Department of Analytical Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan.
Mol Genet Metab. 2011 May;103(1):26-32. doi: 10.1016/j.ymgme.2011.01.013. Epub 2011 Feb 2.
Small molecules including imino sugars are expected to act as chaperones for a mutant α-galactosidase A (GLA), which will be useful for pharmacological chaperone therapy for Fabry disease. However, there is little detailed information about the molecular mechanism. We paid attention to an M51I mutant GLA which had been reported to strongly react to an imino sugar. The predicted structural change caused by this amino acid substitution is very small and located on the surface of the molecule. We produced the mutant enzyme in yeast, and determined its enzymological characteristics. The enzymological parameter values are almost the same as those of the wild-type GLA, although the mutant enzyme is unstable not only under neutral pH conditions but also under acidic ones. Then, we directly examined the effect of imino sugars including 1-deoxygalactonojirimycin and galactostatin bisulfite on the purified mutant enzyme. The imino sugars apparently improved the stability of the mutant enzyme under both neutral and acidic pH conditions. The results of surface plasmon resonance biosensor assaying suggested that the imino sugars retained their binding activity as to the mutant enzyme under both neutral and acidic pH conditions. This information will facilitate improvement of pharmacological chaperone therapy for Fabry disease.
小分子，包括亚氨基糖，有望作为突变型α-半乳糖苷酶 A（GLA）的伴侣分子，这对于法布里病的药理学伴侣治疗将是非常有用的。然而，关于其分子机制的详细信息却很少。我们关注了一个已报道与亚氨基糖强烈反应的 M51I 突变型 GLA。该氨基酸取代所导致的预测结构变化非常小，并且位于分子表面。我们在酵母中产生了该突变酶，并确定了其酶学特性。尽管突变酶不仅在中性 pH 条件下不稳定，而且在酸性条件下也不稳定，但酶学参数值几乎与野生型 GLA 相同。然后，我们直接检查了包括 1-脱氧半乳糖氮己糖和半乳糖托品双硫酸盐在内的亚氨基糖对纯化的突变酶的影响。亚氨基糖明显改善了突变酶在中性和酸性 pH 条件下的稳定性。表面等离子体共振生物传感器分析的结果表明，在中性和酸性 pH 条件下，亚氨基糖对突变酶仍保持其结合活性。这些信息将有助于改善法布里病的药理学伴侣治疗。
