Hogan Marie C, Manganelli Luca, Woollard John R, Masyuk Anatoliy I, Masyuk Tatyana V, Tammachote Rachaneekorn, Huang Bing Q, Leontovich Alexey A, Beito Thomas G, Madden Benjamin J, Charlesworth M Cristine, Torres Vicente E, LaRusso Nicholas F, Harris Peter C, Ward Christopher J
Division of Nephrology & Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
J Am Soc Nephrol. 2009 Feb;20(2):278-88. doi: 10.1681/ASN.2008060564. Epub 2009 Jan 21.
Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.
与常染色体显性和隐性多囊肾病相关的蛋白质（多囊蛋白 -1、多囊蛋白 -2 和纤维囊泡蛋白）定位于各种亚细胞区室，但它们的功能位点被认为在初级纤毛上。在 Pkhd1(del2/del2) 小鼠中，PC1 + 囊泡围绕着纤毛，这促使我们详细分析这些结构。我们对尿外泌体样囊泡（ELV）进行了亚分级，并分离出了一个富含多囊蛋白 -1、纤维囊泡蛋白（裂解形式）和多囊蛋白 -2 的亚群。这去除了主要污染物 Tamm-Horsfall 蛋白，并将 ELV 亚分级为至少三个不同群体，以水通道蛋白 -2、多囊蛋白 -1 和足细胞蛋白的存在来区分。对 PKD ELV 的蛋白质组学分析鉴定出 552 种蛋白质（232 种尚未在尿蛋白质组数据库中），其中许多与信号传导有关，包括 Smoothened 分子。我们还检测到了与囊性疾病相关基因的另外两种蛋白质产物：小鼠 cpk 基因座的产物胱氨酸，以及人类巴德 - 比德尔综合征基因（BBS3）的产物 ADP 核糖基化因子样 6。我们的蛋白质组学分析证实，多囊蛋白 -1 和纤维囊泡蛋白在体内发生裂解，其方式与多囊蛋白 -1 中 GPS 位点以及纤维囊泡蛋白中前体蛋白转化酶位点的裂解一致。在体外，这些 PKD ELV 以快速且高度特异性的方式优先与肾和胆管上皮细胞的初级纤毛相互作用。这些数据表明，PKD 蛋白在尿液中的膜颗粒中脱落，并且这些颗粒与初级纤毛相互作用。
