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Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation.

ABSTRACT: The quality and quantity of high-density lipoprotein (HDL) in blood plasma are important for preventing coronary artery disease. ATP-binding cassette protein A1 (ABCA1) and apolipoprotein A-I (apoA-I) play essential roles in nascent HDL formation, but controversy persists regarding the mechanism by which nascent HDL is generated. In the "direct loading model", apoA-I acquires lipids directly from ABCA1 while it is bound to the transporter. By contrast, in the "indirect model", apoA-I acquires lipids from the specific membrane domains created by ABCA1. In this study, we found that trypsin treatment causes rapid release of phosphatidylcholine (PC) and cholesterol from BHK/ABCA1 cells, and that the time course of lipid release coincides with those of trypsin digestion of extracellular domains (ECDs) of surface ABCA1 and of release of ECD fragments into the medium. This trypsin-dependent lipid release was dependent on ABCA1 ATPase activity, and did not occur in cells that express ABCG1, which exports lipids like ABCA1 but does not have large ECDs. These results suggest that the trypsin-sensitive sites on the cell surface are the large ECDs of ABCA1, and that lipids transported by ABCA1 are temporarily sequestered within the ECDs during nascent HDL formation.

SUBMITTER: Ishigami M

PROVIDER: S-EPMC5906560 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation.

Ishigami Masato M Ogasawara Fumihiko F Nagao Kohjiro K Hashimoto Hidehiko H Kimura Yasuhisa Y Kioka Noriyuki N Ueda Kazumitsu K

Scientific reports 20180418 1

The quality and quantity of high-density lipoprotein (HDL) in blood plasma are important for preventing coronary artery disease. ATP-binding cassette protein A1 (ABCA1) and apolipoprotein A-I (apoA-I) play essential roles in nascent HDL formation, but controversy persists regarding the mechanism by which nascent HDL is generated. In the "direct loading model", apoA-I acquires lipids directly from ABCA1 while it is bound to the transporter. By contrast, in the "indirect model", apoA-I acquires li ...[more]

PMID: 29670126

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Project description:ATP-binding cassette protein A1 (ABCA1) is a cholesterol transporter that contributes to the active transport/removal of excess cellular cholesterol. ABCA1 expression is up-regulated when cells accumulate cholesterol. AIMS:The purpose of this study was to determine any correlation between extracellular phospholipid levels and ABCA1 expression and function. METHODOLOGY:Human foreskin fibroblasts were incubated with cholesterol alone or cholesterol and phosphatidylcholine. Total RNA was isolated and subjected to end-point RT-PCR to compare ABCA1 transcript levels. Cell lysates were subjected to Western blot analysis to compare ABCA1 protein levels. Cells were loaded with radiolabeled cholesterol and cellular cholesterol efflux was measured in the presence and absence of apoE, a cholesterol acceptor. ApoE-dependent efflux was calculated as a measure of ABCA1-mediated efflux. RESULTS:Here we show that incubation of cholesterol-loaded human skin fibroblasts with L-?-phosphatidylcholine (PC) decreases ABCA1 mRNA and protein levels by 93% and 57%, respectively, compared to cells loaded with cholesterol alone. Similarly, PC treatment results in a 25% reduction in ABCG1 mRNA levels compared to cells treated with cholesterol alone, but there is no change in SR-BI transcript levels. Subsequent incubation of phospholipid-treated cells with a cholesterol acceptor such as apoE for 24 hours shows a 65% reduction in ABCA1-mediated cholesterol efflux compared to efflux in cells not treated with PC. During the lipid treatment itself, there is a 2.7-fold greater loss of cholesterol from PC treated cells compared to cells treated with cholesterol alone. Measurement of cholesterol in cellular lipid extracts reveals that cells incubated in the presence of phosphatidylcholine are significantly depleted of cholesterol having only 20% of the cholesterol compared to cells loaded with cholesterol alone. CONCLUSION:Thus, phosphatidylcholine facilitates removal of cellular cholesterol, thereby negating the cholesterol-dependent induction of ABCA1 message, protein and function.

Dataset Information

Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation.

Publications

Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation.

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