Davidson W S, Lund-Katz S, Johnson W J, Anantharamaiah G M, Palgunachari M N, Segrest J P, Rothblat G H, Phillips M C
Medical College of Pennsylvania, Department of Biochemistry, Philadelphia 19129.
J Biol Chem. 1994 Sep 16;269(37):22975-82.
The influence of apolipoprotein conformation on the ability of high density lipoprotein (HDL) to remove cellular free cholesterol (FC) has not been studied in detail. To address the effects of amphipathic alpha-helix structure on cellular FC efflux, three class A helical peptides and apolipoprotein (apo) AI were complexed to dimyristoyl phosphatidylcholine (DMPC) to make discoidal complexes that were used as acceptors of cell cholesterol. The peptides consisted of an 18-amino acid, amphipathic, alpha-helical peptide with the sequence DWLKAFYDKVAEKLKEAF (18A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content than the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipoproteins and form discoidal complexes with DMPC that are similar in diameter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts; efflux of FC from both the plasma membrane and the lysosomal pools was examined. For each of the acceptors, the removal of cholesterol from the plasma membrane and lysosomal pools was equally efficient. All four discoidal complexes were equally efficient cell membrane FC acceptors when compared at saturating acceptor concentrations of > 200 micrograms of DMPC/ml of medium. However, at the same lipid concentration, protein-free DMPC small unilamellar vesicles (SUV) were significantly less efficient. The initial rates of FC removal from cells at saturating concentrations of acceptor particles (Vmax) were 12, 10, 10, and 11% per h, respectively, for the complexes containing either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher Vmax for the apoprotein/peptide-containing acceptors was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the lipid packing characteristics in such a way as to increase the rate of FC desorption from the cell surface. This interaction required amphipathic alpha-helical segments, but it was not affected by the length, number, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helical segments which suggests that this interaction is not mediated by a specific cell surface binding site.(ABSTRACT TRUNCATED AT 400 WORDS)
载脂蛋白构象对高密度脂蛋白（HDL）清除细胞游离胆固醇（FC）能力的影响尚未得到详细研究。为了探讨两亲性α-螺旋结构对细胞FC流出的影响，将三种A类螺旋肽和载脂蛋白（apo）AI与二肉豆蔻酰磷脂酰胆碱（DMPC）复合，制成盘状复合物，用作细胞胆固醇的受体。这些肽包括一种18个氨基酸的两亲性α-螺旋肽，序列为DWLKAFYDKVAEKLKEAF（18A），一个由脯氨酸残基共价连接的18A二聚体（37pA），以及乙酰-18A-酰胺（Ac-18A-NH2），其α-螺旋含量高于未封闭的18A分子。这三种肽强烈模拟载脂蛋白两亲性片段的脂质结合特性，并与DMPC形成盘状复合物，其直径（11-12nm）与以2.5:1（w:w）磷脂与蛋白质比例重构时人apoAI形成的复合物相似。在使用培养的小鼠L细胞成纤维细胞的实验中，比较了这些复合物去除放射性标记FC的能力；研究了FC从质膜和溶酶体池的流出情况。对于每种受体，从质膜和溶酶体池去除胆固醇的效率相同。当在饱和受体浓度>200μg DMPC/ml培养基下比较时，所有四种盘状复合物作为细胞膜FC受体的效率相同。然而，在相同脂质浓度下，无蛋白的DMPC小单层囊泡（SUV）的效率明显较低。对于含有18A、Ac-18A-NH2、37pA或apoAI的复合物，在受体颗粒饱和浓度下从细胞中去除FC的初始速率分别为每小时12%、10%、10%和11%，而对于DMPC SUV，每小时仅为1%的细胞FC。含载脂蛋白/肽的受体的Vmax高10倍，可能是由于载脂蛋白或肽与质膜的可逆相互作用，改变了脂质堆积特性，从而提高了FC从细胞表面解吸的速率。这种相互作用需要两亲性α-螺旋片段，但不受螺旋长度、数量或脂质结合亲和力的影响。此外，流出效率不依赖于螺旋片段的氨基酸序列，这表明这种相互作用不是由特定的细胞表面结合位点介导的。（摘要截断于400字）
