(Received for publication, May 20, 1994; and in revised form, December 12, 1994)
From the
The effect of high density lipoprotein composition on the rates
of unesterified cholesterol exchange between low density lipoproteins
(LDL) and well-defined homogeneous discoidal lipoproteins (LpA-I)
reconstituted with phosphatidylcholine, cholesterol, and apolipoprotein
A-I (apoA-I) has been investigated. LpA-I containing cholesterol and 2,
3, and 4 apoA-I molecules per particle differed in their ability to
accept or donate cholesterol. A significant cholesterol exchange occurs
between LDL and Lp2A-I (7.8 and 9.6 nm), while there is little or no
cholesterol exchange detectable between LDL and Lp3A-I (10.8 and 13.4
nm) and Lp4A-I (17.0 nm) complexes. The cholesterol transfer from LDL
to the cholesterol-free Lp2A-I (9.6 nm), Lp3A-I (13.4 nm), and Lp4A-I
(17.0 nm) particles also shows significant cholesterol transfer to
Lp2A-I, while there is no detectable transfer to Lp3- and 4A-I
particles. The rates of cholesterol transfer to cholesterol-free and
cholesterol-containing Lp2A-I appear to differ significantly.
Cholesterol transfer from LDL to cholesterol-free Lp2A-I is zero order
with respect to acceptor concentrations when the Lp2A-I/LDL ratio is
above 10. Transfer rates from LDL to cholesterol-free Lp2A-I are faster
for the smaller Lp2A-I (8.5 nm) than to the larger Lp2A-I (9.7 nm) and
exhibit half-times (t) at 25 °C of 4.0 and
5.3 h, respectively. In contrast, cholesterol transfer from LDL to
cholesterol-containing Lp2A-I remains dependent upon acceptor
concentrations to an acceptor/donor particle ratio of 80. In addition,
transfer from LDL to cholesterol-containing Lp2A-I is faster to the 9.6
nm than to 7.8 nm particles, with t
of 1.4 and
2.3 h, respectively. The rates of cholesterol transfer from Lp2A-I to
LDL are higher than in the opposite direction, in particular for the
small Lp2A-I (7.8 nm), which has a t
of
approximately 50 min. The results show that changes in the composition
and structure of apoA-I-containing particles have a significant effect
on inter-lipoprotein exchange of cholesterol. This suggests that the
kinetics of cholesterol transfer to and from reconstituted discoidal
LpA-I particles cannot be fully explained by passive aqueous diffusion.
HDL ()is involved in the reverse transport of
cholesterol from peripheral tissues to liver, and apoA-I containing
lipoproteins have been shown to be preferred acceptors of cellular
cholesterol efflux. Cholesterol present in different HDL subfractions
originates from the de novo secretion of lipoproteins as well
as from transfer from cell membranes and from apoB-containing
lipoproteins
(LDL)(1, 2, 3, 4, 5) .
Recent studies by Fielding and colleagues indicate that cellular
cholesterol may first transfer to a small pre-
-HDL and
then be transferred to larger pre-
- and
pre-
-HDL, where the esterification of cholesterol
occurs(6, 7) . The same group has also reported that
LDL-cholesterol is transferred to and esterified in
-HDL
(8, 9) . A recent study by
another group showed that only a fraction of the cellular cholesterol
transferred to the pre-
-HDL pathway is esterified while the
majority recycles through
-HDL, LDL, and
pre-
-HDL(10) .
Cholesterol exchange between lipoproteins and cells is an important mechanism involved in cholesterol redistribution, transport, and metabolism in vivo (reviewed in (11) ). A bidirectional flux of cholesterol molecules occurs between HDL and LDL particles when they are incubated in vitro(12) . Studies done by Phillips and co-workers (13, 14, 15) have shown that the physical state of cholesterol molecules in the phospholipid/water interface of serum lipoprotein particles differs on LDL, HDL, and small unilamellar vesicles (SUV) in a manner that parallels differences in cholesterol flux between these particles(16) . In contrast, the kinetics of cholesterol exchange between HDL and LDL (12) and between SUV (16) are similar and appear to be consistent with a passive aqueous diffusion mechanism(15, 16, 17, 18) . In addition, the presence of apolipoproteins A-I, A-II, and B-100 in lipid vesicles enhances the rate of cholesterol exchange from SUV(19) . These observations indicate that cholesterol transfer between two lipid-containing structures is affected by the presence of lipophilic surface proteins. In the case of lipoproteins, interfacial surfaces reflect both the intrinsic structure of the apolipoproteins and that of the amphipathic surface lipids. Therefore, the transfer of cellular cholesterol or LDL cholesterol to specific plasma lipoprotein subclasses that contain apoA-I appears to be guided by the molecular properties of these particles.
We have previously reported that cholesterol transfer from cells to discoidal LpA-I and from discoidal LpA-I to other plasma lipoproteins varies with particle size and number of apoA-I molecules(20) . To understand the factors that control transfer of cholesterol to apoA-I-containing lipoproteins, we have systematically measured cholesterol exchange between LDL and several well defined, reconstituted discoidal LpA-I particles. The results show that a significant time- and acceptor concentration-dependent cholesterol exchange occurs between LDL and several different Lp2A-I discoidal particles, while there is little or no cholesterol transfer occurring between LDL and Lp3A-I and Lp4A-I particles.
Pure
apoA-I was prepared as described earlier(22) . Reconstituted
discoidal LpA-I particles containing cholesterol (LpA-I)
were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)
(Sigma), cholesterol (Sigma), and apoA-I at an initial molar ratio of
120:6:1 or 88:44:1 by cholate dialysis following previously published
procedures(23, 24, 25) . Homogeneous
lipoprotein containing 2, 3, or 4 apoA-I molecules per particle (Lp2-,
Lp3-, or Lp4A-I
) were purified by gel filtration on two
serial agarose Bio-Gel 5M columns (95
2.5 cm) (Bio-Rad
Laboratories) equilibrated with TBS-EDTA buffer. The
cholesterol-containing Lp2A-I
(9.6 nm), Lp3A-I
(13.4 nm), and Lp4A-I
(17.0 nm) particles were
purified from the lipoprotein population obtained with the initial
molar ratio of 120:6:1 and Lp2A-I
(7.8 nm) and
Lp3A-I
(10.8 nm) particles were purified from the
lipoprotein population obtained with the initial molar ratio of
88:44:1. The chemical compositions of these particles were similar to
those reported in previous studies(25) . Lp2A-I (9.6 nm),
Lp3A-I (13.4 nm), and Lp4A-I (17.0 nm) without cholesterol were
prepared at an initial POPC/apoA-I molar ratio of 120:1 as described
earlier(20) . Lp2A-I with different POPC/apoA-I molar ratios
without cholesterol were prepared by a cholate dispersion method of
Sparks et al.(26) . The homogeneous particles
produced by this method were centrifuged at KBr density (1.063 g/ml) to
remove free lipids. Size characterization of LpA-I particles was as
described(27) . The number of apoA-I molecules per LpA-I
particle was determined by chemical cross-linking with dimethyl
suberimidate(28) . The concentration of protein was measured by
Lowry assay (29) and cholesterol and POPC by enzymatic kits
(Boehringer Mannheim GmbH, Mannheim, Germany).
The determination
of cholesterol transfer from LpA-I particles to LDL was essentially the
same as above, except that LpA-I rather than LDL was labeled with
[H]cholesterol using the same procedure. The
amount of LpA-I as cholesterol donor was constant at 1 µg per tube,
and the amounts of LDL as cholesterol acceptor were varied.
In the
validation of the assay using I-labeled LpA-I, we
observed that as reported previously for HDL
(12) ,
60% of Lp4A-I co-precipitated with LDL. Co-precipitation of other LpA-I
was substantially less than that for Lp4A-I: 5% for Lp2A-I (7.8 nm),
33% for Lp2A-I (9.6 nm), 20% for Lp3A-I (10.8 nm), and 47% for Lp3A-I
(13.4 nm). These values are constant and reproducible between assays
(S.D. < 1%), are not time or acceptor dose-dependent, and were
therefore used to correct the rates (x) of cholesterol
transfer in all assays. In addition, all transfer rates were also
corrected by subtraction of the background values obtained in the
absence of acceptor lipoproteins.
In some cholesterol transfer
assays, LDL and LpA-I were also separated by KBr gradient
ultracentrifugation. At the end of the incubation, the mixtures were
adjusted to the final density of 1.063 g/ml and transferred into mini
tubes for Beckman TLA-100 rotor with a total volume of 250 µl per
tube and ultracentrifuged at 60,000 rpm for 200 min. The contents in
these tubes were fractionated every 50 µl from the top to the
bottom and counted in a -counter to monitor the movement of
[
H]cholesterol. LDL was recovered in the top
50-µl fraction, and LpA-I was recovered in the bottom 100 µl.
The kinetics of cholesterol transfer between LDL and LpA-I were calculated as described by Lund-Katz and colleagues(12, 31) .
To measure cholesterol
transfer from LDL to LpA-I particles, donor LDL labeled
with [
H]cholesterol were incubated with acceptors
Lp2A-I
(9.6 nm), Lp3A-I
(13.4 nm), and
Lp4A-I
(17.0 nm) at different LpA-I
/LDL
particle ratios and incubated for the period up to 90 min. Transfer of
cholesterol from LDL to these LpA-I
particles is expressed
by the rates of [
H]cholesterol moving into
LpA-I
, and the representative data are shown in Fig. 1A. The cholesterol transfer from LDL to
Lp2A-I
(9.6 nm) increases with increasing incubation time,
and the maximum transfer is observed at 60 min. The rate of cholesterol
transfer at 60 min at a ratio of 80 particles of Lp2A-I
(9.6 nm) to 1 LDL particle are 23%. Surprisingly, under the same
experimental conditions, the cholesterol transfers from LDL to
Lp3A-I
(13.4 nm) and Lp4A-I
(17.0 nm) are not
significant and not time- and acceptor concentration-dependent.
Figure 1:
Cholesterol exchange
between LDL and LpA-I. Data presented in this figure are
means from three independent assays. Error bars indicate S.D. A, cholesterol transfer from LDL to LpA-I
. Data
represent cholesterol transfer rates from LDL to Lp2A-I
(9.6 nm) (
), Lp3A-I
(13.4 nm) (
), and
Lp4A-I
(17.0 nm) (
) at LpA-I
/LDL particle
ratios of 80 and as a function of time. B, cholesterol
transfer from LpA-I
to LDL. Data represent cholesterol
transfer rates from Lp2A-I
(9.6 nm) (
),
Lp3A-I
(13.4 nm) (
), and Lp4A-I
(17.0 nm)
(
) at a LpA-
/LDL particle ratio of 0.6 and as a
function of time.
Cholesterol transfer in the opposite direction from LpA-I particles to LDL were determined in similar experiments.
LpA-I
particles were labeled with
[
H]cholesterol and incubated with acceptor LDL.
Significant transfer of cholesterol from Lp2A-I
(9.6 nm) to
LDL has been observed. The maximum transfers are about 36% at a
Lp2A-I
/LDL particle ratio of 0.6 at 90 min of incubation.
There is no further increase of transfer with the incubation extended
to 120 min. Interestingly, cholesterol transfer from Lp3A-I
(13.4 nm) and Lp4A-I
(17.0 nm) to LDL is not
significant and is not acceptor concentration-dependent (Fig. 1B). Therefore, the same differences are observed
in the transfers of cholesterol between LDL and Lp2A-I
(9.6
nm) and Lp3- and 4A-I
in both directions.
Cholesterol
transfer from LDL to Lp2A-I (9.6 nm) has been studied in
detail by varying LpA-I
/LDL particle ratios and as a
function of time between 0 and 90 min. The transfer of cholesterol from
LDL to Lp2A-I
(9.6 nm) is both time- and acceptor (LDL)
dose-dependent when the acceptor/donor ratio is above 5 (Fig. 2A). There is no detectable cholesterol transfer
when the Lp2A-I
(9.6 nm)/LDL particle ratio is below 2.5.
No significant cholesterol transfer from LDL to either Lp3A-I
(13.4 nm) or Lp4A-I
(17.0 nm) has been observed under
all of the conditions tested above.
Figure 2:
Cholesterol transfer from LDL to
cholesterol-containing Lp2A-I (9.6 and 7.8 nm). For each
data point, S.D. ranges from 0.1 to 2.9% which were generated from 4
independent assays. A, cholesterol transfer from LDL to
Lp2A-I
(9.6 nm). Data shown in the figure and the inset are the transfer rates at acceptor/donor particle ratios of 80
(
), 40 (
), 20 (
), and 10 (
), respectively. B, cholesterol transfer from LDL to Lp2A-I
(7.8
nm). Data shown in the figure and inset are the transfer rates
at acceptor/donor particle ratios of 80 (
), 40 (
), 20
(
), and 10 (
). All data are representatives of three
independent assays using the Lp2A-I
particles prepared from
initial POPC/cholesterol/AI molar ratio of 120:6:1 and 88:44:1 as
described in the text. Insets, linear regression plots of the
percentage of [
H]cholesterol radioactivity which
remains in LDL as a function of time are shown. The correlation
coefficients (r) are >0.93 in all
instances.
Further experiments were done to
measure cholesterol transfer between LDL and smaller Lp2- and
Lp3A-I particles, that is Lp2A-I
(7.8 nm) and
Lp3A-I (10.8 nm). The rates of cholesterol transfer from LDL to
Lp2A-I
(7.8 nm) are lower than those to larger Lp2A-I
(9.6 nm), but are significant and time- and acceptor
concentration-dependent. The maximum transfer rate of cholesterol from
LDL to Lp2A-I
(7.8 nm) is approximately 16% at an
acceptor/donor ratio of 80 and at 60 min (Fig. 2B). The
transfer of cholesterol from LDL to Lp2A-I
(7.8 nm) reaches
saturation between 90 and 120 min of incubation (data not shown). As
seen with Lp3A-I
(13.4 nm), there is no detectable
cholesterol transfer from LDL to Lp3A-I
(10.8 nm) at all
conditions tested (data not shown).
The time curves of cholesterol
transfer from Lp2A-I (7.8 nm and 9.6 nm) to LDL are shown
in Fig. 3. Cholesterol transfer was measured at
LpA-I
/LDL particle ratios between 0.6 and 200 and at
different time intervals up to 90 min. In contrast to the transfer out
of LDL, the reverse transfer to LDL is faster from Lp2A-I
(7.8 nm) than that from Lp2A-I
(9.6 nm) to LDL. There
is no detectable cholesterol transfer from Lp2A-I
(9.6 nm)
to LDL when the Lp2A-I
/LDL ratio is above 5 (Fig. 3A). However, the cholesterol flux from
Lp2A-I
(7.8 nm) to LDL is still significant at an
acceptor/donor ratio of 20 and at 60 min of incubation (Fig. 3B).
Figure 3:
Cholesterol transfer from
cholesterol-containing Lp2A-I (9.6 and 7.8 nm) to LDL. All
data are the means from three independent assays where the
concentration of donor (LpA-I
containing
[
H]cholesterol) was constant and the
concentration of acceptor LDL varied (LpA-I
/LDL particle
ratios of 0.6 to 200). For each data point, S.D. ranges from 0.05 to
3.8%. A, cholesterol transfer from Lp2A-I
(9.6 nm)
to LDL. Data presented here are cholesterol transfer rates at
LpA-I
/LDL ratios of 0.6 (
), 1.2 (
), and 2.4
(
). B, cholesterol transfer from Lp2A-I
(7.8
nm) to LDL. Data are the cholesterol transfer rates at a
LpA-I
/LDL particle ratio of 0.6 (
), 2.4 (
), 5
(
), and 20 (
). The transfer of cholesterol from LDL to
Lp2A-I
(10.8 nm) reaches saturation between 60 and 90 min
of incubation. Insets, linear regression plots of the
percentage of [
H]cholesterol radioactivity which
remains in Lp2A-I
as a function of time are shown. The
correlation coefficients (r) are >0.96 for Lp2A-I
(7.8 nm) and >0.93 for Lp2A-I
(9.6
nm).
To determine whether the POPC content and size of LpA-I affect their ability to accept cholesterol from LDL, two Lp2A-I particles without cholesterol and with different POPC contents, Lp2A-I (9.7 nm, PC/A-I = 95) and Lp2A-I (8.5 nm, PC/A-I = 67), were used as acceptors for LDL cholesterol. Time- and acceptor concentration-dependent increases in cholesterol transfer from LDL to both of these two Lp2A-I particles were, again, observed (Fig. 4). However, there is some difference between these two acceptors with different POPC contents. Cholesterol transfer from LDL to the larger Lp2A-I (9.7 nm, PC/A-I = 95) is slower and does not reach saturation at most acceptor concentrations tested up to 90 min of incubation (Fig. 4A), whereas transfers to Lp2A-I (8.5 nm, PC/A-I = 67) are faster at most acceptor/donor ratios tested, and the difference is most significant for the shorter time (15 min) (Fig. 4B).
Figure 4:
Comparison of cholesterol transfer from
LDL to cholesterol-free Lp2A-I (9.7 and 8.5 nm) with different PC/apoAI
molar ratios. Homogeneous Lp2A-I particles without cholesterol and with
different PC/A-I molar ratios were prepared using a method (26) different from that used (23, 24) for the
experiments in Fig. 1to 3. The final preparations of Lp2A-I
(9.7 nm) and Lp2A-I (8.5 nm) have POPC/apoA-I molar ratios of 95:1 and
67:1, respectively. Data in this figure are means of three independent
assays using LDL and LpA-I particles from different preparations. The
S.D. values of each data point range between ±0.01-3.5%.
The cholesterol transfer rates are presented at LDL/Lp2A-I particle
ratios of 80 (), 40 (
), 10 (
), and 2.5 (
). A, cholesterol transfer from LDL to Lp2A-I (9.7 nm, PC/apoA-I
= 95). B, cholesterol transfer from LDL to Lp2A-I (8.5
nm, PC/apoAI = 67). Insets, linear regression plots of
the percentage of [
H]cholesterol radioactivity
which remains in LDL as a function of time are shown. The correlation
coefficients (r) are >0.96 for Lp2A-I (9.7 nm) and >0.98
for Lp2A-I (8.5 nm).
Figure 5:
Measurement of cholesterol transfer from
LDL to LpA-I separated by ultracentrifugation. Lp2A-I
(9.6 nm), Lp3A-I
(13.4 nm), and Lp4A-I
(17.0 nm) were incubated with
[
H]cholesterol-labeled LDL at an acceptor/donor
particle ratio of 40 for 30 min. The incubation mixtures were adjusted
to KBr density of 1.063 g/ml, transferred into microtubes, and
centrifuged in a Beckman Optima TL ultracentrifuge using a TLK-100
rotor at 4 °C, 60,000 rpm for 200 min. The contents in the tubes
were collected in 50-µl fractions from the top to the bottom and
counted in a
-counter. Data in this figure represent means of
quadruplicates, and the error bars indicate S.D. values of
quadruplicates.
Figure 6:
Kinetics of cholesterol exchange from LDL
to Lp2A-I and Lp2A-I. The rate constants (k) of
cholesterol transfer from LDL to different Lp2A-I particles were
calculated by k = -(slope)x
,
where the slopes were obtained by least squares linear regression
analysis from ln(1 [[[- x/x
) versus t at different acceptor/donor particle ratios as
presented in Fig. 2and Fig. 4and described in the
text(12) . Data represent k values of cholesterol
transfer from LDL to cholesterol containing Lp2A-I
, 7.8 nm
(
) and 9.6 nm (
), and to cholesterol-free Lp2A-I, 8.5 nm
(
) and 9.7 nm (
), at different Lp2A-I/LDL particle ratios.
The k values were calculated from 3 to 4 assays and S.D.
ranged between ±0.05-0.39
10
.
The ability of
the cholesterol-containing Lp2A-I (9.6 nm) to accept
cholesterol from LDL differs substantially from the cholesterol-free
Lp2A-I particles ( Fig. 6and Table 2). While initial
cholesterol transfer rates for Lp2A-I
are substantially
less than that shown for the cholesterol-free particles, transfer rates
are dependent on acceptor concentrations up to an acceptor/donor ratio
of 80 and do not reach saturation for either Lp2A-I
(9.6
nm) or Lp2A-I
(7.8 nm). This appears to correspond to an
almost 4-fold increased rate of cholesterol transfer to the
Lp2A-I
relative to that for the cholesterol-free Lp2A-I.
For a small Lp2A-I
(7.8 nm), the k values are 5.0
10
for an acceptor/donor ratio of 80 and
correspond to t
of 140 min. The t
values obtained here are similar to those
obtained by Lund-Katz and Phillips(12) .
Cholesterol
transfer from Lp2A-I (7.8 nm and 9.6 nm) to LDL was also
studied at 25 °C with constant Lp2A-I
concentrations
but with varying acceptor LDL concentrations (Lp2A-I
/LDL
ratios from 0.6 to 100). The rate of cholesterol transfer to LDL is
dependent upon acceptor concentration for both donor
Lp2A-I
s; however, the kinetics of cholesterol transfer from
Lp2A-I
(7.8 nm) and Lp2A-I
(9.6 nm) to LDL
differ significantly. With the small Lp2A-I
(7.8 nm) as
cholesterol donor particles, rate constants, k, increase
sharply at a Lp2A-I
/LDL ratio of 2.5 and then plateau. With
the larger Lp2A-I
(9.6 nm), the rate constants are slower
and do not plateau. The rate constants and t
are
generally constant with the studies of Letizia and
Phillips(19) .
Estimation of the interfacial flux of
cholesterol from the surface of LDL particles into the aqueous phase
varies in incubations with different acceptor particles (Table 2). For acceptor particles devoid of cholesterol, maximum
interfacial cholesterol flux ranged from 0.5 to 0.4 mol/10 nm h for the small and large Lp2A-I (8.5 and 9.7 nm), respectively.
Interfacial flux values for cholesterol-containing Lp2A-I
acceptor particles reach maximum values of 0.9 molecule/10
nm
/h for small Lp2A-I
(7.8 nm) and 2.45
molecules/10 nm
/h for the larger Lp2A-I
(9.6
nm). This sensitivity of interfacial flux to acceptor particle
structure may explain why these values are slightly less than that
previously shown for exchanges between LDL and native HDL acceptor
particles (4 mol/10 nm
/h)(12) . Interfacial
cholesterol flux from Lp2A-I
is considerably less than that
from LDL and is 0.27 molecule/10 nm
/h from the smaller
Lp2A-I
(7.8 nm) and 0.04 mol/10 nm
/h from the
larger Lp2A-I
(9.6 nm) (Table 3).
This study shows that changes in the composition of various
kinds of reconstituted discoidal LpA-I particles has a direct effect on
the exchange of cholesterol with LDL particles. Previous studies have
also shown that different native (12) or reconstituted LpA-I (19) vary in their ability to allow for the desorption of
cholesterol; however, the mechanism that regulates this phenomenon is
unclear. Investigations with small unilamellar vesicles (SUV) have
shown that increasing the number of apoA-I molecules on their surfaces
will increase the rate of cholesterol desorption(19) . This
prompted the authors to propose that apoA-I may perturb the
interactions between cholesterol and phospholipid and promote a
transition state that allows for cholesterol desorption(19) .
In the same study, discoidal LpA-I particles exhibited an almost 6-fold
increased rate of cholesterol desorption as compared to SUV and also
showed a similar relationship between phospholipid:apoA-I ratio and
cholesterol desorption. An increased propensity for cholesterol
desorption was associated with a reduced phospholipid:apoA-I ratio in
LpA-I discoidal complexes. This is consistent with that observed in the
present study for Lp2A-I particles: a decrease in
phospholipid:apoA-I ratio in Lp2A-I
is associated with an
increased rate of cholesterol desorption. Similarly, Lp3A-I
and Lp4A-I
generally exhibit high phospholipid:apoA-I
ratios and very low rates of cholesterol transfer from LDL to
LpA-I
. An exception, however, is the cholesterol-containing
Lp3A-I
(10.8 nm) which does not exhibit a high
phospholipid:apoA-I ratio but which is a very poor acceptor of
cholesterol. Low rates of cholesterol transfer to this complex may be
related to its very high content of cholesterol(34) . A recent
study by Jonas et al.(35) is also consistent with a
cholesterol transfer that is faster to Lp2A-I
(9.6 nm),
slower to Lp2A-I
(7.8 nm), and slowest to Lp3A-I
(10.8 nm) after correction of the background at zero time.
The
increased desorption of cholesterol from smaller discoidal particles
can result from perturbations in phospholipid-cholesterol interactions
that arise from changes in the order of phospholipid acyl
chains(36, 37) . A low phospholipid:apoA-I ratio in
discoidal Lp2A-I is also associated with a reduction in the content and
stability of apoA-I -helices and with an increase in the density
of negative surface charge(26) . Such changes in apoA-I
conformation may also affect cholesterol desorption. An increased
thermodynamic instability that results from a particular apoA-I
conformation may affect the association of apoA-I, phospholipid, and
cholesterol in boundary regions immediately adjacent to apoA-I
molecules(38) .
It is evident that variations in the
structural properties of different LpA-I may directly affect the
ability of these lipoproteins to receive cholesterol. Changes in the
structure of reconstituted LpA-I not only affect the desorption of
cholesterol from these particles, but also indirectly affect the
adsorption of cholesterol transferred from donor LDL particles. This
study shows that the presence of a small amount of cholesterol in
Lp2A-I promotes a 2- to 3-fold increase in the rate of
cholesterol transfer from LDL compared to cholesterol-free LpA-I. The
specificity of cholesterol transfer is also made more complex by the
effect of particle size which has inverse effects in cholesterol-free
or -containing particles: with cholesterol containing
Lp2A-I
, smaller particles are poorer acceptors of
cholesterol than larger ones, while with cholesterol-free Lp2A-I, small
particles are better than the larger ones. This differential effect may
be a result of unique structures of the different LpA-Is or may be due
to a distinct effect of cholesterol on the different particles.
Previous studies have shown that addition of cholesterol to a
reconstituted discoidal LpA-I
directly affects both the
conformation and charge of apoA-I and thereby significantly modifies
the physical properties of the LpA-I particles(39) . As such,
it is possible that cholesterol-containing Lp2A-I
may have
unique molecular properties that may stimulate cholesterol desorption
from LDL by changing the interfacial interactions between LpA-I
and LDL. It is of note that both a reduction in
phospholipid:apoA-I ratio and an increase in cholesterol content
increase the negative surface potential on
LpA-I
(26, 39) . A change in the surface
charge on an LpA-I particle may affect the characteristics of the
unstirred water layer and/or the ionic double layer around the particle
and may thereby indirectly affect interfacial interactions on the
particle surface such as cholesterol exchange. Further, it is possible
that changes in specific interactions between apoA-I and cholesterol in
an LpA-I
particle may also affect cholesterol exchange.
Addition of cholesterol to an LpA-I
complex causes a
significant reduction in the amount of
-helical structure in
apoA-I, but increases the stability of the remaining
helices(39) . Studies (38, 39) have shown that
cholesterol may exist in two domains in a LpA-I
particle:
1) in close contact with apoA-I and 2) in the bulk phase of
phospholipid. It appears that a small amount of cholesterol will
preferentially associate with apoA-I and will reduce the
helix-phospholipid interactions in the LpA-I
particle. This
structural change may decrease the hydrophobic solvation of surface
phospholipid by apoA-I and may allow for an increased accessibility of
the bulk phospholipid to cholesterol. A reduction in
phospholipid-apoA-I interactions may therefore increase the ability of
the Lp2A-I particles to receive cholesterol from LDL by reducing a
competition between apoA-I and cholesterol for the solvation of
phospholipid(38, 39) . The increased transfer of LDL
cholesterol to Lp2A-I
compared with that to
cholesterol-free Lp2A-I observed here may be explained by the two
cholesterol domain hypothesis. Future studies should aim to test this
hypothesis.
Studies by several groups (12, 16, 17, 40) have provided
evidence that the kinetics of cholesterol exchange between lipoproteins
are consistent with a mechanism of exchange in which cholesterol
molecules must diffuse freely through an aqueous phase. The only
activation energy required in this process appears to be the desorption
of cholesterol from the interfacial lipid phase. In the present study
centered on transfers between LDL and model discoidal LpA-I, the rate
of cholesterol flux to cholesterol-free Lp2A-I is independent of
acceptor concentrations above acceptor:donor ratios of 10. A zero order
rate of exchange with respect to acceptor concentration suggests that
the frequency of collisions between donor and acceptor particles is not
rate-determining for cholesterol transfer to cholesterol-free Lp2A-I.
This is similar to that observed in other studies and appears to be
consistent with the aqueous diffusion model proposed by Phillips et
al.(11, 12, 16, 17) .
Cholesterol transfer to Lp2A-I, however, is first order
with respect to acceptor Lp2A-I
concentrations, even when
acceptor concentrations are increased 80-fold. It therefore appears
that the frequency of collisions between donor and acceptor may be
rate-limiting for cholesterol transfer from LDL to the
cholesterol-containing Lp2A-I
. This experimental data
appear to be inconsistent with an aqueous diffusion mechanism of
cholesterol exchange and instead is supportive of a mechanism that may
involve the transient fusion of surface monolayers following the
collision of two particles(41) . It is apparent that an aqueous
diffusion mechanism may not adequately describe all of the factors that
are involved in the transfer of cholesterol between plasma lipoproteins
at least for a number of model discoidal particles studied here.
Consolidating these mechanistic interpretations into one comprehensive
model will require further experiments that need to address how changes
in the structure and perhaps charge of different kinds of lipoprotein
particles may modulate both lipoprotein-lipoprotein and
lipoprotein-cell interactions.
In conclusion, studies of cholesterol
exchange between plasma LDL and several structurally well-defined
reconstituted LpA-I particles containing different numbers of apoA-I
molecules show that unesterified cholesterol exchange is highly
sensitive to changes in the compositional and thereby structural
properties of the different lipoproteins. Increases in the number of
apoA-I molecules in LpA-I significantly inhibit their ability to
exchange cholesterol with LDL. In addition, significant differences in
cholesterol exchange kinetics between cholesterol-free and
cholesterol-containing Lp2A-I acceptors indicate that the presence of
cholesterol in Lp2A-I particles also directly influences
the function of these particles. Since other studies have shown that
variations in LpA-I phospholipid:apoA-I ratio or cholesterol content
have direct effects on the conformation and charge of
apoA-I(26, 38) , it appears that the physical
properties of apoA-I may be critical to the function of HDL and
metabolism of cholesterol.