Inhibition of cholesteryl ester transfer protein by apolipoproteins, lipopolysaccharides, and cholesteryl sulfate

B I ( K ' H I M I C A ET B I O P H Y S I C A AC'['A

ELSEVIER

BBI I Biochimica et Biophysica Acta 1304 (1996) 145-160

Inhibition of cholesteryl ester transfer protein by apolipoproteins, lipopolysaccharides, and cholesteryl sulfate Daniel T. Connolly *, Elaine S. Krul, Deborah Heuvelman, Kevin C. Glenn Cardiovascular Diseases Research Department, Searle, 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA

Received 7 February 1996; accepted 27 June 1996

Abstract Cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl esters and triglycerides between lipoproteins in the plasma. In studies dealing with the mechanism of CETP-mediated lipid transfer, we have examined the effects of several classes of biomolecules, including apolipoproteins and related synthetic peptides, cholesteryl sulfate, and lipopolysaccharides. In all cases, the molecules were inhibitory and their effects were associated with modifications of either HDL, LDL, or both. However, the probable mechanisms were distinct for each class of inhibitor. Inhibition of lipid transfer activity by apolipoprotein A-I was correlated with an increase in the apolipoprotein A-I content of HDL but not LDL, whereas the primary effect of cholesteryl sulfate was associated with modification of LDL, and only modest alteration of HDL. Lipopolysaccharides were found to modify the size and charge properties of both LDL and HDL over the same concentration ranges that affected CETP activity, but might also interact directly with CETP. It is suggested from the present studies that a variety of biomolecules that can interact with lipoproteins under natural or pathological situations have the potential to modify CETP activity, which in turn could affect normal lipoprotein composition and distribution. Keywords: Cholesteryl ester transfer protein; Lipid transfer protein; Apolipoprotein; Amphipathic helix; Cholesteryl sulfate; Recessive X-linked ichthyosis; Sperm acrosome; Lipopolysaccharide

1. Introduction

Abbreviations: CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein; LDL, low density lipoprotein; LPS, lipopolysaccharide; 18A, peptide 18A; 18R, peptide 18R; 18AProI8A, peptide 18AProI8A; CE, cholesteryl ester; TG, triglyceride; GGE, gradient gel electrophoresis; Tes, Tris/EDTA/saline buffer; CS, cholesteryl sulfate; CG, cholesteryl /~-o-glucuronide. * Corresponding author. Fax: + I (314) 6948462.

The m o v e m e n t o f neutral lipids and phospholipids b e t w e e n lipoprotein particles is mediated by cholesteryl ester transfer protein, a 70 kDa glycoprotein found in plasma [1]. The CETP-mediated exchange o f cholesteryl ester (CE) and triglyceride (TG) is thought to be important in determining plasma levels and composition o f LDL, V L D L and HDL. Although it has been suggested that the mechanism o f lipid transfer by C E T P might involve ternary

0005-2760/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0005-2760(96)001 15-4

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D. T. Connolly et al. / Biochimica et Biophysica A cta 1304 (1996) 145-160

complex formation with lipoprotein particles [2], there is substantial evidence indicating that CETP mediates the transfer of lipid between lipoprotein particles by a carrier or 'ping-pong' mechanism. According to this model, which is analogous to a bimolecular group transfer enzyme reaction, the respective donor and acceptor lipoprotein particles can be regarded as substrates for CETP. This model is supported by binding studies [3,4], kinetics [4], the precise stoichiometry of CE and TG exchange [5], the specificity for certain lipids [6-9], preferential inhibition of either CE or TG by monoclonal antibody [10] or other inhibitors [9], and physical studies with lipid interfaces [11]. It is therefore likely that CETP-mediated lipid transfer involves at least three distinct events that are not very well characterized at present, including recognition of the lipoprotein particles by CETP [12], recognition and extraction of specific lipid substrates from within the lipoprotein particles by CETP [6-9], and dissociation or exchange of lipid from CETP to acceptor lipoprotein particles. The present studies with CETP were undertaken to test the effects of various biomolecules that are known to interact with lipoproteins under normal or pathological conditions. It was hypothesized that modifications of the lipoprotein substrates would alter CETPmediated lipid transfer. Apolipoproteins were studied because they are natural constituents of lipoprotein particles and have been observed to influence CETP activity [13-17]. Model peptides that have previously been reported to mimic natural apolipoprotein function [18,19] were also studied, and like the intact apolipoproteins, are shown to inhibit CETP activity. We also show that the amphipathic lipids cholesteryl sulfate (CS), cholesteryl /3-D-glucuronide (CG) and lipopolysaccharide (LPS) are potent CETP inhibitors. Low concentrations of (CS) are found normally in certain tissues and in blood [20]. However, in the human aryl sulfatase deficiency disease known as recessive X-linked ichthyosis, CS becomes highly elevated, particularly in association with LDL [21,22]. Also, both CS and CETP have been independently implicated in sperm capacitation [23-26]. Since CS is known to alter lipid physicochemical environments, it could potentially influence CETP activity. Likewise, LPS was studied here because of potential interactions with lipoprotein particles during sepsis [27-31] and because CETP shares structural homology with

lipopolysaccharide binding protein and with bactericidal permeability enhancing protein [32,33].

2. Materials and methods

2.1. LDL, [3H]CE-labeled HDL, and CETP assay CETP was obtained from the serum-free conditioned medium of CHO cells transfected with a cDNA for CETP (generously provided by Dr. Alan Tall, Columbia University). CETP activity was determined by three different methods. A differential precipitation assay was used to measure the rate of [3H]CE transfer from HDL to LDL as previously described [34]. Briefly, in a volume of 200 /zl, CETP, [3H]CElabeled HDL, LDL, and TES assay buffer (50 mM Tris, pH 7.4; 150 mM NaC1; 2 mM EDTA; 1% bovine serum albumin) were incubated for 2 h at 37°C in 96-well filter plates. LDL was then differentially precipitated by the addition of 50 /_tl of 1% ( w / v ) dextran sulfate/0.5 M MgC12. After filtration, the radioactivity present in the precipitated LDL was measured by liquid scintillation counting. Correction for non-specific transfer or precipitation was made by including samples that did not contain CETP. As previously shown [4,34], the rate of [3H]CE transfer using this assay was linear with respect to time and CETP concentration. For experiments in which inhibitors were included in the assay, the order of addition into sample wells was: buffer, [3H]CElabeled HDL, LDL, inhibitor, CETP. CETP activity was also measured using two methods that did not involve differential precipitation. In the first assay, the incubation conditions were identical to those described above, but separation of LDL acceptor particles from [3H]CE-labeled HDL donor particles was accomplished by size exclusion chromatography on tandem columns of Superose 6, followed by liquid scintillation counting of fractions to determine the amount of [3H]CE associated with LDL and HDL. The amount of transfer measured by this method was in excellent agreement with the precipitation assay. The third assay for CETP activity measured the rate of CETP-mediated transfer of the fluorescent analog NBD-cholesteryl linoleate (NBD-CE) from an egg phosphatidyl choline emulsion to VLDL. This

D.T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160

assay takes advantage of the fact that NBD-CE is self-quenched when in the emulsion, and becomes fluorescent when transferred to VLDL. The assay was carried out according to the manufacturer's instructions (Diagnescent Technologies, Yonkers, NY, USA). Fluorescence measurements were taken using an SLM 8000C spectrophotofluorometer (Milton Roy, Rochester, NY, USA) using 465 nm and 535 nm for excitation and emission wavelengths, respectively.

2.2. Apolipoproteins, anti-apolipoprotein antibodies and synthetic peptides Human apolipoproteins A-I, A-II, C-I, and C-III were purchased from Cappel Research Products, Durham, NC, USA. Human apo D and anti-apo D antiserum were the kind gifts of Dr. William Dilley (Department of Surgery, Washington University School of Medicine). Rabbit anti-human apolipoprotein antisera, characterized by double immunodiffusion a n d / o r Western blotting, were generously provided by Dr. Gustav Schonfeld, Lipid Research Center, Washington University School of Medicine. IgGs were purified by protein A affinity chromatography. Peptides 18A, 18A-Pro-18A, and 18R [35] were synthesized by solid state methods at Research Genetics (Huntsville, AL, USA) and purified in our laboratory by HPLC. Structures were confirmed by amino-acid analysis and mass spectroscopy. Melittin [36], cecropin A [37], magainin 1, and magainin 2 [38] were purchased from Bachem California (Torrance, CA, USA). Baboon apo C-II_3S [39] was prepared by Genosys (Woodlands, TX, USA) using solid-phase synthesis.

2.3. ApoA-I content of HDL An ELISA assay for human apolipoprotein AI (apoA-I) was developed using commercially available reagents. Briefly, Nunc MaxiSorp TM microtiter plates were coated with sheep anti-human apoA-I (The Binding Site, Cat. No. PC085, Birmingham, UK). After blocking the plates with 3% BSA in 50 mM Tris-saline, 0.05% Tween-20, pH 8.0, standard purified human apoA-I (Organon Teknika, Cat. No. 59414, Durham, NC, USA) or samples were added to the wells in the same buffer containing 1% BSA. Wells were washed with Tris-saline, 0.05% Tween-

147

20, pH 8.0 and the amount of apoA-I bound to the first antibody was detected with mouse anti-human apoA-I (Calbiochem, Cat. No. 178472, La Jolla, CA, USA) followed by a goat anti-mouse IgG alkaline phosphatase conjugate (Promega, Cat. No. $372B, Madison, WI, USA). Enzyme activity was quantitated after color development with p-nitrophenyl phosphate substrate and measuring absorbance at 405 nm on a Ceres 900UV900HDi plate reader (BioTek Instruments, Winooski, VT, USA). ApoA-I in the samples was quantitated by comparison to the standard curve generated with the purified apoA-I. The molar ratios of apoA-I to HDL were calculated from the concentrations of apoA-I and total cholesterol as follows. The M r for apoA-I and HDL were taken as 28016 and 175000, respectively [40]. Molar concentrations of HDL were calculated from the total cholesterol concentrations by assuming that 10.1% of the weight of HDL 3 was cholesteryl ester and unesterified cholesterol [41]. For example, for control HDL, the apoA-I concentration was 104 /xg/ml and total cholesterol was 46 /zg/ml. Therefore, the apoA-1 molar concentration was 104 × 103/28016 = 3.7 /xM and that of HDL was (46/0.101) × 103/175000 = 2.6 /xM. The apoAI / H D L ratio is thus 3.7/2.6 mol/mol, or 1.4.

2.4. LPS LPS from Escherichia coli Serotype O26:B6, Pseudomonas aeruginosa Serotype 10, Salmonella enteridis, and Serratia marcescens were used. These lyophilized samples prepared by phenolic extraction [42] and purified by column chromatography were obtained from Sigma Chemical Company (St. Louis, MO, USA). Samples were prepared one hour before the assay by dissolving the lyophilized LPS in Tes buffer (0.01 M Tris buffer, pH 7.4, 0.15 M NaC1, 1 mM ethylenediaminetetraacetic acid) containing 0.1% ( w / v ) NaN 3 and vortexing.

2.5. Agarose gel electrophoresis of LDL The test material was prepared at twice the indicated concentration in Tes prior to mixing 1:1 with purified human LDL [34]. LDL was diluted with Tes to 200 / z g / m l cholesterol prior to mixing with test material. The resulting mixture was incubated at 37°C

148

D.T. Connolly et al./ Biochimica et Biophysica Acta 1304 (1996) 145-160

for 2 h. A sample of each of the incubated mixtures (5 /zl) was applied to a lane of a Paragon ® thin-layer agarose gel (Beckman, Brea, CA, USA), and allowed to be fully absorbed into the gel (approx. 15 min at room temperature), prior to electrophoresis, fixing, and staining according to the manufacturer's instructions.

2.6. Non-denaturing polyacrylamide electrophoresis (GGE) of LDL

Tris buffer pH 7.4, 0.15 M NaC1, 1 mM ethylenediaminetetraacetic acid) containing 0.1% ( w / v ) NaN 3.

2.8. Other materials Cholesteryl 3-sulfate and cholesteryl /3-Dglucuronide were purchased from Sigma (St. Louis, MO, USA).

gradient gel

Test material was prepared at twice the indicated concentration in Tes prior to mixing with purified human LDL [34]. LDL was diluted to 1.125 m g / m l cholesterol in Tes prior to mixing with the test material. Following incubation at 37°C for 2 h, each LDL solution was mixed 1:1 with Sample Buffer containing 0.18 M Tris (pH 8.3), 0.16 M boric acid, 0.005 M EDTA, 20% ( v / v ) glycerol, 0.01% ( w / v ) Bromophenol blue. Non-denaturing polyacrylamide gradient gel electrophoresis (GGE) of LDL was performed essentially as previously described [43]. Briefly, to each lane of a 2 - 1 6 % GGE gel (Jule, New Haven, CT, USA) was added 25 /~1 of sample mixture. GGE gels were subjected to electrophoresis in Running Buffer containing 0.09 M Tris (pH 8.3), 0.08 M boric acid, 0.0026 M EDTA for 12 to 14 h at 150 V. GGE gels were subsequently stained for 1 to 2 h at room temperature with Staining Solution (40% ( v / v ) methanol, 7% ( v / v ) acetic acid, 0.025% ( w / v ) Coomassie brilliant blue R250 (Bio-Rad, Richmond, CA, USA) and then destained with multiple changes of the same buffer lacking Coomassie R250 on a rocking platform until the gel background was colorless. The stained GGE gels were incubated in water containing 20% glycerol and 10% ( v / v ) acetic acid for 1 h at room temperature prior to drying with a Novex Gel Drying Frame (Novex, San Diego, CA, USA) according the manufacturer's instructions.

2.7. FPLC size-exclusion chromatography of lipoproteins Size-exclusion chromatography of lipoproteins was performed using dual Superose 6 HR 1 0 / 3 0 (25 ml) columns (Pharmacia Biotech, Uppsala, Sweden) connected in tandem. Injection volumes were 0.5 ml, with a flow rate of 0.5 m l / m i n of Tes buffer (0.01 M

3. Results

3.1. Effect of apolipoproteins, anti-apolipoprotein antibodies, and amphipathic helix-forming synthetic peptides on CETP actic, ity Fig. l shows that exogenous apolipoproteins A-I, A-II, C-I, C-III and D were all inhibitory to CETPmediated CE transfer in vitro. The ICso values for the apolipoproteins and other peptides (see below) are summarized in Table 1. ICs0 values varied from about 3 /~g/ml for apo C-I to about 100 p~g/ml for apo D. On a molar basis, apo A-I, A-II, and C-I were approximately equipotent, with ICso values of about 0.4-0.5 p~M. Apo C-III was slightly less potent

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Fig. 1. Inhibition of CETP-mediated [3H]CE transfer from HDL to LDL by apolipoproteins. Different concentrations of apolipoprotein A-I, A-II, C-I, C-III or D were included in a [3H]CE transfer assay. All samples contained donor HDL particles labeled with [3H]CE, acceptor LDL particles and CETP. The amounts of [3H]CE transferred during the 2 h incubation were compared with control samples without exogenously added apolipoprotein. Data points represent the means of triplicate determinations. Standard deviations (not shown) were less than _+5%.

D. T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160

(ICso = 0.8 /zM), and apo D was almost an order of magnitude less potent (ICso = 3.4/xM). The lipid transfer process involves binding of CETP to lipoproteins. In order to determine whether apolipoproteins are essential for CETP binding to lipoprotein particles, antibodies against the various apolipoproteins present in HDL and LDL were tested as inhibitors of CETP-mediated lipid transfer. If apolipoprotein recognition were essential for CETP activity, then antibodies directed against apolipoproteins would be expected to inhibit CETP activity. Purified IgGs against apolipoproteins A-I, A-II, A-IV, B, C-II, C-III, D, and E were tested in the CE transfer assay at concentrations between 1 and 200 /xg/ml. None of the immunoglobulins had any effect on CETP-mediated CE transfer activity (data not shown). However, TP-2, a monoclonal IgG directed against CETP, inhibited transfer at concentrations between 5 and 50 /zg/ml, as previously described [10]. Synthetic peptides that form class A amphipathic helix structures have been reported to mimic certain apolipoprotein functions through their propensity to interact with phospholipid [18,19]. Peptide 18A is an 18 amino-acid helix-forming peptide. Peptide 18APro-18A is a dimeric form of 18A (two identical 18A sequences connected by a helix-breaking proline), and peptide 18R contains the same amino acids, but in an altered sequence that renders the peptide less effective for phospholipid interaction. Fig. 2 shows that 18A and 18A-Pro-18A were potent CETP in-

Table 1 Summary of CETP inhibition by apolipoproteins and peptides Mr

IC 50 Ixg/ml

Apo A-I Apo A-II Apo C-I Apo C-III Apo D Peptide 18a-Pro- 18A Peptide 18A Peptide 18R Baboon Apo C-I~_38 Melittin Cecropin A Magainin 1 Magainin 2

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Fig. 2. Inhibition of CETP-mediated [3H]CE transfer from HDL to LDL by amphipathic helix-forming peptides. Different concentrations of synthetic peptides were included in a [3H]CE transfer assay. All samples contained donor HDL particles labeled with [3H]CE, acceptor LDL particles and CETP. The amounts of [3H]CE transferred during the 2 h incubation were compared with control samples without added peptide. Data points represent the means of triplicate determinations. Standard deviations (not shown) were less than _+5%.

hibitors, with ICs0 values of 8 /~M and 0.4 /zM, respectively. The dimeric form of the peptide was thus 20-fold more potent on a molar basis than the monomeric peptide. Moreover, peptide 18A-Pro- 18A inhibited with the same potency as apo A-I, apo A-II and apo C-I, and was more potent than apo C-III and apo D. Peptide 18R was ineffective at inhibiting CETP-mediated lipid transfer ( < 50% inhibition at 100 /zM). ApoC-I~_38, a 38-amino-acid peptide from baboon apo C-I, has been reported to be a CETP inhibitor [39]. This peptide was relatively ineffective in the present study, with an ICso = 100 /xM. Melittin, cecropin A, magainin 1 and magainin 2 are bioactive peptides that interact with membranes via amphipathic helix motifs [36-38]. These peptides, however, did not show CETP inhibitory activity at concentrations up to 10-60 /zM (Table 1).

3.2. Effect of apolipoprotein A-I on the substrate properties of HDL and LDL In order to determine if the inhibitory effect of the apolipoproteins was due to an interaction with one of the lipoprotein components in the assay, apoA-I (0.5 mg) was pre-incubated with either [3H]CE-HDL (0.29 mg total cholesterol) or with LDL (2.6 mg total

150

D.T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160

cholesterol). These concentrations were chosen because they corresponded to the ratio of exogenous apoA-I to either H D L cholesterol or L D L cholesterol that yielded 50% inhibition in the transfer assay. After 1 hour at 37°C, the lipoproteins were re-purified by size exclusion chromatography. The elution of apoA-I treated H D L and L D L was identical to control preparations, indicating that the molecular weights of the particles were not significantly altered by the treatment. E L I S A analysis of apoA-I content yielded a ratio ( w / w ) of apoA-I:cholesterol = 2.3 for the control (mock treated) H D L preparation and 4.3 for the apoA-I treated H D L preparation. Thus, on average, the control sample contained about 1.4 mole of apoA-I per mole H D L and the treated sample contained about 2.7 mole of apoA-I per mole HDL. The incubation with apoA-I doubled the apoA-I content relative to the control HDL. In contrast to the results with HDL, negligible amounts of apoA-I became associated with L D L upon incubation with exogenous apoA-I. The ELISA values for control L D L yielded a ratio ( w / w ) of apo A-I:cholesterol = 0.00011, corresponding to approximately 1 mole of apoA-I per 338 moles LDL. The treated L D L yielded a ratio ( w / w ) of apoA-I:cholesterol = 0.00046, corresponding to about 1 mole apo A-I per 82 moles LDL. So, even though the apoA-I content of L D L was increased, only a very small proportion of the L D L particles carried apoA-I. The modified [3H]CE-HDL or L D L were then tested in the [3H]CE transfer assay as donors or acceptors, respectively. In the experiment shown in Fig. 3A, different concentrations of C E T P were used to test either control or apo A-I treated [3H]CE-HDL particles. The rate of transfer of [3H]CE to L D L was significantly less for the apoA-I-modified HDL than for either control H D L or H D L that had been pre-incubated without apoA-I and then re-isolated. In Fig. 3B, the C E T P concentrations and L D L acceptor particle concentrations were held constant, but the donor [ 3 H ] C E - H D L particle concentrations were varied. Again, at all concentrations, the H D L that had been modified with apoA-I was a poorer substrate for C E T P in comparison with the control preparation. The double-reciprocal plot shown in Fig. 3C indicates that the K m was not changed, but that the V,.... was reduced about 2-fold for the H D L that contained elevated apoA-I content. In contrast, the substrate

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Fig. 3. Comparison of transfer rates of [3H]CE from HDL modified with apoA-I vs. control HDL. Donor HDL particles modified with apoA-I were prepared by incubating [3H]CElabeled HDL and apoA-I together for 1 h at 37°C and then re-isolating the HDL by size-exclusion chromatography, as described in the text (apoA-i-treated HDL). Control HDL was also prepared in the same way, but apoA-I was omitted from the incubation (mock-treated HDL). The rates of [3H]CE transfer from HDL to LDL wer then measured. Untreated HDL was also tested as a control. (A) The amount of CETP in the assay was varied using the usual assay configuration. (B) Donor [3H]CEHDL (apoA-1 treated, B, or mock control, • ) was varied. The CETP concentration was 500 ng/ml. The amount of acceptor LDL in the assay was held constant (0.1 mg/ml cholesterol). (C) Double-reciprocal plot of the data in panel B.

D.T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160 120

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were incubated with or without apoA-I (100 / z g / m l ) for 2 h at 37°C. After chromatography, the amount of [3H]CE transferred to L D L was determined. In the absence o f apoA-I, 30% o f the [3H]CE was transferred to LDL, but in the presence o f apoA-I, only 1% of the [3H]CE was transferred, representing 97% inhibition. ApoA-I was also tested in the fluorescence quenching assay that measures the rate of transfer of fluorescent lipid substrate from a micro emulsion to V L D L acceptor. No inhibition was observed using this assay. This is consistent with the above results that suggest that the effects of apoA-I are associated with the interaction with HDL.

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Fig. 4. Inhibition of CETP-mediated [3H]CE transfer from HDL to LDL by cholesteryl sulfate and cholesteryl /3-o-glucuronide. Different concentrations of CS or CG were included in a [3H]CE transfer assay. The amounts of CE transferred during the 2 h incubation were compared with control samples without added inhibitor. All samples contained donor HDL particles labeled with [3H]CE, acceptor LDL particles and CETP. Data points and error bars represent the mean_+S.D. of triplicates.

3.3. Effect of cholesteryl sulfate (CS) and cholesteryl t~-D-glucuronide (CG) on CETP activity Concentration-dependent inhibition of C E T P activity was observed when CS or CG were added to the C E T P assay mixture containing LDL, [3H]CE-labeled HDL, and C E T P (Fig. 4). The ICs0 for inhibition was approx. 5 /zM for both compounds using the L D L precipitation assay. This concentration corresponds to approx. 3.4 mole percent relative to the total L D L and H D L cholesterol concentration ( 1 4 6 / z M ) present in in this assay. The slopes of the inhibitor curves for CS and CG are relatively steep in comparison with the curves for apolipoproteins or peptides (Figs. 1 and 2). Thus the CS concentration producing essentially no inhibition only differed from the CS concen-

properties o f apoA-I treated L D L acceptor particles were not different from control (data not shown). All of the above results utilized differential precipitation of L D L acceptor particles to separate them from [ 3 H ] C E - H D L donor particles. In order to verify that the inhibitory effects o f apoA-I were not the result of artifactual modification of the precipitation step of the assay by apoA-I, FPLC size-exclusion chromatography was used instead to separate L D L from HDL. [ 3 H ] C E - H D L (7.1 / z g / m l cholesterol), L D L ( 5 7 / ~ g / m l cholesterol), and C E T P (1 / z g / m l )

LDL •~ - 669K 440K

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Fig. 5. Effect of cholesteryl sulfate on electrophoretic mobility of LDL. CS was incubated with purified human LDL for 2 h at 37°C prior to subjecting the samples to thin-layer agarose gel electrophoresis (A) or non-denaturing polyacrylamide gradient gel electrophoresis (GGE) (B). The agarose gel was stained with Sudan black and the GGE gel was stained with Coomassie blue to visualize the migration of LDL. Lane 1, DMSO control; lane 2, 50 /xM CS; lane 3, 10 /xM CS; lane 4, 2 /xM CS; lane 5, 0.4/xM CS.

152

D.T. Connolly et al./ Biochimica et Biophysica Acta 1304 (1996) 145-160

tration producing complete inhibition by only approx. 4- to 8-fold in several experiments. Also, the apparent transfer at 50 /xM CG falls to a negative value, then appears to rebound. This effect is likely to be due to extensive modification of lipoprotein structure at these concentrations. In order to verify that the inhibitory effects of CS and CG observed using the differential L D L precipitation assay were not due to artifactual influences on the precipitation properties of the lipoproteins, CS was also tested using a fluorescence assay that does not utilize precipitation. The ICs0 value obtained by this method, 15 /xM (average of 2 assays), was in close agreement with the value obtained by the precipitation method.

3.4. Effect of cholesteryl sulfate on electrophoretic mobility of LDL

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Native agarose gel electrophoresis or polyacrylamide gradient gel electrophoresis (GGE) were employed to determine if CS affected the charge or size properties of LDL, respectively. Incubation of CS with purified human L D L under conditions similar to those used for the C E T P lipid transfer assay significantly altered the electrophoretic migration pattern of L D L on both thin layer agarose and G G E gels (Fig. 5). The modified migration of L D L on both gel systems suggests that CS altered both the net charge and the size of LDL. At 50 p,M CS, L D L did not leave the origin on agarose gels and could not be visualized on the G G E gel. Even at 10 /xM CS, L D L was more electronegative on the agarose gel relative to control LDL.

3.5. Effect of cholesteryl sulfate on the substrate properties of LDL and HDL [3H]CE-HDL (1.4 mg cholesterol) or L D L (6.2 mg cholesterol) were incubated with inhibitory concentrations of CS (2 mM) for 2 hours at 37°C in order to determine if loading of the lipoproteins with CS would alter their substrate properties toward CETP, and thereby the rate of lipid transfer. The concentrations of CS relative to total cholesterol correspond to 11 mol% for L D L and 35 mol% for HDL, and are above the concentrations required for 50% inhibition o f transfer (3.4 tool%). The modified lipoproteins

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Fig. 6. Comparison of transfer rates of [3H]CE using cholesteryl sulfate-modified donor HDL particles or acceptor LDL particles. CS (2 mM) was incubated for 2 h at 37°C with either [3H]CE-HDL donor particles (1.4 mg/ml cholesterol) or LDL acceptor particles (6.2 mg/ml cholesterol). Control lipoproteins were prepared by incubating with the equivalent amount of DMSO, the solvent used to dissolve CS. The lipoproteins were re-isolated by size exclusion chromatography, and the rates of [3H]CE transfer from HDL to LDL determined. ( • ) Control lipoprotein (mock treated); (O) lipoprotein treated with CS. Panel A, the acceptor LDL concentrations (CS-treated or control) were varied. The concentration of CETP was constant at 500 ng/ml and the donor [3H]CE-HDL was held constant at 40 /.tg/ml cholesterol. Panel B, double reciprocal plot of the data in Panel A. Panel C, the donor [3H]CE-HDL concentrations (CS-treated or control) were varied. The concentration of CETP remained constant at 500 ng/ml and the acceptor LDL concentration was also constant at 100 /xg/ml cholesterol.

D.T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160

were then re-isolated by size-exclusion chromatography, and independently tested in the [3H]CE transfer assay. LDL that had been pre-treated with CS was a substantially poorer substrate at all tested snbstrate concentrations (Fig. 6A). The double-reciprocal plot (Fig. 6B) shows that the K m for LDL was lowered by approx. 2-fold. In contrast, the data in Fig. 6C show that pre-incubation with CS had no effect on the substrate properties of [3H]CE-HDL when tested at low substrate concentration (20 / z g / m l or lower). Thus, recognition by CETP was not altered in the CS-modified [3H]CE-HDL. However, a modest reduction in transfer (about 20%) was observed at the highest CS-modified [3H]CE-HDL concentration (50 /zg/ml). This latter effect, which was also noted with LPS (see below), is interpreted to indicate that a minimal concentration of CS is required for inhibition of [3H]CE transfer when the CS is introduced into the assay via HDL. 3.6. Effect of lipopolysaccharide on CETP actiuity

153

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The effect of LPS on CETP-mediated [3H]CE transfer was determined by adding different concen-

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trations of LPS from four different bacterial sources into the in vitro assay mixture (Fig. 7). LPS from all four species were inhibitory when assayed by the differential LDL precipitation assay. The most potent LPS was from Pseudomonas aeruginosa, which showed significant inhibition at concentrations as low as 1 ~ g / m l . The IC50 values for the four LPSs were: Pseudomonas aeruginosa, 2 /zg/ml; Salmonella enteridis, 20 ~ g / m l ; Serratia marcescens, 30 /xg/ml; Escherichia coli, 200 /xg/ml. LPS's are complex, highly amphipathic molecules that contain lipid a moieties linked to polysaccharide. In order to determine whether the lipid portion of LPS alone is responsible for the inhibitory activity, diphosphoryl lipid a from E. coli was also tested but found to be inactive (Fig. 7), indicating that intact LPS is required for inhibition of CETP. In other control experiments, it was determined that the inhibitory effects of LPS were not due to artifactual alteration of the lipoprotein precipitation properties. LPS added after the transfer portion of the assay was complete, but prior to the precipitation step, did not prevent the precipitation of LDL. Simi-

D.T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160

154

lar results were reported by Mascucci-Magoulas et al. [44]. This was also verified by testing Pseudomonas aeruginosa LPS in the fluorescence assay that does not involve a precipitation step. The ICs0 obtained in this assay was 18 /xg/ml (average of 2 experiments).

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3.7. Effect of lipopolysaccharide on electrophoretic mobility of LDL LPS from all four bacterial species was incubated with LDL under conditions identical to those used in the CETP lipid transfer assay in order to determine if the LPS altered the net charge a n d / o r size of the lipoprotein. Electrophoretic migration patterns on thin layer agarose gels are shown in Fig. 8. Concentrations of LPS from Serratia and Escherichia of 1000 /~g/ml generated multiple species of LDL, as indicated by that altered electrophoretic mobility and diffuse banding relative to control LDL. LPS from Salmonella and Pseudomonas were even more potent, producing grossly altered mobility at concentrations of 100 / x g / m l or greater.

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Fig. 9. Comparison of transfer rates of [3H]CE using lipopolysaccharide-modified HDL donor particles or LDL acceptor particles. LPS from Pseudomonas aeruginosa (5 m g / m l ) was incubated with either [3H]CE-HDL (1.4 m g / m l cholesterol) or LDL (6.2 m g / m l cholesterol) for 2 h at 37°C. Control lipoprotein particles (mock treated) were prepared by incubating with TESA buffer alone. The lipoproteins were re-isolated by size-exclusion chromatography and the rates of transfer of [3H]CE from HDL to LDL determined. ( l l ) Control lipoprotein (mock treated); ( 0 ) lipoprotein treated with LPS. Panel A, the concentrations of LPS-treated or mock-treated donor [3H]CE-HDL particles were varied. The concentration of CETP remained constant at 500 n g / m l . Acceptor LDL concentration (not treated) was held constant at 100 / x g / m l cholesterol. Panel B, the LPS-treated or mock-treated acceptor LDL concentration was varied. The concentration of CETP was constant at 500 n g / m l and the donor [3H]CE-HDL (non-treated) was held constant at 40 / x g / m l cholesterol.

[3H]CE-HDL (1.4 m g / m l ) or LDL (6.2 m g / m l ) were incubated with inhibitory concentrations of LPS from Pseudomonas aeruginosa (5 m g / m l ) for 2 h at 37°C in order to pre-load the lipoproteins with LPS. After re-isolation by size exclusion chromatography, the lipoproteins were tested in the transfer assay. Experiments in which the concentrations of either LPS-treated [3H]CE-HDL or LPS-treated LDL were varied show that LPS had minimal effect on either substrate when tested at low concentrations. This result implies that recognition by CETP was not altered for either of the LPS-modified lipoproteins. However, the rate of [3H]CE transfer dropped at the higher LPS-modified lipoprotein concentrations in comparison to controls (Fig. 9A and B), suggesting that a minimal LPS concentration was required for inhibition. Thus at low [3H]CE-HDL or LDL concentrations, the lipoproteins were indistinguishable from control preparations, but at higher LPS-modified [3H]CE-HDL or LDL concentrations, less transfer occurred relative to controls. This result suggests that a minimal LPS concentration is required for inhibition in this assay.

4. Discussion

The present experiments were designed to determine whether various peptides and other compounds

D. T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160

that alter lipoprotein structure under normal or pathological conditions affect CETP activity. Apolipoproteins and related model peptides represent a class of molecules that naturally associate with lipoproteins, primarily HDL. Cholesteryl sulfate and cholesteryl /3-D-glucuronide represent natural cholesterol analogs that are normally found in low concentrations in the blood and tissues. Lipopolysaccharides are bacterialderived lipids that are not normally found in the blood but that can accumulate to appreciable concentrations under conditions of sepsis. All three classes of compounds were found to inhibit CETP activity in vitro, although the mechanisms of inhibition were distinct for the three classes of inhibitors.

4.1. Apolipoproteins and amphipathic peptides There have been several reports describing either stimulation CETP activity or inhibition of CETP activity by apolipoproteins. Guyard-Dangremont et al. [13] observed stimulation of CETP activity at low concentrations of apoA-I and apoA-IV, and inhibition at higher concentrations. In a study using micro emulsions rather than natural lipoproteins, apoA-I and apoA-II were found to be stimulatory [15]. Lagrost et al. [14] have studied the properties of HDL that contained different ratios of apoA-I to apoA-II, and concluded that apoA-II has the effect of an uncompetive inhibitor. Hayek et al. [16] have studied CETP-mediated transfer in transgenic mice that contained human CETP and a second transgene for human apoA-I. It was concluded that human CETP interacts better with the human apoA-I HDL than with the mouse apoA-I HDL. Kinoshita et al. [17] have studied CETP-mediated lipid transfer using VLDL from a subject with apoE deficiency, and concluded that apoE enhances the transfer process. It is thus evident that the apolipoprotein content can have profound influence on CETP-mediated lipid transfer. In the present experiments, stimulation of CETP activity was not observed, possibly indicating that the HDL donor particles used here already contained naturally optimized concentrations of apolipoproteins. However, inhibition of CETP-mediated CE transfer was observed at concentrations of apo A-I, apo A-II, apo C-I and apo C-Ill between 1 and 100 / z g / m l

155

(IC5os = 0.4-3.4/xM). Although lipoprotein populations are heterogeneous with regard to size and composition, it is estimated that the molar HDL and LDL concentrations used in the present assays were roughly 0.15 /zM and 0.08 /zM, respectively. Thus, the most effective apolipoprotein inhibitors had ICs0s that were only about 2- to 3-fold higher than the total lipoprotein concentration used in the assay. Recognition of HDL or LDL by CETP might involve binding of CETP to apo A-I or to another apolipoprotein residing on a lipoprotein particle. The direct interaction of CETP with apo A-I or apo A-II has been shown in chemical cross-linking experiments demonstrating the formation of heterotypic complexes during incubation with HDL [13]. It is therefore evident that apolipoproteins play a role in CETP-mediated lipid transfer. However, there are also data suggesting that apolipoproteins are not necessary for CETP activity, and that the inhibitory effects are not specific. First, anti-apolipoprotein antibodies used in the present studies did not inhibit CETP activity, so it is unlikely that direct binding of CETP to an apolipoprotein portion of the lipoprotein particle is essential for transfer activity. This is consistent with previous studies showing that CETPmediated lipid transfer occurs using protein-free liposomes, emulsions or monolayers [7,9-12]. Secondly, all five of the apolipoproteins tested here were inhibitory, implying a lack of specificity. Finally, synthetic peptides designed to model the generic class a amphipathic helix motif common to the apolipoprotein family were also inhibitory. Peptides 18A and 18A-Pro- 18A share only remote sequence identity with natural apolipoproteins. However, their physical properties have been shown to closely resemble those of the amphipathic helix portions of apolipoproteins [18,19]. Thus, the peptides are able to mimic certain apolipoprotein functions. The peptides have been shown to mobilize lipid into HDL-like particles [35], and to stimulate LCAT much like the enzyme's natural cofactor, apo A-I [45]. Peptide 18A has been shown to displace apo A-I from HDL [35]. The potency of 18A-Pro-18A as a CETP inhibitor (IC5o = 0.4/zM) was equal to that of apo A-l, apo A-II and apo C-I in this assay system. The enhanced activity of 18A-Pro-18A relative to the 18A monomer is probably the result of its ability to form a stable, cooperative helix that can interact more

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effectively with phospholipid [35] since only a 2-fold increase in potency would be expected if peptide concentration alone is considered. Interaction with the lipoprotein particle via the class A amphipathic helix motif [ 18,19] was evidently essential for effective inhibition since the control peptide 18R, which would display a reverse, unfavorable charge distribution along the helix-phospholipid interface, was only marginally inhibitory. The other helix-forming peptides that were tested, Melittin, cecropin A, and magainin [36-38], were devoid of inhibitory activity at similar concentrations, even though these peptides derive their bioactivity through association with membranes. This suggests that lipid-associating peptide helices per se are not sufficient for CETP inhibitory activity, but that particular structures associated with class A amphipathic helices are essential. Together, these results suggest that the apolipoproteins or peptides inhibited lipid transfer by physical modification of the lipoprotein particles, perhaps by occupation of the phospholipid surface sites on the lipoprotein particles that normally provide CETP with access to the lipid core. Another peptide that was tested is the baboon apo C-I~_3s peptide. Although this peptide was reported to have inhibitory activity in an assay system using baboon lipoproteins and baboon lipoprotein-depleted serum as a source of CETP [39], it was relatively ineffective in our assay system which utilized human CETP, LDL and HDL. Intact human apo C-I was found to be an effective inhibitor here, suggesting that species differences might account for the difference in effectiveness of the baboon peptide in the two studies. There have been several reports in the literature describing the properties and partial purification of a natural CETP inhibitor from plasma, lipid transfer inhibitor protein (LTIP) [46-49]. Amino-acid sequence data for LTIP have not been presented. However, the physical properties of LTIP, including the apparent molecular weight of about 30000 and apparent p I of 3.7-3.8, are similar to several apolipoproteins. In terms of potency, all five of the apolipoproteins tested here inhibited CETP at concentrations similar to the concentration reported for highly purified LTIP [48,49]. Confirmation of the possibility that LTIP may be related to a freely exchangeable apolipoprotein will require additional studies.

In experiments designed to determine whether the inhibition by apolipoproteins was due to direct interaction with CETP or due to modification of the lipoprotein substrates, HDL or LDL were pre-incubated with inhibitory concentrations of apoA-I, and then re-purified. The apoA-I content of HDL was increased about 2-fold under these conditions, whereas apoA-I did not become associated with LDL. When the substrate properties of these preparations were compared with controls, it was observed that rate of [3H]CE transfer from the modified HDL was substantially decreased, and that the K m for the modified HDL was about 2-fold higher. Thus the affinity of CETP for the apoA-I modified HDL was lower than that of control HDL. The substrate properties of LDL were not altered by apo A-I, consistent with the observation that negligible amounts of the apolipoprotein became associated with LDL upon addition of exogenous apo AI.

4.2. Cholesteryl sulfate Cholesteryl sulfate (CS) is a natural component of tissues and blood. When added to erythrocytes, CS stabilizes against hemolysis [20]. CS also inhibits Sendai virus fusion with erythrocytes [50,51], inhibits adrenal mitochondrial cholesterol side-chain cleavage [52], mitochondrial steroidogenesis [53] and inhibits the enzyme lecithin-cholesterol acyltransferase [54]. Thus CS has been shown to have dramatic effects when added to a variety of biological membrane systems, probably by alteration of the physical properties of the membranes. CS may also play important roles in natural processes, including endometrial implantation [55], sperm capacition [23-26], and in X-linked ichthyosis disease [21,22]. Reports from the literature describing the presence of CS in the sperm acrosomal membrane [23] and of CETP in follicular fluid [24,25] are of particular interest. It is thought that the high concentration of CS in the acrosomal membrane inhibits the membrane fusion process, and that enzymatic hydrolysis of the sulfate moiety facilitates capacitation. CETP has been isolated from follicular fluid and has been shown to stimulate sperm capacitation, possibly by altering membrane lipid composition [24-26]. In light of the present results demonstrating that CS is a

D.T. Connolly et al. / Biochimica et Biophysica Acta 1304 (1996) 145-160

potent inhibitor of CETP, new roles for both CETP and CS should be considered. In addition to physical stabilization of the acrosomal membrane, CS could also serve as an effective inhibitor of CETP stimulated sperm capacitation. Conversion of CS to cholesterol by removal of the sulfate group would have the dual effect of increasing membrane fluidity and decreasing CETP inhibition. Furthermore, the transformation from inhibitory to non-inhibitory amounts of CS in the acrosomal membrane would be expected to be abrupt, since the concentration curve for inhibition of CETP by CS is extremely steep (Fig. 4). Recessive X-linked ichthyosis is an inherited metabolic disease caused by a deficiency in aryl sulfatase, an enzyme that removes sulfate from sulfated steroid hormones and CS. Cholesteryl sulfate levels are elevated approx. 20-fold in the plasma of these individuals, with approx. 70% of the excess CS residing in the LDL fraction [22]. It can be calculated from the data presented by Nakamura et al. [21] that the concentration of CS in the LDL fraction of ichthyosis individuals was as high as 2.3 mole percent relative to cholesterol. In the present experiments, the IC50 for CS inhibition of CETP was 5 /zM, or approx. 3.4 mole percent relative to total cholesterol. This would suggest that levels of CS in individuals with ichthyosis could be high enough to inhibit CETP activity, Consistent with this, the LDL from individuals with ichthyosis contained decreased CE and increased TG relative to control subjects [21]. In addition, the L D L from ichthyosis individuals was shown to become more anionic [21,22], similar to the LDL that was treated with CS in the current studies (Fig. 4). The shift in electrophoretic mobility observed here corresponded closely with the inhibition of CETP activity, consistent with reports by others that the charge properties of lipoproteins can influence CETP activity [56]. When LDL or HDL were incubated separately with inhibitory concentrations of CS, there was little affect on the substrate properties of HDL, whereas LDL became a distinctly poorer substrate for CETP. Since the actual concentration of CS in the respective lipoprotein fractions were not measured, it is not clear if the difference lies in preferential association of CS with LDL relative to HDL, or if some other modification specific to LDL is responsible for the effect. Nevertheless, the data clearly indicate that the

157

inhibitory effects of CS involve preferential modification of LDL.

4.3. Lipopolysaccharides The deduced amino-acid sequence of CETP from cDNA clones [57] indicates that CETP is structurally similar to two proteins that bind LPS. Bactericidal permeability increasing protein, a 5 5 - 6 0 kDa protein produced by polymorphonuclear leukocytes, has 22% amino-acid sequence identity with CETP, with another 30% of the amino acids containing conserved substitutions [32]. LPS binding protein, a 60 kDa glycoprotein found in normal plasma, is 23% identical to CETP with another 23% of the amino acids having structurally conserved substitutions [33]. This high degree of homology implies that the three proteins share structural similarity which could include the lipid binding sites. We therefore hypothesized that LPS might inhibit lipid transfer by binding to the lipid-binding site on CETP. LPS from four different strains of bacteria were, in fact, found to be potent inhibitors of CETP-mediated lipid transfer in vitro, confirming the results by [44] using E. coli derived LPS. In the present study, there was an approx. 200-fold difference in potency between the four types of LPS which could be reflective of differences in affinity for the CETP lipid binding site. However, in control experiments, it became evident that LPS drastically modified the structure of lipoprotein particles. The higher concentrations of LPS, where gross alterations in lipoprotein structure were observed, were correlated with complete inhibition of CETP activity. Thus one mechanism whereby LPS inhibits CETP evidently involves gross alteration of lipoprotein structure, especially at high LPS concentrations. A second mechanism of CETP inhibition may involve direct interaction with CETP. Significant inhibition of CETP activity was observed at 10 t t g / m l Pseudomonas LPS and 100 / , g / m l Serratia LPS, but gross alterations in LDL structure were not observed at these concentrations. Although this might simply indicate that the electrophoretic method was not sensitive enough to detect modification, it might also indicate that another mechanism contributes to inhibition. Futher support for this interpretation is derived from experiments in which HDL and LDL

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were pre-incubated with LPS and then re-isolated. In these experiments, only intact LDL or HDL that eluted from the size-exclusion FPLC column were used. Thus, particles that might have had extreme modification were not tested. The substrate properties of these LPS-modified lipoproteins were then examined. At low HDL or LDL concentrations, the substrate properties of the modified lipoproteins were not distinguishable from controls implying that the ability of the particles to interact with CETP had not been altered. However, at higher lipoprotein concentrations, decreased transfer was observed. One interpretation of this result is that at higher lipoprotein concentrations, more LPS was able to diffuse out of the LDL or HDL and interact directly with CETP. Further studies will be needed to resolve this. It is of interest to note that the concentrations of LPS required for detectable changes in the electrophoretic mobility of LDL and for inhibition of CETP activity are quite low relative to the amount of total lipid in the assay. The ICso for Pseudomonas LPS was approximately 2 /xg/ml. LPS structure is heterogeneous, but if an average molecular weight of about 15 000 is assumed [58], then it can be estimated that the molar concentration of LPS in the assay was about 0.1 /xM. Since the total LDL and HDL cholesterol in the assay was 146 /zM, the relative LPS concentration was about 0.07 mole percent. LPS was thus about 50-fold more potent than CS in this assay system. While it is difficult to directly extrapolate the present in vitro results to in vivo conditions, the inhibitory concentrations of LPS in the present studies (10 to 100 /zg/ml) fall within the ranges that are likely to be reached in many in vivo animal sepsis models. LPS treatment has been shown to have dramatic effects on lipid metabolism, especially at higher doses. These effects include decreases in the concent, ations of CETP mRNA and mass [44], as well as alterations in the concentrations of LDL, HDL, and triglycerides [59,60]. The specific effects triggered by LPS are very complicated and are dependent upon the dose of LPS administered. Although LPS activity is usually attributed to induction of potent cytokines, the present data, as well as the data of MascucciMagoulas et al. [44], raise the possibility that inhibition of CETP could contribute to the pathology of

LPS by preventing the normal redistribution of neutral lipids between lipoprotein classes.

5. Conclusions Apolipoproteins and related peptides, CS, CG and LPS were all lbund to inhibit CETP-mediated lipid transfer in vitro. Inhibition by apoA-I was correlated with modification of HDL structure that could reduce CETP recognition or affinity. Further study will be needed to determine the exact mechanism, but could involve displacement of CETP or other lipoproteins from the HDL surface. Inhibition by CS was correlated with modification of LDL. The lipopolysaccharides caused gross lipoprotein modification at high concentrations, but also might interact directly with CETP. The concentrations of each class of inhibitor were higher than the concentrations that would be encountered under normal physiological conditions. However, pathological elevation of these agents, such as might occur in hyperlipoproteinemias, ichthyosis or sepsis, would be expected to influence CETP activity in vivo. The present data also suggest a novel role for CS and CETP in the regulation of the sperm acrosomal reaction.

Acknowledgements We would like to thank Bev Kekec for expert technical assistance, Charles Lewis for expert cell culture assistance, and Michele Melton for providing the lipoproteins used in these studies. We also thank Maribeth Babler and Jon McIntyre for providing fluorescence assay data.

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