的C3氨基酸序列，并将这些C3与不同C3结合蛋白的结合能力联系起来，可以确定C3的结构特征，这些结构特征对其功能很重要。本研究除了为C3的系统发育历史提供新的信息外，还将为C3的结构特征与其多种功能之间的关系提供基本信息。

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.