Neufeld Edward B, Sato Masaki, Gordon Scott M, Durbhakula Vinay, Francone Nicolas, Aponte Angel, Yilmaz Gizem, Sviridov Denis, Sampson Maureen, Tang Jingrong, Pryor Milton, Remaley Alan T
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proteomics Core, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Biology (Basel). 2019 Jul 12;8(3):53. doi: 10.3390/biology8030053.
We describe simple, sensitive and robust methods to monitor lipoprotein remodeling and cholesterol and apolipoprotein exchange, using fluorescent Lissamine Rhodamine B head-group tagged phosphatidylethanolamine (*PE) as a lipoprotein reference marker. Fluorescent Bodipy cholesterol (*Chol) and *PE directly incorporated into whole plasma lipoproteins in proportion to lipoprotein cholesterol and phospholipid mass, respectively. *Chol, but not *PE, passively exchanged between isolated plasma lipoproteins. Fluorescent apoA-I (*apoA-I) specifically bound to high-density lipoprotein (HDL) and remodeled *PE- and *Chol-labeled synthetic lipoprotein-X multilamellar vesicles (MLV) into a pre-β HDL-like particle containing *PE, *Chol, and *apoA-I. Fluorescent MLV-derived *PE specifically incorporated into plasma HDL, whereas MLV-derived *Chol incorporation into plasma lipoproteins was similar to direct *Chol incorporation, consistent with apoA-I-mediated remodeling of fluorescent MLV to HDL with concomitant exchange of *Chol between lipoproteins. Based on these findings, we developed a model system to study lipid transfer by depositing fluorescent *PE and *Chol-labeled on calcium silicate hydrate crystals, forming dense lipid-coated donor particles that are readily separated from acceptor lipoprotein particles by low-speed centrifugation. Transfer of *PE from donor particles to mouse plasma lipoproteins was shown to be HDL-specific and apoA-I-dependent. Transfer of donor particle *PE and *Chol to HDL in whole human plasma was highly correlated. Taken together, these studies suggest that cell-free *PE efflux monitors apoA-I functionality.
我们描述了简单、灵敏且稳健的方法，用于监测脂蛋白重塑以及胆固醇和载脂蛋白交换，使用荧光丽丝胺罗丹明B头部基团标记的磷脂酰乙醇胺（PE）作为脂蛋白参考标记。荧光硼二吡咯胆固醇（Chol）和PE分别按脂蛋白胆固醇和磷脂质量比例直接掺入全血浆脂蛋白中。Chol可在分离的血浆脂蛋白之间被动交换，但PE不行。荧光载脂蛋白A-I（apoA-I）特异性结合高密度脂蛋白（HDL），并将PE和Chol标记的合成脂蛋白-X多层囊泡（MLV）重塑为含有PE、Chol和apoA-I的前β-HDL样颗粒。荧光MLV衍生的PE特异性掺入血浆HDL，而MLV衍生的Chol掺入血浆脂蛋白的情况与直接掺入Chol相似，这与apoA-I介导荧光MLV重塑为HDL并伴随脂蛋白间Chol交换一致。基于这些发现，我们开发了一个模型系统来研究脂质转移，通过将荧光PE和Chol标记物沉积在硅酸钙水合物晶体上，形成致密的脂质包被供体颗粒，可通过低速离心轻易地与受体脂蛋白颗粒分离。已证明PE从供体颗粒转移到小鼠血浆脂蛋白是HDL特异性且依赖apoA-I的。供体颗粒PE和Chol向全人血浆中HDL的转移高度相关。综上所述，这些研究表明无细胞*PE流出可监测apoA-I的功能。
