Bruce C, Davidson W S, Kussie P, Lund-Katz S, Phillips M C, Ghosh R, Tall A R
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
J Biol Chem. 1995 May 12;270(19):11532-42. doi: 10.1074/jbc.270.19.11532.
The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and is associated with high density lipoproteins (HDL). To understand the mechanism of interaction of CETP with HDL, we studied the binding of pure recombinant CETP to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal particles. Separating bound from free CETP using native gradient gel electrophoresis, complexes of CETP with 10-nm hydrodynamic diameter discoidal particles migrated with a diameter of 12-16 nm, compared with approximately 7.5 nm for CETP. At lower ratios of CETP to discs, CETP bound to discs without displacement of apoA-I. CETP alone was unable to generate discoidal complexes. Cross-linking and fluorescence resonance energy transfer experiments indicated that CETP bound to discs as monomers. Cross-linking of CETP to apoA-I in discs suggested proximity of apoA-I and CETP. By negative-stain electron microscopy, discoidal complexes containing CETP and CETP monoclonal antibody showed localization of antibody molecules to the disc edge, suggesting that CETP was bound to the disc edge. The binding of CETP to discs of different composition or size was studied. Discs (10-nm Stokes diameter) prepared with either apoA-I or apoA-II had a similar Kd (120 nM). Inclusion of 1 mol % cholesteryl oleate, 5 mol % cholesterol, or 6 mol % phosphatidylinositol increased the binding affinity of CETP 3-10 times (20-30 nM). In comparison, plasma HDL3 had a Kd of approximately 450 nM. For POPC/apoA-I discs, 10-nm discs bound CETP with much higher affinity than smaller 7.8-nm discs (Kd = 1-2 microM). 7.7-nm hydrodynamic diameter POPC/apoA-I spherical particles containing either triolein or cholesteryl oleate in their core bound CETP with higher affinity (Kd = 50-100 nM) than 7.8-nm POPC/apoA-I discs. Thus, CETP appears to bind to the perimeter of discoidal particles, possibly in a process in which flexible segments in apoA-I or apoA-II accommodate CETP at the disc edge. The binding of CETP to HDL is markedly influenced by overall particle size and shape and by lipid composition, and the increased binding affinity for cholesterol- and cholesteryl ester-containing discs suggests a higher affinity of CETP for nascent than mature HDL.
血浆胆固醇酯转运蛋白（CETP）介导脂蛋白之间中性脂质的转运，且与高密度脂蛋白（HDL）相关。为了解CETP与HDL的相互作用机制，我们研究了纯重组CETP与1-棕榈酰-2-油酰磷脂酰胆碱（POPC）/载脂蛋白A-I盘状颗粒的结合。使用天然梯度凝胶电泳分离结合态和游离态的CETP，与直径约7.5nm的CETP相比，CETP与流体动力学直径为10nm的盘状颗粒形成的复合物迁移时直径为12 - 16nm。在较低的CETP与盘状颗粒比例下，CETP结合到盘状颗粒上而不置换载脂蛋白A-I。单独的CETP无法形成盘状复合物。交联和荧光共振能量转移实验表明，CETP以单体形式结合到盘状颗粒上。CETP与盘状颗粒中的载脂蛋白A-I交联表明载脂蛋白A-I和CETP接近。通过负染电子显微镜观察，含有CETP和CETP单克隆抗体的盘状复合物显示抗体分子定位于盘状边缘，表明CETP结合在盘状边缘。研究了CETP与不同组成或大小的盘状颗粒的结合。用载脂蛋白A-I或载脂蛋白A-II制备的盘状颗粒（斯托克斯直径10nm）具有相似的解离常数（Kd）（120nM）。加入1mol%油酸胆固醇、5mol%胆固醇或6mol%磷脂酰肌醇可使CETP的结合亲和力提高3 - 10倍（20 - 30nM）。相比之下，血浆HDL3的Kd约为450nM。对于POPC/载脂蛋白A-I盘状颗粒，10nm的盘状颗粒比7.8nm的小盘状颗粒结合CETP的亲和力高得多（Kd = 1 - 2μM）。核心含有三油精或油酸胆固醇的流体动力学直径7.7nm的POPC/载脂蛋白A-I球形颗粒比7.8nm的POPC/载脂蛋白A-I盘状颗粒结合CETP的亲和力更高（Kd = 50 - 100nM）。因此，CETP似乎结合在盘状颗粒的周边，可能是载脂蛋白A-I或载脂蛋白A-II中的柔性片段在盘状边缘容纳CETP的过程。CETP与HDL的结合受到颗粒总体大小和形状以及脂质组成的显著影响，并且对含胆固醇和胆固醇酯的盘状颗粒结合亲和力的增加表明CETP对新生HDL的亲和力高于成熟HDL。
