Hofmann Sabine, Philbrook Christine, Gerbitz Klaus-Dieter, Bauer Matthias F
Institut für Diabetesforschung, Akademisches Lehrkrankenhaus Muenchen-Schwabing, Koelner Platz 1, 80804 Muenchen, Germany.
Hum Mol Genet. 2003 Aug 15;12(16):2003-12. doi: 10.1093/hmg/ddg214.
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 approximately 400 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.
WFS1基因突变会导致沃尔弗勒姆综合征，这是一种罕见的隐性疾病，其特征为早发性、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。WFS1基因编码沃尔弗勒姆蛋白，一种内质网中假定的多跨膜糖蛋白。沃尔弗勒姆蛋白的功能完全未知。为了表征沃尔弗勒姆蛋白，我们针对该蛋白的两个亲水性末端产生了多克隆抗体。发现沃尔弗勒姆蛋白在全身广泛表达，在脑、胰腺、心脏和胰岛素瘤β细胞系中表达水平最高。对其结构特征的分析提供了实验证据，表明沃尔弗勒姆蛋白含有九个跨膜区段，并以N（胞质）/C（腔）拓扑结构嵌入膜中。沃尔弗勒姆蛋白在膜中组装成约400 kDa的更高分子量复合物。脉冲追踪实验表明，在成熟过程中，沃尔弗勒姆蛋白进行N-糖基化，但缺乏蛋白水解加工。此外，N-糖基化似乎对沃尔弗勒姆蛋白的生物合成和稳定性至关重要。在这里，我们首次研究了导致受影响个体中沃尔弗勒姆蛋白功能丧失的分子机制。在携带无义突变的患者中，突变的沃尔弗勒姆蛋白完全缺失是由WFS1无义转录本的不稳定性和快速降解引起的。在一名携带复合杂合错义突变R629W的患者中，我们发现沃尔弗勒姆蛋白的稳态水平显著降低。在COS-7细胞中表达的突变型沃尔弗勒姆蛋白的脉冲追踪实验表明，R629W突变导致沃尔弗勒姆蛋白不稳定并使其半衰期大幅缩短。因此，本文研究的患者中的沃尔弗勒姆综合征是由蛋白质剂量减少而非突变型沃尔弗勒姆蛋白功能障碍引起的。
