title: An acetylated 120-kDa lysosomal transmembrane protein is absent from mucopolysaccharidosis IIIC fibroblasts: a candidate molecule for MPS IIIC.
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.
authors.affiliation: Hôpital Sainte-Justine and Département de pédiatrie, Université de Montréal, Montréal, Que., Canada.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Ausseil
authors.forename: Karine
authors.initials: K
authors.lastname: Landry
authors.forename: Volkan
authors.initials: V
authors.lastname: Seyrantepe
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Trudel
authors.forename: Alexander
authors.initials: A
authors.lastname: Mazur
authors.forename: Fanny
authors.initials: F
authors.lastname: Lapointe
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Pshezhetsky
mesh_terms: D000105:Acetyl Coenzyme A; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D009084:Mucopolysaccharidosis III / Q000201:enzymology* / Q000235:genetics; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011506:Proteins / Q000737:chemistry; D040901:Proteomics
chemical_list: D011506:Proteins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases