MOLECULAR DETERMINANTS OF PLASMA CHOLESTERYL ESTER TRANSFER PROTEIN-BINDING TO HIGH-DENSITY-LIPOPROTEINS

The plasma cholesteryl ester transfer protein (CETP) mediates the tran sfer of neutral lipids between lipoproteins and is associated with hig h density lipoproteins (HDL), To understand the mechanism of interacti on of CETP with HDL, we studied the binding of pure recombinant CETP t o 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal part icles, Separating bound from free CETP using native gradient gel elect rophoresis, complexes of CETP with 10-nm hydrodynamic diameter discoid al particles migrated with a diameter of 12-16 nm, compared with simil ar to 7.5 nm for CETP, At lower ratios of CETP to discs, CETP bound to discs without displacement of apoA-I. CETP alone was unable to genera te discoidal complexes, Cross-linking and fluorescence resonance energ y transfer experiments indicated that CETP bound to discs as monomers, Cross-linking of CETP to apoA-I in discs suggested proximity of apoA- I and CETP, By negative-stain electron microscopy, discoidal complexes containing CETP and CETP monoclonal antibody showed localization of a ntibody molecules to the disc edge, suggesting that CETP was bound to the disc edge, The binding of CETP to discs of different composition o r size was studied, Discs (10-nm Stokes diameter) prepared with either apoA-I or apoA-II had a similar K-d (120 nM), Inclusion of 1 mol % ch olesteryl oleate, 5 mol % cholesterol, or 6 mol % phosphatidylinositol increased the binding affinity of CETP 3-10 times (20-30 nar), In com parison, plasma HDL(3) had a K-d of similar to 450 nM, For POPC/apoA-I discs, 10-nm discs bound CETP with much higher affinity than smaller 7.8-nm discs (K-d = 1-2 mu M), 7.7-nm hydrodynamic diameter POPC/apoA- I spherical particles containing either triolein or cholesteryl oleate in their core bound CETP with higher affinity (K-d = 50-100 mu M) tha n 7.8-nm POPC/apoA-I discs, Thus, CETP appears to bind to the perimete r of discoidal particles, possibly in a process in which flexible segm ents in apoA-I or apoA-II accommodate CETP at the disc edge, The bindi ng of CETP to HDL is markedly influenced by overall particle size and shape and by lipid composition, and the increased binding affinity for cholesterol- and cholesteryl ester containing discs suggests a higher affinity of CETP for nascent than mature HDL.