Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm
Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm
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DOI：
10.1111/j.1523-1755.2004.00844.x
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发表时间：
2004-10-01
影响因子：
19.6
通讯作者：
Onuchic, LF
中科院分区：
文献类型：
--
作者：
Menezes, LFC;Cai, YQ;Onuchic, LF
背景PKHD 1是常染色体隐性遗传性多囊肾病（ARPKD）基因，编码多种可变剪接转录本，预测可产生膜结合和分泌蛋白。最长的开放阅读框编码多导管蛋白（纤维囊蛋白），一个推定的4074个氨基酸的蛋白质，具有单个跨膜结构域和细胞内C-末端。为了表征PKHD 1产物及其表达谱，我们针对多导管蛋白的不同部分制备了多克隆抗体，并使用各种方法分析了不同器官。蛋白质印迹分析表明，成人肾脏、肝脏和胰腺中的特异性条带>440 kD，肾脏和肝脏中的特异性条带与230 kD相似，主要在膜组分中观察到。从肾脏免疫沉淀>440-kD推定的膜蛋白，随后使用两种不同的抗血清通过Western印迹检测。亲和纯化的抗血清主要在可溶性组分中特异性识别类似于140 kD的额外产物。免疫组织化学研究显示，在皮质和髓质集合管和厚的Henle（TALH）上升肢体的具体染色。连续切片用针对水通道蛋白-2和Tamm-Horsfall蛋白的抗体染色以确认肾单位节段定位。胆管和胰管上皮也检测到阳性染色。对小鼠发育组织的分析表明，输尿管芽支、肝内和肝外胆管、胰管和唾液腺中有特异性染色。内髓集合管培养细胞的免疫荧光研究和髓集合管的免疫电镜分析表明，该蛋白定位于初级纤毛。阳性信号主要分布于细胞顶膜和细胞质中。结果表明，多导管蛋白是初级顶纤毛中表达的多囊肾病（PKD）相关蛋白质组的一部分。我们的数据还表明，除了可能参与纤毛功能，多导管蛋白可能在其他亚细胞功能的作用。使用两种抗血清检测三种不同的产品，有证据表明不同的亚细胞定位，表明PKHD 1编码膜结合和可溶性亚型。
Background. PKHD1, the autosomal-recessive polycystic kidney disease (ARPKD) gene, encodes multiple alternatively spliced transcripts predicted to generate membrane-bound and secreted proteins. The longest open reading frame encodes polyductin (fibrocystin), a putative 4074 amino acid protein with a single transmembrane domain and an intracellular C-terminus.Methods. To characterize the PKHD1 products and their expression profile, we raised polyclonal antibodies against different portions of polyductin and analyzed different organs using various methods.Results. Western blot analyses demonstrated specific bands of >440 kD in human adult kidney, liver, and pancreas and similar to230 kD in kidney and liver, predominantly observed in membrane fractions. The >440-kD putative membrane protein was immunoprecipitated from kidney and subsequently detected by Western blotting using two distinct antisera. An additional product of similar to140 kD was specifically recognized by affinity-purified antisera predominantly in soluble fractions. Immunohistochemistry studies revealed specific staining in cortical and medullary collecting ducts and thick ascending limbs of Henle (TALH). Serial sections were stained with antibodies against aquaporin-2 and Tamm-Horsfall protein to confirm the nephron segment localization. Positive staining was also detected in biliary and pancreatic duct epithelia. Analyses of mouse developing tissues showed specific staining in the ureteric bud branches, intra- and extrahepatic biliary ducts, pancreatic ducts, and salivary glands. Immunofluorescence studies in inner medullary collecting duct cultured cells and immunoelectron microscopy analysis of medullary collecting ducts demonstrated that the protein localizes to the primary cilium. Positive signal was also detected in the apical membrane and in cytoplasm.Conclusion. The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins ex-pressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.