(Received for publication, April 3, 1995; and in revised form, August 10, 1995)
From the
Alterations in high density lipoprotein (HDL) composition that
occur in dyslipidemic states may modulate a number of events involved
in cholesterol homeostasis. To elucidate the details of how HDL-core
composition can affect the molecular structure of different kinds of
HDL particles, the conformation and stability of apoA-I have been
investigated in homogeneous recombinant HDL particles (LpA-I)
containing palmitoyloleoyl phosphatidylcholine (POPC), triolein (TG),
and/or cholesteryl linoleate (CE). In a discoidal particle containing
two molecules of apoA-I and 85 molecules of POPC, apoA-I exhibits an
-helix content of 70% and a free energy of stability of its
-helical segments (
G
) of 2.2
kcal/mol. Inclusion of eight molecules of TG into the complex
significantly reduces the
-helix content and stability of apoA-I,
whereas inclusion of four molecules of CE into the complex has an
opposite effect in that the
-helix content is significantly
reduced and the stability of the remaining
-helical structure of
apoA-I is increased. Neutral lipids have a different effect on apoA-I
conformation in spherical LpA-I particles. In a sonicated-spherical
LpA-I particle containing two molecules of apoA-I and 70 molecules of
POPC, apoA-I exhibits an
-helix content of about 60% and a
G
of 1.2 kcal/mol apoA-I. Inclusion of
either 10 molecules of TG or six molecules of CE into such a particle
increases both the
-helix content and stability of apoA-I.
Increasing the CE/TG ratio in LpA-I particles that contain both neutral
lipids enhances the stability of the
-helical segments. ApoA-I
molecules tend to dissociate and cause particle instability when
G
for the lipid-bound
-helices is
less than that for helices in the lipid-free state. The stabilities of
both discoidal and spherical LpA-I particles are relatively low when
the only neutral lipid present is TG but the particle stability is
enhanced by the presence of CE molecules. Such dissociation of apoA-I
molecules from LpA-I particles that have a low CE/TG ratio would be
promoted in the hypertriglyceridemic state in vivo.
High density lipoproteins (HDL) ()comprise a
heterogeneous class of particles that contain apolipoprotein A-I
(apoA-I) (LpA-I) or apoA-I and A-II (LpA-I,A-II) as their primary
protein constituents(1) . The central role that HDL plays in
cholesterol metabolism is thought to involve the transport of
cholesterol from peripheral tissues to the liver(2) . Several
studies have suggested that the efficiency of HDL in mediating this
flux may be impaired in hypertriglyceridemic patients and that this
effect may be related to modifications in HDL composition and size (for
review, see (3) ). Investigations in a variety of laboratories
have shown that changes in HDL size and composition can lead to altered
interactions between HDL and lecithin:cholesterol
acyltransferase(4, 5, 6) , cholesteryl ester
transfer protein(7, 8, 9) , and cell
surfaces(10, 11) . There is evidence that an increased
number of small, neutral lipid-poor, HDL particles in
hypertriglyceridemic patients may directly affect cholesterol transport
by stimulating the production of cholesteryl ester within the HDL pool (12) and by promoting an enhanced transfer and potentially
atherogenic accumulation of these lipids in apoB-containing
lipoproteins(3, 13) .
Alterations in HDL composition also give rise to specific changes in the conformation and charge of the primary protein of HDL, apoA-I (14, 15, 16, 17) , and it appears that these changes in the molecular properties of HDL closely correlate with the altered function of these lipoprotein particles(6, 14, 18, 19) . While a substantial amount of information about the organization of apoA-I molecules in discoidal particles is now available (for review, see (20) and (21) ), very little information exists that describes the structure of apoA-I in spherical HDL particles. In recent studies with reconstituted HDL particles (LpA-I), we showed that the overall conformation of human apoA-I is significantly different in spherical particles containing a cholesteryl ester core than in discoidal complexes that do not contain a neutral lipid core(16, 17) . These studies also showed that the surface charge and secondary structure of apoA-I are significantly different on spherical particles and appear to be modulated by changes in composition in a different manner than for apoA-I on discoidal LpA-I particles(17) . In this study, we further show that the type and amount of neutral lipid in reconstituted discoidal and spherical LpA-I directly affects the surface charge and structural characteristics of the lipoprotein particles. The results indicate that cholesteryl ester and triglyceride have distinct effects on the physical properties of apoA-I that are specific to the kind, spherical or discoidal, of LpA-I particle. A decrease in the cholesteryl ester content in the LpA-I particles is associated with a reduction in the structural integrity of these lipoprotein structures. Such particles are very similar to the abnormal HDL particles found in the plasma of hypertriglyceridemic subjects(1, 3, 13) , and it is probable that the unusual charge and structural characteristics of small, neutral lipid-poor, HDL give rise to differences in the functional properties of these lipoproteins.
Figure 1: Densitometer profiles of LpA-I electrophoresed in 8-25% nondenaturing gradient gels. Profiles for discoidal (D) and spherical (S) LpA-I after reisolation by gel filtration are shown. Final molar ratios of POPC, cholesteryl ester (CE), and triglyceride (TG) per two molecules of apoA-I are indicated beside each profile. Stokes diameters were determined as described previously(16) .
Figure 2:
Photographic copies of negatively stained
electron micrographs of discoidal and spherical Lp2A-I particles.
Electron micrographs of reconstituted discoidal (panel A) and
sonicated-spherical (panels B and C) Lp2A-I are
shown. Final molar ratios of POPC and triolein/two molecules of apoA-I
are as follows: panel A, 90:8; panel B, 70:0; and panel C, 82:10. Final magnification is 520,000 (1 cm
= 19 nm). Mean particle dimensions of 100 particles were
determined from each negative and are similar to the hydrodynamic
diameters shown in Table 1.
Inclusion of up to 9 mol % neutral lipids in the discoidal particle (Table 1) has no detectable effect on the particle morphology or
size. In this study, the amount of cholesteryl ester incorporated into
the discoidal complex is very close to the reported solubility of
cholesteryl ester in bilayer phases of egg lecithin(34) . In
contrast, considerably more triglyceride can be incorporated into a
discoidal complex. The amount of triglyceride incorporated into the
discoidal particle is substantially greater than the solubility
expected (3 mol %) for triglyceride in lecithin
bilayers(35) . This suggests that the physical characteristics
of phospholipid in a discoidal complex differs from that in a typical
liquid-crystalline bilayer because of the presence of apoA-I around the
edge of the disc. It is possible that differences in the incorporation
of cholesteryl ester and triglyceride in discoidal particles may be
partially due to the previously reported ability of triglyceride to
interact with phospholipid acyl chains to a greater extent than
cholesteryl ester can(35, 36) .
Similar to the
situation with discs, inclusion of neutral lipids in sonicated
complexes also has no significant effect on the particle size or
spherical (circular) morphology evident in electron micrographs. These
lipoprotein structures appear to be similar in size to small HDL particles(28) . Similarly sized LpA-I complexes have been
shown to be a major component of nascent HDL particles secreted by
HepG2 cells (37) and also have been isolated from
normolipidemic plasma (sm-LpA-I, (38) ) and from cholate
dialysis preparations (rHDL1, (14) ). However, it should be
noted that the reconstituted spherical LpA-I described here have
different hydrated densities than HDL
particles. The LpA-I
prepared by sonication exhibit peak hydrated densities of
1.10-1.13 g/ml for neutral lipid-rich and -poor particles,
respectively. This hydrated density estimated for the various
reconstituted LpA-I is consistent with the lipid/protein ratio of these
complexes. The protein content of the spherical LpA-I particles listed
in Table 1averages about 43% by weight, which is comparable with
that observed for native HDL
particles (1) but
lower than for HDL
particles (50-60% by weight).
Figure 3:
Effect of GdnHCl concentration on the
molar ellipticity of apoA-I on discoidal and spherical LpA-I. Aliquots
of discoidal (POPC:CE:TG molar ratios: , 85:0:0;
, 78:0:8;
, 80:2:0;
, 92:2:4:2) and spherical (
, 70:0:0)
LpA-I were incubated with from 0-6 M GdnHCl in 0.05 M phosphate buffer for 72 h at 4 °C. CD spectra were
measured at 24 °C in a 0.1 cm pathlength quartz cell with sample
protein concentrations of 33.3 µg/ml phosphate buffer, and four
scans from 230 to 200 nm were collected and averaged at each GdnHCl
concentration. Inset, linear regression plots are shown of the
observed free energies of denaturation
(
G
) against RTln(1 + ka) for
discoidal LpA-I. The free energy of denaturation at zero GdnHCl
concentration (
G
) is estimated from
the intercept on the vertical axis and the moles of GdnHCl bound
(
n) are computed from the slopes of the regression
lines(17) .
Fig. 4A shows GdnHCl denaturation curves for spherical
LpA-I with no core lipid and containing either cholesteryl ester or
triglyceride. Addition of cholesteryl ester or triglyceride to the
particle is associated with an increase in the amount of -helix in
apoA-I, but only cholesteryl ester increases the stability of apoA-I
-helices as indicated by an increase in
G
values (Table 3). In Fig. 4B, it is evident that increasing the cholesteryl
ester content in reconstituted lipoprotein complexes containing both
cholesteryl ester and triglyceride further increases the resistance of
apoA-I to denaturation. Analysis of the GdnHCl denaturation curves
shows that, in contrast to the discoidal particles, changes in the
G
of apoA-I parallel D
values ( Table 3and Fig. 4, A and B, insets). It is evident from the insets of Fig. 4, A and B, that
including or increasing the cholesteryl ester content in a spherical
LpA-I particle corresponds to a significant increase in the
G
. Increasing the cholesteryl ester
content in spherical LpA-I, therefore appears to increase the
conformational stability of apoA-I. This observation seems to be
contrary to a recent report by Rye et al.(39) showing
that a small cholesteryl ester-poor particle is more stable to
denaturation with GdnHCl than a larger cholesteryl ester-rich particle.
Interpretation of this observation is, however, complex as changes in
cholesteryl ester content of the different reconstituted complexes
described were accompanied by major changes in all of the other lipid
constituents. For example, the cholesteryl ester-poor particle in the
study also had a significantly reduced free cholesterol content and an
increased POPC:apoA-I ratio. Investigations in both this laboratory and
that of Jonas and co-workers have shown that similar changes in LpA-I
cholesterol (40) and phospholipid (17, 41) content also modulate the stability of apoA-I
to denaturation.
Figure 4:
Effect of GdnHCl concentration on the
molar ellipticity of apoA-I on spherical LpA-I. Aliquots of spherical
LpA-I (POPC:CE:TG molar ratios: panel A, , 70:0:0;
, 86:0:10;
, 84:6:0 and panel B,
, 70:0:0;
, 80:4:6;
, 82:10:12) were incubated with 0-6 M GdnHCl, and CD spectra were measured as described in Fig. 3. Inset, linear regression plots are shown of the
observed free energies of denaturation
(
G
) against RTln(1 + ka) for
spherical LpA-I.
In the present study, low levels of cholesteryl
ester in spherical LpA-I are associated with a reduced structural
stability, and what appears to be an increased propensity of the
complex to reorganize into a more thermodynamically stable structure.
Characterization of both discoidal and spherical LpA-I with low
thermodynamic stabilities shows that these particles tend to become
more heterogeneous with increased storage time. Fig. 5shows
nondenaturing gradient gel profiles of four different LpA-I particles
after 1 month of storage at 4 °C. Comparison with profiles from
gels run immediately after reisolation (Fig. 1) shows that no
significant changes are apparent in profiles for the most stable,
cholesteryl ester-containing, particles, while triglyceride-containing
particles appear to have partially reformed into different sized
particles and lipid-poor apoA-I. Storage of the triglyceride-containing
disc gives rise to an increase in the particle heterogeneity due to the
formation of both larger and smaller LpA-I particles. An increased
heterogeneity is also observed after storage of the more unstable
spherical LpA-I particles and appears to be primarily due to the
formation of a larger particle with a hydrodynamic diameter of 10.3 nm.
SDS-polyacrylamide gels of these LpA-I after chemical cross-linking
shows that this heterogeneity is associated with an increased amount of
trimeric apoA-I (data not shown). This suggests that the larger
particle formed from the metastable, cholesteryl ester-poor, spherical
LpA-I contains three molecules of apoA-I. Integration of the spherical
LpA-I densitometer profiles shows that the amount of apoA-I associated
with the newly formed particle directly parallels the stability of
apoA-I on the LpA-I particle. A decrease in
G
of from 2.0 to 1.2 kcal/mol for the
cholesteryl ester-rich and -poor particles corresponds to an increase
of from 2 to 15% in the relative amount of apoA-I associated with the
10.3 nm particle and a similar increase in the amount of apoA-I
associated with the lipid-free apoA-I.
Figure 5: Densitometer profiles of LpA-I electrophoresed in 8-25% nondenaturing gradient gels. Profiles for discoidal (D) and spherical (S) LpA-I that were electrophoresed immediately after preparation (f) or after storage at 4 °C for one month (s) are shown. Stokes diameters were determined as described previously(16) .
Figure 6:
Effect of LpA-I surface potential on
-helix stability. ApoA-I
-helix stability
(
G
) in spherical LpA-I (
),
discoidal LpA-I (
, (17) and (40) ), and
discoidal LpA-I containing cholesteryl ester or triglyceride (
)
is plotted against the particle surface potential (Table 2). A
strong positive correlation is demonstrated between LpA-I surface
potential and apoA-I conformational stability for discoidal LpA-I (r = 0.93) but not for spherical LpA-I (r = 0.48).
ApoA-I structure appears to be extremely sensitive to the
neutral lipid content of discoidal particles. Thus, addition of
cholesteryl ester or triglyceride to a discoidal complex is associated
with a reduction in the content of -helical segments in apoA-I by
about 10-15% (Table 1). The observed helix contents suggest
that, in the presence of a neutral lipid, apoA-I maintains five to six
22-residue helical segments on the discoidal LpA-I particle. It follows
that a minimum of five to six helical segments are required to maintain
the structural integrity of a discoidal complex that normally has about
eight helical segments on its surface(14) . Inclusion of
neutral lipid in a discoidal complex results in a reduction in
-helix content and stability but still leaves the complex more
stable than a sonicated particle. This suggests that some of the apoA-I
helical structure in a discoidal complex is not functioning to maintain
the particle integrity. It is possible that this component of apoA-I
helical structure is not localized at the edge of the disc but rather
is associated with the phospholipid head groups on the face of the
disc. This is consistent with previous studies that suggested that
amphipathic helical peptides may be able to interact with both the edge
and face of a discoidal complex(42) .
Inclusion of
cholesteryl ester or triglyceride in a sonicated POPC:apoA-I complex is
also associated with changes in both the content and stability of the
amphipathic -helical segments in apoA-I. However, in contrast to
the situation with discoidal LpA-I, the inclusion of up to 10 molecules
of neutral lipid in sonicated complexes is associated with a
significant increase in the
-helix content of apoA-I (Table 1). The changes in secondary structure correspond to
increased involvement of between 15 and 30 amino acids in the
-helical structure of apoA-I. If it is assumed that the
-helical structure of apoA-I is comprised of 22-residue
-helix segments, the lowest
-helical estimate (58%) for the
sonicated POPC:apoA-I particle corresponds to six to seven such
helices. The observed increase in
-helicity associated with the
presence of cholesteryl ester would correspond to an increase of
approximately one, 22-residue, helical segment. Increases in the
-helical content of apoA-I may also involve the formation of new
short segments(17) , and it appears that the formation of
shorter helical segments is consistent with the smaller increase in
-helix content in apoA-I that results when triglyceride is
incorporated into the LpA-I complex.
Incorporation of neutral lipids
in discoidal and spherical particles has distinct and different effects
on the content and stability of the amphipathic -helix secondary
structure of apoA-I. The study shows that variations in LpA-I
triglyceride content have different effects on apoA-I stability in
discoidal particles relative to that in spherical LpA-I particles. In
contrast, an increase in LpA-I cholesteryl ester content is positively
related with an increase in the free energy of stability of apoA-I
-helices, in both discoidal and spherical particles (Fig. 7). Both cholesteryl ester and triglyceride reduce the
-helix content of apoA-I in a disc, but while triglyceride reduces
-helix stability on the disc, cholesteryl ester increases the
helix stability. This may be the result of cholesteryl ester coming
into close contact with apoA-I in a similar manner to that which has
been proposed for cholesterol(40) ; inclusion of a small amount
of cholesterol in a discoidal complex also gives rise to a reduction in
-helix content with a concomitant increase in the helix stability.
This implies that the steroid ring structure of either a cholesterol or
cholesteryl ester molecule may interact with apoA-I and induce a
conformational change. Incorporation of cholesteryl ester and
triglyceride in sonicated, spherical LpA-I is associated with increases
in the stability of apoA-I
-helices to denaturation by GdnHCl (Table 3). The observation that increased helical stability
parallels increases in
-helix content is consistent with previous
investigations (17) and suggests that it may be an increase in
helix-helix interactions that stabilizes the secondary structure of
apoA-I. Conversely, increasing the cholesteryl ester content to 10
molecules in an LpA-I complex also containing 12 molecules of
triglyceride significantly reduces the amount of
-helical
structure in apoA-I, while at the same time increasing the helical
stability. An increase in
-helix stability that is concomitant
with a reduction in helix content has also been observed when the
cholesterol content of a reconstituted LpA-I particle is
increased(40) . It appears that both cholesterol and
cholesteryl ester may compete with apoA-I for ``hydrophobic
solvation'' by the phospholipid acyl chains. In this manner, these
lipids may be able to displace apoA-I from the phospholipid interface
and promote a reorganization of the remaining helical segments into a
more stable conformation. This novel conformation is associated with an
increased stability of apoA-I
-helices and appears to be
concomitant with an increase in the structural integrity of the
lipoprotein complex.
Figure 7:
Effect of cholesteryl ester content on the
apoA-I -helix stability. The free energy of denaturation of apoA-I
-helical structure (
G
, Table 3) in spherical (
) and discoidal (
) LpA-I is
plotted against the particle cholesteryl ester content (Table 1).
The
values of G
listed in Table 3suggest that the presence of eight molecules of
triglyceride in the discoidal LpA-I complex (
G
= 1.8 kcal/mol) induces apoA-I dissociation, whereas the
presence of four molecules of cholesteryl ester (
G
= 2.9 kcal/mol) stabilizes the particle. The presence of a
mixture of cholesteryl ester and triglyceride molecules also stabilizes
the particle. Consistent with this, the discoidal particles that
contain cholesteryl ester are stable upon storage at 4 °C, whereas
those containing triglyceride are not (Fig. 5). In contrast, the
spherical LpA-I particles that contain cholesteryl ester and/or
triglyceride are thermodynamically unstable
(
G
values are in the range
1.4-2.0 kcal/mol, Table 3). While this maximum change in
free energy, for apoA-I on the cholesteryl ester and triglyceride
containing sphere, is less than that observed for apoA-I in a
lipid-free state, no substantial apoA-I dissociation is evident even
after storage for 1 month (data not shown). This suggests that kinetic
effects are involved in maintaining the structural integrity of small
spherical LpA-I. However, when the cholesteryl ester content of the
spherical LpA-I particle falls below five molecules/mol of apoA-I these
lipoprotein particles tend to fuse and shed apoA-I (Fig. 5). The
triglyceride-containing particles are somewhat less stable than the
cholesteryl ester-containing particles as expected from the
G
values (Table 3).
Results from this study have shown that LpA-I with a low thermodynamic stability tend to become more heterogeneous with increased storage time (cf. Fig. 1and Fig. 5). This heterogeneity appears to be concomitant with the formation of particles containing three molecules of apoA-I from complexes that have only two molecules initially. Therefore, it appears that unstable LpA-I particles containing two molecules of apoA-I can reorganize and form new more thermodynamically-stable complexes and, in the process, liberate a molecule of lipid-poor apoA-I (Fig. 8). Although this process appears to occur on a relatively slow time scale in vitro, the rate of the process may be enhanced by the actions of different plasma proteins. A recent report by Nishida and co-workers (43) suggests that the remodeling of HDL by phospholipid transfer protein may stimulate the formation of larger HDL particles (from 8.3 to 10.7 nm) and the concomitant liberation of apoA-I. Other investigations have also shown that changes in the neutral lipid core of HDL can also result in the dissociation of apoA-I. Thus, studies in the laboratory of Barter and co-workers (44) have shown that hydrolysis of HDL-surface and core lipids by hepatic lipase and neutral lipid transfer by cholesteryl ester transfer protein (39) can promote the structural destabilization of HDL and the liberation of lipid-poor apoA-I.
Figure 8: Possible mechanism for the generation of lipid-poor apoA-I from unstable LpA-I particles. Thermodynamically unstable LpA-I particles containing two molecules of apoA-I and a reduced content of cholesteryl ester relative to triglyceride tend to reorganize and form novel more-stable complexes and, in the process, liberate a molecule of lipid-poor apoA-I.