Chiba Yasunori, Sakuraba Hitoshi, Kotani Masaharu, Kase Ryoichi, Kobayashi Kazuo, Takeuchi Makoto, Ogasawara Satoshi, Maruyama Yutaka, Nakajima Tasuku, Takaoka Yuki, Jigami Yoshifumi
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan.
Glycobiology. 2002 Dec;12(12):821-8. doi: 10.1093/glycob/cwf096.
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-PMan(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.
酿酒酵母结合细菌α-甘露糖苷酶在糖蛋白中构建了类似哺乳动物的糖部分，从而为法布里病患者开发出一种更经济的酶替代疗法。我们将人α-半乳糖苷酶A（α-GalA）基因导入了一株N-连接甘露聚糖外链缺陷的酿酒酵母突变体中。重组α-GalA含有中性（Man(8)GlcNAc(2)）和酸性（Man-PMan(8)GlcNAc(2)）糖链。由于α-GalA有效掺入人细胞溶酶体需要甘露糖-6-磷酸（Man-6-P）残基被特定受体识别，我们用新分离的细菌α-甘露糖苷酶修剪α-GalA的糖链。用α-甘露糖苷酶处理α-GalA后，在去除磷酸二酯连接的非还原端甘露糖后，寡糖链的非还原端暴露了一个Man-6-P残基。处理后的α-GalA能有效掺入法布里病患者来源的成纤维细胞中。摄取量比未处理的α-GalA高3至4倍，并且添加5 mM Man-6-P可抑制摄取。掺入的α-GalA靶向溶酶体，并在5天后水解法布里成纤维细胞中积累的神经酰胺三己糖苷。该方法为包括法布里病在内的许多溶酶体疾病提供了一种有效且经济的治疗方法。
