Article

An acetylated 120-kDa lysosomal transmembrane protein is absent from mucopolysaccharidosis IIIC fibroblasts: A candidate molecule for MPS IIIC

Université de Montréal, Montréal, Quebec, Canada
Molecular Genetics and Metabolism (Impact Factor: 2.63). 02/2006; 87(1):22-31. DOI: 10.1016/j.ymgme.2005.09.021
Source: PubMed

ABSTRACT

Genetic deficiency of the lysosomal enzyme, acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase), which catalyses the transmembrane acetylation of heparan sulfate results in severe neurodegenerative disease, mucopolysaccharidosis IIIC. N-Acetyltransferase has never been characterized structurally and its gene has never been identified. We combined traditional methods of enzyme purification with organellar proteomics, isolating lysosomal membranes from mouse liver using differential centrifugation and osmolysis, followed by detergent extraction and purification of N-acetyltransferase by liquid chromatography. Partially purified enzyme had a molecular mass of 240 kDa and pI of 7.4 by gel filtration and chromatofocusing. Its specific activity varied with protein concentration typical of oligomeric enzymes or multienzyme complexes. Incubation of N-acetyltransferase with acetyl[14C]CoA in the absence of the acceptor of the acetyl group resulted in radioactive labeling of a 120-kDa polypeptide, suggesting that it represents a subunit containing the enzyme active site. Furthermore, following acetyl[14C]-labeling, the 120-kDa protein was present in the lysosomal membranes purified from the normal human skin fibroblasts but absent in those from the skin fibroblasts of MPS IIIC patients.

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    • "The reaction was terminated by adding 1.98 ml of 0.5 M Na2CO3/NaHCO3, pH 10.7, and fluorescence was measured and used to calculate the specific activity. Lysosomal β-hexosaminidase activity was measured as previously described [24]. Protein concentration was measured according to the method of Bradford [25] using a commercially available reagent (BioRad). "
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    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: 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.
    Full-text · Article · Oct 2009 · PLoS ONE
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    • "The gene was cloned only recently by two independent laboratories [Fan et al., 2006; Hřebíček et al., 2006]. Based on our previous studies that defined the chromosomal localization of the MPS IIIC allele [Ausseil et al., 2004] and molecular properties of HGSNAT [Hopwood et al., 1983; Pohlmann et al., 1981; Ausseil et al., 2006], we searched the centromeric region of human chromosome 8 for a gene encoding a protein with multiple transmembrane domains and a molecular weight of 100 kDa, which allowed us to exclude the majority of the genes with exception of the gene initially called TMEM76 [Hřebíček et al., 2006]. Furthermore, of all the genes present in the candidate interval only TMEM76 showed a statistically significant reduction of the transcript level in the cells of two MPS IIIC patients. "
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    ABSTRACT: Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N-acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes 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 severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT.
    Full-text · Article · Jul 2009 · Human Mutation
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    • "The gene was cloned only recently by two independent laboratories [Fan et al., 2006; Hřebíček et al., 2006]. Based on our previous studies that defined the chromosomal localization of the MPS IIIC allele [Ausseil et al., 2004] and molecular properties of HGSNAT [Hopwood et al., 1983; Pohlmann et al., 1981; Ausseil et al., 2006], we searched the centromeric region of human chromosome 8 for a gene encoding a protein with multiple transmembrane domains and a molecular weight of 100 kDa, which allowed us to exclude the majority of the genes with exception of the gene initially called TMEM76 [Hřebíček et al., 2006]. Furthermore, of all the genes present in the candidate interval only TMEM76 showed a statistically significant reduction of the transcript level in the cells of two MPS IIIC patients. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N-acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes 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 severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT.
    Full-text · Article · Jun 2009 · Human Mutation
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