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Glycobiology, 2002, Vol. 12, No. 12 821-828
© 2002 Oxford University Press

Production in yeast of {alpha}-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease

Yasunori Chiba2, Hitoshi Sakuraba3, Masaharu Kotani3, Ryoichi Kase3, Kazuo Kobayashi4, Makoto Takeuchi4, Satoshi Ogasawara5, Yutaka Maruyama5, Tasuku Nakajima5, Yuki Takaoka2 and Yoshifumi Jigami1,2

2 Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; 3 Department of Clinical Genetics, Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo 113-8613, Japan; 4 Central Laboratory, Kirin Brewery Co., Ltd., Yokohama 236-0004, Japan; and 5 Division of Applied Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan

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 (Man8GlcNAc2) and acidic ([Man-P]1–2Man8GlcNAc2) 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.

1 To whom correspondence should be addressed; E-mail: jigami.yoshi@aist.go.jp


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