©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of the Cholesterol Content of Reconstituted LpA-I on Lecithin:Cholesterol Acyltransferase Activity (*)

(Received for publication, July 25, 1994; and in revised form, December 2, 1994)

Daniel L. Sparks (1)(§) G. M. Anantharamaiah (2) Jere P. Segrest (2) Michael C. Phillips (3)

From the  (1)Lipoproteins and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4E9 Canada, the (2)Departments of Medicine and Biochemistry and the Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294, and the (3)Department of Biochemistry, The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(max)) 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(3) (12-21 molecules/particle) also significantly increases the V(max) 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(max) 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.


INTRODUCTION

Lecithin:cholesterol acyltransferase (LCAT) (^1)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(3) particles stimulate LCAT activity(5) , while the HDL(2) 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.


EXPERIMENTAL PROCEDURES

Materials

Cholesterol and triolein were purchased from Sigma. 1-Palmitoyl-2-oleoyl phosphatidylcholine was obtained from Avanti Polar Lipids (Birmingham, AL). All other reagents were analytical grade.

Methods

Isolation and Enrichment of HDL(3) with Cholesterol

HDL(3) (density 1.125-1.21 g/ml) was isolated from fresh plasma of normolipidemic donors by sequential ultracentrifugation(18) . The cholesterol content of HDL(3) was increased by incubation with cholesterol-enriched Celite(19) . Briefly, 3 mg of cholesterol in CHCl(3) was dried onto 75 mg of Celite 545 under N(2) and then incubated with 1.5 mg (protein) of HDL(3) overnight at 37 °C. Control HDL(3) was incubated similarly but with cholesterol-free Celite. Cholesterol-enriched and control HDL(3) were labeled by drying [^3H]cholesterol in CHCl(3) under nitrogen, adding HDL(3), and by brief co-sonication under nitrogen for 3 times 30 s punctuated by 1-min cooling periods using a Branson 450 sonicator and 1/8-inch tapered microtip probe (with maximal output of 40 watts). Labeled HDL was reisolated by size exclusion chromatography on a 28 times 1.8 cm Superose 6 column at a flow rate of 0.75 ml/min. Approximately 60% of the labeled cholesterol was recovered with the HDL(3) preparation after reisolation.

Preparation and Characterization of Recombinant Lp2A-I Complexes

Purified apoA-I was isolated from delipidated HDL by size exclusion chromatography(20) . Discoidal Lp2A-I complexes were prepared from POPC and cholesterol, at initial POPC/cholesterol molar ratios ranging from 80:2 to 80:12 (per mol of apoA-I) by the cholate-Biobead removal technique previously described(21) . Core-containing recombinant LpA-I were prepared from POPC, triolein, cholesterol, and apoA-I (molar ratios of 120:12:5:2, 120:12:15:2, and 120:12:20:2, respectively) by a co-sonication technique similar to that originally described by Scanu and colleagues(22, 23) . Briefly, purified lipids in chloroform were dried under nitrogen, and 1.0 ml of Tris/saline, pH 8, was added. All sonications were performed in the 12 times 75-mm test tube suspended in a 15 °C water bath and under nitrogen. The lipids-buffer solution was initially sonicated for 1 min using a Branson 450 sonicator and 1/8-inch tapered microtip probe. This suspension was then incubated for 30 min at 37 °C and sonicated again for 5 min using a 95% duty cycle. ApoA-I (2 mg of a 1.4 mg of protein/ml of Tris/saline solution) was added to the lipid suspension and the protein-lipid mixture was sonicated for 4 times 1 min (with maximal output of 40 watts and 90% duty cycle) punctuated by 1-min cooling periods. LpA-I complexes were then filtered through a 0.22 µm syringe tip filter.

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) .

Purification of LCAT

LCAT was purified as described previously (28) from normolipidemic human plasma. After ultracentrifugation at 1.21 g/ml, the d < 1.21 lipoproteins were removed, and the upper 50% of the d > 1.21 infranate (albumin poor) was applied to an Affi-Gel Blue column. The nonretained fraction was chromatographed on DE52 cellulose and eluted with a linear gradient of 50-250 mM NaCl. Prior to use, LCAT was further chromatographed on hydroxylapatite and eluted with a 10-60 mM phosphate gradient. LCAT activity was characterized using a stock substrate (POPC/apoA-I ratio of 160:2, mol/mol) and quantitated into units of enzyme activity, where 1 unit = 1 nmol cholesterol esterified/mg of protein/h. The specific activity of the purified LCAT preparation varied between 4000 and 6000 units.

Assay of LCAT Activity

Native and reconstituted Lp2A-I were characterized as substrates for purified LCAT using a standard assay system, similar to that described by others(28, 29) . Each enzyme assay contained the lipoprotein substrate, 0.3 units of purified LCAT, bovine serum albumin (2.5 mg), beta-mercaptoethanol (2.5 mM), and Tris/NaCl buffer (final volume, 500 µl). Incubations were carried out for 10 min at 37 °C and were terminated with the addition of 2 ml of ethanol. Reaction products were extracted in hexane, and then the amount of ^3H associated with cholesteryl ester was determined by thin layer chromatography using a solvent system composed of hexane/diethyl ether/acetic acid, 90:10:1 (v/v/v). Under these conditions, initial rates were estimated with minimal substrate conversion; less than 5% of Lp2A-I cholesterol was esterified at enzyme saturation.


RESULTS

Characterization of Lp2A-I Particles

Gradient gel electrophoresis of the chromatographically reisolated discoidal Lp2A-I containing different amounts of cholesterol reveals single homogeneous profiles with sizes ranging from 9.7 to 10.2 nm ( Fig. 1and Table 1). In contrast to previous studies(30) , no smaller secondary band (9.8 nm) is evident in the discoidal preparations. This appears to be a result of the concentration of the apoA-I stock solution prior to addition to the phospholipid/cholate dispersion. In previous experiments, apoA-I stock solutions were generally between 2 and 3 mg/ml. In developmental characterizations we have found that using apoA-I at lower stock concentration (1-1.5 mg/ml) results in improved homogeneity of the final complexes, in particular when particles contain cholesterol. Lp2A-I prepared with lower apoA-I stock concentrations appear slightly smaller (0.2-0.5 nm) than particles prepared previously. An increase in cholesterol content gives rise to increases in particle size and is associated with the appearance of a faint secondary band at 7.5 nm and also with an increased intensity of the band at the buffer front, free apoA-I (Fig. 1). In contrast, increasing the cholesterol content in core-containing Lp2A-I has only a small effect on particle size (Table 1). All sonicated preparations exhibit a single main band on nondenaturing gradient gels with a mean size ranging from 7.3 to 7.5 nm and a small band corresponding to free apoA-I ( Fig. 1and Table 1). Since previous studies have shown that is not possible to obtain homogeneous discoidal particles with greater than 30 molecules of cholesterol/particle(30) , only discoidal and core-containing Lp2A-I containing less than 30 molecules of cholesterol were characterized for structure and LCAT activation. Structural analysis after reisolation showed both discoidal and core-containing Lp2A-I to have similar lipid compositions as previously reported (22, 30) and to contain 2 molecules of apoA-I/particle (Table 1). Consistent with previous studies(30) , this investigation has also shown that increasing discoidal particle cholesterol content gives rise to increases in particle negative surface charge. Increasing the cholesterol content of a discoidal Lp2A-I complex from 4 to 26 molecules is associated with significant increases in the particle negative surface potential (Table 1). Similarly, increasing the cholesterol content of core-containing Lp2A-I from 4 to 18 molecules/particle also seems to be associated with a small increase in the particle negative surface potential, from -10.7 to -11.0 mV.


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) .





Effect of Discoidal Lp2A-I-Cholesterol Content on LCAT Activity

The effect of increasing the cholesterol content in Lp2A-I particles on cholesterol esterification by LCAT has been investigated in incubations with the purified human enzyme. Fig. 2A shows that increasing Lp2A-I cholesterol content corresponds to significant increases in the rate of esterification of complex cholesterol by LCAT. Decreasing phospholipid/cholesterol molar ratios from 44 to 7 are associated with an almost 4-fold increase in initial velocity at maximum substrate concentrations. This result is in agreement with observations from experiments with phosphatidylcholine vesicles (9, 10) and also with discoidal Lp2A-I particles(11) . In this latter study, the investigators also observed an increase in initial velocity as the cholesterol content was increased from 2 to 20 molecules/particle, but observed no further increase in esterification rates when the cholesterol content was increased over 20 molecules.


Figure 2: The effect of discoidal Lp2A-I composition on the rate of cholesterol esterification by LCAT. PanelA, the esterification of [^3H]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 [^3H]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(max)) 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(max)) 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(m)) is similar for the four particles containing various amounts of cholesterol. In contrast, the apparent LCAT K(m) for cholesterol increases almost 4-fold as Lp2A-I cholesterol content increases. As such, the catalytic efficiency of LCAT (V(max)/K(m)) for cholesterol falls with increasing Lp2A-I cholesterol content, while the V(max)/K(m) 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: (circle) 178:4:2; (bullet) 188:8:2; (up triangle) 182:18:2; and () 182:26:2.





Effect of Core-containing Lp2A-I-Cholesterol Content on LCAT Activity

The effect of increasing the cholesterol content in Lp2A-I particles containing a neutral lipid (triolein) core on LCAT activation has also been investigated. Fig. 4shows that increasing core-containing Lp2A-I cholesterol content corresponds to significant increases in the rate of esterification of complex cholesterol by LCAT. Decreasing phospholipid/cholesterol molar ratios from 17 to 4 is associated with a significant increase in initial velocity. Lineweaver-Burk kinetic analysis of this data (Fig. 4, inset) shows that the increase in initial velocities corresponds to an increase in the V(max) for LCAT of from 2.8 to 7.7 nmol of cholesteryl ester/h/unit LCAT (Table 2). As with the discoidal Lp2A-I, it is evident that the concentration of phospholipid (or apoA-I) required for a half-maximal velocity (K(m)) is also similar for the three different core-containing particles, while the apparent LCAT K(m) for cholesterol increases over 4-fold as the core-containing Lp2A-I cholesterol content increases (Table 2).


Figure 4: Effect of the cholesterol content of core-containing Lp2A-I on LCAT activation. The esterification of [^3H]cholesterol by LCAT in different core-containing Lp2A-I particles having the following phospholipid/triolein/cholesterol/apoA-I stoichiometries: (bullet) 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).



Effect of HDL(3)-Cholesterol Content on LCAT Activity

In an attempt to explore the effect of cholesterol on LCAT reactivity with native lipoprotein substrates, incubations were undertaken with native HDL(3) that was enriched in cholesterol. The sonication method utilized to incorporate radioactive cholesterol into HDL(3) had no effect on the charge, size, and size distribution of the lipoprotein particles. After incubation with cholesterol-loaded Celite, the cholesterol content of HDL(3) was increased from 4.2 to 6.0% by weight relative to a control preparation incubated with cholesterol-free Celite. While no other significant changes in the lipid or protein composition or in nondenaturing gradient gel profiles were evident between the two preparations of HDL(3), significant differences in surface charge were identified. Increasing the cholesterol content of HDL(3) by 1.5-fold is associated with an increase in particle negative surface potential from -11.0 to -11.7 mV (Table 3). This is consistent with the changes in surface potential observed in both core-containing and discoidal Lp2A-I with increasing cholesterol content.



Incubations of both control and cholesterol-enriched HDL(3) with LCAT show that cholesterol enrichment has a significant effect on LCAT activity. Fig. 5shows that a 1.5-fold increase in HDL(3) 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(max) for LCAT from 0.5 to 3.6 nmol cholesteryl ester/h and a smaller increase in apparent K(m) from 7.3 to 8.5 µM cholesterol (Table 3). Increasing the HDL(3) cholesterol content from approximately 12 to 21 molecules/particle is therefore associated with a 6-fold increase in the catalytic efficiency (V(max)/K(m)) of LCAT.


Figure 5: Effect of HDL(3)-cholesterol content on LCAT activation. The esterification of [^3H]cholesterol in native HDL particles by LCAT is shown. The cholesterol content of native HDL(3) particles was increased from 12 () to 21 () molecules/particle as described in the text, and then the enriched particles were compared with control HDL(3) 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).




DISCUSSION

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(3) 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(m) 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(max)/K(m) 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(max) values. Since we have observed similar V(max) values for core-containing and discoidal complexes, it appears that the substantially different V(max) 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(m) values for apoA-I may closely reflect the interfacial binding affinity of LCAT to an LpA-I particle. Since K(m) 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(m) cholesterol. As a consequence, the overall catalytic efficiency of LCAT (V(max)/K(m)) for cholesterol falls with increasing Lp2A-I cholesterol content. In contrast, K(m) phospholipid (or apoA-I) values are essentially constant even when V(max) 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(max) to apparent K apoA-I values is plotted against the cholesterol content (Table 1) of both discoidal () and core-containing (bullet, 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(m) 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. (^2)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(max) values. LCAT reaction V(max) 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(max) 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.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Canada and Grants HL22633 and HL34343 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Lipoproteins and Atherosclerosis Group, University of Ottawa Heart Inst., 1053 Carling Ave., Ottawa, Ontario K1Y 4E9 Canada.

(^1)
The abbreviations used are: LCAT, lecithin:cholesterol acyltransferase; apoA-I, apolipoprotein A-I; FC, free (unesterified) cholesterol; HDL, high density lipoproteins; Lp2A-I, HDL containing 2 molecules of apoA-I; POPC, palmitoyloleoyl phosphatidylcholine.

(^2)
D. L. Sparks, unpublished observations.


ACKNOWLEDGEMENTS

We thank Hilary Kinnear, Tracey Neville, Margaret Nickel, Faye Baldwin, and Dr. Quiang-Hua Meng for expert technical assistance.


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