Human Molecular Genetics Advance Access originally published online on July 1, 2003
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Human Molecular Genetics, 2003, Vol. 12, No. 16 2003-2012
DOI: 10.1093/hmg/ddg214
© 2003 Oxford University Press
Wolfram syndrome: structural and functional analyses of mutant and wild-type wolframin, the WFS1 gene product
1Institut für Diabetesforschung and 2Institut für Klinische Chemie, Molekulare Diagnostik und Mitochondriale Genetik, Akademisches Lehrkrankenhaus Muenchen-Schwabing, Koelner Platz 1, 80804 Muenchen, Germany
Received April 9, 2003; Accepted June 19, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 ß-cell lines. Analysis of the structural features provides experimental evidence that wolframin contains nine transmembrane segments and is embedded in the membrane in an Ncyt/Clum topology. Wolframin assembles into higher molecular weight complexes of
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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Wolfram syndrome (OMIM 222300), first described by Wolfram and Wagener in 1938 (1), is a rare, autosomal recessive disorder defined by the association of early-onset, insulin-dependent diabetes mellitus and progressive optic atrophy. The syndrome is also known as DIDMOAD, an acronym summarizing the most frequent clinical symptoms diabetes insipidus, diabetes mellitus, optic atrophy and deafness. Additional neurological manifestations such as ataxia, urinary-tract atony, peripheral neuropathy and psychiatric illness may be present (2). Wolfram syndrome is a progressive neurodegenerative disorder. Atrophy and death of specific cell systems, involving both the neuronal network and the endocrinum, appear to be the morphological hallmarks: (i) post-mortem and MRI studies showed atrophy and cell damage of various brain regions, explaining the severe course and complexity of the syndrome (3,4); and (ii) non-autoimmune diabetes mellitus, the first and one of the essential clinical features in Wolfram syndrome, is caused by selective damage of pancreatic ß-cells (5,6).
The nuclear gene mutated in Wolfram syndrome was identified in 1998 using genetic mapping and candidate gene approaches by an American/Japanese collaboration (7) and by our group (8). The Wolfram syndrome (WFS1) gene encodes wolframin, a protein with 890 amino acid residues and an apparent molecular mass of 100 kDa. Secondary structure predictions identified three structural domains: a hydrophobic central domain comprising 9–10 membrane-spanning segments flanked by a hydrophilic domain at the N-terminus and a hydrophilic carboxy tail. No significant homologies were found in the databases, thus defining wolframin as a member of a novel family of multispanning transmembrane proteins. The function of wolframin is completely unknown, so far.
Genetic analyses in Wolfram syndrome patients have identified a wide spectrum of mutations. In many patients, loss-of-function mutations such as stop, frame-shift and splice site mutations were found, and missense mutations were detected in 35% of the cases (7–11). A recent survey of reported WFS1 mutations (12) revealed no obvious hot spots or clustering and mutations appeared to be distributed randomly throughout the entire coding sequence of the gene. Moreover, analysis of the mutations did not identify any specific domains or essential amino acids which could provide any clue to wolframin function. Many of the missense mutations are located in the C-terminal hydrophilic part of the protein. Even mutation of the last seven amino acids leads to a full-blown disease phenotype underlining the functional importance of the C-terminus of wolframin (10,13).
A recent study localized wolframin to the endoplasmic reticulum (ER) by means of subcellular fractionation and immunofluorescence studies (14). Based on this observation, a possible role of wolframin in protein biosynthesis, modification/folding, trafficking and/or regulation of Ca2+ homeostasis was speculated. As a further step toward characterization of wolframin, we generated antibodies against the hydrophilic N- and C-termini of human wolframin. Insulinoma ß-cell lines and transiently transfected cells were used to study the structural and functional features of wolframin. The data show that the wolframin is a resident ER membrane glycoprotein with an odd number of transmembrane helices in an Ncyt/Clum orientation. N-glycosylation plays an important role for the synthesis and/or stability of the protein. Structural analyses indicate that wolframin is organized in higher molecular weight complexes. In order to assess the effect of WFS1 mutations on protein function, mutant cell lines were analyzed. We show for the first time that loss-of-function mutations of WFS1 lead to a complete absence of wolframin due to the instability of nonsense transcripts. It is concluded that the pathogenic mechanism underlying a missense mutation, R629W, involves a diminished half-life of the mutant protein causing a protein dosage effect.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Wolframin is highly expressed in neuronal and neuroendocrine tissues
Wolfram syndrome is a disorder primarily affecting neuronal and neuroendocrine cells. Previous northern blot analyses showed an ubiquitous expression of the WFS1 gene with the highest transcript levels in pancreas and heart (7,8). Here, we investigated the tissue-specific steady-state levels of the wolframin protein which is encoded by the WFS1 gene. Polyclonal antibodies were generated against the N-terminal (residues 1–285) and C-terminal (residues 663–890) hydrophilic domains of wolframin (Fig. 1A). To characterize these antibodies, western blot analyses were performed on COS-7 cells transfected with the human WFS1 cDNA. Immunodecoration with the antibody against the N-terminus (anti-WoN) gave a strong signal at
100 kDa in crude extracts of WFS1 transfected COS cells but not in mock treated cells (Fig. 1B). A specific signal corresponding to a molecular weight of 100 kDa was also detected when the antibody against the C-terminus of wolframin (anti-WoC) was used. However, the anti-WoC antibody exhibited a reduced sensitivity compared to anti-WoN (Fig. 1B).
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Due to its higher sensitivity anti-WoN was used to analyze the protein levels of wolframin in various mouse tissues. Wolframin appeared to be ubiquitously expressed in all tissues investigated (Fig. 2A). However, steady-state levels varied significantly among organs with the highest levels in brain, pancreas, heart and muscle followed by liver and low abundance of wolframin in kidney and spleen.
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Analysis of wolframin levels in a variety of mammalian cell lines showed highest expression in insulinoma cells being
50-fold higher than that of human fibroblasts or human neuroblastoma cells (Fig. 2B). This is in accordance with the previous observation that wolframin mRNA expression is high in pancreas and restricted to the islet cells of the pancreas (7).
Membrane topology of wolframin
Based on hydropathy plots, wolframin is a hydrophobic protein with multiple transmembrane segments (TMs) and large hydrophilic regions at both termini. Computer-based topology prediction methods suggested nine or 10 transmembrane segments. A putative membrane topology comprising nine transmembrane segments is favored by most investigators (8,10,12), although location and length of transmembrane segments differ between the suggested models. We have, therefore, investigated the membrane topology of wolframin by protease protection assays. When total membrane fractions prepared from WFS1 transfected COS cells were treated with 30 µg/ml trypsin, the lumenal ER component protein disulfide isomerase (PDI) was protected from digestion indicating that the ER membranes remained intact (data not shown). Under these conditions, the N-terminus of wolframin was sensitive to digestion with trypsin as indicated by the complete loss of immunoreactivity of the anti-WoN antibody (Fig. 3). No immunoreactive bands of lower molecular weights were detected demonstrating that the N-terminal domain was completely degraded. In contrast, the C-terminus of wolframin remained intact when probed with the anti-WoC antibody. The generation of two major fragments which retained immuno-reactivity suggested that the C-terminus is exposed into the lumen of the endoplasmic reticulum and protected from protease digestion (Fig. 3). The generated immunoreactive fragments corresponded to molecular masses of 49 kDa and 37 kDa and resulted from clipping in hydrophilic loops exposed at the cytoplasmic face of the ER. Together, these data suggest an Ncyt/Clum membrane topology of wolframin implying a number of nine transmembrane segments.
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Glycosylation is important for the stability of wolframin
Recently, it was demonstrated that the transmembrane protein wolframin is post-translationally modified (14). Human wolframin harbors five consensus sites for N-linked glycosylation. According to the nine-transmembrane segment model only two of these sites are predicted to be located in the ER lumen and would, therefore, be able to function as oligosaccharide acceptors in vivo.
To support this hypothesis, we have investigated the molecular weight shift produced by the N-glycosylation of wolframin in transfected COS and in RIN cells. Both cell lines were treated with endoglycosidase H (Endo H) and analyzed by immunoblotting after separation on high-resolution 3–8% NuPage gels. Overexpressed and endogeneous wolframin showed the same shift when treated with Endo H (Fig. 4A). In both cases, the molecular weight was reduced by 5 kDa which would be in good accordance with a shift produced by removal of two oligosaccharide side chains.
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To follow the kinetics of N-glycosylation in more detail, we performed pulse-chase experiments on heterologously expressed human wolframin. WFS1 transfected COS cells were metabolically labeled with [35S]cysteine/methionine for 15 min and chased with media containing unlabeled amino acids for 0, 1 or 6 h. Directly after labeling (chase 0 h) two forms of wolframin were immunoprecipitated differing in their molecular weight by
5 kDa (Fig. 4B). After chase periods of 1 and 6 h only the wolframin species with the higher molecular weight was detected. The pulse-chase experiments indicated that wolframin is not proteolytically processed. Moreover, the higher molecular weight form was shifted to the lower form upon treatment with Endo H, whereas Endo H had no effect on the lower form. Thus, the lower band represents an unglycosylated form of wolframin which is rapidly matured to the fully N-glycosylated form during synthesis. Furthermore, wolframin appears not to become phosphorylated. It was neither sensitive to treatment with lambda phosphatase nor was the protein found in the phosphorylated protein fraction after affinity chromatography using a phosphoprotein purification column (data not shown). Thus, N-glycosylation appears to be the only form of post-translational modification of wolframin. Carbohydrate side-chains are known to influence the conformational stability of proteins and protect them against uncontrolled proteolysis. We have, therefore, investigated the effect of N-linked glycosylation on the intracellular levels of wolframin. Tunicamycin, an inhibitor of N-glycosylation, was added to COS cells 0, 6 and 24 h after transfection with human wolframin. Subsequently, the steady-state levels of wolframin were analyzed in cell lysates after a total culture time of 30 h. Untreated cells were used as a control. Application of tunicamycin during the entire culture period (30 h) led to the almost complete absence of wolframin (Fig. 4C). When tunicamycin was added later after transfection at time points 6 or 24 h, wolframin became detectable, however, the steady-state levels were reduced to 10 or 50% of the control, respectively. It appears likely, that the detected amounts have been synthesized primarily during the tunicamycin-free time interval and not after addition of tunicamycin. Together, these data demonstrate that tunicamycin treatment severely reduces the steady-state levels of wolframin suggesting that inhibition of glycosylation affects biogenesis and/or stability of the protein.
Wolframin assembles into higher molecular weight complexes
To investigate the native molecular mass of wolframin, RIN cells were solubilized in 1% Triton X-100 and subjected to gel filtration analysis on a Superose 12 column under native conditions. Immunoblot analysis of the eluted material showed that wolframin eluted in a fraction corresponding to a molecular mass of 400 kDa (Fig. 5A). These data show that wolframin is able to form higher molecular weight complexes. Similar data were obtained when human WFS1 transfected COS cells were analyzed: heterologously expressed wolframin eluted predominantly with a molecular mass of 400 kDa suggesting that, also under conditions of overexpression, wolframin assembles into higher molecular weight complexes; only a small amount of wolframin monomer (100 kDa) was found.
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The composition of these oligomeric complexes is not known. To characterize proteins in contact with wolframin we performed crosslinking studies. RIN cells and WFS1 transfected COS cells were harvested by scraping and whole cells were either left untreated or incubated with the membrane-permeable crosslinker MBS (m-maleimido-benzoyl-N-hydroxysuccinimide-ester) for 30 min at room temperature. The crosslinking was performed on whole cells, so that the crosslinker is assumed to preferentially stabilize pre-existing complexes within the intact cell. Crosslink adducts were then analyzed using separation on high resolution 3–8% NuPage gels under reducing conditions, followed by immunoblotting using anti-WoN. In the absence of crosslinker, wolframin migrated primarily as a monomer of 100 kDa whereas with MBS both, endogeneous and heterologously expressed wolframin, were found in higher molecular weight forms corresponding to
300–400 kDa (Fig. 5B). No molecular species of intermediate molecular weights were detectable. Crosslinking was also performed on metabolically labeled, living cells. WFS1 transfected COS cells were pulsed with [35S]cysteine/methionine for 2 h, then washed in 1xPBS (phosphate buffered saline) and MBS was added for 30 min. Cell lysates were prepared and immunoprecipitated material was analyzed by SDS–PAGE and autoradiography. In the absence of crosslinker, immunoprecipitated wolframin migrates as three bands: the wolframin monomer (100 kDa) and oligomeric forms of 200 kDa and 300–400 kDa (Fig. 5B). Treatment with crosslinker shifted all the precipitated material into the 300–400 kDa band indicating that this molecular species is preferentially stabilized by the crosslinker.
Taken together, gel filtration analysis and crosslinking experiments suggest that wolframin is organized in higher molecular weight assemblies. There is no experimental evidence that other, so far unknown, proteins contribute to these complexes. The complex size of 400 kDa being just 4-fold the size of a wolframin monomer suggests that these structures might represent homo-oligomers.
WFS1 gene mutations lead to loss-of-function of wolframin
Numerous disease-causing mutations in the WFS1 gene have now been recognized comprising loss-of-function mutations such as stop, frameshift, splice site and missense mutations. It is unknown, so far, whether truncated or mutated wolframin species expressed from these alleles accumulate in the patient's cells. We, therefore, investigated wolframin protein levels in mutant fibroblast cells derived from two patients with mutations in the WFS1 gene (Table 1). Patient 1 carried a homozygous C-insertion at nucleotide position (np) 1029 (1029insC) generating a frameshift downstream to codon 343 leading to a stop at codon 395. Patient 2 harbored compound heterozygous mutations: a G to A mutation at np 1112 which creates a premature stop at codon 371 (W371X) and a heterozygous C to T exchange at np 1885 leading to an arginine to tryptophan exchange at codon 629 (R629W). The pathogenic state of this latter mutation was additionally assessed by mutational analysis of the genomic DNA from the mother, father and the unaffected sister. The sister of patient 2 was found to carry the R629W mutation in a heterozygous state (Table 1).
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The frameshift mutation of patient 1 and the heterozygous stop mutation of patient 2 would give rise to truncated proteins of about 43 and 41 kDa, respectively. However, none of these truncated wolframin species were detected by immunoblotting using the anti-WoN (Fig. 6A). These data indicate that nonsense or frameshift mutations lead to a complete absence of wolframin. A markedly reduced amount of wolframin (
5% of control) was found in patient 2 carrying the compound heterozygous mutation (stop and missense). This amount is much lower than that expected from such a heterozygous state.
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The majority of nonsense transcripts are known to be rapidly degraded. In order to investigate whether the absence of truncated wolframin proteins is caused by instability of the mutated transcripts, we performed semi-quantitative RT–PCR analysis. In patient 1 carrying the homozygous C-insertion, nonsense-mediated messenger decay was demonstrated by the complete absence of WFS1 transcripts (Fig. 6B). In the compound heterozygous patient 2, the steady-state level of WFS1 transcripts was reduced to about 50% when compared to control fibroblasts. Sequencing confirmed that these WFS1 transcripts are exclusively derived from the missense allele (data not shown). Thus, missense mutations appear not to affect stability of WFS1 mRNA whereas nonsense transcripts are rapidly degraded thereby causing the complete absence of the encoded protein.
A rather low amount of wolframin is expressed from the missense allele (R629W) although significant transcript levels were detected. This suggested a reduced stability of the encoded mutant protein. To assess the in vivo stability of the wolframinR629W, pulse-chase experiments were performed. COS cells transiently transfected with either wild-type or mutant WFS1 were pulse-labeled for 15 min and subsequently chased for 0, 1, 6 or 24 h. Wild-type wolframin remained stable up to 6 h chase and was decreased by about 20% after 24 h (Fig. 7A). In contrast, the mutant wolframin appeared to be rapidly degraded. After 1 h of chase the amount of labeled wolframinR629W was about 50% of wild-type wolframin; mutant wolframin was nearly completely disappeared after 6 h of chase (Fig. 7A). This indicates that the R629W mutation leads to instability of wolframin thereby decreasing its half-life when compared to wild-type wolframin. In addition, steady-state levels of wolframinR629W protein were assessed by the immunoblotting of transfected cells analyzed in the pulse-chase experiment. Protein amounts of wolframinR629W were found to be reduced substantially compared to wild-type wolframin (Fig. 7B). Thus, a protein dosage effect rather than a functional defect of wolframin is associated with the missense mutation R629W. It appears likely that mutant cells are not able to accumulate normal levels of wolframin due to rapid degradation of the translation product.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the WFS1 gene are associated with a severe disorder in humans, the Wolfram syndrome. It is characterized by juvenile, non-autoimmune diabetes mellitus and optic atrophy; in addition, diabetes insipidus, sensorineural deafness, ataxia or atonic bladder is observed. The clinical phenotype reflects a progressive neurodegeneration which predominantly affects the central nervous system and the endocrinum.
Since the identification of the disease gene in 1998, only a single study investigating the WFS1 gene product was published (14). It was shown that WFS1 encodes wolframin, a novel transmembrane glycoprotein of 100 kDa located in the ER. The exclusive localization of wolframin in the ER argues against the earlier clinical hypothesis that Wolfram syndrome is a mitochondrial disease (15). It was speculated that wolframin might play a role in membrane trafficking, protein processing or regulation of ER calcium homeostasis (14). However, there is yet no experimental evidence for the involvement of wolframin in one of these processes and its physiological function is still completely unclear.
We have assessed the protein expression profiles in mammalian organ systems and cell lines using antibodies raised against the hydrophilic N- and C-termini of the protein. Immunoblot analysis of mouse tissues demonstrated that wolframin is an abundant protein in pancreas, brain, heart and muscle. Smaller amounts are present in liver and low levels are found in kidney and spleen. Protein levels found in the respective tissues correlate well with the transcript levels detected by northern blot analyses (7,8). In brain, however, wolframin is found at higher steady-state levels than expected from the relatively low amounts of WFS1 transcripts. This suggests that accumulation of functional wolframin might be critical for its function in neuronal cells. A more detailed northern blot analysis of pancreas showed that the WFS1 expression is restricted to pancreatic islet cells and is absent in the exocrine pancreas (7). Accordingly, we found wolframin to be more highly expressed in insulinoma ß-cell lines being about 50-fold higher when compared to various other cell lines. Together, these data confirm the fact that wolframin plays an important functional role in pancreatic ß-cells and support the fact that loss of wolframin function contributes to islet ß-cell damage and insulin-dependent diabetes mellitus. So far, it is not known which cellular mechanisms, common to these two cell types—neurons and ß-cells—involve the action of wolframin. Both cell types share a number of similarities regarding function and metabolism (16,17). For example, proteins such as glutamic acid decarboxylase, glutamate receptor, neurofilament proteins, receptors for neurotrophins and many others are found in neuronal cells and ß-cells. Further studying these similarities might explain why these two cellular systems are selectively affected in Wolfram syndrome and will help to unravel the wolframin function.
In the present study, we have analyzed the structural organization and functional features of wild-type and mutant wolframin. A first aim was to provide experimental data on the membrane topology of human wolframin in order to define the orientation of functional hydrophilic domains. Using the well-established protease-protection assay on total membrane fractions we showed that immunoreactivity against the N-terminus of wolframin was completely lost upon protease digestion. Identical results were obtained irrespective of whether total membranes isolated from transfected COS cells or intact microsomes isolated from mouse liver (data not shown) were digested indicating that the N-terminus of wolframin extends into the cytoplasma. In contrast, the carboxy tail of wolframin was protected from protease treatment. Upon digestion with trypsin, wolframin was clipped into two major anti-WoC reactive fragments with estimated molecular weights of 49 and 37 kDa. This suggests that the C-terminus of wolframin extends towards the lumenal side of the ER membrane. The 49 kDa band most likely represents a fragment generated by clipping at arginines R456 and/or R457; the 37 kDa fragment can be attributed to clipping at position 558. The data imply that these amino acid residues are exposed to the cytoplasmic face of the ER membrane placing R456/R457 between TM 4 and 5 and R558 between TM 6 and 7. The resulting model is consistent with our previously published data (8) and confirms that wolframin contains nine transmembrane segments, with the N-terminus localized in the cytoplasma and the C-terminus in the ER lumen.
Wolframin is a glycoprotein. Our data suggest that N-glycosylation is the only post-translational modification; in particular, wolframin does not become phosphorylated or proteolytically processed. The primary amino acid sequence of mammalian wolframin contains five consensus sites for asparagine-linked glycosylation (NXS/T) at N28, N335, N500, N661 and N746. These sites are conserved in human, mouse and rat wolframin. However, not all of these consensus sites are necessarily used. In order to function as an acceptor for oligosaccharides, potential N-glycosylation sites have to be exposed to the lumenal side of the ER and the size of the adjacent hydrophilic loop must be at least 33 amino acid residues in length (18,19). According to the nine-transmembrane model of wolframin, only the consensus sites at N661 and N746 are located within the lumenal hydrophilic domains. The addition of carbohydrates at N661 and N746 would be consistent with the gel size shift of 5 kDa observed upon Endo H treatment.
Pulse-chase experiments suggested that glycosylated, mature wolframin is relatively stable. The half-life time of the radiolabeled wolframin in these experiments was estimated to be about 2 days. It is known from many glycoproteins that N-glycosylation confers important physicochemical properties to the molecule such as facilitation of proper folding, stabilization of the native conformation and solubility, or stabilization against proteolysis (20). When N-glycosylation is inhibited by tunicamycin, the intracellular level of wolframin was severely reduced. It appears that tunicamycin treatment prevents stable expression of wolframin. This may either be caused by affecting a proper synthesis or rapid degradation of unglycosylated wolframin.
In the present study, we provide evidence that the native structure of wolframin is a higher molecular weight complex. Firstly, gel filtration analysis after detergent solubilization of membrane proteins revealed that wolframin elutes in a fraction corresponding to an apparent molecular mass of 400 kDa; secondly, crosslinking shifted the wolframin monomer to a high molecular weight form with an estimated molecular mass of 300–400 kDa. The same oligomerization was observed with WFS1 transfected COS cells and with RIN cells containing endogeneous wolframin suggesting that this higher molecular weight complex represents the physiological form and is not caused by unspecific aggregation of overexpressed wolframin. It remains to be established whether this high molecular weight form represents a homo-oligomeric assembly of wolframin or whether other, so far unknown, proteins contribute to this complex. It seems likely that wolframin homo-oligomerizes: firstly, the molecular mass of the higher molecular weight form correspond well to a wolframin tetramer; and, secondly, the gel filtration behavior remains unchanged upon massive overexpression of wolframin in COS cells which should otherwise affect the stoichiometry and/or correct assembly of the complex.
A world-wide genetic survey of Wolfram patients (12) showed the following distribution of WFS1 mutation types: nonsense (25%), frameshift (21%), splice site mutations (2%), in frame deletions/insertions (13%) or missense mutations (35%). Loss-of-function mutations of WFS1 such as stop, frameshift and splice site mutations predict truncated proteins. However, it is not known which of these truncated proteins are indeed expressed. In particular, the pathogenic status of many missense mutations is unclear due to lack of functional data.
Here, we investigated for the first time the effect of WFS1 mutations on wolframin expression and stability in mutant cell lines. Protein analysis of fibroblast cell lines of Wolfram patients indicated that stop and frameshift mutations of WFS1 cause complete absence of the wolframin protein rather than synthesis of truncated species. Furthermore, RT–PCR data provided strong evidence that nonsense WFS1 transcripts are unstable in vivo. These transcripts seem to be recognized and degraded by the cell via a common pathway known as nonsense-mediated mRNA decay (21). Thus, similar to what is known from many other inherited genetic disorders, degradation of nonsense WFS1 transcripts prevents the synthesis of truncated translation products and is the molecular mechanism underlying the loss-of-function of wolframin in these patients.
In mutant cell lines harboring a missense mutation (R629W), an unexpectedly low steady-state level of wolframin was observed. Pulse-chase experiments demonstrated a markedly reduced half-time of the wolframinR629W suggesting that protein instability is responsible for the low wolframin levels in these cells. Thus, the molecular mechanism of the missense mutation, R629W, involves a protein dosage effect rather than a functional defect of expressed mutant wolframin. The precise mechanism by which the R629W mutation leads to instability of wolframin remains to be established. It can be speculated that some structural requirements of stable membrane insertion are altered due to R629W mutation which cause rapid degradation. It is known from other membrane proteins that even single amino acid substitution or small deletions can induce structural changes that lead to accelerated intracellular degradation of mutant proteins (22,23). The amino acid residue, R629, is located within the last cytoplasmic loop just prior to TM 9. Thus, the exchange of this positively charged amino acid may be critical for the correct positioning of transmembrane segments or stabilization of correctly folded wolframin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies and cells
A WFS1 cDNA fragment encoding the N-terminal amino acids 1–285 was subcloned into the BamHI/HindIII site of the pMal-cRI vector (New England Biolabs) to generate a maltose binding protein (MBP)-wolframin(1–285) fusion protein. Likewise, a cDNA fragment corresponding to amino acids 663–890 of wolframin was cloned into the pMal-cRI vector to produce chimeric MBP-wolframin(663–890). The service offered by Pineda (Berlin, Germany) was used to produce rabbit polyclonal antibodies against the recombinantly expressed N- and C-terminal wolframin fusion protein, which were termed anti-WoN and anti-WoC, respectively. Antibodies were further affinity-purified using glutathione s-transferase (GST)-wolframin(1–285) fusion protein or GST-wolframin (663–890) covalently coupled to Sulpho-link (Pierce). Anti-ß actin and anti-PDI mAbs were purchased from Sigma. Human skin fibroblasts, COS-7L cells, rat insulinoma cell lines RIN-5AH and INS-1, as well as the human neuroblastoma cell line LAN-5 were grown in high glucose Dulbecco's modified eagle medium (DMEM; Life Technologies) supplemented with 2 mM glutamine and 10% fetal bovine serum (Promocell).
Cell extraction and membrane preparation
Crude extracts from mouse tissues were prepared by homogenization of tissue samples in 1x PBS in the presence of protease inhibitors. The extracts were cleared by centrifugation (10 min, 850g) and an aliquot of total protein, as determined by the Bio-Rad Protein Assay (Bio-Rad), was separated by SDS–PAGE. Crude cellular extracts from cultured cells were prepared by directly boiling cell pellets (5x105 cells) in Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 5% ß-mercaptoethanol and bromophenol blue) for 5 min at 95°C. An aliquot of cells was then separated by SDS–PAGE.
For preparation of total membranes, pelleted cells were homogenized in 20 mM HEPES, pH 7.4, 150 mM NaCl, 250 mM sucrose in the presence of protease inhibitors (complete) (Roche) by repeated passages through a 27-gauge needle and then centrifuged at 850g to remove unbroken cells. Membranes were collected by centrifugation at 100 000g at 4°C for 30 min, resuspended in homogenization buffer and left untreated or subjected to protease digestion in 30 µg/ml trypsin with 30 min at 4°C followed by incubation with 500 µg/ml soybean trypsin inhibitor (5 min, 4°C). Membranes were collected by recentrifugation at 100 000g and analyzed by SDS–PAGE and western blotting.
Western blot analysis
Proteins were separated on 3–8% NuPage Tris-acetate gels (Invitrogen) or on 10% SDS polyacrylamide gels unless otherwise indicated and electrotransferred to 0.2 µm nitrocellulose membranes. The membranes were blocked with 5% milk powder in Tris-buffered saline containing 0.1% Tween-20, and probed with the indicated primary antibody by incubation at 4°C overnight. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Dako) were used as secondary antibodies. Antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL, Amersham) and exposed onto Super RX films (Fuji).
Transfection of COS cells
A WFS1 cDNA fragment was PCR-amplified from human first-strand cDNA using specific oligonucleotide primers (WFS-f: 5'-atg gac tcc aac act gct ccg-3'; WFS-r: 5'-tca ggc cgc cga cag gaa tgg-3') and subcloned into the EcoRI/XmaI site of the mammalian expression vector pIRES2 (Clontech). Overlap extension PCR was performed to construct the WFS1R629W using mutagenic primers 5'-gaa gtc cct gac gtg gag ct-3' and 5'-agc tcc acg tca ggg act tc-3' and flanking primers WFS-f and WFS-r. Bold underlined letters indicate the altered nucleotide. The mutation was verified by direct sequencing of the resulting PCR fragment. The fragment was then cloned into the EcoRI/XmaI site of pIRES2. COS cells (3x104) were seeded in 12-well plates and cultured in DMEM supplemented with 10% fetal calf serum and 2 mM glutamine. COS cells were transiently transfected with the WFS1 or the WFS1R629W constructs using METAFECTANE (Biontex) according to the procedure recommended by the manufacturer. When indicated, cells were grown in the presence of 5 µg/ml tunicamycin. At 24 h post-transfection, cells were used for metabolic labeling experiments or harvested by scraping, pelleted and stored frozen at -80°C until needed to perform the experiments.
Metabolic labeling of transfected COS cells
COS cells (3x104) were seeded in 12-well plates and transfected 1 day prior to the labeling experiment. Transfected COS cells were then washed with 1x PBS, incubated for 30 min in cysteine/methionine- and serum-free medium and labeled with 100 µCi/ml Expre35S35S Protein Labeling Mix (NEN Life Science) for the indicated time periods at 37°C. Cells were then either incubated at 37°C for the indicated time periods in medium containing 10% fetal calf serum (chase) or directly harvested by scraping and lysed in 0.5 ml IP buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol) containing 1% Triton X-100 and protease inhibitors for 30 min at 0°C. When indicated, cells were subjected to crosslinking prior to lysis, as described below. Cell lysates were cleared by centrifugation and supernatants were incubated for 16 h with 25 µl Protein A sepharose beads (Pierce) and 10 µl affinity-purified anti-WoN antibody. Immunoprecipitates were washed twice with IP buffer containing 0.2% Triton X-100, and eluted by 50 µl Laemmli buffer with subsequent SDS–PAGE. In some experiments, immunocomplexes were eluted in 50 µl denaturating buffer (0.5% SDS, 1% ß-mercaptoethanol) and processed for digestion with Endo H (see below). Probes were separated by SDS–PAGE as indicated, transferred to nitrocellulose membrane and exposed to Biomax MR X-ray film (Kodak).
Crosslinking and protein studies
Transfected COS cells were metabolically labeled with 100 µCi/ml Expre35S35S Protein Labeling Mix (NEN Life Science) for 2 h at 37°C and then harvested by scraping in ice-cold 1x PBS. Whole cells were collected by low-speed centrifugation (10 min, 850g) and crosslinked with 1 mM of the membrane-permeant crosslinker MBS (Pierce) in 0.5 ml 1x PBS at room temperature for 30 min. The reaction was stopped by the addition of 100 mM Tris–HCl pH 7.4 and, after incubation for a further 15 min, cells were lysed by the addition of 1% Triton X-100 in the presence of protease inhibitors (complete) (Roche) for 30 min at 0°C. After a clarifying spin, cell lysates were subjected to immunoprecipitation, as described above. Precipitates were separated on precasted 3–8% NuPage gels, transferred to nitrocellulose and analyzed by autoradiography. Crosslinking of non-metabolically labeled cells was performed as described above, except that after crosslinking, cellular proteins were trichlor acetic precipitated, resuspended in 1x Laemmli buffer, and analyzed by SDS–PAGE (3–8% NuPage gels) and immunoblotting.
For deglycosylation experiments, immunoprecipitated protein from pulse-chase experiments or total cell pellets were subjected to digestion with 1 µl Endo H (New England Biolabs) for 1 h at 37°C. For dephosphorylation, lambda protein phosphatase (New England Biolabs) was used at 1 U/ml for 30 min at 30°C. For further detection of the phosphorylation of wolframin, RIN cells (5x106) were lysed in detergent-containing buffer and the cleared cell lysate was chromatographed on a PhosphoProtein Purification column (Qiagen). The flow-through fraction and the elution fraction (containing phosphorylated proteins) were analyzed by western blotting using the anti-WoN antibody.
Gel filtration analysis
RIN cells (1x107) or COS cells (5x104) transfected with human WFS1 cDNA were solubilized with 1% Triton X-100 in 0.5 ml of column buffer (1x PBS, 10% glycerol, 1 mM PMSF) for 30 min at 0°C. After centrifugation at 100 000g (30 min), the clear supernatant was subjected to a Superose 12 gel filtration column (Amersham Biosciences). Fractions (0.5 ml each) were trichlor acetic (TCA) precipitated and analyzed by separation on 10% SDS–PAGE and immunoblotting using the anti-WoN antibody. As calibration standards, apoferritin (440 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa) were used.
Molecular genetic analysis
For mutation analysis of the WFS1 gene, exons 2 to 8 were PCR-amplified on genomic DNA using specific primers as described previously (8). Amplified gene fragments were sequenced directly using the BigDye Terminator Kit on an ABI310 (PE Applied Biosystems). RNA from human fibroblast cells was extracted using peqGOLD RNApure (Peqlab). First-strand cDNA was synthesized from total RNA using oligo (d)T primers and Superscript II (Invitrogen) according to standard procedures. RT–PCR amplification was performed using WFS1-specific primers (5'-gaa gtc cct gac gtg gag ct-3', 5'-tca tgc cgc cga cag gaa tg-3') and GAPDH-specific primers (5'-ttg gta tcg tgg aag gac tca tg-3', 5'-gat gtc atc ata ttt ggc agg ttt-3') as a control.
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ACKNOWLEDGEMENTS |
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We thank Bettina Treske and Eva-Maria Wagner for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (grant number Ho2374/1 to S.H. and Ba1438/4 to M.F.B.).
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +49 8930793134; Fax: +49 893081733; Email: sabine.hofmann{at}lrz.uni-muenchen.de
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