(Received for publication, July 25, 1994; and in revised form, December 2, 1994)
From the
The production of cholesteryl ester (CE) by lecithin:cholesterol
acyl transferase (LCAT) is elevated significantly in hyperlipidemic
subjects at high risk for coronary artery disease. To elucidate the
molecular events involved, the relationship between LCAT activation and
apolipoprotein (apo) A-I charge and structure in high density
lipoproteins (HDL) has been studied in both native HDL and homogeneous
recombinant HDL (Lp2A-I) particles containing apoA-I, palmitoyloleoyl
phosphatidylcholine and cholesterol. Increasing the cholesterol content
of discoidal Lp2A-I from 4 to 26 molecules/particle raises the maximum
rate of cholesterol esterification by LCAT (V)
from 3.1 to 9.2 nmol CE/h/unit of LCAT and increases the apparent K
from 0.5 to 3.5 µM cholesterol. Similarly, increasing the cholesterol content in
triolein core-containing Lp2A-I (4-18 molecules/particle) and in
native HDL
(12-21 molecules/particle) also
significantly increases the V
for LCAT
(2.8-7.7 and 0.5-3.6 nmol CE/h, respectively) and raises
the K
values (7.6-36.9 and
7.3-8.5 µM cholesterol, respectively). In contrast,
changes in the cholesterol content of native and recombinant HDL have
no significant effect on the apparent K
values when expressed in terms of the concentration of
either apoA-I or palmitoyloleoyl phosphatidylcholine. This appears to
indicate that interfacial cholesterol content has no effect on the
binding affinity of LCAT to different LpA-I particles but directly
affects catalysis by modulating the interaction of cholesterol
molecules with the active site of LCAT. Increasing the cholesterol
content of the different HDL particles progressively increases the
particle net negative charge, and these changes in apoA-I charge are
strongly correlated with both the V
and apparent K
values for LCAT. This suggests that the
conformation and charge of apoA-I play a central role in LCAT
activation and that these parameters are influenced by the amount of
cholesterol in the surface of HDL particles.
Lecithin:cholesterol acyltransferase (LCAT) ()is the
plasma enzyme responsible for the production of the majority of
cholesteryl esters via transfer and esterification of sn-2
fatty acids from phosphatidylcholine to the 3-hydroxyl group of
cholesterol (for review, see (1) and (2) ). It has
been shown that hyperlipidemic patients with high risk of vascular
disease have a significantly increased rate of production of
cholesteryl esters in their HDL pool by LCAT(3) . LCAT activity
appears to be a stimulated in hyperlipidemic patients by a change in
the structure of HDL(3) . Increased LCAT activity was shown to
be directly related to an increased amount of small HDL
particles but was inversely related to the amount of HDL
in the plasma(3, 4) . Other investigations have
shown that small HDL
particles stimulate LCAT
activity(5) , while the HDL
subclass may be
inhibitory to the enzyme (6) .
Several investigators have established that apoA-I is the principal activator of cholesterol esterification by LCAT under in vitro conditions(1, 2) . A number of studies have shown that differences in apoA-I structure in reconstituted HDL particles (LpA-I) correlate with LCAT activation (for review, see (2) ). Reports from experiments with native (5, 6) and reconstituted (7) particles have identified a correlation between HDL particle size and its ability to interact with LCAT. Some studies further suggest that both surface and core lipid content of HDL may be of particular importance in the interactions between HDL and LCAT(7, 8) . Some investigations have attempted to determine the effect that surface cholesterol may have on the rate of esterification by LCAT. Studies using vesicular and discoidal substrates have identified a strong relationship between surface phosphatidylcholine/cholesterol ratios and LCAT activity(9, 10) . Another report, however, suggests that the cholesterol content of discoidal LpA-I is only related to the rate of cholesterol esterification by LCAT at low cholesterol concentrations, where the substrate concentration is rate limiting(11) .
In the present study we have undertaken a detailed characterization of the effect of variations in the cholesterol content of LpA-I containing two molecules of apoA-I (Lp2A-I) on the activity of LCAT. The results show a strong relationship between the cholesterol content of native and reconstituted Lp2A-I particles and LCAT function. This relationship is shown to be independent of direct substrate effects and instead is related to changes in the structure of the Lp2A-I particle. Together with previous studies(8, 12, 13) , this investigation provides further evidence to suggest that the activation of LCAT by HDL may be closely linked to the conformation and charge of apoA-I. This study suggests that an apoprotein conformation-dependent surface charge affects the catalytic ability of LCAT. Other studies have shown that LCAT regulation may involve electrostatic interactions that are highly sensitive to the interfacial environment of the surface of LpA-I particles(12, 14, 15) . In addition, a similar electrostatic sensitivity has been observed with another interfacially-active protein, cholesteryl ester transfer protein(16, 17) . This appears to indicate that lipoprotein surface charges may play important roles in regulating interfacial interactions.
After reconstitution, both core-containing and discoidal complexes were reisolated by chromatography on a Superose 6 column. The size and homogeneity of apoA-I complexes were estimated by nondenaturing gradient gel electrophoresis (24) on precast 8-25% acrylamide gels (Pharmacia Biotech Inc. PhastGel) after protein staining and densitometric scanning. Lipoprotein particle electrophoretic mobility, valence and surface charge characteristics were determined by electrophoresis on precast 0.5% agarose gels (Beckman, Paragon Lipo kit) as described previously(25) . The number of molecules of apoA-I/particle was determined by apoprotein cross-linking with dimethyl suberimidate as described by Swaney(26) . Total cholesterol and phospholipid were determined enzymatically using Boehringer Mannheim kits. Proteins were determined by the Lowry method as modified by Markwell et al.(27) .
Figure 1: Densitometer profiles of discoidal and core-containing Lp2A-I subjected to electrophoresis in 8-25% nondenaturing gradient gels. The molar ratios of POPC, cholesterol and triolein of each Lp2A-I (discs D4-D26 and spheres S4-S18) were determined after chromatographic reisolation and are indicated in Table 1. Stokes' diameters were determined by comparison to high molecular weight standards as described previously(16) .
Figure 2:
The effect of discoidal Lp2A-I composition
on the rate of cholesterol esterification by LCAT. PanelA, the esterification of
[H]cholesterol in four different reconstituted
Lp2A-I particles by LCAT is shown. Discoidal Lp2A-I were prepared with
varying amounts of cholesterol (stoichiometries after reisolation are
shown) and were incubated with purified LCAT as described in the text.
Values are the mean of triplicate determinations and are representative
of three different preparations of Lp2A-I particles. PanelB, data from the incubations described in A are
replotted to show the effect of increasing the number of molecules of
cholesterol/particle on the esterification of
[
H]cholesterol by LCAT. To illustrate the effect
of cholesterol at constant protein concentrations, individual curves
were composed to contain data from four different Lp2A-I particles
containing various amounts of cholesterol.
Since apoA-I/phospholipid and apoA-I/particle ratios are essentially constant for the various Lp2A-I particles, plots of reaction velocity against phospholipid concentration or particle number are similar to that shown in Fig. 2A. These substrate curves are representative of the reaction rates of LCAT for different kinds of Lp2A-I particles containing various amounts of cholesterol. To illustrate the effect of cholesterol directly on the initial velocities of LCAT, the data in Fig. 2A have been regraphed to show reaction rates as a function of particle cholesterol content. Fig. 2B shows that the activity of LCAT increases as the number of cholesterol molecules per Lp2A-I particle increases from 4 to 26. LCAT reaction rates also increase as a function of the number of Lp2A-I particles, up to an apoA-I concentration of 1.14 mM where the enzyme appears to be saturated with substrate (Fig. 2, A and B).
Initial velocities shown in Fig. 2were
estimated with less than 5% substrate conversion to avoid conditions
where the substrate concentration would become rate-limiting. The
increased saturation points for the different curves therefore
correspond to significant increases in the maximum velocity (V) of cholesterol esterification by LCAT. Fig. 3shows double-reciprocal plots of initial velocities as a
function of substrate concentration for both of the enzyme substrates,
cholesterol, and phospholipid, and also for apoA-I. Apparent
Michaelis-Menten kinetic constants were similar when estimated from
double-reciprocal plots or from a rectangular hyperbola curve fitting
equation and are shown in Table 2. Increasing the cholesterol
content in these Lp2A-I complexes is associated with an almost 3-fold
increase in the maximum velocity (V
) of
cholesterol esterification by LCAT (Table 2). It is also evident
in both Fig. 3and Table 2that the concentration of
apoA-I or phospholipid required for a half-maximal velocity (K
) is similar for the four particles containing
various amounts of cholesterol. In contrast, the apparent LCAT K
for cholesterol increases almost 4-fold as
Lp2A-I cholesterol content increases. As such, the catalytic efficiency
of LCAT (V
/K
) for
cholesterol falls with increasing Lp2A-I cholesterol content, while the V
/K
for phospholipid
increases slightly.
Figure 3:
Double-reciprocal plots of cholesterol
esterification in rLpA-I by LCAT. Data from the incubations described
in Fig. 2are presented as double-reciprocal plots where
reciprocals of initial velocities (V,
nmol/h) are plotted against reciprocals of apoA-I (A),
phospholipid (B), and cholesterol (C) concentrations.
The best fit lines are computer-generated linear regressions for four
different Lp2A-I particles having the following
phospholipid/cholesterol/apoA-I stoichiometries: (
) 178:4:2;
(
) 188:8:2; (
) 182:18:2; and (
)
182:26:2.
Figure 4:
Effect of the cholesterol content of
core-containing Lp2A-I on LCAT activation. The esterification of
[H]cholesterol by LCAT in different
core-containing Lp2A-I particles having the following
phospholipid/triolein/cholesterol/apoA-I stoichiometries: (
)
68:8:4:2; (
) 62:8:8:2; and (
) 74:8:18:2 is shown. Lp2A-I
were prepared by co-sonication as described in the text and were
incubated with purified LCAT. Values are the average of duplicate
determinations and are representative of three different preparations
of HDL particles. Inset, double-reciprocal plots are shown
where reciprocals of initial velocities (V
, nmol/h) are plotted against
reciprocals of particle phospholipid concentrations
(µM).
Incubations of both control and cholesterol-enriched
HDL with LCAT show that cholesterol enrichment has a
significant effect on LCAT activity. Fig. 5shows that a
1.5-fold increase in HDL
cholesterol content is concomitant
with a significant increase in the rate of cholesterol esterification
by LCAT. Lineweaver-Burk kinetic analysis of this data (Fig. 5, inset) shows the increase in initial velocities to correspond
to an increase in the V
for LCAT from 0.5 to 3.6
nmol cholesteryl ester/h and a smaller increase in apparent K
from 7.3 to 8.5 µM cholesterol (Table 3). Increasing the HDL
cholesterol content
from approximately 12 to 21 molecules/particle is therefore associated
with a 6-fold increase in the catalytic efficiency (V
/K
) of LCAT.
Figure 5:
Effect of HDL-cholesterol
content on LCAT activation. The esterification of
[
H]cholesterol in native HDL particles by LCAT is
shown. The cholesterol content of native HDL
particles was
increased from 12 (
) to 21 (
) molecules/particle as
described in the text, and then the enriched particles were compared
with control HDL
in incubations with purified LCAT. Values
are the average of duplicate determinations and are representative of
three different preparations of HDL particles. Inset,
double-reciprocal plots are shown where reciprocals of initial
velocities (V
, nmol/h) are plotted
against reciprocals of particle phospholipid concentrations
(µM).
Changes in the structural properties of HDL particles that
result from variations in cholesterol content appear to have major
effects on the interfacial interactions that modulate the metabolism of
HDL ``in vivo.'' Studies have shown that the neutral
lipid transfer and exchange catalyzed by cholesteryl ester transfer
protein is affected by the amount of cholesterol on the surface of HDL
particles(31, 32) . Similarly, investigations with
hepatic lipase (19) have shown that the lipolytic actions of
this enzyme are also sensitive to the cholesterol content of
lipoprotein particles. Investigations with LCAT have shown the
catalytic activity of this enzyme is also sensitive to the amount of
cholesterol in the surface of vesicular and discoidal
particles(9, 10) . In contrast, another study has
suggested that the rate of cholesterol esterification in discoidal
LpA-I particles is independent of their cholesterol
contents(11) . This conclusion was based upon the observation
that the rate of cholesterol esterification by LCAT was independent of
LpA-I cholesterol content above 20 molecules/particle. Interpretation
of these experimental results, however, is difficult as more recent
studies suggest that the high cholesterol contents incorporated into
these complexes may have given rise to heterogeneous LpA-I
preparations(30) . In contrast to their observation with
complexes containing greater than 20 molecules of cholesterol, Jonas et al.(11) showed that LCAT activation was directly
affected when LpA-I cholesterol content was increased from 2 and 20
molecules/particle. In their study, the association of LCAT activity
and LpA-I cholesterol content was proposed to be a consequence of a
rate-limiting concentration of cholesterol in the lipoprotein particle.
In the present study, we have also shown that LCAT activity is
stimulated when Lp2A-I cholesterol content is increased from 4 to 26
molecules. In this study, however, we have shown that this sensitivity
of LCAT to Lp2A-I cholesterol content is significant when substrate
concentrations are not limiting. Compositional analysis of native HDL
particles has shown that the amount of cholesterol in native HDL particles varies between 8 and 15
molecules/particle(33) . This appears to suggest that the
sensitivity that LCAT displays to LpA-I cholesterol contents is likely
physiologically relevant to the in vivo regulation of the
enzyme.
Kinetic determinations in this study have shown that the
maximum velocities of cholesterol esterification by LCAT are similar
for discoidal and core-containing LpA-I particles and are proportional
to the particle content of cholesterol. In contrast, the K values for the core-containing LpA-I complexes
are from 5- to 20-fold greater than that for discoidal particles. As
such, the catalytic efficiency of LCAT (V
/K
of apoA-I) is over
10-fold greater for discoidal particles than for spheres (Fig. 6). This finding is similar to that observed previously by
Jonas and colleagues(8) ; however, in their study, differences
in LCAT activation were primarily associated with changes in V
values. Since we have observed similar V
values for core-containing and discoidal
complexes, it appears that the substantially different V
values reported by Jonas et al.(8) for discs and spheres may have been related to the
very different composition and stoichiometry of the core-containing
LpA-I relative to that of the discoidal particles. In the present
study, it is evident that the significant differences in LCAT
reactivity between discs and core-containing particles is primarily a
function of differences in substrate affinity. Surface cholesterol in
core-containing LpA-I appears to be up to 20-fold less available to
LCAT than in a discoidal complex containing an equivalent amount of
cholesterol. This difference may be due to an inaccessibility of
cholesterol in core-containing LpA-I resulting from differences in the
packing of cholesterol molecules relative to that on discoidal
particles. Previous studies have shown that while cholesterol molecules
are localized on the surface of HDL particles with no core lipids, as
much as 40% of the cholesterol molecules in a particle with a neutral
lipid core may be associated with the apolar lipids(34) . This
difference in cholesterol localization may also indirectly affect
interfacial interactions by modifying surface phospholipid packing.
Investigations by Bolin and Jonas (35) have recently shown that K
values for apoA-I may closely reflect the
interfacial binding affinity of LCAT to an LpA-I particle. Since K
values for apoA-I and phospholipid are up to
20-fold higher for core-containing particles relative to discs, it is
possible that LCAT may bind with a much lower affinity to
core-containing LpA-I complexes. In both core-containing and discoidal
Lp2A-I particles, an increase in the maximum velocity of LCAT is
directly associated with substantial increases in K
cholesterol. As a consequence, the overall catalytic efficiency
of LCAT (V
/K
) for
cholesterol falls with increasing Lp2A-I cholesterol content. In
contrast, K
phospholipid (or apoA-I) values are
essentially constant even when V
values increase
by 3-fold. This results in an increased catalytic efficiency for
phospholipid, which is similar to that observed for apoA-I (Fig. 6). Varying the concentration of cholesterol in LpA-I
particles therefore has unique and different effects on the
interactions between LCAT and the two different substrates of the
enzyme.
Figure 6:
The effect of increasing Lp2A-I
cholesterol content on the catalytic efficiency of LCAT. LCAT reaction
kinetics were estimated from the curves shown in Fig. 2and Fig. 4(Table 2). The ratio of V to
apparent K
apoA-I values is plotted
against the cholesterol content (Table 1) of both discoidal
(
) and core-containing (
, inset)
Lp2A-I.
Studies have shown that increasing the cholesterol content
in reconstituted discoidal LpA-I particles reduces the phospholipid
motional freedom and increases phospholipid acyl chain
order(36) . These interfacial effects appear to directly affect
both the conformation and charge of apoA-I (30) and result in
an impaired interfacial penetration by apoA-I molecules(37) .
It is possible that the increased negative surface charge on apoA-I may
modify the surface environment of the Lp2A-I in a manner that may have
specific effects on the interfacial interactions between Lp2A-I and
LCAT. In this study, however, the K values for
apoA-I or phospholipid remain essentially constant when LpA-I
cholesterol content is increased. This suggests that the binding
affinity of LCAT to the lipoprotein surface is unaffected by changes in
complex cholesterol content. Instead, the significant reduction in LCAT
affinity for cholesterol observed as LpA-I cholesterol content is
increased may suggest that altered LpA-I interfacial properties promote
a conformational change in LCAT that directly affects interaction of
cholesterol molecules with the active site.
Previous studies have
suggested that LCAT activity may be sensitive to the size of HDL
particles; small HDL particles appear to be the preferred substrate of
this enzyme(3, 4, 5) . In this study,
however, increasing cholesterol content corresponds to both an increase
in particle size and also to an increase in LCAT activity. This
indicates that it is not particle size that modulates LCAT activity but
that LCAT is affected by some other factor(s) that changes in parallel
to particle size. Lipoprotein surface charge would be a good candidate
for such a factor. A reduction in particle size usually corresponds to
an increase in the density of negative surface charge and a concomitant
increase in negative surface potential(38) . Increasing the
cholesterol content in an LpA-I particle, however, increases the
magnitude of the negative surface potential in a similar manner as
would reducing the particle size but by increasing the net negative
charge on apoA-I(30) . In this study, we have correlated the
effects that cholesterol has on the particle surface charge to the
substrate specificity of LCAT. Fig. 7shows that the maximum
velocity of LCAT esterification is strongly related (r = -0.97) to the surface charge of apoA-I on both
core-containing and discoidal LpA-I particles containing increasing
amounts of cholesterol. A stimulation of LCAT activity appears to be
associated with an increase in the magnitude of negative surface charge
on apoA-I. This observation, however, appears to be inconsistent with
other reports(35, 39) , which suggest that increasing
HDL negative surface charge through the addition of charged lipids
(phosphatidylserine or free fatty acid) is inhibitory to LCAT. Indeed,
similar observations have been made in this laboratory with another
negatively charged lipid, phosphatidylinositol, which also mildly
inhibits LCAT activity. ()It follows that it is probably a
protein conformation-dependent surface charge rather than a global
interfacial charge that regulates the activity of LCAT. This is
consistent with previous studies that showed that modification of
apoA-I-Lys residues with reagents that modify apoA-I conformation and
charge directly affects the activation of LCAT(12) .
Figure 7:
The
relationship between Lp2A-I surface charge and estimated LCAT V values. LCAT reaction V
values were estimated as described in the text and are plotted
against discoidal (
) and core-containing (
) Lp2A-I
particle surface potential values (Table 1). An inverse
relationship is demonstrated between LCAT V
values and particle surface potential for both core-containing
and discoidal Lp2A-I containing increasing amounts of
cholesterol.
This investigation has shown that the cholesterol content of HDL particles directly affects the rate of cholesterol esterification by LCAT. As such, it appears that cholesterol plays a physiologically important role in the in vivo regulation of this enzyme. The regulatory effect of cholesterol appears to be indirect and conferred through changes in the conformation and charge of apoA-I. The study suggests that cholesterol-induced changes in the interfacial properties of LpA-I particles have minimal effects on the binding affinity of LCAT for the particle surface but modulate the enzyme through changes in the active site exposure.