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.

Keywords: ATP binding cassette transporter A1; POLARIC; apolipoprotein A-I; cholesterol efflux; high density lipoprotein; hydrophobicity-sensitive fluorescence probe.

Fig. 1.

Labeling of apoA-I-V53C variant by POLARIC-maleimide. A: Effect of POLARIC-labeling on ABCA1-dependent cholesterol efflux. BHK/ABCA1 cells were treated with (filled bars) or without (open bars) 10 nM mifepristone for 20 h and incubated with indicated acceptors (5 μg/ml) for 6 h. Cholesterol content in the medium was determined using a fluorescence enzyme assay. B: Fluorescence emission spectra of apoA-I-POLARIC in the lipid-free state (dotted line) or dHDL particles (solid line). Protein concentrations were 5 μg/ml.

Fig. 2.

Detection of HDL formation by apoA-I-POLARIC in BHK/ABCA1 cells. BHK/ABCA1 cells were treated with or without 10 nM mifepristone for 20 h and incubated with the indicated concentrations of apoA-I-POLARIC for 6 h. A: Fluorescence intensity of the medium was determined. No cells (open diamonds), DMSO-treated cells (open circles), mifepristone-treated cells (closed circles). B: Cholesterol efflux from DMSO-treated cells (open circles) and mifepristone-treated cells (closed circles) was determined. Increase in fluorescence intensity of apoA-I-POLARIC was calculated by subtracting the value of no-cells from that of DMSO-treated cells (open squares) or that of mifepristone-treated cells (closed squares).

Fig. 3.

Separation of HDL particles by gel filtration. BHK/ABCA1 cells were treated without (A) or with (B, C) 10 nM mifepristone for 20 h and incubated with 5 μg/ml apoA-I-POLARIC for 6 h. Concentrated medium was separated by gel filtration chromatography [Superdex 200 column (16/60 PG)] as described in the Materials and Methods. Fluorescence intensity (solid line) and cholesterol content (dotted line) in each fraction were determined (A–C). apoA-I protein in fractions 55–78 was detected with anti-apoA-I antibody (upper panel), and the amount of apoA-I (bars) was analyzed using a Fujifilm LAS-4000 mini imaging system (C). ▽, 8.3 nm; ▼, 10.0 nm; WB, Western blot.

Fig. 4.

Detection of HDL formation by apoA-I-POLARIC in THP-1 macrophages. A: THP-1 macrophages were treated with 10 μM TO901317 for 24 h to induce the expression of ABCA1. Cell lysates (10 μg) were separated by 7% polyacrylamide gel electrophoresis, and ABCA1 and vinculin (loading control) were detected using the indicated antibodies. The amount of ABCA1 was normalized against vinculin. B, C: THP-1 macrophages were treated with (filled bars) or without (open bars) 10 μM TO901317 for 24 h and incubated with or without apoA-I-POLARIC (2.5 μg/ml) for 6 h. Cholesterol content in the medium (B) and fluorescence intensity of the medium (C) were determined (n = 3). ***P < 0.001; WB, Western blot.

Fig. 5.

Inhibition of HDL formation by cyclosporine A. BHK/ABCA1 cells were treated with (filled bars) or without (open bars) 10 nM mifepristone for 20 h and incubated with 2.5 μg/ml apoA-I-POLARIC in the presence of the indicated concentrations of cyclosporine A for 6 h. Fluorescence intensity of the medium was determined. CsA, cyclosporine A. **P < 0.01; ***P < 0.001; n.s., not significant.

Fig. 6.

Effect of cell death on the detection of HDL formation by apoA-I-POLARIC. BHK/ABCA1 cells were treated with (filled bars) or without (open bars) 10 nM mifepristone for 20 h and incubated with 2.5 μg/ml apoA-I-POLARIC in the presence or absence of 0.1 mg/ml CaCl₂ and 0.1 mg/ml MgCl₂6H₂O for 6 h. Cholesterol content in the medium (A) and fluorescence intensity of the medium (B) were determined. **P < 0.01; ***P < 0.001; n.s., not significant.