Coppola G, Yan Y, Hantzopoulos P, Segura E, Stroh J G, Calhoun D H
Biochemistry Program, City University of New York, City College of New York, NY 10031.
Gene. 1994 Jul 8;144(2):197-203. doi: 10.1016/0378-1119(94)90378-6.
Fabry disease is an X-linked inborn error of glycolipid metabolism caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GalA; EC 3.2.1.22). In order to obtain large quantities of this human enzyme for physical characterization and for the development of new approaches for enzyme therapy, we constructed derivatives of the Autographa californica nuclear polyhedrosis virus that produce the human enzyme. The recombinant GalA (re-GalA) is produced at high levels, and is active with both the artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, and the natural in vivo substrate, trihexosylceramide. The purified re-GalA is glycosylated and is taken up by normal and Fabry fibroblasts in cell culture. Mass spectral analysis of total monosaccharides released by hydrazinolysis indicates that it contains fucose, galactose, mannose and N-acetylglucosamine. Amino-acid sequence analysis of six proteolytic peptides corresponded to sequences predicted by the cDNA. The molecular masses of the purified enzyme, estimated by electrospray mass spectroscopy and laser desorption time-of-flight analysis are 46.85 and 46.62 kDa, respectively, approx. 10% greater than the polypeptide portion predicted by the cDNA. The recombinant enzyme retains significant catalytic activity after modification with poly(ethylene glycol), a treatment which decreases the immunogenicity and increases the circulation life of many proteins used therapeutically.
法布里病是一种X连锁的糖脂代谢先天性缺陷疾病，由溶酶体酶α - 半乳糖苷酶A（GalA；EC 3.2.1.22）缺乏所致。为了获得大量这种人类酶用于物理特性分析以及开发酶疗法的新方法，我们构建了能产生这种人类酶的苜蓿银纹夜蛾核型多角体病毒衍生物。重组GalA（re - GalA）产量很高，对人工底物4 - 甲基伞形酮基 - α - D - 吡喃半乳糖苷和天然体内底物三己糖神经酰胺均有活性。纯化后的re - GalA进行了糖基化修饰，在细胞培养中能被正常和成纤维细胞摄取。对肼解释放的总单糖进行质谱分析表明，它含有岩藻糖、半乳糖、甘露糖和N - 乙酰葡糖胺。对六个蛋白水解肽段的氨基酸序列分析与cDNA预测的序列相符。通过电喷雾质谱和激光解吸飞行时间分析估算，纯化酶的分子量分别为46.85和46.62 kDa，比cDNA预测的多肽部分大约大10%。用聚乙二醇修饰后，重组酶保留了显著的催化活性，这种处理降低了免疫原性并延长了许多治疗用蛋白质的循环寿命。
