Investigating polymeric antibody assembly, structure, function and therapeutic potential

本提案旨在研究聚合物(P)的组装机制、结构和功能。 分布在粘膜上的免疫球蛋白(Ig)。这些猪是在脊椎动物中发现的，它们在结构上一起形成了一个 不同的抗体群。它们包括几个Ig重链类别，包括哺乳动物的IgA和IgM， 通常含有两到五个免疫球蛋白单体和一个连接链(JC)；然而，潜在的 与JC组装和/或组装成不同大小的聚合物因脊椎动物种类和免疫球蛋白的不同而不同 沉重的链条课程。组装后，猪通过聚合免疫球蛋白受体(PIgR)被运送到粘膜。 在粘膜中，pIgR胞外结构域，称为分泌成分(SC)，保持结合，复合体是 简称分泌型(S)Ig。SIgA是哺乳动物的主要粘膜抗体；它通常见于 二聚(D)形式；然而，高阶聚合物，如四聚物在功能上是相关的。SIGA关联 与单体循环抗体相比，它具有独特的效应器功能；它可以包被、交联和 凝集共生抗原和致病抗原，并介导与宿主和 微生物细胞。尽管意义重大，但PIG组装和SIG功能的结构基础仍然很差 通过几十年的免疫学研究得到了理解。2020年的低温电子显微镜结构 SIgM、SIgA和二聚体(D)IgA前体的发表揭示了前所未有的分子洞察 这些关键的复合体为新的问题和结构指导的实验打开了大门。这个 二聚型和二聚型SIgA的结构揭示了两个IGA通过JC连接形成假对称， 一种弯曲构象，似乎限制了抗原结合片段(Fabs)的位置并促进了访问 到受体结合部位。SC不对称地结合到一侧，并且可以通过溶剂访问，这表明它 可能促进尚未确定的与宿主或微生物因子的相互作用。这些观察结果提高了 猪之间的结构差异是如何产生的(例如，二聚体与四聚体，JC与NO JC)，组件的弯曲、不对称排列是如何诱导和维持的，以及它是如何贡献的 才能发挥作用。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将确定免疫球蛋白重链残基、结构基序和/或构象变化 促进猪的组装，控制猪的聚合状态，同时也确定了JC- 独立的清管器组装和功能。目标2将描述JC特有的猪组装和 它对DiGa准对称构象的结构贡献。目标3将描述功能 意义SC及其与微生物配体的结合能力。这些研究将提供全面的机制 生猪装配模型，生成新的生猪结构，并报告新的SIG结构-功能关系。这 这一结果将提高人们对粘膜免疫的认识，为工程猪和Sig. 以探索其正常功能和治疗潜力。

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.