Expression of five iduronate-2-sulfatase site-directed mutations.
Expression of five iduronate-2-sulfatase site-directed mutations.
复制标题
五种艾杜糖醛酸-2-硫酸酯酶定点突变的表达。
DOI：
10.1016/s0925-4439(00)00006-5
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
P. di Natale
中科院分区：
文献类型：
--
作者：
G. Villani;A. Daniele;N. Balzano;P. di Natale
在COS细胞中表达先前在意大利Hunter患者的艾杜糖醛酸-2-硫酸酯酶（IDS）基因中鉴定的5个点突变（R88 H、R88 P、T118 I、959 delT、R468 Q），以评估它们对酶活性、加工和细胞内定位的功能后果。88精氨酸残基属于所有人硫酸酯酶中保守的CXPSR五肽，其中半胱氨酸修饰为甲酰甘氨酸是酶活性所需的。用组氨酸残基取代精氨酸导致13.7%的残留酶活性，表观Km值（133 μM）低于正常酶（327 μM），表明对底物的亲和力更高;用脯氨酸取代精氨酸导致完全不存在残留活性，与携带R88 H和R88 P突变的患者中观察到的表型一致。对于这四个错义突变，脉冲追踪标记实验显示出明显正常的成熟，然而，亚细胞分级显示出向溶酶体的运输较差。因此，残基88、118和468似乎对于加工不是必需的，但对于IDS构象是重要的。
Five point mutations (R88H, R88P, T118I, 959delT, R468Q) previously identified in the iduronate-2-sulfatase (IDS) gene of Italian Hunter patients were expressed in COS cells to evaluate their functional consequence on enzyme activity, processing and intracellular localization. The 88 arginine residue belongs to the CXPSR pentapeptide conserved in all human sulfatases, where cysteine modification to formylglycine is required for enzyme activity. Substitution of arginine with histidine residue resulted in 13.7% residual enzyme activity, with an apparent Kmvalue (133 μM) lower than that found for the normal enzyme (327 μM), indicating a higher affinity for the substrate; substitution of arginine with proline resulted in total absence of residual activity, in agreement with the phenotypes observed in patients carrying R88H and R88P mutations. For the four missense mutations, pulse-chase labelling experiments showed an apparently normal maturation; however, subcellular fractionation demonstrated poor transport to lysosomes. Therefore, residues 88, 118 and 468 appear to be not essential for processing but important for IDS conformation.
溶酶体水解酶的“假性缺陷”。
影响因子：
9.8
作者：
Thomas,GH
通讯作者：
Thomas,GH
影响因子：
2.7
作者：
S. Nordeen
通讯作者：
S. Nordeen