Protein misfolding as an underlying molecular defect in mucopolysaccharidosis III type C

Abstract

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: alpha-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 alpha-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.

Figure 1. Enzymatic activity and expression of HGSNAT mutants.

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 Materials and Methods. A. N-acetyltransferase activity is shown as a fraction of the activity measured in the cells transfected with the wild-type HGSNAT-TAP plasmid. Values represent means ± S.D. of three independent experiments. B. Blot shows a representative image of triplicate experiments.

Figure 2. Deglycosylation of HGSNAT by endoglycosidase H, PNGase F and tunicamycin treatment.

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 Materials and Methods.

Figure 3. Analysis of the wild-type recombinant HGSNAT by anion-exchange FPLC.

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 Materials and Methods. Graph shows N-acetyltransferase activity (nmol/hr ml) in the collected fractions. Dashed line represents the NaCl gradient. An aliquot from each fraction was analyzed by Western blot using anti-CBP antibodies (inset).

Figure 4. 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 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 Figure S2 for the data on other mutants.

Figure 5. Partial refolding of HGSNAT mutants by glucosamine.

A. Fifty percent confluent immortalized skin fibroblasts from a MPS IIIC patient homozygous for N273K mutation were cultured in the presence or absence of 14 mM D-(+)-glucosamine hydrochloride. Medium was replaced every day and at the indicated time intervals cells were harvested and assayed for N-acetyltransferase activity. N-acetyltransferase activity is shown as a fraction of that measured in non-treated cells after 24 h of culturing. Data show mean values and standard error of 2 independent experiments. ** Significantly different (p<0.01) from non-treated cells according to repeated measurements ANOVA. B. Same cells were cultured in the presence of increasing glucosamine concentrations (0–14 mM) for 5 days, harvested and assayed for N-acetyltransferase activity or β-hexosaminidase activity. Data show mean values and standard error of 2 independent experiments. C. Primary skin fibroblasts of the MPS IIIC patients carrying the missense HGSNAT mutations: L137P/S518F (P1), P283L/R344C (P3), S518F/S518F (P4), N273K/N273K (P6), R344C/R344C (P7), S518F/S518F (P8) and E471K/D562V (P9) or a missense mutation in combination with a splice site (S541L/c.234+1G>A; P2) or nonsense (R344H/R384X, P5) mutation were cultured for 2 (P1, P2, P5, P6, P8, P9) or 3 (P3, P4, P7) days in the absence (open bars) or presence (filled bars) of 7 mM (P3-P9) or 14 mM (P1, P2) glucosamine, harvested and assayed for N-acetyltransferase activity. Data show mean values and standard error of 2 independent experiments. The residual N-acetyltransferase activity detectable in untreated cells most likely represents background chemical or enzymatic reactions occurring with the substrate in the presence of cell homogenates. Significantly (*, p<0.05; **, p<0.01; ***, p<0.001) different from non-treated cells according to non-parametric t-test.

Figure 6. Distribution of missense mutations in HGSNAT protein.

Visual representation of HGSNAT membrane topology was created using the TMRPres2D software . The deduced amino acid sequence of HGSNAT predicts 11 transmembrane domains and five potential N-glycosylation sites oriented towards the lysosomal lumen (shown in blue). Mutations that result in production of misfolded proteins are shown in red. Polymorphisms are shown in green. Figure was adapted from our previous work .