BIOLOGY OF NEPHRITOGENIC GLOMERULAR EPITHELIAL ANTIGEN

这是一项研究肾小球结构生物学的建议。 内脏上皮细胞(GEC)特异性质膜的分析 作为导致肾病的单抗mAb5-的靶标的蛋白质 1比6。在组织切片中，mAb5-1-6仅与大鼠肾小管上皮细胞结合， 特别是在GEC狭缝隔膜附近，它沉淀了一个 51 kDa蛋白来自正常大鼠肾小球。在活体内单次 注射，mAb5-1-6定位于GEC足突并引起即刻， 大量蛋白尿，缺乏补体或炎性细胞。 同时，抗体：抗原复合体似乎重新分布在 GEC表面可被内吞。有人认为，这种抗原 MAb5-1-6识别的(P51)可能是一种完整的跨膜蛋白。 裂隙横隔膜及其细胞外抗体结合 结构域改变了构成正常的 正常工作的狭缝隔膜。另一种可能性是二价 抗体结合导致p51从基底外侧表面移位。 足细胞连接到缝隙隔膜，在那里复合体可能干扰 细胞质和未知物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，以 确定p51的主要氨基酸组成和结构，鉴定 推测的肾病致病结构域，并寻找小鼠和人类的同源物。 在特定目的#2中，抗p51抗体和分离的肾小球细胞 短期培养将用于：检测正常合成、细胞 P51的分布和组织；并在细胞生物学中解释 术语，当p51被抗体结合时会发生什么。一次尝试也将是 旨在建立表达p51的大鼠肾小管上皮细胞系，以促进这些 学习。 具体目标#3旨在：定义p51和其 成人正常发育过程中裂孔与横隔膜的关系 GEC；并确定mAb5-1-6在体内和体内的结合如何改变这种情况 某些蛋白尿状态，在这些状态下，可以预测并得到很好的记录 缝隙隔膜发生了变化。免疫组织学将被用于 确定p51的分布和免疫印迹分析 膜相关p5l。代谢标记研究和信使核糖核酸水平将 提供新的p5l合成的证据。P5l齐聚作用的变化 状态将通过分离的细胞的细胞表面标记来研究 肾小球受伤和恢复。 确定这种蛋白质的分子结构，以及它的 体外和体内抗体结合的细胞组织和反应 Vivo，将让我们深入了解它在维护 GEC是一种通透性屏障，也许能揭示分子 裂隙隔膜的组成及其在蛋白尿中的变化 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.