©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Defective Binding of Neutral Lipids by a Carboxyl-terminal Deletion Mutant of Cholesteryl Ester Transfer Protein
EVIDENCE FOR A CARBOXYL-TERMINAL CHOLESTERYL ESTER BINDING SITE ESSENTIAL FOR NEUTRAL LIPID TRANSFER ACTIVITY (*)

(Received for publication, May 11, 1994; and in revised form, October 7, 1994)

Suke Wang (1) (2)(§) Paul Kussie (2) Liping Deng (2) Alan Tall (2)

From the  (1)Schering-Plough Research Institute, Kenilworth, New Jersey 07033 and the (2)Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The plasma cholesteryl ester transfer protein (CETP, 476 amino acids) transfers cholesteryl ester (CE) from high density lipoprotein (HDL) to triglyceride-rich lipoproteins and plays a major role in HDL catabolism. Using deletional and site-directed mutagenesis, we previously showed that the carboxyl terminus of human CETP comprises the epitope of a neutralizing monoclonal antibody and is necessary for neutral lipid transfer activity. To assess the nature of the involvement of the COOH terminus in cholesteryl ester transfer activity, we characterized a deletion mutant of CETP lacking amino acid residues 470-475 in terms of CE transfer kinetics, association with HDL, and capacity to bind CE, triglyceride (TG), and phosphatidylcholine (PC). Kinetic analysis indicated a major catalytic defect of the deletion mutant, as shown by markedly decreased maximum cholesteryl ester transfer activities (apparent V(max)) for donor (HDL) and acceptor (low density lipoprotein (LDL)) lipoproteins but there were no significant changes of concentrations of the donor and acceptor at 50% V(max) (apparent K). The binding of CETP to HDL, as determined by native gel electrophoresis, was similar for wild-type and mutant protein. When egg PC/CE vesicles were incubated with wild type CETP and then separated by gel filtration chromatography, there was maximum binding of about 1 mol of CE/mol of CETP. Under similar conditions the mutant CETP bound 0.09-0.37 mol of CE/mol of protein. Similarly, when egg PC/TG vesicles were incubated with the CETP proteins, there was a maximum binding of 0.5 mol of triglyceride/mol of wild-type CETP, whereas there was only 0.00-0.07 mol of TG/mol of deletion mutant. The binding of phosphatidylcholine was similar for wild-type and the deletion mutant. The studies suggest that amino acids 470-475 (forming part of a COOH-terminal amphipathic helix) are involved in CE and TG binding by CETP but are not required either for the binding of PC by CETP or the association of CETP with HDL. The COOH terminus of CETP may comprise a neutral lipid binding site directly involved in the lipid transfer mechanism.


INTRODUCTION

The plasma cholesteryl ester transfer protein (CETP) (^1)facilitates net transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to low density lipoprotein (LDL) and very low density lipoprotein (VLDL) and the reciprocal transfer of triglyceride (TG) from LDL and VLDL to HDL(1, 2) . The plasma HDL cholesterol level may be influenced by plasma CETP activity, as shown by increased HDL levels in humans with genetic CETP deficiency (3, 4) and decreased HDL levels in transgenic mice expressing human CETP(5, 6) . Reducing plasma CETP levels could be beneficial for coronary heart disease because of an anticipated increase in anti-atherogenic HDL and a decrease in atherogenic VLDL and LDL(7) .

CETP is a hydrophobic glycoprotein of 476 amino acids with a molecular mass of about 68,000-70,000 daltons in SDS gels(8, 9, 10) . CETP has binding sites for CE, triglyceride (TG), and phospholipids (11) and is thought to transfer lipids through carrier mediated and/or collisional mechanisms(12, 13) . Both the binding of CETP to its lipoprotein substrates and the ability of CETP to bind neutral lipid have been suggested to be essential events in the neutral lipid transfer mechanism(11, 14) . The COOH terminus of the protein is important for the neutral lipid transfer activity as demonstrated by experiments using monoclonal antibodies (mAb) that bind at the COOH terminus and neutralize neutral lipid transfer activity (15) and by deletional and point mutagenesis of the COOH-terminal region(16, 17, 18) .

Nested deletions at the COOH terminus reveal that amino acids 468 to 475 are necessary for binding mAb TP2 and for neutral lipid transfer activity. Characterization of the mutant protein (Delta470-475) showed a normal overall folding pattern, markedly defective CE and triglyceride transfer activity, and normal or increased phospholipid exchange activity. Point mutagenesis of the COOH-terminal region suggested that the amino acid residues on the polar face of a putative COOH-terminal amphipathic helix constitute the epitope of mAb TP2, whereas residues on the hydrophobic face, as well as a nearby hydrophobic region, are involved in CE transfer activity(18) . In the present investigation we have carried out more detailed biochemical characterization of the COOH-terminal deletion mutant (Delta470-475) in order to examine the hypothesis that this region of CETP forms part of a neutral lipid binding site.


MATERIALS AND METHODS

HDL(3) (d = 1.12-1.21 g/ml), HDL(3) containing [^3H]cholesteryl ester ([^3H-CE]HDL), and low density lipoprotein (d = 1.02-1.063 g/ml) were prepared and characterized as described previously(19) . Wild-type (WT) CETP and the COOH-terminal deletion mutant lacking amino acids 470-475 were obtained by stably expressing the cDNAs in Chinese hamster ovary cells as described previously(17) . In some experiments, WT and mutant proteins were purified to homogeneity by hydrophobic then ion exchange chromatography. Tissue culture media (Ex-cell 301, JRH Biosciences, Lenexa, KS) containing recombinant CETP were adjusted to 1 M NaCl and applied to a column containing a hydrophobic resin (Toyopearl Butyl-650M, Tosohaas, Montgomeryville, PA), and the column was washed successively with 750 and 200 mM NaCl. The CETP was then eluted with 2 mM EDTA(24) . Fractions were tested for transfer activity, and active fractions were applied to a Mono Q-Sepharose (Pharmacia Biotech Inc.) anion exchange column. The CETP was the first peak eluted from the Mono Q column with a gradient of 10 mM KH(2)PO(4) to 200 mM KH(2)PO(4) (pH 7.4). The CETP was concentrated using Centricon-30 concentrators (Amicon, Beverly, MA). The mass of the CETP proteins secreted into the media or present in column fractions was determined by RIA (7) or by enzyme-linked immunosorbent assay (ELISA) using an anti-CETP mAb which recognizes a non-COOH-terminal epitope (TP14). The purity of the CETP proteins was judged by SDS-polyacrylamide gel electrophoresis, and the mass of purified WT and mutant CETP was determined by the method of Lowry(25) . Concentration of phosphatidylcholine was measured with a kit from Wako Pure Chemical Industries (Osaka, Japan, Catalog number 996-54001), based on the choline content released by phospholipase D(28) .

Cholesteryl Ester Transfer Activity Assay and Kinetic Measurements

To assay CE transfer activity, CETP, [^3H-CE]HDL, and LDL were mixed in buffer containing 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, and 1 mM EDTA (TSE buffer). The mixture was incubated at 37 °C for up to 2 h with slow shaking. At the end of incubation, the transfer reaction was stopped by adding the TSE buffer to a final volume of 1 ml. A precipitation mixture (0.35 ml) was then added, and LDL was separated by centrifugation in a Microfuge for 15 min at 4 °C. One ml of the supernatant was mixed with 4 ml of scintillation fluid and counted(20) . The transfer kinetic measurements were always in the linear range of the assay such that the counts transferred were less than 20% of the initial counts in the radiolabeled HDL. The counts remaining in the supernatant were converted to transfer rate (nanomoles of CE transferred per h/ng of CETP) as described by Ihm et al.(13) . The exchange rate (R) was related to the experimental parameters as following,

where C() = C(0)[M/(M + M)] and M is the concentration of HDL or LDL (nanomoles of CE/ml), t is incubation time (hours), C is the counts/min of [^3H]CE in LDL at time t or (at equilibrium), and C is the counts/min of [^3H]CE in HDL at t = 0. The apparent K(m) and V(max) were determined by fitting data to Lineweaver-Burk linear transform as described by Cleland(21) .

Binding of WT CETP and Deletion Mutant Delta470-475 to HDL

WT or deletion mutant CETP (at 1.9 µg/ml) along with varying amounts of HDL were incubated at 37 °C for 1 h in the presence of 0.25 mM dithiothreitol in TSE buffer in a final volume of 40 µl. Control runs were performed to assure the CETP concentration used in the assay was in the linear range for the Western blot detection. After being mixed with 16 µl of loading buffer containing 40% (w/v) sucrose and 0.04% (w/v) bromphenol blue, each sample was loaded in a native polyacrylamide gradient gel (4-30%) and run in TBE buffer (90 mM Tris, 80 mM borate, 2.5 mM EDTA (pH 8.4)) in a Hoefer gel apparatus. To locate the position of HDL in the gel, 15 µl of HDL (2.4 µg of cholesterol) was mixed with 5 µl of Sudan black mix, composed of 5 parts of 1% (w/v) Sudan black in ethylene glycol and 3 parts of 40% (w/v) sucrose, and run in parallel with the other samples. The electrophoresis was run at 200 V for 15 h at 20 °C with TBE as the running buffer. After the electrophoresis, the gel was transferred onto nitrocellulose filter paper. WT and mutant CETP were detected using mAb TP14 as the first antibody and sheep anti-mouse IgG antibody conjugated with horseradish peroxidase as secondary antibody with enhanced chemiluminescence detection(17) .

Binding of Cholesteryl Ester to CETP

The binding of CE to CETP was assessed by the procedure of Swenson et al.(11) . Egg PC vesicles (12 nmol) containing 1.5 mol % cholesteryl oleate and 0.09 µCi of [^3H]cholesteryl oleate (egg PC/[^3H]CE) were incubated with CETP in 10 mM Tris-Cl, 1 mM EDTA (TE buffer), and 2 mg of BSA at 37 °C for 1 h in a total volume of 1 ml. The mixture was loaded onto a Sephadex G-200 column (18 mm times 70 cm) controlled by a fast protein liquid chromatography apparatus and equilibrated with the same buffer. The vesicles and CETP were separated at a flow rate of 6 ml/h at room temperature. Fractions of 3 ml were collected and assayed for [^3H]CE profile, CE transfer activity, and CETP mass (ELISA). The CE transfer activity in the column fractions was assayed with a scintillation proximity assay (SPA) utilizing [^3H-CE]HDL, biotin-LDL, and avidin-fluoromicrosphere (Amersham Corp.). To prepare the vesicles, 5.4 µmol of egg PC, 0.082 µmol of cholesteryl oleate, 40 µCi of [^3H]cholesteryl oleate (71 Ci/mmol, Amersham) were mixed and dried in a glass tube under a stream of nitrogen. The lipids were suspended in 7 ml of buffer containing 10 mM Tris-Cl, 1 mM EDTA (pH 7.4) by vortexing. The solution was sonicated 4 times 12-min periods at 4 °C with a Ultrasonic W-385 sonicator with a microtip at a setting of 3. The lipid solution was then centrifuged in a clinical centrifuge for 5 min and chromatographed over a Sepharose 4B column (2.6 x 50 cm) at a flow rate of 1 ml/min. The central one-third of the included radioactive peak was pooled and used for experiments.

Binding of Triglyceride and Phosphatidylcholine to CETP

Egg PC vesicles containing either ^14C-triolin alone or in combination with [^3H]dipalmitoyl phosphatidylcholine were made similarly to the vesicles containing [^3H]cholesteryl oleate. Briefly, 2.7 µmol of egg PC (Avanti Polar Lipids, Inc.), 0.0405 µmol of triolin (TG/PC = 0.015:1, mol/mol) and 18 µCi of [^14C]triolin (Amersham, 110 mCi/mmol) were mixed, and the organic solvents were evaporated under a stream of nitrogen. Seven ml of TE buffer was added and incubated in a water bath set at 37 °C for 15 min. Lipids were suspended by vortexing, followed by sonication for 10 min at 15 °C, with the setting at 2.5 using a macrotip and a glass container connected to water circulation. The solution was centrifuged for 10 min at 4 °C at a speed of 2500 rpm in a clinic centrifuge followed by centrifugation at 140,000 times g at 30 °C for 1 h. The small unilamellar vesicles in the supernatant was used for experiments for TG binding to CETP. Vesicles containing both [^14C]triolin and [^3H]PC were prepared similarly to the [^14C]TG-labeled vesicles preparation except both [^14C]TG and [^3H]PC (36 µCi of [^3H]dipalmitoyl phosphatidylcholine, 42 Ci/mmol, Amersham) were added at the beginning of the procedure. Concentrations of all the components were the same in the vesicles containing either single or dual labels.

Similar conditions were used in the binding assays for TG and PC binding to CETP proteins. Purified CETP was incubated with egg PC (4.7 nmol) vesicles containing 1.5% (mol/mol) ^14C-TG (0.037 µCi) or with egg PC vesicles containing [^14C]TG and [^3H]PC (0.128 µCi), 1.5 mg/ml BSA in a total volume of 1 ml of 10 mM Tris-Cl, at 37 °C for 1 h. The mixture was analyzed the same way as described in the analysis for [^3H]CE binding to CETP by using Sephadex G-200 chromatography, scintillation counting, CETP ELISA, and SPA.

Determination of CE, TG, and Phosphatidylcholine Bound to Purified CETP

Approximately 1 mg of WT CETP was purified from CHO cell medium through hydrophobic and ion exchange chromatography, as described above. The lipids were extracted with organic solvents according to the procedure of Bligh and Dyer(29) . The organic solvents were dried under a stream of nitrogen and separated on TLC with a solvent system consisting chloroform:methanol:water (65:25:4, v/v/v). The plate was visualized with iodine vapor for spots of CE, TG, and phospholipids. Phosphatidylcholine extracted from another batch of 1 mg of CETP was then quantitated by assaying the choline content released from the PC(28) .


RESULTS

CE Transfer Kinetics of WT CETP and Deletion Mutant CETP (Delta470-475)

In previous experiments we showed that the deletion mutant (Delta470-475) had markedly impaired neutral lipid transfer activity(17) . The defect of CE transfer activity could be due to a general reduced binding of mutant CETP to lipoprotein substrates or a selective inability of mutant CETP to bind and transfer CE. To assess the two possibilities, we first conducted measurements to characterize the kinetic parameters describing CETP-mediated CE exchange between HDL and LDL. Kinetic analysis with the Michaelis-Menten treatment for enzyme-catalyzed reactions has been used to study CETP mechanism of action(12, 13) . The apparent K(m) and V(max) were determined by holding one lipoprotein at constant concentration while varying the concentration of the other lipoprotein. When HDL concentration was varied, the deletion mutant CETP displayed markedly reduced apparent V(max) and the reduction of CE transfer by the deletion mutant could not be overcome by increasing HDL to high concentrations (Fig. 1). The apparent K(m) values for both WT and deletion CETPs were similar (Table 1). Comparable results were also obtained when the LDL concentration was varied (Fig. 2, Table 1); the apparent V(max) of the deletion mutant was much lower than that of WT CETP, whereas the apparent K(m) of the deletion mutant was somewhat higher than that of the WT CETP. The large differences in maximum transfer velocities suggests that the COOH terminus is involved in the CE transfer process (Table 1). The parallel decrease in V(max) for both HDL and LDL suggests a defect in an event common to both HDL and LDL rather than a defect in an event associated exclusively with LDL or HDL. The small difference in k(m)(app) suggests that binding to lipoprotein substrates might be essentially normal for the mutant.


Figure 1: CE transfer kinetics of WT CETP and mutant CETP (Delta470-475) with HDL as variable substrate. R represents the initial CE transfer velocity (nanomoles of CE transferred in 1 h by 1 ng of CETP at 37 °C). LDL was held constant at 647 nmol/ml (LDL cholesterol), whereas the concentration of HDL (nanomoles of cholesterol/ml) was varied (top panel). The bottom panel represents the double-reciprocal plot of the data from which apparent V(max) and K values were derived. box, WT CETP; up triangle, deletion mutant Delta470-475. Values are means ± S.D. (n = 4-6).






Figure 2: Determination of apparent K and V for LDL. HDL was kept at 27.1 nmol of cholesterol/ml and LDL was varied (shown as nanomoles of LDL cholesterol/ml). Top panel, initial velocities of CE transfer as a function of LDL concentration. Bottom panel, double-reciprocal plot used to obtain apparent V(max) and K values. box, WT CETP; up triangle, deletion mutant Delta470-475. Values are means ± S.D. (n = 4-6).



Binding of WT CETP and Deletion Mutant Delta470-475 to HDL

The ability of the COOH-terminal mutant to bind to HDL was directly assessed by incubating WT and the mutant proteins with HDL followed by separation by native polyacrylamide gel electrophoresis. Fig. 3shows the binding of WT and mutant CETP to increasing concentration of HDL. In the absence of HDL, WT and mutant CETP ran primarily as monomer with some dimer formation; although more self-association of WT is apparent in Fig. 3, differences in the self-association of WT and mutant CETP were not consistently observed. The binding to HDL could be most clearly assessed from the intensity of the monomer. By this criterion, both WT and the deletion mutant CETP bound to HDL similarly at all concentrations of HDL and no free CETP was visible at 79 nmol/ml (total HDL cholesterol) or higher concentrations of HDL. The apparent dissociation constant (K(d)(app) values), as judged by locating the concentration with half-free and half-bound CETP, were approximately 8-24 nmol of HDL cholesterol/ml for both WT and mutant CETP, comparable with the apparent K(m) values determined in kinetic experiments (Table 1). This suggests that the apparent K(m) values are largely determined by the binding of CETP to lipoproteins. The results indicate that removal of amino acid residues 470-475 did not significantly alter the ability of CETP to bind HDL.


Figure 3: HDL binding of WT CETP and mutant CETP (Delta470-475) analyzed by native polyacrylamide gel electrophoresis. HDL at indicated concentrations (nanomoles/ml total cholesterol) were incubated with WT CETP, deletion mutant CETP (Delta470-475), or medium obtained from mock-transfected cells and subjected to gel electrophoresis. The gel was then Western blotted, stained with monoclonal antibody TP14, and visualized by the chemiluminescence method(17) . The position of HDL(3) was identified in a parallel run of Sudan black stained HDL(3) and is indicated by the bracket at the right. The minor high molecular weight band apparent in lanes containing the mutant (Delta470-475) is due to nonspecific binding of TP14.



Binding of Cholesteryl Ester to CETP

The possibility that the deletion of COOH-terminal amino acids may affect the ability of CETP to bind CE was tested using a previously described CE binding assay(11) . Small unilamellar egg PC vesicles containing 1.5 mol % [^3H]cholesteryl oleate were incubated with purified WT or mutant CETP in the presence of 2 mg/ml BSA and low ionic strength buffer then separated by gel filtration. The distribution of CETP in the column profile was determined with an ELISA assay using a non-COOH-terminal mAb (TP14) and by SPA assay of transfer activity in individual fractions. The distribution of [^3H]CE and CETP mass are shown in Fig. 4for WT CETP (A) and deletion mutant CETP (B). The wild-type CETP bound most of the CE in the vesicles and formed a CEbulletCETP complex, as shown by the large radioactive peak at an elution volume of 84 ml (Fig. 4A). The small radioactive peak at an elution volume of 50 ml was the unbound CE remaining in the PC vesicles eluting in the void volume, at the same position as control vesicles (not shown). By contrast, the deletion mutant CETP bound only small amounts of [^3H]CE, shown by the small radioactive peak at elution volume 81 ml. Most CE remained associated with the PC vesicles in the void volume (at 50 ml) (Fig. 4B). In an experiment under the condition described in Fig. 3A where WT CETP was preincubated with mAb TP2 at room temperature for 20 min, the large [^3H]CE peak of the CEbulletCETP complex shown in Fig. 3A was abolished (not shown), as described previously(11) .


Figure 4: Cholesteryl ester binding of WT CETP (A) and mutant CETP (B). Twelve µg of purified WT or deletion mutant CETP (Delta470-475) were incubated with [^3H]cholesteryl oleate containing egg PC vesicles (12 nmol) and CEbulletCETP complex was separated from the vesicles by gel filtration on a 70-cm Sephadex G-200 column. The radioactivity of [^3H]CE () and CETP mass (box) in the fractions were determined by scintillation counting and ELISA, respectively.



The stoichiometry of CE and CETP in the CEbulletCETP complex was calculated using the peak fraction containing the CEbulletCETP complex (Table 2). In several different experiments, variable amounts of CETP were incubated with a constant amount of egg PC/CE vesicles and analyzed for CEbulletCETP complex formation. As the incubation ratio of vesicles to CETP was decreased, less CE was bound to both WT CETP and the deletion mutant CETP. However, when compared at similar ratios of CETP to egg PC vesicles, WT CETP bound several times more CE than the deletion mutant CETP (Delta470-475).For example, when 12 µg of protein was used, about one CE molecule was bound to one WT CETP molecule (CE/CETP = 1.08), whereas an average of 0.3 molecules of CE were bound to one molecule of CETP (Delta470-475) molecule (CE/CETP = 0.30) (Table 2). Similarly, when 60 µg of CETP was used, the deletion CETP showed only about 25% of the WT CETP CE binding capacity (Table 2). The column fractions were also assayed for CE transfer activity for both WT and the deletion mutant Delta470-475. There was only one active peak eluting at the same elution volume as the [^3H]CEbulletCETP complex (not shown). The average CE transfer specific activity of WT CETP in the experiments shown in Table 2was 434 ± 197 cpm/ng (mean ± S.D., n = 4), compared with that of the deletion mutant, 47 ± 14 cpm/ng (mean ± S.D., n = 4) (Table 2).



Triglyceride and Phosphatidylcholine Binding to CETP

Since CETP transfers TG and PC, as well as CE, between lipoproteins, we tested the role of COOH terminus of CETP in the involvement in binding of TG and PC to CETP by using the same procedure used for the binding of CE to CETP described above. Small unilamellar egg PC vesicles containing either [^14C]TG alone or containing both [^14C]TG and [^3H]PC were incubated with wild-type CETP or the deletion mutant CETP in the presence of low ionic strength buffer containing 1.5 mg/ml BSA. The distribution of [^14C]TG and [^3H]PC in the eluent fractions is show in Fig. 5. The counts of [^14C]TG and [^3H]PC eluting at the void volume (45 ml) represent the unbound TG and PC remaining in the egg PC vesicles, and the radioactive counts eluting at 78 ml represent the [^14C]TG or [^3H]PC associated with CETP. WT CETP bound TG normally as shown by the [^14C]TG peak at 78 ml (Fig. 5A), whereas this peak was not detectable when the COOH-terminal deletion mutant was incubated with the vesicles (Fig. 5B). Comparable CETP masses for both WT and mutant CETP were found at elution volumes between 65 and 85 ml, corresponding to the [^14C]TGbulletCETP complex. In contrast, the [^3H]PCbulletCETP complex appeared similar elution for both WT and deletion mutant (Fig. 5, A and B). The data indicate an impaired TG but unaffected PC binding by CETP of the COOH-terminal deletion.


Figure 5: Triglyceride and phosphatidylcholine binding by WT CETP (A) and mutant CETP (B). Purified WT CETP (30 µg) or mutant CETP (34 µg) was incubated with egg PC vesicles containing [^14C]TG and [^3H]PC and the [^14C]TG-[^3H]PCbulletCETP complex was separated from the vesicles with a 70-cm Sephadex G-200 column. The radioactivity of [^14C]TG (circle) and [^3H]PC (up triangle) in the fractions were determined by scintillation counting of 0.5-ml aliquots.



Several Sephadex G-200 experiments with various amounts of WT or deletion mutant CETP mass were performed and the stoichiometry of TG in the TGbulletCETP complex and PC in the PCbulletCETP complex were determined as summarized in Table 3. The WT CETP bound 0.081-0.502 mol of TG/mol of CETP, whereas the deletion mutant CETP only bound 0.001-0.027 mol of TG/mol of CETP (Table 3). The results for binding of TG to WT were similar to those reported previously(11) ; the binding of less than equimolar TG was thought to represent nonspecific losses on the column. The ability of WT CETP to bind TG from the egg PC vesicles was clearly stronger (0.227 ± 0.150, mean ± S.D., n = 6) than that of the deletion mutant (0.025 ± 0.033, mean ± S.D., n = 4), representing about 11% of the binding ability of WT CETP. In contrast, the binding of PC to CETP was not changed or was even slightly increased for the deletion mutant; 6.1-8.5 molecules of PC were bound per mol of WT CETP (7.18 ± 1.16, mean ± S.D., n = 4), whereas 9.8-38.0 mol of PC were bound per mol of deletion mutant CETP (19.2 ± 12.9, mean ± S.D., n = 4). Therefore, the TG/PC mole ratio was 0.021 ± 0.007 (mean ± S.D., n = 4) for WT and 0.001 ± 0.001 (mean ± S.D., n = 4) for the deletion mutant (Table 3).



To determine if the recombinant CETP used in the lipid binding experiments retained bound lipid prior to incubation with vesicles, about 1 mg of WT CETP was extracted by the Bligh and Dyer procedure (29) , then analyzed by thin layer chromatography. The only bound lipid co-migrated with PC; there was no bound neutral lipid or phospholipid. Quantitation of the amount of PC in the lipid extract by choline assay indicated approximately 1 mol of PC/mol of CETP. The COOH-terminal deletion mutant could not be purified in sufficient amount to do a similar analysis.


DISCUSSION

Earlier mutagenesis experiments established that the COOH terminus of CETP (amino acids 468-476) comprises the epitope of a neutralizing mAb (TP2) and is necessary for normal neutral lipid transfer activity (17) . Intensive point mutagenesis between amino acid 446-476 demonstrated that substitutions involving bulky hydrophobic amino acid residues (particularly Leu,Phe,Leu,Phe, and Phe) led to defects in neutral lipid transfer and suggested that the general hydrophobicity of this region was important for neutral lipid transfer activity(18) . The finding that distinct amino acids were essential for lipid transfer activity or for binding the neutralizing mAb TP2 suggested the existence of an amphipathic helix between amino acids 465 and 476 with the polar face binding the mAb and the non-polar face involved in the neutral lipid transfer activity. However, these studies did not define the mechanism of impaired neutral lipid transfer activity of COOH-terminal deletion or substitution mutant proteins. The present detailed characterization of a representative COOH-terminal deletion mutant protein (Delta470-475) provides direct evidence for involvement of this region in neutral lipid binding by CETP, suggesting that this region forms part of a neutral lipid binding site on CETP.

Several possibilities were considered to explain the impairment of neutral lipid transfer of the COOH-terminal deletion mutant (Delta470-475). These included a global defect in conformation, an inability to bind to lipoproteins, a defect in dimerization, and an inability to bind to neutral lipids. Different properties of the COOH-terminal deletion mutant suggest that it does not have a global defect in conformation. The evidence includes relatively normal secretion levels of the deletion mutant by COS or CHO cells (which appear to have sensitive mechanisms to detect local or global alteration of structure of CETP(17) ), a similar pattern of fragments produced for WT and deletion mutant by partial protease digestion, and normal recognition of the COOH-terminal deletion mutant by CETP mAbs with non-COOH-terminal epitopes(17) .

Various lines of evidence indicate that the COOH-terminal deletion mutant does not have a major defect in binding to lipoprotein substrates. A binding defect seems to be unlikely in view of the normal or increased phospholipid transfer activity of the mutant protein(17) . Also, the mAb TP2 does not inhibit the binding of CETP to lipoproteins; in fact, this antibody tends to increase the binding of CETP to lipoproteins(22) . In the present study, kinetic evidence revealed similar apparent K(m) values for mutant and WT proteins for HDL, and only slightly higher apparent K(m) values for LDL. Direct binding assays confirmed normal binding of the COOH-terminal mutant CETP to HDL (Fig. 3). There is also no apparent indication for a major interaction between the PC portion of the liposome and the polar face of the amphipathic alpha-helix at the COOH terminus: 1) deletion of COOH-terminal amino acids did not have reduced PC binding, suggesting normal interaction between PC and CETP ( Table 3and Fig. 5); 2) WT CETP and the deletion mutant CETP bind HDL normally (Fig. 3); and 3) earlier studies suggested involvement of positive charges on CETP and negative charges on lipoproteins (presumably the phosphate at the head group of PC) in CETP-lipoprotein interactions(26) . In the deleted region there is no positive charge (-Asp-Phe-Leu-Gln-Ser-Leu-). These findings suggest that there is no major defect in binding of the mutant to its substrates, although a slight defect in binding to LDL is a possibility. In another study, several deletion mutants involving large segments of the COOH terminus of CETP were found to have impaired binding to HDL(16) . Thus these mutants have different properties than the COOH-terminal deletion mutant (Delta470-475) characterized herein, suggesting either that the large deletions produced global conformational effects on CETP or that adjoining regions of the CETP sequence outside amino acids 470-475 are involved in binding to HDL.

The recent discovery of a human CETP missense mutation with partially dominant effects on CETP and HDL levels suggest that there may be multimeric forms of CETP in vivo. For example, CETP may become multimeric within the secretory pathway(23) . However, gel filtration chromatography (Fig. 4) and chemical cross-linking with dimethyl suberimidate (not shown) indicated no major difference between WT and mutant CETP in terms of formation of self-associated species. Thus, it is unlikely that the COOH terminus constitutes a dimerization domain of CETP.

In earlier studies, the mAb TP2 was shown to cause decreased binding of neutral lipid by CETP, suggesting that the COOH terminus of CETP might form part of a neutral lipid binding site(11) . However, the surface area of a mAb is large relative to a molecule the size of CETP and point mutagenesis studies showed that the amino acids comprising the epitope of TP2 are different from those involved in CE transfer activity, indicating that the effects of the mAb are indirectly mediated(18) . The present study provides the first direct evidence for involvement of the COOH terminus in binding of neutral lipid by CETP. This was shown in CE and TG binding assays using egg PC/CE and egg PC/TG vesicles as neutral lipid donor(11) . At several different vesicle/CETP ratios there were major defects of CE and TG binding by the mutant CETP ( Fig. 4and Fig. 5, Table 2and Table 3), consistent with the kinetic studies showing a marked decrease in apparent V(max) for the deletion mutant ( Fig. 1and Fig. 2, Table 1). The residual neutral lipid binding and activity of the COOH-terminal mutant suggests either functional redundancy in the structure or a compensatory change in the mutant.

The common defect in binding CE and TG of the COOH-terminal deletion mutant suggests that CE and TG share a common binding mechanism and/or site on CETP. This view is supported by the observation that CE and TG compete for transfer, as shown using donors of variable CE/TG ratio (27) . By contrast, the normal or increased binding of PC by the COOH-terminal deletion mutant indicates different binding sites for neutral lipids and PC. Although it is not clear why CETP possesses multiple binding sites for PC, one can speculate that the multiple binding sites are involved in the binding of CETP to lipoproteins, a process involving CETP-PC interactions(26) . Purified recombinant CETP, derived by cell expression then hydrophobic and ion exchange chromatography contained a single PC molecule, apparently tightly bound. This single high affinity site could be mediating PC exchange activity. The other lower affinity sites may be occupied by PC molecules binding to less hydrophobic sites on the surface of CETP. This would be analogous to the PC transfer protein which has a single internal PC binding site mediating PC transfer activity(30) .

Thus, the hydrophobic COOH-terminal region of CETP, as deduced by point mutagenesis and CD spectroscopy of peptide(18) , amino acids 466-476 may form part of a neutral lipid binding site. This amphipathic helix at the C terminus 1) may form part of a lipid binding site on CETP or 2) help to mediate entry of a neutral lipid molecule into a nearby binding site. The COOH-terminal helix could form a flexible tail which penetrates the surface of the lipoprotein subsequent to lipoprotein binding. The hydrophobic face of the helix may come in contact with a neutral lipid molecule and bind it in a pocket comprised of the COOH-terminal helix and contiguous hydrophobic region. In a more general sense, the present findings substantiate the evidence that the binding of neutral lipid by CETP is an essential event in the lipid transfer mechanism(11, 14) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: CETP, cholesteryl ester transfer protein; CE, cholesteryl ester; TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; mAb, monoclonal antibody; WT, wild-type; SPA, scintillation proximity assay; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.


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