Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity:: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin
Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity:: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin
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DOI：
10.1042/bj20070479
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发表时间：
2007-09-01
影响因子：
4.1
通讯作者：
Fan, Jian-Qiang
中科院分区：
文献类型：
--
作者：
Ishii, Satoshi;Chang, Hui-Hwa;Fan, Jian-Qiang
法布里病是由α-半乳糖苷酶A（α-Gal A）活性缺乏引起的溶酶体贮积症。为了理解在具有残余酶活性的法布里病患者中α-Gal A缺乏的分子机制，从转染的COS-7细胞中纯化具有不同错义突变的酶，并表征其生化性质。在变异型患者中检测到的突变酶（A20 P、E66 Q、M72 V、191 T、R112 H、F113 L、N215 S、Q279 E、M2961、M296 V和R3101 Q）和在轻度经典型患者中多见的突变酶（A97 V、A156 V、L166 V和R356 W）的Km和Vmax值均正常。所有突变体（E59 K除外）的降解均被Kifunensine（ER（内质网）α-甘露糖苷酶I的选择性抑制剂）处理部分抑制。在表达L166 V和R301 Q α-Gal A突变体的COS-7细胞中的代谢标记和亚细胞分级分离研究表明，突变蛋白保留在ER中，并且在没有加工的情况下降解。将DGJ（1-脱氧半乳糖野尻霉素）添加到用大量错义突变体α-Gal A cDNA转染的COS-7细胞的培养基中有效地增加了酶活性和蛋白质产量。DGJ能够使在经典（L166 V）和变体（R301 Q）法布里病患者中发现的突变体a-Gal A的细胞内加工正常化。此外，来自携带各种错义突变的经典和变异型半合子法布里病患者的成纤维细胞或成淋巴细胞中的残留酶活性可以通过用DGJ培养细胞而显著增加。这些结果表明，在具有残留酶活性的患者中，大部分突变酶具有动力学活性。ER的过度降解可能是体内酶活性缺乏的原因，DGJ方法可能广泛适用于具有错义突变的法布里病患者。
Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, 191T, R112H, F113L, N215S, Q279E, M2961, M296V and R3101Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K-m and V-max values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum)alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.