Investigating polymeric antibody assembly, structure, function and therapeutic potential

研究聚合抗体的组装、结构、功能和治疗潜力

• 批准号: 10339172
• 负责人: Beth M. Stadtmueller
• 金额: $ 53.3万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10339172
• 关键词: Antibodies; Antigens; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Birds; Cells; Clostridium difficile; Complex; Cryoelectron Microscopy; Data; Electrons; Engineering; Experimental Models; Fab Immunoglobulins; Family suidae; Foundations; Heavy-Chain Immunoglobulins; Human; Immune response; Immunity; Immunoglobulin A; Immunoglobulin M; Immunoglobulins; Immunologics; Immunology; In Vitro; Investigation; Knowledge; Ligand Binding; Ligands; Mammals; Mediating; Methods; Modeling; Modification; Molecular; Molecular Conformation; Mucosal Immunity; Mucous Membrane; Mus; Mutation; Outcome; Pathogenicity; Play; Polymeric Immunoglobulin Receptors; Polymers; Positioning Attribute; Predictive Factor; Process; Publishing; Reporting; Reptiles; Research; Role; Salmonella enterica; Secretory Component; Secretory Immunoglobulin A; Side; Solvents; Spectrum Analysis; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Testing; Therapeutic; Vertebrates; Wing; Work; X-Ray Crystallography; base; biophysical techniques; commensal microbes; crosslink; dimer; experimental study; flexibility; functional outcomes; host-microbe interactions; improved; insight; microbial; monomer; pathogen; pathogenic bacteria; polymeric IgA; polymerization; porcine model; preservation; programs; receptor; receptor binding; teleost; teleost fish

This proposal aims to investigate the assembly mechanisms, structures, and functions of polymeric (p) immunoglobulins (Ig) that populate the mucosa. The pIgs are found in vertebrates and together form a structurally diverse group of antibodies. They comprise several Ig heavy chain classes, including mammalian IgA and IgM, which typically contain between two and five Ig monomers and one joining chain (JC); however, potential to assemble with the JC and/or to assemble into polymers of different size varies with vertebrate species and Ig heavy chain class. Following assembly, pIgs are transported to the mucosa by the polymeric Ig receptor (pIgR). In the mucosa, the pIgR ectodomain, called secretory component (SC), remains bound and the complex is referred to as a secretory (S) Ig. SIgA is the predominant mucosal antibody in mammals; it is typically found in dimeric (d) forms; however higher order polymers such as tetramers are functionally relevant. SIgA is associated with unique effector functions compared to monomeric, circulatory antibodies; it can coat, cross-link and agglutinate commensal and pathogenic antigens and also mediate interactions with receptors on host and microbial cells. Despite significance, the structural basis for pIg assembly and SIg functions remained poorly understood through decades of immunological research. In 2020 the cryo-electron microscopy structures of SIgM, SIgA and a dimeric (d) IgA precursor were published revealing unprecedented molecular insights into these crucial complexes and opening the door to new questions and structure-guided experiments. The structures of dIgA and dimeric forms of SIgA revealed two IgAs joined through the JC to form a pseudosymmetric, bent conformation that appears to restrict the positions of antigen-binding fragments (Fabs) and promote access to receptor-binding sites. The SC is asymmetrically bound to one side and is solvent accessible, suggesting it may promote yet uncharacterized interactions with host or microbial factors. These observations raise the questions of how structural differences among pIg are generated (e.g dimer versus tetramer and JC versus no JC), how the bent, asymmetric arrangement of components is induced and maintained, and how it contributes to function. The proposed research program will use structural and biophysical approaches to target these questions. Aim 1 will identify Ig heavy chain residues, structural motifs and/or conformational changes that promote pIg assembly and control pIg polymeric state, while also determining the structural basis for JC- independent pIg assembly and function. Aim 2 will characterize JC-specific mechanisms of pIg assembly and its structural contributions to the pseudosymmetric conformation of dIgA. Aim 3 will characterize the functional significance SC and its capacity to bind microbial ligands. These studies will deliver comprehensive mechanistic models for pIg assembly, generate new pIg structures and report new SIg structure-function relationships. This outcome will improve knowledge of mucosal immunity and provide a foundation for engineering pIg and SIg in order to explore their normal functions and therapeutic potential.

该提案旨在研究聚合物（p）的组装机制、结构和功能 粘膜中存在的免疫球蛋白 (Ig)。猪 Igs 存在于脊椎动物中，并共同形成结构 不同的抗体组。它们包含多种 Ig 重链类别，包括哺乳动物 IgA 和 IgM， 通常包含 2 到 5 个 Ig 单体和一个连接链 (JC)；然而，有潜力 与 JC 组装和/或组装成不同尺寸的聚合物，随脊椎动物物种和 Ig 的不同而变化 重链类。组装后，pIg 通过聚合 Ig 受体 (pIgR) 转运至粘膜。 在粘膜中，pIgR 胞外域（称为分泌成分 (SC)）保持结合，并且该复合物 称为分泌型 (S) Ig。 SIgA 是哺乳动物中主要的粘膜抗体；它通常出现在 二聚体(d)形式；然而，更高阶的聚合物（例如四聚体）在功能上是相关的。 SIgA 相关 与单体循环抗体相比，具有独特的效应功能；它可以涂覆、交联和 凝集共生抗原和致病抗原，还介导与宿主和宿主上受体的相互作用 微生物细胞。尽管具有重要意义，但 pIg 组装和 SIg 功能的结构基础仍然很差 通过数十年的免疫学研究得到了理解。 2020年冷冻电子显微镜结构 SIgM、SIgA 和二聚体 (d) IgA 前体的发表揭示了前所未有的分子见解 这些关键的复合体并为新问题和结构引导实验打开了大门。这 dIgA 和 SIgA 二聚体的结构揭示了两个 IgA 通过 JC 连接形成假对称， 弯曲构象似乎限制了抗原结合片段 (Fab) 的位置并促进访问 到受体结合位点。 SC 不对称地结合到一侧并且易于溶剂接触，这表明它 可能促进与宿主或微生物因素的尚未表征的相互作用。这些观察结果提出了 猪猪之间的结构差异如何产生的问题（例如二聚体与四聚体以及 JC 与无 JC），如何引发和维持组件的弯曲、不对称排列，以及它如何发挥作用 发挥作用。拟议的研究计划将使用结构和生物物理方法来针对这些 问题。目标 1 将鉴定 Ig 重链残基、结构基序和/或构象变化， 促进 PIG 组装并控制 PIG 聚合状态，同时确定 JC- 的结构基础 独立的猪组装和功能。目标 2 将描述 pIg 组装和 JC 特异性机制的特征 它对 dIgA 假对称构象的结构贡献。目标 3 将表征功能 意义 SC 及其结合微生物配体的能力。这些研究将提供全面的机制 pIg 组装模型，生成新的 pIg 结构并报告新的 SIg 结构-功能关系。这 结果将提高对粘膜免疫的认识，并为工程化猪免疫球蛋白和SIg提供基础 以探索其正常功能和治疗潜力。