Biochemical characterization of bona fide polycystin-1 in vitro and in vivo.
Biochemical characterization of bona fide polycystin-1 in vitro and in vivo.
复制标题
真正的多囊蛋白-1 的体外和体内生化表征。
DOI：
10.1053/ajkd.2001.29282
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Germino,GG
中科院分区：
文献类型：
--
作者：
Boletta,A;Qian,F;Onuchic,LF;Bragonzi,A;Cortese,M;Deen,PM;Courtoy,PJ;Soria,MR;Devuyst,O;Monaco,L;Germino,GG
常染色体显性多囊肾病（PKD）的最常见形式是由染色体16p13.3上的PKD 1基因突变引起的。该基因编码一个14 kb的信使RNA，预计表达一个462 kd的膜蛋白。基因产物，多囊蛋白-1，有一个大的细胞外部分组成的蛋白质-蛋白质相互作用域的一种新的组合，并假定是一个质膜受体参与细胞-细胞/基质相互作用。然而，在多囊蛋白-1的特性或其功能的测定方面进展缓慢。事实上，该蛋白在组织和细胞系中以非常低的水平表达，并且先前针对重组蛋白表达的努力在很大程度上是不成功的。我们最近开发了全长人PKD 1互补（cDNA），可以在哺乳动物细胞中以稳定和瞬时的方式表达的结构。我们使用这些系统来表征我们的抗体并在体内追踪蛋白质。我们在这里报告的重组多囊蛋白-1的第一个生化特性，并表明该蛋白质是一个520 kD的糖基化的多肽与460 kD的非糖基化的核心。亚细胞分级分离以及生物素化研究证实，该蛋白质是质膜相关的。此外，我们发现，重组蛋白定位于极化的madin达比犬肾细胞的细胞连接处，如间接免疫荧光所示。我们的数据代表了在高度受控条件下进行的多囊蛋白-1的首次表征。© 2001由国家肾脏基金会，公司。
The most common form of autosomal dominant polycystic kidney disease (PKD) results from mutation of the PKD1 gene on chromosome 16p13.3. The gene encodes a 14-kb messenger RNA that is predicted to express a 462-kd membrane protein. The gene product, polycystin-1, has a large extracellular portion composed of a novel combination of protein-protein interacting domains and is postulated to be a plasma membrane receptor involved in cell-cell/matrix interactions. However, slow progress has been made in the characterization of polycystin-1 or the determination of its function. In fact, the protein is expressed at very low levels in tissues and cell lines and previous efforts directed at expression of recombinant protein had been largely unsuccessful. We have recently developed constructs of full-length human PKD1 complementary (cDNA) that can be expressed in both a stable and transient fashion in mammalian cells. We used these systems to characterize our antibodies and to track the protein in vivo. We report here the first biochemical characterization of recombinant polycystin-1 and show that the protein is a 520-kd glycosylated polypeptide with an unglycosylated core of 460 kd. Subcellular fractionation as well as biotinylation studies confirmed that the protein is plasma-membrane associated. Furthermore, we show that the recombinant protein localizes to cell-cell junctions in polarized madin darby canine kidney cells as revealed by indirect immunofluorescence. Our data represent the first characterization of polycystin-1 performed under highly controlled conditions. © 2001 by the National Kidney Foundation, Inc.