Corrective effect on Fabry mice of yeast recombinant human alpha-galactosidase with N-linked sugar chains suitable for lysosomal delivery.
We have previously reported the production of a recombinant alpha-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant alpha-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. However, cleavage of the accumulated CTH in the dorsal root ganglia was insufficient. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant alpha-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease.
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan. sakuraba@rinshoken.or.jp.
Hitoshi
H
Sakuraba
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, 305-0044, Japan.
Yasunori
Y
Chiba
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan.
Masaharu
M
Kotani
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan.
Ikuo
I
Kawashima
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan.|CREST, JST, 4-1-8 Hon-machi, Kawaguchi, 332-0012, Japan.
Mai
M
Ohsawa
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan.
Youichi
Y
Tajima
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, 305-0044, Japan.
Yuki
Y
Takaoka
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, 305-0044, Japan.
Yoshifumi
Y
Jigami
Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan.
Hiroshi
H
Takahashi
Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan.
Yukihiko
Y
Hirai
Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan.
Takashi
T
Shimada
Supra-Biomolecular System Research Group, RIKEN Frontier Research System, 2-1 Hirosawa, Wako, 351-0198, Japan.
Yasuhiro
Y
Hashimoto
Supra-Biomolecular System Research Group, RIKEN Frontier Research System, 2-1 Hirosawa, Wako, 351-0198, Japan.
Kumiko
K
Ishii
Supra-Biomolecular System Research Group, RIKEN Frontier Research System, 2-1 Hirosawa, Wako, 351-0198, Japan.
Toshihide
T
Kobayashi
Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, 2-6 Musashidai, Fuchu, 183-8526, Japan.
Kazuhiko
K
Watabe
Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8520, Japan.
Tomoko
T
Fukushige
Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8520, Japan.
Tamotsu
T
Kanzaki
D000818:Animals; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000795:Fabry Disease / Q000628:therapy*; D005347:Fibroblasts / Q000187:drug effects; D006028:Glycosphingolipids / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000627:therapeutic use; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution; D000519:alpha-Galactosidase / Q000008:administration & dosage* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics / Q000494:pharmacology*
D003470:Culture Media; D006028:Glycosphingolipids; D011994:Recombinant Proteins; C088880:ceramide trihexoside; D000519:alpha-Galactosidase