title: Transport, enzymatic activity, and stability of mutant sulfamidase (SGSH) identified in patients with mucopolysaccharidosis type III A.
abstract: Mucopolysaccharidosis type IIIA (MPSIIIA) is an autosomal recessive lysosomal storage disease caused by mutations in the N-sulfoglucosamine sulfohydrolase gene (SGSH; encoding sulfamidase, also sulphamidase) leading to the lysosomal accumulation and urinary excretion of heparan sulfate. Considerable variation in the onset and severity of the clinical phenotype is observed. We report here on expression studies of four novel mutations: c.318C>A (p.Ser106Arg), c.488T>C (p.Leu163Pro), c.571G>A (p.Gly191Arg), and c.1207_1209delTAC (p.Tyr403del), and five previously known mutations: c.220C>T (p.Arg74Cys), c.697C>T (p.Arg233X), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) identified in MPSIIIA patients. Transient expression of mutant sulfamidases in BHK or CHO cells revealed that all the mutants were enzymatically inactive with the exception of c.318C>A (p.Ser106Arg), which showed 3.3% activity of the expressed wild-type enzyme. Western blot analysis demonstrated that the amounts of expressed mutant sulfamidases were significantly reduced compared with cells expressing wild type. No polypeptides were immunodetectable in extracts of cells transfected with the cDNA carrying the c.697C>T (p.Arg233X) nonsense mutation. In vitro translation and pulse-chase experiments showed that rapid degradation rather than a decrease in synthesis is responsible for the low, steady-state level of the mutant proteins in cells. The amounts of secreted mutant precursor forms, the cellular stability, the proteolytic processing, and data from double-label immunofluorescence microscopy suggest that the degradation of the majority of newly synthesized c.220C>T (p.Arg74Cys), c.571G>A (p.Gly191Arg), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) mutant proteins probably occurs in the ER, whereas c.488T>C (p.Leu163Pro) mutant protein showed instability in the lysosomes.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Hospital Hamburg Eppendorf, Hamburg, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Muschol
authors.forename: Stephan
authors.initials: S
authors.lastname: Storch
authors.forename: Diana
authors.initials: D
authors.lastname: Ballhausen
authors.forename: Clare
authors.initials: C
authors.lastname: Beesley
authors.forename: Jan-Christoph
authors.initials: JC
authors.lastname: Westermann
authors.forename: Andreas
authors.initials: A
authors.lastname: Gal
authors.forename: Kurt
authors.initials: K
authors.lastname: Ullrich
authors.forename: John J
authors.initials: JJ
authors.lastname: Hopwood
authors.forename: Bryan
authors.initials: B
authors.lastname: Winchester
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009084:Mucopolysaccharidosis III / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D006867:Hydrolases; C022708:N-sulfoglucosamine sulfohydrolase