PODOCALYXIN--ANALYSIS OF NUCLEIC ACID/PROTEIN STRUCTURE

肾病综合征影响儿童和成人，并可能导致 肾功能的永久性丧失和死亡。颈椎的解剖异常 与肾病综合征相关的足细胞已被确认为更多 30多年来，但肾小球上皮细胞 细胞病理导致蛋白质渗漏通过滤过屏障 人们对此仍知之甚少。多阴离子分子对 肾小球滤过的电荷选择性。Kerjaschki、Farquhar和 同事们提供的关键信息表明，Podocalyx是 肾小球上皮细胞表面的主要多阴离子分子， 它在足突结构的维持中起着关键作用 和功能。尽管它很重要，但详细的分子结构 泊多卡林的含量尚不清楚。此应用程序的目标是 描述分子的结构、功能和调控 Podocalyin的表达。在初步研究中，PodocalyxMcClone 抗体已被用于从兔肾中克隆重叠的cDNA 皮质c DNA文库。兔Podocalyin的全长cDNA将被 克隆并用于预测蛋白质的结构和功能。这个 该cDNA序列与正品Podocalysin蛋白的关系 将通过部分测序纯化的Podocalysin蛋白和 将该序列与核苷酸序列进行比较。初步数据 表明Podocalysin蛋白在不同细胞类型中的异质性 纸巾。核苷酸水平上这种异质性的可能原因 将通过寻找多个基因和替代剪接来探索。 不同细胞类型中Podocalysin蛋白结构的异质性 和组织将通过蛋白质印迹分析，测量 电泳迁移率和等电点，并通过测定 酶促脱糖和脱糖对凝胶流动性和蛋白质组分的影响 抗Podocalyx单抗的结合。我们预计会有一个 从分子水平上理解豆腐果苷的结构和调控 Level将为肾小球的调节提供新的见解 上皮细胞的结构和功能，并可能促进发展 肾病综合征的新治疗策略。

The nephrotic syndrome affects both children and adults and may lead to permanent loss of renal function and death. Anatomic abnormalities of the podocyte associated with nephrotic syndrome have been identified for more than 30 years, but the molecular mechanisms by which glomerular epithelial cell pathology causes proteins to leak through the filtration barrier are still poorly understood. Polyanionic molecules are important for the charge selectivity of glomerular filtration. Kerjaschki, Farquhar and colleagues have provided critical information showing that podocalyxin is the major polyanionic molecule of the glomerular epithelial cell surface, and that it plays a key role in the maintenance of foot process structure and function. In spite of its importance, the detailed molecular structure of podocalyxin is not known. The goal of this application is to characterize the molecular structure, function and regulation of expression of podocalyxin. In preliminary studies, podocalyxin monoclonal antibodies have been used to clone overlapping cDNAs from a rabbit renal cortical cDNA library. The full length cDNA of rabbit podocalyxin will be cloned and used to predict the proteins' structure and function. The relationship of the cDNA sequence to the authentic podocalyxin protein will be confirmed by partially sequencing purified podocalyxin protein and comparing the sequence to the nucleotide sequence. Preliminary data indicates heterogeneity of podocalyxin protein in different cell types and tissues. The possible causes of this heterogeneity at the nucleotide level will be explored by looking for multiple genes and alternative splicing. The heterogeneity of podocalyxin protein structure in different cell types and tissues will be determined by Western blot analysis, measurement of electrophoretic mobility and isoelectric point, and by determining the effects of enzymatic desialylation and deglycosylation on gel mobility and binding of anti-podocalyxin monoclonal antibodies. We anticipate that an understanding of podocalyxin structure and regulation at the molecular level will provide new insight into the regulation of glomerular epithelial cell structure, and function and may facilitate development of new treatment strategies for the Nephrotic Syndrome.