Wolfram syndrome:: structural and functional analyses of mutant and wild-type wolframin, the WFS1 gene product

被引:164
作者
Hofmann, S
Philbrook, C
Gerbitz, KD
Bauer, MF
机构
[1] Akad Lehrkrankenhaus Muenchen Schwabing, Inst Diabet Forsch, D-80804 Munich, Germany
[2] Akad Lehrkrankenhaus Muenchen Schwabing, Inst Klin Chem Mol Diagnost & Mitochondriale Gene, D-80804 Munich, Germany
关键词
TRANSMEMBRANE PROTEIN; DIABETES-MELLITUS; OPTIC ATROPHY; MUTATIONS; CELLS; GLYCOPROTEINS; EXPRESSION; RETICULUM; DISEASE;
D O I
10.1093/hmg/ddg214
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:2003 / 2012
页数:10
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