VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation

Abstract

Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL<VLDL<VLDL+lipoprotein lipase. Moreover, the conformation of apoE4 depended on the lipoprotein fraction: VLDL-associated apoE4 had a more linear conformation than apoE4 associated with LDL or HDL. Further, by changing VLDL fluidity, VLDL lipolysis products significantly altered apoE4 into a more expanded conformation. Our studies indicate that after every meal, VLDL fluidity is increased causing apoE4 associated with VLDL to assume a more expanded conformation, potentially enhancing the pathogenicity of apoE4 in vascular tissue.

Fig. 1.

A: Structure of spin-labeled cholestanol-3-[17-(1,5dimethyl-hexyl)-3-hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenantren-2-ylidenethyl]2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radical. Its calculated molecular weight based on the elemental analysis (C₃₆H₆₀NO₂) is 538.88. Schematic representation of apoE4 (B) and apoE3-like (C) protein structure. In both apoE4 and apoE3, Ala-76 and Ser-241 were mutated to cysteines and labeled with nitroxyl spin label. The dotted line in represents a salt bridge between Arg-61 (R61) and Glu-255 (E255) of apoE4 showing domain interaction. In C, the salt bridge is not shown since Arg-61 is mutated to Thr (R61T), which prevents the domain interaction and is a model for apoE3.

Fig. 2.

A: Effect of fasting and postprandial plasma (from volunteer 7135) on the EPR spectra of spin- labeled apoE3-like protein and apoE4. ApoE3-like or apoE4 were incubated with pre- or postprandial plasma at 37°C for 1 h before scanning. Each spectrum represents the same number of spins from a spin-labeled apoE3 or apoE4 at a concentration of 0.2 mg/ml. B: Distribution of spin-labeled apoE3-like or apoE4 associated with plasma lipoproteins. The protein samples were incubated at 37°C for 1 h with fasting plasma samples from healthy human subjects. The lipoproteins were separated by gel electrophoresis and stained with Fat Red 7b. The VLDL, LDL, and HDL bands were excised and solubilized into 4.5 mol/l guanidine isocyanate by incubating at 65°C for 3 min. The gel extracts were subjected to EPR spectroscopy. Data represent the average of six independent measurements. Error bars represent SD. *P < 0.001 for significantly different treatments within each apoE (E3/E4) protein group. ^#P < 0.001 for significantly different treatments within each lipoprotein class.

Fig. 3.

Conformations of apoE3-like protein and apoE4 on VLDL, LDL, or HDL. A: EPR spectra of apoE4 and apoE3-like protein after incubation with VLDL or HDL (from volunteer 7135) or with buffer alone. B: The average change in the central (M_I = 0) line width estimated by the peak intensity of the spin-labeled apoE when combined with lipoprotein fractions from three healthy human subjects. Values are the mean ± SD of six independent measurements. Different letters are significantly different from each other (P < 0.001)

Fig. 4.

Postprandial apoE conformation is modulated by VLDL but not HDL. Spectra of apoE4 or apoE3-like protein containing spin labels at positions 76 and 241 in combination with HDL or VLDL fractions isolated from volunteers at either the fasting P(0) or postprandial time points (3.5 and 6 h). Insets: Lipolysis-induced structural changes in apoE-associated with VLDL. The indicated protein was incubated with VLDL or LpL-treated VLDL at 37°C for 1 h before scanning. An incubation of the protein in buffer alone is also shown as reference. All samples contained 0.2 mg/ml apoE.

Fig. 5.

Lipid fluidity of classes of lipoproteins. VLDL, LDL, and HDL were isolated from fasting plasma by density gradient centrifugation, mixed with nitoxyl spin-labeled stearic acid, and subjected to EPR spectroscopy. A, 2 µmol/l 5-DSA; B, 2 µM 12-DSA; and C, 2 µM 16-DSA.

Fig. 6.

Effect of lypolysis on fluidity of TGRL. A: The effect of lipolysis on the EPR spectra of doxyl-labeled stearic acid in VLDL (top three sets) or chylomicrons (bottom set). In each experiment, an equal amount of TGRL sample (200 mg/dl TG) was pretreated with LpL for 30 min to generate lypolysis products prior to addition of the 16-DSA spin label. The arrows in the chylomicron spectrum highlight the increase in the population of highly mobile lipids when the sample is pretreated with LpL (red tracing) as opposed to without LpL pretreatment (black tracing) indicating an increase in lipid fluidity after LpL treatment. B: Effect of lipolysis spin-labeled cholestanol was added slowly while stirring the lipoprotein sample at 37°C and incubated for 2 h prior to scanning. C: Domain interaction of apoE on synthetic lipid particles containing two different concentrations of cholesterol (9 mg/dl or 24 mg/dl). Particles were prepared from synthetic TG rich emulsions as described in Materials and Methods and then incubated with spin-labeled apoE4 or the E3-like protein for 1 h at 37°C, followed by EPR spectroscopy.

Fig. 7.

Schematics of apoE3-like and apoE4 conformations on in a lipid-free environment and associated with HDL and VLDL. The level of spectral broadening in spin-labeled apoE can be explained by the proximity of the C-terminal and N-terminal domains, which differs, depending on the isoform, lipoprotein association, and particle fluidity. The size of the arrows indicates the level of the distribution of species. The predominant species found in each condition is indicated by the larger arrow and vice versa.