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Interactions of apolipoprotein A-I with high-density lipoprotein particles.

ABSTRACT: Although the partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between high-density lipoprotein (HDL)-bound and -unbound states is an integral part of HDL metabolism, the factors that control binding of apoA-I to HDL particles are poorly understood. To address this gap in knowledge, we investigated how the properties of the apoA-I tertiary structure domains and surface characteristics of spherical HDL particles influence apoA-I binding. The abilities of (14)C-labeled human and mouse apoA-I variants to associate with human HDL and lipid emulsion particles were determined using ultracentrifugation to separate free and bound protein. The binding of human apoA-I (243 amino acids) to HDL is largely mediated by its relatively hydrophobic C-terminal domain; the isolated N-terminal helix bundle domain (residues 1-190) binds poorly. Mouse apoA-I, which has a relatively polar C-terminal domain, binds to human HDL to approximately half the level of human apoA-I. The HDL binding abilities of apoA-I variants correlate strongly with their abilities to associate with phospholipid (PL)-stabilized emulsion particles, consistent with apoA-I-PL interactions at the particle surface being important. When equal amounts of HDL2 and HDL3 are present, all of the apoA-I variants partition preferentially to HDL3. Fluorescence polarization measurements using Laurdan-labeled HDL2 and HDL3 indicate that PL molecular packing is looser on the more negatively charged HDL3 particle surface, which promotes apoA-I binding. Overall, it is clear that both apoA-I structural features, especially the hydrophobicity of the C-terminal domain, and HDL surface characteristics such as the availability of free space influence the ability of apoA-I to associate with HDL particles.

SUBMITTER: Nguyen D

PROVIDER: S-EPMC3603221 | biostudies-other | 2013 Mar

REPOSITORIES: biostudies-other

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Project description:High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL's journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand-receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.

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Interactions of apolipoprotein A-I with high-density lipoprotein particles.

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