Gillotte K L, Davidson W S, Lund-Katz S, Rothblat G H, Phillips M C
Department of Biochemistry, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
J Biol Chem. 1996 Sep 27;271(39):23792-8. doi: 10.1074/jbc.271.39.23792.
The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.
高密度脂蛋白（HDL）及其主要蛋白质成分载脂蛋白（apo）A-I在促进从培养细胞中清除过量胆固醇方面的作用已得到充分证实；然而，其发生机制尚未完全明确。为了研究apoA-I修饰对细胞内未酯化（游离）胆固醇（FC）流出的影响，将三种重组人apoA-I缺失突变体和血浆apoA-I与1-棕榈酰-2-油酰磷脂酰胆碱（POPC）和FC结合，制成重组高密度脂蛋白（rHDL）盘状复合物。对这些颗粒进行了结构表征，并检测了它们作为小鼠L细胞成纤维细胞胆固醇受体的效率。缺失突变蛋白缺乏apoA-I的氨基末端（apoA-I（Delta44-126））、中央区域（apoA-I（Delta139-170））或羧基末端（apoA-I（Delta190-243））结构域。这三种缺失突变体均表现出脂质结合能力，并形成了盘状复合物，当以100:5:1（POPC:FC:蛋白）的摩尔比重组时，其主要直径（13.2±1.5nm）与人apoA-I形成的复合物相似。凝胶过滤图谱表明，用apoA-I（Delta190-243）制备的样品中存在未反应的蛋白质，这与apoA-I的羧基末端部分是脂质结合的重要决定因素一致。对蛋白质的α-螺旋含量百分比以及每个rHDL颗粒中的蛋白质分子数量进行测量，可了解缺失突变蛋白在盘状复合物中的排列情况。含有缺失突变体的rHDL颗粒比含有完整apoA-I的颗粒含有更多的蛋白质分子，以至于每个盘状复合物中掺入的螺旋片段数量相似。将实验测定的螺旋片段数量与可用空间估计值进行比较表明，缺失突变蛋白在rHDL边缘周围的排列可能比apoA-I更松散。在使用培养的小鼠L细胞成纤维细胞的实验中，比较了复合物去除放射性标记FC的能力。当以50μg磷脂/ml的受体浓度进行比较时，所有四种盘状复合物从L细胞膜中去除FC的能力相似。因此，本研究中引入的任何缺失都没有显著改变rHDL颗粒参与细胞内FC流出的能力。这些结果表明，有效的apoA-I介导的FC流出需要两亲性α-螺旋片段的存在，但不依赖于特定的螺旋片段。
