STRUCTURE-FUNCTION ANALYSIS OF C3 IN DIFFERENT SPECIES

C3在补体激活的两种途径中起关键作用， 与其与许多其他补体蛋白结合的能力有关。 在 此外，它与几种细胞表面受体的相互作用使其 是吞噬和免疫调节过程的关键参与者， 与外来病原体蛋白质的相互作用可能提供了一种机制， 这些微生物通过其逃避补体中和。 的 阐明与这些C3相关的分子特征 需要进一步分析其结构。 才是目的 研究人C3与CR 1，CR2，CR 3， H、B、I和P的嵌合和突变的C3分子， 其他物种C3的结构和功能。 我们的研究 实验室表明，有可能确定结构 通过比较C3的功能，C3的重要功能 物种之间的氨基酸序列，“天然类似物”，以及相关的 这些信息与这些C3的能力，以结合不同的C3- 结合蛋白 首先，我们将从人（Hu）、非洲爪蟾（Xenopus） (Xe)和鳟鱼（Tr）血浆，并测试它们与人 和自体补体蛋白CR 1、CR2、CR 3、H、B、I和P。 人和自体C3结合蛋白与不同C3的结合将 通过直接结合ELISA，通过结合C3片段包被的 红细胞，并通过抑制测定。 为了将函数 C3-配体与C3结构相互作用的保守性，我们将 通过克隆和测序，获得Tr和TcC 3的一级结构 他们的cDNA。 评价因子I的底物特异性及其在细胞内的表达， 依赖于各种辅助因子介导不同的C3 b 我们将突变人C3中的因子I切割位点， 分析I在存在下对突变的C3 b片段的切割， H、CR 1或CR2。 表征CR 1、CR2、H和B结合结构域 以及与我们将设计的每个域相关的功能 通过在Hu， 和Tr C3和CVF。 为了定位C3中的CR 3结合位点，我们将 产生C3片段，通过酶/化学降解和cDNA 表达和合成肽，并测试它们结合 CR 3和/或抑制iC 3b与CR 3的结合。 我们的初步 实验表明，残基His 1431和Ser 1432是 参与C3肽与备解素的结合。 我们将评估 这些残基在C3 b-P相互作用中的作用通过突变His 1431和 人C3的Ser 1432与α 2-巨球蛋白中发现的残基N和Q; 巨球蛋白不结合P，与Hu C3在氨基酸上不同 1424-1432，仅在这两个残基中。 C3-CR 1/CR2的生物学作用 和C3-B/P相互作用将在体外和体内进行分析， 评价合成的和/或表达的C3片段 干扰抗体反应并抑制另一种 使用缺血/再灌注的骨骼肌通路介导的损伤 小鼠模型。 除了拟议的研究外， C3的系统发育数据，将提供结构上的基本信息， C3的特点，因为它们涉及其多种功能，并有助于 设计特异性补体抑制剂， 重要.

C3 plays a critical role in both pathways of complement activation due to its ability to bind to numerous other complement proteins. In addition, its interactions with several cell surface receptors make it a key participant in phagocytic and immunoregulatory processes, while its interactions with proteins from foreign pathogens may provide a mechanism by which these microorganisms evade complement neutralization. The elucidation of the molecular features related to these C3 associated functions requires further analysis of its structure. It is the purpose of this project to study the interactions of human C3 with CR1, CR2, CR3, H, B, I and P by engineering chimeric and mutated C3 molecules based on the structure and function of C3 from other species. Studies from our laboratory 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, "natural analogs", and correlating this information with the ability of these C3s to bind the different C3- binding proteins. First, we will purify C3 from Human (Hu), Xenopus (Xe), and Trout (Tr) plasma and test their ability to react with human and autologous complement proteins CR1, CR2, CR3, H, B, I, and P. The binding of human and autologous C3-binding proteins to different C3s will be analyzed by direct binding ELISA, by binding to C3 fragment-coated erythrocytes, and by inhibition assays. To correlate the functional conservation of the C3-ligand interactions with the C3 structure we will obtain the primary structure of Tr and Xe C3 by cloning and sequencing their cDNA. To evaluate the substrate specificity of factor I and its dependence on the various cofactors in mediating the different C3b cleavages we will mutate the factor I cleavage sites in human C3 and analyze the cleavage of the mutated C3b fragment by I in the presence of H, CR1 or CR2. To characterize the CR1, CR2, H and B binding domain(s) in C3b and the functions associated with each domain we will engineer chimeric molecules by exchanging the ligand binding domains between Hu, Xe, and Tr C3 and CVF. To localize the CR3 binding site(s) in C3 we will generate C3 fragments, by enzymatic/chemical degradation and cDNA expression, and synthetic peptides and testing their ability to bind to CR3 and/or to inhibit the binding of iC3b to CR3. Our preliminary experiments have suggested that the residues His1431 and Ser1432 are involved in the binding of C3 peptides to properdin. We will assess the role of these residues in C3b-P interaction by mutating His1431 and Ser1432 of human C3 to residues N and Q found in a2-macroglobulin; a2- macroglobulin does not bind P and differs from Hu C3, within amino acids 1424-1432, only in these two residues. The biological role of C3-CR1/CR2 and C3-B/P interactions will be analyzed in vitro and in vivo by evaluating the ability of synthetic and/or expressed C3 fragments to interfere with the antibody responses and to inhibit the alternative pathway mediated injury of skeletal muscle using an ischemia/reperfusion mouse model, respectively. The proposed studies, in addition to the phylogenetic data on C3, will provide basic information on the structural features of C3 as they relate to its multiple functions and assist in the design of specific complement inhibitors which may become medically important.