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. 1992 Jun 15;284(Pt 3):789–794. doi: 10.1042/bj2840789

Correction of human mucopolysaccharidosis type-VI fibroblasts with recombinant N-acetylgalactosamine-4-sulphatase.

D S Anson 1, J A Taylor 1, J Bielicki 1, G S Harper 1, C Peters 1, G J Gibson 1, J J Hopwood 1
PMCID: PMC1132608  PMID: 1320379

Abstract

A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.

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Selected References

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  1. Barton N. W., Furbish F. S., Murray G. J., Garfield M., Brady R. O. Therapeutic response to intravenous infusions of glucocerebrosidase in a patient with Gaucher disease. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1913–1916. doi: 10.1073/pnas.87.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bielicki J., Freeman C., Clements P. R., Hopwood J. J. Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties. Biochem J. 1990 Oct 1;271(1):75–86. doi: 10.1042/bj2710075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brooks D. A., Gibson G. J., McCourt P. A., Hopwood J. J. A specific fluorogenic assay for N-acetylgalactosamine-4-sulphatase activity using immunoadsorption. J Inherit Metab Dis. 1991;14(1):5–12. doi: 10.1007/BF01804381. [DOI] [PubMed] [Google Scholar]
  4. Brooks D. A., McCourt P. A., Gibson G. J., Hopwood J. J. Immunoquantification of the low abundance lysosomal enzyme N-acetylgalactosamine 4-sulphatase. J Inherit Metab Dis. 1990;13(1):108–120. doi: 10.1007/BF01799338. [DOI] [PubMed] [Google Scholar]
  5. Conary J. T., Hasilik A., von Figura K. Synthesis and stability of steroid sulfatase in fibroblasts from multiple sulfatase deficiency. Biol Chem Hoppe Seyler. 1988 Apr;369(4):297–302. doi: 10.1515/bchm3.1988.369.1.297. [DOI] [PubMed] [Google Scholar]
  6. Cullen B. R. Expression of a cloned human interleukin-2 cDNA is enhanced by the substitution of a heterologous mRNA leader region. DNA. 1988 Nov;7(9):645–650. doi: 10.1089/dna.1988.7.645. [DOI] [PubMed] [Google Scholar]
  7. Fratantoni J. C., Hall C. W., Neufeld E. F. The defect in Hurler's and Hunter's syndromes: faulty degradation of mucopolysaccharide. Proc Natl Acad Sci U S A. 1968 Jun;60(2):699–706. doi: 10.1073/pnas.60.2.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibson G. J., Saccone G. T., Brooks D. A., Clements P. R., Hopwood J. J. Human N-acetylgalactosamine-4-sulphate sulphatase. Purification, monoclonal antibody production and native and subunit Mr values. Biochem J. 1987 Dec 15;248(3):755–764. doi: 10.1042/bj2480755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hasilik A., Neufeld E. F. Biosynthesis of lysosomal enzymes in fibroblasts. Synthesis as precursors of higher molecular weight. J Biol Chem. 1980 May 25;255(10):4937–4945. [PubMed] [Google Scholar]
  10. Hopwood J. J., Elliott H., Muller V. J., Saccone G. T. Diagnosis of Maroteaux-Lamy syndrome by the use of radiolabelled oligosaccharides as substrates for the determination of arylsulphatase B activity. Biochem J. 1986 Mar 15;234(3):507–514. doi: 10.1042/bj2340507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hopwood J. J., Muller V., Harrison J. R., Carey W. F., Elliott H., Robertson E. F., Pollard A. C. Enzymatic diagnosis of the mucopolysaccharidoses: experience of 96 cases diagnosed in a five-year period. Med J Aust. 1982 Mar 20;1(6):257–260. [PubMed] [Google Scholar]
  12. Jezyk P. F., Haskins M. E., Patterson D. F., Mellman W. J., Greenstein M. Mucopolysaccharidosis in a cat with arylsulfatase B deficiency: a model of Maroteaux-Lamy syndrome. Science. 1977 Nov 25;198(4319):834–836. doi: 10.1126/science.144321. [DOI] [PubMed] [Google Scholar]
  13. LEABACK D. H., WALKER P. G. Studies on glucosaminidase. 4. The fluorimetric assay of N-acetyl-beta-glucosaminidase. Biochem J. 1961 Jan;78:151–156. doi: 10.1042/bj0780151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Litjens T., Morris C. P., Gibson G. J., Beckmann K. R., Hopwood J. J. Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones. Biochem Int. 1991 May;24(2):209–215. [PubMed] [Google Scholar]
  16. Peters C., Schmidt B., Rommerskirch W., Rupp K., Zühlsdorf M., Vingron M., Meyer H. E., Pohlmann R., von Figura K. Phylogenetic conservation of arylsulfatases. cDNA cloning and expression of human arylsulfatase B. J Biol Chem. 1990 Feb 25;265(6):3374–3381. [PubMed] [Google Scholar]
  17. Singh H., Derwas N., Poulos A. Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes. Arch Biochem Biophys. 1987 Dec;259(2):382–390. doi: 10.1016/0003-9861(87)90504-2. [DOI] [PubMed] [Google Scholar]
  18. Steckel F., Hasilik A., von Figura K. Biosynthesis and maturation of arylsulfatase B in normal and mutant cultured human fibroblasts. J Biol Chem. 1983 Dec 10;258(23):14322–14326. [PubMed] [Google Scholar]
  19. Taylor J. A., Gibson G. J., Brooks D. A., Hopwood J. J. Human N-acetylgalactosamine-4-sulphatase biosynthesis and maturation in normal, Maroteaux-Lamy and multiple-sulphatase-deficient fibroblasts. Biochem J. 1990 Jun 1;268(2):379–386. doi: 10.1042/bj2680379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yanagishita M., Hascall V. C. Proteoglycans synthesized by rat ovarian granulosa cells in culture. Isolation, fractionation, and characterization of proteoglycans associated with the cell layer. J Biol Chem. 1984 Aug 25;259(16):10260–10269. [PubMed] [Google Scholar]

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