Structural/Functional Analysis of Chicken and Lamprey C3

由于C3能与其他补体蛋白结合，并与多种细胞表面受体相互作用，因此在补体激活过程中起着关键作用。要阐明与这些C3相关功能相关的分子特征，还需要进一步分析其结构。本项目的目的是分析鸡和七鳃鳗两个关键物种C3的结构和功能，并将这些信息与哺乳动物C3的已知信息进行比较和关联，以加深我们对C3分子的理解。在蛋白质水平上，目标是从鸡和七鳃鳗的血浆中提纯C3，并测试它们与人类补体蛋白CR1、CR2、CR3、C5、H、B、I和P以及与自体补体受体反应的能力。将使用先前建立的净化程序。C3结合蛋白与来自这两个物种的C3的结合将通过直接结合的酶联免疫吸附试验、与C3片段包裹的红细胞的结合以及抑制试验来分析。不同C3的氨基酸序列将通过对C3的蛋白分解片段进行测序来获得。为了获得由自体C3转化酶和因子I(在H存在下)产生的片段的N末端氨基酸序列，这两种生物的血清将被酵母多糖激活，结合的C3片段被洗脱，用SDS-PAGE分离，然后将其印迹到PVDF膜上。不同的C3片段将直接从PVDF膜中测序。碳水化合物和硫代酯键的存在也将被确定。根据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肝文库，以获得各种C3s的完整一级结构。(已经从这两个物种中制备了Lambda gt11的肝脏文库，到目前为止已经分离到四个鸡C3的候选克隆。)获得的序列将被比较并与功能数据相关联。特别令人感兴趣的是已知的调节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，通过比较不同物种之间的C3氨基酸序列，并将这一信息与这些C3与不同的C3结合蛋白结合的能力相关联，可以识别对其功能重要的C3的结构特征。拟议中的研究，除了提供关于C3系统发育历史的新信息外，还将提供关于C3的结构特征如何与其多重功能相关的基本信息。

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.