Lipid Sensing Apolipoprotein A-I For Novel High-Throughput Lipidation Assays

Apolipoprotein A-I (ApoA-I) is the primary protein component of high density lipoproteins (HDL). ApoA-I plays an important role in cholesterol metabolism by mediating the formation of nascent HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by the ATP-binding cassette A1 (ABCA1). Insufficient ABCA1 activity my lead to reduced HDL formation, reduced cholesterol efflux and the development of arteriosclerosis. The standard radioactive assay for measuring cholesterol transport to lipid-free ApoA-I has low through-put, poor dynamic range and fails to measure phospholipid transferred with cholesterol. We describe the development of two sensitive, non-radioactive high-throughput assays that report on the lipidation state of ApoA-I and may have applications for studying ABCA1 function and HDL metabolism: a homogenous assay based on the Time Resolved FRET (TR-FRET) and a discontinuous assay that uses the Epic. The TR-FRET assay employs a fluorescent ApoA-I where Cysteine is labeled with the FRET acceptor HiLyte-Fluor-647 and an N-terminal Biotin-AviTag is bound to the streptavidin-Terbium conjugate. When this ApoA-I was incorporated into recombinant HDL, TR-FRET decreased proportionally to the increase in the ratio of lipid to ApoA-I in agreement with the expansion of the surface area of lipids concomitant with the increase in separation of N-terminal and central regions of the protein and demonstrated that the HTRF assay was sensitive to the amount of lipid associated with ApoA-I. The Epic is a label-free platform that allows for the observation of direct biomolecular interactions via a resonant wavelength shift which is proportional to the mass bound to the surface. In the Epic assay, biotinylated ApoA-I was captured on streptavidin-coated sensor. The response was proportional to the amount of lipids associated with ApoA-I indicating that the assay could sense lipidation of ApoA-I.