title: Production in yeast of alpha-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease.
abstract: A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ([Man-P](1-2)Man(8)GlcNAc(2)) sugar chains. Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase. Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.
authors.affiliation: Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Chiba
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kotani
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Kase
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ogasawara
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Maruyama
authors.forename: Tasuku
authors.initials: T
authors.lastname: Nakajima
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takaoka
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Jigami
mesh_terms: D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D000795:Fabry Disease / Q000188:drug therapy*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000627:therapeutic use; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D000519:alpha-Galactosidase