This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 51, 2010
Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
Hideg, D. M. Hatters, K. H. Weisgraber, J. C. Voss, and J. C.
Rutledge. VLDL lipolysis products increase VLDL fl uidity
and convert apolipoprotein E4 into a more expanded con-
formation. J. Lipid Res. 2010. 51: 1273–1283.
Supplementary key words lipid fl uidity • postprandial state • struc-
tural conformation • very low density lipoprotein
Apolipoprotein E (apoE), a 34 kDa protein that is im-
portant in lipid metabolism and cholesterol transport, has
three common alleles ( ? 2, ? 3, and ? 4). ApoE polymor-
phisms infl uence the risk of atherosclerotic cardiovascular
disease and neurodegenerative disorders ( 1 ). ApoE3 binds
preferentially to HDL and apoE4 to VLDL ( 2 ). ApoE con-
tains a 22 kDa N-terminal domain (residues 1–191) and a
10 kDa C-terminal domain (residues 222–299) separated
by a protease-sensitive loop ( 3 ). ApoE4 shows a more pro-
nounced domain interaction or closed conformation than
the other apoE isoforms because it has Arg-112, which en-
ables Arg-61 in the N-terminal domain to interact with
Glu-255 in the C-terminal domain, a feature responsible
for the preferential association of apoE4 with VLDL ( 4, 5 ).
Upon binding to lipid, apolipoproteins undergo con-
formational rearrangements ( 6, 7 ) that affect their
function. The association of apoE isoform–dependant
postprandial lipoprotein metabolism with vascular disease
is not well understood. Previously, we reported that li-
polytic products of VLDL reduce the intermolecular inter-
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 par-
ticle fl uidity, 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 ex-
amined by electron paramagnetic resonance spectroscopy
using targeted spin probes on cysteines introduced in the
N-terminal (S76C) and C-terminal (A241C) domains. Fur-
ther, we synthesized a novel nitroxyl spin-labeled cholesterol
analog, which gave insight into lipoprotein particle fl uidity.
Our data revealed that the order of lipoprotein fl uidity was
HDL~LDL<VLDL<VLDL+lipoprotein lipase. Moreover, the
conformation of apoE4 depended on the lipoprotein frac-
tion: VLDL-associated apoE4 had a more linear conforma-
tion than apoE4 associated with LDL or HDL. Further, by
changing VLDL fl uidity, VLDL lipolysis products signifi -
cantly altered apoE4 into a more expanded conformation.
Our studies indicate that after every meal, VLDL fl uidity is
increased causing apoE4 associated with VLDL to assume a
more expanded conformation, potentially enhancing the
pathogenicity of apoE4 in vascular tissue. —Tetali, S. D., M.
S. Budamagunta, C. Simion, L. J. den Hartigh, T. Kalai, K.
The authors acknowledge the support of the Treadwell Innovative Research
Grants Program, the Center for Health and Nutrition Research, and National
Institutes of Health Grants HL-HL71488, HL-55667, RO1 AG 028793, and
R01 AG029246. The authors also acknowledge that part of the work is sup-
ported by the Hungarian National Research Fund (OTKA T048334). Part of
this investigation was conducted at a facility constructed with support from Re-
search Facilities Improvement Program Grant C06 RR-12088-01 from the Na-
tional Center for Research Resources, National Institutes of Health. Its contents
are solely the responsibility of the authors and do not necessarily represent the
offi cial views of the National Institutes of Health or other granting agencies.
Manuscript received 28 July 2009 and in revised form 3 December 2009.
Published, JLR Papers in Press, December 3, 2009
VLDL lipolysis products increase VLDL fl uidity
and convert apolipoprotein E4 into a more
Sarada D. Tetali, 1, * Madhu S. Budamagunta, † Catalina Simion, § Laura J. den Hartigh , §
Tamás Kálai, ** Kálmán Hideg, ** Danny M. Hatters, †† Karl H. Weisgraber, §§ John C. Voss, †
and John C. Rutledge §
Department of Plant Sciences,* School of Life Sciences, University of Hyderabad , Hyderabad 500 046, India ;
Department of Biochemistry and Molecular Medicine † and Department of Internal Medicine, § School of
Medicine, University of California , Davis, CA 95616; Institute of Organic and Medicinal Chemistry,**
University of Pécs , H-7624 Pécs, Hungary ; Department of Biochemistry and Molecular Biology, †† University
of Melbourne , VIC, Australia ; Gladstone Institutes of Cardiovascular and Neurological Diseases , §§ San
Francisco, CA 94158
Abbreviations: apo, apolipoprotein; DMPC, dimyristoylphosphati-
dylcholine; DSA, doxyl stearic acid; EPR, electron paramagnetic reso-
nance; LpL, lipoprotein lipase; TG, triglyceride.
1 To whom correspondence should be addressed.
e-mail: firstname.lastname@example.org or email@example.com
at Carlson Health Sci Library on July 7, 2010
1274Journal of Lipid Research Volume 51, 2010
were isolated by sequential fl otation with minor modifi cations
( 11 ). Plasma was transferred into ultracentrifuge tubes (Beckman-
Coulter), and 0.01% (w/v) NaN 3 was added as a preservative.
Plasma was diluted 1:2 in 196 mM NaCl and 0.25 mM EDTA
( ? = 1.0063) and centrifuged at 63,000 g for 30 min at 14°C to
allow chylomicron fl otation. The remaining plasma was spun at
285,000 g for 18 h at 14°C to isolate VLDL. The density of the
remaining plasma was adjusted to 1.063 to isolate LDL and to
1.21 to isolate HDL and spun at 285,000 g for 18 h at 14°C for
each lipoprotein fraction. Lipid fractions were dialyzed against
150 mM NaCl + 0.25 mM EDTA overnight at 4°C. Plasma and
lipid fractions were stored at 4°C and used within 3 days after
isolation. Triglyceride rich lipoprotein (TGRL) containing both
chylomicrons and VLDL were isolated by centrifuging the plasma
samples at 285,000 g for 18 h at 14°C.
Lipoproteins were lipolyzed by incubation with bovine milk
lipoprotein lipase (LpL) (3 U/ml) at 37°C for 30 min and as-
sayed for nonesterifi ed fatty acids. LDL cholesterol, HDL choles-
terol, total cholesterol, triglycerides (TGs), and nonesterifi ed
fatty acids were quantifi ed with an autoanalyzer.
Separation of lipoproteins by Titan gel electrophoresis
Equal amounts of spin-labeled apoE4 or apoE3-like protein
(0.2 mg/ml) were incubated with fasting plasma, postprandial
VLDL, or LpL-treated postprandial VLDL at 37°C for 1 h and
applied (2 µl/lane) to Titan-agarose precasted gels. To assay the
association of apoE with each lipoprotein class, lipoprotein bands
on the gel were visualized by staining and destaining ( 8 ). HDL,
LDL, and VLDL bands were excised from the gel, solubilized in
4.5 mol/l guanidine isothiocyanate at 65°C, and analyzed by EPR
spectroscopy, and the quantity of spin-labeled apoE associated
with the VLDL fraction was determined ( 8 ).
Samples with spin-labeled lipids
The 5 doxyl-, 12 doxyl-, and 16-doxyl stearic acid (DSA) spin
probes solubilized in ethanol were added to lipoproteins and
mixed extensively by pipetting to ensure uniform equilibration
of the probe with the sample. The fi nal concentration of the 5-,
12-, and 16-DSA probes in the lipoprotein samples was 2 µmol/l.
Spin-labeled probe/total cholesterol ratios were maintained the
Synthesis of spin-labeled cholestanol
The synthesis of SL-cholesterol 3-[17-(1,5dimethyl-hexyl)-3-
hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[ a ]phenantren-
2-ylidenethyl]2,2,5,5-tetramethyl-2,5-dihydro-1 H -pyrrol-1-yloxyl
radical cholestanol ( Fig. 1A ) is described as follows. To a solution of
5 ? -cholestan-3-one (3.86 g, 0.01 mol) in methanol (30 ml), 10%
NaOH solution (2 ml), and aldehyde (1.68 g, 0.01 mol) were
added. The mixture was stirred at room temperature for 3 h,
acidifi ed with 5% H 2 SO 4 , and extracted with CHCl 3 (3 × 20 ml).
The organic phase was washed with brine (2 × 20 ml), dried (MgSO 4 ),
and evaporated. The residue was purifi ed by fl ash chromatography
with hexane/ethanol to give the ketone 17-(1,5-dimethyl-hexyl)-
2-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1 H -pyrrol-3-ylmethylene)-
10,13-dimethyl-hexadecahydro-cyclopenta[ a ]phenantren-3-one
radical. To a solution of ketone (2.69 g, 5.0 mmol) in absolute etha-
nol (20 ml), NaBH 4 (0.19 g, 5 mmol) was added at 0°C. The reac-
tion mixture was allowed to warm up to room temperature. After
1 h at room temperature, the mixture was quenched with water
(10 ml), the alcohol was evaporated, and the aqueous phase was
extracted with CHCl 3 (3 × 20 ml). The organic phase was dried
(MgSO 4 ), evaporated, and purifi ed by fl ash chromatography to
give the fi nal product. The yield was 1.93 g (72%); mp 187–188°C;
R f 0.60 (CHCl 3 /Et 2 O 2:1); MS, m/z (%) = 538 (M + , 7), 508 (7),
465 (10), and 43 (100). Elemental analysis calculated for
action of apoE4, i.e., the self-association of apoE4 via its C
terminus ( 8 ); however, the mechanism for such effect was
unknown. In this study, we used electron paramagnetic
resonance (EPR) spectroscopy to investigate intramolecu-
lar interaction, i.e., domain interaction in apoE4 affected
by VLDL lipolysis products. Our fi ndings show that the
conformation of apoE4 is modulated postprandially and is
mediated by VLDL particle fl uidity. These conformational
changes may be important in lipoprotein-vascular cell
binding and possibly in vascular injury.
MATERIALS AND METHODS
Materials for this work are as follows: streptokinase, ICN Phar-
maceuticals (Costa Mesa, CA); vacutainers and venipuncture sup-
plies, Fisher Scientifi c International (Hampton, NH); glass
capillaries, VitroCom (Mountain Lakes, NJ); fl at cell (catalog no.
ES-LC11), JEOL (Tokyo, Japan); lipoprotein lipase, 5-doxy and
16-doxylstearic acid spin probes, and guanidine thiocyanate, Sigma-
Aldrich (St. Louis, MO); (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-
methyl) methanethiosulfonate and 12-doxylstearic acid, Toronto
Research Chemicals (Toronto, Canada); titan gel electrophoresis
supplies, Helena Laboratories (Beaumont, TX); dimyristoylphos-
phatidylcholine (DMPC), egg yolk phosphatidylcholine, extruder
were from Avanti Polar Lipids (Alabaster, AL); and trioelin, cho-
lesteryl oleate, cholesterol, and free fatty acids were from Nu-
Chek-Prep (Elysian, MN). ? -Thrombin was from Hematologic
Technologies (Essex Junction, VT), and Slide-A-Lyzer dialysis cas-
settes (MWCO 10,000) were from Pierce (Rockford, IL).
Expression and site-directed spin-labeling of apoE
Recombinant plasmids containing thioredoxin-his tagged
apoE sequences in pET32a-NT were expressed in BL21 (DE3)
Escherichi coli cells as described previously but with modifi cations
( 9, 10 ). The protein was purifi ed with a Ni-affi nity column, com-
plexed with DMPC vesicles, and extruded through 100-nm poly-
carbonate membrane fi lters. The thioredoxin-his tag was cleaved
from apoE with human ? -thrombin. The apoE/DMPC complex
was lyophilized, dispersed in 50 ml of methanol, and centrifuged
at 6,000 g for 20 min at 4°C to remove the DMPC. The pelleted
protein was dissolved in denaturation buffer (6 M guanidine and
2× TBS), purifi ed by a second Ni-affi nity column step to remove
the N-terminal tag, labeled with a methanethiosulfonate spin la-
bel, renatured, and assayed for protein content as described ( 10 ).
The purity and integrity of the protein was tested by SDS-PAGE.
The apoE mutants used for this study were apoE4 with two
cysteine moieties substituted at amino acid positions 76 and 241
(76C-241C) and apoE3-like protein (R61T-76C-241C).
Human plasma samples
Healthy human subjects were recruited from the University of
California, Davis. The study was approved by the Human Subjects
Research Committee of the University of California, Davis. Writ-
ten informed consent was obtained from each participant. The
volunteers were fed a moderately high-fat meal (40% calories from
fat), and blood was obtained by venipuncture into Vacutainer
tubes containing streptokinase (1,500 units) or EDTA, before
(fasting, 0 h) and 3.5 and 6 h after ingestion of the test meal ( 8 ).
Lipoprotein isolation by NaCl density gradient
Blood samples were centrifuged at 1,750 g for 10 min at 4°C to
separate plasma from cellular blood constituents. Lipoproteins
at Carlson Health Sci Library on July 7, 2010
Lipid fl uidity affects structural conformation of apoE4 1275
C-terminal domain helix. This intermolecular interface
is largely disrupted when apoE4 binds lipids, as measured
by the loss dipolar interaction of spin labels placed at po-
sition 264 ( 8, 10, 16 ). The reduced intermolecular inter-
action as measured by spin-labeled apoE4(264C) proved
to be a useful marker for apoE4 binding to plasma lipids
collected pre- and postprandially ( 8 ). To explore the in-
fl uence of lipolysis on the conformation and distribution
of apoE4, we used a previously developed intramolecular
domain interaction indicator construct (76C-241C) to
monitor conformational differences between apoE3 and
apoE4 that are predicted to be important in mediating
their functional differences ( 10 ). In artifi cial lipid sys-
tems [DMPC, emulsions, and dipalmitoylphosphatidyl-
choline (DPPC)], where elevated concentrations facilitate
spin dilution, no evidence of intermolecular spin interac-
tion (arising from labels within 2.0 nm of one another) is
observed from either position 76 or position 241 ( 10,
In the lipid-free state, apoE4 is distinguished by do-
main interaction between the N-terminal bundle and the
C-terminal helix, which is responsible for its binding
preference for VLDL ( 10, 16 ). Lipid-bound apoE has an
extensive helical structure but adopts alternate confor-
mations depending on lipid composition and particle
size. For example, lipid-bound apoE3 with extended heli-
cal segments has been found on the edge of phospho-
lipid (DMPC or 1-palmitoyl-2-oleoylphosphatidylcholine)
particles ( 17, 18 ). A similar conformation of apoE4 has
been found on TG-rich emulsions ( 10 ). However, as
shown by X-ray crystallography ( 19, 20 ), apoE4 on DPPC
particles has a double-helix structure with a hairpin near
the midpoint of the protein sequence. EPR measure-
ments showed that proximity of amino acids 76 and 241,
indicating domain interaction in the lipid-free protein, is
maintained in the DPPC-bound state ( 16 ). Thus, the 76-
241 pair is useful for assessing domain interaction in the
lipid-free protein and for distinguishing between a hair-
pin or extended helical conformation in the lipid-bound
The indicator constructs, which contain cysteines at
amino acids 76 and 241, detect the differences in the dis-
tance between the two regions of apoE when these sites
are covalently modifi ed with spin labels and assessed by
EPR spectroscopy ( 10 ). Because apoE3 has an endogenous
cysteine that would react with a spin label, we used an
apoE3 indicator construct with an R61T mutation ( 10 ).
This construct behaves structurally and functionally like
apoE3 because the mutation abolishes domain interaction
( 21 ). Specifi c labeling of apoE4 (which has no cysteines) is
achieved by reacting the sulfhydryl-specifi c nitroxide label
(MTS-SL) with apoE4 containing the substitutions S76C
and A241C ( Fig. 1B ). Based on the level of dipolar interac-
tion evident in the EPR spectrum in apoE4 containing
these two spin-labeled side chains, the relative distance be-
tween the N- and C-terminal domains is evident. Because
apoE3 has Cys-112, we used an apoE3-like protein ( Fig.
1C ) to determine the isoform-dependence of apoE’s re-
sponse to lipolysis ( 4, 10 ).
C 36 H 60 NO 2 (538.88), C 80.24, H 11.22, N 2.60% found C 80.20, H
11.25, and N 2.58%.
To label lipoproteins, the spin-labeled cholestanol probe of 8
mmol/l was prepared in dimethyl formamide and slowly stirred
into the lipoprotein sample to a fi nal concentration of 20 ? mol/l
in a fl at-bottom glass vial with continuous moderate stirring at
37°C for 2 h before the EPR study. In this case, the spin-labeled
probe/total cholesterol ratio was 1:65. Signifi cant signal-to-noise
issues arise if the spin-labeled probe/total cholesterol ratio is
<1:65. The fi nal concentration of the vehicle in the sample did
not exceed 0.5% and was kept constant across the samples.
Preparation of synthetic TG-rich emulsions
The TG-rich emulsions were prepared as previously described
( 12 ). Briefl y, lipids composed of 69% triolein, 22% egg yolk phos-
phatidylcholine, 6% cholesteryl oleate, and varied concentrations of
cholesterol (2–8%) were mixed together and then dried under a
stream of nitrogen. The dried mixtures were resuspended in a small
aliquot of 10 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl
and 1 mM EDTA and then emulsifi ed in a bath sonicator at 50°C for
an hour or until the homogenous emulsion was formed. Then, the
emulsions were extruded using an extruder to form particles by
passing 15 to 20 times through polycarbonate membrane of 100 nm
pore size. After the extrusion, particles were assayed for their total
triglyceride and cholesterol content. Further, the extruded particles
were used for apoE binding assay on the day of preparation.
EPR measurements of apoE4 were performed with a JEOL
X-band spectrometer fi tted with a loop-gap resonator ( 8 ). Spin-
labeled apoE in TBS (10 mM Tris, pH 7.4, 150 mM sodium chloride,
and 0.005% sodium azide) to a fi nal concentration of 0.2 mg/ml
protein was added to plasma or lipoprotein fractions or VLDL
spiked, as described in Ref. 8 , with 0.5 mM of fatty acids [stearic
(18:0), oleic (18:1), linoleic (18:2), or linolenic (18:3) acids], and
the ratio between fatty acid and cholesterol content of lipoprotein
was maintained at 1:2.6. The samples were loaded into one-sided
sealed glass capillaries, incubated at 37°C for 1 h, and scanned by
EPR. For all the samples, vehicle controls were used. The spectra
were obtained by an average of three scans (2 min each) over 100
G at a microwave power of 2 mW and a modulation amplitude of 1
G at room temperature (20–22°C) or at 37°C. To assess the signal
content in guanidine-extracted gel samples, a quartz fl at cell was
used, and the signal-averaged spectra from six 20-G scans (20 s
each) over the central (m I = 0) line were recorded.
Order parameters for the spectra of samples containing spin-
labeled lipids were calculated as described ( 13 ). For samples con-
taining the 5-DSA label, the order parameter S was calculated as
S = [(T || – T ? ? C)/(T || + 2T ? + 2C)]*1.723, where C = 1.4 – 0.053
(T || – T ? ). For the unresolved T || in the 12- and 16-DSA spectra,
T || was estimated from T || = 44.5 – 2T ? .
All statistical analyses were performed using ANOVA as guided by
SigmaStat software, with pairwise comparisons made using the
Holm-Sidak method. Where applicable, data are reported as mean ±
SD. Statistical signifi cance was reported for P < 0.001, as indicated.
Selection of apoE4 (76C-241C) to study conformational
changes of apoE
Mutagenesis studies ( 14, 15 ) have demonstrated that
apoE self-associates via a hydrophobic face along the
at Carlson Health Sci Library on July 7, 2010
1276 Journal of Lipid Research Volume 51, 2010
were separated by agarose gel electrophoresis. The ex-
tent of binding and relative distribution of exogenously
added apoE3-like protein or apoE4 with plasma lipopro-
tein fractions, VLDL, LDL, or HDL, was determined by
assaying the amount of spin-labeled proteins by EPR
spectroscopy ( 8 ). Like the native proteins, apoE3-like
protein bound preferentially to HDL (49% vs. 27% to
LDL and 31% to VLDL) ( Fig. 2B ), and apoE4 bound pre-
dominantly to VLDL (56% vs. 16% to HDL and 27% to
Comparison of apoE conformations on lipoprotein
To assess the conformation of apoE on lipoprotein frac-
tions, spin-labeled apoE4 and apoE3-like protein were in-
cubated with VLDL, LDL, or HDL at 37°C for 1 h before
scanning. Incubation of apoE3-like protein with HDL or
VLDL elicited similar reductions in spectral broadening,
resulting in spectra with larger amplitudes ( Fig. 3A ). In
contrast, HDL produced only moderate narrowing of the
apoE4 spectrum, suggesting that the conformation of the
lipid-free protein is largely retained in HDL. Although
VLDL generated a greater reduction in broadening than
HDL, the broadening did not approach apoE3-like levels
(see lipolysis results below).
To control for individual variations in the properties of
lipoproteins from each volunteer, we averaged the change
in the center peak (m I = 0) line height amplitude for both
apoE4 and the apoE3-like proteins when combined with
VLDL, LDL, or HDL from six volunteers ( Fig. 3B ). For the
apoE3-like protein, all the lipoprotein fractions induced
ApoE4 undergoes a greater spectral change upon
incubation with postprandial plasma
The dipolar interaction between the spin labels at two
sites can be qualitatively determined by the amount of
spectral broadening. Therefore, we examined the double-
spin-labeled apoE4 and apoE3-like proteins after incu-
bation in postprandial plasma ( Fig. 2A ). The spectra
in Fig. 2A are plotted so that each represents the same
number of spins. The R61T mutation increased the dis-
tance between the domains in the lipid-free protein, re-
sulting in less spectral broadening, as shown previously
( 10 ). Postprandial plasma decreased the dipolar broad-
ening of apoE4. The effect was greatest at 3.5 h, but the
broadening was less than that in the apoE3-like sample.
This suggests that this population of apoE4 retains some
of the conformational features of its lipid-free state in
plasma. In contrast, the apoE3-like protein maintained a
more open conformation both in the lipid-free state and
in plasma. This difference allows a systematic investiga-
tion of apoE isoform-specifi c behavior in the postpran-
dial state. Fasting and postprandial serum lipid values of
six of the healthy volunteers who participated in this
study are listed in Table 1 .
Preferential distribution of spin-labeled apoE3-like
protein and apoE4 with plasma lipoproteins
To determine if the apoE3-like and apoE4 mutants
have binding preferences similar to those of the native
proteins, we incubated fasting plasma from healthy vol-
unteers with spin-labeled apoE3-like protein (61T-76C-
241C) or apoE4 (76C-241C) for 1 h at 37°C. The samples
Fig. 1. A: Structure of spin-labeled cholestanol-3-[17-(1,5dimethyl-hexyl)-3-hydroxy-10,13-dimethyl-hexa-
decahydro-cyclopenta[ a ]phenantren-2-ylidenethyl]2,2,5,5-tetramethyl-2,5-dihydro-1 H -pyrrol-1-yloxyl rad-
ical. Its calculated molecular weight based on the elemental analysis (C 36 H 60 NO 2 ) 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 B 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
at Carlson Health Sci Library on July 7, 2010
Lipid fl uidity affects structural conformation of apoE4 1277
bound apoE4 ( Fig. 3 ). These data suggest that the confor-
mation of apoE4 is more affected on VLDL than on HDL
or LDL. Using synthetic lipids, Hatters et al. ( 10 ) found
similar apoE4 conformational changes. However, this is
the fi rst report to show the differences in apoE4 confor-
mation on native lipoproteins.
an increase of ? 30% over the lipid-free protein. However,
for apoE4, the increase in the center peak amplitude was
much higher for apoE4 bound to VLDL (2.15-fold) than
for apoE4 bound to either HDL (1.8-fold) or LDL (1.7-
fold). The proximity of the spin labels in the two domains
in apoE4 was most similar to that of apoE3 on VLDL-
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 associ-
ated with plasma lipoproteins. The protein samples were incubated at 37°C for 1 h with fasting plasma sam-
ples 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 isocya-
nate 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 signifi cantly different
treatments within each apoE (E3/E4) protein group. # P < 0.001 for signifi cantly different treatments within
each lipoprotein class.
TABLE 1. Lipid values of healthy human volunteers recruited for the study (mg/dl)
Preprandial (0 h) Postprandial (3.5 h)Postprandial (6 h)
TG TCLDL HDLTGTC LDL HDLTG TC LDLHDL
153 ± 15
169 ± 6
203 ± 23
192 ± 10
134 ± 18
34.5 ± 0.5
39 ± 4
124 ± 7
311 ± 70
290 ± 40
207 ± 24
191 ± 5
134 ± 20
127 ± 8
40 ± 4
35 ± 0
191 ± 34
208 ± 22
206 ± 30
198 ± 6
136 ± 25
133 ± 8
40 ± 6
34.5 ± 0.5
Volunteers 9126 and 2206 were reinvited for the study; therefore, the data from those volunteers are averages of two independent measurements.
The data from volunteers 7125, 2654, 2020, and 6341 are from a single experiment. TG, total TGs; TC, total cholesterol; LDL, direct measurement
of LDL-cholesterol; HDL, direct measurement of HDL-cholesterol.
at Carlson Health Sci Library on July 7, 2010
1278 Journal of Lipid Research Volume 51, 2010
37°C before scanning. Compared with lipid-free apoE4
in buffer, apoE4 incubated with postprandial VLDL gen-
erated a 2.7-fold greater signal intensity, which increased
to 3.5-fold when apoE4 was incubated with LpL-treated
VLDL ( Fig. 4, inset). ApoE3-like protein showed a 1.4-
fold increase in signal intensity when treated with VLDL
and a 1.8-fold increase when treated with LpL-treated
To determine if lipolyzed VLDL has higher affi nity
for apoE4, we used agarose gel electrophoresis to exam-
ine apoE4 that had been incubated with VLDL or LpL-
treated VLDL. The VLDL bands on the gels were
visualized by staining, and the gel slices were solubilized
in 4.5 mol/l guanidine isothiocyanate by incubating
at 65°C for 3 min. We measured the signal from the
samples to determine the amount of exogenously added
protein associated with VLDL or LpL-treated VLDL.
Lipolysis did not signifi cantly alter the association of
VLDL with either apoE4 or apoE3-like protein. Varia-
tions in association of apoE4 with VLDL or LpL-treated
VLDL were negligible. Therefore, the differences in the
EPR signal amplitude refl ect changes in apoE4 conforma-
tion, not the amount of apoE4 bound to the lipoproteins.
Lipid fl uidity of lipoproteins
Next, we used DSA probes to investigate whether differ-
ences in lipoprotein particle fl uidity infl uence the struc-
Dependence of apoE conformation on postprandial HDL
To confi rm that the decrease in apoE spin interaction
after exposure to postprandial plasma can be attributed to
the VLDL-associated protein, we incubated apoE4 and
apoE3-like proteins with lipoprotein fractions isolated
from fasting plasma (P0) or postprandial plasma obtained
at 3.5 (P3.5) or 6 h (P6). P6 plasma served as an internal
control to P0 plasma. ApoE4 maintained the proximity of
the 76 and 241 probes to a larger extent on HDL ( Fig. 4 ).
In contrast with the effect seen when combined with the
mixture of lipoproteins in plasma ( Fig. 2A ), postprandial
HDL did not infl uence the conformation of apoE4, as
judged by the spectrum of the doubly labeled protein ( Fig.
4 ). Similarly, the conformation of the apoE3-like protein
was not altered by incubation with HDL fractions from the
fasting (P0) or postprandial states (P3.5 and P6). How-
ever, both spin-labeled apoE4 and the apoE3-like proteins
displayed signifi cant spectral narrowing after exposure to
P3.5 VLDL ( Fig. 4 ).
VLDL lipolysis affects the conformation of apoE
Because increased lipolytic activity is associated with
the postprandial state, we examined the effect of VLDL
lipolysis on apoE conformation. Equal amounts of spin-
labeled apoE3-like protein or apoE4 were incubated with
LpL-treated or untreated postprandial VLDL for 1 h at
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 signifi cantly different from each other ( P < 0.001)
at Carlson Health Sci Library on July 7, 2010
Lipid fl uidity affects structural conformation of apoE4 1279
assess the mobility of cholesterol-rich domains. Lipolysis
increased the fl uidity TGRL (both chylomicrons and
VLDL) ( Fig. 6B ). The infl uence of deceased TGRL fl uidity
was also explored by preparing triolein-phosphatidylcho-
line emulsions of differing cholesterol content, which is
expected to reduce lipid fl uidity at higher levels. As shown
in Fig. 6C , apoE4 displays a slightly higher level of domain
interaction when incubated with TGLR mimics containing
the higher cholesterol level, while no difference was de-
tected for the apoE3-like sample. In summary, the data
with various lipid probes indicate that lipolysis increases
the fl uidity of lipoproteins, and when added to the previ-
ous data, suggest that the proximity of positions in apoE
indicative of domain interaction provide a marker of lipol-
ysis activity and thereby lipoprotein fl uidity.
To further establish the relationship between lipopro-
tein fl uidity and effects on the conformation of apoE4, we
spiked VLDL with fatty acids (fi nal concentration, 0.5
mmol/l) of 18-carbon chain with different degrees of un-
saturation. Linoleic (18:2) and linolenic (18:3) fatty acids
(28% and 35%) were more effective than stearic (18:0)
and oleic (18:1) fatty acids (12% and 18%) in reducing
domain interaction of apoE4 (data not shown).
Finally, to evaluate the compositional changes in VLDL
after lipolysis, we assayed the free fatty acid content of post-
prandial VLDL (350 mg/dl TG) in the absence of LpL treat-
ment. Lipolysis of VLDL (3 U/ml) increased the levels of
NEFAs by 15-fold (from 0.04 to 0.655 mmol/l). Total NEFA
ture of apoE. To measure particle fl uidity, we added 5-DSA,
12-DSA, or 16-DSA (2 µmol/l) to HDL, LDL, VLDL, or
LpL-treated VLDL and subjected the lipoproteins to EPR
spectroscopy. The spin labels in each probe showed re-
stricted movement upon equilibration with LDL or HDL
but relatively free mobility upon equilibration with VLDL
( Fig. 5 ). Increased mobility of the spin label, indicated by
a narrower and sharper spectrum, can be quantifi ed by a
decrease in the order parameter S ( Table 2 ). These results
show that VLDL is more fl uid than LDL and HDL.
Effect of lipolysis on TGRL fl uidity
We investigated the role of LpL in the increased fl uidity
of postprandial lipoproteins. The effect of LpL treatment
on the lipid order within VLDL and chylomicrons also was
examined using spin-labeled fatty acids ( Fig. 6A ). Both the
12- and 16-DSA probes displayed clear narrowing of the
lines and lowered order parameters ( Table 2 ) with lipoly-
sis treatment, revealing greater mobility, and increased
rates of motion, indicating a more fl uid environment. Li-
polysis had little effect on the 5-DSA probe, suggesting
that the headgroup region is less affected by lipolysis.
To investigate the actions of lipolysis on TGRL fl uidity,
we used fatty acid spin probes (doxyl stearic acid) and a
cholesterol spin probe (spin-labeled cholestanol) to study
lipid fl uidity changes. We incubated TGRL, untreated or
treated with LpL, with the spin-labeled cholestanol spin
probe (20 µmol/l) and performed EPR spectroscopy to
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.
at Carlson Health Sci Library on July 7, 2010
1280 Journal of Lipid Research Volume 51, 2010
than LDL and HDL, and VLDL-associated apoE4 had a
more extended conformation than when associated with
LDL or HDL. Our fi ndings suggest a model for apoE as-
sociation with lipid particles ( Fig. 7 ). These conforma-
tional changes may be important in lipoprotein-vascular
cell binding and possibly in vascular injury.
Lipid association induces structural rearrangements in
apoE. The conformation of apoE4 differs, depending on
whether it is complexed to phospholipid alone or TG-rich
emulsions ( 10 ). Plasma lipoproteins are highly variable in
size and composition, and apoE4 and apoE3 have differ-
ent preferences for plasma lipoproteins. However, no re-
ports examine these lipoproteins for different effects on
apoE conformation. ApoE4 showed different conforma-
tions on VLDL versus HDL and LDL, showing a more dra-
matic effect on VLDL compared with HDL or LDL. ApoE3
conformation was relatively unchanged by association with
these different lipoprotein classes. The reason for the
apoE4 structural conformational change is not known.
Studies with model lipoproteins have shown that molecu-
lar packing of lipids and the structures of lipids and apoli-
poproteins are important determinants of the equilibrium
binding and kinetics of transfer of apolipoproteins ( 23–
26 ). Among lipoproteins, surface lipid fl uidity increased
in the order HDL < LDL < VLDL and varied inversely with
their (protein + cholesterol/phospholipid) ratios ( 27 ).
We used the EPR mutant clones of apoE [apoE4 (76C-
241C) and apoE3-like protein (R61T, 76C, 241C)] to mon-
itor conformational effects. Like the native apoE proteins,
spin-labeled apoE4 preferentially associated with VLDL
and apoE3-like protein with HDL after a moderately high-
fat meal. Thus, the mutations in these proteins did not af-
fect their structural integrity or their preference for plasma
Our fi ndings are consistent with earlier work by Massey
and Pownall ( 27 ), who used fl uorescent probes to fi nd a
similar fl uidity for HDL and LDL but a more fl uid envi-
ronment for VLDL. They showed a positive correlation
between surface fl uidity of lipoprotein particles with re-
activity of phospholipase A2. Gorshkova, Menschikowski,
and Jaross ( 28 ) showed that phospholipase A 2 treatment
increases order at the headgroup (as detected by 5-DSA).
This is consistent with the insignifi cant change in VLDL
fl uidity by the 5-DSA probe. Thus, the surface density may
be relatively unaffected, while the core becomes much
more fl uid. Foucher et al. ( 29 ) found that PUFAs make
lipoproteins more fl uid than saturated. Previously, we
showed that adding palmitate to VLDL reduced C-terminal
(i.e., intermolecular) interaction and promoted the transi-
tion of apoE4 from a tetrameric state to a monomeric state
content in fasting and postprandial VLDL were not signifi -
cantly different. However, upon lipolysis fasting, VLDL
NEFA content increased about 5- to 7-fold, whereas lipolysis
of postprandial VLDL yielded a 12- to 18-fold increase in
NEFA content. Changes in total TG content could not be
detected by our assay, which could be explained because the
TG assay measures glycerol, and glycerol is the surrogate for
TG measurement, no change in TG is expected. Postpran-
dial free fatty acid content in human plasma can be as high
as 0.35–0.65 mmol/l and depends strongly on diet ( 22 ).
Thus, NEFA levels may play an important role in altering
TGRL fl uidity and apoE conformation.
In this study, we used EPR spectroscopy to investigate
conformational changes of apoE4 induced by VLDL lipol-
ysis products. Our fi ndings show that the conformation of
apoE4 is modulated postprandially and is mediated by
VLDL particle fl uidity. ApoE4 was more sensitive to sur-
face lipid fl uidity than apoE3, and lipid fl uidity was in-
versely related to the proximity of regions in the N- and
C-terminal domains of apoE4. VLDL was much more fl uid
Fig. 5. Lipid fl uidity of classes of lipoproteins. VLDL, LDL, and
HDL were isolated from fasting plasma by density gradient centrifu-
gation, 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
TABLE 2. Order parameters for spin-labeled fatty acids in isolated
plasma lipid fractions
Fraction 5-DSA12-DSA 16-DSA
VLDL + LpL
at Carlson Health Sci Library on July 7, 2010
Lipid fl uidity affects structural conformation of apoE4 1281
the mechanisms are not known with certainty. Our studies
indicate that the postprandial state can signifi cantly alter
the structural conformation of apoE4. Further research is
necessary to determine whether postprandial conforma-
tional changes in apoE4 affect its interactions with cells and
contribute to atherogenicity. Postprandial hyperlipemia
occurs several times daily, and postprandial lipoproteins
could injure arterial endothelium and can cause repetitive
arterial injury ( 32, 33 ). Because postprandial hyperlipemia
has been implicated in the development of atherosclerosis
via repetitive injury to the arterial endothelium ( 34 ), un-
derstanding the role of the apoE isoforms and their confor-
mation in modulating pro-infl ammatory processes may be
very important in atherogenesis.
Our study provides direct evidence that apoE assumes
different conformations, depending on its association with
lipoprotein species. ApoE4 preferentially binds to VLDL,
but the reasons for its preference are not known. We
showed that apoE4 assumes a more linear conformation
on VLDL particles. The increase in free fatty acid content
in VLDL due to lipolysis is accompanied by an increase in
lipid fl uidity, which affects apoE4 conformation ( Fig. 7 ).
From these results, we demonstrate that the conformation
of apoE4 is infl uenced by lipoprotein classes and lipid fl u-
idity. More fl uid particles may promote the partition of
apoE side chains (favoring lipid-protein vs. protein-protein
interactions). Such modulations in apoE4 structure due to
changes in lipoprotein fl uidity, especially during postpran-
dial state, may regulate lipoprotein binding and uptake by
cellular receptors. Additional studies are needed to under-
( 8 ). To establish the relationship between lipoprotein fl u-
idity and conformational changes in of apoE4, we tested
the effect of 18 carbon fatty acids with different degrees of
unsaturation. Linoleic (18:2) and linolenic (18:3) fatty ac-
ids showed a greater effect than stearic (18:0) and oleic
(18:1) fatty acids in reducing the proximity of the N- and
C-terminal regions of apoE4.
Our previous article ( 8 ) showed a dramatic change in
apoE4 conformation 3.5 h after consumption when
compared with fasting apoE4 conformation in healthy
humans. Our goal was to determine the mechanism of this
clinically relevant change in apoE4 conformation in the
postprandial state. Previous studies, including our own,
suggested that VLDL lipolysis products could mediate this
change. Since the postprandial state is a period of height-
ened lipolysis, we sought to model the postprandial state
by treating postprandial VLDL with lipoprotein lipase.
Our studies mimic the clinical fi ndings in the postpran-
dial state and provide strong evidence that VLDL lipolysis
products mediate the change in particle fl uidity that cause
apoE4 conformational changes in the postprandial state.
During lipolysis, fatty acids accumulate at the lipopro-
tein surface because the rate of fatty acid transfer is much
slower than lipolysis ( 30, 31 ). Accumulation of fatty acids
on the lipoprotein surface could mediate changes in li-
poprotein fl uidity. Therefore, we investigated lipolysis-
induced changes in the fl uidity of the particle and its
association with structural conformation of apoE.
ApoE4 is pro-infl ammatory and associated with increased
risk of atherosclerosis and Alzheimer’s disease; however,
Fig. 6. Effect of lypolysis on fl uidity of TGRL. A:
The effect of lipolysis on the EPR spectra of doxyl-
labeled stearic acid in VLDL (top three sets) or chylo-
microns (bottom set). In each experiment, an equal
amount of TGRL sample (200 mg/dl TG) was pre-
treated 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 trac-
ing) indicating an increase in lipid fl uidity after
LpL treatment. B: Effect of lipolysis spin-labeled
cholestanol was added slowly while stirring the lipo-
protein sample at 37°C and incubated for 2 h prior to
scanning. C: Domain interaction of apoE on synthetic
lipid particles containing two different concentra-
tions 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 incu-
bated with spin-labeled apoE4 or the E3-like protein
for 1 h at 37°C, followed by EPR spectroscopy.
at Carlson Health Sci Library on July 7, 2010
1282 Journal of Lipid Research Volume 51, 2010
lipoproteins and lipolysis products. Supplies and facilities for
part of this study were provided by the Western Human
Nutrition Research Center, Ragel Facility, and the Clinical
Nutrition Research Unit at the University of California, Davis.
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work. The authors also would like to acknowledge Theresa
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Fig. 7. Schematics of apoE3-like and apoE4 conformations on in a lipid-free environment and associated with HDL and VLDL. The level
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