Structural and biochemical studies on Pompe disease and a "pseudodeficiency of acid α-glucosidase"
Structural and biochemical studies on Pompe disease and a "pseudodeficiency of acid α-glucosidase"
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DOI：
10.1007/s10038-007-0191-9
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发表时间：
2007-11-01
影响因子：
3.5
通讯作者：
Sakuraba, Hitoshi
中科院分区：
文献类型：
--
作者：
Tajima, Youichi;Matsuzawa, Fumiko;Sakuraba, Hitoshi
我们通过同源建模构建了人野生型酸性α-葡萄糖苷酶和具有氨基酸取代的酶的催化结构域和周围区域的结构模型，并检查氨基酸取代是否引起酶蛋白的结构和生化变化。包括p.R600C、p.S619R和p.R437C在内的错义突变预计会引起明显的结构变化。p.C103X的无义突变终止了酸性α-葡糖苷酶在其生物合成中途的翻译，并推断不允许形成活性位点口袋。在庞贝氏症患者中发现的这些错义和无义突变导致的突变蛋白质是可预见的不稳定的，并且在细胞中迅速降解。预测由p.G576S引起的结构变化很小，并且来自该氨基酸取代纯合的受试者的细胞在Western印迹上分别表现出人工底物和糖原的正常酶活性水平的15%和11%，以及相应量的酶蛋白。在受试者的器官（包括骨骼肌）中未发现糖原蓄积，因此残留的酶活性可保护细胞免于糖原蓄积。另一方面，被称为中性多态性的p.E689K几乎不影响酸性α-葡萄糖苷酶的三维结构。结合生物化学研究，对突变型酸性α-葡萄糖苷酶的结构进行计算机模拟研究有助于了解庞贝氏症的分子病理学。
We constructed structural models of the catalytic domain and the surrounding region of human wild-type acid alpha-glucosidase and the enzyme with amino acid substitutions by means of homology modeling, and examined whether the amino acid replacements caused structural and biochemical changes in the enzyme proteins. Missense mutations including p.R600C, p.S619R and p.R437C are predicted to cause apparent structural changes. Nonsense mutation of p.C103X terminates the translation of acid alpha-glucosidase halfway through its biosynthesis and is deduced not to allow formation of the active site pocket. The mutant proteins resulting from these missense and nonsense mutations found in patients with Pompe disease are predictably unstable and degraded quickly in cells. The structural change caused by p.G576S is predicted to be small, and cells from a subject homozygous for this amino acid substitution exhibited 15 and 11% of the normal enzyme activity levels for an artificial substrate and glycogen, respectively, and corresponding amounts of the enzyme protein on Western blotting. No accumulation of glycogen was found in organs including skeletal muscle in the subject, and thus the residual enzyme activity could protect cells from glycogen storage. On the other hand, p.E689K, which is known as a neutral polymorphism, little affected the three-dimensional structure of acid alpha-glucosidase. Structural study on a mutant acid alpha-glucosidase in silico combined with biochemical investigation is useful for understanding the molecular pathology of Pompe disease.