突变NPHS2基因在真核细胞中的表达

Expression of V165X and R168H mutant podocins in mammalian cells

目的通过在真核细胞中表达突变基因,探讨NPHS2基因突变对podocin分子表达和分布的影响。方法构建分别含有野生型NPHS2编码区cDNA、467-468insT和503G>A突变NPHS2编码区cDNA的表达载体,分别转染人胚肾细胞(HEK293)后用抗podocin氨基端多克隆抗体(P21)和抗podocin羧基端多克隆抗体(P35)进行免疫荧光染色,在共聚焦显微镜下观察podocin在细胞内的分布。结果与野生型NPHS2编码的podocin分子的表达和分布比较,抗podocin氨基端抗体染色显示,两个突变NPHS2表达的podocin与野生型的一样均为阳性;抗podocin羧基端抗体染色显示503G>A突变NPHS2表达的podocin与野生型的一样为阳性,而467-468insT突变NPHS2表达的podocin呈阴性。野生型NPHS2表达的podocin不但分布在胞核的周围,而且主要在细胞膜上呈连续性的线状分布,而两个突变NPHS2表达的podocin主要分布在胞核周围。结论NPHS2的467-468insT突变将严重影响podocin的分子结构和在细胞内的正常分布。NPHS2的503G>A突变即使没有严重影响podocin的结构,但仍然导致podocin不能正常到达细胞膜,因而不能行使正常功能。podocin的正常功能不但有赖于分子结构也有赖于分子分布。

Objective To investigate the effect of NPHS2 gene mutation of both V165X（467- 468insT） and R168H （503G〉A） on the expression and distribution of podocins in HEK293 cells. Methods The wild-type, V165X and R168H mutant cDNAs in the expression plasmids were transfected into HEK293 cells, and the subcellular localization of the wild-type, mutant podocins was studied by immunofluoreseence staining with a specific podocin N-terminal antibody （P21） and a specific podocin C-terminal antibody （P35）, immunolabehng and confocal microscopy. Results The fluorescence stainings of the wild-type, V165X and R168H mutant podocins with antibody P21 were positive. The fluorescence stainings of the wad-type and R168H mutant podocins with antibody P35 were also positive, whereas the staining of V165X mutant podocin with P35 was negative. The staining for wild-type podocin was distributed around nuclei and mainly on the cell membrane surface in a filamentous pattern, whereas V165X and R168H mutant podocins staining localized predominantly around nuclei with a loss of surface expression. Conclusions Both the molecular structure and the subcellular localization of V165X mutant podocin are changed evidently, so is the suboellular localization of R168H mutant podocin, whereas the molecular structure of R168H mutant podocin is little changed. The normal biological functions of the wild podocin rely on its normal molecular structure and subcellular localization as well.