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Division of Cell and Molecular Pathology, Department of Pathology, University of Zurich, Zurich, Switzerland
1Correspondence: Division of Cell and Molecular Pathology, Department of Pathology, University of Zurich, Schmelzbergstr. 12, CH-8091 Zurich, Switzerland. E-mail: juergen.roth{at}usz.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase A activity in lysosomes resulting in an accumulation of glycosphingolipid globotriosylceramide (Gb3). Some classical Fabry hemizygotes and all cardiac variants have residual -galactosidase A activity, but the mutant enzymes are unstable. Such mutant enzymes appear to be misfolded, recognized by the ER protein quality control, and degraded before sorting into lysosomes. Hence, correction of the trafficking defect of mutant but catalytically active enzyme into lysosomes would be beneficial for treatment of the disease. Here we show that a nontoxic competitive inhibitor (1-deoxygalactonojirimycin) of -galactosidase A functions as a chemical chaperone by releasing ER-retained mutant enzyme from BiP. The treatment with subinhibitory doses resulted in efficient, long-term lysosomal trafficking of the ER-retained mutant -galactosidase A. Successful clearance of lysosomal Gb3 storage and a near-normal lysosomal phenotype was achieved in human Fabry fibroblasts harboring different types of mutations. Small molecule chemical chaperones will be therapeutically useful for various lysosomal storage disorders as well as for other genetic metabolic disorders caused by mutant but nonetheless catalytically active enzymes.—Yam, G. H.-F., Zuber, C., Roth, J. A synthetic chaperone corrects the trafficking defect and disease phenotype in a protein misfolding disorder.
Key Words: chemical chaperone • protein misfolding disease • Fabry disease • -galactosidase A • lysosomes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. In the endoplasmic reticulum (ER), various molecular chaperones, including BiP (4
5
6)
assist proteins to fold. Misfolded proteins will be retained by the quality control machinery, retrotranslocated to the cytoplasm, and degraded by the proteasome (7
8
9)
.
Deficiency of a protein due to mutations is a frequent cause of inherited diseases. However, mutated proteins may be functional, and efforts have been made to adjust their misfolding and subsequent proteasomal degradation in order to therapeutically correct protein misfolding diseases (10
11
12)
. Fabry’s disease is an inherited deficiency of lysosomal -galactosidase A (-Gal A) that causes progressive lysosomal glycosphingolipid accumulation (mainly globotriosylceramide Gb3) preferentially in vascular endothelia, neurons, and smooth muscle cells and results in the failure of vital organs (13)
. However, residual -Gal A activity indicating functional mutant enzyme has been observed in classical Fabry hemizygotes and atypical cardiac variants (14
15
16)
. In line with this, cardiac variant-causing mutant enzymes (R301Q and Q279E) in vitro degrade Gb3 similar to the wild-type but are less stable, particularly at neutral pH (17
, 18)
. The activity of mutant -Gal A in vitro at neutral pH could be stabilized with the small molecular mass nontoxic competitive inhibitor 1-deoxygalactonorijimycin (DGJ). Residual cellular enzyme activity in lymphoblasts from Fabry patients and in tissues of transgenic R301Q -Gal A knockout mice was enhanced by treatment with DGJ (19)
. It was also reported that subinhibitory dose of N-(n-nonyl)deoxynojirimycin increased the activity of lysosomal ß-glucosidase in Gaucher fibroblasts (20)
and that a galactose derivative enhanced the activity of lysosomal ß-galactosidase in GM1-gangliosidosis fibroblasts and tissues of knockout transgenic mice (21)
.
Mechanistic insight into how DGJ acts potentially as a chemical chaperone is missing and most important clearing of the lysosomal storage has not been demonstrated. Here, we provide evidence that DGJ efficiently promotes trafficking of ER-retained mutant -Gal A to lysosomes, where it is active, and that this effect is stableover long periods of time. Most important, we demonstrate that DGJ treatment of fibroblasts from Fabry patients harboring different mutations and containing low residual -Gal A activity resulted in a correction of the lysosomal storage phenotype. Together, our results strongly indicate the feasibility of this approach as a novel therapeutic strategy for Fabry’s disease and other genetic metabolic disorders caused by mutant but nonetheless catalytically active enzymes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Gal A, respectively) (19)
were cultured in McCoy’s 5A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen). Human Fabry R301Q and Q357X fibroblasts were cultured in modified Eagle’s medium (Invitrogen) with 10% fetal bovine serum. Cells from similar passages were used in the experiments. The culture media containing different DGJ concentrations (20 µM to 160 µM; Sigma, St. Louis, MO, USA) were freshly prepared and replenished daily.
Enzyme activity assay
Cellular -Gal A activity was determined according to Mayes et al. (22)
. The reaction product was measured fluorimetrically and -Gal A activity was expressed as the synthesis of 4-methyl umbelliferone per microgram total soluble protein as measured by the Bradford method.
Double labeling immunofluorescence and quantification
TgN and TgM cells grown to near-confluence on glass coverslips were fixed with 2% freshly prepared formaldehyde (Fluka, Buchs, Switzerland) and saponin-permeabilized (23)
. A rabbit polyclonal antibody against human -Gal A (17)
(provided by Dr. J. Q. Fan, New York, NY, USA) was diluted to 50 ng/mL and a rat monoclonal antibody against mouse lysosomal-associated membrane protein 1 (LAMP1; Research Diagnostic Inc., NJ, USA) was diluted to 0.5 µg/mL. They were simultaneously applied to transgenic mouse fibroblasts, followed by Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes Inc., Eugene, OR, USA) and Red X-conjugated Fab goat anti-rat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Human Fabry fibroblasts fixed and permeabilized as described were incubated sequentially with mouse monoclonal anti-Gb3 antibody (diluted to 0.6 µg/mL; Seikagaku, Tokyo, Japan), Alexa 488-conjugated goat anti-mouse IgG, mouse monoclonal anti-human LAMP1 antibody (diluted to 0.5 µg/mL; Research Diagnostic Inc.), and Red X-conjugated Fab goat anti-mouse IgG (Jackson Immunoresearch Laboratories). All samples were stained with Hoechst 33258 to visualize nuclei and examined by confocal laser scanning microscopy (CLSM SP2, Leica, Wetzlar, Germany).
For semiquantitative evaluation of lysosomal localization of -Gal A, a minimum of 200 cells per time point in each DGJ treatment protocol was analyzed from eight randomly chosen optical fields. We identified punctate -Gal A labeling as lysosomal when it codistributed with LAMP1 fluorescence and graded it from 0 to 4+: 0, no codistribution; 1+, less than 5 discrete double fluorescent spots per cell; 2+, 6 to 10 double fluorescent spots per cell; 3+, more than 10 double fluorescent spots per cell; 4+, extensive punctate double fluorescence.
The scale of Gb3 staining in human Fabry fibroblasts was evaluated by analyzing a minimum of 200 cells for each time point in each DGJ treatment protocol. Lysosomal Gb3 labeling was graded from not detectable to 3+ to indicate increasing intensity of staining. The Gb3 staining index was calculated using 4 x (3+)% + 2 x (2+)% + 1 x (1+)% and plotted against time of treatment.
Quantitative immunoelectron microscopy and transmission electron microscopy
TgN and TgM cells were fixed in 2% formaldehyde-0.05% glutaraldehyde (EM Science, Gibbstown, NJ, USA), embedded in low-melting agarose (Cambrex, Corp., East Rutherford, NJ, USA), and infiltrated with a sucrose-polyvinylpyrolidone mixture (24
, 25)
. Ultrathin cryosections were incubated with rabbit anti-human -Gal A (diluted to 0.5 µg/mL) and rat anti-mouse LAMP1 (diluted to 10 µg/mL), followed by 6 nm and 12 nm gold-labeled secondary reagents according to standard protocols (26
, 27)
, and examined with an EM912 AB transmission electron microscope (Zeiss, Oberkochen, Germany).
Immunogold labeling for lysosomal -Gal A was quantified on ultrathin sections of low temperature Lowicryl K4M embedded cells (26
, 27)
. Regions of cytoplasm containing a minimum of three lysosomes were sampled. The intensity of -Gal A immunolabeling was expressed as the number of gold particles/µm2 of lysosomes.
Lysosomal morphology in human Fabry fibroblasts was analyzed on ultrathin sections of cells fixed in situ with glutaraldehyde-osmium tetroxide and embedded in Epon 812.
Subcellular density fractionation, Western blot analysis, and mannose-6-phosphate incubation
Sucrose density differential centrifugation of cell lysate was performed according to standard protocol. Density fractions were resolved by 10% SDS-PAGE. Western blot analysis was done with anti-human -Gal A (diluted to 100 ng/mL) and anti-mouse LAMP1 (diluted to 1 µg/mL) antibodies, followed by appropriate amounts of horseradish peroxidase-conjugated secondary antibodies, and detected by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK).
To interfere with the lysosomal targeting of -Gal A, cells were simultaneously incubated in 20 µM DGJ and 5 mM mannose-6-phosphate (Sigma) for 4 days and analyzed.
Combined immunoprecipitation and Western blot analysis
Cells were washed twice with ice-cold PBS, resuspended at a concentration of 107 cells/mL in 0.25 M sucrose isotonic buffer containing 10 mM PIPES (pH 6.8, Sigma), 100 mM KCl, 3 mM MgCl2, 10 mM CaCl2, protease inhibitor cocktail (EDTA-free, Roche Diagnostics, Basel, Switzerland), and PMSF. The cells were Dounce-homogenized for 2 min on ice. After spinning, clear supernatant was collected and incubated with protein A-conjugated Dynabeads (DYNAL GmbH, Hamburg, Germany) that had been preincubated with polyclonal antibody to -Gal A for one hour at 4°C. After thorough washing in ice-cold PBS, the beads were boiled at 95°C in 0.5% SDS denaturation buffer containing 50 mM Tris-HCl(pH 6.8) and 0.25 M ß-mercaptoethanol for 10 min and spun. Western blot analysis of clear supernatant was done with monoclonal anti-mouse BiP (diluted to 1 µg/mL; BD Biosciences, San Jose, CA, USA) and detected by enhanced chemiluminescence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Gal A activity-Gal A knockout mice overexpressing either wild-type (TgN) or R301Q (TgM) human -Gal A (19
, 28)
. Activity of -Gal A in R301Q mutant TgM fibroblasts was increased by DGJ treatment in a time- and dose-dependent manner (Fig. 1
). After 6 days of treatment with 20 µM DGJ, it was 4-fold increased and remained 2-fold increased over 60 days of treatment.
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DGJ redistributes R301Q -Gal A to lysosomes
By confocal double immunofluorescence, DGJ-enhanced -Gal A activity was correlated with -Gal A trafficking to LAMP1-positive lysosomes. In TgN cells, -Gal A was predominantly detected in lysosomes (Fig. 2
A) but, in untreated TgM cells, predominantly in an ER-like pattern (Fig. 2B
). Treatment of TgM cells with DGJ resulted in the presence of -Gal A immunoreactivity in LAMP1-positive lysosomes (Fig. 2C
). This finding was substantiated by double immunogold electron microscopy (Fig. 3
A–C). By confocal immunofluorescence, the number of labeled lysosomes per cell and the number of cells with lysosomal -Gal A staining were significantly increased, and the effect was maintained over 60 days of treatment. In contrast to TgN cells, overall colocalization scores in untreated TgM cells were minimal (Fig. 2D
). Upon DGJ treatment, TgM cells with lysosomal -Gal A staining were observed from the second day onward. The DGJ effect reached a peak on day 6 of treatment (Fig. 2D
and Fig. 4
). Upon continuous treatment for 60 days, the colocalization scores stabilized at about one-third of that in TgN cells (Fig. 2D
). Treatment for 60 days with higher DGJ concentrations did not improve colocalization scores (Fig. 4)
. By quantitative immunogold electron microscopy, lysosomes of TgN cells and DGJ-treated TgM cells showed similar labeling profiles spanning the same range of labeling densities (Fig. 3D
). This indicates that the lysosomal level of -Gal A in DGJ-treated TgM cells resembles that of TgN cells.
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To obtain more insight into the maturation of R301Q -Gal A and its delivery to lysosomes, Western blot analysis of lysosome-enriched fractions was performed (Fig. 5
A). TgN cells showed -Gal A-positive bands with molecular masses from 45 to 52 kDa. In contrast to the faint immunoreactivity in untreated TgM cells, strong bands at 46 kDa (arrowhead in Fig. 5A
) and 51 kDa were detected after DGJ treatment. Band intensity of the mature 46 kDa -Gal A of TgM cells increased and resembled that of TgN cells (Fig. 5A
, bottom panel). Since sorting of lysosomal enzymes to lysosomes is mediated by the mannose-6-phosphate (Man-6-P) receptor, we studied the influence of an excess of free Man-6-P on the DGJ-induced lysosomal routing of R301Q -Gal A. TgM cells treated with DGJ in the presence of free Man-6-P had a strongly reduced lysosomal content of -Gal A (Fig. 5A
). This result was corroborated by immunofluorescence analysis. In both TgN and DGJ-treated TgM cells, the punctate lysosomal staining for -Gal A had disappeared after Man-6-P treatment (Fig. 5B-F
).
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DGJ clears lysosomal storage of Gb3 in human Fabry cells
On the basis of these results from transgenic mouse cells, we investigated the consequence of DGJ-enhanced -Gal A on lysosomal storage of Gb3 in fibroblasts from Fabry patients. Fabry fibroblast lines with missense mutations (R301Q and Q357X), which contained low residual enzyme activity, were analyzed. All Fabry fibroblasts showed extensive lysosomal Gb3 accumulation as detected by confocal double immunofluorescence (Fig. 6
A, B) and typical multilamellar lysosomal inclusions as shown by transmission electron microscopy (Fig. 6G, H
). After treatment with 20 µM DGJ for up to 100 days, the absence or a drastic reduction of lysosomal Gb3 immunostaining was observed (Fig. 6C, D
). In parallel, multilamellar inclusions of lysosomes were ultrastructurally undetectable or greatly reduced (Fig. 6I, J
). Quantification of confocal immunofluorescence over 100 days of treatment revealed that in about one-third of cells, lysosomal Gb3 accumulation became undetectable (Fig. 6E
). The majority of the remaining cells displayed a greatly reduced intensity of Gb3 staining. This effect was observed in the Fabry fibroblast lines studied irrespective of the type of mutation. The severity of the lysosomal storage was evaluated by the Gb3 staining index. In the studied Fabry fibroblast lines, the indices were dramatically reduced after 6 days of treatment and remained at low levels close to that of normal human MRC5 fibroblasts over the entire treatment period of 100 days (Fig. 6F
).
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DGJ releases mutant -Gal A from BiP
We next sought to obtain mechanistic insight into how DGJ affects the unproductive folding cycles of misfolded -Gal A. Since -Gal A lacks N-glycans within the first 50 amino acid residues at its N terminus (29)
, preferential interaction with BiP has to be expected (6)
. Complex formation of -Gal A with BiP was studied by combined immunoprecipitation and Western blot analysis. In contrast to TgN cells, samples immunoprecipitated with -Gal A antibodies from untreated TgM cells contained considerable amounts of BiP (Fig. 7
). A significant reduction of -Gal A/BiP complexes occurred after DGJ treatment (Fig. 7)
and, as shown above, was paralleled by trafficking of mutant -Gal A into lysosomes. This indicates that mutant -Gal A had escaped the quality control. The binding of the specific inhibitor may have increased the native state stability of mutant -Gal A, similar to the effect of small molecules on other misfolded proteins (11
, 20)
. Binding of DGJ to -Gal A occurs at neutral pH and the complexes dissociate at acidic pH (19)
, which are the pH conditions found in the ER and lysosomes, respectively. The acidic pH of the lysosomes stabilizes the conformation of the mutant lysosomal enzymes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Gal A, induces targeting of ER-retained R301Q -Gal A to lysosomes of transgenic mouse fibroblasts, where it was catalytically active (Fig. 8
). DGJ appears to positively influence the folding state of the mutant enzyme resulting in its release from BiP. Most important, we demonstrate that DGJ treatment results in efficient clearance of lysosomal Gb3 storage in human Fabry cells having low (2–7%) residual -Gal A activity. Thus, our data provide proof-of-concept that a specific enzyme inhibitor, used at subinhibitory concentrations, can reverse the Fabry lysosomal phenotype to near-normal. This is supported by a recent report that an inhibitor of lysosomal ß-galactosidase decreases GM1 and GA1 accumulations in GM1-gangliosidosis fibroblasts and neurons in short-term experiments (21)
.
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Our quantitative analyses unequivocally indicated significant lysosomal targeting of mutant -Gal A in transgenic TgM cells upon DGJ induction. Notably, the scale of increment was comparable to the level present in TgN cells expressing wild-type -Gal A. Upon long-term treatment, substantial enzyme activity levels were maintained that would be expected to be adequate for proper glycosphingolipid catabolism. Indeed, we observed an efficient unloading of Gb3 stored in the lysosomes of human Fabry fibroblasts as early as 6 days after DGJ treatment and the effect was maintained upon treatment for as long as 100 days. It is known that the substrate turnover rate in lysosomes correlates with the residual enzyme activity and that activity above a threshold level of 10–15% of normal can result in normal substrate turnover (30)
. As a case in point, Fabry patients with varying levels of residual enzyme activity have been reported to develop either no clinical signs of the disease or late-onset, mild forms with restricted organ involvement. Even classical hemizygotes may have residual -Gal A activity (13
, 31
, 32)
. In our study, the enzyme enhancement effected by the DGJ treatment was sufficient to clear the lysosomal storage of Gb3 in such Fabry fibroblasts.
Furthermore, our study shows that the DGJ inductive effect is not restricted to a specific type of -Gal A mutation. It efficiently recovered the normal lysosomal phenotype in Fabry fibroblasts harboring different types of mutations, such as single nucleotide substitutions and premature termination (present data) as well as frameshifting (unpublished results). This suggests the applicability of DGJ treatment to a wide range of mutant -Gal A exhibiting residual enzyme activity. A positive effect on the growth kinetics of the Fabry fibroblasts and no adverse effects on their cellular fine structure were observed upon continuous DGJ treatment. It had been shown before that mice treated with DGJ for several months showed no signs of toxicity, weight loss, or change of behavior (19)
.
In conclusion, DGJ will be a convenient and cost-efficient alternative to enzyme replacement therapy of Fabry disease. Cellular uptake of DGJ is not dependent on a specific receptor and, as a small molecular mass compound, diffuses freely in tissues. In principle, the use of small molecule enzyme inhibitors will be applicable to other lysosomal storage diseases and to congenital metabolic disorders exhibiting residual enzyme activities.
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ACKNOWLEDGMENTS |
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-Gal A antibody; J. S. Bonifacino (NIH, Bethesda, MD, USA), A. von Eckardstein (University Hospital Zurich), P. Komminoth (Cantonal Hospital Baden, Switzerland), P. M. Lackie (Southampton, UK), and T. Stallmach (University Hospital Zurich) for useful discussions and comments on the manuscript; and the team of the Central Laboratory for Electron Microscopy of the University of Zurich for access to the confocal laser scanning microscope. Supported by the Wolfermann-Nägeli Stiftung, the Swiss National Science Foundation, and the Canton of Zurich. Received for publication May 21, 2004. Accepted for publication August 27, 2004.
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REFERENCES |
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-Galactosidase A deficiency: Fabry disease. Scriver, C. Beaudet, A. Sly, W. Valle, D. eds. The Metabolic and Molecular Bases of Inherited Disease III,3733-3774 McGraw-Hill New York.
-galactosidase A in Fabry’s disease. Biochem. Genet. 13,615-628[CrossRef][Medline]
-galactosidase A activity. Am. J. Hum. Genet. 33,71A
-galactosidase A activity and genetic mutations on clinical course. Medicine 81,122-138[CrossRef][Medline]
-galactosidase A mutants (Q297E and R301Q) found in an atypical variant of Fabry disease. Biochim. Biophys. Acta 1501,227-235[Medline]
-galactosidases in human tissues and its application to Fabry’s disease. Clin. Chim. Acta 112,247-251[CrossRef][Medline]
-Galactosidase transgenic mouse: heterogeneous gene expression and posttranslational glycosylation in tissues. Glycoconj. J. 15,591-594[CrossRef][Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and, being a misfolded glycoprotein, eventually becomes a substrate for ER-associated degradation (ERAD). Due to the trafficking defect, lysosomes lack