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Originally published In Press as doi:10.1074/jbc.M609304200 on January 21, 2007

J. Biol. Chem., Vol. 282, Issue 12, 9150-9161, March 23, 2007
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Pyrimethamine as a Potential Pharmacological Chaperone for Late-onset Forms of GM2 Gangliosidosis*

Gustavo H. B. Maegawa{ddagger}§1, Michael Tropak§, Justin Buttner§, Tracy Stockley||, Fernando Kok**, Joe T. R. Clarke{ddagger}§, and Don J. Mahuran§{ddagger}{ddagger}2

From the {ddagger}Division of Clinical and Metabolic Genetics, Departments of Pediatrics and ||Pediatric Laboratory Medicine, §Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, the Institute of Medical Sciences and {ddagger}{ddagger}Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada and the **Department of Neurology, Faculdade de Medicina, University of Sao Paulo, 01246903 Brazil

Received for publication, October 2, 2006 , and in revised form, January 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Late-onset GM2 gangliosidosis is composed of two related, autosomal recessive, neurodegenerative diseases, both resulting from deficiency of lysosomal, heterodimeric beta-hexosaminidase A (Hex A, {alpha}beta). Pharmacological chaperones (PC) are small molecules that can stabilize the conformation of a mutant protein, allowing it to pass the quality control system of the endoplasmic reticulum. To date all successful PCs have also been competitive inhibitors. Screening for Hex A inhibitors in a library of 1040 Food Drug Administration-approved compounds identified pyrimethamine (PYR (2,4-diamino 5-(4-chlorophenyl)-6-ethylpyrimidine)) as the most potent inhibitor. Cell lines from 10 late-onset Tay-Sachs (11 {alpha}-mutations, 2 novel) and 7 Sandhoff (9 beta-mutations, 4 novel) disease patients, were cultured with PYR at concentrations corresponding to therapeutic doses. Cells carrying the most common late-onset mutation, {alpha}G269S, showed significant increases in residual Hex A activity, as did all 7 of the beta-mutants tested. Cells responding to PC treatment included those carrying mutants resulting in reduced Hex heat stability and partial splice junction mutations of the inherently less stable {alpha}-subunit. PYR, which binds to the active site in domain II, was able to function as PC even to domain I beta-mutants. We concluded that PYR functions as a mutation-specific PC, variably enhancing residual lysosomal Hex A levels in late-onset GM2 gangliosidosis patient cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GM23 gangliosidosis (GM2, OMIM 230700 [OMIM] ), is a clinically heterogeneous inherited neurodegenerative disorder characterized by progressive deterioration of motor, cerebral, and spinocerebellar function caused by deficiency of lysosomal beta-hexosaminidase A. Normal human tissues contain two major beta-hexosaminidase (Hex) isozymes, Hex A and Hex B. Hex A is a heterodimer made up of {alpha} and beta subunits. These subunits have nearly identical three-dimensional structures and similar active sites. They are encoded by two evolutionarily related genes, HEXA (15q23-q24) and HEXB (5q13), respectively. Hex B is a homodimer made up of two identical beta-subunits. A third minor, unstable Hex isozyme, Hex S, is comprised of two {alpha}-subunits and is only unequivocally detectable in tissues from patients with the Sandhoff disease variant (SD, OMIM 268800 [OMIM] ) of GM2. SD results from HEXB mutations producing abnormal or deficient beta-subunits. Thus SD is associated with combined deficiency of both Hex A ({alpha}beta) and Hex B (betabeta) activities. On the other hand, Tay-Sachs disease variant (TSD; OMIM 272800 [OMIM] ) is caused by HEXA mutations resulting in abnormal or deficient {alpha}-subunits, which only affects Hex A levels. Mutations affecting GM2A (5q31.3-q33.1), encoding the non-catalytic GM2 activator protein (Activator), results in the third very rare AB-variant form of GM2 gangliosidosis (OMIN 272750) (1).

In humans, only the Hex A isozyme catalyzes the removal of the beta-GalNAc residue from the non-reducing terminal end of GM2 ganglioside, but it requires the Activator as a substrate-specific co-factor for the reaction (1). The synthetic substrate, 4-methylumbelliferyl-(2-acetamido-2-deoxy)-beta-D-glucopyranoside (MUG), is hydrolyzed by both the {alpha}- and beta-active sites and therefore, is used to measure total Hex activity. A newer, more specific synthetic substrate is 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy)-beta-D-glucopyranoside (MUGS). Its negatively charged 6-sulfate group has been shown to interact with the same positively charged binding pocket, found only in the {alpha}-active site, that binds the sialic acid residue of GM2 ganglioside (2-4). Thus, this substrate most closely mimics the natural substrate. However, MUGS hydrolysis is Activator-independent and thus, it is turned over more rapidly by Hex S (in SD samples) than by Hex A (5).

GM2 gangliosidosis is characterized by a wide spectrum of clinical presentations. The most severe forms are the infantile or acute TSD and SD, associated with <0.5% of normal Hex A activity, resulting in rapid neurodegeneration, and culminating in death in infancy. At the other end of the spectrum are the late-onset forms, which are subdivided into juvenile or subacute and adult or chronic forms (6). These are usually associated with residual Hex A activities, ~1-10% of normal (7). Patients with juvenile GM2 gangliosidosis usually present with evidence of neurodeterioration starting after 1 year of age, experiencing a slower rate of progression than patients with the infantile forms (8). Patients with adult-onset forms may present with spinocerebellar, psychiatric, and/or peripheral neuropathies, which do not significantly decrease life expectancy in some cases (9). The rate of disease progression and severity has been found to correlate roughly with the level of residual Hex A activity. Generally, a clinical disease does not develop unless residual Hex A activity is <10% of normal (10). Thus, only a low level of residual Hex A activity is apparently needed to prevent or reverse substrate storage in this condition.

Pharmacological chaperones (PC) are low molecular weight compounds that stabilize the native conformation of a mutant enzyme in the ER, allowing it to escape aggregation and premature degradation by the ER-associated degradation pathway. The properly folded mutant enzyme, stabilized by the PC can then be transported to the lysosome, increasing the residual enzyme activity of the cells (11). Most PCs have also been competitive inhibitors of their target enzyme (12). Once the PC-enzyme complex reaches the lysosome, the large amounts of stored substrate(s) are believed to displace the PC and take over stabilization of the mutant enzyme (13). The PC approach has been shown to enhance the residual activity levels of five different mutant lysosomal enzymes causing chronic forms of the lysosomal storage diseases, GM2 gangliosidosis (14), GM1 gangliosidosis (15), Fabry (16), Gaucher (11), and Morquio B diseases (17).

A major challenge to the exploitation of this phenomenon for the treatment of disease is identifying potential PCs from among the thousands of existing chemicals and drugs. High throughput screening of small molecule libraries has been used to identify specific enzyme inhibitors with PC potential (18, 19).

We report here the application of a small molecule library screening approach to search for novel inhibitors of lysosomal Hex A with the potential to treat late-onset variants of GM2 gangliosidosis by functioning as PCs. We undertook a manual screening for Hex inhibitors in a 1040-compound library of Food Drug Administration (FDA)-approved drugs obtained from NINDS (National Institutes of Health). Pyrimethamine (PYR) was discovered to have considerable PC potential. The evaluation of PYR as a PC was performed using fibroblast cell lines from 17 patients with juvenile and adult GM2 gangliosidosis. Ten of the 11 different HEXA mutations, and 5 of the 9 different HEXB mutations that we identified in patients were reported previously. Each of the mutant cell lines from our patients responded differently to PYR, as well as to a carbohydrate-based PC, N-acetylglucosamine thiazoline (NGT), which we reported previously (14). In addition, the different responses of mutants to PYR brought interesting insights into the role of the 2 domains present in each subunit ({alpha} and beta) in the folding and assembly of the Hex dimers.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Study Subject—Fibroblast cell lines were obtained from 17 patients with late-onset forms of GM2 gangliosidosis. Patients were from two Genetic Metabolic centers, and informed consents and assents approved by relevant research ethic boards were obtained from each patient.

Chemical Reagents and Antibodies—The NINDS National Institutes of Health drug library was received from MicroSource Discovery Systems in a 96-well format; one drug per well at a concentration of 10 mM diluted in dimethyl sulfoxide (Me2SO), sealed under nitrogen. Plates were stored at -20 °C. The following fluorogenic substrates, all purchased from Sigma, 4-methylumbelliferyl-beta-D-galactopyranoside (MUbetaGal) and 4-methylumbelliferyl phosphate, MUG and MUGS were used to assay the lysosomal enzymes beta-galactosidase, acid phosphatase, total Hex, and Hex A/Hex S, respectively. Enzymatic reactions were stopped using 2-amino-2-methyl-1-propanol (at 0.1 M, pH 10.5). Purified Hex A and Hex B, which were used in kinetic characterization of PYR, were extracted from human placenta as described previously (21). Qiagen kits for DNA and RNA extraction from fibroblasts were used. Molecular kits for PCRs and reverse transcriptase-PCRs were purchased from Invitrogen Inc. Specific oligonucleotides for sequencing of genomic and cDNA were synthesized by The Center of Applied Genomics from the Hospital for Sick Children. Rabbit polyclonal antibodies against human Hex A (Western blot) and sheep polyclonal IgG against human beta-subunit (used for immunoprecipitation and cell immunofluorescence) were prepared as previously described (22). Mouse IgG monoclonal against Lamp-1 was purchased from the Iowa Hybridoma Bank. Secondary antibodies, donkey anti-sheep IgG, and chicken anti-mouse IgG were purchased from Molecular Probes Inc. Stock solutions of NGT, provided by Dr. S. Withers, University of British Columbia, were prepared by dissolving the compound in Me2SO, (4 mg/ml) or water (10 mg/ml). PYR, purchased from Sigma, was dissolved in Me2SO (stock solution of 4 mg/ml), or ethanol (ETOH) (stock solution of 0.1 mg/ml). For IP, Gamma beads were purchased from Amersham Biosciences (UK). Steel wool number 0000 (International Steel Wool, Mexico), FeCl2 and FeCl3 (Sigma), and dextran T4000 (Amersham Biosciences, UK) were used to prepare lysosomes by magnetic chromatography. Tritiated GM2 ganglioside, [3H]GM2 (10 mol %), cholesterol (20 mol %), phosphatidylinositol (20 mol %), phosphatidylcholine (50 mol %), polycarbonate Liposo-Fast filter (Avestin 100 nm), AG3X4 (acetate form) resin (Bio-Rad), concanavalin A spheroid beads (Amersham Biosciences), and recombinant Activator were used for the natural substrate assay. Chemical cross-linking was performed using dithiobis(succinimidylpropionate) (DSP) as a cross-linker reagent. Stock solution of DSP was dissolved in Me2SO at 6 mM.

Screening for Competitive Hex Inhibitors—The NINDS National Institutes of Health library of 1040 FDA-approved compounds was screened to identify potential PCs for Hex A. The screening was performed using wild-type Hex B, and the common fluorescent MU-based artificial substrate, MUG. The library was screened in duplicate; with test compounds at a final concentration of 20 µM. From the original 96-well plates, compounds were initially diluted to 10 mM by addition of Me2SO. Real-time Hex assays were performed in final volumes of 100 µl, containing 1 mM of each drug, 12 ng of purified Hex B diluted in citrate phosphate buffer (CP) (pH 4.1) containing 0.025% human serum albumin, and 25 µl of MUG (0.4 mM). Real-time fluorometric assays were performed using a Gemini EM Microplate Spectrofluorometer (Molecular Devices), with excitation at 345 nm and emission at 450 nm, every 2 s during the 20-min incubation period at 37 °C. Mean Vmax was then obtained and expressed in relative fluorescence units/s using standardized SoftMax® Pro Software coupled to the spectrofluorometer. The values described on a replicate screen plot represent ratios related to mean of the Vmax obtained from the control Me2SO sample (Fig. 1).


Figure 1
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  		FIGURE 1.
Replicate plot of a screen of the NINDS FDA-approved library of 1,040 small molecules for Hex A inhibitors. The figure represents a replicate plot of residual Hex A activity in the presence of individual compounds from the NINDS National Institutes of Health library, i.e. each set of replicates define an X,Y coordinate. The zone outlined (dashed line) at 40% was set as the "inhibitor compound area," which contained 4 compounds.

 
Mutation Identification—Most mutations were identified and reported in previous studies from our group (20, 23, 24). For mutations of cell lines, 26649, 32429, and 36986, total genomic DNA and cDNA were isolated from fibroblasts by routine techniques (25). PCR amplification was performed using primers described previously (24, 26). Reverse transcriptase-PCR from extracted total RNA followed by specific PCR amplification from cDNA with oligonucleotides previously described were performed (26). Fragments were sequenced with the use of ABI 377 and 3700 sequence analyzers.

Tissue Culture Conditions and Enzyme Assays—Fibroblast cell lines were cultured to confluency. The culture medium used, {alpha}-minimal essential medium with 10% of fetal calf serum and antibiotics, was then replaced by the same medium containing filter-sterilized (Millipore-0.45 µm) PYR or NGT at the described concentrations. For the initial experiments, 10-cm culture plates of each cell line were treated with PYR and NGT diluted in Me2SO at final concentrations of 20, 10, and 5 µg/ml. In follow-up experiments, each cell line was treated with PYR at 3.0, 1.5, 0.5, and 0.1 µg/ml, and NGT, at 300, 150, and 75 µg/ml. Control plates containing only solvents, ETOH (used to dissolve PYR), and water (used to dissolve NGT) added to culture media were also established. After 5 days of incubation in drug-containing media, culture medium was removed; cells were washed twice with phosphate-buffered saline and harvested. Cell pellets were re-suspended in NaH2PO4 (10 mM, pH 6.0), containing 5% glycerin, and lysed by freezing-thawing on dry ice. Hex assays using MUG or MUGS were performed. Lysates were diluted 10-fold by addition of 20 mM CP buffer (pH 4.1). For all lysosomal enzyme assays, stock solutions of the substrates dissolved in the same CP buffer of MUG (3.2 mM), MUGS (3.2 mM), 4-methylumbelliferyl phosphate (10 mM), and MUbetaGal (0.56 mM) were used. Assays were carried out by addition of 100 µl of substrate solution (final volume 200 µl), and incubation at 37 °C for 1 h for all artificial substrates used, except from MUG where the incubation time was 15 min. Reactions were stopped by addition of 1.5 ml of 0.1 M 2-amino-2-methyl-1-propanol (pH 10.5), and fluorescence was measured as described above (22). Enzyme activities were calculated in nanomoles of MU hydrolyzed/h/mg of protein. The relative activities of total Hex, Hex A, and Hex S were expressed as ratios of corresponding samples from treated versus control cells. Residual Hex A % activities were calculated based on those of the wild-type cell line lysate, assayed concomitantly with the mutant cell lines (range of Hex A activity 3,500-8,500 MU of nmol/h/mg of protein).

Immunoselection Assays for Hex A and Hex S—For measurement of residual Hex activities in SD cell lines (beta-mutants), Hex A and Hex B (residual Hex B activity is generally undetectable in SD cells) were immunoprecipitated by solid-phase IP with polyclonal sheep anti-beta-subunit IgG as previously described (22, 27, 28). Residual Hex A activity was determined by measurement of Hex activity from the immunoprecipitated phase (containing antibody bound to Hex A) with MUGS as substrate. Hex S activity was determined by measurement of enzyme activity in the IP supernatant, also using MUGS as substrate.

Western Blot Analysis—The total protein contained in clarified lysates was determined by the Lowry method (29). Aliquots of lysates containing 20 µg of total protein were diluted 1:1 with 1x standard Laemmli buffer containing 50 mM dithiothreitol and heating at 65 °C for 15 min. Each sample was then subjected to SDS-PAGE on a 10% bisacrylamide gel, and transferred to nitrocellulose. The nitrocellulose was then incubated with a rabbit anti-human Hex A antibody as previously described (23). Blots were developed using chemiluminescent substrate according to the manufacturer's protocol (Amersham Biosciences). Bands were visualized, recorded, and their optical density quantitated using a high sensitivity documentation system (Fluorchem 8000) consisting of a cooled CCD camera coupled with software from Alpha Innotech Corp.

Purification of Iron-dextran-labeled Lysosomes by Magnetic Chromatography—Lysosomal fractions were prepared from fibroblasts grown for 5 days in media lacking or containing PYR, 3 µg/ml, using a previously described procedure (14, 30). Solid-phase IP of the LYSO and postnuclear supernatant (PNS) fractions were performed as described above.

Immunofluorescence Labeling—Cells were grown on coverslips, fixed with 100% methanol for 15 min, and then blocked with 10% fetal calf serum for 30 min. After washing twice with phosphate-buffered saline, the cells were incubated in the presence of anti-beta-subunit (diluted 1:400) and anti-Lamp-1 (diluted 1:500) antibodies at room temperature for 1 h. The cells were then immunostained with suitable secondary antibodies as cited earlier (Molecular Probes) at room temperature for 1 h. Confocal laser scanning microscopy on the Zeiss LSM 510 confocal system was performed. All images were taken with 100 x 1.4 numerical apertures (NA) and 63 x 1.4 NA Apochromat objective (Zeiss). All image processing was performed using the Zeiss LSM 5 image examiner software.


Figure 2
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  		FIGURE 2.
A and B, characterization of two inhibitors identified in the screen. The IC50 curves for the two "hits," PYR (A) and thioguanine (B) are shown. C, PYR behaves kinetically as a classical competitive inhibitor of Hex. D, maximal inhibition of Hex A by PYR was at pH 6.5, whereas the transition state analog, NGT, showed a maximum inhibition at pH of 4.5.

 
Natural Substrate Assay—Tritiated GM2 ganglioside, [3H]GM2, containing liposomes were prepared as previously described (4). To concentrate Hex A, as well as other soluble lysosomal enzymes, 3.5-3.8 mg of total cell lysate protein was incubated with concanavalin A beads (30 µl of drain solution) overnight at 4 °C. Beads were then washed with phosphate-buffered saline three times and assayed in a final volume of 100 µl, which contained CP (20 mM; pH 4.1), bovine serum albumin (50 µg/ml), 2.5 µg of recombinant Activator isolated from transformed Escherichia coli as described (31), and 20 nmol of [3H]GM2 contained in negatively charged liposomes. The incubation period of 18 h, as well as further procedures for stopping the reaction and preparation for liquid scintillation counting analysis, were performed as previously described (4).

Chemical Cross-linking with DSP—Total lysate protein from mutant cells grown in medium containing PYR (3.0 µg/ml) or solvent (ETOH) were adjusted to a concentration of 0.5 mg/ml with NaH2PO4 (10 mM, pH 6.0), containing sufficient DSP to give a final concentration of 0.9 mM. After 15 min incubation at 37 °C, the reaction was quenched by the addition of 2.2 volumes of Tris-HCl (pH 7.5) to bring the mixture to 100 mM. The protein samples (5 µg, normal control, 10 or 20 µg of mutant cell lines) were then mixed with the 2x Laemmli sample buffer (1:1, containing no reducing agent), heated at 65 °C for 15 min, and separated by SDS-PAGE (10% gel) for Western blot analysis and quantitation (see above).


Figure 3
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  		FIGURE 3.
PYR enhances Hex A enzyme levels in some TSD patient cell lines. Two mutant TSD fibroblast cell lines, {alpha}G269S/IVS6 + 1G>A (A) and {alpha}R178H/R178H (B), were treated with PYR and NGT, at 20, 10 and 5 µg/ml, dissolved in Me2SO. Hex A activities using MUGS as a substrate were determined. The relative fold-increase of Hex A activity was calculated based on the activity of cell lines treated only with Me2SO. The mature (lysosomal) {alpha}-subunit protein levels seen in Western blots (below histograms) correlated with the relative increase of Hex A observed in the {alpha}G269S/IVS6 + 1G>A mutant (A). However, the cells containing the {alpha}R178H/R178H active site mutations failed to show any increases in relative Hex A activity or changes in {alpha}-subunit protein levels (B). {alpha}p, indicates the {alpha}-subunit precursor, {alpha}m, the mature lysosomal {alpha}-subunit. *, p < 0.01; **, p < 0.001.

 


Figure 4
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  		FIGURE 4.
Specificity of PYR as a PC for Hex A. Relative levels of MU-substrate hydrolysis by acid phosphatase (AcPhos, substrate 4-methylumbelliferyl phosphate), Hex A (substrate, MUGS), and beta-galactosidase (substrate, MUbetaGal) in one of the TSD cell lines, {alpha}G269S/c1278insTACT, incubated with increasing concentrations of PYR (units in millimolar).

 
Heat Denaturation Assays—The effect of PYR on the stability of the Hex A containing a mutant beta-subunit (betaR505Q) was determined by heat denaturation experiments. Lysates from mutant cell lines containing 100-150 µg of protein were added to preheated CP buffer (20 mM; pH 4.1) with 0.3% of heat-treated bovine serum albumin (Sigma) and PYR at a final concentration of 3.0 µg/ml. Aliquots of 100 µl were removed at fixed intervals of 0, 5, 10, and 30 min at 37 °C and put on ice. Aliquots from each fraction of heated and non-heated lysates were put in tubes with anti-beta antibodies bound to Gamma beads, and the IP procedure was followed (as described above). Mutant lysate samples containing only ETOH (PYR solvent) were also tested as a control for the PYR-exposed lysates.

Statistical Analysis—The statistical test Z' factor was used to measure the quality of the assay and its applicability to screening (32). This single statistic takes into consideration both signal-to-noise and reproducibility. Assays with a Z'-statistic >0.5 are robust enough to identify enhancement of enzyme activity reliably (32). Where applicable, data are expressed as the mean ± S.E. Comparisons of parametric data were analyzed in the use of conventional parametric statistical methods as two-tailed Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Characterization of PYR as a Hexosaminidase Inhibitor—The results of screening each of the 1040 small molecules in the NINDS National Institutes of Health library, done in duplicate, for inhibition of Hex activity, are shown in Fig. 1. Each of the two replicates is defined by a X and Y pair of coordinates on the graph. Thus, consistent replicates fall on, or near the diagonal line (Z'= 0.54) (32). Compounds within the box were considered "hits," potentially effective as PCs, and were selected for secondary screening.

Two inhibitory compounds were identified as potential PCs, PYR and thioguanine (TGN) (Fig. 2). Secondary screening of PYR showed it had an IC50 of 5-13 µM for the Hex isozymes at pH 4.3 (Fig. 2A). On the other hand, TGN had a significantly higher IC50 of 170 µM (Fig. 2B). Unlike PYR, TGN was toxic to the fibroblast cell lines tested at the concentrations needed for it to act as a PC. Thus, PYR was chosen for further study. Kinetic examinations demonstrated that PYR behaved as a competitive inhibitor of Hex A with a Ki of 13 µM at pH 4.5 (Fig. 2C). Interestingly, PYR was found to have a pKHA of 6.5 with an IC50 ~ 2 µM at pH 6.5 (Fig. 2D). Thus, PYR would be least effective as an inhibitor in the acidic environment of the lysosome, but would bind maximally at the neutral pH of the ER, where optimal PC activity is desired. Other compounds considered to be possible PC candidates failed to show competitive inhibition properties in a secondary screening and were not studied further (Fig. 1).


Figure 5
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  		FIGURE 5.
Missense mutations identified in TSD and SD patients localized onto a three-dimensional Hex A ribbon diagram (2). Amino acid residue alterations are highlighted in color and shown in a space filled format. Two novel mutations (betaC137Y and betaT150P) are located in domain I (betaA50-P201; light gray) of the beta-subunit (blue). NGT is shown as a red stick format bound in the active site of the {alpha}-subunit (cyan).

 
PYR as a Pharmacological Chaperone for Hex A—Initially, two fibroblast cell lines from TSD patients were selected for testing of PYR, {alpha}G269S/IVS6 + 1G>A (number 32540) and {alpha}R178H/R178H (number 27986) (Table 1). The total relative Hex (A and B) activity (MUG) showed no significant increases in both treated cell lines (data not shown). However, when MUGS was used as the substrate, the {alpha}G269S/IVS6 + 1G>A cell line showed a statistically significant increase in Hex A activity, over 3-fold, with the highest concentration of PYR added to the culture media, whereas NGT at the same concentration (20 µg/ml) showed a smaller increase (Fig. 3A). As expected, the {alpha}R178H/R178H cell mutant, which carries an active site mutation affecting substrate binding, also known as the B1 variant (33, 34), showed no effect above that of the Me2SO control for either PYR or NGT (Fig. 3B). Interestingly, Me2SO was found to be a weak competitive inhibitor of Hex (Ki of 170 ± 20 µM), and to have mild chaperone activity, complicating the initial interpretation of the data. For this reason, we used ETOH as a solvent of PYR in further experiments. The final ETOH concentration was 0.03% in culture media, and did not affect the Hex activity ("Experimental Procedures"). The PC effect of PYR was also specific for Hex; the activities of acid phosphatase and beta-galactosidase were not enhanced in the {alpha}G269S/ins1278TACT mutant cell line, whereas Hex A levels were enhanced at levels ≥0.1 mM (Fig. 4).


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  		TABLE 1
Fibroblast cell lines tested with their respective mutations in HEXA (TSD variant), HEXB (SD variant) genes, phenotype, and residual Hex A activities

 
Assembly, Genotyping, and Enzymatic Phenotyping of Fibroblast Cell Lines from TSD and SD Patients—The genotypes of the various cell lines used to evaluate PYR are shown in Table 1. The localizations in the crystallographic structure of Hex A of the different missense mutations examined in this study are shown in Fig. 5 (2). Clinical features of the juvenile GM2 gangliosidosis patients were reported previously by our group (20). The genotype and residual Hex A activity against MUGS of each cell line are given in Table 1. These activities, with the exception of the betaP504S-containing line, were within the expected range, based on previous correlations made between natural substrate hydrolysis and clinical phenotypes (7).

Responses of Different TSD and SD Mutants to PYR and NGT at Concentrations Known to Be Non-toxic—At concentrations of PYR achievable in cerebrospinal fluid, thus available to neuronal cells of patients receiving therapeutic doses of the drug for malaria and toxoplasmosis, residual Hex A levels of two late-onset TSD cell lines, {alpha}G269S/c.1278insTACT and {alpha}IVS9 + 1G>A/IVS8-7G>A, were enhanced (Fig. 6, A and B, respectively). The {alpha}G269S mutants showed lower relative increases (Fig. 6A) than those observed using higher concentrations of PYR (Fig. 3A). On other hand, NGT, at 300 µg/ml, which is non-toxic in mice,4 showed up to an 8-fold relative increase (Fig. 6A). The residual Hex A from the {alpha}IVS9 + 1G>A/IVS8-7G>A cell line was also chaperoned by both PYR and NGT (Fig. 6B). Interestingly, with this cell line the relative fold-increases with PYR was higher than the one obtained with 100-fold higher concentrations of NGT (Fig. 6B). The increase in the Hex A activity was confirmed by the increased levels of the mature {alpha}-subunit ({alpha}m) in the respective Western blots shown (Fig. 6, A and B). Maturation of the Hex subunits has been shown to occur after dimerization in the late endosome/lysosome (35-37).


Figure 6
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  		FIGURE 6.
TSD or {alpha}-mutant cell lines that responded to PYR and NGT. The {alpha}G269S/c1278insTACT (A) and {alpha}IVS9 + 1G>A/IVS8-7G>A (B) are shown with their relative Hex A activity and {alpha}-subunit protein levels at different treatment regimens of PYR and NGT. The fold-increase in activities was calculated based on the activity measured in control cell lines treated only with the dissolvent, i.e. ETOH for PYR-treated and H2O, for NGT-treated cells (under "Experimental Procedures"). {alpha}p, indicates the {alpha}-subunit precursor; {alpha}m, the mature, lysosomal {alpha}-subunit. *, p < 0.01; **, p < 0.001.

 
Some {alpha}-mutants, {alpha}R178H, {alpha}R499H, and {alpha}R499C (allelic to R178H mutation), show no increase of Hex A when treated with either PYR or NGT (Fig. 7, A and B). The {alpha}R499H (Fig. 7A), a common mutation in Western Europeans, is associated with an {alpha}-subunit mutant with decreased solubility that tends to aggregate in the ER (38). Western blots confirmed that there were also no increases in the protein level of mature {alpha}-subunit (Fig. 7, A and B).

All seven of the late-onset SD cell lines showed some degree of relative increase in residual Hex A (and Hex S) activity with PYR or NGT (Table 2 and Fig. 8). IP was used to separate the residual Hex A from Hex S in lysates obtained from treated beta-mutants ("Experimental Procedures"). The highest relative increase of Hex A with PYR was seen in cells with the betaR505Q mutant allele. The betaR505Q/{Delta}16kb mutant showed the best response to PYR of all 7 SD cell lines (Fig. 8). The response to PYR was even greater than the response to NGT (Fig. 8). Interestingly, the betaR505Q/IVS11 + 5G>A cell line showed a smaller, although significant increase (up to 4-fold) of residual Hex A with PYR (Table 2). The cell line betaC137Y/C137Y also showed a significantly large increase in relative Hex A activity with PYR as compared with NGT (Table 2). This cell line also had the lowest starting residual Hex A activity (Table 1). On the other hand, a cell line with a mutation in the adjacent residue, betaP504S/{Delta}16kb, showed higher relative Hex A increases with NGT than with PYR. The cell line betaT150L/P417L showed a 2.7-fold relative increase in Hex A activity with PYR, which was a better response than the 1.6- and 1.5-fold relative increases observed for the betaP417L/{Delta}16kb and betaG353R/IVS12-26G>A cell lines, respectively (Table 2). Data from Western blots correlated with the increased Hex A activities as shown in the beta-mutants from Table 2 (data not shown).


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  		TABLE 2
Six of seven of the SD or beta-mutant cell lines with their relative activities of Hex A and Hex S after treatment with PYR or NGT

 
Hex A Is Increased in a Lysosomal Enriched Fraction of PYR-treated Cells—To confirm the cellular localization of Hex A in PYR-treated and untreated cell lines, we loaded lysosomes with ferrous-dextran colloid, and performed a magnetic fractionation from which we obtained a PNS, and an enriched lysosomal (LYSO) fraction, as previously described (14, 30). After separating Hex A from Hex S by IP, the LYSO as well PNS fractions of one PYR-treated cell line (betaC137Y/C137Y) showed significantly higher absolute Hex A and Hex S activities than the untreated controls (Table 3).


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  		TABLE 3
Increased Hex A and S activities in the enriched lysosomal fraction, LYSO, in relation to postnuclear supernatant (PNS) fraction in a domain I beta-mutant, betaC137Y/C137Y, cell line treated with PYR

 
Mutant Hex beta-Subunits of Hex A Are Present in the Lysosomes of PYR-treated SD Cells—To further confirm that enhanced Hex A levels in SD cells was lysosomal, we localized Hex A by virtue of its mutant beta-subunit in one of our high responding SD cell lines, betaR505Q/IVS11 + 5G>A, treated with PYR (3.0 µg/ml). Immunofluorescence microscopy, using sheep anti-beta-subunit IgG and mouse anti-Lamp-1 IgG, co-localization the beta-subunit (green) of Hex A with Lamp1 (red) only in treated cells (Fig. 9, A-F). In control cells lines treated only with solvent (Fig. 9, G-L), beta-subunit staining was difficult to detect even in the ER.

PYR Enhances Natural Substrate Hydrolysis—PYR treatment was confirmed to enhance both natural substrate (Activator-[3H]GM2 complex) and MUGS hydrolysis in a {alpha}G269S chronic TSD cell line (Fig. 10).

PYR Mechanism of Function on Hex—To investigate the mechanism in which PYR promotes the observed increases in Hex A activity, two experiments were conducted. First, lysates from two mutants, {alpha}G269S, a mutation that is located well away from the subunit-subunit interface, and betaR505Q, located at the interface (Fig. 5), were exposed to a cross-linking reagent, DSP. PYR was shown to stabilize dimers and monomers in both {alpha}- and beta-mutant cell lines (Fig. 11A). Optical density analysis ("Experimental Procedures") documented a 2-fold increase in both dimer and monomer bands in the treated {alpha}G269S TSD cells (Fig. 11A). However, the treated betaR505Q/{Delta}16kb cells showed a larger, 15-fold increase in their Hex dimer as compared with a 4-fold increase in their monomer bands. These data indicate that in the former case PYR (and NGT) are primarily stabilizing mutant monomers, whereas in the later case the effect is primarily on dimers. Second, NGT has previously been shown to protect {alpha}G269S Hex A from heat denaturation,5 (14, 39), and we now demonstrate that PYR (3 µg/ml) also increases the thermostability the Hex A containing the betaR505Q mutation, from a half-life of 7.6 ± 3.7 min at 37 °C to 24.9 ± 5.3 min (Fig. 11B).


Figure 7
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  		FIGURE 7.
Some TSD or {alpha}-mutants showed no relative increase in residual Hex A activity when exposed to PYR. PYR treatment had no effect on cell lines containing {alpha}R499H, {alpha}R499C, and {alpha}R178H mutations. The {alpha}R499H/c.1278insTACT mutant cell line (A) and {alpha}R178H/c.1510delC (B) are shown here to illustrate this observation. {alpha}p, indicates the {alpha}-subunit precursor; {alpha}m, the mature lysosomal {alpha}-subunit in Western blots.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrated that PYR functions as a PC for several {alpha}- and beta-mutants affecting Hex A (Figs. 6 and 8, Table 2). The {alpha}G269S mutant, the most prevalent mutation encountered in late-onset, adult GM2 gangliosidosis (TSD) (40, 41), showed significant response to PYR. An early pulse-chase study demonstrated that the majority of mutant {alpha}G269S precursors remain primarily as monomers in patients cells and are degraded. Only low level mature {alpha}-subunits were found in the lysosome where they were always associated with beta-subunits (35), confirming that dimerization is necessary for transport from the ER to lysosomes. It was later demonstrated that this apparent defect in association could be substantially overcome by overexpressing the mutant {alpha}-subunit along with the normal human beta-subunit in co-transfected COS cells, but the resulting Hex A was unstable at 37 °C. Additionally, when the same mutation was made in the aligned Gly of the more stable beta-subunit, there was little effect on the levels of expression of Hex B (22). Taken along with the new crystal structure of Hex A (2), these data indicate that the mutation destabilizes the folded {alpha}-monomer, accelerating its clearance by the ER-associated degradation pathway, which results in a diminished pool of {alpha}-monomers available for heterodimer formation. We also noted that, at high PYR concentrations, >0.1 mM, the inhibitory effects of the drug on Hex became evident, whereas at the same time, there was no effect on the activities of two other lysosomal enzymes (Fig. 4). This indicates that, when applied to patients, dosages must be carefully evaluated to maximize the PC activity of PYR while minimizing its inhibitory effect.

Another {alpha}-mutant, {alpha}IVS9 + 1G>A/IVS8-7G>A, showed a small, but significant increase in Hex A levels with PYR (Fig. 6B). Interestingly, in these cells, PYR treatment produced a higher relative level of Hex A activity than NGT, an effect that was not observed with {alpha}G269S mutants (Fig. 6A). This observation suggests that, in addition to missense mutations, some splice mutants may be treatable by PC therapy. Moreover, the {alpha}IVS9 + 1G>A mutation has been shown to produce no normal mRNA (42, 43), so all the residual Hex A activity in this cell line originates from the novel {alpha}IVS8-7G>A mutation we have identified. Because properly spliced mRNA from this allele would have the wild-type sequence, we concluded that stabilizing the pool of normal {alpha}-monomer in the ER, i.e. increasing the half-life and thus the concentration of {alpha}-monomers, leads to an increase in heterodimer formation. This model is also consistent with the previously published biochemical data on the in vivo effects of the {alpha}G269S mutant (see above) and the cross-linking studies we now report (Fig. 11A).

On other hand, the Hex A levels of other {alpha}-mutant cell lines from our collection, {alpha}R178H, {alpha}R499H (Fig. 7), and {alpha}R499C (data not shown), fail to be enhanced by PYR treatment. The {alpha}R178H mutation, i.e. the B1 variant affects an active site residue (Fig. 5) (33, 44) and would not be expected to respond. The {alpha}R499H and {alpha}R499C mutants were previously shown to be retained and form aggregates in ER (38, 45). This illustrates that PCs can be mutation-specific, regardless of the mutant proteins common degradation pathway.


Figure 8
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  		FIGURE 8.
PYR increases the enzyme activity and protein level of the {alpha}- and beta-subunits of Hex A in the betaR505Q/{Delta}16kb cell line. Histograms show relative activity enhancement of the lysate (total Hex A and S), as well as Hex A and S separated by IP ("Experimental Procedures"). Western blots of the samples treated with PYR and NGT also document an increase in protein levels of the mature, lysosomal {alpha}- and beta-subunits in the total lysate of treated cells. {alpha}/betap, indicates the {alpha}- and beta-subunit precursors; {alpha}m and betam indicate the mature (lysosomal) {alpha}- and beta-subunits, respectively. *, p < 0.05; **, p < 0.01.

 
Among the beta-mutants, the betaR505Q mutation is located at or near the subunit-subunit interface (Fig. 5) (3). When betaR505Q occurs with the beta{Delta}16kb mutation (cell line 36986), the highest relative increase of residual Hex A activity with PYR treatment was seen (Fig. 8). However, the betaR505Q/IVS11 + 5G>A mutant (number 32045) showed a smaller increase in both Hex A and S activities (Table 2). In addition, the fibroblast cell line from an affected sibling of this patient produced the same levels of enhancement (data not shown). Thus, the observed differences in response to PC treatment may not be solely due to the identity of the beta-mutations, but may also be influenced by other factors, e.g. components of the ER quality control system.

Treatment of the betaP504S/{Delta}16kb cell line with PYR resulted in a ~2-fold increase of Hex A activity. However, NGT produced an even higher level of enhancement in this cell line, 4.7-fold. The betaP504S mutation is unique in its effect on Hex A. It was shown to reduce the specific activity of the mutant Hex A toward its natural substrate by ~3-fold as compared with MUGS; thus explaining its apparently high residual MUGS activity (23). Interestingly, betaP504S is adjacent to betaR505Q, which is enhanced better by PYR than NGT. Both residues are near the subunit-subunit interface contained in domain II, which buries a surface area of 2694 Å2 in each monomer and forms a large groove into which the Activator-GM2 ganglioside complex can be docked. Pro504 introduces a kink into helix {alpha}8, which is required for proper packing of the helix against two loops that interact directly with the docked Activator (3). Arg505 forms a salt bridge and some hydrogen bonds, which are needed to stabilize the surfaces of the dimer interface once they are buried (2). Like the {alpha}G269S substitution, betaP504S and also betaR505Q generate a more heat labile form of mutant Hex A (46), which indicates that monomer stability also affects the stability of the dimer. Such mutations appear to respond well to PYR treatment.

The betaG353R mutation found in one of the beta-mutant cell lines (number 30037) is located in a very well conserved amino acid sequence, GGDE, found in all members of family 20 glycoside hydrolases (47). The Glu residue in this sequence is the catalytic acid group, and the Asp residue is involved in stabilizing the reaction intermediate (47). This cell line had betaIVS12-26G>A as the second allele, which was shown to result in inefficient splicing in another juvenile SD cell line, reported to produce ~3% of normal Hex A activity (48, 49). Thus, it is likely that the 3.5% of normal Hex A activity that we found in cells (Table 1) of the patient is the result of the splice junction mutation and not the missense mutation.


Figure 9
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  		FIGURE 9.
Cellular localization of the mutant beta-subunit-containing Hex isozymes in a patient fibroblast cell line, betaR505Q/IVS11 + 5G>A. Cells were grown in the presence of PYR (3.0 µg/ml; A-F) or the solvent (ETOH) alone (control; G-L). The images in panels D-F represent selected areas of the same cell seen in panels A-C, but with higher magnification to demonstrate co-localization of the beta-subunit of Hex (soluble protein in the lysosomal lumen) and Lamp-1 (integral lysosomal membrane protein) in lysosomes, versus the control cell, (G-I and J-L at higher magnification). Scale bars, red (10 µm) and magenta (2 µm).

 


Figure 10
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  		FIGURE 10.
Natural and artificial substrate activities are increased in a {alpha}G269S mutant TSD cell line grown in the presence of PYR (3 µg/ml). The level of Hex enhancement as measured by natural substrate, [3H]GM2 ganglioside and recombinant Activator (gray bars), hydrolysis was similar to that observed with the {alpha}-active site-specific artificial substrate, MUGS (white bars).

 


Figure 11
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  		FIGURE 11.
Effects of growth in PYR on the levels of monomeric and dimeric subunits of Hex. A, Western blots from a normal, an {alpha}-G296S/null, and a betaR505Q/{Delta}16kb mutant cell line grown with or without PYR in their media. B, log scale of the percentage of remaining Hex A activity from betaR505Q/{Delta}16kb mutant cell lysate exposed to PYR or solvent (ETOH) over a course of 30 min at 37 °C.

 
One interesting observation was the different responses of Hex A and Hex S to PC treatment (Table 2). In general, cells treated with PYR showed higher relative increases of Hex S, as compared with Hex A, again suggesting that PYR is more effective than NGT in stabilizing the wild type {alpha}-subunit. As a consequence, the {alpha}-subunit could then act as a chaperone for the mutant beta-subunit, rather than the other way around as with the wild-type subunits (35, 37).

One SD cell line was homozygous for a novel betaC137Y mutation in domain I (Fig. 5). This cell line responded to PYR treatment with a relatively high increase in residual Hex A activity. Because protein domains are believed to fold independently of each other (50-52), and domain II contains both the active site and the subunit-subunit interface, it is possible that the heterodimer can form without domain I. If this is the case, the increased stability of the dimer resulting from PC treatment might allow domain I additional time to achieve its proper conformation before being directed to ER-associated degradation pathway. Regardless of the mechanism, this is the first example of a PC appearing to enhance the folding of a domain adjacent to the domain containing its site of binding. This phenomenon was not observed in the betaT150P cell line that also had a novel domain I mutation in heterozygozity with the previously reported betaP417L (24, 26) (Fig. 5 and Table 2). In previous reports (24, 26), the betaP417L allele was present with the {Delta}16kb null allele and was shown to affect mRNA splicing. Thus, it conferred the residual Hex A activity we observed (24, 26). Testing the betaP417L/{Delta}16kb cell line with PYR or NGT resulted in similar enhancements (Table 2) as were achieved in the betaT150P/P417L cells. Thus, as for domain II mutants, not all domain I mutants can benefit from PC treatment.

PYR was originally developed as a dihydrofolate reductase inhibitor, which is used for treatment of parasitic diseases, including chloroquine-resistant malaria and toxoplasmosis (53, 54). PYR is an orally administered drug, with a well studied pharmacokinetic profile (55). Studies have shown that 12-26% of serum levels cross the blood-brain barrier (39). Thus to test PYR as a PC for mutant forms of Hex A, we used PYR concentrations corresponding to levels achieved in the nervous system by administration of routine therapeutic doses. This is a critical observation, as in late-onset GM2 gangliosidosis, along with many other lysosomal storage diseases, symptoms are principally related to the effect of the metabolic abnormality on the brain; the blood-brain barrier still remains the main obstacle for different types of emerging therapeutic interventions for these inherited metabolic diseases (12).

The potential clinical impact of the PYR acting as a PC may be estimated from the level of enhancement of the residual Hex A activity in tissues from the patients compared with the critical threshold approximation of 5-10% of normal (10). In Table 4, we calculate the % of residual Hex A based on the maximum increases that were achieved with either PYR or NGT treatment. Five mutant cell lines, {alpha}G269S/c1278insTACT, {alpha}G269S/IV6 + 1G>A, betaP504S/{Delta}16kb, betaR505Q/IVS11 + 5G>A, and betaR505Q/{Delta}16kb, showed increases in residual Hex A activity over the 10% critical threshold. Similar treatment of other cell lines significantly increased residual Hex A levels, but failed to produce levels over 10% of normal. Given the wide range of clinical phenotypes associated with a very narrow range of residual enzyme activities, even these smaller enhancements might be expected to be clinically relevant and slow the rate of progressive neurodeterioration in GM2 gangliosidosis. NGT, in general, performed better as a PC, but at much higher concentrations than PYR. However, whereas PYR is an FDA-approved drug with extensive data about its pharmacokinetics and safety, studies on the toxicity of NGT in animals are still in progress.


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  		TABLE 4
Fibroblast cell lines with relative increases of residual Hex A activity in the presence of PYR and NGT

 
In conclusion, PYR was identified, and shown to function as a PC for mutant cell lines from patients presenting with a late-onset form, i.e. juvenile or adult, of GM2 gangliosidosis. Our studies on the effects of PYR treatment on {alpha}- and beta-mutants have provided interesting insights into the independence of protein domains during the folding and subunit assembly stages of Hex in the ER. We have shown that screening of a library of FDA-approved drugs for competitive inhibitors of lysosomal enzymes is a feasible and practical approach to find potential PC for mutants causing devastating inherited metabolic disorders, such as GM2 gangliosidosis. The drugs represented in the library are already in current use for other medical purposes, indicating that they already passed extensive testing for toxicity, both in animals and humans. This considerably decreases the time and expense associated with conventional drug development, bringing potentially effective therapies to patients more rapidly. Clinical trials using such drugs could also provide a proof-of-principle for the concept of PC therapy, which would encourage investments into development and testing more optimized PC molecules.


   FOOTNOTES
 
* This work was supported in part by the Canadian Institutes of Health Research, the National Institutes of Health, and the Uger Estate and Life for Luke Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a donation from Amicus Therapeutics USA. Back

2 To whom correspondence should be addressed: Rm. 9146, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6161; Fax: 416-813-8700; E-mail: hex{at}sickkids.ca.

3 The abbreviations used are: GM2, GalNAcbeta4(Neu5Ac{alpha}3)Galbeta4Glc-ceramide; GM1, Galbeta3GalNAcbeta4(Neu5AcAc{alpha}3)Galbeta4Glc-ceramide; Activator, GM2 activator protein; CP, citrate phosphate buffer; DSP, dithiobis(succinimidylpropionate); FDA, Food Drug Administration; GalNAc, N-acetylgalactosamine; [3H]GM2, tritiated GM2 ganglioside; Hex, hexosaminidase; IP, immunoprecipitation; Lamp-1, lysosomal associated membrane protein-1; LYSO, lysosomal fraction; PC, pharmacological chaperone; PNS, postnuclear supernatant; PYR, pyrimethamine; MU, methylumbelliferone; MUbetaGal, 4-methylumbelliferyl-beta-D-galactopyranoside; MUG, 4-methylumbelliferyl-(2-acetamido-2-deoxy)-beta-D-glucopyranoside; MUGS, 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy)-beta-D-glucopyranoside; NGT, N-acetylglucosamine thiazoline; NINDS, National Institute of Neurological Disorders and Stroke; SD, Sandhoff disease; TGN, thioguanine; TSD, Tay-Sachs disease; ER, endoplasmic reticulum. Back

4 B. Rigat, M. Tropak, S. Withers, and D. J. Mahuran, unpublished data. Back

5 Tropak, M. B., Blanchard, J., Withers, S. G., Brown, E., and Mahuran, D. (2007) Chem. Biol., in press. Back


   ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of Amy Leung, Daphne Benedict, and Scott Bukovac. We thank Richard Bagshaw and Dr. John Callahan for helpful and critical suggestions. We thank Dr. Corien Verschuuren-Bemelmans from Groningen, Netherlands, who made available one of the cell lines studied. We also thank one of the patients, who donated his own cell line, banked by Dr. John O'Brien, Mayo Clinic, Rochester, MN.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hirabayashi, Y., Li, Y. T., and Li, S. C. (1983) J. Neurochem. 40, 168-175[CrossRef][Medline] [Order article via Infotrieve]
  2. Lemieux, M. J., Mark, B. L., Cherney, M. M., Withers, S. G., Mahuran, D. J., and James, M. N. (2006) J. Mol. Biol. 359, 913-929[CrossRef][Medline] [Order article via Infotrieve]
  3. Mark, B. L., Mahuran, D. J., Cherney, M. M., Zhao, D., Knapp, S., and James, M. N. (2003) J. Mol. Biol. 327, 1093-1109[CrossRef][Medline] [Order article via Infotrieve]
  4. Sharma, R., Bukovac, S., Callahan, J., and Mahuran, D. (2003) Biochim. Biophys. Acta 1637, 113-118[Medline] [Order article via Infotrieve]
  5. Hou, Y., Tse, R., and Mahuran, D. J. (1996) Biochemistry 35, 3963-3969[CrossRef][Medline] [Order article via Infotrieve]
  6. Gravel, R., Kaback, M., Proia, R., Sandhoff, K., Suzuki, K., and Suzuki, K. (2001) in The Metabolic and Molecular Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) 8th Ed., pp. 3827-3876, McGraw-Hill, New York
  7. Leinekugel, P., Michel, S., Conzelmann, E., and Sandhoff, K. (1992) Hum. Genet. 88, 513-523[Medline] [Order article via Infotrieve]
  8. Maegawa, G. H., Stockley, T., Tropak, M., Banwell, B., Blaser, S., Kok, F., Giugliani, R., Mahuran, D., and Clarke, J. T. (2006) Pediatrics 118, e1550-1562[Abstract/Free Full Text]
  9. Neudorfer, O., Pastores, G. M., Zeng, B. J., Gianutsos, J., Zaroff, C. M., and Kolodny, E. H. (2005) Genet. Med. 7, 119-123[Medline] [Order article via Infotrieve]
  10. Conzelmann, E., and Sandhoff, K. (1983) Dev. Neurosci. 6, 58-71[Medline] [Order article via Infotrieve]
  11. Sawkar, A. R., Cheng, W. C., Beutler, E., Wong, C. H., Balch, W. E., and Kelly, J. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15428-15433[Abstract/Free Full Text]
  12. Desnick, R. J., and Schuchman, E. H. (2002) Nat. Rev. Genet. 3, 954-966[CrossRef][Medline] [Order article via Infotrieve]
  13. Desnick, R. J. (2004) J. Inherit. Metab. Dis. 27, 385-410[Medline] [Order article via Infotrieve]
  14. Tropak, M. B., Reid, S. P., Guiral, M., Withers, S. G., and Mahuran, D. (2004) J. Biol. Chem. 279, 13478-13487[Abstract/Free Full Text]
  15. Tominaga, L., Ogawa, Y., Taniguchi, M., Ohno, K., Matsuda, J., Oshima, A., Suzuki, Y., and Nanba, E. (2001) Brain Dev. 23, 284-287[CrossRef][Medline] [Order article via Infotrieve]
  16. Fan, J. Q., Ishii, S., Asano, N., and Suzuki, Y. (1999) Nat. Med. 5, 112-115[CrossRef][Medline] [Order article via Infotrieve]
  17. Matsuda, J., Suzuki, O., Oshima, A., Yamamoto, Y., Noguchi, A., Takimoto, K., Itoh, M., Matsuzaki, Y., Yasuda, Y., Ogawa, S., Sakata, Y., Nanba, E., Higaki, K., Ogawa, Y., Tominaga, L., Ohno, K., Iwasaki, H., Watanabe, H., Brady, R. O., and Suzuki, Y. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15912-15917[Abstract/Free Full Text]
  18. Verkman, A. S., Lukacs, G. L., and Galietta, L. J. (2006) Curr. Pharm. Des. 12, 2235-2247[CrossRef][Medline] [Order article via Infotrieve]
  19. Arnold, L. A., Estebanez-Perpina, E., Togashi, M., Shelat, A., Ocasio, C. A., McReynolds, A. C., Nguyen, P., Baxter, J. D., Fletterick, R. J., Webb, P., and Guy, R. K. (2006) Sci STKE 2006, pl3[Abstract/Free Full Text]
  20. Maegawa, G. H. B., Stockley, T., Tropak, M., Banwell, B., Blaser, S., Kok, F., Giugliani, R., Mahuran, D., and Clarke, J. T. R. (2006) Pediatrics 118, e1550-e1562[Abstract/Free Full Text]
  21. Mahuran, D., and Lowden, J. A. (1980) Can. J. Biochem. 58, 287-294[CrossRef][Medline] [Order article via Infotrieve]
  22. Brown, C. A., and Mahuran, D. J. (1993) Am. J. Hum. Genet. 53, 497-508[Medline] [Order article via Infotrieve]
  23. Hou, Y., McInnes, B., Hinek, A., Karpati, G., and Mahuran, D. (1998) J. Biol. Chem. 273, 21386-21392[Abstract/Free Full Text]
  24. McInnes, B., Potier, M., Wakamatsu, N., Melancon, S. B., Klavins, M. H., Tsuji, S., and Mahuran, D. J. (1992) J. Clin. Investig. 90, 306-314[Medline] [Order article via Infotrieve]
  25. Sambrook, J., and David, W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  26. Wakamatsu, N., Kobayashi, H., Miyatake, T., and Tsuji, S. (1992) J. Biol. Chem. 267, 2406-2413[Abstract/Free Full Text]
  27. Sagherian, C., Poroszlay, S., Vavougios, G., and Mahuran, D. (1993) Biochem. Cell Biol. 71, 340-347[Medline] [Order article via Infotrieve]
  28. Sagherian, C., Thorner, P., and Mahuran, D. (1994) Biochem. Biophys. Res. Commun. 204, 135-141[CrossRef][Medline] [Order article via Infotrieve]
  29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  30. Rodriguez-Paris, J. M., Nolta, K. V., and Steck, T. L. (1993) J. Biol. Chem. 268, 9110-9116[Abstract/Free Full Text]
  31. Smiljanic-Georgijev, N., Rigat, B., Xie, B., Wang, W., and Mahuran, D. J. (1997) Biochim. Biophys. Acta 1339, 192-202[CrossRef][Medline] [Order article via Infotrieve]
  32. Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) J. Biomol. Screen 4, 67-73[Abstract/Free Full Text]
  33. Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318[Medline] [Order article via Infotrieve]
  34. Tanaka, A., Ohno, K., Sandhoff, K., Maire, I., Kolodny, E. H., Brown, A., and Suzuki, K. (1990) Am. J. Hum. Genet. 46, 329-339[Medline] [Order article via Infotrieve]
  35. d'Azzo, A., Proia, R. L., Kolodny, E. H., Kaback, M. M., and Neufeld, E. F. (1984) J. Biol. Chem. 259, 11070-11074[Abstract/Free Full Text]
  36. Hasilik, A., and Neufeld, E. F. (1980) J. Biol. Chem. 255, 4937-4945[Free Full Text]
  37. Proia, R. L., d'Azzo, A., and Neufeld, E. F. (1984) J. Biol. Chem. 259, 3350-3354[Abstract/Free Full Text]
  38. Paw, B. H., Moskowitz, S. M., Uhrhammer, N., Wright, N., Kaback, M. M., and Neufeld, E. F. (1990) J. Biol. Chem. 265, 9452-9457[Abstract/Free Full Text]
  39. Weiss, L. M., Harris, C., Berger, M., Tanowitz, H. B., and Wittner, M. (1988) J. Infect. Dis. 157, 580-583[Medline] [Order article via Infotrieve]
  40. Kaback, M. M. (2000) Eur. J. Pediatr. 159, Suppl. 3, S192-S195
  41. Navon, R., and Proia, R. L. (1989) Science 243, 1471-1474[Abstract/Free Full Text]
  42. Akerman, B. R., Zielenski, J., Triggs-Raine, B. L., Prence, E. M., Natowicz, M. R., Lim-Steele, J. S., Kaback, M. M., Mules, E. H., Thomas, G. H., Clarke, J. T., and Gravel, R. A. (1992) Hum. Mutat. 1, 303-309[CrossRef][Medline] [Order article via Infotrieve]
  43. Akli, S., Chomel, J. C., Lacorte, J. M., Bachner, L., Kahn, A., and Poenaru, L. (1993) Hum. Mol. Genet. 2, 61-67[Abstract/Free Full Text]
  44. Kytzia, H. J., and Sandhoff, K. (1985) J. Biol. Chem. 260, 7568-7572[Abstract/Free Full Text]
  45. Mules, E. H., Hayflick, S., Dowling, C. E., Kelly, T. E., Akerman, B. R., Gravel, R. A., and Thomas, G. H. (1992) Hum. Mutat. 1, 298-302[CrossRef][Medline] [Order article via Infotrieve]
  46. Bolhuis, P. A., Ponne, N. J., Bikker, H., Baas, F., and Vianney de Jong, J. M. (1993) Biochim. Biophys. Acta 1182, 142-146[Medline] [Order article via Infotrieve]
  47. Henrissat, B., and Bairoch, A. (1993) Biochem. J. 293, 781-788
  48. Neote, K., McInnes, B., Mahuran, D. J., and Gravel, R. A. (1990) J. Clin. Investig. 86, 1524-1531[Medline] [Order article via Infotrieve]
  49. Nakano, T., and Suzuki, K. (1989) J. Biol. Chem. 264, 5155-5158[Abstract/Free Full Text]
  50. Murzin, A. G., Brenner, S. E., Hubbard, T., and Chothia, C. (1995) J. Mol. Biol. 247, 536-540[CrossRef][Medline] [Order article via Infotrieve]
  51. Apic, G., Gough, J., and Teichmann, S. A. (2001) Bioinformatics 17, Suppl. 1, S83-S89[Abstract]
  52. Teichmann, S. A., Rison, S. C., Thornton, J. M., Riley, M., Gough, J., and Chothia, C. (2001) Trends Biotechnol. 19, 482-486[CrossRef][Medline] [Order article via Infotrieve]
  53. Leport, C., Chene, G., Morlat, P., Luft, B. J., Rousseau, F., Pueyo, S., Hafner, R., Miro, J., Aubertin, J., Salamon, R., and Vilde, J. L. (1996) J. Infect. Dis. 173, 91-97[Medline] [Order article via Infotrieve]
  54. Weiss, L. M., Luft, B. J., Tanowitz, H. B., and Wittner, M. (1992) Am. J. Trop. Med. Hyg. 46, 288-291[Abstract/Free Full Text]
  55. Buxton, I. L. O. (2006) in Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11 Ed., McGraw-Hill Books, New York

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