Protein Misfolding as an Underlying Molecular Defect in Mucopolysaccharidosis III Type C

Department of Medical Genetics, CHU Sainte-Justine University of Montreal, Montreal, Canada.
PLoS ONE (Impact Factor: 3.23). 10/2009; 4(10):e7434. DOI: 10.1371/journal.pone.0007434
Source: PubMed


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, 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.

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Available from: Alexey Pshezhetsky, Oct 30, 2014
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    • "HGSNAT transfers an acetyl group from cytoplasmic acetyl-CoA to terminal N-glucosamine residues of heparan sulphate within the lysosomes (Klein et al., 1978). To date, more than 80 HGSNAT mutations have been identified including 18 missense which all result in misfolding of the mutant enzyme (Feldhammer et al., 2009). "
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    ABSTRACT: Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email:
    Full-text · Article · Jan 2015 · Molecular Genetics and Metabolism
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    • "The pathogenic effect may be as simple as a build-up of a toxic substrate or dearth of a critical product in a metabolic pathway; in such cases, means of reducing the former and increasing the latter are obvious therapeutic approaches. For example, the zinc deficiency observed in acrodermatitis enteropathica can be addressed by dietary supplementation [64,65], while the accumulation of a toxic metabolite may be addressed by dietary means, such as the phenylalanine restriction used to treat phenylketonuria [62]. Where dietary modification is either not possible or insufficient, modulation of the activity of an alternate pathway may be employed as a possible adjunctive therapy. "
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    ABSTRACT: With the advent of next-generation DNA sequencing, the pace of inherited orphan disease gene identification has increased dramatically, a situation that will continue for at least the next several years. At present, the numbers of such identified disease genes significantly outstrips the number of laboratories available to investigate a given disorder, an asymmetry that will only increase over time. The hope for any genetic disorder is, where possible and in addition to accurate diagnostic test formulation, the development of therapeutic approaches. To this end, we propose here the development of a strategic toolbox and preclinical research pathway for inherited orphan disease. Taking much of what has been learned from rare genetic disease research over the past two decades, we propose generalizable methods utilizing transcriptomic, system-wide chemical biology datasets combined with chemical informatics and, where possible, repurposing of FDA approved drugs for pre-clinical orphan disease therapies. It is hoped that this approach may be of utility for the broader orphan disease research community and provide funding organizations and patient advocacy groups with suggestions for the optimal path forward. In addition to enabling academic pre-clinical research, strategies such as this may also aid in seeding startup companies, as well as further engaging the pharmaceutical industry in the treatment of rare genetic disease.
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    • "This hypothesis would be strengthened by the severe neurodegenerative phenotype of the Cln10 mouse model deficient in the aspartyl protease cathepsin D, which mediates protein degradation and activates precursor forms of proteases [55]. Disease-causing mutations in polytopic lysosomal membrane proteins , such as CLN3 and HGSNAT, are typically retained in the endoplasmic reticulum due to misfolding of the proteins [56] [57]. However, we and others have shown that several disease-causing mutations in the CLN7/MFSD8 gene did not alter subcellular localization of CLN7 in lysosomal compartments [9] [16], suggesting that these mutations cause alterations in protein stability and/or biological function in lysosomes, rather than retention of misfolded CLN7 proteins in the endoplasmic reticulum or intracellular missorting. "
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    ABSTRACT: CLN7 is a polytopic lysosomal membrane glycoprotein of unknown function and is deficient in variant late infantile neuronal ceroid lipofuscinosis. Here we show that full-length CLN7 is proteolytically cleaved twice, once proximal to the used N-glycosylation sites in lumenal loop L9 and once distal to these sites. Cleavage occurs by cysteine proteases in acidic compartments and disruption of lysosomal targeting of CLN7 results in inhibition of proteolytic cleavage. The apparent molecular masses of the CLN7 fragments suggest that both cleavage sites are located within lumenal loop L9. The known disease-causing mutations, p.T294K and p.P412L, localized in lumenal loops L7 and L9, respectively, did not interfere with correct lysosomal targeting of CLN7 but enhanced its proteolytic cleavage in lysosomes. Incubation of cells with selective cysteine protease inhibitors and expression of CLN7 in gene-targeted mouse embryonic fibroblasts revealed that cathepsin L is required for one of the two proteolytic cleavage events. Our findings suggest that CLN7 is inactivated by proteolytic cleavage and that enhanced CLN7 proteolysis caused by missense mutations in selected luminal loops is associated with disease.
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