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: Yasunori Chiba, Hitoshi Sakuraba, Masaharu Kotani, Ryoichi Kase, Kazuo Kobayashi, Makoto Takeuchi, Satoshi Ogasawara, Yutaka Maruyama, Tasuku Nakajima, Yuki Takaoka, Yoshifumi Jigami
• Journal: Glycobiology (Glycobiology) Vol. 12 Issue 12 Pg. 821-8 (Dec 2002) ISSN: 0959-6658 [Print] England
• PMID: 12499404 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Oligosaccharides
• Recombinant Proteins
• alpha-Galactosidase

Topics

• Carbohydrate Sequence
• Cloning, Molecular
• Fabry Disease (drug therapy)
• Humans
• Lysosomes (enzymology)
• Molecular Sequence Data
• Oligosaccharides (chemistry)
• Recombinant Proteins (chemistry, therapeutic use)
• Saccharomyces cerevisiae (enzymology)
• alpha-Galactosidase (chemistry, genetics, therapeutic use)