Disease-causing missense mutations in NPHS2 gene alter normal nephrin trafficking to the plasma membrane
Disease-causing missense mutations in NPHS2 gene alter normal nephrin trafficking to the plasma membrane
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DOI：
10.1111/j.1523-1755.2004.00898.x
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发表时间：
2004-11-01
影响因子：
19.6
通讯作者：
Yan, K
中科院分区：
文献类型：
--
作者：
Nishibori, Y;Liu, L;Yan, K
背景Podocin是一种膜整合蛋白，位于肾小球裂孔隔膜并直接与nephrin相互作用。编码podocin的基因NPHS 2在常染色体隐性遗传的类固醇耐药肾病综合征（SRN）患者中发生突变。为了研究SRN患者大量蛋白尿的潜在病理机制，我们研究了5种常见致病错义突变体的转运和亚细胞定位。应用定点突变来产生编码人podocin的五种不同错义突变（P20 L、G92 C、R138 Q、V180 M和R291 W）的cDNA构建体。为了鉴定转染的人胚肾（HEK）293细胞中每个突变体的亚细胞定位，我们已经产生并表征了兔抗人podocin的多克隆抗体。通过光学显微镜和免疫电子显微镜以及使用人肾小球的免疫印迹分析来确定抗体的特异性。应用共聚焦显微镜来确定野生型和突变的podocin分子以及野生型nephrin在转染细胞中的亚细胞定位。通过免疫沉淀和pull-down研究podocin突变体和野生型nephrin的相互作用。免疫荧光和共聚焦显微镜显示，野生型podocin在HEK 293细胞中表达时定位于质膜。两个错义突变，P20 L和G92 C，位于分子的N-末端部分，也存在于质膜上，表明这些突变不影响突变的podocin分子的亚细胞定位。与此相反，位于近端C-末端的蛋白质的一部分的其他三个错义突变体的亚细胞定位急剧改变，其中R138 Q被保留在内质网（ER），V180 M在细胞质中形成包涵体，和R291 W突变体被困在ER和小的细胞内囊泡。有趣的是，podocin错义突变体的这种异常亚细胞定位也通过两种分子之间的强蛋白结合导致共转染细胞中野生型nephrin蛋白运输的改变。在SRN患者中，NPHS 2基因中的一些错义突变不仅导致突变的podocin的错误折叠和错误定位，而且它们还可以通过改变nephrin向质膜的适当运输来干扰狭缝隔膜结构和功能。
Background. Podocin is a membrane-integrated protein that is located at the glomerular slit diaphragm and directly interacts with nephrin. The gene encoding podocin, NPHS2, is mutated in patients with autosomal-recessive steroid-resistant nephrotic syndrome (SRN). In order to study a potential pathomechanism of massive proteinuria in patients with SRN, we have investigated the trafficking and subcellular localization of five common disease-causing missense mutants of human podocin.Methods. Site-directed mutagenesis was applied to generate cDNA constructs encoding five different missense mutations of human podocin (P20L, G92C, R138Q, V180M, and R291W). To identify the subcellular localization of each mutant in transfected human embryonic kidney (HEK) 293 cells, we have generated and characterized a rabbit polyclonal antibody against the human podocin. Specificity of the antibody was determined by light and immunoelectron microscopy, as well as immunoblot analysis using human glomeruli. Confocal microscopy was applied to determine subcellular localization of the wild-type and the mutated podocin molecules, as well as wild-type nephrin in transfected cells. Immunoprecipitation and pull-down studies were carried out to investigate the molecular interaction of podocin mutants and wild-type nephrin.Results. Immunofluorescence and confocal microscopy showed that wild-type podocin located to the plasma membrane when expressed in HEK293 cells. Two missense mutations, P20L and G92C, located at the N-terminus part of the molecule, were also present at the plasma membrane, indicating that these mutations did not affect the subcellular localization of the mutated podocin molecules. In contrast, subcellular localization of three other missense mutants located in the proximal C-terminus part of the protein was drastically altered, in which R138Q was retained in the endoplasmic reticulum (ER), V180M formed inclusion bodies in the cytoplasm, and the R291W mutant was trapped both in the ER and in small intracellular vesicles. Interestingly, this abnormal subcellular localization of podocin missense mutants also resulted in alteration in protein trafficking of wild-type nephrin in cotransfected cells through the strong protein binding between both molecules.Conclusion. In patients with SRN, some missense mutations in the NPHS2 gene not only lead to misfolding and mislocalization of the mutated podocin, but they can also interfere with slit diaphragm structure and function by altering the proper trafficking of nephrin to the plasma membrane.