BIOLOGY OF NEPHRITOGENIC GLOMERULAR EPITHELIAL ANTIGEN

这是一个研究肾小球结构生物学的建议， 内脏上皮细胞（GEC），通过分析GEC特异性质膜 蛋白质，其是致肾炎单克隆抗体mAb 5- 1-6.在组织切片中，mAb 5-1-6仅与大鼠GEC结合， 特别是在GEC狭缝光阑附近， 来自正常大鼠肾小球的51 kDa蛋白。经过一次体内 注射后，mAb 5-1-6定位于GEC足突， 缺乏补体或炎性细胞的大量蛋白尿。 同时，抗体：抗原复合物似乎重新分布在 GEC表面并可被内吞。提示该抗原 (p51)由mAb 5-1-6识别的一种整合的跨膜蛋白可能是 以及抗体与其细胞外结合 结构域改变了分子排列， 功能狭缝光阑。另一种可能性是， 抗体结合导致p5 l从细胞的基底外侧表面转移， 足细胞到狭缝隔膜，在那里复合物可能干扰 细胞质和未知细胞质之间的正常分子排列 跨膜蛋白 在具体目标#1中，提出了一系列补充方法， 确定p51的主要氨基酸组成和结构， 推定的致肾炎结构域，并寻找小鼠和人类同源物。 在具体的目标#2中，抗-p51抗体和分离的肾小球细胞在 短期培养将用于：检查正常合成，细胞 p51的分布和组织;并解释，在细胞生物学 术语，当p51被抗体结合时会发生什么。还将尝试 为了开发大鼠GECs的p51表达系， 问题研究 具体目标#3旨在：定义p51的精确位置及其 与正常成熟发育中的狭缝光阑的关系 GEC;并确定mAb 5-1-6在体内和体内结合如何改变这一点。 某些蛋白尿状态，其中可预测和有据可查 在狭缝光阑中发生变化。免疫组织学将用于 确定p5 l的分布和免疫印迹分析， 膜相关p5 l。代谢标记研究和mRNA水平将 提供新的p5 l合成的证据。p5 l寡聚化的变化 状态将通过分离的细胞的细胞表面标记来研究， 损伤和恢复肾小球。 定义这种蛋白质的分子结构，以及它的 细胞组织和抗体结合反应的体外和体内研究 体内，将深入了解其在保持完整性的作用， GEC作为渗透性屏障，可能有助于阐明 组成的狭缝隔膜，以及它是如何改变蛋白尿 疾病

This is a proposal to study the structural biology of the glomerular visceral epithelial cell (GEC) by analyzing a GEC-specific plasma membrane protein that is the target of a nephritogenic monoclonal antibody, mAb 5- 1-6. In tissue sections, mAb 5-1-6 binds exclusively to rat GECs, especially in the vicinity of GEC slit diaphragms, and it precipitates a 51 kDa protein from normal rat glomeruli. After a single in vivo injection, mAb 5-1-6 localizes to GEC foot processes and causes immediate, massive proteinuria in the absence of complement or inflammatory cells. Simultaneously, the antibody:antigen complex appears to redistribute on the GEC surface and may be endocytosed. It is suggested that the antigen (p51) recognized by mAb 5-1-6 may be an integral transmembrane protein of the slit diaphragm and that binding of antibody to its extracellular domain alters the molecular arrangement that constitutes a normally functioning slit diaphragm. An alternative possibility is that divalent antibody binding causes p5l to shift from the basolateral surface of the podocyte to the slit diaphragm where the complex might interfere with the normal molecular arrangement between cytoplasmic and as yet unknown transmembrane proteins. In specific aim #1, a series of complementary methods are proposed to define the primary amino acid composition and structure of p51, identify putative nephritogenic domains and seek mouse and human homologues. In specific aim #2, anti-p51 antibodies and isolated glomerular cells in short term culture will be used to: examine the normal synthesis, cellular distribution and organization of p51; and explain, in cell biological terms, what happens when p51 is bound by antibody. An attempt will also be made to develop a p51-expressing line of rat GECs to facilitate these studies. Specific aim #3 is designed to: define the precise location of p51 and its relationship to the slit diaphragm in the normal mature and developing GEC; and determine how this is altered by mAb 5-1-6 binding in vivo and in certain proteinuric states in which predictable and well documented changes occur in the slit diaphragm. Immunohistology will be used to define the distribution of p5l and immunoblot analysis the amount of membrane-associated p5l. Metabolic labelling studies and mRNA levels will provide evidence of new p5l synthesis. Changes in p5l oligomerization status will be studied by cell surface labeling of cells isolated from injured and recovering glomeruli. Defining the molecular structure of this protein, together with its cellular organization and response to antibody binding in vitro and in vivo, will give insight into its role in maintaining the integrity of the GEC as a permeability barrier and perhaps shed light on the molecular composition of the slit diaphragm and how it is altered in proteinuric diseases.