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

A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial α-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human α-galactosidase A (α-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant α-GalA contained both neutral (Man₈GlcNAc₂) and acidic ([Man-P]_1-2Man₈GlcNAc₂) sugar chains. Because an efficient incorporation of α-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 α-GalA by a newly isolated bacterial α-mannosidase. Treatment of the α-GalA with the α-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 α-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 α-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated α-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.