ApoA-I-Mediated Lipoprotein Remodeling Monitored with a Fluorescent Phospholipid
Edward B. Neufeld,
Masaki Sato,
Scott M. Gordon,
Vinay Durbhakula,
Nicolas Francone,
Angel Aponte,
Gizem Yilmaz,
Denis Sviridov,
Maureen Sampson,
Jingrong Tang,
Milton Pryor,
Alan T. Remaley
Affiliations
Edward B. Neufeld
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Masaki Sato
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Scott M. Gordon
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Vinay Durbhakula
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Nicolas Francone
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Angel Aponte
Proteomics Core, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Gizem Yilmaz
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Denis Sviridov
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Maureen Sampson
Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA
Jingrong Tang
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Milton Pryor
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Alan T. Remaley
Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
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.
