Xie B, Rigat B, Smiljanic-Georgijev N, Deng H, Mahuran D
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Biochemistry. 1998 Jan 20;37(3):814-21. doi: 10.1021/bi971211s.
The function of the GM2 activator protein is to act as a substrate-specific cofactor in the hydrolysis of GM2 ganglioside by beta-hexosaminidase A. Mutations in the gene encoding it result in the AB variant form of GM2 gangliosidosis. One such mutation, Cys138 Arg, results in the mutant protein being retained and degraded in the endoplasmic reticulum of mammalian cells. In order to characterize the biochemical effects of this substitution, we expressed the mutant protein in transformed bacteria. We first compared the wild-type protein produced by two bacterial expression methods, one requiring protein refolding, with activator purified from the medium of transfected CHO cells. The "activity" and circular dichroism spectrum (alpha-helical content) of all three proteins were similar, justifying the use of refolded activator from transformed bacteria in structure/function studies. Second, the mutant protein was expressed in both bacterial systems and in each retained approximately 2% of the wild type's specific activity. The presence of even this small amount of activity in the mutant protein coupled with a calculated alpha-helical content nearly identical to the wild type, strongly suggest that no major tertiary or secondary structural changes, respectively, had occurred due to the mutation. However, we demonstrate that its heat stability at 60 degrees C is reduced 14-fold, suggesting some localized change in tertiary structure. The loss of a disulfide loop was confirmed by reacting the mutant protein with Ellman's reagent. A kinetic analysis detected a large increase in the apparent K(m) of beta-hexosaminidase A for the mutant; however, there was no apparent change in Vmax. A fluorescence dequenching assay was used to evaluate the ability of the mutant protein to transport lipids and bind GM2 ganglioside. These assays detected no difference between the wild-type and mutant proteins, indicating that the Cys138 Arg substitution has no effect on these functions. We conclude that the mutation specifically affects a domain in the activator protein that is responsible for the recognition of the activator-GM2 ganglioside complex by beta-hexosaminidase A.
GM2激活蛋白的功能是在β-己糖胺酶A水解GM2神经节苷脂的过程中作为底物特异性辅因子。编码该蛋白的基因突变会导致GM2神经节苷脂贮积症的AB变异型。其中一种突变，即Cys138 Arg，会导致突变蛋白在哺乳动物细胞的内质网中滞留并降解。为了表征这种替代的生化效应，我们在转化细菌中表达了突变蛋白。我们首先比较了两种细菌表达方法产生的野生型蛋白，其中一种需要蛋白质重折叠，与从转染的CHO细胞培养基中纯化的激活剂。这三种蛋白的“活性”和圆二色光谱（α-螺旋含量）相似，证明在结构/功能研究中可以使用来自转化细菌的重折叠激活剂。其次，突变蛋白在两种细菌系统中均有表达，且每种表达形式保留的活性约为野生型的2%。突变蛋白中即使存在这么少量的活性，再加上计算得出的α-螺旋含量与野生型几乎相同，强烈表明分别没有发生主要的三级或二级结构变化。然而，我们证明其在60℃时的热稳定性降低了14倍，表明三级结构发生了一些局部变化。通过使突变蛋白与埃尔曼试剂反应，证实了二硫键环的丢失。动力学分析检测到β-己糖胺酶A对突变体的表观K(m)大幅增加；然而，Vmax没有明显变化。使用荧光猝灭测定法评估突变蛋白转运脂质和结合GM2神经节苷脂的能力。这些测定未检测到野生型和突变蛋白之间的差异，表明Cys138 Arg替代对这些功能没有影响。我们得出结论，该突变特异性地影响激活蛋白中负责β-己糖胺酶A识别激活剂-GM2神经节苷脂复合物的结构域。
