PODOCALYXIN--ANALYSIS OF NUCLEIC ACID/PROTEIN STRUCTURE

肾病综合征影响儿童和成人，并可能导致 肾功能永久丧失和死亡。解剖异常 与肾病综合征相关的足细胞已经被确定为更多 但是肾小球上皮细胞的分子机制 细胞病变导致蛋白质通过过滤屏障渗漏， 仍然知之甚少。聚阴离子分子对于 肾小球滤过的电荷选择性。Kerjaschki，Farquhar和 同事们提供了关键信息，表明足糖萼蛋白是 肾小球上皮细胞表面的主要聚阴离子分子， 在足突结构的维持中起着关键作用 和功能尽管它的重要性，详细的分子结构 podocalyxin是未知的。此应用程序的目标是 表征分子结构、功能和调节 podocalyxin的表达。在初步研究中，足糖萼蛋白单克隆 已使用抗体从兔肾细胞克隆重叠cDNA， 皮层cDNA文库。兔足糖萼蛋白的全长cDNA将被克隆。 克隆并用于预测蛋白质的结构和功能。的 cDNA序列与真正足糖萼蛋白的关系 将通过部分测序纯化的足糖萼蛋白来证实， 将该序列与核苷酸序列进行比较。初步数据 表明足糖萼蛋白在不同细胞类型中的异质性， 组织中这种异质性在核苷酸水平上的可能原因 将通过寻找多基因和选择性剪接来探索。 足糖萼蛋白在不同细胞类型中的结构异质性 和组织将通过蛋白质印迹分析来测定， 电泳迁移率和等电点，并通过测定 酶促去唾液酸化和去糖基化对凝胶流动性的影响， 抗足糖萼蛋白单克隆抗体的结合。我们预计， 了解足糖萼蛋白的分子结构和调控 水平将提供新的见解肾小球的调节， 上皮细胞结构和功能，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.