Fc receptor/Ig interactions and trafficking

描述(由申请人提供)：我们试图阐明跨细胞免疫球蛋白Fc受体(FCRs)的识别和跨细胞机制：聚合免疫球蛋白受体(PIgR)，它将聚合免疫球蛋白A(PIgA)通过粘膜上皮运输到粘膜分泌物中。PIgR代表了一个新的方向，它是对我们之前对新生儿Fc受体(FcRN)的补充，FcRN是一种跨细胞的FCR，它将母亲的免疫球蛋白G(IgG)通过上皮转移到胎儿或哺乳的新生儿。在前一个资助周期中，我们使用生化/生物物理和结构方法(X射线结晶学和单粒子电子显微镜)表征了FcRN和其他FCRs。我们还开发了通过电子断层扫描(ET)显示上皮细胞内运输FcRN-Ig G复合体的囊泡的方法，获得了跨细胞过程中动态事件的高分辨率三维快照。我们将应用这些方法来研究黏膜分泌物中的主要免疫球蛋白IgA，它以聚合物(PIGA)的形式存在于与pIgR的复合体中，pIgR是一种多域受体，介导PIgA通过极化的上皮细胞基侧到顶端的跨细胞传递，将pIgR-PIGA复合体运送到黏膜分泌物，在那里它们结合宿主和病原体蛋白。虽然大多数相互作用保护宿主上皮屏障，但与一些病原体蛋白(如肺炎链球菌的蛋白)结合可通过反向跨细胞作用增强疾病的毒力，从而促进细菌入侵。尽管PIGA和pIgR在免疫系统功能中的基本作用已经确立，但其调控的分子机制和结构在很大程度上仍不清楚。我们建议使用X射线结晶学、补充的生化实验和电子显微镜来表征溶液和细胞内与PIGA和病原体蛋白的pIgR复合体：既有单颗粒EM来派生大的蛋白质复合体的结构，也有ET来描绘三维上皮细胞中的跨细胞途径。由此产生的单个蛋白质、复合体和运输细胞的结构模型将揭示PIGA、pIgR及其与病原体蛋白质的复合体的结构，以及控制它们在健康和疾病期间的相互作用、功能和运输的分子机制。这些研究产生的模型将为深入了解跨细胞受体介导的PIGA过程提供基本的见解，这对于理解粘膜和体液免疫以及疾病病理学以及开发现有的和新的免疫疗法是必要的。我们的结果也将是普遍的细胞生物学兴趣，因为他们解决的分子机制，通过细胞内运输机制进行识别和适当的分类内化的货物。

DESCRIPTION (provided by applicant): We seek to elucidate recognition and transcytosis mechanisms for a transcytotic immunoglobulin Fc receptor (FcRs): the polymeric Ig receptor (pIgR), which transports polymeric IgA (pIgA) across mucosal epithelia into mucosal secretions. pIgR represents a new direction that is complementary to our previous studies of the neonatal Fc receptor (FcRn), a transcytotic FcR that transfers maternal immunoglobulin G (IgG) across epithelia to the fetus or suckling newborn. In the previous funding cycle, we characterized FcRn and other FcRs using biochemical/biophysical and structural approaches (X-ray crystallography and single particle electron microscopy). We also developed methods to visualize vesicles transporting FcRn-IgG complexes inside epithelial cells by electron tomography (ET), deriving high resolution 3-D snapshots of dynamic events during transcytosis. We will apply these methods to study IgA, the predominant immunoglobulin in mucosal secretions where it exists as a polymer (pIgA) in complex with pIgR, a multi-domain receptor that mediates basolateral-to-apical transcytosis of pIgA across polarized epithelial cells to deliver pIgR-pIgA complexes to mucosal secretions where they bind host and pathogen proteins. While most interactions protect the host epithelial barrier, binding to some pathogen proteins (e.g., from S. pneumoniae bacteria) enhances disease virulence through reverse transcytosis that can facilitate bacterial invasion. Despite well-established, fundamental roles in immune system function, the molecular mechanisms and architecture governing pIgA and pIgR remain largely uncharacterized. We propose to characterize pIgR complexes with pIgA and pathogen proteins in solution and inside cells using X-ray crystallography, complimentary biochemical experiments, and electron microscopy: both single particle EM to derive structures of large protein complexes, and ET to delineate transcytotic pathways in 3-D in epithelial cells. Resulting structural models of individual proteins, complexes, and transporting cells will reveal the architecture of pIgA, pIgR, and their complexes with pathogen proteins as well as the molecular mechanisms governing their interactions, function, and transport during health and disease. Models resulting from these studies will provide insights into fundamental transcytotic receptor-mediated pIgA processes, which are necessary to understand mucosal and humoral immunity as well as disease pathology, and to develop existing and novel immunotherapies. Our results will also be of general cell biological interest, since they address a molecular mechanism by which recognition and proper sorting of internalized cargo is performed by intracellular trafficking machinery.