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

说明(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(经典途径、凝集素途径和替代途径)激活，导致逐步放大的炎症级联反应。补体系统的激活通常由一系列向下调节因子严格控制，以将宿主组织的损害降至最低。然而，当无调控的补体激活发生时，它在包括心肌梗死、类风湿性关节炎和移植排斥反应在内的广泛的炎症性疾病过程中促进组织损伤。这项研究的长期目标是阐明补体在C1q(经典途径)和MBL(凝集素途径)复合体最初识别配体时激活的分子基础，作为开发治疗方法以减轻或防止非调节补体激活对宿主组织破坏的先决条件。我们先前发现，人类星状病毒外壳蛋白(CP)分别在C1q和MBL水平上有效地抑制补体的经典途径和凝集素途径。最近，从野生型CP中提取的一种30个氨基酸的多肽被鉴定出来，它介导了这种活性。此外，初步数据表明，这种外壳蛋白多肽(CPP)衍生物与C1q结合，以抑制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体的功能提供了新的试剂。我们的具体假设是，CPP直接与C1q和MBL的胶原样区域结合，这对启动经典和凝集素途径活性所需的相关丝氨酸蛋白酶之间的相互作用以及CRT/cC1qR相互作用至关重要。本应用的实验重点是确定CPP与C1q/MBL之间精确的相互作用以抑制补体激活，以及CPP抑制C1q/MBL-CRT/cC1qR相互作用对细胞功能的功能影响。具体目标1将描述CPP与C1q/MBL之间的相互作用，并通过以下方式测量对同源丝氨酸蛋白酶激活的功能影响：(A)通过结合(ELISA)分析、功能分析和利用CPP的合成衍生物确定CPP/C1q和CPP/MBL的相互作用区域，(B)通过表面等离子体共振和恒温滴定热法表征CPP与C1q/MBL结合的动力学，以及(C)利用本实验室建立的竞争结合分析方法(ELISA法)，分析CPP与C1q相互作用改变与其同源丝氨酸蛋白酶复合体相互作用的程度。具体目标2将评估CPP改变中性粒细胞与C1q-CRT/cC1qR相互作用的程度及其对细胞功能的影响，方法是：(A)利用流式细胞术测定CPP抑制中性粒细胞上C1q与CRT/cC1qR结合的能力；以及(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动的生物学影响。 公共卫生相关性：该研究项目的成功实施将明确CPP抑制补体级联的C1和MBL激活的机制，以及阻止C1q-CRT/cC1qR相互作用对中性粒细胞功能的影响。补体激活失调会导致类风湿性关节炎、心肌梗塞和移植排斥等炎症性疾病，但目前用于治疗的补体抑制剂很少。了解这种新型多肽补体抑制剂的作用机制将为其成为预防补体介导的免疫病理的治疗化合物铺平道路。

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. PUBLIC HEALTH RELEVANCE: Successful execution of this research project will define both the mechanism of CPP inhibition of C1 and MBL activation of the complement cascade and the consequence of impeding C1q-CRT/cC1qR interaction on neutrophil function. Dysregulated complement activation contributes to inflammatory diseases in humans such as rheumatoid arthritis, myocardial infarction and transplant rejection, however very few complement inhibitors are currently available for therapeutic use. Understanding the mechanism of action of this novel peptidic complement inhibitor will pave the way for its development as a therapeutic compound to prevent complement- mediated immunopathology.