Human Molecular Genetics, 2002, Vol. 11, No. 24 3097-3105
© 2002 Oxford University Press
Intracellular retention of mutant retinoschisin is the pathological mechanism underlying X-linked retinoschisis
1Department of Medical Genetics and Cambridge Institute for Medical Research, University of Cambridge, CB2 2XY, UK and 2Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, CB2 2XY, UK
Received August 14, 2002; Accepted September 20, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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X-linked retinoschisis results in visual loss in early life with splitting within the inner retinal layers. Many missense and protein truncating mutations of the causative gene RS1 (encoding retinoschisin) have been identified but disease severity is not mutation-dependent. Retinoschisin is a soluble secretory protein predicted to have a globular conformation. Missense mutations would be expected to interfere with protein folding leading to an abnormal conformation and intracellular retention and elimination. To test this hypothesis we have expressed seven pathological RS1 mutations (L12H, C59S, G70S, R102W, G109R, R141G and R213W) in COS-7 cells and investigated their intracellular processing and transport. Using immunoblotting and confocal fluorescent immunocytochemistry we show normal secretion of WT RS1, but either reduced (C59S and R141G) or absent (L12H, G70S, R102W, G109R and R213W) secretion of mutant RS1 and intracellular retention. In addition, we show that L12H RS1 is degraded by proteasomes and in vitro transcription/translation revealed the defects in both cleavage of its signal peptide and translocation into the endoplasmic reticulum. Our results indicate the pathological basis of RS1 is intracellular retention of the majority of mutant proteins, which may explain why disease severity is not mutation-specific. Furthermore, we have shown that in vitro expression of RS1 may be a useful functional assay to investigate the pathogenicity of sequence changes within the RS1 gene.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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X-linked recessive juvenile retinoschisis (RS, MIM#312700) is the leading cause of juvenile macular degeneration in males and is characterized by microcystic-like changes of the macular region of the retina and schisis or splitting within the inner retinal layers, leading to visual deterioration (1–4). Peripheral retinal lesions are also present in
50% of cases (3). Most patients present with progressive visual impairment at school age, but a proportion of patients present in infancy with squint, nystagmus and bilateral bullous retinoschisis (5). Complications of disease may occur later in life and include vitreal haemorrhage, choroidal sclerosis, retinal detachment and occasionally retinal atrophy resulting in blindness. Carrier females remain asymptomatic and have no clinical features of the condition (2,4). The disease is variable even within families and severity is thus not determined solely by the specific mutation (6). The gene causing X-linked juvenile retinoschisis, RS1, maps to Xp22 and was identified by positional cloning (7). Numerous disease-causing mutations have now been recognized (8) (www.dmd.nl/rs/rs1.html). The majority of these are missense mutations, although nonsense mutations, deletions, insertions and splice site mutations have all been found. RS1 encodes the protein retinoschisin, a 224 amino acid protein which contains a signal sequence with a signal peptidase cleavage site and otherwise consists almost entirely of a discoidin domain (7). Discoidin domains are present in a family of extracellular or transmembrane proteins implicated in cell adhesion or cell–cell interactions (9,10). Retinoschisin is secreted from photoreceptors and bipolar cells and appears to form an oligomeric complex which may be disulphide bonded (11,12). Mutations cluster within the discoidin domain suggesting that it is essential for the normal function of retinoschisin (8).
Recently a knockout mouse RS model has been generated by disrupting the mouse RS1 gene Rs1h by introducing a lacZ reporter gene in frame into exon 3 (13). The Rs1h mouse has a retinal phenotype which closely resembles the human disease and shows a generalized disruption of retinal cell layer architecture affecting the inner retinal layers in particular. These observations support a role for retinoschisin in the maintenance of retinal cell architecture through mediating interactions between cell and extracellular matrix or between different cells.
Retinoschisin contains 10 cysteine residues, is secreted and the discoidin domain is predicted to have a globular conformation (11). Missense mutations would be expected to interfere with protein folding in the endoplasmic reticulum (ER) leading to an abnormal conformation. Such misfolded proteins are rapidly eliminated from cells by intracellular protein degradation which can result in disease phenotypes (14). Cystic fibrosis, for example, is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator) and mutant misfolded CFTR is not transported to the cell surface, but is degraded by the ubiquitin–proteasome pathway (15,16). Intracellular degradation and lack of secretion of mutant retinoschisin could explain the lack of correlation between mutation type and disease severity, as different mutations would result in the same outcome. We set out to test this hypothesis by expressing a series of known pathological RS1 mutations in COS-7 cells and investigating their intracellular processing. Here we demonstrate complete or partial intracellular retention of most mutant retinoschisin forms. We found that only one retained mutant form of retinoschisin was unstable and degraded by proteasomes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RS1 mutations reduce retinoschisin secretion
To investigate the expression of wild-type (WT) and mutant retinoschisin (RS1) we generated expression constructs in pcDNA3.1 vectors for WT RS1 and seven different RS1 mutations, L12H, C59S, G70S, R102W, G109R, R141G and R213W, using site directed mutagenesis. The presence of each mutation was confirmed by DNA sequencing. These mutations represent the whole range of missense mutations identified in RS1: within the signal peptide (L12H); occurring N-terminal of the discoidin domain (C59S); and common disease-associated mutations spread throughout the discoidin domain (G70S, R102W, G109R, R141G and R213W). Mutations known to be pathogenic were chosen to investigate the fate of mutant protein known to cause the X-linked RS. The pcDNA3.1 constructs were used to transfect COS-7 cells and protein expression was induced. Non-transfected COS-7 cells do not express retinoschisin (Fig. 1A). In the transfected cells, WT retinoschisin was found in whole cell lysates and in the medium from the cell culture (Fig. 1B), confirming that retinoschisin is secreted by these cells as it is in the retina and in Weri-Rb1 cells (11). The secretion of mutant retinoschisin was either reduced (C59S and R141G) or absent (L12H, G70S, R102W, G109R and R213W; Fig. 1B).
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Intracellular location of mutant retinoschisin
We therefore examined subcellular localization of RS1 WT and mutant proteins by cell fractionation. This revealed WT and mutant RS1 mainly in the membrane fraction, which includes the ER and Golgi complex (Fig. 2). A minor fraction of WT and mutant protein was detected in the cytosol, which may be a result of retrograde translocation from the lumen of the ER due to overexpression of transfected protein or may be due to disruption of secretory transport vesicles or ER during the homogenizing process. The amount of protein detected for L12H appeared less than for all other mutant forms of RS1 and this was consistent when the experiment was repeated three times. In addition the band for L12H showed reduced mobility on SDS gels compared with WT and the other mutants (Fig. 2). The expression of the L12H mutant appeared to compromise the viability of the cells as there was more cell death of the transfected L12H cells than of those from WT and the other mutants.
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We further investigated the localization of retinoschisin by confocal fluorescent microscopy using the RS1 antibody and antibodies against an ER resident protein, BiP, or Golgi resident protein, GM130. The staining pattern for WT retinoschisin was reticular (Fig. 3A, top) and WT RS1 colocalized with BiP in the ER consistent with retinoschisin localization within the ER. In addition, WT RS1 colocalized with the Golgi protein GM130, suggesting that WT retinoschisin was present throughout the secretory pathway (Fig. 3B, top). For those mutants which were secreted (C59S, Fig. 3 and R141G, not shown), the fluorescence pattern was similar to WT. The non-secreted mutants G70S, R102W, G109R and R213W showed colocalization with BiP in the ER (Fig. 3A for R102W, others not shown) and also some colocalization with GM130 (Fig. 3B for R102W, others not shown), indicating that some mutant protein was reaching the Golgi complex. L12H RS1, however, did colocalize partially with BiP (Fig. 3A), but showed no colocalization with GM130 (Fig. 3B), suggesting that this form of RS1 is fully retained at the ER and not transported to the Golgi complex, but may undergo reverse translocation into the cytosol for degradation.
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L12H prevents cleavage of the signal peptide and entry into the ER
The nature of the L12H mutation, which lies within the retinoschisin signal peptide and the size of the L12H band on SDS gels suggested that the mutation prevented cleavage of the signal peptide. We investigated this using in vitro transcription/translation (Fig. 4A) of WT retinoschisin and all seven mutants. When microsomal membranes were added to the reaction signal cleavage was observed for all RS1 proteins (WT, C59S, G70S, R102W, G109R, R141G and R213W) except L12H RS1 (Fig. 4B).
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We used a protease protection assay to investigate whether L12H retinoschisin is translocated into the ER lumen (Fig. 4C). The addition of Proteinase K to the in vitro transcription/translation reactions containing membranes led to digestion of the precursor WT RS1 with intact signal peptide, but the signal peptide cleaved WT RS1 was protected within the microsomes. The in vitro translated L12H RS1 was fully digested by the proteinase K, even in the presence of microsomes, indicating that most of this protein is not fully translocated into the ER. Thus in addition to preventing signal peptide cleavage, this signal peptide mutation interferes with entry into the ER.
L12H RS1 is degraded by ubiquitin–proteasomal pathway
Given the low amounts of L12H RS1 intracellularly, we suspected that this mutation led to L12H RS1 degradation. Cytosolic proteins are degraded by proteasomes and misfolded secretory proteins retained within the ER are often transported back to the cytosol for proteasome degradation. We incubated transfected cells with the proteasome inhibitor lactacystin (Fig. 5A and B). In cells transfected with L12H RS1 the addition of lactacystin resulted in markedly increased levels of retinoschisin over time (over 4-fold increase by 6 h, Fig. 5B), indicating that in the absence of lactacystin this mutant protein is degraded by proteasomes. We did not detect significant changes in the levels of WT and other mutant retinoschisins in the presence of lactacystin, even for those mutations causing complete intracellular retention of retinoschisin (Fig. 5A and B).
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Misfolded proteins that escape the quality control machinery in the ER can be directed in the Golgi complex to lysosomes for degradation. Since we observed some transport to the Golgi complex of most mutant RS1 forms we considered the lysosomal pathway as an alternative route of intracellular protein degradation of these mutant proteins (17). We investigated whether any retinoschisin forms were subject to lysosomal degradation by incubating transfected cells with the lysosomal protease inhibitor leupeptin. We found no change in the levels of protein for cells transfected with WT RS1 or any of the mutant forms of RS1 (data not shown), leading us to conclude that lysosomes do not play a major role in the degradation of mutant RS1 and, with the exception of L12H RS1, intracellularly retained mutant RS1 forms are stable.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the gene RS1 lead to the disease X-linked juvenile retinoschisis (7,8). RS1 encodes retinoschisin, a secretory retinal protein which contains a discoidin domain and is predicted to have a highly folded globular conformation (7,11,12). Patients with mutations in RS1 have very variable phenotypes and disease severity is not mutation-dependent (3,6). We postulated that mutant RS1 proteins would be retained intracellularly rather than secreted, which could explain why the phenotypes are not dependent on the position or type of mutation. To test this hypothesis we expressed seven different disease-causing missense RS1 mutations in COS-7 cells: RS1 gene with a mutation occurring within the signal peptide; a mutation upstream of the discoidin domain; and five mutations within the discoidin domain of the protein. Each of these mutations was found originally in patients with RS and, apart from R141G, had occurred in more than one family (8). R141G was identified in a UK family and has since not been seen in other RS families, but other missense mutations affecting the same codon have been identified (8). The missense mutations in the discoidin domain occurred within conserved residues (8). Each of these mutations could thus be predicted to lead to large conformational changes in the protein. Our results indicated that six of these seven mutations led to intracellular retention of the mutant protein. The only mutation which had little effect on protein secretion was C59S, a missense mutation in exon 3 of the gene just 5' of the discoidin domain. The results indicate that X-linked retinoschisis is primarily caused by intracellular protein retention and our experiments suggest that in vitro expression of RS1 may be a useful functional assay to investigate the pathogenicity of sequence changes within the RS1 gene.
Proteins with abnormal conformations are often retained intracellularly and degraded. Most intracellular protein degradation (up to 80–90%) occurs via the ubiquitin–proteasome system [for review see (17)] with the remaining 10–20% using the lysososmal degradation pathway. Proteins which are retained in the ER, such as mutant CFTR (15,16) and the mutant Z form of 
1-antitrypsin (18,19), are transported from the ER to the cytosol and degraded by proteasomes. Our results suggest that mutant L12H retinoschisin is degraded by proteasomes. The use of lactacystin, which inactivates proteasomes, led to an accumulation of this mutant form of retinoschisin, indicating that proteasomes are responsible for its degradation. However, we saw no accumulation of any other mutant form of RS1 in the presence of lactacystin. An alternative explanation would be degradation of these mutants via the lysosomal pathway as for gap junction proteins (20). Our results indicate this is unlikely as the treatment of transfected cells with leupeptin (a lysosomal protease inhibitor) did not lead to an increase in mutant RS1 within those cells. The proteasomes and lysosomes do not play a major role in the degradation of these mutant forms of RS1 and it seems most likely that these are retained intracellularly and cycle between ER and Golgi like mutant forms of the gap junction protein connexin 32 (20).
The L12H mutation was predicted to interfere with the function of the signal peptide and we found that it affected both signal peptide cleavage and entry into the ER. We did detect co-localization of this mutant with an ER resident protein, indicating association of L12H RS1 with the ER. The viability of the cells expressing this mutant form of RS1 was compromised in contrast to those expressing either WT or the other mutant forms of RS1. The L12H RS1 signal peptide may gain entry into the protein conducting channel in the ER membrane (21), but failure to cleave the signal sequence may abort subsequent translocation of the L12H RS1. The mutant protein may, to some extent, block the channel, interfere with the translocation of other secretory proteins and thus affect the health of the cells.
A recent report describes the histological phenotype of an enucleated eye from a patient with X-linked retinoschisis caused by the RS1 mutation R102W (22). Immunostaining with an antibody against RS1 revealed negative staining in the atrophic central retina and reduced staining in the peripheral retina. The authors suggest this can be explained by a dysfunctional protein with a reduced half-life and defective cellular adhesive function. The data we present here, which include results from this mutation, indicate an alternative explanation: the reduced staining is due to a lack of secretion of the mutant retinoschisin hence the RS1 antibody is detecting intracellular retinoschisin only. Thus there is no signal in the atrophic central retina and a reduced signal in the well-preserved peripheral retina.
One mutant form of retinoschisin, C59S, was secreted from the cells. C59S occurs just prior to the start of the discoidin domain within retinoschisin. The conformational change, if any, in this mutant must be minimal as the protein is successfully secreted from the cell and may suggest that this cysteine is not paired in a disulphide bond. This is therefore the only mutant we identified in which the secreted protein may by dysfunctional and it will be useful for future functional studies of retinoschisin binding to its receptor, once its binding partners have been identified. Unfortunately, we have no clinical details about patients with this mutation and to our knowledge there are no reports in the literature. We would speculate that the phenotype may be milder than with other RS1 mutations but this will of course depend on the resulting conformation and the subsequent interactions with the retinoschisin receptor(s).
It is interesting to consider our results with a recent report describing work investigating collagen binding epitopes within discoidin domain receptor 1 (DDR1) (23). DDR1 with a similar protein DDR2 form a transmembrane tyrosine receptor subfamily and contain a discoidin domain within their extracellular regions. Activation of the DDR1 intracellular tyrosine kinase domain is achieved by collagens. The results described by Curat et al. (23) indicate that the discoidin domain of DDR1 is essential for collagen binding and subsequent tyrosine phosphorylation. To investigate this further the authors compared the sequence of the DDR1 discoidin domain with that of retinoschisin and used site-directed mutagenesis to insert nine disease-causing RS1 mutations into the respective positions in DDR1. The interactions of these mutant DDR1 proteins with collagen were studied. Some DDR1 mutants had reduced collagen binding and some showed reduced tyrosine kinase activation when stimulated. One of these had normal binding to collagen, suggesting that some residues are essential for both collagen binding and transmembrane signal transduction whereas others are necessary for only collagen binding or signal transduction. We have expressed and investigated three of the mutations in the report from Curat et al.: G36S (described here as G70S); R36W (described here as R102W); and R179W (described here as R213W). In each case the mutant retinoschisin was retained within the cell. A degree of collagen activation was seen with the DDR1 mutants studied by Curat et al. (23), implying that at least some protein was targetted correctly to the cell membrane, but the reduction in collagen binding or activation of these DDR1 mutants could be explained by a reduction in the proportion of mutant DDR1 being inserted appropriately into the cell membrane. It would be interesting to investigate this further. Retinoschisin and DDR1 are very different proteins, although they do both contain a discoidin domain. These domains are not identical but share similarity and conserved residues. It may be that the RS1 mutations affect the folding of retinoschisin to a much greater degree than the same mutations expressed in the different discoidin domain of DDR1. Expressing RS1 mutations within the corresponding residues of DDR1 may be of great value once the binding partner(s) of retinoschisin have been identified. As most of the mutant forms of RS1 are retained, this approach may help define the crucial residues for interactions between these proteins.
The results of this study indicate that X-linked retinoschisis is primarily caused by intracellular protein retention. One striking feature of this disease is the variable phenotype and the lack of correlation between mutation type and position within the gene and disease severity. Retinoschisin mutations resulting in intracellular retention of mutant protein will cause affected males to have little or no detectable retinoschisin within their retinae. Their phenotype may depend on the secretory capacity of their cells and the degree to which they may induce the unfolded protein response. This mechanism has been shown to influence disease severity in 1-antitrypsin deficiency (24). This disease is associated with retention in the ER of the mutant form (Z) of a secretory protein (1-antitrypsin). These patients develop early-onset emphysema and a small proportion (10–15%) also have liver disease. The patients with liver disease have been shown to have slower degradation of mutant 1-antitrypsin from the ER and hence an increase in intracellular accumulation of the mutant protein (24). It would be interesting to investigate this as a mechanism in RS patients with severe and mild disease from the same family and thus with the same RS1 mutation. An alternative mechanism, which might explain the disease variation within a family, would be the presence of polymorphisms or variations within other extracellular matrix molecules within the retina. Manipulating these mechanisms might lead to a future therapy for these patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of mutant RS1 by site-directed mutagenesis
Constructs of RS1 mutations were generated by site-directed mutagenesis using primers containing the desired mutations (Table 1), with full-length WT RS1 cDNA in pBluescript (a gift from Dr BHF Weber, Würzberg, Germany) as a template. For mutations of C59S, G70S, R102W, R141G, R213W, primary PCR was performed using the mutant primer and T7 or T3 primer, depending on the site of the mutations. These PCR products were used as a mega-primer in secondary PCR reactions with the T3 or T7 primer to produce full-length mutant RS1 cDNA, which were cloned into pGEM-T Easy vector (Promega) and the mutations confirmed by DNA sequencing. Expression vectors encoding WT and mutant RS1 were made by sub-cloning into pcDNA 3.1 at the EcoR1 site. For mutations L12H and G109R, mutations were generated using QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene) with WT RS1 in pcDNA 3.1 as template. All the cDNA sequences in expression vectors were confirmed by DNA sequencing.
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Cell culture and transfection
COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin/streptomycin, 2 mML-glutamine at 37°C with 5% CO2. For transfection,
1x106 cells/well were seeded in a six-well plate the day before transfection. Transfection was performed using LipofectAMINE (Invitrogen) according to manufacture's instructions. Briefly, a mixture of 2 µg of Endo-free plasmid DNA and 10 µl of LipofectAMINE in 1 ml of OPTI-MEN I medium (Invitrogen) was applied to COS-7 cells at about 80% confluence. After 5 h incubation at 37°C the transfection mixture was replaced by complete culture medium and incubated for 24–72 h after transfection. For lactacystin and leupeptin treatment, two sets of COS-7 cells were transfected in 24-well plates with 0.5 µg of WT and mutant RS1 in pcDNA 3.1. Twenty hours after transfection, lactacystin (Calbiochem) or leupeptin (Calbiochem) was added to one set of the cells to a final concentration of 10 or 20 µM, respectively. Lactacystin was refreshed after 24 h. Whole cell lysates were prepared at 6, 12, 24 and 30 h time points for lactacystin and 6 and 24 h for leupeptin. Equal amounts of cell lysates were used for western blot analysis. Empty EGFP-N1 vectors (Clontech; 0.2 µg/well) were co-transfected with RS1 to serve as transfection/loading control.
Western blot analysis
For western blot analysis, whole cell lysates were prepared by lysing the transfected cells in Leammli buffer (62.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 5% ß-mercaptoethanol and bromophenol blue). Membrane and cytosol fractions were prepared by lysing the transfected cells in Tris-sucrose buffer (10 mM Tris-base, pH 7.4, 1 mM EDTA, 0.1 mM sucrose, protease inhibitors). The cells were homogenized on ice using Pestles in 1.5 ml tubes then centrifuged at 1500 g, 4°C for 10 min to get rid of nuclei and un-broken cells. The supernatant was centrifuged at 100 000 g, 4°C for 1 h, resulting in the cytosolic fraction (supernatant) and membrane fraction (pellet resuspended in Tris–sucrose buffer). Protein concentration was quantified by Bio-Rad Protein Assay reagent (Bio-Rad) and 5 µg protein subjected to 10% SDS–polyacrylamide gel (SDS–PAGE) electrophoresis and transferred onto nitrocellulose membrane (Amersham Biosciences Inc.). The membrane was incubated with primary antibodies [1:\500 dilution for RS (11) and 1:2000 dilution for GFP, Affinity purified rabbit polyclonal, Abcam]. Blots were then probed with peroxidase-labelled anti-rabbit (DACO) secondary antibodies at 1:2000 dilutions. Finally the signals were visualized using enhanced chemiluminescence reagent (ECL, Amersham Biosciences Inc.) and exposed onto ECL Hyperfilm (Amersham Biosciences Inc.).
Immunocytochemistry
COS-7 cell were grown on cover slips in six-well plates and transfected as described above. Twenty-four hours after transfection, cells were washed with 1xPBS, fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, quenched with 0.25% NH4Cl for 5 minx4, and permeabilized with 0.1% Triton-X for 10 min. After PBS washing, non-specific antibody binding was blocked with blocking buffer (PBS containing 5% fetal calf serum). Cells were then incubated with primary antibodies [1 : 500 dilution for RS1 (11); 1 : 100 dilution for both ER marker BiP (C-20), Santa Cruz Biotechnology Inc., and golgi marker GM130, BD Biosciences] in blocking buffer for 1 h at room temperature, washed with PBS, and then incubated with Alexa Fluor 488-labelled anti-rabbit immunoglobulin secondary antibody (Molecular Probe, for RS1), Alexa Fluor 594-labelled anti-goat immunoglobulin secondary antibody (for ER) and Texas red-labelled anti-mouse immunoglobulin secondary antibody (for Golgi) for 1 h. After an additional wash with PBS, the cover glasses were mounted in Cityfluor (Citifluor Ltd, London) containing 3 µg/ml DAPI for nucleus staining. Immunostaining was visualized under a confocal microscope (ZEISS 510).
In vitro transcription/translation
One microgram of plasmid DNA of RS1 WT and mutant DNA constructs in pcDNA 3.1 were used for in vitro transcription/translation using TNT Quick Coupled Transcription/Translation System (Promega). L-[35S] methionine was used as the labelled precursor. The in vitro synthesised proteins were analysed by 14% SDS–PAGE and autoradiography. To study signal peptide cleavage, 1.5 µl of canine pancreatic microsomal membranes (Promega) were included in the in vitro transcription/translation reaction.
For protease protection of proteins translated in the presence of membranes, aliquots from the in vitro translation with microsomes were digested with 0.1 mg/ml of proteinase K (Roche) on ice for 15 min. The reaction was stopped by adding trichloracetic acid to a final concentration of 10% and incubated further 30 min on ice. The precipitated proteins were pelleted by centrifugation (13 000 g, 5 min, 4°C) and the pellets washed by cold acetone and resuspended in Leammli buffer. Proteins were then separated by 14% SDS–PAGE followed by autoradiography.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Gudrun Ihrke (Department of Clinical Biochemistry, University of Cambridge, UK) for helpful experimental advice and to Dr Bernard H.F. Weber (Universtät Wurzburg, Germany) for providing the RS1 cDNA. This study was funded by the Medical Research Council, UK.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Hills Rd, Cambridge, CB2 2XY, UK. Tel: +44 1223331139; Fax: +44 1223331206; Email: dt207{at}cam.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Kellner, U., Brummer, S., Foerster, M.H. and Wessing, A. (1990) X-linked congenital retinoschisis. Graefes Arch. Ophthal., 228, 432–437.
2
George, N.D., Yates, J.R. and Moore, A.T. (1995) X linked retinoschisis. Br. J. Ophthal., 79, 697–702.
3
George, N.D., Yates, J.R. and Moore, A.T. (1996) Clinical features in affected males with X-linked retinoschisis. Arch. Ophthal., 114, 274–280.
4 Sieving, P. (1998) Juvenile Retinoschisis. In Traboulsi, E. (ed.), Genetic Diseases of the Eye. Oxford University Press, New York, pp. 347–355.
5
George, N.D., Yates, J.R., Bradshaw, K. and Moore, A.T. (1995) Infantile presentation of X linked retinoschisis. Br. J. Ophthal., 79, 653–657.
6
Eksandh, L.C., Ponjavic, V., Ayyagari, R., Bingham, E.L., Hiriyanna, K.T., Andreasson, S., Ehinger, B. and Sieving, P.A. (2000) Phenotypic expression of juvenile X-linked retinoschisis in Swedish families with different mutations in the XLRS1 gene. Arch. Ophthal., 118, 1098–1104.
7 Sauer, C.G., Gehrig, A., Warneke-Wittstock, R., Marquardt, A., Ewing, C.C., Gibson, A., Lorenz, B., Jurklies, B. and Weber, B.H. (1997) Positional cloning of the gene associated with X-linked juvenile retinoschisis. Nat. Genet., 17, 164–170.[Web of Science][Medline]
8
The Retinoschisis Consortium (1998) Functional Implications of the spectrum of mutations found in 234 cases with X-linked juvenile retinoschisis (XLRS). Hum. Mol. Genet., 7, 1185–1192.
9 Baumgartner, S., Hofmann, K., Chiquet-Ehrismann, R. and Bucher, P. (1998) The discoidin family revisited: New members from prokaryotes and a homology based fold prediction. Prot. Sci., 7, 1626–1631.[Web of Science][Medline]
10
Vogel, W. (1999) Discoidin domain receptors: structural relations and functional implications. FASEB J., 13, S77–S82.
11
Grayson, C., Reid, S.N., Ellis, J.A., Rutherford, A., Sowden, J.C., Yates, J.R., Farber, D.B. and Trump, D. (2000) Retinoschisin, the X-linked retinoschisis protein, is a secreted photoreceptor protein, and is expressed and released by Weri-Rb1 cells. Hum. Mol. Genet., 9, 1873–1879.
12
Molday, L.L., Hicks, D., Sauer, C.G., Weber, B.H. and Molday, R.S. (2001) Expression of X-linked retinoschisis protein RS1 in photoreceptor and bipolar cells. Invest. Ophthal. Vis. Sci., 42, 816–825.
13
Weber, B.H., Schrewe, H., Molday, L.L., Gehrig, A., White, K.L., Seeliger, M.W., Jaissle, G.B., Friedburg, C., Tamm, E. and Molday, R.S. (2002) Inactivation of the murine X-linked juvenile retinoschisis gene, Rs1h, suggests a role of retinoschisin in retinal cell layer organization and synaptic structure. Proc. Natl Acad. Sci. USA, 99, 6222–6227.
14 Aridor, M. and Balch, W.E. (1999) Integration of endoplasmic reticulum signaling in health and disease. Nat. Med., 5, 745–751.[Web of Science][Medline]
15 Ward, C.L., Omura, S. and Kopito, R.R. (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell, 83, 121–127.[Web of Science][Medline]
16 Jensen, T.J., Loo, M.A., Pind, S., Williams, D.B., Goldberg, A.L. and Riordan, J.R. (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell, 83, 129–135.[Web of Science][Medline]
17 Lee, D.H. and Goldberg, A.L. (1998) Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol., 8, 397–403.[Web of Science][Medline]
18
Teckman, J.H., Burrows, J., Hidvegi, T., Schmidt, B., Hale, P.D. and Perlmutter, D.H. (2001) The proteasome participates in degradation of mutant alpha 1-antitrypsin Z in the endoplasmic reticulum of hepatoma-derived hepatocytes. J. Biol. Chem., 276, 44865–44872.
19
Qu, D., Teckman, J.H., Omura, S. and Perlmutter, D.H. (1996) Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. J. Biol. Chem., 271, 22791–22795.
20
VanSlyke, J.K., Deschenes, S.M. and Musil, L.S. (2000) Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins. Mol. Biol. Cell., 11, 1933–1946.
21 Matlack, K.E., Mothes, W. and Rapoport, T.A. (1998) Protein translocation: tunnel vision. Cell, 92, 381–390.[Web of Science][Medline]
22
Mooy, C.M., Van Den Born, L.I., Baarsma, S., Paridaens, D.A., Kraaijenbrink, T., Bergen, A. and Weber, B.H. (2002) Hereditary X-linked juvenile retinoschisis: a review of the role of Muller cells. Arch. Ophthal., 120, 979–984.
23
Curat, C.A., Eck, M., Dervillez, X. and Vogel, W.F. (2001) Mapping of epitopes in discoidin domain receptor 1 critical for collagen binding. J. Biol. Chem., 276, 45952–45958.
24
Wu, Y., Whitman, I., Molmenti, E., Moore, K., Hippenmeye, P. and Perlmutter, D.H. (1994) A lag in intracellular degradation of mutant 1-antitrypsin correlates with the liver disease phenotype in homozygous PiZZ 1-antitrypsin deficiency. Proc. Natl Acad. Sci. USA, 91, 9014–9018.
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