Human Molecular Genetics, 2001, Vol. 10, No. 3 291-299
© 2001 Oxford University Press
Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of 
-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation
Departments of 1Human Genetics and 2Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA and 3Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia, Australia
Received 30 November 2000; Revised and Accepted 5 December 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hurler syndrome is the most severe form of a lysosomal storage disease caused by loss of the enzyme
-L-iduronidase (encoded by the IDUA gene), which participates in the degradation of glycosaminoglycans (GAGs) within the lysosome. In some populations, premature stop mutations represent roughly two-thirds of the mutations that cause Hurler syndrome. In this study we investigated whether the aminoglycoside gentamicin can suppress stop mutations within the IDUA gene. We found that a Hurler syndrome fibroblast cell line heterozygous for the IDUA stop mutations Q70X and W402X showed a significant increase in -L-iduronidase activity when cultured in the presence of gentamicin, resulting in the restoration of 2.8% of normal -L-iduronidase activity. Determination of -L-iduronidase protein levels by an immunoquantification assay indicated that gentamicin treatment produced a similar increase in -L-iduronidase protein in Hurler cells. Both the -L-iduronidase activity and protein level resulting from this treatment have previously been correlated with mild Hurler phenotypes. Although Hurler fibroblasts contain a much higher level of GAGs than normal, we found that gentamicin treatment reduced GAG accumulation in Hurler cells to a normal level. We also found that a reduced GAG level could be sustained for at least 2 days after gentamicin treatment was discontinued. The reduction in the GAG level was also reflected in a marked reduction in lysosomal vacuolation. Taken together, these results suggest that the suppression of premature stop mutations may provide an effective treatment for Hurler syndrome patients with premature stop mutations in the IDUA gene. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recent studies have shown that aminoglycosides can suppress premature stop mutations in mammalian transcripts both in vitro and in vivo at levels that restore physiologically relevant amounts of functional protein. The utility of this approach was previously demonstrated with the autosomal recessive disease cystic fibrosis (CF), where the aminoglycosides gentamicin and G418 were shown to suppress nonsense mutations in the CF transmembrane conductance regulator (CFTR) gene (1,2). These compounds were shown to suppress a genomic CF nonsense mutation in a human bronchial epithelial cell line, restoring both CFTR protein localized to the apical plasma membrane and its cAMP-activated chloride channel activity. The specificity of aminoglycoside action for nonsense suppression was shown in these studies since cells homozygous for the
F508 CFTR allele did not respond to aminoglycoside treatment. More recently, clinical data obtained in pilot studies with CF patients carrying nonsense mutations indicated that topical or intravenous gentamicin can partially restore CFTR activity in vivo (3,4). Another study found that gentamicin can also suppress a nonsense mutation in the dystrophin (Dmd) gene of the mdx mouse, which represents an animal model for Duchenne muscular dystrophy (5). The partial restoration of dystrophin expression was accompanied by a significant decrease in muscular deterioration in treated animals.
The term mucopolysaccharidoses describes a broad class of genetic disorders that are characterized by the excessive accumulation of glycosaminoglycans (GAGs) within the lysosomes of various tissues. Among these disorders, mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease caused by a loss of the enzyme -L-iduronidase, which participates in the degradation of GAGs within the lysosome. MPS I can be further subdivided into three categories: Hurler (MPS I-H), the most severe form; Scheie (MPS I-S) a mild form; and Hurler/Scheie (MPS I-H/S), an intermediate form. Hurler syndrome is characterized by a near total absence of -L-iduronidase activity, leading to the accumulation of both dermatan and heparan sulfate within the lysosomes (6). Physical symptoms of the disease include stiffness in joints, skeletal abnormalities and corneal clouding. Progression of Hurler syndrome results in heart and liver disease as well as mental deterioration, with death usually occurring in childhood (7). The two most frequent mutations found in MPS I patients with Hurler syndrome, the Q70X and W402X nonsense mutations, are present in 
70% of patients of European descent (8). Significantly, the other forms of MPS I present clinically with milder symptoms, suggesting that much of the disease phenotype can be alleviated by as little as 1% of normal -L-iduronidase activity (9–11). This well-defined correlation between enzymatic activity and disease severity makes Hurler syndrome a good candidate disease to examine whether the level of protein expression restored by aminoglycoside suppression of stop mutations can reverse the biochemical defects associated with a human genetic disease. Our results indicate that the suppression of nonsense mutations by gentamicin can reverse these biochemical defects, suggesting that aminoglycoside therapy may provide an effective treatment for many patients with Hurler syndrome.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Hurler Q70X and W402X premature stop mutations are susceptible to gentamicin-mediated suppression
The suppression of stop mutations in mammalian cells is dependent on the context surrounding the stop codon (12,13). To determine whether aminoglycosides can suppress naturally occurring premature stop mutations that cause Hurler syndrome, IDUA cDNA templates containing the Q70X or W402X mutations were expressed in a rabbit reticulocyte lysate-coupled transcription/translation system in the presence of increasing concentrations of gentamicin. We were able to successfully visualize the IDUA W402X truncated peptide in the mammalian translation system expressed from the human IDUA cDNA construct. A dose-dependent increase in the amount of full-length
-L-iduronidase protein produced from the IDUA W402X cDNA was observed on addition of gentamicin to the translation mixture, with suppression of the premature stop codon occurring at a frequency of 4.6% in the presence of 10 µg/ml gentamicin (Fig. 1A). However, we were unable to visualize the truncated Q70X product due to its small size and lack of methionine codons. To determine whether gentamicin can suppress the Q70X mutation, we introduced the Q70X mutation and the six upstream and downstream codons into a construct previously developed to quantitate aminoglycoside-mediated suppression of stop mutations (Fig. 1B) (12). In this reporter system, efficient translation termination at the Q70X stop codon resulted in the production of a 26 kDa polypeptide and suppression of the Q70X mutation allowed the synthesis of a 37 kDa protein. We found that gentamicin could suppress the Q70X mutation to a level as high as 10.9%. These results demonstrate that both the IDUA Q70X and W402X premature stop mutations are susceptible to suppression by gentamicin.
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-L-iduronidase activity and protein are partially restored in Hurler fibroblasts following gentamicin treatmentTo determine whether the Q70X/W402X premature stop mutations in the IDUA gene can be suppressed in intact cells, Hurler fibroblasts (P4) were cultured in the presence of gentamicin for 24 h. Cellular extracts were then prepared and an
-L-iduronidase activity assay was performed. We found that gentamicin treatment increased the -L-iduronidase-specific activity to 0.89 nmol/h/mg protein (Fig. 2A). This resulted in 2.8% of the -L-iduronidase specific activity that was measured in normal fibroblasts, a level previously reported to be sufficient to reduce or prevent the Hurler phenotype (10,11).
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During the initial stages of this study we made the unexpected observation that the gentamicin-mediated increase in
-L-iduronidase activity was reduced when Hurler fibroblasts had undergone higher cell passages (Fig. 2B). For example, cells in passage 4 (P4) routinely exhibited 2.5–3% of wild-type -L-iduronidase activity after incubation for 1 day in the presence of 200 µg/ml gentamicin. By P6, an average of only 0.6% of normal -L-iduronidase activity was observed in cells cultured for 1 day with 600 µg/ml gentamicin. Only 0.1% of normal -L-iduronidase activity was observed by P8, even when the cells were cultured for 1 day in the presence of 1000 µg/ml gentamicin. None of these cells had previously been exposed to aminoglycosides, so an adaptation to gentamicin could not have occurred. This gradual reduction in susceptibility to gentamicin-induced suppression of stop mutations could conceivably be related to the absence of -L-iduronidase activity in these cells. However, it is more likely that this trend is caused by changes in gentamicin permeability (or efflux) that may occur as primary fibroblast cell lines age (14). If so, this would not reflect an adaptation that would occur during in vivo gentamicin administration. Because of this observation, all subsequent experiments in this study were carried out using fibroblasts cultured at P6 or earlier.
We next asked whether the increase in -L-iduronidase activity observed after gentamicin treatment could also be correlated with an increase in the amount of -L-iduronidase protein. Hurler fibroblasts (P6) were cultured in the presence or absence of gentamicin and the amount of -L-iduronidase protein present in cell extracts was immunoquantified using a polyclonal–monoclonal sandwich immunoassay (10,15). We found that the amount of -L-iduronidase protein also increased significantly in gentamicin-treated Hurler fibroblasts (Fig. 3). The level of -L-iduronidase protein observed in cells cultured in the absence of gentamicin (0.039 ng/mg total protein) is generally associated with a severe Hurler phenotype. In contrast, the amount of -L-iduronidase in the gentamicin-treated Hurler cells was 0.41 ng/mg total protein, a level previously associated with a mild Hurler phenotype (10,15). Taken together, the results of both enzymatic and protein quantification assays indicate that gentamicin can suppress premature stop mutations in the IDUA gene and restore a functionally significant level of -L-iduronidase activity and protein in cultured Hurler fibroblasts.
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Gentamicin-treated Hurler fibroblasts have decreased GAG retention
Previous studies have shown that Hurler fibroblasts accumulate a much higher level of GAGs than normal fibroblasts. This occurs because
-L-iduronidase activity is essential for lysosomal GAG degradation (6,16). To determine whether aminoglycoside-mediated suppression of stop mutations can restore a level of -L-iduronidase activity that is sufficient to reduce GAG accumulation, normal and Hurler Q70X/W402X fibroblasts (P6) were cultured with 35SO4 for 3 days to label the sulfated GAGs synthesized during this period. The cells were then cultured in medium lacking 35SO4 for 2 days in the presence or absence of gentamicin. After this chase period, the GAGs were precipitated and the total 35S incorporated into precipitable counts was quantitated. This value was then expressed relative to the total protein recovered in the precipitant. Under these conditions, untreated Hurler fibroblasts accumulated almost 4-fold more 35S-labeled GAGs than normal fibroblasts (Fig. 4A). However, the amount of 35S-precipitable counts in the gentamicin-treated Hurler fibroblasts was reduced to a level similar to that observed in normal fibroblasts. These results indicate that the low level of -L-iduronidase activity restored by gentamicin treatment can dramatically decrease the steady-state GAG level in Hurler fibroblasts.
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Gentamicin mediates the suppression of stop mutations by binding to the decoding site of the small subunit rRNA (17). Based on this mechanism of action, the synthesis of full-length
-L-iduronidase should be maintained in Hurler fibroblasts only as long as gentamicin is available to facilitate the suppression of stop mutations in the IDUA gene. However, -L-iduronidase activity should persist until the protein synthesized during gentamicin treatment is degraded. To determine whether a reduced GAG level can be sustained following the cessation of gentamicin treatment, we labeled normal and Hurler fibroblasts for 72 h with 35SO4 (days 1–3) followed by a 48 h non-labeling (chase) period in the presence or absence of gentamicin (days 4–5). This was then followed by incubation for an additional 48 h (days 6–7) with 35SO4 to label newly synthesized GAGs (Fig. 4B). Following this procedure, the level of GAGs was determined. Using this protocol, we found that the level of GAGs in Hurler fibroblasts was 1.6-fold higher than normal when cells were cultured in the absence of gentamicin. When gentamicin treatment was present continuously during days 4–7, the GAG level in Hurler fibroblasts was maintained at a normal level. Finally, when gentamicin was present during days 4–5 but omitted during days 6–7, we observed an intermediate level of GAG accumulation (1.3-fold higher than normal) in Hurler fibroblasts. Since this level was significantly lower than that observed in the same cells cultured in the absence of gentamicin, we conclude that gentamicin-treated cells can retain enough -L-iduronidase activity to partially reduce GAG levels for at least 2 days following the cessation of gentamicin treatment.
Gentamicin treatment restores normal lysosome distribution and morphology in Hurler fibroblasts
Hurler cells exhibit an increased abundance of lysosomes (termed vacuolation) and an abnormal lysosomal morphology as observed by light and electron microscopy (18). Since this morphological change is thought to occur as a direct consequence of the accumulation of GAGs within lysosomes, we next examined whether gentamicin treatment could reverse this atypical lysosomal morphology. Normal and Hurler fibroblasts were cultured on glass coverslips in the presence or absence of gentamicin for 2 days. The cells were then incubated for 1 h at 37°C with LysoTracker Red, a fluorescent dye that is endocytosed into lysosomes. We found that untreated Hurler fibroblasts contain more lysosomes than normal cells and these compartments appeared smaller in size than the lysosomes observed in normal cells (Fig. 5). In contrast, the majority of gentamicin-treated Hurler fibroblasts contained fewer lysosomes that were normal in appearance. The staining pattern observed in the gentamicin-treated Hurler fibroblasts resembled the pattern observed in wild-type cells in 
70% of cells examined, indicating that gentamicin treatment largely restores a normal pattern of lysosome distribution and morphology in Hurler fibroblasts.
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Gentamicin concentrations that suppress premature stop mutations do not induce a strong stress response
A major concern related to the pharmacological suppression of premature stop mutations is the possibility that native stop codons present at the end of normal cellular mRNAs may also be suppressed. The global suppression of stop codons at the ends of genes could be expected to produce many proteins with C-terminal extensions that may lead to protein misfolding. Such widespread protein misfolding should induce a stress response, leading to an increase in the steady-state level of the molecular chaperone Hsp70. Hsp70 is induced during many different conditions and acts to prevent the aggregation of misfolded proteins that may accumulate as a result of cellular stress (19).
To determine whether the gentamicin concentrations used in this study can induce a stress response, we cultured normal human fibroblasts (P6) in the presence of increasing levels of gentamicin and monitored the induction of Hsp70 by western blot analysis. A parallel flask of cells was subjected to heat shock to determine the maximal Hsp70 level obtained during a full-scale stress response (Fig. 6). We observed small progressive increases in Hsp70 levels with increasing gentamicin concentrations. We found a 1.2-fold increase in the Hsp70 level in cells cultured in the presence of 200 µg/ml gentamicin, a 1.3-fold increase in cells cultured in the presence of 600 µg/ml gentamicin and a 2.7-fold increase in cells cultured with 1000 µg/ml gentamicin. However, this maximal level of Hsp70 remained 10-fold below the level of Hsp70 observed in cells exposed to heat shock. Consistent with these results, we also found that this range of aminoglycosides did not have a significant effect on cell viability or total protein synthesis rates (data not shown). These results indicate that gentamicin treatment under the conditions used in this study induce only a very modest stress response.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Three approaches are currently considered promising avenues for the treatment of Hurler syndrome patients: bone marrow transplantation, enzyme replacement therapy and gene therapy. Hurler syndrome patients who received allogenic bone marrow transplantation exhibited a significant increase in serum
-L-iduronidase activity, a marked decrease in urinary GAG levels and a significant improvement in survivability. Diverse symptoms of the somatic disease such as liver and heart disease, hearing problems and dysmorphic features were also greatly diminished in treated patients. Skeletal abnormalities generally persisted but neurological improvement occurred in some patients (20–23). Although these results are extremely promising, the potential complications that accompany the use of immunosuppressive drugs to prevent graft rejection make this approach less than ideal. Another promising treatment for patients with Hurler syndrome is enzyme replacement therapy, or the periodic intravenous administration of purified -L-iduronidase. Recently, Hurler patients administered weekly infusions of recombinant -L-iduronidase were found to exhibit a marked reduction of GAG excretion and liver disease and improvements in joint mobility and heart function (24). Possible drawbacks to this approach include the lack of normal post-translational modification on the recombinant protein used, which could reduce cellular uptake and increase the chance of an immune response. Although limited immune responses have occurred in some treated patients, these responses were not severe enough to force the termination of treatment and this approach continues to show promise. Finally, gene therapy has also been explored as a treatment for Hurler syndrome patients. Hurler fibroblasts and CD34+ bone marrow cells transduced with recombinant adeno-associated virus or retroviral constructs containing the human IDUA cDNA showed high, extended -L-iduronidase expression and clearance of GAGs in vitro (25–27). However, the use of gene therapy approaches as a standard treatment for Hurler syndrome patients may remain years away.
Our results suggest that nonsense-suppression therapy may provide a novel, non-invasive option for the treatment of Hurler syndrome patients carrying premature stop mutations in the IDUA gene. Previous studies have shown that low levels of -L-iduronidase activity and protein are correlated with a less severe MPS I phenotype (10,11,15). In the current study, we found that gentamicin treatment was capable of restoring 2.5–3% of normal -L-iduronidase activity in cultured Hurler fibroblasts. We also found that the level of -L-iduronidase protein in gentamicin-treated Hurler cells was 0.41 ng/mg total protein, a level of enzyme that has been correlated with a mild Hurler phenotype (10,15). Thus, both enzymatic and immunological criteria suggest that gentamicin treatment can restore a sufficient level of -L-iduronidase to reduce the severity of the MPS I phenotype in cultured cells from Hurler patients that harbor premature stop mutations in the IDUA gene.
Another advantage of aminoglycoside therapy is that proteins produced by this approach should transit normally through the secretory pathway, resulting in normal glycosylation of the protein within the ER and Golgi apparatus. When combined with the overall low level of -L-iduronidase produced by the suppression of a premature stop mutation, the possibility of an immune response induced by the restored expression of -L-iduronidase should be low in MPS I patients. Since the protein will probably not carry the normal amino acid at the position where the stop mutation occurred, it is also possible that the half-life of the protein may be altered. However, we found that cells labeled for 2 days after the removal of gentamicin could sustain reduced GAG levels. This indicates that the -L-iduronidase produced by readthrough is relatively stable during this period. One potential limitation of this approach as a treatment for Hurler syndrome is the low permeability of gentamicin across the blood–brain barrier, which may prohibit the correction of the neurological manifestations of the disease (28). The establishment of an animal model will be necessary to determine whether gentamicin-mediated suppression of stop mutations in the IDUA gene can reduce GAG levels in the brain.
In studies over the last few years, it was found that aminoglycoside permeability varies significantly between different cell types. This is readily apparent from previous in vitro studies with CF and muscular dystrophy models, where the range of aminoglycoside concentrations used varied from 10 to 1000 µg/ml (1,2,5). In this study, we obtained evidence suggesting that the permeability or efflux of gentamicin in primary fibroblasts may change with increasing passage number. It has been reported that changes in membrane permeability occur with continued passage of cultured primary fibroblasts (14). In an attempt to bypass these difficulties, we examined whether commonly used permeabilizing agents could increase the entry of gentamicin into Hurler fibroblasts. Unfortunately, neither dimethylsulfoxide (DMSO) nor mannitol was found to increase the level of -L-iduronidase activity produced by gentamicin treatment (data not shown). However, we did find that the addition of poly-L-aspartate to the growth medium increased the ability of gentamicin to stimulate -L-iduronidase activity in Hurler fibroblasts at high gentamicin concentrations. This suggests that poly-L-aspartate may be capable of stimulating the uptake of aminoglycosides into the cell, possibly through a mechanism involving fluid phase endocytosis. Because of these permeability problems associated with cultured primary cells, the aminoglycoside concentrations routinely used in these in vitro studies generally exceeded the concentrations that would be useful in a clinical setting. However, pilot studies have shown that aminoglycosides can partially restore CFTR expression through the suppression of stop mutations when administered at clinically relevant doses (3,4). When combined with the low threshold for correction that appears to be associated with Hurler syndrome, these findings indicate that this approach may provide a viable treatment for MPS I patients with Hurler syndrome.
A hurdle to long-term gentamicin therapy is the nephrotoxicity and ototoxicity that can be associated with aminoglycoside treatment in some patients. However, several studies have shown that the cause of aminoglycoside-induced toxicity appears to be unrelated to their ability to suppress translation termination and our finding that gentamicin treatment does not induce a stress response supports those results (29,30). Numerous studies have reported that the co-administration of polyanionic compounds appears to reduce aminoglycoside-induced nephrotoxicity in rats (31,32) and antioxidant compounds have been found to relieve ototoxicity in guinea pigs (33,34). In addition, structural changes within aminoglycosides (including gentamicin) have been shown to reduce their nephrotoxic effects in a rat model (35,36). This suggests that it may be possible to design new compounds that may be able to suppress premature stop mutations without inducing the toxic side effects associated with aminoglycosides. Additional studies are needed to determine whether aminoglycoside suppression of premature stop mutations can be developed into a successful long-term treatment for patients with Hurler syndrome.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In vitro transcription/translation reactions
Human
-L-iduronidase (IDUA) cDNAs containing either the W402X (TGG
TAG) or Q70X (CAG TAG) mutation were expressed from the SP6 promoter in a rabbit reticulocyte lysate coupled transcription/translation system (Promega). Optimal gentamicin concentrations used for the suppression of stop mutations were similar to those described by Manouvakova et al. (12). The addition of 35S-labeled methionine to the translation reaction allowed the analysis of the translation products by SDS–PAGE and quantitation by PhosphorImager analysis. The level of suppression of the stop mutation was expressed as the amount of full-length protein produced relative to the sum of the truncated and full-length proteins. When necessary, corrections were also made for the difference in the number of methionine residues present in the truncated and full-length translation species. The readthrough reporter plasmid pDB650, used to examine the suppression of the IDUA Q70X mutation, was derived from plasmid pDB603 (12). To make this plasmid, a HindIII site beyond the polylinker was first changed to an NsiI site (ATGCAT). This was done using a QuikChange mutagenesis kit (Stratagene) and the primer DB843 (5'-GTC GAC CTG CAG CCC ATG CAT GGC GTA ATC ATG GTC-3') and its complement, DB844. A synthetic restriction fragment containing the Q70X TAG stop mutation and six codons of flanking upstream and downstream IDUA context was then introduced between the unique BamHI and HindIII sites located in the readthrough cassette. The synthetic DNA fragment was made by annealing the DNA oligonucleotides DB863 (5'-GAT CCT ACG TCC TCA GCT GGG ACT AGC AGC TCA ACC TCG CCT ATG CA-3') and DB864 (5'-AGC TTG CAT AGG CGA GGT TGA GCT GCT AGT CCC AGC TGA GGA CGT AG-3').
Cell culture
A primary human skin fibroblast cell line heterozygous at the IDUA locus (Q70X/W402X) and a normal fibroblast control that had been cultured a similar number of passages (P4–P6) were used in this study. Cells were cultured using Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum at 37°C with 5% CO2. All experiments were conducted with fibroblasts at 50–70% confluency. The concentration of gentamicin used in this study varied from 200 to 1000 µg/ml, with higher concentrations used with cells at a higher passage number.
-L-iduronidase activity and immunoquantification assays
Hurler fibroblasts (P4–P6) were grown in the presence of gentamicin (Gibco BRL) for 24 h. The cells were lysed using M-Per Mammalian Protein Extraction Reagent (Pierce) and the total protein concentration of each cellular extract was measured using the BCA method (Pierce). The assay measuring -L-iduronidase activity was adapted from Hopwood et al. (37). The enzymatic activity from cell extracts containing 20 µg of protein from Hurler cells and 2.5 µg of protein from normal fibroblast extracts was measured following the addition of 80 nmol of 4-methyl-umbelliferone iduronide (FMU) (Calbiochem) substrate in a 50 µl reaction mixture. The reaction was incubated for 1 h at 37°C after which the reaction was quenched by the addition of 1 ml of glycine–NaOH buffer pH 10.8. The fluorescence of the cleaved free FMU molecule was immediately measured at 365 nm excitation and 450 nm emission using a Shimadzu fluorometer and the amount of active iduronidase was expressed as nmol FMU cleaved/h/mg protein. A blank control demonstrated no change in fluorescence with the addition of the protein extraction reagent. The immuno-quantification assay was performed as previously described by Ashton et al. (10). The amount of -L-iduronidase protein present in the Hurler cells was determined by interpolation using a standard curve generated from cell lysates prepared from normal human fibroblasts (where 30 ng of -L-iduronidase protein was present per mg of total cell protein).
Measurement of cellular GAG levels
The GAG labeling assay was adapted from Thompson et al. (16). Normal and Hurler (P6) cells were cultured and labeled by the addition of 4 µCi/ml 35SO4 to the culture medium. Radiolabeled extracts were prepared and subjected to serial hot EtOH extractions to determine the total counts incorporated into macromolecules (primarily protein and GAGs). Unlabeled control samples processed in parallel were used to determine the total protein recovered from the precipitation procedure.
Visualization of lysosomal abundance in intact cells
Normal and Hurler fibroblasts (P6) were grown on glass coverslips in polystyrene culture dishes in the presence or absence of 1000 µg/ml gentamicin for 48 h. Following the addition of 40 mM HEPES to buffer the culture medium, 50 mM LysoTracker Red (Molecular Probes) was added for 1 h at 37°C. The coverslips were then mounted on an Olympus inverted fluorescence microscope without fixation and visualized at 100x magnification.
Hsp70 western blot
Normal fibroblasts were grown in the presence or absence of gentamicin for 24 h. Cellular extracts were then prepared by lysis in the presence of SDS sample buffer. Following a brief spin in a microfuge to remove insoluble debris, 25 µg of total protein was loaded onto an SDS–PAGE gel. Protein was transferred from the gel to Immobilon paper (Millipore) and incubated with rabbit anti-human Hsp70 antibody (StressGen) followed by incubation with [125I]Protein A (Amersham). The abundance of Hsp70 present in each sample was quantitated by PhosphorImager analysis. A positive control for a maximal stress response was prepared by subjecting cells to a heat shock at 45°C for 2 h followed by an additional 2 h incubation at 37°C prior to harvesting.
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ACKNOWLEDGEMENTS |
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The authors thank Dr Susan Jackson for help with the immunoquantification assay and Albert Tousson of the UAB Imaging Facility for help with fluorescence miscroscopy.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Microbiology, BBRB 432/Box 8, 1530 Third Avenue South, The University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA. Tel: +1 205 934 6593; Fax: +1 205 975 5482; Email: dbedwell@uab.edu
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