Molecular Medicine

, Volume 18, Issue 1, pp 76–82 | Cite as

Efficient Uptake of Recombinant α-Galactosidase A Produced with a Gene-Manipulated Yeast by Fabry Mice Kidneys

  • Takahiro Tsukimura
  • Ikuo Kawashima
  • Tadayasu Togawa
  • Takashi Kodama
  • Toshihiro Suzuki
  • Toru Watanabe
  • Yasunori Chiba
  • Yoshifumi Jigami
  • Tomoko Fukushige
  • Takuro Kanekura
  • Hitoshi Sakuraba
Research Article


To economically produce recombinant human a-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-l,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.



We wish to thank Ashok B Kulkarni (National Institutes of Health) and Toshio Oshima (Waseda University) for providing the Fabry mice. We also thank Yoshiko Tanabe for typing the manuscript. This work was supported by the Program for Research on Intractable Diseases of Health and Labor Science Research Grants (H Sakuraba); the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (ID: 09-15, H Sakuraba); the JAPS Asia/Africa Scientific Platform Program (H Sakuraba); the Japan Society for the Promotion of Science (JSPS ID: 21390314, H Sakuraba); and the High-Tech Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan (S0801043, H Sakuraba).


  1. 1.
    Spada M, et al. (2006) High incidence of later-onset Fabry disease revealed by newborn screening. Am. J. Hum. Gene. 79:31–40.CrossRefGoogle Scholar
  2. 2.
    Hwu WL, et al. (2009) Newborn screening for Fabry disease in Taiwan reveals a high incidence of the later-onset GLA mutation c.936+919G>A (IVS4+919G>A). Hum. Mutat. 30:1397–405.CrossRefGoogle Scholar
  3. 3.
    Lin HY, et al. (2009) High incidence of the cardiac variant of Fabry disease revealed by newborn screening in the Taiwan Chinese population. Circ. Cardiovasc. Gene. 2:450–6.CrossRefGoogle Scholar
  4. 4.
    Desnick RJ, Ioannou YA, Eng CM. (2001) Alpha-galactosidase A deficiency: Fabry disease. In: The Metabolic and Molecular Bases of Inherited Disease. 8th ed. Scriver CR, Beaudet AL, Sly WS, Valle D (eds.). McGraw-Hill, New York, pp. 3733–74.Google Scholar
  5. 5.
    Aerts JM, et al. (2008) Elevated globotriaosylsphingosine is a hallmark of Fabry disease. Proc. Natl. Acad. Sci. U. S. A. 105:2812–7.CrossRefGoogle Scholar
  6. 6.
    Togawa T, et al. (2010) Plasma globotriaosylsphingosine as a biomarker of Fabry disease. Mol. Genet. Metab. 100:257–61.CrossRefGoogle Scholar
  7. 7.
    Togawa T, et al. (2010) Tissue and plasma globotriaosylsphingosine could be a biomarker for assessing enzyme replacement therapy for Fabry disease. Biochem. Biophys. Res. Commun. 399:716–20.CrossRefGoogle Scholar
  8. 8.
    Schiffmann R, et al. (2000) Infusion of α-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease. Proc. Natl. Acad. Sci. U. S. A. 97:365–70.CrossRefGoogle Scholar
  9. 9.
    Eng CM, et al. (2001) A phase 1/2 clinical trial of enzyme replacement in Fabry disease: pharmacokinetic, substrate clearance, and safety studies. Am. J. Hum. Genet. 68:711–22.CrossRefGoogle Scholar
  10. 10.
    Eng CM, et al. (2001) Safety and efficacy of recombinant human α-galactosidase A replacement therapy in Fabry’s disease. N. Engl. J. Med. 345:9–16.CrossRefGoogle Scholar
  11. 11.
    Kornfeld S, Sly WS. (2001) I-cell disease and pseudo-Hurler polydystrophy: disorders of lysosomal enzyme phosphorylation and localization. In: The Metabolic and Molecular Bases of Inherited Disease. 8th ed. Scriver CR, Beaudet AL, Sly WS, Valle D (eds.). McGraw-Hill, New York, pp. 3469–82.Google Scholar
  12. 12.
    Rosenfeld EL, Belenky DM, Bystrova NK. (1986) Interaction of hepatic asialoglycoprotein receptor with asialoorosomucoid and galactolyzed lysosomal alpha-glucosidase. Biochim. Biophys. Acta. 883:306–12.CrossRefGoogle Scholar
  13. 13.
    Mehta A, et al. (2009) Fabry Outcome Survey investigators: enzyme replacement therapy with agalsidase alfa in patients with Fabry’s disease: an analysis of registry data. Lancet. 374:1986–96.CrossRefGoogle Scholar
  14. 14.
    Fervenza FC, Torra R, Warnock DG. (2008) Safety and efficacy of enzyme replacement therapy in the nephropathy of Fabry disease. Biologics. 2:823–43.PubMedPubMedCentralGoogle Scholar
  15. 15.
    Thurberg BL, et al. (2009) Cardiac microvascular pathology in Fabry disease: evaluation of endomyocardial biopsies before and after enzyme replacement therapy. Circulation. 119:2561–7.CrossRefGoogle Scholar
  16. 16.
    Salviati A, Burlina AP, Borsini W. (2010) Nervous system and Fabry disease, from symptoms to diagnosis: damage evaluation and follow-up in adult patients, enzyme replacement, and support therapy. Neurol. Sci. 31:299–306.CrossRefGoogle Scholar
  17. 17.
    Sakuraba H, et al. (2006) Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice. J. Hum. Genet. 51:180–8.CrossRefGoogle Scholar
  18. 18.
    Ioannou YA, Zeidner KM, Gordon RE, Desnick RJ. (2001) Fabry disease: preclinical studies demonstrate the effectiveness of α-galactosidase A replacement in enzyme-deficient mice. Am. J. Hum. Genet. 68:14–25.CrossRefGoogle Scholar
  19. 19.
    Chiba Y, et al. (2002) Production in yeast of α-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease. Glycobiology. 12:821–8.CrossRefGoogle Scholar
  20. 20.
    Sakuraba H, et al. (2006) Corrective effect on Fabry mice of yeast recombinant human α-galactosidase with N-linked sugar chains suitable for lysosomal delivery. Hum. Genet. 51:341–52.CrossRefGoogle Scholar
  21. 21.
    Kuroda K, et al. (2006) Production of Man5GlcNAc2-type sugar chain by the methylotrophic yeast Ogataea minuta. FEMS Yeast Res. 6:1052–62.CrossRefGoogle Scholar
  22. 22.
    Odani T, Shimma Y, Tanaka A, Jigami Y. (1996) Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae. Glycobiology. 6:805–10.CrossRefGoogle Scholar
  23. 23.
    Odani T, Shimma Y, Wang XH, Jigami Y. (1997) Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae. FEBS Lett. 420:186–90.CrossRefGoogle Scholar
  24. 24.
    Ishii S, Kase R, Sakuraba H, Suzuki Y. (1993) Characterization of a mutant α-galactosidase gene product for the late-onset cardiac form of Fabry disease. Biochem. Biophys. Res. Commun. 197:1585–9.CrossRefGoogle Scholar
  25. 25.
    Akeboshi H, et al. (2007) Production of recombinant β-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta. Appl. Environ. Microbiol. 73:4805–12.CrossRefGoogle Scholar
  26. 26.
    Mayes JS, Scheerer JB, Sifers RN, Donaldson ML. (1981) Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry’s disease. Clin. Chim. Acta. 112:247–51.CrossRefGoogle Scholar
  27. 27.
    Akeboshi H, et al. (2009) Production of human β-hexosaminidase A with highly phosphorylated N-glycans by the overexpression of the Ogatae minuta MNN4 gene. Glycobiology. 19:1002–9.CrossRefGoogle Scholar
  28. 28.
    Ohshima T, et al. (1997) α-Galactosidase A deficient mice: a model of Fabry disease. Proc. Natl. Acad. Sci. U. S. A. 94:2540–4.CrossRefGoogle Scholar
  29. 29.
    Ohshima T, et al. (1999) Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice. Proc. Natl. Acad. Sci. U. S. A. 96:6423–7.CrossRefGoogle Scholar
  30. 30.
    Kotani M, et al. (1994) Generation of one set of murine monoclonal antibodies specific for globoseries glycolipids: evidence for differential distribution of the glycolipids in rat small intestine. Arch. Biochem. Biophys. 310:89–96.CrossRefGoogle Scholar
  31. 31.
    Kawashima I, et al. (2006) Phospholipid storage in the myocardium of a unique Japanese case of idiopathic cardiomyopathy. Clin. Chim. Acta. 372:154–7.CrossRefGoogle Scholar
  32. 32.
    Tajima Y, et al. (2009) Use of a modified α-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease. Am. J. Hum. Genet. 85:569–80.CrossRefGoogle Scholar
  33. 33.
    Zoon K. (2003) To manufacturers of biological products (Letter). Rockville, MD: U.S. Department of Health and Human Services, Center for Biologics Evaluation and Research.Google Scholar
  34. 34.
    European Medicines Agency (EMEA). (2003) Note for guidance on minimising the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products (EMEA/410/01). Revised. London, UK: EMEA.Google Scholar
  35. 35.
    Shibata N, Suzuki A, Kobayashi H, Okawa Y. (2007) Chemical structure of the cell-wall mannan of Candida albicans serotype A and its difference in yeast and hyphal forms. Biochem. J. 404:365–72.CrossRefGoogle Scholar
  36. 36.
    Faille C, et al. (1990) Immunoreactivity of neoglycolipids constructed from oligomannosidic residues of the Candida albicans cell wall. Infect. Immun. 58:3537–44.PubMedPubMedCentralGoogle Scholar
  37. 37.
    Ballou CE. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol. 185:440–70.CrossRefGoogle Scholar
  38. 38.
    Vedder AC, et al. (2007) Treatment of Fabry disease: outcome of a comparative trial with agalsidase alfa or beta at a dose of 0.2 mg/kg. PLoS One. 2:e598.CrossRefGoogle Scholar
  39. 39.
    Vedder AC, et al. (2008) Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3. Mol. Genet. Metab. 94:319–25.CrossRefGoogle Scholar
  40. 40.
    Lee K, et al. (2003) A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease. Glycobiology. 13:305–13.CrossRefGoogle Scholar
  41. 41.
    Sly WS, et al. (2006) Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors. Proc. Natl. Acad. Sci. U. S. A. 103:15172–7.CrossRefGoogle Scholar
  42. 42.
    Takamatsu S, et al. (2004) Monitoring of the tissue distribution of fibroblast growth factor containing a high mannose-type sugar chain produced in mutant yeast. Glycoconj. J. 20:385–97.CrossRefGoogle Scholar
  43. 43.
    Zhu Y, et al. (2009) Glycoengineered acid α-glucosidase with improved efficacy at correcting the metabolic aberrations and motor function deficits in a mouse model of Pompe disease. Mol. Ther. 17:954–63.CrossRefGoogle Scholar
  44. 44.
    Zhou Q, et al. (2011) Strategies for neoglycan conjugation to human acid α-glucosidase. Bioconjug. Chem. 22:741–51.CrossRefGoogle Scholar
  45. 45.
    Tsuji D, et al. (2011) Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis. Ann. Neurol. 69:691–701.CrossRefGoogle Scholar

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Authors and Affiliations

  • Takahiro Tsukimura
    • 1
  • Ikuo Kawashima
    • 2
    • 3
  • Tadayasu Togawa
    • 1
  • Takashi Kodama
    • 1
  • Toshihiro Suzuki
    • 1
  • Toru Watanabe
    • 4
  • Yasunori Chiba
    • 2
    • 4
  • Yoshifumi Jigami
    • 4
  • Tomoko Fukushige
    • 5
  • Takuro Kanekura
    • 5
  • Hitoshi Sakuraba
    • 1
    • 2
  1. 1.Department of Analytical BiochemistryMeiji Pharmaceutical UniversityKiyose, TokyoJapan
  2. 2.Department of Clinical GeneticsMeiji Pharmaceutical UniversityTokyoJapan
  3. 3.Department of Molecular Medical ResearchThe Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical ResearchTokyoJapan
  4. 4.Research Center for Medical GlycoscienceNational Institute of Advanced Industrial Science and TechnologyTsukubaJapan
  5. 5.Department of DermatologyKagoshima University Graduate School of Medical and Dental SciencesKagoshimaJapan

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