From the
A series of monoclonal antibodies against epitopes spanning
different domains of apoA-I have been tested for their effects on
unesterified cholesterol transfer between low density lipoprotein (LDL)
and well-defined homogenous lipoproteins reconstituted with
phosphatidylcholine, cholesterol, and apoA-I (LpA-I). Antibodies 2G11
(reacting between residues 25 and 110), A05 (residues 25-82), A03
(residues 135-140), A44 and r5G9 (residues 149-186), and
4A12 (residues 173-205) significantly inhibit cholesterol
transfer from LDL to Lp2A-I while they enhance transfer in the opposite
direction, thus causing an increased net transfer to LDL. Most of these
monoclonal antibodies (mAbs) also enhance phospholipid transfer to LDL
but in a lesser and variable proportion relative to cholesterol. Their
epitopes are mainly contained within domains that are predicted to be
amphipathic
The efflux of unesterified (free) cholesterol from cells to
acceptor lipoproteins has been well studied and appears to be
satisfactorily explained by the mechanism of aqueous diffusion (for
review, see Ref. 1). However apolipoprotein A-I-containing lipoproteins
(LpA-I)
We propose that the specificity of the collision at
the interface must depend on an apolipoprotein specificity that
implicates the interaction of defined and possibly different domains
with each donor and acceptor system. The complementarity of such
domains, which may depend on charge
(8, 9) , would
differ as a function of the interface involved. This hypothesis can be
tested using monoclonal antibodies against apoA-I as probes to modify
the interactions between the LpA-I and other cholesterol-carrying
structures. We have selected LDL and discoidal LpA-I particles as a
simpler and well defined system
(10, 11, 12, 13, 14) , compared
with the cell-lipoprotein interface, and have used a selection of mAbs
reacting with epitopes distributed over most of apoA-I sequence
(15) to test the directionality of the interaction between these
two types of lipoproteins. The results obtained in this study are
compatible with a concept of directional specificity in the interaction
of lipoproteins but more importantly demonstrate the involvement of
specific domains of apoA-I in the control of cholesterol binding and
desorption. However the unidirectional effect of the active mAbs on
cholesterol transfer and their increase of the
For cholesterol transfer from LDL to LpA-I, LDL was
labeled with [
Cholesterol transfer rate as a function of incubation time was
calculated essentially as described by others
(10) . The
negative and positive values represent inhibitory and enhancement
effects of the mAbs on cholesterol transfer, respectively.
Since specific anti-apoA-I mAbs both
stimulate the transfer of cholesterol from Lp2A-I to LDL and inhibit
transfer in the opposite direction, net mass transfer of cholesterol
from LDL to LpA-I must take place. Indeed cholesterol mass in the
supernatant fractions (LpA-I compartment) is significantly reduced
after incubation with LDL in the presence of mAbs such as A05, A03,
A44, and 4A12, but not with 4H1 or control mAb 2H2 (Fig. 3).
The effects of these anti-apoA-I mAbs on cholesterol transfer to and
from LDL were also studied using other Lp2A-I particles of different
sizes. Very similar results were observed, and the same mAbs inhibited
transfer from LDL to Lp2A-I and enhanced transfer in the opposite
direction (). In contrast, these antibodies have no effect
on the transfers to and from Lp2A-I (7.8 nm) (not illustrated), the
smallest and most cholesterol enriched particle in the Lp2A-I class.
This particle also is unstable and tends to release free apoA-I from
which it is difficult to separate. We attribute the lack of antibody
effect on transfer reactions with this particle to its extreme
composition and its unstability.
We have also observed no effect by
anti-apoA-I mAbs on cholesterol transfer between LDL and either Lp3A-I
or Lp4A-I (not illustrated). The kinetics of these transfer have been
studied in details elsewhere
(28) . Lp3A-I or -4A-I of a size
and composition identical to these studied here were found to exchange
very little cholesterol with LDL
(28) , whereas Lp2A-I was the
most active donor and acceptor. We attribute the lack of the effect of
the mAbs on cholesterol transfer to the large particles to the slow
transfer rates.
Only antibody A44 causes a significant increase in
the precipitation of
The observation that specific anti-apoA-I antibodies inhibit
cholesterol transfer from LDL to LpA-I and enhance transfer in the
opposite direction, resulting in a net loss of cholesterol from LpA-I,
was unexpected and incompatible with the initial hypothesis that formed
the basis of our experimental approach. Indeed such effects cannot be
related to a simple steric hindrance but must also imply specific
conformational modifications of the Lp2A-I particle that modify
cholesterol binding.
To rule out any possibility of artifact, the
cholesterol transfer assay has been extensively verified and validated,
especially when carried out in the presence of antibodies. The
antibodies have been shown not to interact with the
heparin-Mn
Both cholesterol and phospholipids have been
proposed to transfer by collision- or diffusion-mediated mechanisms
(6, 7) , and simultaneous transfers of these lipids have
been demonstrated from cells to apolipoproteins
(34) . This
suggested to us that mAbs could have simultaneous effects on the
transfer of both lipids. However, when we analyzed the effects of mAbs
on the transfer of other components of LpA-I, phospholipids, and
apoA-I, the picture of transfer that emerges is one that appears to
vary between mAbs and affect each component individually. Among all
antibodies tested, only A44 enhances transfer of
As shown by the ellipticity of the immunecomplexes,
most antibodies increase the
The difference
between antibodies in eliciting either simultaneous or individual
transfer of cholesterol and phospholipids suggests that the
distribution of these lipids in relation to apoA-I could vary between
domains or that different domains compete differently for binding to
the phospholipids. Calorimetric and solubility studies of reconstituted
HDL with varying cholesterol have shown that cholesterol is excluded
from about 55% of the phospholipid molecules bound to the
apolipoprotein in a nonmelting state, which constitutes the
phospholipid boundary layer
(36) . Incorporation of fluorescent
labeled cholesterol and phospholipids in reconstituted HDL has also
demonstrated that the fraction of cholesterol adjacent to apoA-I is
less than that of phospholipids
(35) , and this has been
interpreted as the result of a competition between apoA-I and
cholesterol for hydrophobic solvation by phospholipids. This concept is
supported by measurements of interfacial tension of phosphatidylcholine
and cholesterol monolayers that exhibit a strong cooperative lateral
interaction between the two lipids. In the presence of apoA-I, this
cooperation is replaced by a strong positive interaction between apoA-I
and phospholipids and a weak lateral interaction between apoA-I and
cholesterol
(37) . These observations could also be compatible
with the notion that different pools of cholesterol may exist within
lipoprotein particles and that their distribution may also vary in
relation to apoA-I domains. This would explain the differential effects
of mAbs on cholesterol and phospholipid transfers, and mAbs against
different regions of apoA-I could affect cholesterol transfer through
different mechanisms.
The specificity of the inhibitory mAbs is made
more significant by the existence of other mAbs that have no effect on
cholesterol transfers between LDL and LpA-I, and it is interesting to
consider the location of their epitopes in relation to what is known of
apoA-I structure. The epitopes of the neutral mAbs are located in two
distinct sequences, 4H1 at the extreme N terminus, residues 2-8,
and 3G10 and 5F6 in a central domain centered around residue 121 and
spanning residues 99-135 (Table I and Fig. 6). These two
domains have been already identified as being closely related, both
structurally and functionally. Our initial competition assays have
shown that the N-terminal domain (identified by mAb A05) is close to
the central domain
(21) , but 4H1, which was then used as
capture antibody, could not be entered in these competition assays.
More recent competition experiments using the capture antibody as a
competitor have brought further evidence showing that these two
domains, including 4H1, are indeed very close in discoidal LpA-I
(Fig. 6). However it should be noted that such composite domain
can be intermolecular rather than intramolecular as depicted here,
i.e. involving the N terminus of one apoA-I and the middle
domain of another. These antibodies also have similar effects on the
cholesterol esterification reaction
(31) ; 4H1 and 5F6 both
enhance lecithin:cholesterol acyltransferase reaction with small Lp2A-I
(7.8 nm), while 4H1, 2G11, and 5F6, enhance lecithin:cholesterol
acyltransferase reaction with small Lp3A-I (10.8 nm). It is therefore
reasonable to conclude that the epitopes for 4H1 and 5F6 are located
within the same domain on apoA-I three-dimensional structure and also
share the same functional specificity, including a similar lack of
effect of mAbs binding to these sites on cholesterol transfer. This
neutral domain separates two lipid binding domains, residues
25-110 or 25-120 and residues 135-205, which, based
on the present results, are those competing with cholesterol for
binding to phospholipids. Thus the binding of mAbs to epitopes located
in these domains decreases cholesterol retention by LpA-I by increasing
apoA-I interaction with phospholipids.
In conclusion, although we need more
information for a complete interpretation of the role of apoA-I domains
in cholesterol transport and metabolism, we have presented evidence for
the direct link between apoA-I structure and cholesterol transfers and
for the existence of apoA-I domains that differ in their interaction
with cholesterol versus phospholipids. Furthermore we can make
several hypothesis for further testing. It is clear that as initially
suggested by others
(14) , the interface between apoA-I and
lipids on the edge of discs introduce incongruities in lipid packing.
We have further defined this concept and provided evidence that
cholesterol binding is linked to defined regions of apoA-I and depends
on the conformation of specific domains. The present report suggests
that cholesterol association and desorbtion from LpA-I may involve
particular domains because mAbs binding to epitopes overlapping
specific amphipathic
Lp2A-I (9.6 nm) labeled with [
-helices. In contrast, mAbs 4H1 (residues 2-8),
3G10 (residues 96-121), and 5F6 (residues 116-141) have
little or no effect on either cholesterol or phospholipid transfer, and
the epitopes for these three mAbs have been shown in earlier studies to
be structurally and functionally related. Their immunoreactivity
responds similarly to variation in lipoprotein cholesterol content, and
the antibodies binding to these sites compete with one another and have
similar effects on the cholesterol esterification reaction. Thus, the
current results are compatible with the hypothesis that they form an
integrated domain with a common function in cholesterol metabolism,
possibly as part of a hinge domain. Most mAbs were found to increase
significantly the
-helicity of apoA-I in the Lp2A-I
immunecomplexes, suggesting that they may increase the stability of the
lipid-bound apoA-I. However, not unexpectedly, there is no correlation
between the effects of mAbs on
-helicity and their effects on
cholesterol or phospholipid transfer since each mAb has a discrete
effect on these transfers. These studies demonstrate the specificity of
LpA-I particles in cholesterol transport and document the existence of
apoA-I domains with different functions in cholesterol transport.
(
)
with a pre
migration are the most
avid acceptors of cellular cholesterol, and the specificity of these
lipoproteins in this pathway also suggests that greater binding (or
affinity) of cholesterol with these lipoproteins provides a
directionality for cholesterol flux
(2, 3) . In
contrast, the transfer of cholesterol between lipoproteins appears to
proceed with a different specificity as demonstrated by the transfer
from LDL to plasma lipoproteins, which is first channeled through
-migrating lipoproteins rather than pre
-lipoproteins
(4, 5) . Therefore, the observed preferential binding
affinity of pre
LpA-I for cholesterol depends on the type of donor
and acceptor involved (the transfer interface). Such a selectivity of
acceptors and interfacial systems suggest that a complex mechanism is
involved, which may include both an activation step, possibly a
desorption step as suggested by McLean and Phillips
(6) or a
unimolecular activation event, perhaps the movement of cholesterol to a
more exposed (hydrated) position, and a collision step
(7) characterized by a directionality specific for each
interface, such as cells to lipoproteins and lipoproteins to
lipoproteins.
-helicity of
several Lp2A-I immunecomplexes suggest that the action of mAbs in
cholesterol transfer is more complex than a simple steric hindrance and
is probably related to specific conformational modifications.
Isolation of Plasma LDL, HDL, and Purification of
apoA-I
Plasma LDL was prepared from pooled plasma from
normolipemic volunteers by sequential flotation ultracentrifugation as
described previously
(16) . The LDL fraction (KBr density
= 1.020-1.063 g/ml) of plasma was dialyzed against
Tris-EDTA buffer containing 10 m
M Tris-HCl, pH 8.0, 1 m
M EDTA, and 1 m
M NaNand stored at 4 °C
under N
for up to 2 weeks. The HDL fraction (KBr density
= 1.063-1.21 g/ml) of plasma was delipidated,
resolubilized in 6
M guanidine-HCl, and chromatographed on two
serial Sephacryl S-200 gel filtration columns (100
2.5 cm)
equilibrated with 3
M guanidine in 10 m
M Tris buffer,
pH 8.0, to isolate apoA-I protein
(17) .
Reconstitution and Purification of Homogeneous LpA-I
Particles
Reconstituted HDL was prepared from
1-palmitoyl-2-oleoylphosphatidylcholine (POPC) (Sigma), cholesterol
(Sigma), and apoA-I at initial molar ratio of 120:6:1 or 88:44:1 by
cholate dialysis following previously published procedures
(18, 19) . Homogeneous lipoprotein particles containing
two, three, or four apoA-I molecules (Lp2A-I, -3A-I, or -4A-I) were
purified by gel filtration on two serial agarose Bio-Gel 5
M columns (95 2.5 cm) (Bio-Rad) equilibrated with Tris-EDTA
buffer. The Lp2A-I (9.6 nm), Lp3A-I (13.4 nm), and Lp4A-I (17.0 nm)
particles were purified from the above reconstituted HDL at an initial
molar ratio of 120:6:1, and Lp2A-I (7.8 nm), Lp2A-I (8.6 nm), and
Lp3A-I (10.8 nm) particles were purified from the above reconstituted
HDL at an initial molar ratio of 88:44:1. The Lp2A-I (10.2 nm)
particles were prepared according to Sparks et al. (20) at an initial POPC/cholesterol/apoA-I molar ratio of
150:6:1. The characteristics of these particles were well defined and
reported in previous studies
(21) . Size characterization of
LpA-I particles was done by native gradient gel electrophoresis using
high molecular calibration kit (Pharmacia Biotech Inc.) with reference
globular proteins (thyroglobulin, 17.0 nm; ferritin, 12.2 nm; lactate
dehydrogenase, 8.16 nm; and albumin, 7.1 nm) as described by others
(22) . The number of apoA-I molecules/LpA-I particles was
determined by the cross-linking with dimethyl suberimidate technique
(23) . The protein concentration of lipoproteins was measured by
Lowry assay
(24) , and that of cholesterol and POPC were
measured by enzymatic kits (Boehringer Mannheim).
Labeling of LDL and LpA-I
To measure cholesterol
transfer from LDL to LpA-I, LDL was prelabeled with
[1,2-H]cholesterol
([
H]cholesterol) (DuPont NEN) using 10
µCi/100 µg LDL protein. Briefly, 10 µCi of
[
H]cholesterol solution in toluene was dried to
completion under N
onto the wall of a glass tube. LDL
protein (100 µg) diluted to 500 ml with 1% bovine serum albumin in
Tris-buffered saline-EDTA was added into the
[
H]cholesterol-coated tube and incubated at 37
°C for 30 min. The incubation mixture was dialyzed against 20
m
M Tris buffer, pH 7.7, containing 150 m
M NaCl (TBS)
in which all cholesterol transfer assays were carried out. The
procedure to label LpA-I with [
H]cholesterol is
essentially the same as above. To monitor the POPC transfer between
lipoproteins, the donor particles were incubated with 10 µCi of
[ oleoyl-1-
C]POPC
([
C]POPC) (DuPont NEN) coated on the glass wall
as described above. To monitor the potential apoA-I movement during the
process of cholesterol transfer assay, apoA-I in LpA-I were iodinated
by Iodo-Bead technique (Pierce).
Monoclonal Antibodies
Antibodies 4H1, 3G10, and
5F6 were prepared in our laboratory
(25) . Antibodies 2G11 and
4A12, characterized by Petit et al. (26) , were
purchased from Sanofi (Paris, France). Antibodies A05, AO3, A44, and
r5G9 are a generous gift of Dr. Fruchart (SERLIA, Institut Pasteur,
Lille). The apoA-I sequence forming the epitopes for each of these mAbs
is indicated in and was previously delineated in our lab
by their immunoreaction to synthetic peptides, -galactosidase
fusion proteins and CNBr fragments of apoA-I molecules
(15) .
Antibody 2H2 against rat synthetic atrial natriuretic factor and
Bsol.12 against solubilized apo B
(27) were used as control.
All of the mAbs used are murine IgG, which were purified on protein G-
or protein A-Sepharose columns (Pharmacia). Fab fragments of these mAbs
were prepared by papain digestion and purified by protein A-Sepharose
affinity chromatography.
Cholesterol Transfer Assay between LDL and LpA-I
Particles
All components for this assay were dialyzed against 4
liters of TBS at 4 °C overnight immediately prior to the
experiment. LpA-I particles diluted in 100 µl of 1% bovine serum
albumin-TBS were preincubated at 37 °C for 30 min with serial
dilution of mAbs in 200 µl of TBS, which gave mAb/apoA-I molar
ratio between 2 and 0.0625. LDL diluted in 160 µl of 1% bovine
serum albumin-TBS was added to initiate the cholesterol transfer
process. The mixtures were incubated at room temperature for 20 min.
The transfer reactions were stopped in an ice bath.
Heparin-Mnprecipitation procedure was employed to
separate LDL and LpA-I as described by others
(6, 10) .
Briefly, 25 µg of carrier LDL in 50 µl of Tris buffer, 100
µl of 0.14% heparin, and 25 µl of 1
M MnCl
were added into each tube. The tubes were vortexed, kept on ice
for 10 min and then centrifuged in a Microfuge (Eppendorf, Westbury,
NY) at 15,000 rpm for 10 min. The radioactivities of precipitates
containing mainly LDL and the supernatants containing LpA-I were
subsequently counted in a scintillation counter. A correction factor
was included in the calculations to correct for the constant amount of
LpA-I, which is co-precipitated and is independent of the presence of
antibodies.
H]cholesterol, and the assays were
done at LpA-I/LDL particle ratio of 40:1. A typical incubation mixture
included 2.1 µg of LpA-I protein, 0.5 µg of LDL protein, and
mAb starting at 22.5 µg/tube with 1:2 serial dilution. For
cholesterol transfer from LpA-I to LDL, LpA-I was labeled with
[
H]cholesterol, and the assays were done at
LpA-I/LDL particle ratios of 2:1. Particle ratio is calculated using
the molar apolipoprotein composition. A typical incubation mixture
includes 1.0 µg of LpA-I protein, 4.5 µg of LDL protein, and
mAb starting at 12 µg/tube with 1:2 serial dilution. These
conditions were determined and chosen within linear range of
cholesterol transfer according to the kinetic studies described
elsewhere
(28) . No significant difference in the effects of the
mAbs tested were observed during incubations varying from 20 to 60 min.
Measurement of Cholesterol Mass Transfer from LDL to
LpA-I in the presence of Anti-apoA-I Antibodies
The transfer
reactions were done under the same conditions as above but without
isotope labeling and scaled up 10-fold in order to obtain sufficient
material for enzymatic assay of cholesterol mass. After incubation for
transfer and precipitation of LDL by heparin-Mn, the
supernatants (LpA-I compartment) were concentrated 5 times by speedvac
concentrator (Savant, Farmingdale, NY), and cholesterol mass in these
supernatants was determined by enzymatic kits (Boehringer Mannheim).
Separation of Lp2A-I (9.6 nm) or Lp2A-I
LpA-I labeled with
[4-mAb Complexes
and LDL by Agarose Gel Electrophoresis
C]cholesterol
([
C]cholesterol) were incubated with appropriate
mAbs in the presence or absence of LDL at the same ratio as for the
transfer assay but in smaller volumes. The reaction mixtures were
applied onto Paragon Lipo agarose gel electrophoresis system (Beckman,
Brea, CA) and run at 100 V for 30 min. The gels were fixed with 10%
acetic acid for 5 min and baked to dryness at 80 °C. The dry gels
were either autoradiographed or sliced into 2-mm fractions for counting
of radioactivity. Pure IgG preparation of some mAbs were also iodinated
and run in agarose gel electrophoresis to compare their electrophoretic
mobility with that of their immunecomplexes.
Separation of Lp2A-I or Lp2A-I
The Lp2A-I, mAbs, and LDL
were incubated as described for cholesterol transfer assay. At the end
of incubation, the mixtures were immediately applied onto precast
native gradient gel (4-20%) (Bio-Rad). The electrophoresis were
run at 100 V at room temperature for 6 h, and the gel was stained with
Coomassie Brilliant Blue. In studies to exclude the possibility of
fusion between LpA-I and LDL particles during the cholesterol transfer
assay, Lp2A-I, -3A-I, and -4A-I were labeled with mAb Complexes from LDL
by Native Gradient Gel Electrophoresis
I,
incubated with LDL as in the transfer assay, and then separated by
native gradient gel electrophoresis. The distribution of radioactivity
was then assessed by autoradiography.
Circular Dichroism of Immunecomplexes of LpA-I
Particles
The effect of anti-apoA-I mAbs on the secondary
structures of apoA-I molecules in the LpA-I/anti-apoA-I immunecomplexes
were studied by subtractive CD on a Jasco J-40A spectropolarimeter. All
samples and mAbs were dialyzed against 0.5 m
M phosphate
buffer, pH 7.4, at 4 °C overnight. LpA-I particles were
preincubated with different mAbs at mAb/apoA-I molar ratio of 1 in
duplicates. Molar ellipticities were read at 222 nm at 24 °C in a
0.1-cm path length quartz cell. Percent -helix of the LpA-I or
their immunecomplexes were calculated from the molar ellipticities at
222 nm
(29) using a mean residue weight for apoA-I of 115.3 and
for mouse IgG of 112.5.
Effects of Anti-apoA-I mAbs on Cholesterol Transfer
between LDL and Lp2A-I
Discoidal LpA-I prepared in the presence
of cholate are well characterized. Variations in the phospholipid,
cholesterol, and apoA-I molar ratios generates distinct populations of
homogeneous particles with different lipid to apoA-I ratios or with
different numbers of apoA-I/particle
(18, 19, 20) . We first studied Lp2A-I (9.6 nm),
which represents the major and most stable particle amongst the Lp2A-I
species
(18, 19, 20) . The acceptor Lp2A-I (9.6
nm) particles were preincubated with serial dilutions of anti-apoA-I
mAbs or control mAbs, and then
[H]cholesterol-labeled LDL were added at
Lp2A-I/LDL particle molar ratio of 40:1 to evaluate the effects of
anti-apoA-I mAbs in the cholesterol transfer from LDL to Lp2A-I. The
apoA-I sequences attributed to each epitope are indicated in
. The results are summarized in Fig. 1and
. Antibodies 2H2 and Bsol.12 are nonrelevant mAbs used as
negative controls. The following are antibodies that strongly inhibited
cholesterol transfer from LDL to Lp2A-I, with the position of their
epitopes indicated in the parenthesis: A05 (residues 25-82), 2G11
(residues 25-110), A03 (residues 135-140), r5G9 (residues
149-186), A44 (residues 149-186), and 4A12 (residues
173-205). The inhibitory effects start at mAb/apoA-I molar ratio
of 0.25 and reach saturation at mAb/apoA-I molar ratio between 0.5 and
1. However, mAbs 4H1 (residues 2-8) and 3G10 (residues
96-121) do not have any significant effect on cholesterol
transfer from LDL to Lp2A-I (9.6 nm) in comparison with the control
mAbs. Antibody 5F6 (residues 116-141) marginally increases the
cholesterol transfer (20%) compared with the variation in controls
(10-15%), but this effect is not mAb dose-dependent. The affinity
of these mAbs for Lp2A-I (9.6 nm) particles were previously determined
by immunoprecipitation using Pansorbin armed with rabbit anti-mouse IgG
and
I-labeled LpA-I
(30, 31) . Using these
parameters, no correlation is seen between the affinity of these mAbs
for LpA-I and their effects on cholesterol transfer.
Figure 1:
Effects of anti-apoA-I monoclonal
antibodies on unesterified cholesterol transfer from LDL to Lp2A-I (9.6
nm). Lp2A-I (9.6 nm) was preincubated with mAbs at mAb/apoA-I molar
ratios between 0.125 to 2, and then LDL was added at Lp2A-I/LDL
particles ratio of 40:1 to initiate the cholesterol transfer process. A
typical incubation mixture included 2.1 µg of LpA-I protein, 0.5
µg of LDL protein, and mAb starting at 22.5 µg/tube with 1:2
serial dilution. The data are means calculated from three independent
assays in duplicate. Anti-apoA-I mAbs tested are 4H1 (), 2G11
(
), A05 (
), 3G10 (▾), 5F6 (
), A03 (
), A44
(
), r5G9 (
), and 4A12 (
). Antibodies Bsol.12
(
) and 2H2 (
) are nonrelevant mAbs used as negative
control.
The effects of
Fab fragments of 3G10, A03, A44, and 4A12 on cholesterol transfer from
LDL to Lp2A-I were also tested. The inhibitory effects of A03, A44, and
4A12 remain but are slightly attenuated (between 60 and 80% at
Fab/apoA-I molar ratio of 4) (Fig. 2). Fabs of 4H1 and 5F6 did
not affect cholesterol transfer significantly (data not shown).
Figure 2:
Effects of Fab fragments of anti-apoA-I
mAbs on cholesterol transfer from LDL to Lp2A-I (9.6 nm). Fab fragments
were prepared from IgG by papain digestion and purified by affinity
chromatography on a protein A-Sepharose column. Purity of these Fab
fragments were proven by SDS-polyacrylamide gel electrophoresis. Fabs
3G10 (▾), A03 (), A44 (
), and 4A12 (
) retain
their specific binding activities to apoA-I as tested by RIA. Fab 2H2
(
) is a negative control. Data are calculated from two repeated
assays.
When
[H]cholesterol-labeled Lp2A-I (9.6 nm) was used
as cholesterol donor to test the influence of mAbs on cholesterol
transfer from Lp2A-I (9.6 nm) to LDL, all of the mAbs that inhibit
cholesterol transfer from LDL to LpA-I do enhance cholesterol transfer
from this Lp2A-I to LDL (). The only difference is observed
with mAb 3G10, which does not significantly affect cholesterol transfer
from LDL to Lp2A-I, but increases the cholesterol transfer from Lp2A-I
(9.6 nm) to LDL by about 70%.
Figure 3:
Effects of mAbs on cholesterol mass in
Lp2A-I. After incubation with LDL, the cholesterol transfer assays were
done at Lp2A-I/LDL particle ratio of 40:1 and mAb/AI molar ratio of 1,
but scaled up to 10-fold compared with the ordinary assay. Cholesterol
mass remaining in the Lp2A-I after the transfer assay by
heparin-Mnprecipitation was measured; data represent
means of two independent assays in quadruplicate. Error bars indicate ± S.D. In comparison to mAb 2H2, the
presence of mAbs A05, A03, A44, or 4A12 significantly reduced the
cholesterol concentration in the Lp2A-I
compartment.
To address the possibility of artifacts related to the binding of
mAbs to heparin or LDL, we have tested the binding of the anti-apoA-I
mAbs to heparin or LDL by radioimmunoassay. The removawells were coated
with heparin-Mnreagent or LDL and incubated with
these mAbs and then with iodinated anti-mouse IgG. None of these mAbs
showed binding affinity for heparin or LDL (data not illustrated).
Effects of mAbs on the Transfer of apoA-I and
Phospholipids from LpA-I to LDL
The effect of mAbs on the
transfer of lipoprotein components other than cholesterol between
Lp2A-I and LDL were also evaluated. Lp2A-I (9.6 nm) particles labeled
with either I-labeled apoA-I or
[
C]POPC were incubated with mAbs and LDL under
conditions similar to those used for the study of cholesterol transfer
and separated from LDL by heparin-Mn
. There is no
significant correlation between the transfers of either
I-labeled apoA-I or [
C]POPC of
Lp2A-I (9.6 nm) to LDL and cholesterol transfers between the same
particles. This demonstrates that LpA-I is not transferred or
precipitated as a whole particle, but that different mAbs appear to
have specific effects on the transfer of cholesterol and phospholipids
().
I-labeled LpA-I (17%) together with
the increased transfer of cholesterol (100%) and phospholipids (18%).
Most mAbs that enhance transfer of cholesterol from LpA-I to LDL, also
significantly enhance phospholipid transfer (2G11, A05, 3G10, A03,
A44), whereas those that have minimal or no effect on cholesterol
transfer have no effect on phospholipids (4H1, 5F6). The exceptions are
4A12, which elicits a highly significant transfer of cholesterol but
not of POPC and apoA-I, and 3G10, which has a weak effect on
cholesterol transfer compared with its effect on POPC and apoA-I
transfer.
Electrophoretic Migration of Lp2A-I after Incubation with
mAbs in the Presence or Absence of LDL
To exclude the
possibility that in the presence of LDL, the binding of mAbs to LpA-I
may induce the destabilization or rearrangement of Lp2A-I and thereby
nonspecific incorporation of Lp2A-I constituents into LDL (fusion of
lipoprotein particles), I-labeled Lp2A-I (9.6 nm) were
prepared and incubated with mAbs in the presence and absence of LDL.
The migration profiles of Lp2A-I particles on agarose gel
electrophoresis are affected differently by the presence of specific
mAbs, and these shifts in migration provide evidence for the binding of
mAbs to Lp2A-I particles. However, the presence or absence of LDL does
not induce any additional change in migration LpA-I or LpA-I
mAb
complexes (Fig. 4, A-C). Therefore, the difference
of mobility of the immunecomplexes reflects the difference in surface
charge of each mAb (Fig. 4 D).
Figure 4:
Agarose electrophoretic migration profile
of mAb-Lp2A-I (9.6 nm) complexes in the presence and absence of LDL.
Panels A, B, and C, anti-apoA-I mAbs were incubated
with I-labeled Lp2A-I (9.6 nm) in the presence or absence
of LDL under the optimal conditions determined for the cholesterol
transfer assay, i.e. 2 µg of
I-labeled
Lp2A-I protein (20,000 cpm), 11.3 µg of mAb, and with or without
0.5 µg of LDL. At the end of incubation, aliquots of the mixtures
were immediately loaded on agarose gel and run at 100 V for 30 min. The
gels were fixed with 10% acetic acid, baked to dryness, and then
subjected to autoradiography. Antibodies 2H2 and Bsol.12 are negative
control. Panel D, agarose gel electrophoretic mobility of
iodinated anti-apoA-I mAbs. All of these mAbs were iodinated by IODO
bead technique (Pierce). The samples were loaded at 100,000 cpm/lane
and run at the same condition as that for
lipoproteins.
The same incubation
mixtures were also applied onto native gradient gel electrophoresis. As
shown in Fig. 5, LDL do not enter the separation gel in a
4-20% gradient gel. In the incubation mixture containing
phosphate-buffered saline or control mAb 2H2, Lp2A-I (9.6 nm) migrate
to their normal position, while in the presence of mAbs 4H1, 2G11, A03,
5F6, 3G10, A44, and 4A12, Lp2A-I formed complexes of different sizes as
indicated by the migration bands with much higher molecular weights.
Immunoblots using anti-mouse IgG and anti-apoAI showed that these high
molecular weight bands are immunecomplexes rather than larger LpA-I
particles (data not shown). As mentioned above, when
I-labeled LpA-I are used in the same experiments, there
is no radioactivity incorporated into LDL zone (data not illustrated).
This demonstrates that there is no particle fusion of LpA-I with LDL
during incubation for cholesterol transfer.
Figure 5:
Native gradient gel electrophoresis
migration pattern of Lp2A-I (9.6 nm) antibody complexes in the presence
of LDL. Anti-apoA-I mAbs were incubated with Lp2A-I (9.6 nm) in the
presence of LDL under the optimal condition as the cholesterol transfer
assay, i.e. 2 µg of Lp2A-I protein, 11 µg of mAb, and
0.5 µg of LDL protein. Antibody 2H2 is a negative control. At the
end of incubation, the mixtures were loaded and run in native
4-20% gradient polyacrylamide gel and run at 100 V for 6 h. The
protein migration bands were visualized with Coomassie Brilliant Blue
staining.
Evaluation of Cholesterol Transfer by Agarose Gel
Electrophoresis
To assess cholesterol transfer by another assay,
we have analyzed by agarose gel electrophoresis the movement of
[C]cholesterol from Lp2A-I (9.6 nm) bands to LDL
bands after incubation in the presence of mAbs. However, the
electrophoretic mobility of Lp2A-I (9.6 nm) complexed with mAbs 4H1,
3G10, A44, and 4A12 overlaps more or less with that of LDL
(Fig. 4), limiting the application of this method to the effect
of mAbs A03, 2G11, and A05 on transfer. In these experiments,
[
C]cholesterol-labeled Lp2A-I were incubated
with the mAbs and LDL, and the incubation mixture was separated by
agarose gel electrophoresis, and the
[
C]cholesterol radioactivity present in the
LpA-I and LDL fractions was measured. Compared with phosphate-buffered
saline and control mAb 2H2, A03, 2G11, and A05, increased cholesterol
transfer from Lp2A-I (9.6 nm) to LDL and only the effect of mAb A05 did
not reach significance (I).
Secondary Structure of apoA-I in LpA-I
To understand the nature of the interference of mAbs
on cholesterol transfer with Lp2A-I, we assessed the effects of the
anti-apoA-I mAbs on the secondary structure of apoA-I in these
particles. Lp2A-I (9.6 nm) was incubated with various anti-apoA-I mAbs
at an antibody/apoA-I molar ratio of 1, and the mAb
Complexes
-helicity of
resulting Lp2A-I
mAb complexes, free Lp2A-I particles, and free
mAbs was measured by circular dichroic spectroscopy at the wavelength
of 222 nm. Nonrelated mAbs against atrial natriuretic peptide or
soluble apoB were used as control. Immunoglobulins contain very little
-helix secondary structure and studies have shown that binding to
an antigen has a negligible effect on the antibody helical structure
(32) . Therefore, the ellipticity values of each mAb were
measured and used as background to correct the CD values of each
corresponding immunecomplexes. In our study, the
-helicity
contents of free Lp2A-I (9.6 nm) is 69.2%, in agreement with earlier
studies by Jonas et al. (33) . Compared with the mean
of control values obtained with irrelevant mAbs preincubated with
Lp2A-I (9.6 nm), the
-helicity of the immunecomplexes formed with
most anti-apoA-I mAbs is significantly increased (). This
suggests that the binding of antibodies to Lp2A-I does not destablilize
apoA-I but, on the contrary, may increase the stability of amphipathic
-helices. However, there is no correlation between the capacities
of mAbs to increase the
-helicity in the Lp2A-I complex and to
interfere in cholesterol transfers involving these particles.
system. Also, the analysis of the
lipoproteins incubated in the presence of the different mAbs
demonstrates the quantitative binding of the antibodies to all LpA-I
particles and provides no evidence for the fusion of LpA-I and LDL in
the presence of mAbs as judged by agarose gel electrophoresis and no
evidence of dissociation of Lp2A-I and generation of lipid-free apoA-I
as judged by gradient gel electrophoresis. In addition to the
demonstration of the stability of LpA-I immunecomplexes in the transfer
assay and to the absence of LDL-LpA-I fusion, it was important to also
show that similar results could be obtained with a totally different
assay, such as by separation of LDL and LpA-I by agarose gel
electrophoresis.
I-labeled apoA-I to LDL. Although this enhanced transfer
represents only 10% above background, it is nevertheless significant
and suggests that A44 may cause the association or coprecipitation with
LDL of about 1 in 10 apoA-I molecules. All but one mAb, which enhances
cholesterol transfer from Lp2A-I to LDL by about 70% or more, also
significantly increases phospholipid transfer by 14-22%. The
exception is 4A12, which although is the strongest stimulator of
cholesterol transfer (150%) elicits no change in phospholipid transfer.
The two mAbs that have no significant effect on cholesterol transfer,
4H1 and 5F6, also have no effect on phospholipid transfer. For those
mAbs, which stimulate cholesterol and phospholipid transfers from
Lp2A-I to LDL, we can calculate from the initial lipid concentrations
in Lp2A-I and from the transfer rates () that under basal
conditions and in the absence of mAbs, 0.76 nmol of cholesterol and
0.23 nmol of phospholipid transfer per hour from Lp2A-I to LDL, a ratio
of about three moles to one; typical stimulatory mAbs increase transfer
to 1.5 mol of cholesterol and 0.26 nmol of phospholipid or a ratio of 6
to 1 mol. The ratio of cholesterol to phospholipid transferred measured
here is comparable with that of 6 mol to 1 between unilamellar donor
vesicles and neutral acceptor vesicles reported by McLean and Phillips
(6) . The net effect of the antibody-mediated stimulation is
therefore to enhance the transfer of cholesterol relative to
phospholipids.
-helicity of apoA-I, suggesting that
they may stabilize the LpA-I. We hypothesize that depending on the
position of epitopes, i.e. specifically for those in
amphipathic
-helices, specific anti-apoA-I may increase the
binding of apoA-I to phospholipid, which would increase the competition
of the amphipathic domain with cholesterol and thus promote cholesterol
desorption and transfer to other lipoproteins. This would be compatible
with the concept proposed earlier by others that apoA-I compete with
cholesterol for solvation in or binding to the phospholipid phase
(35) . The absence of correlation between mAb effects on
-helicity and lipid transfers should not be unexpected since
apoA-I structure is complex, and different domains are predicted to
have different interactions with lipids. As discussed below, mAb 4H1
reacts with an epitope that is close to the central domain. While 4H1
epitope does not contain any predicted
-helix, this antibody
enhances
-helicity, and we propose that it does exert this effect
indirectly through its overlap with other regions predicted to have
significant
-helicity, such as that for 5F6.
Figure 6:
Model of apoA-I structure on discoidal
LpA-I and location of epitopes recognized by anti-apoA-I mAbs
inhibitory or enhancing for cholesterol transfer between LDL and Lp2A-I
(9.6 nm). The different amphipathic -helices (represented by
rectangles) run parallel to the axis of the phospholipid disc,
are antiparallel to each other, and linked by coiled regions and
-turns (represented by a string). The areas identifying
the epitopes are represented by different shades of gray beneath the motifs of secondary structure. The epitopes for mAbs
without effect on cholesterol transfers are represented in areas of
darkest gray.
Our conclusions are in
general compatible with recent results of others on the effects of
specific mAbs on cellular cholesterol efflux, which we also interpret
as defining a neutral central domain separating two domains where the
binding of mAbs influence cholesterol transfer. Luchoomun and
co-workers
(38) identified two mAbs reacting at residues
25-82 (A05) and at residues 149-186 (A44) that do inhibit
cellular cholesterol transfer to LpA-I, while mAbs reacting between
residues 99-132 and 135-140 do not. Fielding et al. (39) have described two mAbs reacting between residues
137-144 and between residues 113-128 . . . 141-148,
the later being a discontinuous epitope that inhibits cellular
cholesterol transfer to pre-LpA-I while mAbs reacting between
93-99 and 167-174 do not. Banka et al. (40) have also observed inhibition of efflux by mAbs reacting
between residues 95-110 and 74-105. While there is a
general concordance in the location of epitopes for the mAbs inhibitory
to cholesterol transfers, there is also uncertainty for the exact
boundaries of these domains. Considering the variation that we observed
ourselves with different Lp2A-I (Tables I and II), we attribute the
variation to the different conformations that apoA-I assumes in these
different lipoproteins. This is particularly true for the different
lipoproteins used in the studies cited above. Luchoomun and colleagues
(38) use discoidal LpA-I; Fielding et al. (39) studied the partially defined plasma
pre
-LpA-I; and Banka et al. (40) used
HDL and apoA-I proteoliposomes.
-helices enhance cholesterol transfer.
Finally, the effects of specific mAbs on the binding of cholesterol to
LpA-I and on its desorption from LpA-I appears to demonstrate that the
cholesterol content of lipoprotein particles is regulated by the
apolipoprotein conformation.
Table:
Effects of antibodies on the transfer of Lp2A-I
(9.6 nm) constituents to LDL
Table:
Effects of anti-apoA-I monoclonal antibodies on
cholesterol transfer between LDL and Lp2A-I (8.6 nm and 10.2 nm)
Table:
Effect of antibodies on cholesterol transfer
from Lp2A-I (9.6 nm) to LDL measured by agarose gel electrophoresis
C]cholesterol
were preincubated with mAbs at mAb/apoA-I molar ratio of 2 at 37 °C
for 30 min, and LDL were added and incubated at 25 °C for another
20 min. LDL and Lp2A-I in incubation mixtures were separated by agarose
gel electrophoresis. Each lane of the gel were sliced into 2-mm
fractions and counted for
-radioactivity. Data represent mean
± S.E. calculated from two experiments.
Table:
Effects of anti-apoA-I monoclonal
antibodies on predicted secondary structure of apoA-I in Lp2A-I (9.6
nm) immunecomplex
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.