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Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I.


ABSTRACT: ABCA1 mediates the efflux of cholesterol and phospholipids into apoA-I to form HDL, which is important in the prevention of atherosclerosis. To develop a novel method for the evaluation of HDL formation, we prepared an apoA-I-POLARIC by labeling the specific residue of an apoA-I variant with a hydrophobicity-sensitive fluorescence probe that detects the environmental change around apoA-I during HDL formation. apoA-I-POLARIC possesses the intact ABCA1-dependent HDL formation activity and shows 4.0-fold higher fluorescence intensity in HDL particles than in the lipid-free state. Incubation of apoA-I-POLARIC with ABCA1-expressing cells, but not ABCA1-non-expressing cells, caused a 1.7-fold increase in fluorescence intensity. Gel filtration analysis demonstrated that the increase in fluorescence intensity of apoA-I-POLARIC represents the amount of apoA-I incorporated into the discoidal HDL particles rather than the amount of secreted cholesterol. THP-1 macrophage-mediated HDL formation and inhibition of HDL formation by cyclosporine A could also be measured using apoA-I-POLARIC. Furthermore, HDL formation-independent lipid release induced by microparticle formation or cell death was not detected by apoA-I-POLARIC. These results demonstrate that HDL formation by ABCA1-expressing cells can be specifically detected by sensing hydrophobicity change in apoA-I, thus providing a novel method for assessing HDL formation and screening of the HDL formation modulator.

SUBMITTER: Omura R 

PROVIDER: S-EPMC4617144 | BioStudies | 2014-01-01T00:00:00Z

REPOSITORIES: biostudies

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