Activation of Fc receptors by antibody and innate acute phase proteins

抗体和先天急性期蛋白激活 Fc 受体

基本信息

批准号：
10014078
负责人：
PETER D. SUN
金额：
$ 49.98万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10014078
关键词：
Acute-Phase Proteins Affinity Amyloid Antibodies Antibody Activation Antigen-Antibody Complex Autoimmune Diseases Bacteria Bacterial Infections Binding Binding Sites Biological Assay C-terminal Carbohydrates Cattle Cells Charge Competitive Binding Complement Complement 1q Complex Crystallization Dissociation Dopamine D2 Receptor Engineering Excision Extracellular Domain Extracellular Structure Face Family Family member Fc Receptor Fc domain Glycosaminoglycans Goals Heparin High Density Lipoproteins Human Humoral Immunities IgA receptor IgG Receptors IgG1 IgG3 IgG4 Immunoglobulin A Immunoglobulin G Infection Inflammation Inflammatory Inflammatory Response Length Ligands Manuscripts Mediating Mediator of activation protein Molecular Molecular Conformation Mutation Mutation Analysis N-terminal Natural Immunity Parasitic infection Pathogenicity Pathway interactions Peptides Phagocytosis Phosphorylation Plasma Proteins Polysaccharides Production Protomer Publications Receptor Activation Resolution Role Secondary Amyloidosis Serum Serum amyloid A protein Site Structure Surface TNF gene Tail Therapeutic Variant Virus Diseases Work amyloid formation amyloid peptide base combat crosslink cytokine design experimental study first responder insight interest macrophage mutant neutrophil novel novel therapeutics receptor receptor binding

项目摘要

Recent evidence suggests that pentraxins interact with Fc receptors raising the possiblity of cross interaction between the complement and the Fc receptor mediated pathways. We are investigating the interaction between SAP/CRP and Fc receptors and determine the structure of their complex. We have recently determined the crystal structure of human SAP in complex with FcRIIa. The 1:1 receptor-SAP recognition is predominantly mediated through the interactions of the ridge helix from two separate SAP protomers with the D1 and D2 domains of the Fc receptor. The complex structure between human SAP and FcRIIa reveals a diagonally bound receptor on each SAP pentamer. Mutational analysis suggests a conserved receptor recognition among pentraxins. The shared binding site for SAP and IgG results in their competition to FcR binding and the inhibition of immune complex-mediated phagocytosis by soluble pentraxins. These results establish the role of innate pentraxins in the FcgR pathway, and have novel therapeutic implications for autoimmune diseases.Unexpectedly, the SAP binding site on FcRIIa overlaps partially with the IgG binding site on the receptor. The solution-based binding experiments confirmed that SAP and CRP competed against IgG for the binding to FcRs. Solutuion binding experiments showed that pentraxins recognize various FcRs and activate FcR-mediated phagocytosis and cytokine secretion. Moreover, soluble SAP and CRP inhibited significantly the immune complex-mediated phagocytosis, indicating a regulatory function for this family of plasma proteins. The result highlights the importance of pentraxins in interfacing between the innate and humoral immunities and suggests new therapeutic applications for pentraxins in autoimmune diseases. We recently identified the major IgA receptor, FcRI as a ligand for pentraxins. We conclude through competitive binding and mutational analysis that CRP binds to a distinct site on FcRI from that of IgA, and that the recognition involves the effector face of CRP in a region overlapping with its C1q and FcR binding site. Furthermore, CRP crosslinking of FcRI resulted in ERK phosphorylation, degranulation and cytokine production in FcRI transfected RBL cells. In neutrophils, CRP binding induced FcRI surface expression and TNF- secretion, and CRP-opsonized bacteria triggered phagocytosis. The impact of this work is two folds. First, the discovery that pentraxins activate FcRI reveals a novel function for pentraxins in inflammation. It implicates a potential pentraxin-mediated synergistic activation of various Fc receptors in neutrophil and macrophage-mediated inflammatory responses. This is particular so since neutrophils and macrophages are the first responders of infection and inflammation. Second, our finding also highlights the innate aspect of antibody receptors that are mediators of humoral immunity. As pentraxins, such as CRP, are acute phase proteins whose expressions are upregulated during infections, we are investigating the potential role of CRP in Fc receptor activations during parasitic and viral infections. Serum Amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined the first structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. The structural-based mutational studies revealed two positive charge clusters, near the center and apex of the hexamer, are involved in SAA association with heparin. The binding of high density lipoprotein (HDL) involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helix 1 and helix 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting the native SAA as non-pathogenic. Further, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various sized structural aggregates containing 5 nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights to the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation. To elucidate the molecular mechanism of its high affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcRI in complex with the Fc domain of human IgG1. FcRI binds to the Fc in the same mode as the low affinity FcRII and FcRIII receptors, and shares the conserved contacts to the lower hinge region of Fc. Unique to the high affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG-loop to enable a charged KHR motif to interact with proximal carbohydrate units on the Fc glycans. Both the length and charge of the FcRI FG-loop are well conserved among FcRI homologs. Ala and Glu mutations of the FG-loop KHR residues showed significant contributions from His 174 and Arg 175 to antibody binding, and the loss of the FG-loop glycan contacting residues resulted in a 20-30 fold decrease in FcRI affinity to IgG1 and IgG3, and a 12 fold decrese to IgG4 affinity. The binding of the mutant FcRI receptors to IgG1 is comparable to that of wild type FcRI to deglycosylated IgG1, demonstrating the involvement of the receptor FG-loop in glycan recognition. These results highlight the unique contribution of glycan recognition to FcRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan-mimics to target FcRI for certain autoimmune diseases. To further characterize the contribution of the FG-loop to the high affinity FcRI binding, we generated a H174R variant of human FcRI. R174 is present in bovine and ovine FcRI sequences. We recently crystallized the FcRI R174 variant in complex with IgG1-Fc and determined the FcRI-Fc complex crystal structure to 2.3 angstrom resolution. The current resolution of the complex is much better than our original 3.5 angstrom structure and it showed clearly a direct contact between the FG-loop and the Fc-glycan. A manuscript is being prepared for publication.

最近的证据表明五聚蛋白与 Fc 受体相互作用，增加了补体和 Fc 受体介导途径之间交叉相互作用的可能性。我们正在研究 SAP/CRP 和 Fc 受体之间的相互作用并确定其复合物的结构。我们最近确定了人 SAP 与 FcRIIa 复合物的晶体结构。 1:1 受体-SAP 识别主要通过来自两个独立 SAP 原聚体的脊螺旋与 Fc 受体的 D1 和 D2 结构域的相互作用来介导。人 SAP 和 FcRIIa 之间的复杂结构揭示了每个 SAP 五聚体上有一个对角结合的受体。突变分析表明五聚蛋白之间存在保守的受体识别。 SAP 和 IgG 的共享结合位点导致它们与 FcR 结合竞争，并抑制可溶性五聚蛋白介导的免疫复合物介导的吞噬作用。这些结果确立了先天五聚蛋白在 FcgR 途径中的作用，并对自身免疫性疾病具有新的治疗意义。出乎意料的是，FcRIIa 上的 SAP 结合位点与受体上的 IgG 结合位点部分重叠。基于溶液的结合实验证实 SAP 和 CRP 与 IgG 竞争与 FcR 的结合。溶液结合实验表明五聚蛋白识别各种 FcR 并激活 FcR 介导的吞噬作用和细胞因子分泌。此外，可溶性 SAP 和 CRP 显着抑制免疫复合物介导的吞噬作用，表明该血浆蛋白家族具有调节功能。该结果强调了五聚蛋白在先天免疫和体液免疫之间相互作用的重要性，并提出了五聚蛋白在自身免疫性疾病中的新治疗应用。我们最近确定了主要的 IgA 受体 FcRI 作为五聚蛋白的配体。我们通过竞争性结合和突变分析得出结论，CRP 结合到 FcRI 上与 IgA 不同的位点，并且识别涉及 CRP 效应器面与其 C1q 和 FcR 结合位点重叠的区域。此外，FcRI 的 CRP 交联导致 FcRI 转染的 RBL 细胞中 ERK 磷酸化、脱粒和细胞因子产生。在中性粒细胞中，CRP 结合诱导 FcRI 表面表达和 TNF 分泌，CRP 调理细菌引发吞噬作用。这项工作的影响有两个方面。首先，五聚蛋白激活 FcRI 的发现揭示了五聚蛋白在炎症中的新功能。它暗示了中性粒细胞和巨噬细胞介导的炎症反应中各种 Fc 受体的潜在五聚蛋白介导的协同激活。这是特别的，因为中性粒细胞和巨噬细胞是感染和炎症的第一反应者。其次，我们的发现还强调了作为体液免疫调节剂的抗体受体的先天性。由于 CRP 等五聚蛋白是急性期蛋白，其表达在感染期间上调，因此我们正在研究 CRP 在寄生虫和病毒感染期间 Fc 受体激活中的潜在作用。血清淀粉样蛋白 A (SAA) 代表进化上保守的炎症急性期蛋白家族。它也是继发性淀粉样变性的主要成分。为了了解其功能和向淀粉样蛋白的结构转变，我们确定了人类 SAA1.1 的两种晶体形式的第一个结构，代表了该家族的原型成员。天然 SAA1.1 以六聚体形式存在，其亚基显示出独特的四螺旋束折叠，并通过其长 C 末端尾部稳定。基于结构的突变研究揭示了六聚体中心和顶点附近的两个正电荷簇参与了 SAA 与肝素的关联。高密度脂蛋白（HDL）的结合仅涉及SAA的顶端区域，并且可以被肝素抑制。肽淀粉样蛋白形成分析鉴定出 N 末端螺旋 1 和螺旋 3 是 SAA1.1 的淀粉样蛋白生成肽。两种肽都隐藏在 SAA1.1 的六聚结构中，表明天然 SAA 是非致病性的。此外，SAA 六聚体的解离似乎不足以引发淀粉样变转变，并且 SAA C 末端尾部的蛋白水解切割或去除导致形成包含 5 nm 规则重复原纤维样单元的各种大小的结构聚集体。结构和功能相结合的研究为糖胺聚糖在 SAA1.1 介导的 AA 淀粉样蛋白形成中的致病作用提供了机制见解。为了阐明其高亲和力 IgG 结合的分子机制，我们确定了人 FcRI 胞外结构域与人 IgG1 Fc 结构域复合物的晶体结构。 FcRI 以与低亲和力 FcRII 和 FcRIII 受体相同的模式与 Fc 结合，并与 Fc 的下铰链区共享保守的接触。然而，高亲和力受体-Fc 复合物的独特之处在于受体 D2 结构域 FG 环的构象，使带电 KHR 基序能够与 Fc 聚糖上的近端碳水化合物单元相互作用。 FcRI FG 环的长度和电荷在 FcRI 同系物中都非常保守。 FG 环 KHR 残基的 Ala 和 Glu 突变显示 His 174 和 Arg 175 对抗体结合有显着贡献，FG 环聚糖接触残基的缺失导致 FcRI 对 IgG1 和 IgG3 的亲和力降低 20-30 倍，并且 IgG4 亲和力降低 12 倍。突变型 FcRI 受体与 IgG1 的结合与野生型 FcRI 与去糖基化 IgG1 的结合相当，证明受体 FG 环参与聚糖识别。这些结果强调了聚糖识别对 FcRI 功能的独特贡献，并开辟了基于抗体聚糖工程或小分子聚糖模拟物以靶向 FcRI 治疗某些自身免疫性疾病的潜在治疗途径。为了进一步表征 FG 环对高亲和力 FcRI 结合的贡献，我们生成了人 FcRI 的 H174R 变体。 R174 存在于牛和羊 FcRI 序列中。我们最近结晶了与 IgG1-Fc 复合的 FcRI R174 变体，并以 2.3 埃分辨率确定了 FcRI-Fc 复合物晶体结构。该复合物的当前分辨率比我们最初的 3.5 埃结构好得多，并且它清楚地显示了 FG 环和 Fc 聚糖之间的直接接触。手稿正在准备出版。