title: Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients.
abstract: A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemical Pathology, Adelaide Medical Centre for Women and Children, South Australia.
authors.forename: D A
authors.initials: DA
authors.lastname: Brooks
authors.forename: P A
authors.initials: PA
authors.lastname: McCourt
authors.forename: G J
authors.initials: GJ
authors.lastname: Gibson
authors.forename: L J
authors.initials: LJ
authors.lastname: Ashton
authors.forename: M
authors.initials: M
authors.lastname: Shutter
authors.forename: J J
authors.initials: JJ
authors.lastname: Hopwood
mesh_terms: D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002803:Chondro-4-Sulfatase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003871:Dermatan Sulfate / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D009083:Mucopolysaccharidoses / Q000201:enzymology / Q000235:genetics*; D010641:Phenotype; D011933:Reagent Kits, Diagnostic; D013379:Substrate Specificity
chemical_list: D000911:Antibodies, Monoclonal; D011933:Reagent Kits, Diagnostic; D003871:Dermatan Sulfate; D002803:Chondro-4-Sulfatase