(Received for publication, May 11, 1994; and in revised form, October 7, 1994)
From the
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) 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
(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.
The plasma cholesteryl ester transfer protein (CETP) ()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 (470-475) 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
(
470-475) in order to examine the hypothesis that this
region of CETP forms part of a neutral lipid binding site.
HDL (d = 1.12-1.21 g/ml),
HDL
containing [
H]cholesteryl ester
([
H-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
PO
to 200 mM KH
PO
(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) .
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
[
H]CE in LDL at time t or
(at
equilibrium), and C
is the counts/min of
[
H]CE in HDL at t = 0. The
apparent K
and V
were
determined by fitting data to Lineweaver-Burk linear transform as
described by Cleland(21) .
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) C-TG (0.037 µCi) or with egg PC
vesicles containing [
C]TG and
[
H]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
[
H]CE binding to CETP by using Sephadex G-200
chromatography, scintillation counting, CETP ELISA, and SPA.
Figure 1:
CE transfer kinetics of WT CETP and
mutant CETP (470-475) 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
and K
values were derived.
, WT CETP;
, deletion
mutant
470-475. 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
and K
values.
, WT CETP;
, deletion mutant
470-475. Values are means ± S.D. (n =
4-6).
Figure 3:
HDL binding of WT CETP and mutant CETP
(470-475) analyzed by native polyacrylamide gel
electrophoresis. HDL at indicated concentrations (nanomoles/ml total
cholesterol) were incubated with WT CETP, deletion mutant CETP
(
470-475), 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
was identified in a parallel run of Sudan black stained HDL
and is indicated by the bracket at the right.
The minor high molecular weight band apparent in lanes containing the
mutant (
470-475) is due to nonspecific binding of
TP14.
Figure 4:
Cholesteryl ester binding of WT CETP (A) and mutant CETP (B). Twelve µg of purified WT
or deletion mutant CETP (470-475) were incubated with
[
H]cholesteryl oleate containing egg PC vesicles
(12 nmol) and CE
CETP complex was separated from the vesicles by
gel filtration on a 70-cm Sephadex G-200 column. The radioactivity of
[
H]CE (
) and CETP mass (
) in the
fractions were determined by scintillation counting and ELISA,
respectively.
The stoichiometry of CE and CETP in the
CECETP complex was calculated using the peak fraction containing
the CE
CETP 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 CE
CETP 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
(
470-475).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 (
470-475) 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
470-475. There
was only one active peak eluting at the same elution volume as the
[
H]CE
CETP 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).
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 [C]TG and
[
H]PC and the
[
C]TG-[
H]PC
CETP
complex was separated from the vesicles with a 70-cm Sephadex G-200
column. The radioactivity of [
C]TG (
) and
[
H]PC (
) 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 TGCETP
complex and PC in the PC
CETP 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.
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
(
470-475) 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
(470-475). 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 values for mutant and
WT proteins for HDL, and only slightly higher apparent K
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
-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 (
470-475)
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 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) .