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Molecular Mechanism of the Blockade of Plasma Cholesteryl Ester Transfer Protein by Its Physiological Inhibitor Apolipoprotein CI

2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês

10.1074/jbc.m504678200

ISSN

1083-351X

Autores

Laure Dumont, Thomas Gautier, Jean-Paul Paı̈s de Barros, Hélène Laplanche, Denis Blache, Patrick Ducoroy, Jamila Fruchart, Jean‐Charles Fruchart, Philippe Gambert, David Masson, Laurent Lagrost,

Tópico(s)

Diabetes, Cardiovascular Risks, and Lipoproteins

Resumo

Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal α-helix with significant electrostratic properties, whereas its N-terminal α-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal α-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers. Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal α-helix with significant electrostratic properties, whereas its N-terminal α-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal α-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers. The cholesteryl ester transfer protein (CETP) 4The abbreviations used are: CETP, cholesteryl ester transfer protein; apo, apolipoprotein; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; CETPTg/apoCI-KO mouse, apoCI-deficient mouse expressing human CETP; NBD, nitrobenz-oxadiazol; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. 4The abbreviations used are: CETP, cholesteryl ester transfer protein; apo, apolipoprotein; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; CETPTg/apoCI-KO mouse, apoCI-deficient mouse expressing human CETP; NBD, nitrobenz-oxadiazol; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. mediates the exchange of neutral lipids, i.e. cholesteryl esters and triglycerides between plasma lipoproteins (1Bruce C. 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Posma J.L. van Tol A. Kastelein J.J. Circulation. 2002; 105: 2159-2165Crossref PubMed Scopus (452) Google Scholar, 9Brousseau M.E. Schaefer E.J. Wolfe M.L. Bloedon L.T. Digenio A.G. Clark R.W. Mancuso J.P. Rader D.J. N. Engl. J. Med. 2004; 350: 1505-1515Crossref PubMed Scopus (705) Google Scholar, 10Clark R.W. Sutfin T.A. Ruggeri R.B. Willauer A.T. Sugarman E.D. Magnus-Aryitey G. Cosgrove P.G. Sand T.M. Wester R.T. Williams J.A. Perlman M.E. Bamberger M.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 490-497Crossref PubMed Scopus (372) Google Scholar). Most interestingly, previous studies (11Boekholdt S.M. Kuivenhoven J.A. Wareham N.J. Peters R.J. Jukema J.W. Luben R. Bingham S.A. Day N.E. Kastelein J.J. Khaw K.T. Circulation. 2004; 110: 1418-1423Crossref PubMed Scopus (188) Google Scholar, 12Curb J.D. Abbott R.D. Rodriguez B.L. Masaki K. Chen R. Sharp D.S. Tall A.R. J. Lipid Res. 2004; 45: 948-953Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 13van der Steeg W.A. Kuivenhoven J.A. Klerkx A.H. Boekholdt S.M. Hovingh G.K. Kastelein J.J. Curr. Opin. Lipidol. 2004; 15: 631-636Crossref PubMed Scopus (53) Google Scholar) in human populations are consistent with the association of high CETP with an increase in coronary heart disease, in particular in subjects with elevated triglycerides.Besides interventional studies with exogenous compounds, a number of studies (14Guyard-Dangremont V. Lagrost L. Gambert P. J. Lipid Res. 1994; 35: 982-992Abstract Full Text PDF PubMed Google Scholar, 15Lagrost L. Perségol L. Lallemant C. Gambert P. J. Biol. Chem. 1994; 269: 3189-3197Abstract Full Text PDF PubMed Google Scholar, 16Kushwaha R.S. Hasan S.Q. McGill H.G. Getz G.S. Dunham R.G. Kanda P. J. Lipid Res. 1993; 34: 1285-1297Abstract Full Text PDF PubMed Google Scholar, 17Cho K.H. Lee J.Y. Choi M.S. Cho J.M. Lim J.S. Park Y.B. Biochim. Biophys. Acta. 1998; 1391: 133-144Crossref PubMed Scopus (29) Google Scholar, 18Connolly D.T. Krul E.S. Heuvelman D. Glenn K.C. Biochim. Biophys. Acta. 1996; 1304: 145-160Crossref PubMed Scopus (20) Google Scholar, 19Wang X. Driscoll D.M. Morton R.E. J. Biol. Chem. 1999; 274: 1814-1820Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) indicated that plasma CETP activity can be modulated by endogenous factors, among them the apolipoprotein components of circulating lipoproteins. Recently, apolipoprotein CI, i.e. a 6.6-kDa HDL apolipoprotein, was identified as a potent CETP inhibitor (20Gautier T. Masson M. Pais de Barros J.P. Athias A. Gambert P. Aunis D. Metz-Boutigue M.H. Lagrost L. J. Biol. Chem. 2000; 275: 37504-37509Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In contrast to other putative apolipoprotein candidates that were identified only through in vitro experiments, the ability of apoCI to decrease specific CETP activity was documented in vivo through studies in CETPTg/apoCI-KO and CETPTg/HuapoCITg (transgenic mouse to both human CETP and human apolipoprotein CI) mice (21Gautier T. Masson D. Jong M.C. Duverneuil L. Le Guern N. Deckert V. Pais de Barros J.P. Dumont L. Bataille A. Zak Z. Jiang X.C. Tall A.R. Havekes L.M. Lagrost L. J. Biol. Chem. 2002; 277: 31354-31363Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22Gautier T. Masson D. Jong M.C. Pais de Barros J.P. Duverneuil L. Le Guern N. Deckert V. Dumont L. Bataille A. Zak Z. Jiang X.C. Havekes L.M. Lagrost L. Biochem. J. 2005; 385: 189-195Crossref PubMed Scopus (17) Google Scholar). Although it was concluded that apoCI constitutes the major physiological inhibitor of CETP in the plasma compartment, the molecular mechanism of the blockade of the cholesteryl ester transfer reaction by apoCI remained to be identified. The CETP-mediated lipid transfer reaction is a complex process, with at least two rate-limiting steps. In the first step, CETP binds to lipoproteins through electrostatic interactions with negative charges localized at the lipoprotein surface (23Nishida H.I. Arai H. Nishida T. J. Biol. Chem. 1993; 268: 16352-16360Abstract Full Text PDF PubMed Google Scholar, 24Sammett D. Tall A.R. J. Biol. Chem. 1985; 260: 6687-6697Abstract Full Text PDF PubMed Google Scholar, 25Masson D. Athias A. Lagrost L. J. Lipid Res. 1996; 37: 1579-1590Abstract Full Text PDF PubMed Google Scholar). In the second step, and after a conformational change of CETP, one neutral lipid molecule binds to an hydrophobic site in the C terminus of the protein prior to be transferred to the lipoprotein acceptor (26Swenson T.L. Brocia R.W. Tall A.R. J. Biol. Chem. 1988; 263: 5150-5157Abstract Full Text PDF PubMed Google Scholar, 27Swenson T.L. Hesler C.B. Brown M.L. Quinet E. Trotta P.P. Haslanger M.F. Gaeta F.C. Marcel Y.L. Milne R.W. Tall A.R. J. Biol. Chem. 1989; 264: 14318-14326Abstract Full Text PDF PubMed Google Scholar). In concordance with the two steps of the CETP-mediated transfer reaction, at least two distinct ways of CETP blockade were reported in previous studies. First, CETP inhibition may result from either insufficient or excessive binding of CETP at the lipoprotein surface in step 1 of the lipid transfer process, and both weak CETP-lipoprotein interaction (25Masson D. Athias A. Lagrost L. J. Lipid Res. 1996; 37: 1579-1590Abstract Full Text PDF PubMed Google Scholar, 28Tall A.R. J. Lipid Res. 1986; 27: 361-367Abstract Full Text PDF PubMed Google Scholar) and strong, sometimes irreversible, CETP-lipoprotein association (10Clark R.W. Sutfin T.A. Ruggeri R.B. Willauer A.T. Sugarman E.D. Magnus-Aryitey G. Cosgrove P.G. Sand T.M. Wester R.T. Williams J.A. Perlman M.E. Bamberger M.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 490-497Crossref PubMed Scopus (372) Google Scholar, 25Masson D. Athias A. Lagrost L. J. Lipid Res. 1996; 37: 1579-1590Abstract Full Text PDF PubMed Google Scholar, 27Swenson T.L. Hesler C.B. Brown M.L. Quinet E. Trotta P.P. Haslanger M.F. Gaeta F.C. Marcel Y.L. Milne R.W. Tall A.R. J. Biol. Chem. 1989; 264: 14318-14326Abstract Full Text PDF PubMed Google Scholar) results in significant inhibition of the lipid transfer reaction. Second, CETP inhibition may result from the blockade of the neutral lipid binding site in step 2 of the lipid transfer process, resulting in an abnormal production of irreversibly associated CETP-lipoprotein complexes in this case (8de Grooth G.J. Kuivenhoven J.A. Stalenhoef A.F. de Graaf J. Zwinderman A.H. Posma J.L. van Tol A. Kastelein J.J. Circulation. 2002; 105: 2159-2165Crossref PubMed Scopus (452) Google Scholar, 27Swenson T.L. Hesler C.B. Brown M.L. Quinet E. Trotta P.P. Haslanger M.F. Gaeta F.C. Marcel Y.L. Milne R.W. Tall A.R. J. Biol. Chem. 1989; 264: 14318-14326Abstract Full Text PDF PubMed Google Scholar, 29Hope H.R. Heuvelman D. Duffin K. Smith C. Zablocki J. Schilling R. Hegde S. Lee L. Witherbee B. Baganoff M. Bruce C. Tall A.R. Krul E. Glenn K. Connolly D.T. J. Lipid Res. 2000; 41: 1604-1614Abstract Full Text Full Text PDF PubMed Google Scholar, 30Epps D.E. Greenlee K.A. Harris J.S. Thomas E.W. Castle C.K. Fisher J.F. Hozak R.R. Marschke C.K. Melchior G.W. Kézdy F.J. Biochemistry. 1995; 34: 12560-12569Crossref PubMed Scopus (29) Google Scholar).In the present study, the effect of apoCI on the lipid transfer process was determined in a systematic way. Concordant in vitro observations indicate that the inhibitory property of apoCI is in a direct link with its electrostatic charge properties and its ability to produce significant changes in CETP-lipoprotein interactions.MATERIALS AND METHODSPlasma Samples—Fresh citrated plasmas from normolipidemic subjects were provided by the Etablissement Français du Sang (Hôpital du Bocage, Dijon, France).Isolation of HDL Particles—Total 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 an L90-K ultracentrifuge (Beckman Instruments). HDL2 and HDL3 were isolated as the 1.070 < d < 1.125 g/ml and the 1.125 < d < 1.210 g/ml fractions, respectively. Isolation of HDL2 and HDL3 was conducted in an NVT-90 rotor in an L90-K ultracentrifuge (Beckman Instruments), with a 2.5-h, 90,000 rpm spin at density 1.070 g/ml, a 3-h, 90,000 rpm spin at density 1.125 g/ml, and a 3.5-h, 90,000 rpm spin at density 1.210 g/ml. Densities were adjusted by the addition of solid KBr. Isolated HDL, HDL2, and HDL3 were dialyzed overnight against 10 mmol/liter Tris-buffered saline, pH 7.4, with 3 mmol/liter NaN3 (TBS buffer).Purification of Apolipoprotein CI and CETP—ApoCI was purified from delipidated HDL apolipoproteins by using the chromatofocusing method of Tournier et al. (31Tournier J-F. Bayard F. Tauber J.-P. Biochim. Biophys. Acta. 1984; 804: 216-220Crossref PubMed Scopus (17) Google Scholar). This method takes advantage of the high isoelectric point of apoCI as compared with other HDL apolipoprotein components. Purified apoCI, which appeared as a homogeneous band on polyacrylamide gel, was dialyzed against TBS buffer. CETP was purified from human plasma by using a sequential chromatography procedure as described previously, and CETP preparation was deprived of both lecithin/cholesterol acyltransferase and phospholipid transfer protein activities (32Lagrost L. Athias A. Gambert P. Lallemant C. J. Lipid Res. 1994; 35: 825-835Abstract Full Text PDF PubMed Google Scholar, 33Guyard-Dangremont V. Lagrost L. Gambert P. Lallemant C. Clin. Chim. Acta. 1994; 231: 147-160Crossref PubMed Scopus (42) Google Scholar).Anti-apoCI Immunoaffinity Chromatography—ApoCI was removed from total human HDL by passage through an anti-apoCI immunoaffinity column as described previously (20Gautier T. Masson M. Pais de Barros J.P. Athias A. Gambert P. Aunis D. Metz-Boutigue M.H. Lagrost L. J. Biol. Chem. 2000; 275: 37504-37509Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). ApoCI-poor HDL that did not bind to the immunosorbent column were washed off with TBS buffer, and their ability to exchange cholesteryl esters was compared with total HDL.Apolipoprotein CI Acetylation—ApoCI was acetylated as described previously (34Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3178-3182Crossref PubMed Scopus (822) Google Scholar). Briefly, 100 μg of purified apoCI was incubated for 1 h at room temperature with a saturated solution of sodium acetate in the presence of increasing amounts of acetic anhydride (range, 0-21 μmol). At the end of the treatment, acetylated preparations were dialyzed overnight against TBS buffer containing 1 mmol/liter EDTA.Isoelectrophoretic Analysis—Native apoCI (5 μg) and apoCI treated with acetic anhydride (5 μg) or protein pI standards (two-dimensional SDS-PAGE; Bio-Rad) (10 μl) were diluted in 300 μl of hydration buffer (8 mol/liter urea, 4% CHAPS, 20 mmol/liter dithiothreitol, 0.2% Bio-Lyte 3-10). After an overnight hydration of 17-cm-long ReadyStrip, pH 3-10 (Bio-Rad), at 50 V in a Protean IEF cell (Bio-Rad), isoelectrofocalization was conducted for 40 kV-h. Strips were stained with Coomassie Brilliant Blue G-250 and analyzed on a GS-800 calibrated densitometer (Bio-Rad).Matrix-assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry of Native and Acetylated ApoCI—Prior to mass spectrometry analysis, native and acetylated apoCI were desalted on ZipTip microcolumns (ZipTip μC18, Millipore) as described by the manufacturer. Briefly, 1 μg of protein in 10 μl was acidified with trifluoroacetic acid (final concentration, 0.1% trifluoroacetic acid). ZipTips were pre-wetted with 10 μl of 50% acetonitrile in MilliQ grade water and equilibrated with 10 μl of 0.1% trifluoroacetic acid. Bound proteins were washed twice with 10 μl of 0.1% trifluoroacetic acid, and they were finally eluted with 4 μl of 0.1% trifluoroacetic acid, 50% acetonitrile in MilliQ grade water. One microliter of eluted protein was mixed with 4 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid (Bruker Daltonique S.A., Wissembourg, France) in a 0.1% acetonitrile/trifluoro-acetic acid (1:2, v/v) solution. Finally, 1 μl of the mixture was spotted on a MTP 384 ground steel target plate (Bruker Daltonique S.A.).The MALDI-TOF mass spectrometric measurements were performed on a Ultraflex II TOF/TOF spectrometer (Bruker Daltonique S.A.) in positive 25-kV linear mode. Insulin (Mr = 5734.56), ubiquitin I (Mr = 88565.89), cytochrome c (Mr = 12361.09), and myoglobin (Mr = 16952.55) were used as external calibration standards (protein calibration standard I, Bruker Daltonique S.A.).Measurement of Cholesteryl Ester Transfer Activity—CETP activity was determined in microplates by a fluorescent method using donor liposomes enriched with nitrobenzoxadiazol (NBD)-labeled cholesteryl esters (phospholipid/cholesterol/NBD-cholesteryl ester molar ratio, 1:1:1) (Roar Biomedical). For measurements of cholesteryl ester transfer activity with isolated CETP, donor liposomes (phospholipids, 5 μmol/liter) and purified CETP (range, 2-16 μg) were incubated in the presence of HDL (range, 0.5-4.0 μmol/liter) or LDL (7 μg of cholesterol), as indicated. For measurement of cholesteryl ester transfer activity in total plasma, incubation media contained donor liposomes (phospholipids, 5 μmol/liter) and 10 μl of plasma. Final volumes were adjusted to 250 μl with TBS (unless specified), and incubations were conducted in triplicate for 3 h at 37 °C in a Victor2 1420 multilabel counter (PerkinElmer Life Sciences). The CETP-mediated transfer of NBD-cholesteryl esters from self-quenched donors to acceptor lipoproteins was monitored by the increase in fluorescence intensity (excitation, 465 nm; emission, 535 nm), and results were expressed in fluorescence arbitrary units after deduction of blank values that were obtained with control mixtures without CETP.SDS-PAGE—HDL apolipoproteins were delipidated with ethanol/ether (3:2), diluted in the sample buffer, and applied on 4-12% NuPAGE® BisTris Novex SDS-polyacrylamide gels as recommended by the manufacturer (Invitrogen). Proteins were stained with Coomassie Brilliant Blue G-250, and the apparent molecular weights of individual protein bands were determined by reference to protein standards (Mark12, Invitrogen).Agarose Gel Electrophoresis—The electrophoretic mobility (U in the following equations) of HDL was determined by electrophoresis on 0.5% agarose gels (Paragon Lipo kit, Beckman Instruments) according to the method of Sparks and Phillips (35Sparks D.L. Phillips M.C. J. Lipid Res. 1992; 33: 123-130Abstract Full Text PDF PubMed Google Scholar). Briefly, gels were cast in a Sebia Tank K20 system, and electrophoresis was performed for 45 min at 100 V in barbital buffer, pH 8.6. After electrophoresis, the gels were successively fixed for 5 min in an ethanol/acetic acid/water 60:10:30 solution, dried, stained for 5 min in a 0.07% solution of Sudan Black B in ethanol/water 55:45, and destained for 10 min with a solution of ethanol/water 45:55. In parallel, gel portions containing purified bovine serum albumin, which was used as an internal standard, were stained with a 0.8 g/liter solution of Coomassie Brilliant Blue G-250 in a methanol/acetic acid/water 10:1:10 solution and destained in a solution of methanol/acetic acid/water 2:3:40. Mean migration distances were obtained by using the GelDoc analysis software (Bio-Rad).Calculation of Electrophoretic Mobility and Surface Potential of HDL Particles—Surface charges of HDL were determined as described previously (35Sparks D.L. Phillips M.C. J. Lipid Res. 1992; 33: 123-130Abstract Full Text PDF PubMed Google Scholar). Briefly, electrophoretic mobilities (U) were calculated by dividing the electrophoretic velocity (mean migration distance (mm) per time in seconds) by the electrophoretic potential (voltage per gel distance in centimeters). To correct the pI-dependent retardation effects, the Equation 1 was applied (35Sparks D.L. Phillips M.C. J. Lipid Res. 1992; 33: 123-130Abstract Full Text PDF PubMed Google Scholar), U{\\ }corrected=(U{\\ }agarose)/1.211 The surface potentials of HDL were calculated by using the Henry's Equation 2 (see Ref. 35Sparks D.L. Phillips M.C. J. Lipid Res. 1992; 33: 123-130Abstract Full Text PDF PubMed Google Scholar), S=U×6πn/D where n is the coefficient of viscosity (0.0089 poise), and D is the solvent dielectric constant.Formation of CETP-Lipoprotein Complexes—Ultracentrifugally isolated HDL were covalently bound to CNBr-activated Sepharose 4B (Amersham Biosciences) at a ratio of 10 mg of HDL proteins per g of gel as recommended by the manufacturer. The HDL-Sepharose phase was resuspended in PBS containing 3 mmol/liter NaN3. Each incubation mixture contained 100 μl of the HDL-Sepharose suspension in PBS, corresponding to 70 μg of HDL cholesterol. In a first step, purified CETP (final concentration, 3.75 mg/liter) was bound to the HDL-Sepharose suspension during a 1-h incubation at room temperature under mild agitation. At the end of the incubation period, the HDL-Sepharose phase with bound CETP was resuspended in 200 μl of PBS, which contained increasing amounts of either purified apoCI, amino acids 4-25 N-terminal apoCI fragment, or amino acids 34-54, C-terminal apoCI fragment (concentration range, 0-2 μmol/liter). Mixtures were incubated for 1 h at room temperature under mild agitation, and supernatants containing unbound CETP that was released from the HDL-Sepharose phase were finally recovered after a gentle, 1-min low speed centrifugation. The amount of CETP released in the supernatant was determined by a specific immunoassay with TP1 anti-CETP monoclonal antibodies. Briefly, proteins in the incubation supernatants were applied to a nitrocellulose membrane (Hybond ECL, Amersham Biosciences) by using a dot blot vacuum system (Bio-Rad). The resulting blots were blocked for 30 min at 37 °C in 3% low fat dried milk in Tris-buffered saline containing 0.1% Tween, and they were washed in Tris-buffered saline/Tween. CETP was revealed by successive incubations with TP1 anti-CETP antibodies (Heart Institute, Ottawa, Canada) and horseradish peroxidase-coupled second antibodies as described previously (36Masson D. Duverger N. Emmanuel F. Lagrost L. J. Biol. Chem. 1997; 272: 24287-24293Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Blots were finally developed with an ECL kit (Amersham Biosciences). Band intensities were measured by using the GelDoc analysis software (Bio-Rad). The amount of CETP in each supernatant was determined by comparison with a calibration curve that was obtained with known amounts of purified CETP that were applied to the membrane together with the samples.Lipid and Protein Analyses—All assays were performed on a Victor2 1420 multilabel counter (PerkinElmer Life Sciences). Total cholesterol was measured by the enzymatic method using the Cholesterol 100 reagent (ABX Diagnostics). Phospholipids and triglycerides were determined by enzymatic methods, as described previously (21Gautier T. Masson D. Jong M.C. Duverneuil L. Le Guern N. Deckert V. Pais de Barros J.P. Dumont L. Bataille A. Zak Z. Jiang X.C. Tall A.R. Havekes L.M. Lagrost L. J. Biol. Chem. 2002; 277: 31354-31363Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Protein concentration was measured by using the bicinchoninic acid reagent (Pierce).Statistical Analyses—Mann-Whitney U test was used to determine the statistical significance between data means.RESULTSComparative Effects of Increasing Levels of Human HDL Versus Human CETP on the ApoCI-mediated Inhibition of the Cholesteryl Ester Transfer Reaction—When the amount of fluorescent liposome donors and HDL acceptors were kept constant in the CETP activity assay, the cholesteryl ester transfer rate measured over a 3-h incubation period increased gradually with the amount of purified CETP added (from 2 μg in Fig. 1A to 16 μg in Fig. 1D). In all cases, and in accordance with previous in vitro and in vivo studies (20Gautier T. Masson M. Pais de Barros J.P. Athias A. Gambert P. Aunis D. Metz-Boutigue M.H. Lagrost L. J. Biol. Chem. 2000; 275: 37504-37509Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21Gautier T. Masson D. Jong M.C. Duverneuil L. Le Guern N. Deckert V. Pais de Barros J.P. Dumont L. Bataille A. Zak Z. Jiang X.C. Tall A.R. Havekes L.M. Lagrost L. J. Biol. Chem. 2002; 277: 31354-31363Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), the addition of apoCI was accompanied by a marked inhibition of the lipid transfer reaction. The inhibitory potential of a given amount of apoCI appeared to be independent of the amount of CETP added, in all cases with a constant ∼70% reduction in cholesteryl ester transfer rate as compared with control mixtures with no apoCI added. Conversely, the inhibitory potential of purified human apoCI was markedly affected by the amount of HDL acceptors that were added to the incubation mixture, with CETP kept constant. In the latter case, the capacity of human apoCI to block the lipid transfer process, approximating 80% of inhibition in incubation mixtures with the lowest HDL levels, was completely abolished with the highest HDL concentration studied (Fig. 2). CETP inhibition by HDL is the consequence of a direct and specific property of apoCI, because apoCI-poor HDL displayed a much weaker ability to block the lipid transfer reaction in the HDL concentration range studied (Fig. 3). The apolipoprotein profiles of apoCI-poor HDL as prepared by anti-apoCI immunoaffinity chromatography and of native HDL differed only by their apoCI content (Fig. 3). Percent composition of the lipid moiety did not differ significantly between total and apoCI-poor HDL from three distinct preparations (total cholesterol %, 35.9 ± 2.3 versus 31.9 ± 1.6, respectively; triglyceride %, 8.2 ± 0.5 versus 9.9 ± 1.1, respectively; and phospholipid %, 55.9 ± 2.4 versus 58.1 ± 0.9, respectively). Overall, the results showed that the capacity of apoCI to block the lipid transfer reaction was independent of the amount of active CETP, but it was tightly dependent on the amount of HDL acceptors in the incubation mixtures.FIGURE 2Effect of HDL concentrations on the inhibitory potential of apoCI. Top panel, cholesteryl ester transfer activity was determined as the rate of transfer of fluorescent NBD-cholesteryl esters from labeled liposome donors (phospholipids, 5 μmol/liter) to HDL acceptors (range, 0.5-4.0 μmol/liter) (A-D) in the presence of purified CETP (8 μg) in a final volume of 200 μl. Incubations were conducted for 3 h at 37 °C in the absence or in the presence of purified apoCI (concentration, 0.5 μmol/liter). Bottom panel, percentage of CETP inhibition (vertical bars A-D) was calculated by comparing the initial transfer rate in the presence of apoCI to the initial transfer rate with no apoCI added (A-D in top panel). Initial transfer rates were determined from the linear, initial portion of the time course curves. Plotted values and vertical bars are the mean ± S.D. of three determinations. *, significantly different from mixtures containing 0.5 μmol/liter of HDL cholesterol, p < 0.05; Mann-Whitney test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Effect of apoCI depletion of HDL on the concentration-dependent inhibition of CETP activity. ApoCI-poor HDL were prepared by anti-apoCI immunoaffinity chromatography; apolipoproteins were analyzed on an SDS-polyacrylamide gradient gel, and cholesteryl ester transfer activity was determined by the fluorescent assay as described under “Materials and Methods.” The extent

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