title: Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.
abstract: alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemical Pathology, Adelaide Children's Hospital, North Adelaide, Australia.
authors.forename: L J
authors.initials: LJ
authors.lastname: Ashton
authors.forename: D A
authors.initials: DA
authors.lastname: Brooks
authors.forename: P A
authors.initials: PA
authors.lastname: McCourt
authors.forename: V J
authors.initials: VJ
authors.lastname: Muller
authors.forename: P R
authors.initials: PR
authors.lastname: Clements
authors.forename: J J
authors.initials: JJ
authors.lastname: Hopwood
mesh_terms: D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007068:Iduronidase / Q000032:analysis* / Q000276:immunology; D008059:Mucopolysaccharidosis I / Q000201:enzymology*
chemical_list: D000911:Antibodies, Monoclonal; D007068:Iduronidase