Structure and function analysis of C1/C1q and MBL using a novel peptide inhibitor

描述（由申请人提供）：补体系统是由血清蛋白组成的先天免疫的关键组成部分，可通过三种不同的途径（经典，凝集素和替代）激活，导致逐步放大的炎症级联。补体系统的激活通常受到一系列下调调控因子的严格控制，以尽量减少宿主组织损伤。然而，当不受调节的补体激活发生时，它会在广泛的炎症疾病过程中导致组织损伤，包括心肌梗死、类风湿关节炎和移植排斥反应。本研究计划的长期目标是阐明补体激活在C1q（经典途径）和MBL（凝集素途径）复合物初始识别配体时的分子基础，作为开发治疗方法以减轻或防止不受调节的补体激活对宿主组织破坏的先决条件。我们之前发现，星状病毒的外衣蛋白（CP）分别在C1q和MBL水平上抑制补体的经典途径和凝集素途径。最近，从野生型CP中提取的30个氨基酸的肽被鉴定出介导这种活性。此外，初步数据表明，该外壳蛋白肽（CPP）衍生物与C1q结合，抑制C1q/MBL受体钙网蛋白（CRT/cC1qR）的相互作用。C1q和MBL激活的小分子抑制剂的发现为破译这些复合物的功能提供了一种新的试剂。我们的具体假设是CPP直接结合到C1q和MBL的胶原样区域，该区域对于启动经典和凝集素途径活性以及CRT/cC1qR相互作用所需的相关丝氨酸蛋白酶之间的相互作用至关重要。本应用的实验重点是确定CPP和C1q/MBL之间抑制补体激活所需的精确相互作用，以及CPP抑制C1q/MBL- crt /cC1qR相互作用对细胞功能的功能后果。Specific Aim 1将描述CPP与C1q/MBL之间的相互作用，并通过以下方法测量同源丝氨酸蛋白酶激活的功能影响：(a)通过结合（ELISA）测定、功能测定和利用合成CPP衍生物的质谱蛋白足迹测定，确定CPP/C1q和CPP/MBL的相互作用域；(b)通过表面等离子体共振和等温滴定量热法表征CPP结合C1q/MBL的动力学；(c)利用我们实验室开发的竞争性结合测定（ELISA），分析CPP与C1q相互作用改变与其同源丝氨酸蛋白酶复合物相互作用的程度。特异性目标2将评估CPP在多大程度上可以改变与C1q-CRT/cC1qR对人中性粒细胞的相互作用及其对细胞功能的影响：(a)利用流式细胞术确定CPP抑制C1q与中性粒细胞与CRT/cC1qR结合的能力；(b)利用可抑制sod的铁细胞色素C还原试验表征CPP破坏C1q-CRT/cC1qR相互作用对中性粒细胞呼吸爆发活性的生物学后果。

DESCRIPTION (provided by applicant): The complement system is a critical component of innate immunity comprised of serum proteins, which can be activated by three different pathways (classical, lectin and alternative) causing a progressively amplifying inflammatory cascade. Activation of the complement system is generally tightly controlled by an array of down- regulators to minimize host-tissue damage. However, when unregulated complement activation occurs, it contributes to tissue damage in a wide range of inflammatory disease processes including myocardial infarction, rheumatoid arthritis and transplant rejection. The long-range goal of this research program is to elucidate the molecular basis of complement activation upon initial recognition of ligand by the C1q (classical pathway) and MBL (lectin pathway) complex as a prerequisite to the development of therapeutic methods to attenuate or prevent host tissue destruction by unregulated complement activation. We previously discovered that the coat protein (CP) of human astrovirus potently suppresses both the classical and lectin pathways of complement at the level of C1q and MBL, respectively. Recently, a 30 amino acid peptide derived from the wild-type CP mediating this activity has been identified. In addition, preliminary data demonstrates that this coat protein peptide (CPP) derivative binds C1q to inhibit interaction with the C1q/MBL receptor, calreticulin (CRT/cC1qR). The discovery of a small molecule inhibitor of C1q and MBL activation provides a novel reagent to decipher how these complexes function. Our specific hypothesis is that CPP directly binds to the collagen-like region of C1q and MBL that is critical for interaction between the associated serine proteases required for initiation of classical and lectin pathway activity as well as CRT/cC1qR interaction. The experimental focus of this application is to define the precise interactions between CPP and C1q/MBL required to inhibit complement activation as well as the functional consequence of CPP inhibition of the C1q/MBL-CRT/cC1qR interaction on cellular function. Specific Aim 1 will characterize the interactions between the CPP and C1q/MBL and measure functional effects on cognate serine protease activation by: (a) defining the interaction domains of CPP/C1q and CPP/MBL via binding (ELISA) assays, functional assays and mass spectrometric protein footprinting utilizing synthetic derivatives of CPP, (b) characterizing the kinetics of CPP binding C1q/MBL by surface plasmon resonance and isothermal titration calorimetry and (c) assaying the extent to which CPP interaction with C1q alters interaction with its cognate serine protease complex by utilizing a competitive binding assay developed in our laboratory (ELISA). Specific Aim 2 will evaluate the extent to which CPP can alter the interaction with C1q-CRT/cC1qR on human neutrophils and its effect on cellular function by: (a) determining the ability of CPP to inhibit C1q binding to CRT/cC1qR on neutrophils utilizing flow cytometry and (b) characterizing the biological consequence of CPP disruption of the C1q-CRT/cC1qR interaction on respiratory burst activity in neutrophils using a SOD-inhibitable ferricytochrome C reduction assay.