Leblond L, Marcel Y L
Laboratory of Lipoprotein Metabolism, Clinical Research Institute of Montreal, Quebec, Canada.
J Biol Chem. 1991 Apr 5;266(10):6058-67.
Nine monoclonal antibodies (mAbs) against apoA-I reacting with distinct but overlapping epitopes covering more than 90% of the sequence have been used to block the interaction of 125I-labeled high density lipoprotein (125I-HDL) with HepG2 cells in order to delineate the cell binding domain of apolipoprotein A-I (apoA-I). While 2 mAbs reacting with epitopes exclusively localized in the N-terminal region (residues 1 to 86) enhanced slightly association of 125I-HDL, all other mAbs, which react with epitopes localized in the regions of amphipathic alpha-helical repeats, inhibited that association by 9 to 15%. Although this inhibition is not significant compared to the effect of an irrelevant mAb, combination of these mAbs could significantly inhibit the association of 125I-HDL (32 to 43%) as could polyclonal antibodies (up to 95%). These results are compatible with the concept of HDL binding to these cells via the nonexclusive interaction of each of the amphipathic alpha-helical repeats of apoA-I. When the same approach was applied to block the association of 3H-cholesteryl ether (CE)-labeled HDL to HepG2 cells, each anti-apoA-I could inhibit by 15 to 25% the cellular association of cholesteryl ether while mAbs in combination or polyclonal antibodies could inhibit this association up to 45% or 60%, respectively. The cholesteryl ether radioactivity that remained associated with the cells (40%) in the presence of polyclonal antibodies could be effectively blocked by addition of an antibody against the receptor binding domain of apoE (1D7). Therefore, the differential cellular association of cholesteryl ether compared to apolipoprotein can be explained by the presence of apoE secreted by HepG2 and apoE or apoB/E receptors. Thus, we conclude that the optimum uptake of both cholesteryl ether and apoA-I of HDL by cells requires the accessibility of the entire apoA-I and the cooperative binding of the amphipathic alpha-helical repeats to HepG2 cell membranes. This type of interaction would explain the competitive binding observed for apoA-I, -A-II, and -A-IV by others.
已使用9种针对载脂蛋白A-I（apoA-I）的单克隆抗体（mAb），它们与不同但重叠的表位反应，覆盖了超过90%的序列，用于阻断125I标记的高密度脂蛋白（125I-HDL）与HepG2细胞的相互作用，以描绘载脂蛋白A-I（apoA-I）的细胞结合域。虽然2种与仅位于N端区域（第1至86位氨基酸残基）的表位反应的mAb略微增强了125I-HDL的结合，但所有其他与位于两亲性α-螺旋重复区域的表位反应的mAb，将这种结合抑制了9%至15%。尽管与无关mAb的作用相比，这种抑制不显著，但这些mAb的组合可显著抑制125I-HDL的结合（32%至43%），多克隆抗体也可如此（高达95%）。这些结果与HDL通过apoA-I的每个两亲性α-螺旋重复的非排他性相互作用与这些细胞结合的概念一致。当应用相同方法阻断3H-胆固醇醚（CE）标记的HDL与HepG2细胞的结合时，每种抗apoA-I可将胆固醇醚的细胞结合抑制15%至25%，而mAb组合或多克隆抗体分别可将这种结合抑制高达45%或60%。在多克隆抗体存在下仍与细胞结合的胆固醇醚放射性（40%）可通过添加针对apoE受体结合域的抗体（1D7）有效阻断。因此，与载脂蛋白相比，胆固醇醚在细胞中的不同结合可通过HepG2分泌的apoE以及apoE或apoB/E受体的存在来解释。因此，我们得出结论，细胞对HDL中胆固醇醚和apoA-I的最佳摄取需要整个apoA-I的可及性以及两亲性α-螺旋重复与HepG2细胞膜的协同结合。这种相互作用类型将解释其他人观察到的apoA-I、-A-II和-A-IV的竞争性结合。
