Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

Osamu Miyazaki,Jun Ogihara,Isamu Fukamachi,Takafumi Kasumi

doi:10.1194/jlr.m041038

Osamu Miyazaki, Jun Ogihara + Show 2 more

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https://doi.org/10.1194/jlr.m041038

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Abstract

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.

Highlights

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane
Cyanogen bromide (CNBr) dissolved in 0.2 ml of 70% trifluoroacetic acid (TFA) to a concentration of 1 mg/ml was added to the apoA-1 solution; the bottle was suffused with nitrogen gas and sealed, followed by incubation at room temperature for 24 h in the dark
In nondenaturing 2D electrophoresis, only pre␤1-HDL spots were detected in the purified fraction (Fig. 1A)

Summary

Introduction

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. The structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. In sandwich ELISA using two anti-pre␤1-HDL MAbs, reactivity with intact plasma pre␤1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. The presence or absence of lipid-free- or -poor apoA-1 in plasma and its composition and structure remain obscure [5]. In fibroblasts obtained from Tangier patients, cholesterol and phospholipid efflux to lipid-free apoA-1 is completely defective, and HDL is not formed [17]. Plasma pre␤1-HDL may be lipid-free or -poor apoA-1, the substrate (precursor) for ABCA1-mediated cholesterol efflux.

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