Wolfram syndrome:: structural and functional analyses of mutant and wild-type wolframin, the WFS1 gene product
Wolfram syndrome:: structural and functional analyses of mutant and wild-type wolframin, the WFS1 gene product
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DOI：
10.1093/hmg/ddg214
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发表时间：
2003-08-15
影响因子：
3.5
通讯作者：
Bauer, MF
中科院分区：
文献类型：
--
作者：
Hofmann, S;Philbrook, C;Bauer, MF
WFS 1基因的突变导致Wolfram综合征，这是一种罕见的隐性疾病，其特征在于早发性非自身免疫性糖尿病、视神经萎缩以及进一步的神经和内分泌异常。WFS 1基因编码钨蛋白，一种推测的内质网多跨膜糖蛋白。Wolframin的功能完全未知。为了表征钨蛋白，我们已经产生了多克隆抗体对两个亲水性末端的蛋白质。发现Wolframin在脑、胰腺、心脏和胰岛素瘤β细胞系中普遍表达，且表达水平最高。结构特征的分析提供了实验证据，钨蛋白包含9个跨膜片段，并嵌入在膜中的N-cyt/C-lum拓扑结构。在细胞膜上，钨蛋白组装成分子量约为400 kDa的高分子复合物。脉冲追踪实验表明，在成熟过程中，钨蛋白是N-糖基化的，但缺乏蛋白水解加工。此外，N-糖基化似乎是必不可少的生物合成和稳定的钨蛋白。在这里，我们调查，第一次，在受影响的个人，导致功能丧失的钨蛋白的分子机制。在携带无义突变的患者中，突变的钨蛋白完全缺失是由WFS 1无义转录物的不稳定和快速衰变引起的。在携带复合杂合错义突变R629 W的患者中，我们发现稳态钨蛋白水平显著降低。在COS-7细胞中表达的突变型钨蛋白的脉冲追踪实验表明，R629 W突变导致钨蛋白的不稳定性和强烈降低的半衰期。因此，Wolfram综合征在这里调查的患者是由减少蛋白质剂量，而不是功能障碍的突变体钨蛋白。
Mutations of the WFS1 gene are responsible for Wolfram syndrome, a rare, recessive disorder characterized by early-onset, non-autoimmune diabetes mellitus, optic atrophy and further neurological and endocrinological abnormalities. The WFS1 gene encodes wolframin, a putative multispanning membrane glycoprotein of the endoplasmic reticulum. The function of wolframin is completely unknown. In order to characterize wolframin, we have generated polyclonal antibodies against both hydrophilic termini of the protein. Wolframin was found to be ubiquitously expressed with highest levels in brain, pancreas, heart and insulinoma beta-cell lines. Analysis of the structural features provides experimental evidence that wolframin contains nine transmembrane segments and is embedded in the membrane in an N-cyt/C-lum topology. Wolframin assembles into higher molecular weight complexes of similar to400 kDa in the membrane. Pulse-chase experiments demonstrate that during maturation wolframin is N-glycosylated but lacks proteolytical processing. Moreover, N-glycosylation appears to be essential for the biogenesis and stability of wolframin. Here we investigate, for the first time, the molecular mechanisms that cause loss-of-function of wolframin in affected individuals. In patients harboring nonsense mutations complete absence of the mutated wolframin is caused by instability and rapid decay of WFS1 nonsense transcripts. In a patient carrying a compound heterozygous missense mutation, R629W, we found markedly reduced steady-state levels of wolframin. Pulse-chase experiments of mutant wolframin expressed in COS-7 cells indicated that the R629W mutation leads to instability and strongly reduced half-life of wolframin. Thus, the Wolfram syndrome in patients investigated here is caused by reduced protein dosage rather than dysfunction of the mutant wolframin.