Lieberman Raquel L, D'aquino J Alejandro, Ringe Dagmar, Petsko Gregory A
Structural Neurology Lab at the Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Biochemistry. 2009 Jun 9;48(22):4816-27. doi: 10.1021/bi9002265.
Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and alpha-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using alpha-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of alpha-Gal A with DGJ. Both GCase and alpha-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in alpha-Gal A are not seen. Thermodynamic parameters obtained from alpha-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and alpha-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological chaperones for lysosomal storage disorders.
人类溶酶体酶酸性β-葡萄糖苷酶（GCase）和酸性α-半乳糖苷酶（α-Gal A）分别水解鞘脂葡萄糖神经酰胺和球三糖神经酰胺，这些酶的突变分别导致脂质代谢紊乱戈谢病和法布里病。我们研究了GCase和α-Gal A在反映内质网的中性pH环境和反映溶酶体的酸性pH环境中的结构和稳定性。这些细节对于戈谢病和法布里病的药理伴侣疗法的发展很重要，在该疗法中，小分子在内质网中结合突变酶，使突变酶能够满足溶酶体运输的质量控制要求。我们报告了pH 4.5、pH 5.5时脱辅基GCase的晶体结构，以及pH 7.5时与药理伴侣异夫糖胺（IFG）形成复合物的晶体结构。我们还使用差示扫描量热法对pH 7.4和5.2时的GCase进行了热稳定性分析。我们将我们的结果与使用α-Gal A和伴侣1-脱氧半乳糖野茉莉霉素（DGJ）的类似实验进行比较，包括α-Gal A与DGJ的首个结构。无论有无各自的亚氨基糖结合，GCase和α-Gal A在溶酶体pH下都更稳定，值得注意的是，GCase-IFG复合物的稳定性对pH敏感。我们表明，GCase中活性位点环的构象对配体结合敏感，但对pH不敏感，而在α-Gal A中未观察到类似的半乳糖或DGJ依赖性构象变化。从α-Gal A解折叠获得的热力学参数表明，在中性和溶酶体pH下，在没有亚氨基糖的情况下为两态范特霍夫解折叠，在有DGJ的情况下为非两态解折叠。综上所述，这些结果深入了解了亚氨基糖如何在热力学上稳定GCase和α-Gal A，并为溶酶体贮积症新药理伴侣的开发提供了策略。
