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.

中文翻译：

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在酵母中产生α-半乳糖苷酶A，一种溶酶体酶，适用于法布里氏病的酶替代疗法。

酿酒酵母与细菌α-甘露糖苷酶联合在糖蛋白中产生了哺乳动物样糖部分，为法布里病患者提供了一种更经济的酶替代疗法。我们将人类的α-半乳糖苷酶A（alpha-GalA）基因引入到S. cerevisiae突变体中，该突变体缺乏N-连接的甘露聚糖的外链。重组alpha-GalA包含中性（Man（8）GlcNAc（2））和酸性（[Man-P]（1-2）Man（8）GlcNAc（2））糖链。由于将α-GalA有效掺入人体细胞的溶酶体中需要特定受体识别的甘露糖6-磷酸（Man-6-P）残基，因此我们通过新的方法修剪了α-GalA的糖链分离的细菌α-甘露糖苷酶。用α-甘露糖苷酶处理α-GalA导致在除去磷酸二酯连接的未还原末端甘露糖后，Man-6-P残基暴露于寡糖链的未还原末端。将经处理的α-GalA有效地掺入源自法布里病患者的成纤维细胞中。摄取量是未经处理的α-GalA的三到四倍，并被添加5 mM Man-6-P抑制。掺入的α-GalA靶向溶酶体，水解的神经酰胺三己糖苷在5天后积聚在法布里成纤维细胞中。这种方法为包括Fabry病在内的许多溶酶体疾病提供了一种有效且经济的疗法。将经处理的α-GalA有效地掺入源自法布里病患者的成纤维细胞中。摄取量是未经处理的α-GalA的三到四倍，并被添加5 mM Man-6-P抑制。掺入的α-GalA靶向溶酶体，水解的神经酰胺三己糖苷在5天后积聚在法布里成纤维细胞中。这种方法为包括Fabry病在内的许多溶酶体疾病提供了一种有效且经济的疗法。将经处理的α-GalA有效地掺入源自法布里病患者的成纤维细胞中。摄取量是未经处理的α-GalA的三到四倍，并被添加5 mM Man-6-P抑制。掺入的α-GalA靶向溶酶体，水解的神经酰胺三己糖苷在5天后积聚在法布里成纤维细胞中。这种方法为包括Fabry病在内的许多溶酶体疾病提供了一种有效且经济的疗法。