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J. Biol. Chem., Vol. 275, Issue 48, 37504-37509, December 1, 2000

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Human Apolipoprotein C-I Accounts for the Ability of Plasma High Density Lipoproteins to Inhibit the Cholesteryl Ester Transfer Protein Activity^*

Thomas Gautier, David Masson, Jean-Paul Pais de Barros, Anne Athias, Philippe Gambert, Dominique Aunis^§, Marie-Hélène Metz-Boutigue^§, and Laurent Lagrost^¶

From the  Laboratoire de Biochimie des Lipoprotéines-INSERM U498, Hôpital du Bocage, BP1542, 21034 Dijon Cedex, France and the ^§ Laboratoire de Biologie de la Communication Cellulaire-INSERM U338, Centre de Neurochimie du CNRS, 67084 Strasbourg Cedex, France

Received for publication, August 9, 2000, and in revised form, September 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The aim of the present study was to identify the protein that accounts for the cholesteryl ester transfer protein (CETP)-inhibitory activity that is specifically associated with human plasma high density lipoproteins (HDL). To this end, human HDL apolipoproteins were fractionated by preparative polyacrylamide gradient gel electrophoresis, and 30 distinct protein fractions with molecular masses ranging from 80 down to 2 kDa were tested for their ability to inhibit CETP activity. One single apolipoprotein fraction was able to completely inhibit CETP activity. The N-terminal sequence of the 6-kDa protein inhibitor matched the N-terminal sequence of human apoC-I, the inhibition was completely blocked by specific anti-apolipoprotein C-I antibodies, and mass spectrometry analysis confirmed the identity of the isolated inhibitor with full-length human apoC-I. Pure apoC-I was able to abolish CETP activity in a concentration-dependent manner and with a high efficiency (IC₅₀ = 100 nmol/liter). The inhibitory potency of total delipidated HDL apolipoproteins completely disappeared after a treatment with anti-apolipoprotein C-I antibodies, and the apoC-I deprivation of native plasma HDL by immunoaffinity chromatography produced a mean 43% rise in cholesteryl ester transfer rates. The main localization of apoC-I in HDL and not in low density lipoprotein in normolipidemic plasma provides further support for the specific property of HDL in inhibiting CETP activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cholesteryl ester transfer protein (CETP),¹ a member of the lipid transfer/lipopolysaccharide-binding protein family promotes the exchange of neutral lipid species, i.e. cholesteryl esters and triglycerides, between plasma lipoproteins. In addition to the concentration of CETP in the plasma compartment, a number of other parameters, including the concentration and composition of lipoprotein substrates, can affect the velocity and the extent of the CETP-mediated neutral lipid transfer reaction. In earlier kinetic studies, Barter and Jones (1) and Ihm and colleagues (2) reported that the interactions of CETP with low density lipoproteins (LDL) versus high density lipoproteins (HDL) markedly differ. Hence, at a constant amount of HDL, the rate of the CETP-mediated cholesteryl ester exchange rises with increasing amounts of LDL until a maximal constant value is reached with high LDL to HDL cholesteryl ester ratios. Although at a fixed amount of LDL the rate of cholesteryl ester exchange also tended to increase with moderate amounts of HDL, a potent and significant inhibition of the CETP-mediated lipid transfer process was observed with higher HDL/LDL cholesterol ratios (1, 2). Initially, the inhibitory effect of HDL has been explained in terms of a greater interaction of CETP with HDL than with LDL, favoring the HDL-HDL exchanges over the LDL-HDL exchanges (1, 2). More recently, purified HDL apolipoproteins were shown to modulate the cholesteryl ester transfer reaction, and in particular apoA-I and apoA-II; i.e. the two major HDL apolipoproteins were alternatively described as neutral, inhibitory, or activating factors of the cholesteryl ester transfer process (3-8). In fact, the quest for a CETP inhibitor in plasma HDL over the last decade led to inconsistent observations, and no clear identification and characterization of an inhibitory protein in human HDL has been provided so far. Comparative studies of the ability of HDL from control mice and transgenic mice expressing human HDL apolipoproteins revealed that the lipid transfer-inhibitory activity associated with control mouse HDL can be completely lost as the result of the apoA-I and apoA-II overexpression in the transgenic mouse models (9). It appears therefore that neither apoA-I nor apoA-II can account for the strong inhibitory activity associated with plasma HDL, and observations in transgenic animals strongly support the existence of a distinct inhibitor protein in the HDL fraction. Recently, Wang et al. (10) identified human apoF as a lipid transfer inhibitor protein. It is noteworthy, however, that apoF is not an HDL apolipoprotein, and no direct evidence for the blockade of lipid transfer-inhibitory activity in HDL with anti-apoF antibodies was provided (10).

Given that the putative lipid transfer inhibitors that were previously proposed are rather heterogenous in terms of molecular mass (ranging from 3 up to 41 kDa (11-16)), the entire spectrum of human HDL apolipoproteins was explored in the present study. This exhaustive experimental approach led to the preparation of up to 30 distinct protein fractions with molecular masses ranging from 2 up to 80 kDa. Among all of the isolated proteins, one single candidate was shown to account for most of the lipid transfer-inhibitory activity that is associated with human plasma HDL. The present work reports the purification and the characterization of the lipid transfer-inhibitory protein in human plasma HDL, providing a new explanation for the known concentration-dependent ability of HDL to inhibit the CETP-mediated lipid transfer process.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plasma Samples

Fresh citrated plasmas from normolipidemic subjects were provided by the Etablissement de Transfusion Sanguine (Hôpital du Bocage, Dijon, France).

Antibodies

The anti-human apolipoprotein C-I antiserum from goats was purchased from Europa Research Products. Affinity-purified anti-human apoC-I immunoglobulins from goats were purchased from Biodesign International. Affinity-purified anti-human apoC-III immunoglobulins were purchased from Rockland.

Isolation of LDL and HDL Particles

LDL were ultracentrifugally isolated from normolipidemic human plasmas as the 1.019 < d < 1.063 g/ml fraction, with one 17-h, 50,000-rpm spin at the lowest density and one 24-h, 50,000-rpm spin at the highest density in a 70.Ti rotor in an L90-K ultracentrifuge (Beckman). HDL were ultracentrifugally isolated from normolipidemic human plasmas as the 1.070 < d < 1.210 g/ml fraction, with one 24-h, 45,000-rpm spin at the lowest density and one 24-h, 50,000-rpm spin at the highest density in a 70.Ti rotor in a L90-K ultracentrifuge. Densities were adjusted by the addition of solid KBr. The isolated lipoproteins were dialyzed overnight against a 10 mmol/liter Tris, 150 mmol/liter NaCl, 3 mmol/liter NaN₃, pH 7.4, buffer (TBS buffer).

Measurement of Cholesteryl Ester Transfer Activity

Cholesteryl ester transfer activity was determined by quantitating the transfer of radiolabeled cholesteryl esters from [³H]CE-LDL to unlabeled acceptor HDL or from [³H]CE-HDL₃ to unlabeled acceptor LDL as described previously (6). Human LDL and HDL were biosynthetically radiolabeled as described previously (17). A protein fraction containing purified CETP activity and devoid of lecithin:cholesterol acyltransferase and phospholipid transfer protein activities was prepared by a sequential chromatographic procedure as described previously (18). In cholesteryl ester transfer assays, donor (cholesterol, 2.50 nmol) and acceptor lipoproteins (cholesterol, 0.62-20.00 nmol) were incubated for 3 h at 37 °C in the presence of partially purified CETP (4.5 µg) in a final volume of 50 µl. Following the incubation, the d < 1.068 and the d > 1.068 g/ml fractions were separated by ultracentrifugation and transferred into counting vials containing 2 ml of scintillation fluid. The radioactivity was assayed for 2 min in a Wallac 1410 liquid scintillation counter (Amersham Pharmacia Biotech). The recovery of total radioactivity in the d < 1.068 and in the d > 1.068 g/ml fractions was greater than 95%. Cholesteryl ester transfer rates were calculated from the known specific radioactivity of the donor and the accumulation of [³H]CE in the d < 1.068 or the d > 1.068 g/ml acceptor fraction, after deduction of blank values from control mixtures that were incubated at 37 °C without CETP. Data were expressed as the amount of cholesteryl ester transferred per ml of incubation mixture per h (nmol/ml/h).

Delipidation of HDL Apolipoproteins

Ultracentrifugally isolated HDL were delipidated using ethanol/acetone (1:1, v/v) according to the general procedure of Jackson and Holdsworth (19). The aqueous solution containing delipidated apolipoproteins was dialyzed overnight against TBS buffer.

Separation of HDL Apolipoproteins by Preparative Electrophoresis

HDL particles (5 mg of protein) were incubated for 15 min at 56 °C with 3 volumes of TBS containing SDS (20 g/liter) and dithiothreitol (33 g/liter). HDL apolipoproteins were separated by SDS electrophoresis in a 100-250 g/liter polyacrylamide gradient gel. Proteins of known molecular weight were electrophoresed in an adjacent well (Low Molecular Weight Calibration Kit; Amersham Pharmacia Biotech). The electrophoresis was performed overnight in a 1 g/liter SDS, 50 mmol/liter Tris, 380 mmol/liter glycine buffer.

Electroelution of HDL Apolipoproteins from the Polyacrylamide Gradient Gel

The distinct HDL apolipoproteins were recovered from the polyacrylamide gradient gel by using the Whole Gel Eluter system (Bio-Rad), which allows proteins to migrate through the thickness of the gel by delivering a perpendicular electric current. HDL apolipoproteins were electroeluted as recommended by the manufacturer into 30 distinct narrow chambers, and they were finally recovered in a nondenaturing elution buffer (60 mmol/liter Tris, 40 mmol/liter CAPS). Each fraction was subsequently concentrated (Microsep 3K, Filtron) to a final volume of 200 µl.

Purification of ApoC-I by Chromatofocusing

Pure apoC-I was obtained by using the chromatofocusing method of Tournier et al. (20). Briefly, delipidated HDL apolipoproteins (up to 50 mg) were dialyzed against a histidine buffer (25 mmol/liter, pH 6.2), and they were applied to a 20 × 1-cm inner diameter column of Polybuffer exchanger 94 (Amersham Pharmacia Biotech) that was preequilibrated at 4 °C with the same buffer. Under these experimental conditions, all of the HDL apolipoproteins bound to the column, with the exception of apoC-I that eluted with the void volume. Finally, pure apoC-I was dialyzed against TBS buffer.

Anti-apoC-I Immunoaffinity Chromatography

Affinity-purified anti-apoC-I antibodies (Biodesign) were covalently bound to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) at a ratio of 4 mg of protein/g of gel as recommended by the manufacturer (21). The maximal binding capacity of the column approximated 0.3 mg of apoC-I. Native HDL particles (approximately 4 mg of protein) were applied at room temperature at a flow rate of 6 ml/h. Particles that did not bind the immunosorbent column were washed off with TBS buffer until the absorbance returned to base line. ApoC-I-containing HDL were subsequently eluted with a 0.1 mol/liter, pH 3.0, acetic acid solution before neutralization by Tris (1 mol/liter). In order to ensure optimal removal of apoC-I-containing particles, HDL from the same batch were passed twice through the immunoaffinity column.

Electrophoretic Analyses

Polyacrylamide Gel Electrophoresis-- Protein samples were diluted 1:4 in TBS buffer containing SDS (25 g/liter), dithiothreitol (33 g/liter), and they were incubated for 15 min at 80 °C. Samples were subsequently applied on SDS-polyacrylamide high density gels (Phastsystem; Amersham Pharmacia Biotech), and migrations were conducted as recommended by the manufacturer. Proteins were subsequently silver-stained by using Merril's method (22). Apparent molecular weights of individual protein bands were determined by reference to protein standards (Ultra Low Range Molecular Weight Markers; Sigma).

Capillary Zone Electrophoresis-- Protein samples (0.3 pg) diluted in borate buffer (50 mmol/liter, pH 9.3) containing SDS (1 g/liter) were pressure-injected in an untreated fused silica capillary (effective length, 50 cm; diameter, 50 µm) and electrophoresed at 30 kV and 25 °C (HP^3D Capillary Electrophoresis System, Hewlett Packard) in the same buffer. Absorbance was continuously monitored at 214 nm.

Western Blot Analyses

Samples were electrophoresed on SDS-polyacrylamide high density gels as described above and were further transferred to a nitrocellulose membrane using a Phast semidry electrophoretic transfer system (Amersham Pharmacia Biotech). The resulting blots were blocked overnight at 4 °C with 10% low fat dried milk in TBS containing 0.1% Tween, and washed with TBS-Tween. The blots were developed by successive incubations with affinity-purified anti-apoC-I antibodies (Biodesign) and with peroxidase-conjugated anti-goat antibodies (Sigma) as described previously (9). Blots were finally developed using an ECL kit (Amersham Pharmacia Biotech).

Amino Acid Sequencing

The N-terminal sequence of protein samples was determined by automatic Edman degradation on an Applied Biosystems 473A microsequencer. Samples purified by high pressure liquid chromatography were loaded on Polybrene-treated and precycled glass fiber filters (23). Phenylthiohydantoin-derivatives were identified by chromatography on a phenylthiohydantoin C18 column (2.1 × 200 mm).

Protein Mass Spectrometry Analyses

Protein mass spectrometry analyses were carried out by using either a MALDI-TOF-MS or a single quadrupole mass detector. In the former case, determination of mass was carried out as described previously (24) on a Brucker Biflex MALDI-TOF-MS equipped with SCOUT High Resolution Optics with X-Y multisample probe, a gridless reflector, and the HIMAS linear detector. In the second case, protein samples (0.3 mg/ml) were diluted in a water/formic acid solution (99:1, v/v). Mass spectrometry was performed on an MSD 1100 (Hewlett-Packard) single quadrupole mass detector using the positive electrospray ionization mode, and injections were carried out as follows: flow injection analysis mode at 100 µl/min; capillary voltage, 4000 V; capillary exit voltage, 150 V; nitrogen drying gas flow, 8 liters/min, 325 °C.

Protein and Lipid Analyses

All chemical assays were performed on a Cobas-Fara Centrifugal Analyzer (Hoffmann-La Roche). Total cholesterol was measured by an enzymatic method using Roche Molecular Biochemicals reagents. Protein concentration was measured using bicinchoninic acid reagent (Pierce) according to Smith et al. (25).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Differential Effects of HDL and LDL on CETP Activity-- Mixtures containing purified human CETP and various amounts of isolated plasma LDL and HDL were incubated for 3 h at 37 °C. As shown in Fig. 1A, the rate of transfer of radiolabeled cholesteryl esters from a constant amount of [³H]CE-HDL₃ (cholesterol, 50 nmol/ml) to LDL increased gradually as the LDL cholesterol levels rose from 12.5 up to 400 nmol/ml. At a constant amount of [³H]CE-LDL (cholesterol, 50 nmol/ml), a rise in cholesteryl ester transfer could be observed only with low amounts of unlabeled HDL acceptors, not exceeding 25 nmol/ml HDL cholesterol. In direct contrast with LDL, and for HDL cholesterol levels greater than 25 nmol/ml, cholesteryl ester transfers were reduced in a concentration-dependent manner (Fig. 1B). An approximately 75% inhibition of CETP activity was reached with the highest HDL cholesterol dose (400 nmol/ml) as compared with the maximal cholesteryl ester transfer rate measured with the optimal 25 nmol/ml cholesterol dose (Fig. 1B).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Differential CETP-inhibitory properties of human plasma LDL and HDL. Mixtures contained a constant amount of radiolabeled [3H]CE-HDL donors (cholesterol, 50 nmol/ml) and increasing amounts of LDL acceptors (cholesterol, 12.5-400 nmol/ml) (Fig. 1A) or a constant amount of [3H]CE-LDL donors (cholesterol, 50 nmol/ml) and increasing amounts of HDL acceptors (cholesterol, 12.5-400 nmol/ml) (Fig. 1B). Mixtures were incubated for 3 h at 37 °C in the presence of partially purified CETP (4.5 µg) in a final volume of 50 µl. Cholesteryl ester transfer rates were determined as described under "Materials and Methods." Each point represents the mean ± S.D. of triplicate determinations.

Isolation and Characterization of a Lipid Transfer Inhibitor from Human Plasma HDL-- Total HDL apolipoproteins from normolipidemic human plasma were separated on a polyacrylamide gradient gel, and up to 30 distinct protein fractions, with mean molecular masses ranging from approximately 80 down to 2 kDa, were finally obtained after electroelution in CAPS buffer (see "Materials and Methods"). Individual protein fractions were then analyzed for their ability to inhibit the CETP-mediated cholesteryl ester transfer reaction as measured from [³H]CE-LDL toward HDL. As shown in Fig. 2, several fractions displayed a substantial inhibitory activity, with fraction 26 showing the strongest inhibition. The nearly complete blockade of the lipid transfer process that was obtained with fraction 26 contrasted clearly with the discrete, approximately 25% inhibition that was observed with the same volume of other protein fractions of larger size (Fig. 2). Fraction 26 was then further purified and concentrated through a second passage on the preparative electrophoresis apparatus. The analysis of the protein composition of fraction 26 by denaturating gradient gel electrophoresis revealed the presence of a major protein component with an apparent molecular mass that was slightly lower than 6.5 kDa (Fig. 2). After automatic Edman degradation, a predominant sequence of 26 residues (XPDVSSALDKLKQFGNTLEDKARELI) was determined, together with a minor sequence resulting from a proteolytic cleavage between P2 and D3. Comparison with sequences included in the SWISS PROT Data Bank was performed using the ClustalV multiple alignment program (26). Thus, the deduced sequence was identified as the N-terminal fragment of the processed apolipoprotein C-I (P 02654; Ref. 27). MALDI-TOF-MS indicated an experimental value of 6627, corresponding to the m/z ratio of processed apoC-I. An N-terminal threonine residue could not be identified, and a glutamine residue in position 13 took the place of a glutamic acid. The inhibitory potency of fraction 26 was completely blocked with specific anti-apoC-I antibodies (Fig. 3). In addition, it is noteworthy that cholesteryl ester transfer activity in control mixtures was even further increased in the presence of anti-apoC-I antibodies, with an approximately 40% increment in cholesteryl ester transfer rates in anti-apoC-I-treated mixtures as compared with controls (p < 0.05; Fig. 3). In contrast to anti-apoC-I antibodies, anti-apoC-III antibodies did not alter the inhibitory effect of fraction 26 (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Determination of the ability of isolated HDL apolipoprotein fractions to inhibit CETP activity. HDL apolipoprotein fractions (numbers 6-30) were obtained by preparative electrophoresis as described under "Materials and Methods." [3H]CE-LDL (50 nmol/ml), HDL (200 nmol/ml), and partially purified CETP (4.5 µg) were incubated for 3 h at 37 °C in the absence or in the presence of a 15-µl aliquot of individual HDL apolipoprotein fraction. Inhibition of CETP activity was expressed as the percentage of reduction in the cholesteryl ester transfer rate relative to control mixtures in which the 15-µl apolipoprotein aliquot was replaced by 15 µl of CAPS buffer. Each point represents the mean ± S.D. of triplicate determinations.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of anti-apoC-I and anti-apo-C-III antibodies on cholesteryl ester transfer inhibition by fraction 26. Mixtures containing [3H]CE-LDL (cholesterol, 50 nmol/ml) and HDL (cholesterol, 200 nmol/ml) were preincubated overnight at 4 °C in the presence of fraction 26 (3.8 µg of protein; + fraction #26 samples), in the presence of anti-apoC-I antibodies (10 µg of IgG; + anti C-I samples), in the presence of anti-apoC-III antibodies (10 µg of IgG; + anti C-III samples), in the presence of both fraction 26 and anti-apoC-I antibodies (+ fraction #26 + anti C-I samples), or in the presence of both fraction 26 and anti-apoC-III antibodies (+ fraction #26 + anti C-III samples). 50-µl mixtures were supplemented with partially purified CETP (4.5 µg) and incubated for 3 h at 37 °C. Cholesteryl ester transfer rates were determined as described under "Materials and Methods," and data were expressed relative to control mixtures containing only CETP and lipoprotein substrates. Each point represents the mean ± S.D. of triplicate determinations (significantly different from control: p < 0.0001 (a), p < 0.01 (b), p < 0.05 (c); significantly different from + fraction #26: p < 0.0001 (d); Mann-Whitney test).

In complementary experiments, apolipoprotein C-I was completely purified by using an independent, chromatofocusing procedure that took advantage of the high isoelectric point of apoC-I as compared with other HDL apolipoprotein components. Purified apoC-I appeared as a homogenous band on polyacrylamide gel with the same apparent molecular weight as the main protein component of fraction 26 (Fig. 4). Its molecular mass determined by the MSD 1100 single quadrupole mass detector was 6630 kDa, and partial analysis of the N-terminal sequence matched the apoC-I sequence (results not shown). In incubation mixtures containing LDL, HDL, and purified CETP, pure apoC-I could completely block cholesteryl ester transfer activity, with an IC₅₀ value of approximately 100 nmol/liter (Fig. 5).

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Polyacrylamide gel electrophoresis and Western blot analysis of fraction 26 and purified apoC-I. Aliquots of fraction 26 and human apoC-I purified by chomatofocusing (0.1 µg of protein) were submitted to polyacrylamide gel electrophoresis prior to being either silver-stained (22) or transferred to a nitrocellulose membrane. The resulting blots were incubated successively with anti-apoC-I antibodies and peroxidase-conjugated anti-goat IgG antibodies. Finally, immunoblots were revealed by using a chemiluminescent substrate (ECL Kit; Amersham Pharmacia Biotech). A, silver-stained molecular weight markers (Sigma); B, silver-stained fraction 26; C, Western blot of fraction 26; D, silver-stained pure apoC-I; E, Western blot of pure apoC-I.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent inhibition of cholesteryl ester transfer activity by apoC-I. 50-µl mixtures containing [3H]CE-LDL (cholesterol, 50 nmol/ml), HDL (cholesterol, 200 nmol/ml) and partially purified CETP (4.5 µg) were incubated for 3 h at 37 °C in the presence of increasing amounts of purified apoC-I (0-3.7 µg/ml). Cholesteryl ester transfer rates were calculated as described under "Materials and Methods," and data were expressed as relative to control. Each point represents the mean ± S.D. of triplicate determinations.

Role of ApoC-I in Regulating the Interaction of CETP with Plasma HDL-- In order to determine the inhibitory effect of apoC-I not only as a pure apolipoprotein but also as one component of the HDL protein moiety, the effect of total delipidated HDL apolipoproteins on CETP activity was further studied. As shown in Fig. 6, total delipidated HDL apolipoproteins could markedly inhibit the rate of transfer of cholesteryl esters. The inhibition could be completely blocked by anti-apoC-I antibodies, whereas anti-apoC-III antibodies were without any effect.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of anti-apoC-I and anti-apoC-III antibodies on cholesteryl ester transfer inhibition by total HDL apolipoproteins. Mixtures containing [3H]CE-LDL (cholesterol, 50 nmol/ml) and HDL (cholesterol, 200 nmol/ml) were preincubated overnight at 4 °C in the presence of HDL apolipoproteins (200 µg of protein; + HDL apo samples), in the presence of HDL apolipoproteins and anti-apoC-I antibodies (10 µg of IgG; + HDL apo + anti C-I samples), or in the presence of HDL apolipoproteins and anti-apoC-III antibodies (10 µg of IgG; + HDL apo + anti C-III samples). 50-µl mixtures were supplemented with partially purified CETP (4.5 µg) and incubated for 3 h at 37 °C. Cholesteryl ester transfer rates were determined as described under "Materials and Methods," and data were expressed relative to control mixtures containing only CETP and lipoprotein substrates. Each point represents the mean ± S.D. of triplicate determinations (significantly different from control: p < 0.0001 (a), p < 0.001 (b); significantly different from + HDL apo: p < 0.05 (c) (Mann-Whitney test)).

Total native plasma HDL were subsequently passed through an anti-apoC-I affinity column. As a result, most of the apoC-I bound to the anti-apoC-I immunoaffinity column, and the unbound HDL fraction retained most of the protein composition of normal HDL, with apoA-I and apoA-II constituting the two major components (results not shown). The bound fraction contained mainly apoC-I, together with apoA-I that constituted the most abundant coeluted protein. Again, native plasma HDL could markedly inhibit CETP activity in a concentration-dependent manner. The inhibitory potency of HDL was strongly diminished as the result of the reduction in the apoC-I content of HDL, and a considerable rise in cholesteryl ester transfer rates was constantly observed along the HDL concentration range studied (results not shown). The removal of most of apoC-I from HDL of six distinct plasma samples was accompanied by a mean 43% increase in the rate of cholesteryl ester transfers that were measured in the presence of 200 nmol/ml HDL cholesterol (p < 0.005) (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effect of apoC-I depletion on the ability of plasma HDL to interact with CETP. Total HDL of six distinct normolipidemic plasmas were submitted to anti-apoC-I affinity chomatography as described under "Materials and Methods." For each plasma, mixtures containing [3H]CE-LDL (cholesterol, 50 nmol/ml) and either control HDL or apoC-I-poor HDL (cholesterol, 200 nmol/ml) were incubated for 3 h at 37 °C in the presence of purified CETP (4.5 µg) in a final volume of 50 µl, and cholesteryl ester transfer rates were determined as described under "Materials and Methods." Open squares represent the mean value of three determinations for one given HDL preparation. The closed circles represent the mean of the six distinct HDL samples (significance of the difference between nontreated HDL and apoC-I-depleted HDL: p < 0.005; Student's t test for paired samples).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In human plasma, one single protein, i.e. the CETP, accounts for the exchange of neutral lipid species between lipoprotein particles. CETP can interact with all the plasma lipoprotein fractions, and the velocity as well as the direction of net mass transfers are actually dependent on several parameters, including the concentration of CETP as well as the relative proportions and compositions of lipoprotein donors and acceptors (28). As shown in the present study, there is a major difference between HDL and LDL in terms of their ability to exchange cholesteryl ester molecules through the CETP-mediated lipid transfer reaction. Unlike LDL, HDL can markedly reduce the lipid transfer reaction in a concentration-dependent manner, leading to the blockade of the CETP-mediated transfer between LDL and HDL in the presence of high HDL concentrations. These observations are in good agreement with former kinetic studies that were conducted by Barter and Jones (1) with lipoprotein-free plasma as a CETP source, and by Ihm et al. (2) by using purified lipid transfer complex. Initially, the HDL-mediated inhibition of CETP activity was explained in terms of a greater interaction of CETP with HDL than with LDL, favoring HDL-HDL transfers at the expense of LDL-HDL transfers (1, 2). Although the greater affinity of CETP for HDL than for LDL comes in support of the "substrate inhibition" concept, the sequestration of CETP by HDL (29) may not constitute a relevant hypothesis to explain the inhibitory property of these particles. Indeed, recent in vitro analysis of the inhibition of CETP activity by HDL from wild-type mice and apoA-I transgenic mice did not come in support of a class effect of HDL versus LDL. Thus, CETP activity was shown to be inhibited by native plasma HDL from wild-type mice but not by native plasma HDL from apoA-I transgenic mice, despite very similar affinities of CETP for either substrate (9). The latter observations rather come in support of an alternative hypothesis according to which a specific factor that is localized in HDL and not in LDL accounts for the inhibition of CETP activity. This specific inhibitor would be displaced from the HDL surface as a result of apoA-I overexpression in transgenic animals (9).

Over the last 2 decades, a number of laboratories sought the lipid transfer inhibitor that is associated with plasma HDL. However, this quest led to inconsistent observations, with the identification of several apolipoprotein components as putative inhibitors of CETP. Among the apolipoprotein candidates, purified apoA-I, apoA-II, apoA-IV, apoE, apoCs, apoD, and apoF were alternatively described as inhibitors of CETP activity (5, 6, 10, 15, 16, 30). It is noteworthy, however, that the inhibitory potency of most of the purified apolipoproteins may not be of physiological relevance, and it might depend mainly on the experimental conditions used. For instance, in incubation mixtures containing lipoprotein donors, lipoprotein acceptors, and purified CETP, the addition of increasing amounts of pure apoA-I was demonstrated to successively activate and inhibit CETP activity, reflecting indirectly the ability of added apoA-I to reassociate or not with co-incubated lipoprotein substrates (5). Whereas the replacement of apoA-I by apoA-II in plasma HDL was shown to significantly reduce the CETP-mediated cholesteryl ester transfers, HDL particles containing only apoA-II and no apoA-I still constituted significant acceptors and donors of neutral lipids (6). In addition, no evidence for CETP inhibition could be brought in double transgenic mice expressing both human CETP and human apoA-II (31), suggesting that apoA-II may not represent a potent and specific inhibitor of CETP in vivo (31). More recently, apoF was presented as a new CETP inhibitor (10). It is noteworthy, however that apoF is bound almost exclusively to LDL, and not to HDL particles, and inhibitory activity could not be suppressed with anti-apoF antibodies (10). Given that apoF has been reported to be mainly involved in the decrease in lipid transfers between very low density lipoprotein and LDL, with the least effect on transfers involving HDL (32, 33, 10), it certainly does not account for the potent inhibitory activity that is specifically associated with HDL (Refs. 1, 2, and 13; present study).

In order to assess the molecular basis for the inhibition of CETP activity by plasma HDL, the complete apolipoprotein pattern of HDL was fractionated by preparative electrophoresis, and resulting individual fractions were explored for their inhibitory potential. The latter experimental approach met two important requisites. First, it allowed us to explore the entire HDL apolipoprotein spectrum, with apparent molecular masses of isolated proteins ranging from approximately 80 down to 2 kDa. Second, the same volumes of individual apolipoprotein fractions, reflecting their real contribution to the total HDL protein moiety, were tested concomitantly for their inhibitory potential. Substantial inhibitory activity was found in a few protein fractions; among them only one, i.e. fraction 26, could completely inhibit the cholesteryl ester transfer reaction. Molecular weight determination by mass spectrometry, amino acid sequence analysis, and immunological characterization of fraction 26 led to the identification of apolipoprotein C-I as the potent inhibitor of CETP activity in plasma HDL. Consistent observations were made whether apoC-I was isolated by either denaturing preparative electrophoresis or chomatofocusing. Pure apolipoprotein C-I was shown to inhibit CETP activity in a concentration-dependent manner, leading to a complete blockade of the cholesteryl ester transfer reaction. As an HDL component, apoC-I was shown in the present study to account for most of the lipid transfer-inhibitory activity that is associated with these particles, and consistent observations were made when HDL were isolated from plasma of distinct normolipidemic subjects. Interestingly, earlier studies reported that approximately 85% of total functional lipid transfer-inhibitory activity in human plasma is localized in the HDL fraction (13), as is apolipoprotein C-I (34). In fact, the bulk of plasma apoC-I (approximately 80%) was shown to associate with HDL in vivo, whereas no apoC-I could be detected in LDL (34). This peculiar distribution of apoC-I provides a direct explanation for the marked discrepancy in the ability of plasma LDL and HDL to inhibit CETP (Refs. 1 and 2; present study). Interestingly, an N-terminal fragment of baboon apoC-I (residues 1-38) was previously reported to suppress CETP activity in vitro (15). However, the IC₅₀ value calculated with the baboon apoC-I fragment (approximately 100 µmol/liter) was considerably higher than the IC₅₀ value reported in the present study with full-length human apoC-I (approximately 100 nmol/liter).

Plasma apolipoprotein C-I contains 57 residues, and it has the highest isoelectric point among the HDL apolipoproteins. ApoC-I can inhibit phospholipase A2 (35) and hepatic lipase (36), and it can stimulate cell growth (20). It can also activate lecithin:cholesterol acyltransferase, however with much less efficiency than apoA-I (37, 38). Among apoCs, apoC-I was shown to be the most potent inhibitor of the apoE-mediated binding of -very low density lipoprotein to the LDL receptor and the LDL receptor-related protein (39-41). Although in vivo studies in apoC-I transgenic mice susbtantiated the former observations, studies in homozygous apoC-I-deficient mice led unexpectedly to opposite conclusions (42-44). In fact, both apoC-I overexpression and apoC-I deficiency led to markedly increased levels of atherogenic very low density lipoprotein- and LDL-like particles (42-44). These observations suggest therefore that the function of apoC-I may not be restricted to the regulation of the cellular uptake of potentially atherogenic lipoproteins. Since the present study ascribed a key role to apoC-I in regulating plasma cholesteryl ester transfer activity, previous in vivo studies in apoC-I transgenic mice and apoC-I knocked out mice may well not have been fully conclusive due to the lack of substantial levels of active CETP in this animal species (45, 46).

In conclusion, results of the present studies demonstrated that apolipoprotein C-I accounts for most of the CETP-inhibitory activity that is associated with human plasma HDL. In contrast to most other putative apolipoprotein inhibitors, apoC-I was proved to meet all the following criteria: (i) apoC-I-inhibitory activity is specifically localized in HDL, and not in LDL; (ii) it constitutes a potent inhibitor of CETP, with the exclusion of activating potential; (iii) a complete blockade of CETP activity can be reached with elevated inhibitor doses; (iv) apoC-I is active not only as an isolated protein, but also as a component of the HDL protein moiety; (v) substantial increment in cholesteryl ester transfer rates can be obtained by the addition of anti-apoC-I antibodies to incubation mixtures containing purified CETP and lipoprotein donors and acceptors; (vi) immunopurified apoC-I-free HDL interact more readily with CETP than native apoC-I-containing particles. The physiological relevance of the role of apoC-I in regulating specific activity of plasma CETP is in the scope of the present study.

	FOOTNOTES

^* This work was supported by the Université de Bourgogne, the Conseil Régional de Bourgogne, and INSERM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 33 3 80 29 38 25; Fax: 33 3 80 29 36 61; E-mail: laurent.lagrost@u-bourgogne.fr.

Published, JBC Papers in Press, September 9, 2000, DOI 10.1074/jbc.M007210

	ABBREVIATIONS

The abbreviations used are: CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein(s); LDL, low density lipoprotein(s); [³H]CE-HDL, high density lipoprotein(s) containing tritiated cholesteryl esters; [³H]CE-LDL, low density lipoprotein(s) containing tritiated cholesteryl esters; apo, apolipoprotein; TBS, Tris-buffered saline; CAPS, 3-(cyclohexylamino)-propanesulfonic acid; MALDI-TOF-MS, matrix-assisted laser desorption ionization time of flight mass spectrometer.

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