Abstract

This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ∼ 12, 10, 7.5, and <6 nm) and composition were purified by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ∼ 12 nm and 10 nm diameter, contained ∼ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ∼ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1.

Highlights

  • This report details the lipid composition of nascent HDL particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I. nascent HDLs (nHDLs) particles of different size and composition were purified by size-exclusion chromatography

  • A purification strategy was investigated for nHDL particles formed from the incubation of lipid-free 125I-apolipoprotein A-I (apoA-I) and ABCA1-expressing Human embryonic kidney (HEK) cells in serum-free media for 18–24 h

  • NHDL lipids We have characterized apoA-I containing nHDL particles purified from ABCA1-expressing HEK cells

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Summary

Introduction

This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ‫ف‬12, 10, 7.5, and

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