Pharmacological Enhancement of β-Hexosaminidase Activity in Fibroblasts from Adult Tay-Sachs and Sandhoff Patients
2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês
10.1074/jbc.m308523200
ISSN1083-351X
AutoresMichael B. Tropak, Stephen P. Reid, Marianne Guiral, Stephen G. Withers, Don J. Mahuran,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoTay-Sachs and Sandhoff diseases are lysosomal storage disorders that result from an inherited deficiency of β-hexosaminidase A (αβ). Whereas the acute forms are associated with a total absence of hexosaminidase A and early death, the chronic adult forms exist with activity and protein levels of ∼5%, and unaffected individuals have been found with only 10% of normal levels. Surprisingly, almost all disease-associated missense mutations do not affect the active site of the enzyme but, rather, inhibit its ability to obtain and/or retain its native fold in the endoplasmic reticulum, resulting in its retention and accelerated degradation. By growing adult Tay-Sachs fibroblasts in culture medium containing known inhibitors of hexosaminidase we have raised the residual protein and activity levels of intralysosomal hexosaminidase A well above the critical 10% of normal levels. A similar effect was observed in fibroblasts from an adult Sandhoff patient. We propose that these hexosaminidase inhibitors function as pharmacological chaperones, enhancing the stability of the native conformation of the enzyme, increasing the amount of hexosaminidase A capable of exiting the endoplasmic reticulum for transport to the lysosome. Therefore, pharmacological chaperones could provide a novel approach to the treatment of adult Tay-Sachs and possibly Sandhoff diseases. Tay-Sachs and Sandhoff diseases are lysosomal storage disorders that result from an inherited deficiency of β-hexosaminidase A (αβ). Whereas the acute forms are associated with a total absence of hexosaminidase A and early death, the chronic adult forms exist with activity and protein levels of ∼5%, and unaffected individuals have been found with only 10% of normal levels. Surprisingly, almost all disease-associated missense mutations do not affect the active site of the enzyme but, rather, inhibit its ability to obtain and/or retain its native fold in the endoplasmic reticulum, resulting in its retention and accelerated degradation. By growing adult Tay-Sachs fibroblasts in culture medium containing known inhibitors of hexosaminidase we have raised the residual protein and activity levels of intralysosomal hexosaminidase A well above the critical 10% of normal levels. A similar effect was observed in fibroblasts from an adult Sandhoff patient. We propose that these hexosaminidase inhibitors function as pharmacological chaperones, enhancing the stability of the native conformation of the enzyme, increasing the amount of hexosaminidase A capable of exiting the endoplasmic reticulum for transport to the lysosome. Therefore, pharmacological chaperones could provide a novel approach to the treatment of adult Tay-Sachs and possibly Sandhoff diseases. GM2 1The abbreviations used are: GM2, N-AcGalβ1,4(NeuAcα2,3)Galβ1,4 Glc-ceramide; TSD, Tay-Sachs disease; ATSD, adult form of Tay-Sachs disease; ITSD, infantile form of Tay-Sachs disease; SD, Sandhoff disease; ISD, infantile form of Sandhoff disease; ASD, adult form of Sandhoff disease; Hex, lysosomal β-N-acetylhexosaminidase; MUG, 4-methylumbelliferyl-7-(2-acetamido-2-deoxy)-β-d-glucopyranoside; MUGS, 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy)-β-d-glucopyranoside; MUbGlr, 4-methylumbelliferyl-β-d-glucuronide; MUbGal, 4-methylumbelliferyl-β-d-galactopyranoside; MUP, 4-methylumbelliferyl phosphate; MU, 4-methylumbelliferone; ER, endoplasmic reticulum; GalNAc, N-acetylgalactosamine; AddNJ, 2-acetamido-1,2-dideoxynojirimycin; AdNJ, 2-acetamido-2-deoxynojirimycin; ACAS, 6-acetamido-6-deoxycastanospermine; DNJ, deoxynojirimycin; NB-DNJ, n-butyl-DNJ; NGT, N-acetylglucosamine-thiazoline; WT, wild type; CAE, cellulose acetate electrophoresis; PBS, phosphate-buffered saline; CP, citrate/phosphate; HB, homogenization buffer; PNS, postnuclear supernatant; α-MEM, α-minimal essential medium. gangliosidosis, arising from the neuronal storage of GM2 ganglioside (GM2), occurs in three variants; Tay-Sachs disease (TSD), Sandhoff disease (SD) and the AB variant. The former two result from mutations in the evolutionarily related HEXA or HEXB genes, encoding the α or β subunits of heterodimeric β-N-acetylhexosaminidase A (Hex A, αβ), respectively (reviewed in Ref. 1Gravel R.A. Clarke J.T.R. Kaback M.M. Mahuran D. Sandhoff K. Suzuki K. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. 2. McGraw-Hill, New York1995: 2839-2879Google Scholar). Two other homodimeric Hex isozymes exist, i.e. Hex B (ββ) and Hex S (αα). Each isozyme has its own characteristic pI and stability with Hex B being both the most stable and basic (pI = 6.9), followed by less stable Hex A (pI = 4.8), and unstable Hex S (pI ∼ 3). In normal human tissue only Hex A and B are readily detectable (1Gravel R.A. Clarke J.T.R. Kaback M.M. Mahuran D. Sandhoff K. Suzuki K. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. 2. McGraw-Hill, New York1995: 2839-2879Google Scholar). Although each subunit contains an active site, dimerization is required for enzymatic activity (2Mark B.L. Mahuran D.J. Cherney M.M. Zhao D. Knapp S. James M.N. J. Mol. Biol. 2003; 327: 1093-1109Google Scholar). However, in the lysosome only Hex A, along with its substrate-specific cofactor the GM2 activator protein, can hydrolyze the terminal, β-linked N-acetylgalactosamine (GalNAc) residue from GM2 ganglioside. Whereas both the α- and/or β-active sites of dimeric Hex can hydrolyze synthetic N-acetylhexosamine terminal substrates, only the α-sites of Hex A and S can efficiently utilize negatively charged substrates, e.g. 6-sulfated GlcNAc (2Mark B.L. Mahuran D.J. Cherney M.M. Zhao D. Knapp S. James M.N. J. Mol. Biol. 2003; 327: 1093-1109Google Scholar, 3Hou Y. Tse R. Mahuran D.J. Biochemistry. 1996; 35: 3963-3969Google Scholar). Therefore, total Hex activity is measured using 4-methylumbelliferyl-β-N-acetylglucosamine (MUG), whereas, 4-methylumbelliferyl-β-N-acetylglucosamine-6-sulfate (MUGS) is used to measure Hex A and Hex S activity. At pH 4.2 the relative MUG/MUGS hydrolysis ratios of the three isozymes are: 150–300/1 for Hex B, 3–4/1 for Hex A, and 1–1.5/1 for Hex S (3Hou Y. Tse R. Mahuran D.J. Biochemistry. 1996; 35: 3963-3969Google Scholar). The more common infantile TSD (ITSD) variant of GM2 gangliosidosis results from absent α subunits and elevated amounts of Hex B such that near normal levels of total Hex activity (MUG) are retained. Less common is acute, infantile SD (ISD), resulting from an absence of β-subunits. Interestingly, despite normal levels of α-mRNA, only 2–4% residual Hex activity, from Hex S, can be detected in ISD patient samples. In contrast to the infantile forms, adult TSD (ATSD) and SD (ASD) are chronic, slowly progressive, neurodegenerative diseases. In many cases these are associated with missense mutations, usually producing thermolabile Hex A with residual activity (MUGS) and protein levels that are 2% of normal. The majority of patients with ATSD possess a missense mutation in exon 7, αG269S (1Gravel R.A. Clarke J.T.R. Kaback M.M. Mahuran D. Sandhoff K. Suzuki K. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. 2. McGraw-Hill, New York1995: 2839-2879Google Scholar). This and similar point mutations do not directly affect the active site of Hex (2Mark B.L. Mahuran D.J. Cherney M.M. Zhao D. Knapp S. James M.N. J. Mol. Biol. 2003; 327: 1093-1109Google Scholar) but are believed to result in increased amounts of misfolded protein in the endoplasmic reticulum (ER), which in turn are retained by its quality control system and degraded (4Mahuran D.J. Biochim. Biophys. Acta. 1991; 1096: 87-94Google Scholar). Only a low level of residual Hex A activity is needed to ameliorate the clinical phenotype in ITSD or ISD, and asymptomatic individuals have been identified with residual activities of ≥10% of normal (5Dlott B. D'Azzo A. Quon D.V.K. Neufeld E.F. J. Biol. Chem. 1990; 265: 17921-17927Google Scholar, 6Cao Z. Petroulakis E. Salo T. Triggs-Raine B. J. Biol. Chem. 1997; 272: 14975-14982Google Scholar). Sandhoff and colleagues (7Conzelmann E. Sandhoff K. Dev. Neurosci. 1984; 6: 58-71Google Scholar) have estimated that 5–10% of normal Hex A levels represents a “critical threshold” for disease. Currently, there is no therapy for any form of GM2 gangliosidosis. Strategies involving bone marrow transplantation and enzyme replacement therapy are unlikely to be successful due to neuronal storage and the inaccessibility imposed by the blood-brain barrier. Substrate deprivation therapy, although feasible, is limited by specificity and toxicity issues. Pharmacological chaperones, small molecules that function either as antagonists (receptors) or competitive inhibitors (enzymes), have been used to “rescue” several misfolded proteins (reviewed in Refs. 8Fan J.Q. Trends Pharmacol. Sci. 2003; 24: 355-360Google Scholar and 9Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Google Scholar) from ER-associated degradation. They apparently act by increasing the stability of the newly synthesized, mutant protein, allowing more of it to exit the ER for transport to its site of action, e.g. lysosome. Because only a modest increase in residual Hex A activity is needed for ATSD and ASD patients to achieve the ∼10% critical threshold, these diseases are candidates for pharmacological chaperone-based therapy. In this report, we evaluate a panel of known Hex inhibitors (see Table I) as potential pharmacological chaperones. We show that the Hex inhibitors can increase levels of lysosomal Hex A activity to 35% of normal levels in fibroblasts from an αG269S homozygous ATSD patient. Additionally, the effects of these compounds on ASD, ISD, and ITSD fibroblasts were examined.Table IList of compounds evaluated for enhancing Hex A activity1 Value determined using human placental HEX.2 Value determined using Jack Bean Hex. Open table in a new tab 1 Value determined using human placental HEX. 2 Value determined using Jack Bean Hex. Reagents—The following fluorogenic substrates, all purchased from Sigma, methylumbelliferyl-β-d-glucuronide (MUbGlr), 4-methylumbelliferyl-β-d-galactopyranoside (MUbGal), 4-methylumbelliferyl phosphate (MUP), MUG, and MUGS were used to assay the lysosomal enzymes β-glucuronidase, β-galactosidase, acid phosphatase, Hex A/B/S, and Hex A/S, respectively. Rabbit polyclonal antibodies against Human Hex A and β-glucosidase/glucocerebrosidase were prepared in our laboratory and have been previously described (10Brown C.A. Mahuran D.J. Am. J. Hum. Genet. 1993; 53: 497-508Google Scholar). Donkey polyclonal antibody developed against a C-terminal calnexin peptide was purchased from Santa Cruz Biotechnology. Castanospermine, deoxynojirimycin (DNJ), 6-acetamido-6-deoxycastanospermine (ACAS) (IRL, New Zealand), 2-acetamido-1,2-dideoxynojirimycin (AddNJ) (TRC, Canada), GalNAc (Sigma) were commercially available; whereas 2-acetamido deoxynojirimycin (AdNJ) and N-acetylglucosamine thiazoline (NGT) were synthesized and purified according to Kappes and Legler (11Kappes E. Legler G. J. Carbohydr. Chem. 1989; 8: 371-388Google Scholar) and Knapp et al. (12Knapp S. Vocadlo D. Gao Z.N. Kirk B. Lou J.P. Withers S.G. J. Am. Chem. Soc. 1996; 118: 6804-6805Google Scholar), respectively. All compounds were dissolved in water and used as 10–25 mg/ml solutions. Steel wool #0000 (International Steel Wool, Mexico), FeCl2 and FeCl3 (Sigma), and dextran T4000 (Amersham Biosciences, UK) were used to prepare lysosomes by magnetic chromatography. Cell Lines—The following fibroblast cell lines were used: 4212 (from hereon referred to as wild type (WT) or unaffected) was from an unaffected individual; 1766 (ATSD) was from a ∼40-year-old female patient diagnosed with the chronic (adult) form of TSD (kindly provided by Dr. J. R. Donat, University of Saskatchewan, Kinsmen Children's Centre, Saskatoon, Saskatchewan, Canada) and found to be homozygous for the mutation 805G→A/805G→A (αG269S) in exon 7 of HEXA (Molecular Diagnostics Laboratory, Hospital For Sick Children, Toronto, Ontario, Canada); 2317 (ITSD) was from a female fetus with the acute (infantile) form of TSD homozygous for a 4-bp insertion mutation 1278insTATC in exon 11 of HEXA (Molecular Diagnostics Laboratory); 3577 (ASD), 3577 was from a ∼30-year-old female heterozygous for a βP504 S missense mutation and a 16-kb 5′ HEXB deletion mutation with an adult Sandhoff phenotype (13Rubin M. Karpati G. Wolfe L.S. Carpenter S. Klavins M.H. Mahuran D.J. J. Neurol. Sci. 1988; 87: 103-119Google Scholar, 14Hou Y. McInnes B. Hinek A. Karpati G. Mahuran D. J. Biol. Chem. 1998; 273: 21386-21392Google Scholar); and 294 (ISD) was from an infant female, homozygous for the 16-kb HEXB deletion mutation with infantile Sandhoff disease (15O'Dowd B. Klavins M. Willard H. Lowden J.A. Gravel R.A. Mahuran D. Freysz L. Dreyfus H. Massarelli R. Gatt S. Enzymes of Lipid Metabolism II. Plenum Publishing Co., New York1986: 779-784Google Scholar, 16Neote K. Brown C.A. Mahuran D.J. Gravel R.A. J. Biol. Chem. 1990; 265: 20799-20806Google Scholar). All cell lines were grown in media containing α-MEM (Invitrogen) supplemented with 10% fetal calf serum (Sigma) and antibiotics penicillin/streptomycin (Invitrogen) at 37 °C in a humidified CO2 incubator. Enzyme Kinetics—Cells were grown in 96-well tissue culture plates (Falcon). Trypsinized cells were diluted to give 50% confluence when plated, and equal numbers of cells were aliquoted into each well of the plate. Cells grown for longer than 5 days were supplemented with fresh media. Compounds were diluted in media, filter-sterilized (Millipore), and evaluated in triplicate, i.e. three different wells. Following 3–7 days of treatment, intracellular Hex A activities were determined. Media was removed, cells were washed twice with phosphate-buffered saline (PBS), and lysed using 60 μl of 10 mm citrate/phosphate (CP; pH 4.2) buffer (CP buffer), containing 0.5% human serum albumin and 0.5% Triton X-100. Aliquots of the lysates were transferred to a 96-well plate, and Hex A activity was measured using 25 μl of 3.2 mm MUGS in CP buffer, with incubation at 37 °C for 1 h. The reaction was stopped by the addition of 200 μl of 0.1 m 2-amino-2-methyl-1-propanol, pH 10.5. Fluorescence was measured using an excitation wavelength of 365 nm and emission wavelength of 450 nm as previously described (14Hou Y. McInnes B. Hinek A. Karpati G. Mahuran D. J. Biol. Chem. 1998; 273: 21386-21392Google Scholar). For experiments in Figs. 2 and 5, 6, 7, the relative increase in MUGS activity was expressed as the average fluorescence reading from three wells of cells (n = 3) grown in the presence of compound, divided by the average fluorescence reading from three wells of cells (n = 3), grown for the same length of time, in the absence of any compound. For untreated cells, fluorescence readings for the individual wells varied by less than 20%. To measure total Hex activity and the activities of the other lysosomal enzymes, the substrates MUG (3.2 mm), MUP (3 mg/ml), MUbGal (0.56 mm) and MUbGlr (2.33 mm) were dissolved in CP buffer and used as described for MUGS.Fig. 5Kinetics of Hex A activity enhancement. a, MUGS activity in ATSD fibroblasts grown in the presence of three different concentrations of NGT over a 9-day period. Shown is the relative increase in MU fluorescence from MUGS hydrolysis (MU from treated/MU from untreated cells grown for the same length of time) in lysates from ATSD fibroblasts grown in different concentrations of NGT (corresponding symbols shown in the left-hand corner of the graph) for increasing periods of time (days). Each MUGS assay was further normalized using the acid phosphatase activity (MUP) of the same lysate. b, demonstrates that MUP hydrolysis is a valid means of normalization, because there were no changes in relative acid phosphatase activity (MUP) in another set of paired treated/untreated cell lysates over the same 9-day period. For each of the graphs, standard deviations are shown above the data points (average MU fluorescence from three wells of treated cells/three wells of untreated cells grown for the same period of time). Dashed lines denote the position at which there is no change in MU fluorescence, i.e. relative increase = 1.View Large Image Figure ViewerDownload (PPT)Fig. 6Hex A activity persists at elevated levels for several days following removal of the inhibitors from the growth media. All panels show the relative increase in MUGS hydrolysis (MU fluorescence, see legend to Fig. 5) by Hex in lysates from ATSD fibroblasts grown in different concentrations of NGT (corresponding symbols shown in the upper left corners) for increasing periods of time (days). Initially all fibroblasts were grown in the presence of NGT (a) or ACAS (b) for 6 days. Afterward fibroblasts continued to be grown in the presence of the drug (filled symbols) or in the absence of the drugs (open symbols) following removal of the original drug containing media on the sixth day. For each of the graphs, standard deviations are shown above the data points (average time-paired values from three wells each of treated/untreated cells). Dashed lines denote the position at which there is no change in MU fluorescence, i.e. relative increase = 1. The titles to the left and bottom of all panels describe the y- and x-axis of all graphs in the figure.View Large Image Figure ViewerDownload (PPT)Fig. 7Effect of NGT treatment on Hex activity in fibroblasts from an unaffected individual and from patients with different forms of TSD and SD. Fibroblasts from an unaffected individual (a), and patients with; ITSD (b), ISD (c), ATSD (d), or ASD (e) were grown in the presence of 4 (a–d) or 3 (e) different concentrations (mm) of NGT for 5 (a–d) or 6 days (e). The relative changes in MU fluorescence due to MUG (filled diamonds) or MUGS (filled squares) hydrolysis are shown (left-most y-axis). Each datum point (average time-paired values from four wells each of treated/untreated cells, i.e. 1 = no change) is shown along with its corresponding standard deviation. Also shown on the graphs is the MUG/MUGS ratio in treated cells (open triangles) and in untreated cells as a single open circle to the left of the other data points (the x-axis does not apply to these points), and the MUG/MUGS ratios for treated cell lysates are given on the right-most axis.View Large Image Figure ViewerDownload (PPT) For Western blot analysis and cellulose acetate electrophoresis (CAE), ATSD fibroblasts were grown for 5–6 days in 6-well plates (Falcon, 40 mm2) using 1.5 ml of medium with or without the compounds to be evaluated. Subsequently, media were removed, and cells were washed twice in PBS and finally scraped into 1 ml of PBS. Following pelleting by microcentrifugation, the cells were resuspended in 10 mm sodium phosphate buffer, pH 6.1, containing 5% glycerol and disrupted by sonication on ice. Cleared lysates were prepared by microcentrifugation for 15 min at 4 °C, and the total protein concentration was determined using BCA protein assay (Pierce) according to manufacturer's instructions. Hex A activities were determined using MUGS substrate at 37 °C for 1 h as described above and expressed as nanomoles of 4-methylumbelliferone (MU) released/h/mg of total protein. Western Blotting—Lysates (5 μg of total protein) were subjected to SDS-PAGE on a 10% bis:acrylamide gel, transferred to nitrocellulose, and processed as previously described (14Hou Y. McInnes B. Hinek A. Karpati G. Mahuran D. J. Biol. Chem. 1998; 273: 21386-21392Google Scholar). Blots were developed using chemiluminescent substrate according to the manufacturer's protocol (Amersham Biosciences, UK) and recorded on BIOMAX x-ray film (Kodak). Blots were probed multiple times following stripping of bound antibody following treatment with RESTORE buffer (Pierce). Cellulose Acetate Electrophoresis—To separate and directly visualize the active Hex isozymes, Hex A (pI = 4.8, αβ), B (pI = 6.9, ββ), and S (pI∼ 3, αα), CAE was performed according to Ref. 17Hoeksema H.L. Reuser A.J. Hoogeveen A. Westerveld A. Braidman I. Robinson D. Am. J. Hum. Genet. 1977; 29: 14-23Google Scholar with modifications. Briefly, 2 μg of lysates protein was spotted onto Sepraphore (Gelman) strips (pre-wetted in 20 mm sodium phosphate buffer, pH 7.0), partially dried, and resolved electrophoretically at 10 mA for 20 min. Strips were overlaid with another cellulose acetate strip soaked in CP buffer containing 3.2 mm MUG, wrapped in plastic wrap, and incubated for 1 h at 37 °C. Subsequently, strips were briefly exposed to ammonium hydroxide vapor, and visualized bands were photographed under UV light (340 nm). Heat Inactivation Assay—Heat inactivation experiments were performed using partially purified Hex A from unaffected or ATSD fibroblasts. Lysates from fibroblasts were prepared in 10 mm sodium phosphate buffer, pH 6.1, 5% glycerol after sonication. DEAE columns were pre-equilibrated with 10 mm sodium phosphate buffer, pH 6.1 (no salt). Lysates were applied, and the column was washed with 10 volumes of 20 mm sodium chloride in 10 mm sodium phosphate buffer, pH 6.1, to elute Hex B. Hex A was eluted using the same buffer containing 100 mm sodium chloride. For heat inactivation experiments equal amounts of total protein (0.1–0.2 μg) from WT and mutant Hex A fractions were diluted in CP buffer, pH 4.2, containing 0.5% human serum albumin. Prior to incubation at 42 °C, diluted samples of the mutant Hex A enzyme lacking or containing Hex inhibitors were left on ice for 15 min. Following incubation of the WT and mutant Hex A enzymes at 42 °C for the requisite time, the heat-treated enzyme was then held on ice until completion of the time series. Time zero time points correspond to samples that were not heat-treated and kept on ice for the duration of the heat inactivation experiment. The heat-treated samples were equilibrated to 37 °C for 10 min, followed by addition of MUGS substrate and incubation at 37 °C for a further 30 min. Purification of Iron-dextran-labeled Lysosomes by Magnetic Chromatography—A lysosomal fraction was prepared from ATSD fibroblasts grown for 6 days in media lacking or containing 250 μg/ml (0.9 mm) of NGT, using a modification of a previously described procedure (18Diettrich O. Mills K. Johnson A.W. Hasilik A. Winchester B.G. FEBS Lett. 1998; 441: 369-372Google Scholar). Briefly, cells from one 150-mm tissue culture plate were incubated for 9 h at 37 °C in an iron-dextran solution (prepared as previously described (18Diettrich O. Mills K. Johnson A.W. Hasilik A. Winchester B.G. FEBS Lett. 1998; 441: 369-372Google Scholar)) containing complete α-MEM (with or without NGT). To chase the iron-dextran into lysosomes, cells were washed twice in PBS and incubated for a further 16 h in complete α-MEM media lacking or containing NGT. Subsequently, cells were trypsinized, pelleted at 100 × g, and then sequentially washed in PBS followed by homogenization buffer (HB), consisting of 4 mm imidazole, pH 7.4, and 0.25 m sucrose. To prepare a postnuclear supernatant (PNS) fraction, the cell pellet was resuspended in HB, homogenized with 10 strokes of a tight fitting Dounce homogenizer and finally centrifuged for 10 min. at 1000 × g. For the steel wool column, a 1-ml syringe fitted with a 23-gauge needle was packed with 60 mg of steel wool and held in place by a rare earth magnet. Prior to use, the column was washed with HB, loaded with the PNS, and then the column was washed with 10 column volumes of HB. To elute the iron-dextran-loaded lysosomes bound to the steel wool, the column was removed from the magnet and loaded with 10 mm sodium phosphate buffer, pH 6, containing 5 mm EDTA and 0.4% Triton X-100. Fractions were collected, and those containing increased MUG activity (“lysosomes”) were identified and stored frozen at –80 °C. Competitive Inhibitors of Hex Attenuate Heat Inactivation of WT and ATSD Mutant Hex A—To assess the chaperoning potential of the inhibitors listed in Table I, the compounds were tested initially for their ability to attenuate heat inactivation of the ATSD mutant Hex A at 42 °C. Greater than 50% of the activity of partially purified Hex A from ATSD fibroblasts (Fig. 1a) was lost after 30 min, as compared with WT fibroblast Hex A whose activity was not significantly reduced. Attenuation of heat inactivation was observed when mutant Hex A was incubated at 42 °C in the presence of GalNAc (Fig. 1b), ACAS (Fig. 1c), and NGT (Fig. 1d) at concentrations that were previously determined to result in a 25–75% reduction in Hex A activity. The degree to which heat inactivation of mutant Hex A was attenuated was directly proportional to the concentration of the inhibitor included during treatment. With a longer period of heating, 60–90 min, similar protective effects of the three compounds were seen with purified placental Hex A (data not shown). ATSD Fibroblasts Treated with Hex Inhibitors Show Increased MUGS Activity—In fibroblasts from the ATSD patient, MUGS activity was found to be ∼10% of normal. When cells were grown for 5 days in the presence of GalNAc, AddNJ, AdNJ, ACAS, or NGT, increased hydrolysis of MUGS was observed (Fig. 2). This effect was dose-dependent, but was limited by the toxicity of GalNAc and ACAS, both of which reduced cell viability above concentrations of 200 and 0.1 mm, respectively. The decline in effectiveness with decreasing concentration of inhibitors was greatest for GalNAc and least for ACAS, which was still effective in enhancing MUGS activity even at concentrations of 5 μm. These results also demonstrated that Hex inhibitors, which attenuated heat inactivation of mutant enzyme, also enhanced Hex A activity in ATSD fibroblasts. Furthermore, because the effective concentration at which the inhibitors enhance MUGS activity correlates with their Ki values (Table I), the observed increases are most likely due to the specific binding of the compounds to the α-active site of Hex A. Two compounds, DNJ and castanospermine, which lacked the 2-acetamido group, did not increase MUGS hydrolysis and were used as negative controls. Treatment of ATSD Fibroblasts Results in Increased Levels of the Lysosomally Processed (Mature) α-Subunit and the Hex A Heterodimer—To directly show that treatment of ATSD fibroblasts with the inhibitory compounds results in increased amounts of the α-subunit in the lysosome, cell lysates were subjected to Western blotting with an anti-Hex A antibody (Fig. 3a). In comparison to untreated cells, increased amounts of a 56-kDa band corresponding to lysosomally processed α-subunit (αm) were seen in cells treated with AddNJ, GalNAc, NGT, or ACAS. The corresponding band was also seen in WT fibroblasts, but not in ITSD fibroblast cells (which do not express the α-subunit). With the exception of cells treated with DNJ, the bands at 28 kDa, corresponding to lysosomally processed β-subunit of Hex (βm), remained largely unaffected by the treatments. As judged from densitometry, the increased levels of these bands (Fig. 3c) closely paralleled the increase in specific MUGS activity from lysates of inhibitor-treated ATSD cells. Of these, ATSD cells treated with 0.9 mm NGT showed the greatest increase in specific MUGS activity (∼3-fold) and levels of mature α-subunit. To rule out the unlikely possibility that the observed increased MUGS hydrolysis in treated cells was solely due to Hex S, the different Hex isozymes were resolved using cellulose acetate electrophoresis combined with MUG zymography (Fig. 3b). Increased amounts of a band that co-migrated with purified placental Hex A, were observed in inhibitor treated cell lysates, but could not be detected in untreated ATSD cells. Hex S (pI ∼ 3.5), which migrates faster than Hex A (pI = 4.8), could not be detected. The increase in α-subunits (Fig. 3a) and the Hex A isozyme (Fig. 3b) paralleled the increase in MUGS hydrolysis (Fig. 3c) indicating that the increased MUGS activity was a direct measure of increased Hex A in ATSD cells. Hex A Activity in NGT-treated ATSD Fibroblasts Is Enriched in Lysosomes—To demonstrate directly that the increased Hex A (MUGS) activity is found in lysosomes, an ER-free lysosomal enriched fraction was prepared by magnetic chromatography of the crude lysates from untreated and NGT-treated ATSD fibroblasts loaded with iron-dextran colloid (18Diettrich O. Mills K. Johnson A.W. Hasilik A. Winchester B.G. FEBS Lett. 1998; 441: 369-372Google Scholar, 19Glombitza G.J. Becker E. Kaiser H.W. Sandhoff K. J. Biol. Chem. 1997; 272: 5199-5207Google Scholar). An ∼10-fold enrichment in Hex A-specific activity was observed in the lysosomal fraction relative to the postnuclear supernatant (Fig. 4a). NGT treatment resulted in a greater than 6-fold increase in intracellular, lysosomal Hex A-specific activity relative to untreated cells. A similar increase in mature α-subunit protein levels was seen in the PNS and lysosomal fractions of NGT-treated cells (Fig. 4b). Although a Hex α-subunit-specific band is observed in the PNS from untreated cells, it corresponds to the α-chain precursor, because it is not found in the lysosomal fraction (Fig. 4b). Both glucocerebrosidase (another lysosomal enzyme) and the β-subunit of Hex are enriched in the lysosomal fraction relative to PNS, confirming the enrichment of lysosomal organelles in the “lysosome” fraction from both treated and untreated cells. Furthermore, these lysosomal-enriched fractions did not
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