Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 2011 Jul 26;50(29):6356-64. doi: 10.1021/bi2006702. Epub 2011 Jun 29.
Apolipoprotein E (apoE) isoforms are known to differentially accumulate in the lysosomes of neuronal cells, and the deleterious effects of the apoE4 isoform in Alzheimer's disease may relate to its properties at the low lysosomal pH. However, the effect of pH on the molecular properties of full-length apoE is unclear. Here we examine the pH dependence of the monomer-dimer-tetramer reaction, of lipid binding, and of the stability of the three major apoE isoforms. Using FRET measurements, we find that the association-dissociation behavior of apoE proteins changes dramatically with changes in pH. At pH 4.5, approximating the pH of the lysosome, rate constants for association and dissociation are 2-10 times faster than those at pH 7.4. Aggregation beyond the tetrameric form is also more evident at lower pH values. Stability, as measured by urea denaturation at pH 4.5, is found to be considerably greater than that at neutral pH and to be isoform dependent. Lipid binding, as measured by turbidity clearance of unilamellar vesicles of DMPC, is faster at acidic pH values and consistent with our previous hypothesis that it is only the monomeric form of apoE that binds lipid tightly. Since apoE is more stable at pH 4.5 than at neutral pH, the more rapid apoE-lipid interactions at low pH are not correlated with the stability of the apoE isoforms, but rather to the faster association-dissociation behavior. Our results indicate that pathological behavior of apoE4 may arise from altered molecular properties of this protein at the acidic pH of the lysosome.
载脂蛋白 E（apoE）异构体已知在神经元细胞的溶酶体中差异积累，apoE4 异构体在阿尔茨海默病中的有害影响可能与其在低溶酶体 pH 下的特性有关。然而，pH 对全长 apoE 分子特性的影响尚不清楚。在这里，我们研究了单体-二聚体-四聚体反应、脂质结合以及三种主要 apoE 异构体的稳定性对 pH 的依赖性。使用 FRET 测量，我们发现 apoE 蛋白的缔合-解离行为随着 pH 的变化而剧烈变化。在 pH 4.5 时，接近溶酶体的 pH，缔合和解离的速率常数比 pH 7.4 时快 2-10 倍。在较低的 pH 值下，也更明显地超过四聚体形式的聚集。在 pH 4.5 时通过脲变性测量的稳定性被发现明显大于中性 pH 值，并且与异构体依赖性有关。如通过 DMPC 的单层囊泡的浊度清除测量的脂质结合，在酸性 pH 值下更快，与我们之前的假设一致，即只有apoE 的单体形式才能紧密结合脂质。由于 apoE 在 pH 4.5 下比在中性 pH 值下更稳定，因此在低 pH 值下更快的 apoE-脂质相互作用与 apoE 异构体的稳定性无关，而是与更快的缔合-解离行为有关。我们的结果表明，apoE4 的病理行为可能源于该蛋白在溶酶体酸性 pH 下的改变的分子特性。
