Transport, enzymatic activity, and stability of mutant sulfamidise (SGSH) identified iin patients with mucopolysaccharidosis type III A

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.Va1379fsX33) 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.Va1379fsX33) mutant proteins probably occurs in the ER, whereas c.488T>C (p.Leu163Pro) mutant protein showed instability in the lysosomes. (C) 2004 Wiley-Liss, Inc.