Homodimerization and heterodimerization of the glomerular podocyte proteins nephrin and NEPH1
Homodimerization and heterodimerization of the glomerular podocyte proteins nephrin and NEPH1
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DOI：
10.1097/01.asn.0000057853.05686.89
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发表时间：
2003-04-01
影响因子：
13.6
通讯作者：
Walz, G
中科院分区：
文献类型：
--
作者：
Gerke, P;Huber, TB;Walz, G
NPHS1和NEPH1的基因产物NePhin和Neg1是Ig超家族的足细胞膜蛋白。与neparin基因敲除类似，缺乏NEPH1的小鼠表现出严重的蛋白尿，导致围产期死亡。为了确定NEPH1的配体，将NEPH1胞外区与人免疫球蛋白融合。这种NEPH1-Ig融合蛋白标记小鼠肾脏的肾小球毛细血管壁的染色模式与NEPH1和neaffin相同，这引发了人们的猜测，即Negh1可能与neaffin形成同源二聚体和/或异源二聚体。在免疫共沉淀和下拉实验中，NEPH1-Ig融合蛋白从高表达的HEK 293T细胞中沉淀出野生型NEPH1。截断分析表明，粘附性并不局限于NEPH1的单一Ig结构域。含有NEPH1两个Ig结构域的融合蛋白足以固定化NEPH1，但它们不能与包含多囊蛋白-1的系统发育相关的PKD重复序列的对照蛋白相互作用。Negh1还沉淀了neparin，这是一种具有8个Ig结构域和一个纤维连接蛋白样结构域的蛋白质。截断分析揭示了一种非常相似的相互作用模式，即两个与人免疫球蛋白Ig融合的结构域可沉淀出neaffin或Negh1，但不能产生对照蛋白。两种蛋白的相互作用都严格依赖于翻译后糖基化，而细菌表达的蛋白不能与其结合。这些发现表明，NEPH1和neparin的Ig结构域形成了混杂的同二聚体和异二聚体相互作用，这可能促进这些分子在肾小球裂隙隔膜的顺式和反式同源二聚化和异二聚化。
Nephrin and NEPH1, the gene products of NPHS1 and NEPH1, are podocyte membrane proteins of the Ig superfamily. Similar to the nephrin knockout, mice lacking NEPH1 show severe proteinuria leading to perinatal death. To identify the ligand of NEPH1, the extracellular domain of NEPH1 was fused to human IgG. This NEPH1-Ig fusion protein labeled the glomerular capillary wall of mouse kidneys in a staining pattern identical to NEPH1 and nephrin, prompting speculation that that NEPH1 might form homodimers and/or heterodimers with nephrin. In coimmunoprecipitation and pull-down assays, the NEPH1-Ig fusion protein precipitated wild-type NEPH1 from overexpressing HEK 293T cells. Truncational analysis revealed that the adhesive properties were not confined to a single Ig domain of NEPH1. Fusion proteins containing two Ig domains of NEPH1 were sufficient to immobilize NEPH1, but they failed to interact with control protein containing the phylogenetically related PKD repeats of polycystin-1. NEPH1 also precipitated nephrin, a protein with eight Ig domains and a fibronectin-like domain. Truncational analysis of nephrin revealed a very similar mode of interaction, i.e., two nephrin Ig domains fused to human IgG precipitated either nephrin or NEPH1, but not the control protein. Both NEPH1 and nephrin interactions were strictly dependent upon posttranslational glycosylation, and bacterially expressed protein failed to bind NEPH1. These findings demonstrate that the Ig domains of NEPH1 and nephrin form promiscuous homodimeric and heterodimeric interactions that may facilitate cis- and trans- homodimerizations and heterodimerizations of these molecules at the glomerular slit diaphragm.