Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study
Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study
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DOI：
10.1016/s1388-1981(99)00196-1
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发表时间：
2000-02-24
影响因子：
4.8
通讯作者：
Tsibulsky, V
中科院分区：
文献类型：
--
作者：
Dergunov, AD;Smirnova, EA;Tsibulsky, V
利用荧光素和丹酚标记的载脂蛋白分子的各种光谱特性，研究了在受控条件下，胍或胆碱诱导变性后，人载脂蛋白E （apoE）在溶液中的缓慢再折叠。结果表明，apoE在溶液中再折叠的最后阶段包括一个缓慢的（在24℃下的几个小时）自相关的“开放”构象相互转化为更密集的“封闭”构象。疏水相互作用是形成这种更紧密的apoE结构的主要原因。为了通过固相结合实验可视化载脂蛋白构象的贡献和/或“活性”脂质结合载脂蛋白分子在与低密度脂蛋白受体（LDLr）结合反应中的数量，我们使用了人血浆载脂蛋白或重组（rec） apoE3与双棕榈酰磷脂酰胆碱（DPPC）或棕榈酰磷脂酰胆碱（POPC）大小不同的复合物。7个血浆蛋白复合物（4个DPPC复合物和3个POPC复合物），最终磷脂酰胆碱(PC)/蛋白摩尔比为117 ~ 279；两种PC的亲和常数K-a取平均值，并根据该比值绘制，突然从3.8 × 10(7)增加到3.8 × 10(8) M-1，过渡中点为150-180 PC/apoE摩尔比。两个DPPC复合物与rec蛋白的结合效率更高。血浆和rec apoE复合物能够与从E3/3表型患者分离的极低密度脂蛋白（VLDL）或低密度脂蛋白（LDL）竞争，与LDLr结合。同样，随着PC含量的增加，竞争效率突然增加，过渡中点为130 PC/apoE，摩尔比。在直接结合和竞争结合实验中观察到的转变可能对应于复合物表面“活性”apoE分子数量的突然增加，伴随着复合物大小和/或形状的变化。比较了apoE和apoB作为VLDL和LDL与LDL受体结合反应中相应的主要配体的效率。VLDL与LDLr的结合遵循简单的相遇复杂模型，而LDL的结合则采用更复杂的两步模型，其中包含额外的异构化步骤。结合数据的分析使我们认为，在富含tg的颗粒表面存在几个（2-3）apoE分子的连续体，导致结合亲和力增加，平均比LDL高3.5倍。提出了apoE的水相和不同的脂质结合形式之间存在复杂的平衡，特别是在体内与LDLr相互作用以及lpl刺激的脂质相的脂质分解过程中形成瞬时圆盘状脂蛋白颗粒结构。(C) 2000 Elsevier Science B.V.版权所有
Slow refolding of human apolipoprotein E (apoE) in solution after guanidine- or cholate-induced denaturation followed by dialysis under controlled conditions was investigated using various spectroscopic properties of fluorescein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow (several hours at 24 degrees C) interconversion of a self-associated 'open' conformer into a more dense 'closed' conformer. The hydrophobic interactions are primarily responsible for the formation of this more compact apoE structure. To visualize the contribution of apolipoprotein conformation and/or the number of 'active' lipid-bound apoE molecules in the reaction of binding to the low density lipoprotein receptor (LDLr) by solid-phase binding assay, the complexes of human plasma apolipoprotein or recombinant (rec) apoE3 with dipalmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidylcholine (POPC) varying in size were used. For seven complexes with plasma protein (four DPPC and three POPC complexes), the final phosphatidylcholine (PC)/protein mole ratio ranged from 117 to 279; affinity constant K-a averaged for both PCs and plotted against this ratio abruptly increased from 3.8 x 10(7) to 3.8 x 10(8) M-1 with a transition midpoint of 150-180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasma and rec apoE were able to compete with very low density lipoproteins (VLDL) or low density lipoproteins (LDL) isolated from patients with E3/3 phenotype, for binding to the LDLr. Again, the competition efficiency abruptly increased at the increase in PC content with a transition midpoint of 130 PC/apoE, mole ratio. The transitions observed both in direct and competitive binding assay probably correspond to the abrupt increase in the number of 'active' apoE molecules on the complex surface accompanying the change in the size and/or in the shape of the complexes. The efficiency of apoE and apoB as the corresponding major ligands in the binding reaction of VLDL and LDL to the LDL receptor was compared. VLDL bind to LDLr following a simple encounter complex model, while LDL binding Was characterized by a more complex two-step model with an additional isomerization step. The analysis of the binding data led us to suggest the existence of the continuum from several (2-3) apoE molecules on the surface of TG-rich particles that resulted in the increased binding affinity, on average 3.5-fold higher, compared to LDL. The existence of a complex equilibrium between aqueous and different lipid-bound forms of apoE is proposed, in particular, the formation of a transient disc-lipoprotein particle structure during the interaction with LDLr in vivo as well as in LPL-stimulated lipolysis Of the lipid phase of the particle. (C) 2000 Elsevier Science B.V. All rights reserved.