Báo cáo Y học: Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions pot

Eur. J. Biochem. 269, 5939–5949 (2002) Ó FEBS 2002

doi:10.1046/j.1432-1033.2002.03319.x

Differences in the binding capacity of human apolipoprotein
E3 and E4 to size-fractionated lipid emulsions
Matthew A. Perugini1, Peter Schuck2 and Geoffrey J. Howlett1
1

Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia; 2Division of
Bioengineering and Physical Science, ORS, OD, National Institutes of Health, Bethesda, MD, USA

We describe sensitive new approaches for detecting and
quantitating protein–lipid interactions using analytical ultracentrifugation and continuous size-distribution analysis
[Schuck (2000) Biophys. J. 78, 1606–1619]. The new methods
were developed to investigate the binding of human apolipoprotein E (apoE) isoforms to size-fractionated lipid
emulsions, and demonstrate that apoE3 binds preferentially
to small lipid emulsions, whereas apoE4 exhibits a preference
for large lipid particles. Although the apparent binding
affinity for large emulsions is similar (Kd % 0.5 lM), the
maximum binding capacity for apoE4 is significantly higher

than for apoE3 (3.0 and 1.8 amino acids per phospholipid,
respectively). This indicates that apoE4 has a smaller binding
footprint at saturation. We propose that apoE isoforms
differentiate between lipid surfaces on the basis of size, and
that these differences in lipid binding are due to a greater
propensity of apoE4 to adopt a more compact closed conformation. Implications for the role of apoE4 in blood lipid
transport and disease are discussed.

Traditional methods for monitoring protein–lipid interactions have employed a variety of lipid systems, including

emulsions, phospholipid vesicles and phospholipid micelles
[1–6]. However, the methodologies and heterogeneity of
lipids employed in these studies do not allow a thorough
investigation into the effect of particle size on protein
binding. This may be important to examine, given that
earlier studies have demonstrated that apolipoprotein E
(apoE) isoforms differ in their propensity to bind to small
and large lipoproteins [7–10]. Experimental approaches that
can therefore measure and quantitate binding to sizefractionated lipid particles may provide insight into the role
of blood lipid transport proteins, such as apolipoprotein E,
in lipid metabolism and disease.
Human apolipoprotein E is a key component of plasma
lipoproteins, exchanging reversibly between chylomicrons,
very low density lipoprotein (VLDL), intermediate density
lipoprotein (IDL), and a subclass of high density lipoprotein
(HDL) [11–13]. It serves as a high-affinity ligand for a
number of cell surface receptors, thereby mediating the
uptake of cholesterol and other complex lipids into the cell
[12,13]. The three common isoforms of apoE, designated
apoE2 (C112/C158), apoE3 (C112/R158) and apoE4

(R112/R158), differ by single amino acid substitutions at
positions 112 and 158 [13–15]. ApoE2 is linked to type III
hyperlipoproteinemia and has low affinity for the low
density lipoprotein (LDL) receptor [13,16]. ApoE3 is the
most common isoform and is associated with normolipidemia [13,17], while the apoE4 isoform is independently
associated with an increased risk for atherosclerosis [17,18]
and late-onset Alzheimer’s disease [19,20]. Recent studies
compare the structure–function relationships of the apoE
isoforms, including their stability [21], self-association in the

presence and absence of phospholipid [4], and their ability to
bind preferentially to different lipoprotein classes [7–10].
ApoE is composed of two independently folded domains.
The 10 kDa COOH-terminal domain (residues 225–299)
possesses high lipid affinity, while the 22 kDa NH2-terminal
domain (residues 1–191) binds weakly to lipid and mediates
receptor interactions [13]. In the absence of lipid, the NH2terminal domain of apoE3 forms an elongated four helicalbundle, stabilized by hydrophobic contacts and intra- and
inter-helical salt bridges [22]. The substitution of cysteine for
arginine at position 112 in apoE4, results in an additional
salt-bridge between Glu109 and Arg112 and the displacement of Arg61 from the surface of the four-helix bundle [8].
The displaced Arg61 side chain forms an interdomain saltbridge with Glu255, an interaction that is critical for
directing the preference of apoE4 to bind large VLDL
particles [7]. In contrast, the apoE3 isoform binds preferentially to smaller HDL particles, with residues NH2terminal to position 244 implicated as the primary HDL
binding determinants [9,10,23].
Recently, a number of studies have shown that the
22 kDa NH2-terminal structure of apoE reorganizes
upon interaction with lipid [3,24–28]. It has been proposed
that the NH2-terminal domain of apoE undergoes conformational changes, converting from a closed receptorinactive conformation in the absence of lipid, to an open

Correspondence to M. Perugini, Department of Biochemistry and
Molecular Biology, The University of Melbourne, Parkville, VIC,
Australia, 3010. Fax: + 61 39347 7730, Tel.: + 61 38344 5911,
E-mail:
Abbreviations: apoE, apolipoprotein E; Myr2Gro-PCho,
dimyristoylglycerophosphocholine; EggPtdCho, egg yolk
phosphatidylcholine; F, fraction; HDL, high density lipoprotein;
IDL, intermediate density lipoprotein; LDL, low density lipoprotein;
TO, triolein; VLDL, very low density lipoprotein.
(Received 25 July 2002, revised 7 October 2002,
accepted 17 October 2002)

Keywords: Alzheimer’s disease; apolipoprotein E; open
conformation; lipid binding; analytical ultracentrifugation.


Ó FEBS 2002

5940 M. A. Perugini et al. (Eur. J. Biochem. 269)

receptor-active conformation in the presence of phospholipid [13,27]. Evidence in support of this model is provided
by recent lipid binding studies employing synthetic lipid
emulsions and intact apoE4, its 22 kDa NH2-terminal
fragment, and the 10 kDa COOH-terminal fragment [28].
At a low surface concentration of protein, the binding
enthalpy of intact apoE4 was consistent with the sum of the
enthalpies for the 22 kDa and 10 kDa derivatives, indicating that both NH2- and COOH-terminal domains bind
to the emulsion surface [28]. At saturation, however, the
enthalpy for intact apoE4 was similar to that of the 10 kDa
fragment, suggesting that only the COOH-terminal domain
of intact apoE4 interacts with the emulsion surface [28]. It is
not known whether the apoE3 isoform displays a similar
phenomenon on the surface of lipid particles.
In the present study we develop methods for the
characterization of lipid emulsions of well-defined composition but different particle size. We compare the binding of
apoE3 and apoE4 to small and large lipid particles, and
show that apoE4 has a greater capacity than apoE3 to bind
large lipid emulsions. These results suggest that at saturation, apoE4 binds primarily in a more compact conformation, whereas apoE3 adopts an expanded conformation on
the lipoprotein surface.

phosphate, pH 7.4. Phospholipid and triacylglycerol concentrations were determined using enzymatic spectrophotometric phospholipid and glycerol assay kits (Roche).

Peptide synthesis and purification
ApoE(263–286) peptide (SWFEPLVEDMQRQWAGLV
EKVQAA, Mr ¼ 2818) [30–32], was synthesized by automated solid phase methods on an Applied Biosystems 431A
synthesizer, starting with HMP resin (NovaBiochem) and
using Fmoc amino acids (AusPep). The peptide was cleaved
from the solid support with trifluoroacetic acid and
deprotected with piperidine. Before purification, the crude
peptide mixture was washed and precipitated in ice-cold
diethyl ether, filtered, then solubilized in 100 mM ammonium bicarbonate, pH 7.8, containing 10% (v/v) acetic acid.
The peptide was purified by reversed-phase FPLC on a
3-mL Resource column with a 10 mL, 0–100% gradient, of
acetonitrile containing 0.1% (v/v) trifluoroacetic acid.
Fractions containing pure peptide, as assessed by matrixassisted laser desorption ion-spray mass spectrometry
(Finnigan Mat) were pooled, lyophilized, solubilized in
distilled water and stored at )20 °C.
Expression and purification of apolipoprotein E3 and E4

EXPERIMENTAL PROCEDURES
Materials
Dimyristoylglycerophosphocholine (Myr2Gro-PCho) and
egg phosphatidylcholine (EggPtdCho) were purchased from
Sigma, St Louis, MO, USA and triolein [1,2,3-tri(cis-9octadecanoyl)glycerol; TO] from Nu-Chek Prep, Elysian,
MN, USA.

Human apoE3 and apoE4 were expressed in Escherichia coli
as fusion proteins with glutathione S-transferase using the
pGEX-3X plasmid, and purified as described previously [4].
The purity of both apoE3 and apoE4 as assessed by SDS/
PAGE was estimated to be > 98%. The molar masses of
purified apoE3 and apoE4 (34 245 Da and 34 297 Da,

respectively) determined by electrospray mass spectrometry
agree well with theoretical values and known differences
between the amino acid compositions of the isoforms [15].

Emulsion preparation and fractionation
Emulsions were prepared as described previously [29] with
some modifications. A mixture of 2 : 1 TO/Myr2Gro-PCho
or 2 : 1 TO/EggPtdCho in chloroform (1.0 or 10.0 mgỈmL)1
total lipid) was dried down under nitrogen, desiccated
overnight, and resuspended in 10 mL of 0.1 M sodium
phosphate buffer, pH 7.4. The suspension was sonicated at
34 °C using a probe sonicator (Soniprep 150, MSE
Scientific Instruments, Sussex, England) at an amplitude
of 10 microns for 5 · 1 min (with 30 s intervals) under a
constant stream of nitrogen. The dispersion was extruded
10–12 times through 2 · 100 nm polycarbonate filters using
a LiposoFast hand-held microextruder (Avestin Inc.,
Ottawa, Ontario, Canada). The density of the extrudate
was increased to 1.038 gỈmL)1 by the addition of sucrose
(10 gỈdL)1) and the sample layered beneath 0.1 M sodium
phosphate buffer, pH 7.4. The sample was centrifuged at
110 000 g for 35 min in a Beckman 70.1 Ti rotor and model
L8-70 ultracentrifuge. After centrifugation, the top 1.5 mL
was collected (unfractionated) and the density adjusted to
1.018 gỈmL)1 with solid sucrose (5 gỈdL)1), before adding a
0–5% (w/v) linear sucrose gradient prepared in 0.1 M
sodium phosphate, pH 7.4. The sample was then centrifuged at 4500 g for 2 h in a Beckman SW-40 rotor and
model L8-70 ultracentrifuge. Fractions (1.0 mL) were
collected from the bottom of the tube using a peristaltic
pump and dialyzed exhaustively against 50 mM sodium

Dynamic light scattering
Dynamic light scattering experiments were conducted with a
Protein Solutions DynaPro instrument with MSTC200
microsampler (Protein Solutions, Charlottesville, VA,
USA). Samples, suspended in 50 mM sodium phosphate,
pH 7.4, were centrifuged for 5 min in a microcentrifuge to
remove dust particles, and 20 lL sample was inserted in the
cuvette with the temperature control set to 20 °C. The light
scattering signal was collected at 90°, and diffusion coefficients and Stokes-radii of the emulsion fractions were
calculated with the instrument software.
Flotation velocity
Flotation velocity experiments were performed using a
Beckman model XL-A analytical ultracentrifuge. Prior to
centrifugation, apoE and emulsion samples were exhaustively dialyzed (> 20 h) against 50 mM sodium phosphate,
pH 7.4. Samples (300–400 lL) and reference (320–420 lL)
solutions were loaded into a conventional double-sector
quartz cell and mounted in a Beckman An-60 Ti rotor.
Experiments were conducted at 20 °C and at a rotor speed
of 5000 r.p.m. Data was collected in continuous mode, at a
single wavelength (230 nm or 250 nm), time interval of 360 s
and a step-size of 0.003 cm without averaging. Multiple
scans at different time points were fitted to a single species or


Ó FEBS 2002

Binding of apolipoprotein E3 and E4 to emulsions (Eur. J. Biochem. 269) 5941

to a continuous size distribution (see below) using the

program SEDFIT (which is available at www.analyticalultra
centrifugation.com). Solvent densities were measured at
20 °C in an Anton Paar model DMA 02C precision density
meter equipped with a water bath, or computed using the
program SEDNTERP [33], kindly supplied by D. Hayes
(Magdalen College, NH, USA), T. Laue (University of New
Hampshire, Durham, NH, USA) and J. Philo (Alliance
Protein Laboratories, Thousand Oaks, CA, USA). Partial
specific volumes () of apoE3 isoforms (0.732 mLỈg)1) and
v
apoE(263–286) (0.738 mLỈg)1) were calculated from amino
acid composition [33].
Size distribution analysis
The size distribution of noninteracting lipid emulsion
particles can be calculated as a flotation coefficient distribution, c(sf), according to Eqn (1):
Z
aðr; tÞ ffi cðsf ÞLðsf ; D; r; tÞdsf
ð1Þ
where a(r,t) denotes the observed optical density at
radius r and time t, c(sf) denotes the differential flotation
coefficient distribution, L(sf,D,r,t) denotes the solution
to the Lamm equation [34], calculated with an adaptation of the moving frame of reference method [35] to
flotation velocity, taking into consideration the rotor
acceleration phase. One feature of boundary modeling
with Eqn (1) is that it allows interconversion of the
flotation coefficient distribution to a molar mass distribution via the Stokes-Einstein and Svedberg equation
[36], upon consideration of the spherical shape and the
size-dependent particle density of the polydisperse solutes. For the functional dependence between the density
and molar mass of the fractionated emulsion particles,
we assumed a spherical monolayer of Myr2Gro-PCho

surrounding a core of TO, supported by transmission
electron microscopy (data not shown). Consequently,
the relationship between density of the particles as a
function of particle mass was determined using values

for M and v reported by [29], calculated from the ratio of
phospholipid to triacylglycerol assuming each phos˚
pholipid molecule occupies an area of 60 A2 in the
monolayer surface, and that each triacylglycerol mole˚
cule occupies a volume of 1610 A3 in the particle core
[37,38]. Linear regression least squares analysis of these
data yields the relationship:

v ẳ 1:042 ỵ 0:0191 ẵM=108 ị1=3

2ị

Similarly, linear regression analysis of size-fractionated
emulsions comprised of EggPtdCho and TO yields the
relationship:

v ẳ 1:014 ỵ 0:0264 ẵM=108 ị1=3

3ị

Subsequently, the molar mass distribution of Myr2GroPCho/TO and EggPtdCho/TO emulsion fractions were
calculated using Eqns (2) and (3), respectively.
For the mixtures of emulsion particles and protein,

because of the unknown contribution of the protein to the v,

a flotation coefficient distribution was calculated by
approximating the diffusion with an average diffusion
coefficient measured by dynamic light scattering. Because of

the size of the emulsion particles, diffusional broadening of
the flotation profiles is not very large, and variation of the
diffusion coefficient throughout the distribution can be
considered a second order effect. However, this method
does not allow the transformation of the flotation coefficient
distribution in a molar mass distribution.
To prevent an ill-conditioned analysis when performing
continuous size-distribution analysis with many species, a
regularization technique was employed that selects the most
parsimonious distribution of species that fits the data within
a predetermined confidence limit. Consistent with observations in previous studies [4,36], this resulted in smooth
distributions. However, in contrast to earlier studies with
sedimentation coefficient distributions of proteins, for which
maximum entropy regularization seemed advantageous
because of its potential to produce sharp peaks for discrete
mixtures [4,36,39], we found the Tikhonov-Phillips regularization with second derivative functional more useful,
because it avoids possible oscillatory artifacts known to be
encountered with the maximum entropy method for broad
distributions [40]. Furthermore, in order to obtain a high
parsimony and stability of the distribution, we applied a
high confidence limit of P ¼ 0.95. Unless stated otherwise,
all size distributions were solved on a grid of 300 radial
values between the meniscus and bottom, a confidence level
of P ¼ 0.95, frictional ratio (f/f0) ¼ 1.0 and at a resolution
(N) of 100 sedimentation coefficients between )1.0 S and
)1000 S, respectively. This resulted usually in residuals with

rms errors < 0.01. Values of f/f0 > 1.0 led to significantly
poorer fits, consistent with the spherical shape of the
emulsion particles. For Monte-Carlo statistical analysis,
1000 synthetic data sets were generated, based on the best-fit
continuous size distribution, each with different normally
distributed noise. For each point in the distribution, the
mean and the quantiles enclosing 95% of the values
from the analyses of the simulated distributions were
determined.
Apolipoprotein-emulsion binding assay
Samples of emulsion alone, protein alone, and emulsion
plus protein at various apoE concentrations were centrifuged using conventional quartz cells in a Beckman model
XL-A analytical ultracentrifuge for up to 4 h, at a rotor
speed of 5000 r.p.m. and a temperature of 20 °C.
Estimates of the signal due to free protein (relative to
the emulsion alone control) were calculated from the
optical density in the infranatant averaged over a radial
range of 0.1 cm in the plateau region using data from the
final radial scan. The average optical density due to free
protein was converted into concentration units via a fivepoint standard curve. The concentration of bound protein
was calculated based on the measured free and the known
total amount of protein. The apparent dissociation
constant (Kd) and maximum binding capacity (Bmax) were
estimated on the basis of a Langmuir isotherm, by
plotting the amount of free protein (Pf), against total
phospholipid concentration (PC) multiplied by the ratio of
free to bound protein (Pf/Pb) [2,6,41]:
Pf ẳ PCPf =Pb ịBmax Kd

4ị

Data was processed and fitted using the program
SIGMAPLOT.


5942 M. A. Perugini et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

RESULTS
Sucrose gradient fractionation of lipid emulsions
Synthetic lipid emulsions comprised of TO and Myr2GroPCho were prepared by sonication, pressure extrusion, and
fractionated by sucrose gradient ultracentrifugation [29]. To
initially examine the size-fractionated lipid emulsions,
phospholipid and triacylglycerol concentrations of each
fraction (F) were determined by enzymatic spectrophotometric analysis and the major lipid-containing samples, F2
to F6, were characterized by dynamic light scattering
(Fig. 1). The relationship of the particle radius, calculated
from the measured diffusion coefficient obtained by
dynamic light scattering, to the TO/Myr2Gro-PCho molar
ratio demonstrates successful sucrose gradient fractionation
of the lipid emulsions (Fig. 1). The larger particles show a
higher TO/Myr2Gro-PCho ratio, as predicted for a model
emulsion comprised of a phospholipid monolayer surrounding a triacylglycerol core. Based on dynamic light
scattering data, the fractionation procedure yielded individual fractions of particles with radii in the range of 38–52 nm.
Flotation velocity analysis of fractionated
lipid emulsions
The solution properties of the major emulsion fractions
were further characterized by analytical ultracentrifugation.
Figure 2 shows flotation velocity data of fractions 2, 4 and 6

at 360 s intervals. A significant time-dependent broadening
of the flotation boundary is observed in each, suggesting the
emulsion fractions are moderately heterogeneous. This
assertion is supported by the poor fits (rmsd ¼ 0.0574)
and nonrandom distribution of residuals, when for example,
the data for fraction 4 is fitted assuming a single species with
the average diffusion coefficient as determined independ-

Fig. 2. Flotation velocity of Myr2Gro-PCho/TO fractionated lipid
emulsions. Absorbance at 250 nm is plotted as a function of radial
position (open circles) for fraction 2 (A), fraction 4 (B) and fraction 6
(C) at t ¼ 78–120 min. The solid lines represent the continuous sizedistribution best-fits. Insets: Residuals (DA) are plotted as a function of
radial position (cm).

Fig. 1. Dynamic light scattering of size-fractionated lipid emulsions. The
TO/Myr2Gro-PCho molar ratio of emulsion fractions (F) 2, 3, 4, 5 and
6 is plotted vs. the particle radius determined by dynamic light scattering. The solid line represents the linear regression best-fit, describing
the relationship between TO/Myr2Gro-PCho molar ratio (m) and
particle radius (r) as, m ¼ 0.18r ) 4.9.

ently by dynamic light scattering (data not shown). Similar
observations were made with fractions 2, 3, 5, and 6. We
therefore sought a method to characterize the residual
polydispersity.
In a previous study [29], dc/dt analysis [42] was employed
to determine the apparent flotation distribution function
g(s*) of fractionated lipid emulsions. In particular for large
particles and when using absorbance optical ultracentrifuge
data, this method is intrinsically limited due to artificial
broadening that is introduced by the finite time-difference

between the scans considered for dc/dt analysis [43]. Therefore, in the present study we took advantage of the continuous size distribution method c(s) and c(M) for direct
boundary modeling [36]. Although the direct boundary


Ó FEBS 2002

Binding of apolipoprotein E3 and E4 to emulsions (Eur. J. Biochem. 269) 5943

method for the apparent flotation coefficient distribution
ls-g*(s) does not generate artificial broadening, the c(s) and
c(M) methods have the additional advantage of the
deconvolution of diffusion effects. Furthermore, compared
to more traditional approaches, such as the van HoldeWeischet method [44], we have recently demonstrated that
better resolution and more detailed distributions can be
obtained by c(sf) analysis for determining the size distributions of fractionated lipid emulsions [45]. The c(sf) analysis
best fits for fractions 2, 4 and 6 are shown in Fig. 2 (solid
lines), which result in a random distribution of residuals
(Fig. 2, insets) and low rmsd values (< 0.005) when
compared to fits assuming a single species. The resulting
c(sf) size-distributions for the major emulsion fractions,
including fractions 2, 4 and 6, are shown in Fig. 3. The data
indicate that the size-fractionated emulsions have wellseparated size-distributions, albeit with some degree of
overlap. The continuous size distributions are broader with
increasing fraction size, suggesting that the larger fractions,
F5 and F6, are more polydisperse than the smaller fractions,
F2 and F3. This may be an artifact of the fractionation

process, as the emulsions are harvested smallest to largest
following sucrose gradient ultracentrifugation. The c(sf)
distribution for unfractionated emulsions is also shown,

demonstrating a high degree of heterogeneity, as expected,
and a double maxima in c(sf) at approximately 200 S and
550 S (Fig. 3). Similar c(sf) distributions were obtained
when the emulsions were prepared and fractionated at a
10-fold higher total lipid concentration of 10 mgỈmL)1 (data
not shown). Furthermore, c(sf) analysis was also employed
to demonstrate that the fractionated lipid emulsions were
stable over a period of 9 days, at pH values in the range of
4.0–10.0, at temperatures of 5–35 °C, and in the presence of
up to 1 M NaCl (data not shown).
Continuous mass, c(M), distributions for the major
Myr2Gro-PCho/TO fractions were also determined. The
molar masses at the ordinate maximum of c(M) for each
fraction are presented in Table 1, demonstrating that the
emulsions range from 9.4 · 107 Da for fraction 2, to
7.00 · 108 Da for fraction 6. Assuming spherical particles,
supported by transmission electron microscopy (data not
shown), the calculated particle radii at maximum c(M)
correspond to 34 nm to 67 nm for fractions 2 and 6,
respectively, in the range of IDL-VLDL particles (Table 1),
and the dynamic light scattering results (Fig. 1). The values
in Table 1 are slightly higher than those obtained by
dynamic light scattering (Fig. 1), an effect attributed to the
skewed continuous size distributions especially for the larger
particles (Fig. 3).
Interaction of ApoE(263–286) with lipid emulsions

Fig. 3. Continuous size-distribution flotation velocity analysis of sizefractionated lipid emulsions. Calculated c(sf) from Eqn (1) is plotted vs.
flotation coefficient, sf, for fractions (F) 2–6 and unfractionated lipid
emulsions. The c(sf) distribution of the unfractionated lipid emulsions

has been arbitrarily scaled.

We initially examined the binding of a synthetic peptide
comprising residues 263–286 of human apoE to the
fractionated lipid emulsions. ApoE(263–286) is amphipathic
in nature, and has previously been reported to bind to
Myr2Gro-PCho bilayers [31] and SDS micelles [32], a
common lipid-mimetic. Figure 4 shows the continuous
flotation, c(sf), distribution of emulsion fraction 4 in the
absence and presence of apoE(263–286), calculated with a
fixed diffusion coefficient of 0.46 · 10)7 cm2Ỉs)1. Relative to
the control, the flotation rate of fraction 4 after the addition
of 1.0 lM peptide is significantly reduced, accompanied also
by an increase in the area under the distribution curve
(Fig. 4A). These changes are attributed to peptide binding
to the emulsion particles. Monte-Carlo analysis demonstrates that the observed increase in area under the curve
and shifts to lower values of sf in the presence of peptide are

Table 1. Hydrodynamic properties of Myr2Gro-PCho/TO size-fractionated lipid emulsions. Lipid emulsions composed of Myr2Gro-PCho and TO
were fractionated and characterized by size-distribution analysis as described in the Materials and methods. The symbols used are sf, flotation
coefficient taken from the ordinate maximum of the best-fit c(sf) distribution calculated according to Eqn (1) (Fig. 3); M, molar mass taken from the

ordinate maximum of the best-fit c(M) distribution (data not shown); v , partial specific volume calculated from M according to Eqn (2); Rs, particle

radii, calculated assuming a spherical particle and using the experimentally determined values for M and v (as above).

Fraction #

TO:Myr2Gro-PCho
molar ratio

sf
(S)

M
(· 108 Da)


v
(mLỈg)1)

RS
(nm)

2
3
4
5
6

2.05
2.62
3.18
3.86
4.29

166
333
506
664

795

0.94
2.3
4.00
5.60
7.00

1.061
1.067
1.072
1.076
1.079

34
46
55
62
67


5944 M. A. Perugini et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 5. Binding of apoE(263–286) peptide to Myr2Gro-PCho/TO
emulsion fraction 6. The amount of bound apoE(263–286) (symbols +
solid line) is plotted as a function of free protein (lg/mL). The concentrations of Myr2Gro-PCho and TO in lipid emulsion fraction
6 are 110 lM and 390 lM, respectively. Binding data was obtained by
analytical ultracentrifugation using the direct binding assay as described in Experimental procedures. Inset: Linearized plot of the

binding data for apoE(263–286) (symbols) shown in panel A. The solid
line represents the linear least-squares fits to the data according to Eqn
(4), where the y-intercept and slope equate to the apparent Kd and
Bmax, respectively (Table 2).

Fig. 4. Continuous size-distribution analysis of fraction 4 in the presence
and absence of apoE(263–286) peptide. (A) The c(sf) distribution calculated using an invariant D ẳ 0.46 Ã 10)7 cm2ặs)1 is plotted as a
function of flotation coefficient for Fraction 4 alone (solid line, no
symbols); fraction 4 + 1.0 lM apoE(263–286) (solid line, open symbols) and fraction 4 + 10.0 lM apoE(263–286) (solid line, solid symbols). (B) Results of Monte-Carlo statistical analysis distributions,
calculated from 1000 synthetic data sets to a confidence level of P ¼
0.95. The lower (0.025) and upper (0.975) quantiles are depicted as
dashed lines, enclosing the mean distribution (solid lines) for fraction 4
alone (labelled 1), and fraction 4 + 1.0 lM apoE(263–286) peptide
(labelled 2).

statistically significant, and cannot be attributed to noise
affecting the data analysis (Fig. 4B). At a 10-fold higher
peptide concentration of 10.0 lM, a greater decrease in
flotation rate is observed, indicating the peptide binds to the
emulsion particles in a saturable manner (Fig. 4A). However, although c(sf) analysis shows detailed changes of the
size-distribution upon peptide binding, it is not directly
possible to quantify the observed binding for the calculation
of a binding constant and maximum capacity. Nevertheless,
this can be accomplished by a direct binding assay. This
method is based on depletion of emulsion-bound protein (or
peptide) from the infranatant region under conditions where
the degree of sedimentation of the unbound protein (or
peptide) is negligible. The binding profile for apoE(263–286)
to Myr2Gro-PCho/TO emulsion fraction 6 is presented in

Fig. 5. As the peptide concentration is increased, larger
amounts of apoE(263–286) bind to the emulsion particles
(Fig. 5), which is consistent with the results of the flotation
velocity analysis (Fig. 4A). The shape of the binding profile
indicates that saturation is approached at a peptide
concentration of 120 lgỈmL)1 (% 50 lM). By plotting the
free protein, Pf, against the phospholipid concentration
multiplied by the ratio of free to bound peptide, a linear plot
results (Fig. 5, inset), yielding an apparent dissociation
constant, Kd, of 75 lM and binding capacity, Bmax, of
approximately four amino acids per phospholipid (Table 2).
Interaction of ApoE3 and ApoE4 with Myr2Gro-PCho/TO
lipid emulsions
To compare the interactions of apoE3 and apoE4 isoforms
with size-fractionated lipid emulsions, flotation velocity
experiments were conducted in the analytical ultracentrifuge. Fraction 2 was employed as a synthetic model for
small lipoprotein particles and fraction 6 for large lipoprotein particles.
The c(sf) distributions of fraction 2 in the absence and
presence of 1.0 lM apoE3 or apoE4 are compared in
Fig. 6A. Relative to the control, the c(sf) distribution of
fraction 2 in the presence of 1.0 lM apoE3 shows an
increase in area under the curve and a shift to lower
flotation coefficients, indicating significant amounts of
apoE3 bind these small emulsions (Fig. 6A). In contrast,
the c(sf) distribution of fraction 2 in the presence of
1.0 lM apoE4 is more similar to the control, indicating
lesser amounts of apoE4 bind the small emulsion particles.
As for apoE(263–286), Monte-Carlo analysis demonstrates

Ó FEBS 2002

Binding of apolipoprotein E3 and E4 to emulsions (Eur. J. Biochem. 269) 5945

Table 2. Parameters for the binding of apoE(263–286), apoE3 and apoE4 to Myr2Gro-PCho/TO emulsion fraction 6. Kd and Bmax values were
calculated according to Eqn (9).
Bmax

Kd
ApoE Isoform
or Peptide

(lgỈmL)1)

(lM)

ApoE/particle

PL/ApoE

Amino acids/PL

ApoE(263–286)
ApoE3
ApoE4

210
15
17

75
0.44
0.51

3.14 · 104
1010
1630

5.9
163
101

4.1
1.83
2.96

that the observed changes in the c(sf) distributions of
fraction 2 in the presence of apoE3 or apoE4 are
statistically significant, and cannot be attributed to noise
affecting the data analysis (Fig. 6B).
Analysis of the c(sf) distributions for large emulsions
(fraction 6) in the presence and absence of 0.5, 1.0 and
2.0 lM apoE3 or apoE4 reveals an opposite trend
(Fig. 7A,B). Although there is evidence that apoE3 interacts
with fraction 6, given by the shift in the distribution to lower
flotation coefficients (Fig. 7A), there is a significantly
greater increase in the ordinate maximum value and area
under the curve for the c(sf) distribution in the presence of
apoE4 at corresponding protein concentrations (Fig. 7B).
This suggests that a higher proportion of apoE4 binds to the

large emulsion particles. As for fraction 2 (Fig. 6B), MonteCarlo analysis of all data sets presented in Fig. 7A and B
demonstrates that the comparative differences observed for
apoE3 and apoE4 are statistically significant (data not
shown).

fraction 2 in the presence of 1.0 lM apoE4 is similar to the
control, suggesting little apoE4 is bound to the fraction 2
particles.
Likewise, the interaction of apoE3 and apoE4 isoforms with large EggPtdCho/TO emulsions was examined.
Figure 7C shows the flotation velocity data of EggPtdCho/
TO fraction 6 in the presence and absence of 1.0 lM apoE3
or apoE4. Consistent with earlier observations (Fig. 7A,B),
a minor shift to smaller flotation coefficients is observed for
EggPtdCho/TO fraction 6 in the presence of both apoE
isoforms. However, a greater increase in the ordinate
maximum and area under the curve is obvious in the
presence of the apoE4 isoform (Fig. 7C, open symbols),
indicating that a greater proportion of apoE4 binds the
larger particles. Together with the results obtained in the
previous section employing Myr2Gro-PCho/TO emulsions,
these data support the conclusion that apoE3 and apoE4
bind preferentially to small and large lipid particles,
respectively.

Interaction of ApoE3 and ApoE4 with EggPtdCho/TO
lipid emulsions

Direct binding analysis of ApoE3 and ApoE4 to large
emulsion particles

The binding of apoE3 and apoE4 to lipid particles was
also assessed using size-fractionated emulsions comprised
of egg yolk phosphatidylcholine (EggPtdCho) and TO.
EggPtdCho is comprised of saturated and unsaturated
phospholipids, which also differ in fatty acyl chain length,
and are therefore more biologically relevant than monolayers comprised of Myr2Gro-PCho alone [46]. The
EggPtdCho/TO emulsions were synthesized by pressure
extrusion and fractionated by sucrose-gradient ultracentrifugation, employing identical procedures to those used for
the synthesis of Myr2Gro-PCho/TO emulsions. Following
fractionation, flotation velocity experiments were performed
to characterize the solution properties of these emulsions,
which are summarized in Table 3. The data presented in
Table 3 demonstrates that the EggPtdCho/TO emulsions
share similar physical properties to the fractionated
Myr2Gro-PCho/TO emulsions (Table 1). Accordingly, flotation velocity experiments were employed to compare the
binding of apoE3 and apoE4 to small (fraction 2) and large
(fraction 6) EggPtdCho/TO emulsions.
As for Myr2Gro-PCho/TO emulsions (Figs 6A,B and
7A,B), identical size-dependent binding preferences were
observed (Figs 6C and 7C). Relative to the emulsion sample
alone, the flotation rate of fraction 2 in the presence of
1.0 lM apoE3 is significantly decreased, particularly evident
for particles with sf < 200 S (Fig. 6C), indicating an appreciable amount of apoE3 is bound to the small lipid particles.
In comparison, the c(sf) distribution of EggPtdCho/TO

To verify and quantify the size-dependent interaction of
apoE3 and apoE4 to fractionated lipid emulsions, we also
employed a direct binding using Myr2Gro-PCho/TO
fraction 6 and physiological concentrations of apoE. The
binding isotherms for the apoE3 and apoE4 isoforms are

presented in Fig. 8A. Both curves show evidence of
saturation, although the proportion of bound apoE4 is
significantly greater at all protein concentrations
employed, particularly in excess of 30 lgỈmL)1 or 1.0 lM
(Fig. 8A). The apparent Kd and Bmax values for apoE3
and apoE4, calculated from the linearized plots according
to Eqn (4) (Fig. 8B), are presented in Table 2. In general,
these values agree well with previous studies employing
purified apoE [2,6]. However, the results of these analyses
indicate that apoE3 and apoE4, though possessing a
similar apparent binding affinity (Kd) for the large lipid
particles, are notably distinct in their maximum binding
capacities (Table 2). In particular, the binding footprint
for the apoE4 isoform, corresponding to 3.0 amino acids
per phospholipid, is almost twofold greater than the
apoE3 isoform (Table 2), suggesting these isoforms differ
in their conformational state when bound to large
synthetic emulsions.

DISCUSSION
Besides size, native lipoproteins differ in density, lipid
dynamics, lipid composition and apolipoprotein content


5946 M. A. Perugini et al. (Eur. J. Biochem. 269)

Fig. 6. Flotation velocity analysis of Myr2Gro-PCho/TO and
EggPtdCho/TO emulsion fraction 2 in the absence and presence of
apoE3 and apoE4 isoforms. The c(sf) distribution calculated using an
invariant D ¼ 0.63 · 10)7 cm2Ỉs)1 is plotted as a function of flotation

coefficient. (A) Myr2Gro-PCho/TO fraction 2 alone (solid line, no
symbols), and Myr2Gro-PCho/TO fraction 2 in the presence of 1.0 lM
apoE3 (solid line, solid symbols) and 1.0 lM apoE4 (solid line, open
symbols). Total lipid concentration in Myr2Gro-PCho/TO fraction 2 ¼ 410 lM, i.e. [Myr2Gro-PCho] ¼ 150 lM + [TO] ¼ 260 lM.
(B) Results of Monte-Carlo statistical analysis distributions, calculated
from 1000 synthetic data sets to a confidence level of P ¼ 0.95. The
lower (0.025) and upper (0.975) quantiles are depicted as dashed lines,
enclosing the mean distribution (solid lines) for Myr2Gro-PCho/TO
fraction 2 alone (labelled 1), Myr2Gro-PCho/TO fraction 2 + 1.0 lM
apoE3 (labelled 2), and Myr2Gro-PCho/TO fraction 2 + 1.0 lM
apoE4 (labelled 3). (C) EggPtdCho/TO fraction 2 alone (solid line, no
symbols); EggPtdCho/TO fraction 2 + 1.0 lM apoE3 (solid symbols
+ line) and EggPtdCho/TO fraction 2 + 1.0 lM apoE4 (open symbols + line). The total lipid concentration in EggPtdCho/TO fraction
6 ¼ 280 lM, i.e. [EggPtdCho] ¼ 135 lM + [TO] ¼ 145 lM.

Ĩ FEBS 2002

Fig. 7. c(sf) distribution analysis of Myr2Gro-PCho/TO and
EggPtdCho/TO fraction 6 in the presence and absence of apoE3 and
apoE4. The c(sf) distributions calculated using an invariant D ẳ
0.38 Ã 10)7 cm2ặs)1 are plotted as a function of flotation coefficient.
(A) Myr2Gro-PCho/TO fraction 6 alone (solid line, no symbols);
Myr2Gro-PCho/TO fraction 6 + 0.5 lM apoE3 (dashed line),
Myr2Gro-PCho/TO fraction 6 + 1.0 lM apoE3 (dashed-dotted line)
and Myr2Gro-PCho/TO fraction 6 + 2.0 lM apoE3 (solid line +
open symbols). The total lipid concentration in Myr2Gro-PCho/TO
fraction 6 ¼ 125 lM, i.e. [Myr2Gro-PCho] ¼ 23 lM + [TO] ¼
102 lM. (B) Myr2Gro-PCho/TO fraction 6 alone (solid line, no symbols); Myr2Gro-PCho/TO fraction 6 + 0.5 lM apoE4 (dashed line),
Myr2Gro-PCho/TO fraction 6 + 1.0 lM apoE4 (dashed-dotted line)
and Myr2Gro-PCho/TO fraction 6 + 2.0 lM apoE4 (solid line +

open symbols). (C) EggPtdCho/TO fraction 6 alone (solid line, no
symbols); EggPtdCho/TO fraction 6 + 1.0 lM apoE3 (solid symbols
+ line) and EggPtdCho/TO fraction 6 + 1.0 lM apoE4 (open symbols + line). The total lipid concentration in EggPtdCho/TO fraction
6 ¼ 94 lM, i.e. [EggPtdCho] ¼ 21 lM + [TO] ¼ 73 lM.


Ó FEBS 2002

Binding of apolipoprotein E3 and E4 to emulsions (Eur. J. Biochem. 269) 5947

Table 3. Hydrodynamic properties of EggPtdCho/TO size-fractionated lipid emulsions. Lipid emulsions composed of EggPtdCho and TO were
fractionated and characterized by size-distribution analysis as described in Experimental procedures. The symbols used are sf, flotation coefficient
taken from the ordinate maximum of the best-fit c(sf) distribution; M, molar mass taken from the ordinate maximum of the best-fit c(M)

distribution employing Eqn (3); v, partial specific volume calculated from M using Eqn (3) (as above); Rs, particle radii, assuming a spherical

particle and using the experimentally determined values for M and v.

Fraction #

TO:EggPtdCho
molar ratio

sf
(S)

M
(· 108 Da)



v
(mLỈg)1)

RS
(nm)

2
3
4
5
6

1.07
1.66
2.18
2.84
3.54

133
313
487
649
806

0.76
2.31
4.05
5.80
7.60

1.038
1.049
1.056
1.061
1.067

31
46
55
62
68

Fig. 8. Binding of apoE3 and apoE4 to Myr2Gro-PCho/TO emulsion
fraction 6. (A) The amount of bound apoE3 (solid symbols + solid
line) and apoE4 (open symbols + solid line) is plotted as a function of
free protein (lgỈmL)1). The concentration of Myr2Gro-PCho and TO
in lipid emulsion fraction 6 ¼ 150 lM and 260 lM, respectively.
Binding data was obtained by analytical ultracentrifugation using
the direct binding assay as described in Experimental procedures.
(B) Linearized plot of the binding data for apoE3 (solid symbols)
and apoE4 (open symbols) shown in panel A. The solid line represents
the linear least-squares fits to the data according to Eqn (4), where the
y-intercept and slope equate to the apparent Kd and Bmax, respectively
(Table 2).

[47–50]. This raises the possibility that any one or a
combination of these properties may influence the binding
of apolipoproteins to lipoprotein particles. The capacity to
fractionate lipid emulsions into relatively homogeneous
particles (Figs 1 and 3) provides an experimental system to

directly examine the effect of particle size on apolipoprotein
binding. Nevertheless, synthetic emulsions can differ markedly in monolayer-core lipid dynamics depending on their
lipid composition [51,52]. Accordingly, we have employed
two different phospholipid-stabilized emulsions to demonstrate that apoE3 and apoE4 discriminate between synthetic
lipid emulsions on the basis of size or surface curvature. The
apoE3 isoform is shown to bind preferentially to small,
highly curved lipid emulsions (Fig. 6); whereas the apoE4
molecule binds preferentially to large, less curved lipid
particles (Fig. 7). This behaviour is consistent with previous
studies showing that apoE3 and apoE4 distribute preferentially with HDL and VLDL, respectively [7–10]. Furthermore, these results may be considered in relation to a
number of in vivo observations. The elevated LDL concentrations reported in subjects with homozygous E4/4 phenotypes [18] may be due to the inability of apoE4 to bind
and initiate the clearance of small lipoproteins in plasma. In
contrast, the observation that chylomicron remnants are
cleared faster in subjects with apoE4, compared to those
with apoE3 [53], may be explained by the superior ability of
apoE4 to bind large lipid particles. Similarly, a sizedependent binding phenomenon may account for the
observation that apoE3 distributes preferentially with small
(density > 1.125 gỈmL)1) and apoE4 with large (density
< 1.00 gỈmL)1) lipoproteins in the cerebrospinal fluid of the
brain [54].
Insight into the structural basis for apoE3 and apoE4
lipid binding preferences is provided by earlier studies,
where it is demonstrated that apoE3 and apoE4 differ in
their NH2- and COOH-terminal domain interactions [7,8].
In addition, it is known that truncation of apoE4 at residue
244 abolishes VLDL binding [7], although the same
truncated variant of apoE3 retains the ability to bind
HDL [23]. This suggests the important determinants for
binding large lipid particles are downstream of residue 244,
in the COOH-terminal region of the protein. More recently,

studies employing fluorescence resonance energy transfer
[24,26], intradomain disulfide bonding [3], nuclear magnetic
resonance [25], and microcalorimetry [28] experiments
demonstrate that the NH2-terminal domain of apoE can
reorganize from a closed to open conformation in the


Ó FEBS 2002

5948 M. A. Perugini et al. (Eur. J. Biochem. 269)

REFERENCES

Fig. 9. A model for the lipoprotein-bound conformations of human
apolipoprotein E isoforms. The COOH-terminal domain is shown
bound to the lipoprotein surface, whilst the NH2-terminal domain
undergoes conformational changes from (A) the closed conformation,
to (B) the open conformation. Model adapted from [27,28].

presence of lipid. In view of these observations, differences
in the propensity for apoE3 and apoE4 to adopt different
conformations on the surface of lipid particles may explain
their ability to distribute preferentially to different sized
particles. The data presented in this study supports this
suggestion.
We used a direct binding assay in the analytical
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However, we have previously demonstrated that both
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likely to provide greater flexibility for interacting with
highly curved lipid surfaces. Differences in the propensity
of apoE3 and apoE4 to adopt open and closed conformations (Fig. 9) may provide insight into the role of
apoE4 in disease.

ACKNOWLEDGEMENTS
We would like to thank Dr Karl Weisgraber (Gladstone Institute of
Cardiovascular Disease, San Francisco, CA, USA) for kindly supplying
the apoE3 and apoE4 pGEX-3X plasmids. We also thank Con
Dogovski, Cait MacPhee and Ben Atcliffe for advice and assistance
during the course of this work. This work was funded by the National

Health and Medical Research Council, Australia.

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