Conceived and designed the experiments: MF SD AVP. Performed the experiments: MF SD. Analyzed the data: MF SD AVP. Wrote the paper: MF SD AVP.
The authors have declared that no competing interests exist.
Mucopolysaccharidosis type IIIC or Sanfilippo syndrome type C (MPS IIIC, MIM #252930) is an autosomal recessive disorder caused by deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT, EC 2.3.1.78), which catalyses transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by α-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death causing neurodegeneration and is accompanied by mild visceral and skeletal abnormalities, including coarse facies and joint stiffness. Surprisingly, the majority of MPS IIIC patients carrying missense mutations are as severely affected as those with splicing errors, frame shifts or nonsense mutations resulting in the complete absence of HGSNAT protein.
In order to understand the effects of the missense mutations in HGSNAT on its enzymatic activity and biogenesis, we have expressed 21 mutant proteins in cultured human fibroblasts and COS-7 cells and studied their folding, targeting and activity. We found that 17 of the 21 missense mutations in HGSNAT caused misfolding of the enzyme, which is abnormally glycosylated and not targeted to the lysosome, but retained in the endoplasmic reticulum. The other 4 mutants represented rare polymorphisms which had no effect on the activity, processing and targeting of the enzyme. Treatment of patient cells with a competitive HGSNAT inhibitor, glucosamine, partially rescued several of the expressed mutants.
Altogether our data provide an explanation for the severity of MPS IIIC and suggest that search for pharmaceutical chaperones can in the future result in therapeutic options for this disease.
Mucopolysaccharidosis III (also called Sanfilippo syndrome) is an autosomal recessive disease caused by lysosomal accumulation of heparan sulfate
Although from the moment of discovery MPS IIIC was recognized as a deficiency of an enzyme that transfers an acetyl group from cytoplasmically derived acetyl-CoA to terminal N-glucosamine residues of heparan sulfate within the lysosomes
The effect of HGSNAT mutations on the enzyme biogenesis and catalytic activity was studied by the transient expression of the mutant cDNA. Mutations were generated by site directed mutagenesis in the pCTAP-HGSNAT construct that expresses human HGSNAT with a C-terminal tandem affinity purification (TAP) tag consisting of a high affinity streptavidin-binding peptide (SBP) and a calmodulin-binding peptide (CBP) to allow purification of the recombinant protein using successively-applied streptavidin-resin and calmodulin-resin affinity purification steps or its detection with anti-CBP antibodies
COS-7 cells were harvested 42 h after transfection with HGSNAT-TAP plasmids bearing missense mutations. Cell homogenates were (A) assayed for N-acetyltransferase activity and (B) analyzed by Western blot using anti-CBP antibody as described in
The expression of HGSNAT mutants was studied by Western blot analysis of cellular homogenates using anti-CBP antibodies (
COS-7 cells expressing either the wild-type HGSNAT or the protein containing C76F, P237Q, G262R or V481L variants were harvested 42 h post-transfection and their homogenates were treated overnight with endoglycosidase H (A) or PNGase F (B). (C) COS-7 cells expressing wild-type HGSNAT and C76F or P237Q variants were cultured for 48 h in the presence or absence of 1 µg/ml of tunicamycin added to the culture medium 5 h after the transfection. The treated and control homogenates were analyzed by Western blot using anti-CBP antibodies as described in
COS-7 cells were harvested 42 h after transfection with the wild-type HGSNAT plasmid, solubilized in a buffer containing 0.1% NP-40, applied to an ion-exhange Mono Q HR 5/5 column and eluted by 0-0.5 M NaCl gradient as described in
The effects of missense mutations on the subcellular localization of the enzyme were studied by confocal immunofluorescence microscopy. Immortalized human skin fibroblasts from a normal control were transfected with constructs expressing each of the missense mutations. The cells were allowed to express the mutant and polymorphic enzymes for 42 hours and were then fixed by paraformaldehyde. To identify the lysosomal-late endosomal compartment, prior to fixation the cells were treated with LysoTracker Red DND-99 dye. After fixation the cells were permeabilized with Triton X-100 and probed with anti-CBP antibodies to localize the HGSNAT and with antibodies against the ER marker calnexin and lysosomal marker LAMP-2.
Distinct punctate staining characteristic of lysosomal targeting of the protein was evident for the recombinant wild-type enzyme and all active mutants. Accordingly, both wild-type recombinant HGSNAT and polymorphic mutants almost completely co-localized with the lysosomal markers LysoTracker Red or LAMP-2 (representative data are shown in
The cells transfected with wild-type or mutant HGSNAT-TAP constructs as indicated were fixed and stained with either mouse monoclonal anti-LAMP-2 antibodies, Lysotracker Red DND-99 or mouse monoclonal anti-calnexin antibodies (red) and rabbit polyclonal anti-CBP antibodies (green) as indicated. Slides were studied on a Zeiss LSM510 inverted confocal microscope. Magnification 630x. Panels show representative images illustrating co-localization of anti-CBP antibodies (green) and lysosomal and ER markers (red) for the wild-type HGSNAT, active enzyme containing P237Q polymorphism and inactive L137P and P571L mutants. From 10 to 15 cells all showing similar localization patterns were studied for each variant. See
Since all the data were consistent with general folding defects and retention in the ER compartment of the HGSNAT mutants (C76F, L137P, G262R, N273K, P283L, R344C, R344H, W403C, G424S, E471K, M482K, A489E, S518F, S539C, S541L, D562V, P571L) we further tested whether competitive inhibitors of the enzyme that mimic the substrate binding in the active site would help to fold the enzyme in the ER so it can be properly modified and exported to the lysosome. We first tested several potential inhibitors of HGSNAT that were not expected to be highly toxic for the cultured cells and found that one of them, D-(+)-glucosamine hydrochloride, was a competitive inhibitor of the enzyme with a KI of 0.28 mM close to the KM value for the 4MU-βGlcN substrate (
A. Fifty percent confluent immortalized skin fibroblasts from a MPS IIIC patient homozygous for N273K mutation
The current work provides an explanation for the severe phenotype of MPS IIIC patients carrying missense mutations in the
Visual representation of HGSNAT membrane topology was created using the TMRPres2D software
Seventeen mutations (C76F, L137P, G262R, N273K, P283L, R344C, R344H, W403C, G424S, E471K, M482K, A489E, S518F, S539C, S541L, D562V and P571L, shown in red in
Six mutations (R344C, R344H, E471K, S539C, S541L and D562V) are found adjacent to the predicted transmembrane domains either on the cytoplasmic (D562V) or on the lumenal (R344C, R344H, E471K, S539C, and S541L) side and 4 mutations (C76F, L137P, N273K, and G424S) reside inside the hydrophilic lumenal domains of the enzyme. In most cases these mutations are predicted to have a drastic effect on protein folding since they involve replacements with amino acids significantly different in hydrophobicity (C76F, D562V, S541L), charge (R344C/H, N273K, E471K) or size (C76F, L137P, G424S). Thus, enzyme folding defects due to missense mutations, together with nonsense-mediated mRNA decay seem to be the major molecular mechanisms underlying MPS IIIC.
For at least 5 of the above changes (N273K, R344C, R344H, S518F and S541L) the active conformation can be stabilized by the competitive inhibitor of HGSNAT glucosamine resulting in part of the enzyme pool being properly processed and targeted to the lysosomes. L137P and P283L mutants may also be stabilized by the glucosamine treatment, however this could not be verified experimentally because in the available patient cell lines they were present together with the responsive mutations S518F and R344C, respectively. Only one cell line carrying E471K and D562V mutations did not show a significant increase in N-acetyltransferase activity in response to glucosamine. Further structural studies are needed to fully understand the difference in the effect of glucosamine on these mutants.
Although the spectrum of mutations in MPS IIIC patients shows substantial heterogeneity, some of the missense mutations have a high frequency within the patient population. Importantly, the two mutations, R344C and S518F, responsive to glucosamine-mediated refolding account for 22.0% and 29.3%, respectively, of the alleles among the probands of Dutch origin
The current study was conducted with ethics approval from the review board of CHU Ste-Justine, University of Montreal.
The wild-type HGSNAT-TAP plasmid was obtained by subcloning the HGSNAT 1992 bp coding sequence into pCTAP vector (Stratagene). Briefly, a 3′ part of human pCMV-Script construct
Skin fibroblast lines of MPS IIIC patients, obtained with a written informed consent
Patient and control skin fibroblasts were cultured as described above in growth media supplemented with various concentrations of D-(+)-glucosamine hydrochloride (Sigma G4875). Medium was replaced every day and at specified time cells were harvested and assayed for N-acetyltransferase activity and β-hexosaminidase activity as described below.
N-acetyltransferase enzymatic activity was measured using fluorogenic substrate, 4-methylumbelliferyl
To remove N-linked glycans from HGSNAT, cell homogenates were treated with recombinant endoglycosidase H, or peptide: N-Glycosidase F (Endo H; PNGase F New England Biolabs). Briefly, the mix consisted of 10 µg of cell homogenate in 25 mM sodium phosphate buffer, pH 7.5, to which 1 µl (500 U) of concentrated Endo H or PNGase F was added before incubating at 37°C overnight and analysis by Western blot.
To inhibit glycosylation of newly-synthesized HGSNAT COS-7 cells transfected with plasmids coding for the wild-type enzyme and the C76F or P237Q variants were treated with 1 µg/ml of tunicamycin (Sigma) added 5 h after transfection and allowed to express the recombinant protein for 48 h in the presence of drug. Media and drug were changed after 24 h and the homogenates were analyzed by Western blot as described below.
Cell homogenates were sonicated and boiled in LDS sample buffer (Invitrogen) in the presence of 25 mM DTT. Proteins were resolved by SDS-polyacrylamide gel electrophoresis using NuPAGE 4–12% Bis-Tris gels (Invitrogen) and electrotransferred to PVDF membrane. Detection of TAP-tagged N-acetyltransferase protein was performed using anti-calmodulin binding peptide epitope tag (CBP) rabbit antibodies (Immunology Consultants Laboratory, dilution 1∶30,000) and the Amersham ECL Western Blotting Detection Reagents (GE Healthcare) in accordance with the manufacturer's protocol.
COS-7 cells were harvested 42 h after transfection with the wild-type HGSNAT plasmid and suspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 0.1% NP-40, 1 mM PMSF and Sigma P8340 protease inhibitor cocktail at 10 µl per 1 ml of cell suspension). The homogenate was sonicated, gently shaked at 4°C for 2 h and centrifuged at 13,000 rpm for 30 min. One ml of the supernatant containing 8 mg of total protein was applied to an ion-exhange Mono Q HR 5/5 column equilibrated with 10 mM Tris buffer, pH 8.2. The column was washed with 4 ml of the same buffer and then eluted using a 20 mL gradient of NaCl (0–0.5 M) at a flow rate of 0.5 ml/min. One ml fractions were collected and assayed for N-acetyltransferase activity. Thirty µl aliquots from each fraction were analyzed by SDS-PAGE and Western blot using anti-CBP antibodies as described above.
Immortalized control human skin fibroblasts were transfected with HGSNAT-TAP or plasmids coding for the HGSNAT mutants using Lipofectamine LTX (Invitrogen) as described in the manufacturer's protocol. Forty-two hours post-transfection cells were incubated for 1 hour with 1 µM Lysotracker Red DND-99 (Invitrogen) and then washed with ice-cold PBS. Cells were fixed with 4% paraformaldehyde, 4% sucrose in PBS for 5 min, and then rinsed 3 times with PBS. Cells were permeabilized by 0.25% Triton X-100 for 10 min and blocked for 1 h in 3% horse serum and 0.1% Triton X-100. Cells were either co-stained with rabbit anti-CBP (Immunology Consultants Laboratory; 1∶400) and mouse monoclonal anti-calnexin (Millipore; 1∶250) antibodies in 3% horse serum, with anti-CBP antibodies and mouse monoclonal antibodies against human LAMP-2 (Developmental Studies Hybridoma Bank; 1∶150), or with anti-CBP antibodies and Lysotracker Red DND-99. Cells were then counterstained with Oregon Green 488-conjugated anti-rabbit IgG antibodies or Texas-Red-conjugated goat anti-mouse antibodies (Molecular Probes; 1∶1000). Slides were studied on a Zeiss LSM510 inverted confocal microscope (Zeiss). Images were processed using the LSM image browser software (Zeiss) and Photoshop (Adobe).
Lineweaver-Burk plot of substrate dependance for partially purified HGSNAT wild-type and P237Q and V481L mutants. COS-7 cells were harvested 42 hrs after transfection with the wild-type or mutant HGSNAT plasmids and suspended in lysis buffer (40 mM Tris-HCl, 300 mM KCl, pH 7.5, 0.1% NP-40, 1 mM PMSF and Sigma P8340 protease inhibitor cocktail at 10 µl per 1 ml of cell suspension). The homogenate was sonicated, gently shaked at 4°C for 2 h and centrifuged at 13,000 rpm for 30 min. The supernatant was first passed through an avidin-agarose column (Sigma A9207) then affinity purification of TAP-tagged HGSNAT was performed using streptavidin resin (Stratagene) according to the manufacturer's protocol. N-acetyltransferase activity was assayed as described in Material and Methods using 0.05 to 2.0 mM 4MU-βGlcN and 18 h incubation time. KM and VMAX values for all 3 enzymes were similar within the statistical error.
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Localization of HGSNAT mutants expressed in cultured human skin fibroblasts by immunofluorescence microscopy. The cells transfected with wild-type or mutant HGSNAT-TAP constructs as indicated were fixed and stained with either mouse monoclonal anti-LAMP-2 antibodies, Lysotracker Red DND-99 or mouse monoclonal anti-calnexin antibodies (red) and rabbit polyclonal anti-CBP antibodies (green) as indicated. Slides were studied on a Zeiss LSM510 inverted confocal microscope. Magnification 630x. Panels show representative images showing co-localization of anti-CBP antibodies (green) and lysosomal and ER markers (red) for the active enzyme containing polymorphisms and all inactive mutants.
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Dixon plot showing the inhibition of HGSNAT by glucosamine. COS-7 cells were harvested 42 hrs after transfection with the wild-type HGSNAT plasmid and N-acetyltransferase activity was measured in the homogenates for 3 h at 37°C in the presence of 2 mM AcCoA, 0.0375 to 1.5 mM 4MU-βGlcN and 0 to 2 mM D-(+)-glucosamine hydrochloride.
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Primers for site-directed mutagenesis of HGSNAT-TAP plasmid.
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The authors thank the patients and their families for participating in our study. We also thank Maryssa Canuel for helpful advice, Mila Ashmarina for critical reading and Eva Lacroix for help in preparation of the manuscript.