Abstract
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.
Highlights
Lipoproteins are composed of a monolayer surface of phospholipids, cholesterol, apolipoproteins and a host of other surface-associated proteins that surround a hydrophobic lipid core of triglycerides and cholesterol ester
We first assessed the effect of labeling isolated lipoprotein subfractions with trace amounts of both fluorescent PE (*PE) and cholesterol (*Chol) by Fast Protein Liquid Chromatography (FPLC) lipoprotein analyses and found that the typical elution profiles for very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were unaltered by the labeling process (Figure 1A–C)
The distribution of *Chol and *PE among lipoproteins detected by agarose gel electrophoresis (Figure 1G) was similar to that observed by FPLC analysis (Figure 1A–C)
Summary
Lipoproteins are composed of a monolayer surface of phospholipids, cholesterol, apolipoproteins and a host of other surface-associated proteins that surround a hydrophobic lipid core of triglycerides and cholesterol ester. Biology 2019, 8, 53 enzymes and transfer proteins, as well as for their potential role in regulating the structural organization of functional lipid–protein assemblies on the surface of lipoproteins [5,6]. Complex interactions of plasma enzymes and cofactors with lipoproteins modify both their surface lipid and protein composition and core lipid composition, enabling lipoproteins to serve their various functions in systemic cholesterol and triglyceride energy homeostasis. Recent evidence suggests that perturbed lipoprotein remodeling and lipid and protein exchange may play a causal role in a variety of disorders, including, diabetes [10,11], obesity [12,13], nephropathy [14,15], and cardiovascular disease [9,11]
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