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INTRODUCTION |
Lipoprotein metabolism is regulated by the coordinated action of
several factors, including lipolytic enzymes, lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein
(CETP)1 and phospholipid
transfer protein (PLTP) (1). Although PLTP was originally described as
a mediator facilitating phospholipid transfer between lipoprotein
particles, it is now recognized as a key factor in the intravascular
metabolism and remodeling of HDL (2, 3). PLTP facilitates the transfer
of different compounds, including phospholipids, lipopolysaccharides,
-tocopherol, and unesterified cholesterol, among lipoprotein classes
and between lipoproteins and cells (2). Besides its transfer activity, PLTP enhances formation of large-sized HDL and pre-
HDL, through apoAI release and HDL fusion (4). Although the physiological role of
PLTP has not been completely defined yet, recent in vivo studies conducted with PLTP transgenic and knock-out mice strongly suggest that PLTP contributes to the control and regulation of HDL
levels and to the generation of pre-
HDL, the initial acceptors of
cellular cholesterol (3).
PLTP belongs to the lipid transfer/lipopolysaccharide binding protein
(LT/LBP) family, together with CETP, lipopolysaccharide binding protein
(LBP), and bactericidal permeability increasing protein (BPI) (5). CETP
transfers neutral lipids, i.e. cholesteryl esters and
triglycerides between various lipoprotein fractions, and has limited
phospholipid transfer activity (6). LBP and BPI bind and transfer
bacterial endotoxins and lipopolysaccharides (LPS) and thus modulate
the host response to Gram-negative bacterial infection (7, 8).
At the sequence level, the four LT/LBP family members share ~20%
identity (5), suggesting a similar tertiary structure (9). The
three-dimensional structure of BPI was recently determined by x-ray
crystallography (10); this protein appears as a boomerang-shaped
molecule, which consists of two symmetrical barrels connected by a
linker region. Each barrel forms a hydrophobic pocket that can
incorporate one phosphatidylcholine molecule. The crystal structure of
BPI provides a useful framework for the modeling of the
three-dimensional structure of the other members of the LT/LBP family,
as well as for the investigation of their functional similarities and
differences. Several structure/function studies were recently aimed at
identifying the functional domains and elucidating the mechanism of
action of LBP, BPI, and CETP (11-15). These results demonstrated that
the activity of the lipopolysaccharide-binding and lipid transfer
proteins depends not only upon their ability to accommodate specific
lipid substrates but also upon their interaction with bacteria and/or
lipoproteins. The molecular interaction of LBP with surface-exposed
lipopolysaccharides on bacteria is critical for its activity (11), and
a cluster of positively charged amino acids (Arg-94, Lys-95, and
Lys-99) was recently identified as the
lipopolysaccharide-binding domain of this protein (16). Based on the
crystal structure of BPI, this cationic cluster is fully exposed at the
N-terminal tip of the boomerang-shaped LBP model (17).
Because PLTP activity and HDL metabolism are closely related (3), a
defective binding of PLTP to its HDL substrate would have direct
physiological consequences, and the factors that regulate the
association of PLTP with HDL are highly relevant to lipoprotein metabolism. Although the molecular and macromolecular specificity of
PLTP has been thoroughly investigated (18-21), the structure/function relationships of this protein have not been completely resolved (22),
and the molecular determinants regulating the association of PLTP with
lipoproteins have not been elucidated yet.
In the present study, we performed multiple sequence alignment between
members of the LT/LBP family and used the coordinates of crystallized
BPI to build a three-dimensional model for PLTP. The results suggest
that in PLTP, a cluster of hydrophobic residues, Tyr-45, Tyr-90,
Trp-91, Phe-92, Phe-93, and Tyr-94, substitutes for the positively
charged LPS-binding patch located on the surface of LBP and BPI. From
the engineered PLTP model and the characterization of PLTP mutants
obtained by site-directed mutagenesis, we propose that this
solvent-exposed hydrophobic cluster is critical for the interaction of
PLTP with its preferred substrate, HDL.
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MATERIALS AND METHODS |
Chemicals
Egg yolk phosphatidylcholine (PC), bovine brain
phosphatidylserine, 2,4,6-trinitrophenyl-phosphatidylethanolamine
(TNP-PE) (used as a quencher of pyrene fluorescence in liposomes), and human serum albumin were obtained from Sigma.
1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine
was purchased from Molecular Probes. Phospholipid concentrations were
measured by an enzymatic method using Biomérieux reagents.
Multiple Sequence Alignment and Modeling of Human PLTP
Thirteen sequences of BPI, LBP, CETP, and PLTP from various
species were aligned using the ClustalW program (23) and the Hidden
Markov Model method SAM-T99 (24, 25), both available on the Web.
The SAM-T99 sequence alignment was plotted using the Alscript software
(26), and the conservation index per residue was calculated using the
AMAS program (27) also available on the Web. The working model for the
human PLTP structure was built based on this alignment, and on the
Protein Data Bank coordinates of the BPI protein (10), by using the
HOMOLOGY software of the Insight II package (Molecular Simulations, San
Diego, CA). The quality of the model was checked using WHATCHECK (28)
and PROCHECK (29). When aligned with the BPI template, the PLTP
sequence contains four deletions defined as loops 1, 3, 4, and 5. They were replaced by the best fitting loops found by an automatic loop
search of a nonredundant Protein Data Bank data base (Fig. 1). Loop 2 contains no insertions or deletions but falls within a sequence region
with weak similarity with BPI; its coordinates were copied from the BPI
template. Nonconserved side chains were replaced by their best-fitting
rotamers using the program SCWRL (30). The model was soaked with water,
and the solvated model was then energy-minimized in steps with
decreasing constraints on the conserved backbone and side chains using
CDiscover (Molecular Simulations) with the CVFF force field and the
cell multipole method. The model was relaxed by steepest descent and
conjugate gradient minimization until a convergence of <0.5
kcal/mol/Å2 was achieved.
The conformation of loops 1, 2, and 4 in the N-terminal "tip" of
PLTP was further studied by molecular dynamics simulation as follows:
all residues within 5 Å of loops 1, 2, and 4 were allowed to move,
while the rest of the PLTP model was fixed. A 16-Å layer of water was
created around the mobile PLTP residues, and the model was
energy-minimized to converge at <0.5 kcal/mol/Å2. During
the molecular dynamics simulation, the system was equilibrated at 900 K
in 5 ps, kept at 900 K for 20 ps, followed by a 10-ps dynamics
simulation at 300 K. The final structure was energy-minimized by
steepest descent and conjugate gradient minimization.
Site-directed Mutagenesis, Subcloning, and Transfection of
COS-1 Cells
Oligonucleotides containing the desired mutations were purchased
from Eurogentec (Seraing, Belgium). Each of the Tyr-45, Tyr-90, Trp-91,
Phe-92, Phe-93, and Tyr-94 residues was mutated to an alanine, and
restriction sites were introduced by silent mutagenesis to verify the
presence of each mutation prior to subcloning.
Mutagenesis of the PLTP cDNA was carried out by using the
Stratagene QuikChange site-directed mutagenesis kit. The PLTP14 vector
was a kind gift from Drs. J. J. Albers and A.-Y. Tu (Seattle, WA)
and was used as the template for PCR. The PCR reactions were set up
according to instructions of the manufacturer using the following
thermal cycling conditions: 95 °C for 1 min, 55 °C for 1 min, and
68 °C for 16 min (2 min/kb of plasmid length). After PCR and
DpnI digestion of the parental dam-methylated template (2 h
at 37 °C), the mutant plasmids were transformed into XL1 blue
Escherichia coli cells and resulting colonies were screened by restriction analysis.
Transient expression of the PLTP cDNA into COS-1 cells was
carried out by transfection with a mixture of 2 µg of the recombinant plasmids and 8.6 µg of LipofectAMINE (Life Technologies, Inc.) in
serum-free DMEM. After 5-h incubation at 37 °C, cells were washed
and grown overnight in complete DMEM. DMEM was then replaced by Optimem
(Life Technologies, Inc.), and media were harvested between 24 and
96 h. After 10-min centrifugation at 1000 rpm, the media were
stored at
20 °C in 1-ml aliquots.
Quantification of Expressed Mutants by Western Blot
Secretion of PLTP mutants into the media of the transfected
COS-1 cells was assessed by SDS-polyacrylamide gel electrophoresis separation in 10% acrylamide and by Western blotting of the media after 3-fold concentration by acetone precipitation. Serial dilutions of a plasma protein fraction with a known PLTP concentration, quantified by a previously described enzyme-linked immunosorbent assay
(31), were used as standards for quantification of PLTP in the cell media.
After electrophoresis, the proteins were transferred to polyvinylidene
difluoride membranes (Immobilon P transfer membranes, Millipore), which
were subsequently blocked with 50 g/liter nonfat dry milk in a
phosphate-buffered saline buffer containing 1 g/liter Tween 20, for
1 h at room temperature. The membranes were incubated overnight at
4 °C with rabbit anti-PLTP antibodies (a generous gift from Dr. L. Lagrost, Dijon, France) diluted in phosphate-buffered saline containing
10 g/liter human serum albumin. After incubation with horseradish
peroxidase-labeled anti-rabbit IgG antibodies (Dako), blots were
revealed by chemiluminescence (ECL kit, Roche Molecular Biochemicals)
and scanned on a densitometer.
Isolation of Lipoproteins
Very-low density (VLDL), low density (LDL), and high density
(HDL) lipoproteins were isolated at d < 1.019 g/ml,
1.019 < d < 1.063 g/ml, and 1.07 < d < 1.21 g/ml, respectively, according to standard
protocols (32). Densities were adjusted by the addition of KBr. All
isolated lipoproteins were dialyzed overnight against a Tris/HCl buffer
(10 mM Tris, 1 mM EDTA, 3 mM
NaN3, pH 7.4).
PLTP Activity Assays
Liposomes/HDL Transfer Assay--
PLTP activity was monitored by
a fluorometric assay (20) in the supernatant of cells transfected with
wild-type (WT) or mutated PLTP, or of nontransfected cells (mock). In
this assay, the rate of phospholipid transfer is monitored by the
increase of pyrene monomer fluorescence intensity upon transfer of
pyrene-labeled phosphatidylcholine from quenched donors to unquenched
acceptors. The molar composition of the donor liposomes consisted of
68% egg yolk PC, 17% phosphatidylserine (PS), 2.5% pyrene-labeled PC
(1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine), and 12.5% TNP-PE (the quencher of pyrene fluorescence). 12 nmol of
donor liposomes containing quenched pyrene-PC-containing liposomes was
mixed with 80 nmol of unlabeled HDL used as acceptors, in the presence
of cell media containing PLTP. Pyrene-PC transfer was accompanied by an
increase of the monomer fluorescence intensity (excitation wavelength,
342 nm; emission wavelength, 378 nm) due to release and dequenching of
the pyrene probe from the liposomes. The initial slope of the
fluorescence intensity increase as a function of time represented a
measure of the PLTP activity in the medium. The activity measured with
mock medium was close to the spontaneous transfer activity and taken as
a blank value for all activity determinations. In this assay, the
volume of cell medium was adjusted to yield a value within the linear
range of the fluorescence curve. The assay was calibrated by measuring the rate of transfer of 14C-radiolabeled
phosphatidylcholine in a plasma sample, which was subsequently used as
a standard for the fluorometric measurements.
Liposomes/VLDL and Liposomes/LDL Transfer Assays--
These
assays were as described above, except that acceptor HDL were replaced
by VLDL or LDL, containing an equivalent amount (80 nmol) of
phospholipids. The volume of medium used for measurements was taken
within the linear range of the activity curve.
HDL/HDL Transfer Assay--
We developed an "inter-HDL"
transfer system in which pyrene-labeled HDLs were used as phospholipid
donors while unlabeled HDLs served as acceptors.
Pyrene-labeled HDL were prepared by ethanol injection of
pyrene-phosphatidylcholine. Briefly, 300 nmol of
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine were dissolved in ethanol and injected into HDL (3 µmol of
phospholipids) under continuous stirring. The mixture was then
incubated for 6 h at 37 °C while mixing, and the HDL were
reisolated by ultracentrifugation between densities 1.07 and 1.21 g/ml.
60-70% of the label was incorporated into HDL, corresponding to 6.5 mol % of HDL phospholipids.
For PLTP activity measurements, pyrene-labeled HDL (40 nmol of
phospholipids) were mixed with unlabeled HDL (160 nmol of
phospholipids) in the presence of either control or PLTP-containing
cell culture media. Fluorescence intensities of the monomer and of the
excimer were recorded at 378 and 475 nm, respectively, after 60-min
incubation at 37 °C. The monomer/excimer (M/E) intensity ratio
increased linearly as a function of both incubation time and PLTP
amount. The increase in the M/E fluorescence intensity ratio thus
represented a reliable measure of "inter-HDL" PLTP transfer
activity. M/E intensity ratios measured with mock medium were used as
blank values and subtracted for specific activity calculations.
HDL Size Conversion Activity of PLTP
Salt-sucrose Density Gradient Ultracentrifugation of HDL-PLTP
Mixtures--
Conversion of HDL particles was analyzed by incubating
either control or PLTP-containing cell culture media with HDL particles (5 µg of protein) for 30 h at 37 °C. The final incubation
volume was 0.6 ml, and the ratio of PLTP activity/HDL concentration was similar to that in plasma. After incubation, HDL subclasses were separated by density gradient ultracentrifugation in a salt-sucrose gradient. The gradient was prepared in 12-ml polyallomer tubes (Beckman) and consisted of 0.5 g of sucrose, 5 ml of 4 M NaCl, and 500 µl of sample, on which 6.2 ml of 0.67 M NaCl was layered. Samples were centrifuged for 66 h
in a SW 41 Ti rotor (Beckman) at 10 °C and 38,000 rpm. Fractions
(500 µl) were collected using an auto-densiflow system
(Searle, Fort Lee, NJ). Apolipoprotein AI (apoAI) was quantified in the
fractions by enzyme-linked immunosorbent assay (33). The percentage of
apoAI released in the bottom fraction (d > 1.20 g/ml)
of ultracentrifuged mixtures was used as a measure of the HDL size
conversion activity of PLTP.
Determination of HDL Size by Native Polyacrylamide Gradient Gel
Electrophoresis--
The size distribution of HDL was determined by
electrophoretic analysis on 4-20% polyacrylamide gradient gels,
according to the general procedure previously described (34). The gel
was run at 70 V during 1 h and then at 150 V for 20 h, in a
90 mM Tris-HCl, 80 mM boric acid, pH 8.3, buffer containing 3 mM Na-EDTA and 3 mM
NaN3. At the end of the electrophoresis, gels were stained with Coomassie Brilliant Blue G. The size distribution profiles of HDL
were obtained by analysis of stained gels on a Bio-Rad GS-670 imaging
densitometer. The apparent diameters of HDL were determined by
comparison with a calibration curve constructed with albumin (7.1 nm),
lactate dehydrogenase (8.2 nm), ferritin (12.2 nm), and thyroglobulin
(17.0 nm).
Statistical Analysis
Data are expressed as mean ± S.D. or mean ± S.E., as
indicated in the legend of the figures. The statistical significance of
differences between data means was determined using the Student's t test.
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RESULTS |
Multiple Sequence Alignment and Building of the PLTP Model--
In
the aligned sequences of human CETP, LBP, BPI, and of pig, mouse, and
human PLTP, shown in Fig. 1, only 22 residues are strictly conserved among all sequences. The sequence
alignment of human PLTP and BPI shown in Fig. 1 is practically
identical to that used by Huuskonen et al. (22) for the
modeling of PLTP. As described by these authors (22), most secondary
structure elements are well conserved within this protein family.
Compared with the human BPI sequence, PLTP contains four deletions,
which occur in the surface loops connecting
-strands and
-helices. Based on the BPI coordinates, a model was built for human
PLTP (Fig. 2A). The central
part of the boomerang-shaped model structure of PLTP, containing the
linker domain and the hypothetical lipid-binding pockets (22), is
better conserved than the boomerang tips, which contain three of
the four deletions. The N-terminal boomerang tip of BPI and LBP,
consisting of loops 1, 2, and 4 and of helix
2 (as defined in Fig.
1), contains several positively charged residues. These residues are
not conserved in PLTP, because the corresponding region contains a
hydrophobic cluster of aromatic residues, Tyr-45, Tyr-90, Trp-91,
Phe-92, Phe-93, and Tyr-94 (Fig. 2A). Fig. 2 (B
and C) shows the electrostatic surface potentials around the
BPI crystal structure and the PLTP model. Fig. 2 (D and
E) shows the corresponding surface hydrophobicity around BPI and PLTP. The most striking difference between the BPI structure and
the PLTP model lies within the N-terminal boomerang tip of the
proteins. In this region, BPI shows a strongly positive electrostatic potential and low surface hydrophobicity (Fig. 2, B and
D), whereas the corresponding region in PLTP has high
surface hydrophobicity and a weakly positive electrostatic potential
(Fig. 2, C and E). This is due to the replacement
of positively charged residues of BPI by hydrophobic residues in PLTP
(Fig. 1). Because the positively charged residues in the N-terminal
boomerang tip of LBP are involved in binding of negatively charged LPS,
we hypothesized that the corresponding hydrophobic region in PLTP might
play a role in substrate binding through hydrophobic interactions. Fig.
3 shows the conformation of the
N-terminal tip of the PLTP model after optimization by molecular
dynamics simulation. The aromatic residues Tyr-45, Tyr-90, Trp-91,
Phe-92, Phe-93, and Tyr-94 are solvent-exposed, with solvent
accessibility values of 0.56, 0.33, 0.59, 0.88, 0.65, and 0.52, respectively. These residues were separately mutated to an alanine.

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Fig. 1.
Alignment of the pig, mouse, and human PLTP
sequences with those of human CETP, LBP, and BPI. Homologous or
conserved residues among the LT/LBP family members are in
gray and black boxes, respectively. Residue
numbers correspond to human PLTP. The positions of the five loops in
the PLTP sequence are indicated by a dotted line. The
position of secondary structure elements are indicated; -helices are
numbered from 1 to 9, and -sheets are drawn in
capital letters. The residues mutated in this study are
indicated with an asterisk under the
alignment.
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Fig. 2.
Structural model of human PLTP and molecular
surface representations of human BPI (B and
D) and PLTP (C and
E). A, PLTP model, showing the
mutated aromatic residues in the N-terminal tip. In B and
C: electrostatic surface potentials, calculated with Delphi
(52). The positive and negative electrostatic surface potentials of BPI
(B) and PLTP (C) are blue and
red, respectively. In D and E: The
residues were colored according to the Wimley and White (41)
hydrophobicity scale. The hydrophilic and hydrophobic surface areas of
BPI (D) and PLTP (E) are blue and
red, respectively. White arrows in B
and E point to the positively charged cluster of BPI and to
the hydrophobic cluster of PLTP, respectively.
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Fig. 3.
Structure of the N-terminal tip of the PLTP
molecule. This figure shows the clustering of aromatic residues
Tyr-45, Tyr-90, Trp-91, Phe-92, Phe-93, and Tyr-94 at the N-terminal
tip of the boomerang-shaped PLTP and the solvent-exposed orientation of
Trp-91, Phe-92, and Phe-93 residues. The indexes of solvent exposure of
the residues are indicated in parentheses.
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Expression of Recombinant Human PLTP by COS-1 Cells--
The
kinetics of PLTP secretion by COS-1 cells were monitored by assaying
PLTP activity in the cell culture media 24, 48, 72, and 96 h after
transfection. As shown in Fig. 4, a
time-dependent increase in PLTP activity was observed in
the culture media of transfected cells, with a maximal increase 72-96
h post-transfection. As PLTP mass was only detected 96 h after
transfection by Western blot analysis of cell culture media, and all
media were harvested 4 days post-transfection. The specific activity of
recombinant wild-type (WT) PLTP expressed as activity/µg of PLTP
protein was ~60 nmol/µg/h, compared with about 100 nmol/µg/h for
plasma PLTP.

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Fig. 4.
Kinetics of PLTP secretion by transiently
transfected COS-1 cells. Aliquots of the cell culture medium of
PLTP-transfected COS-1 cells were removed 24, 48, 72, and 96 h
post-transfection. The secretion of PLTP by the transfected cells was
followed by measuring phospholipid transfer activity from quenched
pyrene-labeled liposomes to acceptor HDL in the presence of control
medium (mock medium) or PLTP-containing medium. PLTP activities
(expressed in nmol/ml/h) are the means ± S.D. of three
determinations and were obtained after subtraction of control
values.
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All PLTP mutants were efficiently expressed, at levels above 65% of WT
PLTP. The concentration of recombinant PLTP in the cell culture media
was around 1 µg/ml (0.9 ± 0.1, 0.6 ± 0.2, 0.6 ± 0.2, 1.0 ± 0.1, 1.0 ± 0.2, 0.9 ± 0.2, and 0.6 ± 0.1 µg/ml for WT, Y45A, Y90A, W91A, F92A, F93A, and Y94A mutants, respectively).
Phospholipid Transfer Activity of WT and PLTP Mutants--
The
kinetic parameters for phospholipid transfer from liposomes to various
lipoprotein substrates were measured for the engineered PLTP mutants.
We first measured PLTP-mediated transfer of pyrene-labeled
phosphatidylcholine from donor liposomes to HDL acceptors. The specific
transfer activities of all mutants were calculated based upon the PLTP
concentration in the media. Except for the Y45A and Y94A variants,
whose specific activity was not statistically different from that of WT
PLTP, all single point mutations substantially decreased PLTP-specific
activity in the liposome/HDL transfer assay (Fig.
5A). The activity of the Y90A
variant decreased most, because it amounted only to 30% that of WT
PLTP. Mutations of residues Trp-91, Phe-92, and Phe-93 decreased the
specific PLTP activity by up to 60%.

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Fig. 5.
Specific phospholipid transfer activity of
wild-type and PLTP mutants toward HDL particles. Phospholipid
transfer activity was measured in the cell medium 96 h
post-transfection, either from liposomes toward HDL (A) or
between HDL particles (B). Specific phospholipid transfer
activities were calculated by taking into account the expression levels
of the various transfectants. The results are mean values ± S.E.
of three independent experiments and are expressed as percentages of
the activity of wild-type PLTP with *, p < 0.05; **,
p < 0.01; and ***, p < 0.005 compared
with WT PLTP activity (Student's t test).
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To determine whether the reduced activity of the mutants was primarily
due to their decreased interaction with liposomes and/or with HDL
particles, we modified the liposome/HDL transfer assay into an HDL/HDL
transfer assay. Pyrene-labeled HDL were used as phospholipid donors,
unlabeled HDL were used as acceptors, and PLTP activity was quantified
by measuring the decrease in excimer/monomer fluorescence intensity in
HDL. The specific activities of WT and PLTP mutants in this assay
system are shown in Fig. 5B. They are well correlated with
the activities measured with the liposomes/HDL transfer system
(r2 = 0.80). Interestingly, the relative
specific activities of the deficient mutants were slightly reduced in
the HDL/HDL compared with the liposomes/HDL assay, suggesting that the
functional defect of the mutants primarily lies in a decreased
interaction with HDL substrates.
To investigate whether these clustered aromatic residues might also be
involved in the interaction of PLTP with VLDL and LDL, the specific
phospholipid transfer activity of the WT PLTP and of PLTP mutants was
determined by using liposomes as donors and VLDL or LDL particles as
acceptors. As shown in Table I, only the
Y90A mutant showed a significantly reduced ability to transfer phospholipids from liposomes to VLDL or LDL; all other PLTP mutants displayed a normal or even slightly increased specific phospholipid transfer activity toward VLDL and LDL, suggesting that mutation of
Trp-91, Phe-92, and Phe-93 specifically impairs the interaction of PLTP
with HDL particles.
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Table I
Relative specific activity (% of wild-type, mean ± S.E.) in cell
media from wild-type and mutant PLTP transfectants
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Effect of Salt Concentrations on PLTP Activity--
To investigate
the contribution of electrostatic and hydrophobic forces to the
activity of WT PLTP and of PLTP mutants, the effect of ionic strength
on phospholipid transfer from donor liposomes to either VLDL, LDL, or
HDL was measured. PLTP activity was hardly detectable when the assay
was performed in 1 mM EDTA, however, it was markedly
enhanced in the presence of 10 mM Tris/HCl buffer, as
previously observed for CETP (35). In assays carried out in the
presence of 10 mM Tris-HCl buffer (Fig.
6A), phospholipid transfer
from liposomes to HDL was markedly increased when the NaCl
concentration was decreased from 150 to 50 mM. Phospholipid transfer from liposomes to VLDL and LDL particles was also enhanced upon decreasing NaCl concentrations, although to a lesser extent than
with HDL acceptors. The effect of decreasing NaCl concentrations on
phospholipid transfer from liposomes to HDL was more pronounced for the
W91A, F92A, and F93A PLTP mutants, compared with WT PLTP (Fig.
6B). The increase in phospholipid transfer from liposomes to
VLDL and LDL was similar for all PLTP variants (results not shown). The
phospholipid transfer activity from liposomes to VLDL, LDL, or HDL
particles measured for the Y94A mutant was similar to that of WT PLTP
(Fig. 6B). In experiments performed with egg PC liposomes
instead of PC/PS liposomes, a similar trend was observed (data not
shown).

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Fig. 6.
Effect of sodium chloride concentration on
PLTP-mediated phospholipid transfer. A, phospholipid
transfer was measured in assay mixtures containing donor liposomes (12 nmol of phospholipids), acceptor lipoproteins (80 nmol of
phospholipids), and control or PLTP-containing cell culture medium.
NaCl concentrations were adjusted with 10 mM Tris/NaCl
solutions. Each point is the mean of two determinations.
PLTP activity in the presence of acceptor VLDL ( ), LDL ( ), and
HDL ( ) particles. B, phospholipid transfer was measured
in assay mixtures containing donor liposomes (12 nmol of
phospholipids), acceptor HDL (80 nmol of phospholipids), and control or
PLTP-containing cell culture medium. The experiment was conducted
either with WT PLTP or with PLTP mutants. Each point is the
mean of two determinations: WT PLTP ( ); W91A mutant (X); F92A mutant
( ); F93A mutant ( ); Y94A mutant ( ).
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HDL Size Conversion by WT and PLTP Mutants--
Changes in the
size distribution of HDL particles by recombinant wild-type and PLTP
mutants were investigated. Isolated plasma HDL were incubated with
either mock- or PLTP-transfected culture media, containing identical
amounts of WT PLTP or PLTP mutants, for 30 h at 37 °C. The
mixtures were then subjected to density gradient ultracentrifugation,
to determine the density of the incubated HDL, together with the amount
of released apoAI. As shown in Fig. 7,
HDL particles incubated in the presence of control medium were isolated
in the 1.07-1.15 g/ml density range; after incubation with PLTP, the
HDL peak was shifted to lower densities (1.05-1.10 g/ml), illustrating
formation of apoAI-depleted, larger-sized HDL particles. Concomitant
release of lipid-poor apoAI was observed in the d > 1.20 g/ml fraction. The apoAI release in the bottom fraction of the
gradient was used as a quantitative measure of PLTP conversion
activity. Table II shows the effect of
PLTP concentration on apoAI release. The experimental data were fitted
to a linear equation (y = 0.41x + 10, r2 = 0.86), which was used to calculate the
specific conversion activities of the PLTP mutants. As shown in Table
III, mutants Y90A, W91A, F92A, and F93A
only displayed 46, 37, 26, and 61% of WT specific conversion activity,
whereas Y45A and Y94A mutations did not markedly impair the HDL size
conversion activity of PLTP. The HDL size conversion was also verified
by native polyacrylamide gradient gel electrophoresis as described by
other groups (36, 37). Changes in HDL size upon incubation with WT PLTP
are represented in Fig. 8. The sizes of
the HDL subfractions measured after incubation with mock medium were:
58% particles with a diameter of 9.6 nm; 42% of 8.5 nm, and no
smaller sized particles (7.8 nm). After incubation with WT PLTP, 63%
of the particles had an average size of 9.6 nm; 25% of 8.5 nm, and
12% had a size of 7.8 nm. This size distribution is in accordance
with the changes in density of the HDL particles as described in
Fig. 7.

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Fig. 7.
Density gradient ultracentrifugation profile
of HDL incubated with mock medium ( ) or with PLTP-containing medium
( ). The position of the HDL peaks was identified by measuring
the apoAI concentration (µg/ml) in each fraction.
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Table II
Size conversion activity of wild-type recombinant PLTP as assessed
by apoAI release
Isolated HDL were incubated with control or PLTP-containing cell
culture media for 30 h at 37 °C. The percentage of apoAI
released in the bottom fraction of ultracentrifuged mixtures was used
to quantitate the HDL size conversion activity of recombinant PLTP.
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Table III
Size conversion activity of wild-type and mutant forms of PLTP as
assessed by apoAI release
Isolated HDL were incubated with control or PLTP-containing cell
culture media for 30 h at 37 °C. The volumes of cell culture
media were adjusted so as to contain the same amounts of WT PLTP or
PLTP mutants. The percentage of apoAI released in the bottom fraction
of ultracentrifuged mixtures was used to quantitate the HDL size
conversion activity of PLTP.
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Fig. 8.
Effect of incubation with PLTP on the HDL
size distribution as determined by native density gradient (4-20%)
gel electrophoresis. HDL were incubated with mock medium or with
medium containing WT PLTP as described under "Materials and
Methods." 1, a size of 9.6 nm; 2, 8.5 nm; and
3, 7.8 nm.
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DISCUSSION |
The molecular and macromolecular specificity of PLTP has recently
been thoroughly investigated (18-21). Phospholipid transfer activity
depends upon the size, composition, fluidity (19, 21), and
electrostatic charge (38) of the lipoprotein substrates. HDL particles
are the preferential substrates for PLTP (21). Because the PLTP
structure has not yet been determined, the molecular basis for PLTP
specificity is not known. The results reported here are a new
experimental link between PLTP structure and macromolecular specificity.
We first performed a multiple sequence alignment to assess the degree
of residue conservation among the members of the LT/LBP family. Residue
conservation within members of this family is low, around 20%.
Residues in the secondary structure elements of BPI are, however, well
conserved, enabling molecular modeling of PLTP, using BPI as a
template. As described by Huuskonen et al. for PLTP (22) and
by Bruce et al. for CETP (15), the major structural elements
and the functional lipid-binding pockets inside the anti-parallel
barrel structures are well conserved. Because our sequence alignments
are practically identical to those of Huuskonen et al. (22),
both PLTP models share high similarity.
In this study, a cluster of positively charged residues was identified
on the N-terminal boomerang tip of BPI. In LBP, these residues were
shown to be critical for its interaction with bacterial lipopolysaccharides (16). The multiple sequence alignment indicated that these residues are not conserved in PLTP. Residues Lys-95, Arg-96,
and Lys-99 in BPI are replaced by Tyr-90, Trp-91, and Tyr-94 in PLTP.
Analysis of the PLTP model showed that these residues, together with
the adjacent residues Phe-92 and Phe-93, as well as Tyr-45, form a
highly hydrophobic patch exposed at the N-terminal tip of the PLTP
molecule. We therefore speculated that at least some of these
hydrophobic residues might play a specific role in the activity of
PLTP, and we tested this hypothesis by site-directed mutagenesis.
Mutation of residues Trp-91, Phe-92, and Phe-93, but not Tyr-45 and
Tyr-94, decreased the specific phospholipid transfer activity of PLTP
from donor liposomes to HDL by up to 60%. The decreased transfer
activity of the PLTP mutants on HDL was confirmed in a new assay
system, where HDL served both as phospholipid donors and acceptors for
PLTP-mediated transfer. In contrast, when VLDL or LDL particles were
used as acceptors in the transfer assay, the specific PLTP activity of
the five mutants was not different from that of WT PLTP. This
observation indicates that the nature of acceptor lipoproteins is
critical for phospholipid transfer by the W91A, F92A, and F93A PLTP
mutants. Mutation Y90A affected PLTP-mediated transfer of phospholipids
to VLDL, LDL, and HDL lipoproteins. Because Tyr-90 is less
solvent-exposed than the other mutated residues in the PLTP model (Fig.
3), this residue is more likely involved in stabilizing hydrophobic
intramolecular interactions, whose impairment might account for the
loss of PLTP activity of the Y90A mutant, in all assays.
Because residues Trp-91, Phe-92, and Phe-93 seem critical for
phospholipid transfer to HDL particles, they might also influence the
HDL size conversion activity of PLTP. We observed a close parallel
between HDL conversion and phospholipid transfer activities of the
mutants, suggesting that both processes are linked. This observation
strongly supports the sequential mechanism recently proposed by Lusa
et al. (39), in which PLTP-mediated phospholipid transfer is
a prerequisite for HDL fusion. According to this hypothesis, redistribution of phospholipid molecules among HDL particles would induce dissociation of apoAI molecules from HDL surface, followed by
destabilization of surface-depleted HDL particles. This would enhance
HDL fusion into larger particles. The cluster of hydrophobic residues
mutated in this study might play a role in the displacement of apoAI
from the HDL surface, possibly by penetrating deeply into the outer
phospholipid layer and increasing surface pressure on HDL.
The detailed mechanism of PLTP-mediated phospholipid transfer is still
unknown. Studies of the mechanism of action of the related CETP protein
(1, 6, 35) have stressed the importance of two events for lipid
transfer activity: (i) interaction of the transfer protein with donor
and acceptor substrate particles and (ii) binding and accommodation of
substrate lipid molecules in CETP. The decreased activity of the PLTP
mutants generated in this study might be due either to decreased
interactions with donor and/or acceptor lipoprotein particles or to a
decreased ability to accommodate phospholipids in the lipid-binding
pockets (22). A decreased interaction of the PLTP mutants with
phospholipids would have affected phospholipid transfer activity from
liposomes to all lipoprotein acceptors. In contrast, the transfer
activity of the mutants was close to that of WT PLTP for liposomes/VLDL and liposomes/LDL assays. This suggests that all mutants (except Y90A)
interact normally with donor liposomes, and efficiently transfer
phospholipids from these particles. Mutation of residues Trp-91,
Phe-92, or Phe-93, which appear as the most protruding and
solvent-exposed residues in the optimized conformation of the PLTP
N-terminal tip (Fig. 3), prevented PLTP-mediated transfer of
phospholipids toward HDL. This observation suggests that these residues
might be specifically involved in hydrophobic interactions between PLTP
and HDL particles.
We previously showed that electrostatic interactions can also
contribute to PLTP-HDL association (38). The relative importance of
electrostatic and hydrophobic interactions for HDL-PLTP association was
investigated by performing phospholipid transfer measurements at
varying salt concentrations. We first observed that addition of
Tris-HCl ions to the assay mixtures increased PLTP transfer activity,
probably through increased hydrophobic interactions (35). Moreover,
increasing electrostatic interactions by decreasing NaCl concentrations
below physiological range also enhanced phospholipid transfer,
especially from liposomes (both PC only and PC/PS liposomes) to HDL.
This effect was most pronounced for the W91A, F92A, and F93A mutants,
where the contribution of hydrophobic interactions with HDL had been
reduced by mutations of the aromatic residues to alanine. These
observations thus support the assumption that Trp-91, Phe-92, and
Phe-93 contribute to hydrophobic interactions between PLTP and HDL
particles, that constitute a rate-limiting factor for PLTP activity at
physiological ionic strength. At lower ionic strength, electrostatic
interactions might compensate for the impaired hydrophobic interactions
in the engineered mutants.
Residues Trp-91, Phe-92, and Phe-93 might play a role in the
interaction of PLTP with either apolipoprotein AI or other HDL protein
components (40). However, because aromatic residues are known to be
frequently involved in protein-lipid interactions (41), the mutated
aromatic residues in PLTP probably contribute to hydrophobic
interactions with HDL lipids. Lookene et al. (42) recently
demonstrated the contribution of aromatic residues for the interaction
of lipoprotein lipase with an interfacial substrate. The interfacial
binding domain of snake venom phospholipase A2 also contains several
solvent-exposed hydrophobic residues. Depending on their location and
side-chain orientation, aromatic Trp, Tyr, and Phe residues can
significantly contribute to interfacial binding (43). Studies of the
PLA2 family showed that, although electrostatic interactions between basic residues and anionic phospholipids account
for high affinity binding of some of these enzymes, hydrophobic residues in the interfacial binding domain enhance interaction with
neutral lipid substrates (44, 45). In analogy with the phospholipase A2
family, the presence of a solvent-exposed hydrophobic cluster is a
specific feature of PLTP within the LT/LBP family, whereas the
corresponding positively charged domains constitute the
lipopolysaccharide-binding domain in LBP and BPI (16).
Activity measurements performed with the PLTP mutants suggest that the
nature of lipoprotein substrates, and especially their physicochemical
properties, determines relative transfer activities. Differences in the
molecular packing of surface lipids were shown to determine the
lipoprotein specificity of apolipoproteins (46, 47) and of lipolytic
enzymes (48, 49). Ibdah et al. (50) demonstrated a denser
phospholipid packing on the surface of LDL relative to HDL particles.
Phospholipid packing might therefore influence the depth of insertion
of the bulky hydrophobic residues of PLTP between the polar headgroups
of the phospholipid monolayer. Binding of lipolytic enzymes to their
substrates can potentially cause protein denaturation. Therefore, the
structure of these proteins is stabilized against denaturation by
disulfide bridges in phospholipases A2 and by buried salt bridges in
fungal lipases. In the PLTP model, a buried salt bridge was detected
between the conserved residues arginine 86 and aspartic acid 94, which
are on the adjacent beta-strands
4 and
5. This salt bridge, which is not conserved in other members of the LT/LBP family, might help to
stabilize the PLTP structure. This hypothesis could be tested
experimentally, by site-directed mutagenesis and expression of the mutants.
In conclusion, the results of the present study show that phospholipid
transfer and size conversion activities of PLTP on HDL are influenced
by mutation of three aromatic residues (Trp-91, Phe-92, and Phe-93),
predicted to be solvent-exposed in an hydrophobic patch located at the
N-terminal tip of the molecule. These protruding residues might be
critically involved in the interaction of PLTP with HDL. Huuskonen
et al. (22) recently demonstrated that the interaction of
PLTP with HDL involves Leu-286, which is located in the central part of
the concave surface of PLTP. In contrast, mutations in the N-terminal
pocket decreased phospholipid transfer activity but not HDL binding
(22). Deletion of the 30 C-terminal residues 464-493 did not affect
secretion of PLTP but decreased the activity (51). The contribution of
Phe-464, located at the entrance of the N-terminal lipid-binding
pocket, for the proper conformation of the C-terminal tail of PLTP was
clearly demonstrated in the same study (22). Taken together, these data
and our present observations support the existence of at least two
distinct HDL-binding sites on PLTP, suggesting that PLTP-mediated lipid
transfer occurs through formation of collisional ternary complexes
rather than through a "shuttle" mechanism. Moreover, PLTP-mediated
size conversion of HDL particles, which results from particle fusion,
might be enhanced by the bridging ability of PLTP through
inter-particle contacts in the aqueous phase. Experimental
determination of the kinetics of PLTP activity could contribute toward
the unraveling of the structure/function relationship of PLTP.