Weers Paul M M, Narayanaswami Vasanthy, Choy Nicole, Luty Robert, Hicks Les, Kay Cyril M, Ryan Robert O
Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA.
Biophys Chem. 2003;100(1-3):481-92. doi: 10.1016/s0301-4622(02)00300-9.
Human apolipoprotein (apo) E exists as one of three major isoforms, E2, E3 or E4. Individuals carrying the epsilon 4 allele have an increased risk of heart disease and premature onset of Alzheimer's disease. To investigate the molecular basis for this phenomenon, the N-terminal domain of apoE3, apoE2 and apoE4 were expressed in bacteria, isolated and employed in lipid binding and stability studies. Far UV circular dichroism spectroscopy in buffer at pH 7 revealed a similar amount of alpha-helix secondary structure for the three isoforms. By contrast, differences were noted in apoE-NT isoform-specific transformation of bilayer vesicles of dimyristoylphosphatidylglycerol (DMPG) into discoidal complexes. ApoE4-NT induced transformation was most rapid, followed by apoE3-NT and apoE2-NT. To determine if differences in the rate of apoE-NT induced DMPG vesicle transformation is due to isoform-specific differences in helix bundle stability, guanidine HCl denaturation studies were conducted. The results revealed that apoE2-NT was the most stable, followed by apoE3-NT and apoE4-NT, establishing an inverse correlation between helix bundle stability and DMPG vesicle transformation rate at pH 7. When the zwitterionic dimyristoylphosphatidylcholine (DMPC) was employed as the model lipid surface, interaction of apoE-NT isoforms with the lipid substrate was slow. However, upon lowering the pH from 7 to 3, a dramatic increase in the rate of DMPC vesicle transformation rate was observed for each isoform. To evaluate if the increased DMPC vesicle transformation rates observed at low pH is due to pH-dependent alterations in helix bundle stability, guanidine HCl denaturation studies were performed. ApoE2-NT and apoE3-NT displayed increased resistance to denaturation as a function of decreasing pH, while apoE4-NT showed no change in stability. Studies with the fluorescent probe, 8-anilino-1-naphthalene sulfonic acid, indicated an increase in apoE hydrophobic surface exposure upon decreasing the pH to 3.0. Taken together, the data indicate that changes in the stability of secondary structure elements in apoE-NT isoforms are not responsible for pH-induced increases in lipid binding activity. It is likely that pH-induced disruption of inter-helical tertiary contacts may promote helix bundle conformational changes that present the hydrophobic interior of the protein to potential lipid surface binding sites.
人类载脂蛋白（apo）E以三种主要异构体E2、E3或E4之一的形式存在。携带ε4等位基因的个体患心脏病和早发性阿尔茨海默病的风险增加。为了研究这一现象的分子基础，apoE3、apoE2和apoE4的N端结构域在细菌中表达、分离并用于脂质结合和稳定性研究。在pH 7的缓冲液中进行的远紫外圆二色光谱显示，三种异构体的α-螺旋二级结构含量相似。相比之下，在将二肉豆蔻酰磷脂酰甘油（DMPG）双层囊泡转化为盘状复合物的过程中，观察到apoE-NT异构体特异性转化存在差异。ApoE4-NT诱导的转化最快，其次是apoE3-NT和apoE2-NT。为了确定apoE-NT诱导DMPG囊泡转化速率差异是否是由于螺旋束稳定性的异构体特异性差异所致，进行了盐酸胍变性研究。结果显示，apoE2-NT最稳定，其次是apoE3-NT和apoE4-NT，这在pH 7时建立了螺旋束稳定性与DMPG囊泡转化速率之间的负相关。当使用两性离子二肉豆蔻酰磷脂酰胆碱（DMPC）作为模型脂质表面时，apoE-NT异构体与脂质底物的相互作用缓慢。然而，将pH从7降至3时，观察到每种异构体的DMPC囊泡转化速率显著增加。为了评估在低pH下观察到的DMPC囊泡转化速率增加是否是由于螺旋束稳定性的pH依赖性改变所致，进行了盐酸胍变性研究。随着pH降低，apoE2-NT和apoE3-NT对变性的抵抗力增加，而apoE4-NT的稳定性没有变化。用荧光探针8-苯胺基-1-萘磺酸进行的研究表明，将pH降至3.0时，apoE的疏水表面暴露增加。综上所述，数据表明apoE-NT异构体二级结构元件稳定性的变化与pH诱导的脂质结合活性增加无关。pH诱导的螺旋间三级接触破坏可能促进螺旋束构象变化，从而将蛋白质的疏水内部呈现给潜在的脂质表面结合位点。
