Fc receptor/Ig interactions and trafficking

描述（由申请人提供）：我们试图阐明一种经细胞免疫球蛋白Fc受体（FcRs）的识别和胞吞机制：聚合Ig受体（pIgR），它将聚合IgA （pIgA）运输过粘膜上皮进入粘膜分泌物。pIgR代表了一个新的方向，是我们之前对新生儿Fc受体（FcRn）的研究的补充，FcRn是一种跨细胞FcR，可将母体免疫球蛋白G （IgG）通过上皮传递给胎儿或哺乳新生儿。在之前的资助周期中，我们使用生化/生物物理和结构方法（x射线晶体学和单粒子电子显微镜）对FcRn和其他fcr进行了表征。我们还开发了通过电子断层扫描（ET）可视化上皮细胞内运输FcRn-IgG复合物的囊泡的方法，获得胞吞过程中动态事件的高分辨率3d快照。我们将运用这些方法来研究IgA， IgA是粘膜分泌物中主要的免疫球蛋白，它作为一种聚合物（pIgA）与pIgR复合物存在，pIgR是一种多结构域受体，介导pIgA在极化上皮细胞中从基底侧到根尖的胞吞作用，将pIgR-pIgA复合物传递到粘膜分泌物，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，但与某些病原体蛋白（例如，来自肺炎链球菌的细菌）结合，通过可促进细菌入侵的反胞吞作用增强疾病毒力。尽管pIgA和pIgR在免疫系统功能中具有公认的基本作用，但它们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射线晶体学、互补生化实验和电子显微镜来表征pIgR复合物与pIgA和病原体蛋白在溶液和细胞内的特征：单颗粒EM可以推导出大蛋白复合物的结构，ET可以描绘上皮细胞的3-D胞吞途径。由此产生的单个蛋白质、复合物和运输细胞的结构模型将揭示pIgA、pIgR及其与病原体蛋白复合物的结构，以及在健康和疾病期间控制其相互作用、功能和运输的分子机制。这些研究产生的模型将提供对经细胞受体介导的pIgA过程的基本见解，这对于理解粘膜和体液免疫以及疾病病理以及开发现有和新的免疫疗法是必要的。我们的研究结果也将具有普遍的细胞生物学意义，因为它们解决了细胞内运输机制对内化货物进行识别和适当分类的分子机制。

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.