Identification of an ApoA-I ligand domain that interacts with high-affinity binding sites on HepG2 cells

We have previously described the presence of two (high- and low-affinity) H DL binding sites on the hepatoma cell line (HepG2) (R. Barbaras, X. Collet, H. Chap, and B. Perret (1994) Biochemistry 33, 2335-2340]. Moreover, apoA- I, the major HDL apolipoprotein, interacts with these two binding sites, wh ile lipid-free apoA-I binds only to the high-affinity sites. Using tryptic HDL fragments and HepG2 cell monolayers as an "affinity matrix," we identif ied an apoA-I peptide of 16 amino acids, spanning between residues 62 and 7 7, as a ligand domain. The corresponding synthetic peptide displays high-af finity (K-d similar to 10(-7) M) and low-capacity (B-max 8 pmol/mg of cell protein) binding components, Competition experiments with this peptide, usi ng I-125-labeled free apoA-I as a ligand, show that this binding correspond s to the high-affinity binding sites already described. In conclusion, we i dentified the apoA-I 62-77 region as a specific high-affinity ligand domain of HDL on HepG2 cells. (C) 2000 Academic Press.