Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.
Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.
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DOI：
10.1016/j.exer.2013.09.019
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发表时间：
2013-11
影响因子：
3.4
通讯作者：
Varadaraj, Kulandaiappan
中科院分区：
文献类型：
--
作者：
Kumari, Sindhu S.;Gandhi, Jason;Mustehsan, Mohammed H.;Eren, Semih;Varadaraj, Kulandaiappan
关键词：
水通道蛋白0 （Aquaporin 0, AQP0）在晶状体纤维细胞中具有双重功能，既是一个水孔，又是一个细胞间的粘附分子。AQP0突变可导致人类和小鼠严重的晶状体白内障。氨基酸33 （R33C）的精氨酸与半胱氨酸错义突变导致一个中国家族五代先天性常染色体显性白内障。我们利用定点诱变技术在野生型（WT-AQP0）人类AQP0 cDNA中重建了该突变体，并在异源表达系统中克隆表达了该突变体AQP0 （AQP0- r33c）。突变体AQP0-R33C表现出与WT-AQP0一样的转运和膜定位。在爪蟾卵母细胞中进行的功能研究显示，AQP0-R33C和WT-AQP0的透水性无显著差异（P>0.05）。然而，细胞聚集和细胞间粘附实验表明，AQP0-R33C与WT-AQP0相比，细胞间粘附性能明显降低（P< 0.001）。使用路西法黄染料的刮擦加载试验显示，影响间隙连接偶联的细胞间粘附减少（P< 0.001）。所提供的数据表明，这种突变可能不会引起蛋白质折叠的重大改变，因为没有阻碍蛋白质运输或水渗透。细胞间粘连的减少和白内障的发展表明细胞外环A的保守正电荷可能在使纤维细胞更紧密的过程中起重要作用。所提出的示意图模型表明，AQP0引起的细胞间粘附对晶状体透明和体内平衡至关重要。
Aquaporin 0 (AQP0) performs dual functions in the lens fiber cells, as a water pore and as a cell-to-cell adhesion molecule. Mutations in AQP0 cause severe lens cataract in both humans and mice. An arginine to cysteine missense mutation at amino acid 33 (R33C) produced congenital autosomal dominant cataract in a Chinese family for five generations. We re-created this mutation in wild type (WT-AQP0) human AQP0 cDNA by site-directed mutagenesis, and cloned and expressed the mutant AQP0 (AQP0-R33C) in heterologous expression systems. Mutant AQP0-R33C showed proper trafficking and membrane localization like WT-AQP0. Functional studies conducted in Xenopus oocytes showed no significant difference (P>0.05) in water permeability between AQP0-R33C and WT-AQP0. However, the cell-to-cell adhesion property of AQP0-R33C was significantly reduced (P< 0.001) compared to that of WT-AQP0, indicated by cell aggregation and cell-to-cell adhesion assays. Scrape-loading assay using Lucifer Yellow dye showed reduction in cell-to-cell adhesion affecting gap junction coupling (P< 0.001). The data provided suggest that this mutation might not have caused significant alterations in protein folding since there was no obstruction in protein trafficking or water permeation. Reduction in cell-to-cell adhesion and development of cataract suggest that the conserved positive charge of Extracellular Loop A may play an important role in bringing fiber cells closer. The proposed schematic models illustrate that cell-to-cell adhesion elicited by AQP0 is vital for lens transparency and homeostasis.
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影响因子：
3.4
作者：
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通讯作者：
STOLS, ALH
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