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Recombinant alpha-L-iduronidase: characterization of the purified enzyme and correction of mucopolysaccharidosis type I fibroblasts. 1994

E G Unger, and J Durrant, and D S Anson, and J J Hopwood
Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia.

Mucopolysaccharidosis type I (MPS I, Hurler and Scheie syndromes) is an autosomal recessive lysosomal storage disorder that results from a deficiency of the hydrolase alpha-L-iduronidase (IDUA) which is involved in the lysosomal degradation of both heparan sulphate (HS) and dermatan sulphate (DS). Patients with MPS I store and excrete large amounts of partially degraded HS and DS. In order to evaluate enzyme replacement therapy for MPS I patients we have expressed human IDUA cDNA in Chinese Hamster Ovary (CHO)-K1 cells utilizing a plasmid vector that places the cDNA under the transcriptional control of the human polypeptide-chain-elongation factor I alpha gene promoter. A clonal cell-line that secreted recombinant IDUA in a precursor form at approximately 2.2 micrograms/10(6) cells per day was identified. This enzyme was shown to be endocytosed into cultured MPS I fibroblasts via mannose-6-phosphate receptors and to correct the storage phenotype of these cells by enabling the lysosomal-digestion of accumulated sulphated glycosaminoglycans. The recombinant IDUA had on SDS/PAGE a molecular mass of 85 kDa and was processed to 74 kDa and smaller forms following its uptake by fibroblasts. Milligram quantities of the recombinant IDUA were immunopurified and the enzyme was shown to have pH optimum and kinetic parameters differing from those of the mature enzyme purified from human liver. The specific activity of the recombinant enzyme was shown to increase on dilution and on incubation with reducing agents. This was in contrast to the mature IDUA form (74 kDa) which did not have its activity stimulated by reducing agents or dilution.

UI MeSH Term Description Entries
D007068 Iduronidase An enzyme that hydrolyzes iduronosidic linkages in desulfated dermatan. Deficiency of this enzyme produces Hurler's syndrome. EC 3.2.1.76. alpha-L-Idosiduronase,alpha-L-Iduronidase,alpha L Idosiduronase,alpha L Iduronidase
D008059 Mucopolysaccharidosis I Systemic lysosomal storage disease caused by a deficiency of alpha-L-iduronidase (IDURONIDASE) and characterized by progressive physical deterioration with urinary excretion of DERMATAN SULFATE and HEPARAN SULFATE. There are three recognized phenotypes representing a spectrum of clinical severity from severe to mild: Hurler syndrome, Hurler-Scheie syndrome and Scheie syndrome (formerly mucopolysaccharidosis V). Symptoms may include DWARFISM; hepatosplenomegaly; thick, coarse facial features with low nasal bridge; corneal clouding; cardiac complications; and noisy breathing. Hurler's Syndrome,Hurler-Scheie Syndrome,Lipochondrodystrophy,Mucopolysaccharidosis V,Pfaundler-Hurler Syndrome,Scheie's Syndrome,Gargoylism,Gargoylism, Hurler Syndrome,Hurler Disease,Hurler Syndrome,Hurler's Disease,Mucopolysaccharidosis 1,Mucopolysaccharidosis 5,Mucopolysaccharidosis I-S,Mucopolysaccharidosis Type I,Mucopolysaccharidosis Type Ih,Mucopolysaccharidosis Type Ih S,Mucopolysaccharidosis Type Is,Scheie Syndrome,alpha-L-Iduronidase Deficiency,Disease, Hurler's,Gargoylisms,Hurler Scheie Syndrome,Hurler Syndrome Gargoylism,Lipochondrodystrophies,Mucopolysaccharidosis I S,Mucopolysaccharidosis Is,Mucopolysaccharidosis Type Ihs,Syndrome, Hurler's,Syndrome, Scheie's,Type Ih, Mucopolysaccharidosis,Type Ihs, Mucopolysaccharidosis,alpha L Iduronidase Deficiency,alpha-L-Iduronidase Deficiencies
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004790 Enzyme Induction An increase in the rate of synthesis of an enzyme due to the presence of an inducer which acts to derepress the gene responsible for enzyme synthesis. Induction, Enzyme
D005347 Fibroblasts Connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules. Fibroblast
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D018076 DNA, Complementary Single-stranded complementary DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase. cDNA (i.e., complementary DNA, not circular DNA, not C-DNA) is used in a variety of molecular cloning experiments as well as serving as a specific hybridization probe. Complementary DNA,cDNA,cDNA Probes,Probes, cDNA

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