The Influence of Cholesteryl Ester Transfer Protein on the Composition, Size, and Structure of Spherical, Reconstituted High Density Lipoproteins

Abstract

The effect of cholesteryl ester transfer protein (CETP) on the size, composition, and structure of spherical, reconstituted HDL (rHDL) which contain apolipoprotein (apo) A-I as their sole apolipoprotein has been studied. Spherical rHDL were incubated with CETP and Intralipid for up to 24 h. During this time CETP promoted transfers of cholesteryl esters (CE) and triglyceride (TG) between rHDL and Intralipid. As a result, the rHDL became depleted of CE and enriched in TG. However, as the loss of CE from the rHDL was greater than the gain of TG, the concentration of core lipids in the rHDL decreased. The decrease in the concentration of rHDL core lipids, which was evident throughout the incubation, was accompanied by a reduction in rHDL diameter from 9.2 to 8.0 nm, the dissociation of apoA-I from rHDL and a decrease in the number of apoA-I molecules, from three/particle in the 9.2-nm rHDL, to two/particle in the 8.0-nm rHDL. Spectroscopic studies showed that the lipid-water interface and phospholipid packing of the 8.0-nm rHDL were, respectively, more polar and less ordered than those of the 9.2-nm rHDL. Quenching studies with KI revealed that the number of exposed apoA-I Trp residues in the 9.2- and 8.0-nm rHDL was two and three, respectively. Circular dichroism established that the 9.2- and 8.0-nm rHDL had identical apoA-I alpha-helical contents. The 9.2- and 8.0-nm rHDL also had identical surface charges as determined by agarose gel electrophoresis. Denaturation studies with guanidine hydrochloride demonstrated that apoA-I is more stable in 8.0-nm rHDL than in 9.2-nm rHDL. It is concluded that CETP converts rHDL to small, TG-enriched, apoA-I-depleted particles with increased lipid-water interfacial hydration and less ordered phospholipid packing. These changes are associated with enhanced stability and minor changes to the conformation of the apoA-I which remains associated with the rHDL.

The Mechanism of the Remodeling of High Density Lipoproteins by Phospholipid Transfer Protein

“…Isolation of CETP-CETP was prepared as described (21 (22,23). The assay was linear when less than 30% of the [ 3 H]CE transferred from HDL 3 to low density lipoproteins.…”

“…The respective diameters of the CE-rHDL and TG-rHDL were 9.3 and 9.5 nm, and their PL/UC/CE/TG/apoA-I molar ratios were 95.3/6.8/ 68.1/1.8/3.0 and 103.2/6.2/54.2/22.9/3.0 (Table I). The small amount of TG in the CE-rHDL probably represents traces of Intralipid co-isolating with the rHDL rather than spontaneous transfer of TG from Intralipid to the rHDL (21). Since crosslinking with bis(sulfosuccinimidyl) suberate showed that the CE-rHDL and TG-rHDL both contained three molecules of apoA-I/particle, the stoichiometries are expressed relative to three molecules of apoA-I.…”

“…The unfolding of apoA-I in CE-rHDL and TG-rHDL was assessed by incubation with 0 -8 M guanidine hydrochloride (GdnHCl) (21,35). The rHDL were added to aliquots of 50 mM Tris-HCl, pH 8.0, containing varying concentrations of GdnHCl.…”

Evidence That Cholesteryl Ester Transfer Protein-mediated Reductions in Reconstituted High Density Lipoprotein Size Involve Particle Fusion

“…All data were acquired at 25°C, with the temperature being maintained by a Lauda RM6T recirculating water bath (Lauda-Königshofen, Germany). Details of the steady-state fluorescence polarization studies using aliquots of rHDL labeled individually with 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH), and 6-propionyl-2-dimethylaminonaphthalene (PRODAN) are described elsewhere (18).…”

“…The preparation used in this study generated 227 nmol CE/ml/h. CETP was also prepared from pooled human plasma as described elsewhere (18) except that the first chromatographic step was carried out on a Macro-Prep hydrophobic interaction gel (Bio-Rad) in an XK 50/30 column at a flow rate of 10 ml/min. The CETP preparation had 12.5 units of activity/ml, where 1 unit is the transfer activity of 1 ml of pooled human lipoprotein-deficient plasma (18).…”