Structure-Function Relationships of C-Reactive Protein

C反应蛋白的结构-功能关系

• 批准号: 6941238
• 负责人: ALOK AGRAWAL
• 金额: $ 25.55万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6941238
• 关键词: CHO cells; Salmonella typhimurium; Streptococcus pneumoniae; acute phase protein; antibody receptor; atherosclerosis; bacteria infection mechanism; binding sites; complement pathway; enzyme linked immunosorbent assay; gene targeting; genetically modified animals; immunoaffinity chromatography; inflammation; laboratory mouse; ligands; low density lipoprotein; macrophage; mutant; pathologic process; phagocytosis; phosphorylcholine; protein binding; protein protein interaction; protein structure function; receptor binding; site directed mutagenesis

DESCRIPTION (provided by applicant): C-reactive protein (CRP) is a major human acute phase protein and a component of the innate immune response. Its serum concentration is increased during inflammatory states, persists for the duration of the inflammatory process and returns to its normal low concentration following subsidence of inflammation. While CRP is felt to play a significant role in inflammation and host defense, the mechanisms by which CRP exerts its effects are unclear. In vitro, CRP binds to phosphocholine (PCh) moieties and can then bind to complement C1q and activate the classical complement pathway. In addition, binding of CRP to phagocytic cells via Fc receptors, with a variety of functional consequences, has been described. Recent publication of the crystal structures of CRP has provided insight into the amino acids that mediate binding of CRP to PCh, to Fc receptors, and to C1q, permitting generation of CRP mutants incapable of binding to PCh and to Fc receptors, as well as incapable of activating complement. Most known functional activities of CRP, in vitro, are associated with ligand-binding and subsequent complement activation or phagocytosis. Accordingly, we will employ such mutants to define the roles of binding to PCh and Fe receptors, and of complement activation in 2 model systems: a) the protective role of CRP in bacterial infections and b) the putative role of CRP in the pathogenesis of atherosclerosis resulting from its ability to bind to enzymatically-degraded LDL (E-LDL). Our specific aims are: 1.To precisely define the ligand-binding sites on CRP required for binding to PCh, FcR and C1q and to generate mutants lacking these critical binding capabilities. 2. To define the role of these 3 binding capabilities in the protective effects of CRP in infection with Streptococcus pneumoniae, known to bind to CRP, and Salmonella typhimuriurn, which does not. We hypothesize that both complement activation and phagocytosis will be found to be involved in CRP-mediated protection of mice from bacterial infections. 3) To define the role of the 3 binding capabilities of interest on CRP-E-LDL interaction, and the role of such interaction in the pathogenesis of atherosclerosis. Our working hypothesis is that all 3 binding sites participate in the pathogenesis of atherosclerosis, by binding to E-LDL and initiating complement activation and uptake of E-LDL by macrophages. We will also determine the effects of injecting wild-type and mutant CRPs on the size of the atherosclerotic lesions formed in ApoE knock-out mice. These studies will provide substantial insight into the mechanisms by which this ancient protein may contribute to host defense, or alternatively, to pathogenesis of disease.

描述（由申请人提供）：C反应蛋白（CRP）是一种主要的人类急性期蛋白，是先天免疫应答的组成部分。其血清浓度在炎症状态期间增加，在炎症过程期间持续存在，并在炎症消退后恢复到正常的低浓度。虽然人们认为C反应蛋白在炎症和宿主防御中发挥重要作用，但C反应蛋白发挥其作用的机制尚不清楚。在体外，CRP与磷酸胆碱（PCh）部分结合，然后可以与补体C1 q结合并激活经典补体途径。此外，已描述了CRP通过Fc受体与吞噬细胞结合，具有多种功能后果。最近发表的CRP晶体结构提供了对介导CRP与PCh、Fc受体和C1 q结合的氨基酸的了解，从而允许产生不能与PCh和Fc受体结合以及不能激活补体的CRP突变体。CRP在体外的大多数已知功能活性与配体结合和随后的补体激活或吞噬作用相关。因此，我们将使用这样的突变体来定义结合PCh和Fe受体的作用，以及在2个模型系统中补体激活的作用：a）CRP在细菌感染中的保护作用和B）CRP在动脉粥样硬化发病机制中的假定作用，这是由于其结合酶降解的LDL（E-LDL）的能力。我们的具体目标是：1.精确确定CRP与PCh、FcR和C1 q结合所需的配体结合位点，并产生缺乏这些关键结合能力的突变体。2.确定这3种结合能力在CRP感染肺炎链球菌（已知与CRP结合）和鼠伤寒沙门氏菌（不与CRP结合）的保护作用中的作用。我们假设补体激活和吞噬作用都参与CRP介导的保护小鼠免受细菌感染。3)确定3种相关结合能力对CRP-E-LDL相互作用的作用，以及这种相互作用在动脉粥样硬化发病机制中的作用。我们的工作假设是，所有3个结合位点参与动脉粥样硬化的发病机制，通过结合E-LDL和启动补体激活和巨噬细胞摄取E-LDL。我们还将确定注射野生型和突变型CRP对ApoE基因敲除小鼠形成的动脉粥样硬化病变大小的影响。这些研究将为这种古老的蛋白质可能有助于宿主防御或疾病发病机制的机制提供实质性的见解。