Influence of ApoE Content on Receptor Binding of Large, Buoyant LDL in Subjects With Different LDL Subclass Phenotypes

From the Life Sciences Division, Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Berkeley.

Correspondence to Ronald M. Krauss, MD, Lawrence Berkeley National Laboratory, Donner Laboratory, Room 465, University of California, One Cyclotron Rd, Berkeley, CA 94720. E-mail rmkrauss{at}lbl.gov

	Abstract

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Abstract—We investigated the influence of apolipoprotein (apo) E–containing particles on LDL receptor binding of large, buoyant LDL subfractions (LDL I) from subjects with predominantly large (phenotype A) and small (phenotype B) LDL particles. Direct binding by human fibroblast LDL receptors was tested at 4°C before and after removal of apoE-containing particles by immunoaffinity chromatography. The binding affinity of total LDL I in phenotype B was greater than that in phenotype A (Kd of 1.83±0.3 and 3.43±0.9 nmol/L, respectively, P<.05). LDL I from phenotype B subjects had a higher apoE to apoB molar ratio than did that from phenotype A (0.16±0.04 versus 0.06±0.02, P<.05). Nondenaturing gradient gel electrophoresis of apoE-containing LDL I isolated by immunoaffinity chromatography revealed a substantially larger peak particle diameter than in apoE-free LDL I, and comparison of LDL I composition before and after immunoaffinity chromatography suggested an increase in triglyceride content of apoE-containing particles. After removal of these particles, there was a greater than twofold reduction in LDL receptor affinity of phenotype B LDL (Kd of 1.83±0.3 to 3.76±0.6, P<.01), whereas in phenotype A no change was observed (Kd of 3.43±0.9 to 3.57±0.4, respectively). The receptor affinity of apoE-free LDL I from phenotype A and B subjects did not differ. These findings confirm that large, buoyant LDL particles from phenotype B subjects have a higher LDL receptor affinity than does LDL I from phenotype A subjects and suggest that this difference is due to an increased content of large, triglyceride-enriched, apoE-containing lipoproteins. It is possible that the accumulation of these particles reflects abnormalities in the metabolism of remnant lipoproteins that contribute to atherosclerosis risk in phenotype B subjects.

Key Words: LDL subclasses • apoE • receptor binding • fibroblasts

	Introduction

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Low density lipoproteins comprise multiple distinct subclasses differing in size, density, chemical composition, and apolipoprotein content.^{1 2} On the basis of differences in LDL subclass profiles as determined by GGE, individuals may be categorized as having a predominance of large (phenotype A) or small (phenotype B) LDL particles.³ Phenotype B is associated with increased plasma triglyceride levels, reduced HDL cholesterol levels, and as much as a threefold increase in risk for coronary artery disease.^{4 5} Phenotype B, at least in part, is genetically determined but is also strongly influenced by sex, age, and environmental factors, including abdominal obesity, oral contraceptive use, and dietary fat and carbohydrate intake.^{6 7 8 9 10}

We recently evaluated whether differences in the binding of LDL to fibroblast LDL receptors might contribute to the different LDL subclass distributions in phenotype A and B subjects.¹¹ In phenotype A subjects, we found that, consistent with other reports,^{12 13 14 15 16 17} LDL receptor binding affinity was higher in fractions containing LDL of mid-range size and density than in fractions containing either large, buoyant LDL or small, dense LDL. In phenotype B, the binding affinities of both mid-range and small LDL were similar to those for phenotype A, but the affinity for large LDL (ie, LDL I) was significantly greater.¹¹ The apoE content of large, buoyant LDL fractions was also higher in LDL I from phenotype B subjects.¹¹ Particles with additional apoE have been shown to have greater LDL receptor binding affinity than do those containing apoB but no apoE, and previous studies have documented that apoE is able to increase receptor uptake of LDL.^{18 19} Moreover, apoE-dependent receptor binding has been reported to be greatest for large LDL particles.²⁰ We therefore hypothesized that the higher receptor binding of LDL I fractions in phenotype B was due to an increase in apoE. Consistent with the hypothesis, we demonstrated that incubation with an apoE-specific monoclonal antibody substantially reduced receptor binding.¹¹

To confirm this hypothesis and directly determine whether the enhanced binding of the LDL I fraction in phenotype B subspecies was due to the presence of a subpopulation of LDL particles containing apoE, we assessed the receptor binding of LDL I subfractions from phenotype A and B subjects before and after absorption of apoE-containing particles by anti-apoE IAC.

	Methods

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Patients
We studied 11 healthy adult male volunteers, aged 37 to 71 years. Exclusion criteria included the presence of the apoE2 isoform, use of drugs known to affect lipid metabolism, and plasma triglyceride concentrations >300 mg/dL. Five subjects were classified as phenotype A and six as phenotype B on the basis of nondenaturing polyacrylamide GGE of whole plasma (see below). Clinical characteristics and the plasma lipid profiles of the subjects are shown in Table 1.

Analytical Methods
Blood samples were collected in tubes containing 1 mmol/L disodium EDTA after an overnight fast. After separation of plasma, Trolox (Aldrich Chemical Co) and aprotinin (Sigma Chemical Co) were added to a final concentration of 1 µmol/L each to inhibit oxidative modification and proteolysis, respectively. Analyses of plasma cholesterol, triglyceride, HDL cholesterol, apoB, LDL composition (triglyceride, free cholesterol, cholesteryl esters, proteins, and phospholipids), and apoE phenotypes were performed by standard laboratory procedures as previously described.^{21 22} LDL cholesterol was calculated by the formula of Friedewald. et al.²³ ApoE concentrations were measured by an antibody sandwich-style ELISA using purified goat anti-human apoE IgG (International Immunology Corp).²⁴ Reference plasma, run as controls in these assays, had intra-assay and interassay coefficients of variation equal to 6.5% and 9.5%, respectively. Assays were carried out in duplicate or triplicate with standards, control, and unknowns on the same plate. The apoB to apoE molecular ratios were estimated using the molecular weights of 550 000 for apoB²⁵ and of 34 000 for apoE.²⁶

Isolation of LDL
LDLs were isolated from plasma by preparative ultracentrifugation between d=1.019 and 1.063 g/mL under standard conditions.²⁷ NaBr was used to adjust densities, and the samples were centrifuged at 10°C in a Beckman 40 Ti fixed-angle rotor at 40 000 rpm.

ApoE IAC of LDL
ApoE-containing particles were removed from whole LDL by IAC.²⁸ In brief, affinity-purified goat anti-apoE IgG (International Immunology Corp) was coupled to Affigel-10 (Bio-Rad Laboratories) as described in the manufacturer's instructions. The immunoabsorbent was poured into a column and washed with equilibration buffer containing 20 mmol/L Tris HCl (pH 8.0), 0.15 mol/L NaCl, and 1 mmol/L disodium EDTA . LDL was dialyzed against the equilibration buffer, added to the column, and incubated with the immunoabsorbent gel overnight at 4°C. ApoE-free LDL was washed from the column with equilibration buffer and immediately concentrated by using centrifugal concentration units with 10 000 molecular weight–cutoff membranes (Amicon). ApoE-containing lipoproteins were eluted with 250 mmol/L acetic acid (pH 3), 0.15 mol/L NaCl, and 1 mmol/L disodium EDTA; immediately adjusted to 20 mmol/L Tris, pH 7.4; and concentrated as described above.

Agarose Electrophoresis and Western Blotting
Samples (1.0 µg) were applied to each lane on Beckman Paragon Lipogels and allowed to penetrate for 5 to 10 minutes before electrophoresis. The samples were then electrophoresed at 100 V for 30 minutes in the barbital buffer supplied in the Beckman Paragon Electrophoresis System kit. The samples were then transferred to nitrocellulose by diffusion. Nitrocellulose blots were then washed in TSE buffer (25 mmol/L Tris Cl, 137 mmol/L NaCl, and 3 mmol/L KCl, pH 8.0), and nonspecific binding sites were blocked by incubation in 5% (wt/vol) nonfat milk in TSE buffer for 2 hours at room temperature. The blots were then washed three times, 10 minutes per wash, in TSE buffer and incubated for 1 hour with goat anti-human apoE antisera (International Immunology Corp) diluted 1:10 000 in TSE buffer. The blots were washed again as described and incubated for 1 hour with alkaline phosphatase–conjugated rabbit anti-goat IgG (Sigma) diluted 1:2000 in TSE buffer. The blots were then washed three times in TSE buffer and incubated with a developing reagent consisting of 25 mg each of ß-naphthyl phosphate, Fast Blue BB salt, and 60 mg MgSO₄ in 50 mL of a stock solution of 1.8 g NaOH and 3.7 g borate per liter.²⁹

Equilibrium Density Gradient Ultracentrifugation
Equilibrium density gradient ultracentrifugation was performed as previously described.²¹ Total and apoE-free LDLs were adjusted to d=1.040 g/mL by dialysis in an NaBr solution at 4°C with repeated changes over a 24- to 48-hour period. The dialyzed LDLs (2.0 mL) were carefully layered above a solution of d=1.054 g/mL (2.5 mL) in a 1/2x31/2-in. Ultraclear Tube (Beckman Instruments), and 2.5 mL of a solution of d=1.0275 g/mL was layered above the LDL. The tubes were centrifuged at 17°C in a Beckman SW45 rotor in a Beckman L5–75 ultracentrifuge. The slowest setting for acceleration was used at the start of the run. After 40 hours at 40 000 rpm the rotor was allowed to coast to a stop without braking, and the tube contents were withdrawn by pipetting. As described previously, LDL I was isolated as the fraction between 0.5 and 2.5 mL³ , corresponding to the density interval 1.025 to 1.032 g/mL.^{2 30} All solutions contained 1 µmol/L Trolox, and fractions were deoxygenated by bubbling with N₂ and stored in the dark until use.

Nondenaturing Polyacrylamide GGE
Nondenaturing polyacrylamide GGE of whole plasma or lipoprotein fractions was performed at 10°C by using 2% to 16% polyacrylamide gradient gels for 24 hours at 125 V in Tris (0.09 mol/L)-boric acid (0.08 mol/L)–disodium EDTA (0.003 mol/L) buffer (pH 8.3) as described elsewhere.² Gels were fixed and stained for lipids in a solution containing oil red O in 60% ethanol at 55°C and for proteins in a solution containing 0.1% Coomassie Brilliant Blue R-250, 50% ethanol, and 9% acetic acid (vol/vol/vol). Gels were scanned at 530 nm (for oil red O staining) or at 555 nm (for Coomassie Blue staining) with a Transidyne RFT densitometer. The migration distance for each absorbance peak was determined, and the molecular diameter corresponding to each peak was calculated from a calibration curve generated from the migration distances of protein standards of known diameter, which included carboxylated latex beads (Duke Scientific), thyroglobulin, and apoferritin (high molecular weight standards, Pharmacia) with molecular diameters of 380 Å, 170 Å, and 122 Å, respectively, and lipoprotein calibrators of previously determined particle size. LDL subclass phenotypes were determined as described previously.⁶

Iodination of LDL Subfractions
LDL samples were radiolabeled with Na[¹²⁵]I (Amersham, Inc) and the method of McFarlane³¹ as modified by Bilheimer et al.³² Iodinated samples were dialyzed extensively against saline/EDTA. Radiolabeled samples were assayed for protein concentration (Bio-Rad DC Kit); trichloroacetic acid–precipitable (>90%) and lipid-extractable (<5%) counts were determined by standard techniques before the samples were used in experiments. To minimize any potential for¹²⁵I-mediated damage of LDL particles, the specific activity was maintained at levels <300 disintegrations per minute per nanogram protein (mean, 191±72). The values did not differ significantly among LDL fractions from phenotype A versus phenotype B subjects and were similar in LDL fractions obtained before and after IAC.

LPDS Preparation
Plasma samples (50 to 100 mL), d>1.21 g/mL, were pooled, transferred to dialysis tubing, and packed in Aquacide III on ice for concentration. The concentrated serum was dialyzed against 4 L saline overnight three times at 4°C. The serum was then prefiltered, and 1 mmol/L CaCl₂ and 50 µg/mL gentamicin (Sigma) were added. The serum was heated at 37°C for 1 hour and then at 56°C for 30 minutes, sterilized by filtration, and stored at -70°C until use. Protein concentration was 60 mg/mL.

Fibroblast LDL Receptor Binding Assays
Human fibroblast cell lines were maintained at 37°C and 5% CO₂ in DMEM containing 10% fetal calf serum and used at passages 9 to 12. Five days prior to the experiment, cells were grown to confluence in six-well tissue culture dishes (Corning) at a density of 50 000 cells per well after the LDL receptors had been upregulated by incubation for 48 hours prior to the experiment with DMEM plus 10% LPDS. The receptor binding assays were based on the methods Campos et al¹¹ and Arnold et al.³³ Eight concentrations between 0.125 and 16 µg/mL were tested for each labeled LDL. Each concentration was assayed in duplicate, and a 90-fold excess of unlabeled LDL was included for each concentration as a nonspecific binding control. These solutions were made in DMEM plus 10% LPDS. Homologous unlabeled LDL was used in one set of experiments, but since it did not appear to affect the results, we used heterologous LDL. We also included labeled, unfractionated LDL (d=1.019 to 1.063 g/mL) from the same donor (LDL predominant peak size, 275 Å) in all the experiments as an internal control. After 4.5 hours on ice at 4°C, the medium was removed and cells were washed and collected as referenced. Counting was done in a Packard Auto-Gamma 800, and protein concentrations were determined using the BCA assay (Pierce Chemical Co). Binding data were analyzed by Scatchard plot.³⁴ The Kd for the control LDL, determined on six assays from two LDL preparations, was 2.02±0.30 nmol/L. One apoE-free LDL I fraction of a phenotype B subject was lost during processing, and therefore, results for this group are from five subjects.

Statistical Analysis
Statistical analysis was performed using StatView II software. Differences between means were evaluated by Student's t test, the Mann-Whitney U test, or Wilcoxon rank test when suitable.

	Results

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Lipids and Lipoproteins
Levels of total and LDL cholesterol were not different between subjects with phenotypes A or B (Table 1); as described previously,⁵ triglyceride levels were significantly higher and HDL cholesterol levels and LDL peak particle diameter lower in phenotype B subjects. There were similar numbers of individuals with apoE3/3 and apoE4/3 isoform phenotypes in each group.

LDL I Composition and Apolipoprotein Content
Following anti-apoE IAC, the isolated LDL I from the unbound fraction was analyzed for apoE concentration by ELISA and Western blotting of agarose gels. ApoE levels were below the level of detection in all subjects. Fig 1 displays representative densitometric tracings from 2% to 14% GGE of total LDL, the total LDL fraction retained by the anti apoE IAC column, and the LDL I ultracentrifugal fraction prepared from the fraction that was not retained on the column. The findings demonstrate in both phenotypes a single, distinct species of particles retained by the anti-apoE column, with a diameter >290 Å, larger than that described for LDL I (264 to 285 Å, see Reference 30³⁰ ) and falling within the range for intermediate density lipoproteins.³⁵ Removal of the apoE-containing particles resulted in a significant reduction of LDL I mass in both groups of subjects (Table 2; from 58.2±7.3 to 51.6±6.9 mg/dL, P<.05, in phenotype A and from 35.3±2.8 to 25.6±3.0 mg/dL, P<.05, in phenotype B). As expected, the mass of total LDL I was significantly greater in phenotype A than phenotype B subjects. However, anti-apoE IAC resulted in only an 11% reduction in the mass of LDL I in phenotype A, whereas there was a 27% reduction in phenotype B (P<.05). Estimation of plasma mass concentrations of apoE-containing LDL I particles showed significantly higher levels in phenotype B versus phenotype A (9.7±0.6 versus 6.6±0.9 mg/dL, P<.05). These differences are consistent with the higher apoE to apoB molar ratio in LDL I from phenotype B compared with phenotype A: 0.16±0.04 versus 0.06±0.02, P<.05. Moreover, in phenotype B, total LDL I and the apoE-free fraction were enriched in triglyceride in comparison with corresponding fractions from phenotype A subjects (Table 2). The apoE-free particles, however, had substantially lower triglyceride and higher cholesteryl ester content than that estimated for apoE-containing LDL I in both phenotypes.

View larger version (23K): [in this window] [in a new window]   Figure 1. GGE (2% to 14%) densitometric tracings of total LDL (A and B), the total LDL fraction retained by the anti-apoE IAC column (apoE-containing LDL, C and D), and the LDL I ultracentrifugal fraction prepared from the fraction that was not retained by the column (apoE-free LDL I, E and F). The results are from representative phenotype A and B subjects. Diameters of major peaks are shown in Ångstroms, and intervals corresponding to the sizes of the major human LDL subclasses I through IV are shown on the x axis.1 Due to variation in the amounts of samples applied to the gels, peak amplitudes do not represent comparable lipoprotein concentrations.

Receptor Binding Affinity of LDL I
Fig 2 shows Scatchard plots for specific binding of LDL I subfractions from phenotype A (A) and phenotype B (B) subjects to fibroblast LDL receptors at 4°C. The slope of the line represents the negative reciprocal of the equilibrium dissociation constant (Kd), and the x-axis intercept represents the number of receptor-bound lipoproteins at receptor saturation (B_max). It is evident that the binding affinity of total and apoE-free LDL I from phenotype A subjects did not differ, whereas in phenotype B, the binding affinity of apoE-free LDL I was substantially reduced in comparison with total LDL I.

View larger version (15K): [in this window] [in a new window]   Figure 2. Scatchard plots of direct binding of 125I-LDL I to fibroblasts at 4°C from 5 phenotype A and 6 phenotype B subjects. LDL receptors were upregulated by incubations for 48 hours prior to the experiment with DMEM plus 10% LPDS. Cells were incubated for 4.5 hours on ice with eight different concentrations (0.125 to 16 µg protein/mL) of DMEM/LPDS containing labeled LDL I. Nonspecific binding was determined by incubation of a 90-fold excess of unlabeled LDL (d=1.019 to 1.063 g/mL). A control sample (unfractionated LDL from one of the authors; predominant LDL peak size, 275 Å; Kd 2.02 nmol/L) was included in each experiment. A, Direct binding of total LDL I (filled squares and solid line) and apoE-free LDL I (filled triangles and dotted line) of phenotype A subjects; B, direct binding of total LDL I (filled squares and solid line) and apoE-free LDL I (filled triangles and dotted line) of phenotype B subjects. Results at each point are shown as the mean±SEM. For some points the SEM was too small to be displayed. Table 3 gives mean values for Kd and Bmax values derived from these data.

The mean values for Kd and B_max for total and apoE-free LDL I from the two groups of subjects are shown in Table 3. Total LDL I from phenotype A subjects had a lower binding affinity than did total LDL I from phenotype B subjects, as documented by a higher Kd, whereas B_max was not significantly different. In phenotype B subjects, removal of apoE-containing particles led to a greater than twofold increase in Kd, again with no change in B_max. In contrast, in phenotype A subjects, no change was observed in either parameter. ApoE-free LDL I from phenotype A or B subjects did not differ significantly with regard to receptor affinity or B_max.

	Discussion

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Subjects with a predominance of small, dense LDL particles (phenotype B) commonly have an array of interrelated metabolic aberrations, including increased levels of triglyceride-rich lipoproteins, reduced levels of HDL, and a relative resistance to insulin-mediated glucose uptake.^{5 36 37} It has been suggested that a shift from larger LDL to smaller LDL particles in individuals with this trait is mediated, at least in part, by conversion of larger to smaller LDL species and that this difference may result from the action of lipases (in particular hepatic triglyceride lipase) on large, triglyceride-enriched LDL particles.^{38 39}

Recent results from our laboratory have indicated that the increased levels of small LDL particles in phenotype B versus A subjects are not associated with reduced receptor-mediated clearance.¹¹ This study confirmed earlier reports that both large, buoyant and small, dense LDLs have reduced receptor affinity in comparison with LDL of mid-range size and density¹² and also showed that there were no differences in the receptor binding of small or midsize LDL in phenotype A versus phenotype B.¹¹ In contrast, the receptor binding affinity of LDL I from phenotype A subjects was found to be significantly lower than that for LDL I from phenotype B.¹¹ This difference was associated with a lower apoE content of LDL I from phenotype A subjects. A critical role for apoE in the enhanced receptor binding of LDL I from phenotype B subjects was suggested by the finding of reduced receptor affinity after incubation with an apoE-specific monoclonal antibody.¹¹ We could not, however, rule out the possibility that the apoE monoclonal antibody sterically interfered with the apoB-mediated binding of the particles. In the present study, therefore, we sought to directly determine by use of anti-apoE IAC whether removal of apoE-containing particles from LDL I of phenotype B subjects reduced its receptor binding affinity. Consistent with our earlier results, we found that in phenotype B subjects there is, on average, 1 molecule of apoE for every 6 LDL I particles, whereas in phenotype A, the ratio is 1 to 17. With essentially complete removal of apoE from the ultracentrifugal subfraction containing LDL I, there was a greater than twofold reduction in receptor binding affinity for phenotype B but no change for phenotype A. The bulk of the LDL retained by the apoE immunoaffinity column consisted of particles with a size distribution larger than what we have typically observed for LDL particles.²

Thus, phenotype B subjects have increased levels of a minor, apoE-enriched species that overlaps in density with LDL I but that has a particle size and an apparent triglyceride content more similar to those found in intermediate density lipoproteins.³⁵ On the basis of the reduction in LDL receptor affinity that we observed after immunochemical adsorption of these particles, we infer that they are responsible for the increased receptor binding of the LDL I fraction from phenotype B subjects. It is not clear, however, why these particles accumulate in the plasma of phenotype B subjects. This accumulation may be due to an increase in their production or greater competition for receptor binding sites due to the increased levels of VLDL and IDL that are found in the plasma of such patients.⁴⁰ It may also be that in vivo clearance of these particles is dependent on other receptors whose function may be impaired in phenotype B subjects.^{41 42} In this regard, our studies using LDL receptors in fibroblasts may not be indicative of LDL receptor function in hepatocytes. It is also possible that other characteristics of apoE-containing LDL I in phenotype B subjects (eg, increased content of apoCIII) may result in impairment in their metabolism or clearance.⁴³

One potential concern regarding the present findings is the possibility of redistribution of apoE among lipoproteins with ultracentrifugation. However, unpublished results from our laboratory have documented an increased content of apoE in large LDL from phenotype B compared with phenotype A subjects when lipoproteins are separated by either gel filtration or IAC rather than ultracentrifugation (P.J. Blanche et al, unpublished data, 1997).

It is noteworthy that the apoE-free LDLs I from phenotype A and B subjects were similar in binding affinity and, except for increased triglyceride content in phenotype B, were similar in chemical composition. The present findings, therefore, do not provide an explanation for the lower concentrations of apoE-free LDL I in phenotype B versus phenotype A subjects.

In conclusion, our findings demonstrate that the most buoyant LDL subfraction from phenotype B subjects includes increased levels of apoE-enriched particles that appear to be responsible for the increased affinity of this subfraction for fibroblast LDL receptors. It is possible that the accumulation of these particles reflects abnormalities in metabolism of remnant lipoproteins that contribute to atherosclerosis risk in these subjects.

	Selected Abbreviations and Acronyms

 

DMEM = Dulbecco's modified Eagle's medium GGE = gradient gel electrophoresis IAC = inmunoaffinity chromatography LPDS = lipoprotein-deficient serum

	Acknowledgments

 
This work was supported by the Director, Office of Energy Research, Office of Health and Environmental Research, Division of the US Department of Energy, under contract No. DE-AC03–76SF00098, National Institutes of Health program project grant HL18574, and a grant from the National Dairy Promotion and Research Board (to R.M.K.), administered in cooperation with the National Dairy Council. C.M.B. was the recipient of grants from Consiglio Nazionale delle Ricerche, Rome, Italy, and from the International Atherosclerosis Society, Visiting Fellowship Award, Houston, Tex. The authors wish to thank Laura Holl for laboratory analysis and Linda Abe for preparation of the figures.

Received June 11, 1997; accepted November 24, 1997.

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