Lipid-sensing high-throughput ApoA-I assays.
Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.
Gilead Sciences Inc., 333 Lakeside Dr., Foster City, CA 94404, USA. anita.majka@gilead.com
Anita
A
Niedziela-Majka
Latesh
L
Lad
Jeffrey W
JW
Chisholm
Leanna
L
Lagpacan
Karen
K
Schwartz
Magdeleine
M
Hung
Debi
D
Jin
Wanchi
W
Fung
Katherine M
KM
Brendza
Xiaohong
X
Liu
Nikos
N
Pagratis
Roman
R
Sakowicz
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000819:agonists / Q000378:metabolism; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050197:Atherosclerosis / Q000378:metabolism / Q000517:prevention & control; D001710:Biotin; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids; D008075:Lipoproteins, HDL / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D019809:Streptavidin
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D005456:Fluorescent Dyes; D008055:Lipids; D008075:Lipoproteins, HDL; D001710:Biotin; D019809:Streptavidin; D002784:Cholesterol