Structure-Function Relationships of C-Reactive Protein

描述（由申请人提供）：C反应蛋白（CRP）是主要的人类急性期蛋白，也是先天免疫反应的组成部分。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 CRP 被认为在炎症和宿主防御中发挥着重要作用，但 CRP 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 C1q 结合并激活经典补体途径。此外，已经描述了 CRP 通过 Fc 受体与吞噬细胞结合，产生多种功能后果。最近发表的 CRP 晶体结构提供了对介导 CRP 与 PCh、Fc 受体和 C1q 结合的氨基酸的深入了解，从而允许产生不能与 PCh 和 Fc 受体结合且不能激活补体的 CRP 突变体。大多数已知的 CRP 体外功能活性与配体结合和随后的补体激活或吞噬作用有关。因此，我们将利用此类突变体来定义与 PCh 和 Fe 受体结合的作用，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，b) CRP 在动脉粥样硬化发病机制中的推定作用，这是由于 CRP 与酶降解的 LDL (E-LDL) 结合的能力所致。我们的具体目标是： 1. 精确定义 CRP 上与 PCh、FcR 和 C1q 结合所需的配体结合位点，并生成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 对肺炎链球菌（已知与 CRP 结合）和鼠伤寒沙门氏菌（不与 CRP 结合）感染中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 介导的小鼠免受细菌感染的保护。 3) 明确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 个结合位点通过与 E-LDL 结合并启动补体激活和巨噬细胞对 E-LDL 的摄取，从而参与动脉粥样硬化的发病机制。我们还将确定注射野生型和突变型 CRP 对 ApoE 敲除小鼠中形成的动脉粥样硬化病变大小的影响。这些研究将为了解这种古老蛋白质可能有助于宿主防御或疾病发病机制的机制提供深入的见解。

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.