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J. Biol. Chem., Vol. 276, Issue 29, 26898-26905, July 20, 2001
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From the § Lipid Research Laboratory, The Hanson Centre,
Adelaide, South Australia 5000, Australia,
Received for publication, November 28, 2000, and in revised form, April 10, 2001
Phospholipid transfer protein (PLTP) remodels
high density lipoproteins (HDL) into large and small particles. It also
mediates the dissociation of lipid-poor or lipid-free apolipoprotein
A-I (apoA-I) from HDL. Remodeling is enhanced markedly in triglyceride (TG)-enriched HDL (Rye, K.-A., Jauhiainen, M., Barter, P. J., and
Ehnholm. C. (1998) J. Lipid. Res. 39, 613-622). This study defines the mechanism of the remodeling of HDL by PLTP and determines why it is enhanced in TG-enriched HDL. Homogeneous populations of
spherical reconstituted HDL (rHDL) containing apoA-I and either cholesteryl esters only (CE-rHDL; diameter 9.3 nm) or CE and TG in
their core (TG-rHDL; diameter 9.5 nm) were used. After 24 h of
incubation with PLTP, all of the TG-rHDL, but only a proportion of the
CE-rHDL, were converted into large (11.3-nm diameter) and small (7.7-nm
diameter) particles. Only small particles were formed during the first
6 h of incubation of CE-rHDL with PLTP. The large particles and
dissociated apoA-I were apparent after 12 h. In the case of
TG-rHDL, small particles appeared after 1 h of incubation, while
dissociated apoA-I and large particles were apparent at 3 h. The
composition of the large particles indicated that they were derived
from a fusion product. Spectroscopic studies indicated that the apoA-I
in TG-rHDL was less stable than the apoA-I in CE-rHDL. In conclusion,
these results show that (i) PLTP mediates rHDL fusion, (ii) the fusion
product rearranges by two independent processes into small and large
particles, and (iii) the more rapid remodeling of TG-rHDL by PLTP may
be due to the destabilization of apoA-I.
Phospholipid transfer protein
(PLTP)1 transfers
phospholipids (PL) between high density lipoproteins (HDL) and very low
density lipoproteins as well as between different particles within the HDL fraction (1, 2). It also remodels HDL into large and small
particles in a process that is accompanied by the dissociation of
lipid-poor or lipid-free apolipoprotein A-I (apoA-I) (3-8). Remodeling
is enhanced markedly in HDL that contain triglyceride (TG) in their
core (9).
Evidence of the importance of PLTP in HDL metabolism comes from studies
of mice transgenic for human PLTP. These animals have increased levels
of pre- The mechanism of the remodeling of HDL by PLTP is poorly understood.
Although there is evidence that particle fusion and the dissociation of
lipid-poor or lipid-free apoA-I are involved (7, 8), nothing is known
about how the interaction of PLTP with HDL is regulated or the events
that occur when HDL are remodeled into large and small particles. In
addition, the reasons why remodeling is enhanced in TG-enriched HDL are
not understood. The present study was undertaken in order to address
these issues. The results show that the PLTP-mediated remodeling of
reconstituted HDL (rHDL) containing cholesteryl esters as the sole core
lipid (CE-rHDL) and rHDL that are enriched with TG (TG-rHDL) involves
the formation of a large, unstable fusion product, which either (i)
rearranges into small particles in a process that is not accompanied by
the dissociation of apoA-I or (ii) loses two molecules of apoA-I to form a more stable, large conversion product. The results also show
that these processes occur independently and that the enhanced PLTP-mediated conversion of TG-rHDL into large and small particles may
be due to the destabilization of apoA-I.
Isolation of ApoA-I--
HDL were isolated from pooled,
autologously donated samples of human plasma (Gribbles Pathology,
Adelaide, South Australia) by ultracentrifugation in the 1.07 < d < 1.21 g/ml density range (15). The HDL were
delipidated by standard techniques (16). The resulting apo-HDL were
subjected to anion exchange chromatography on a column of Q Sepharose
Fast Flow (Amersham Pharmacia Biotech) attached to an FPLC system
(Amersham Pharmacia Biotech) (17). This gave homogeneous preparations
of apoA-I, which appeared as a single band after electrophoresis on a
20% SDS-polyacrylamide gel (Phast System; Amersham Pharmacia Biotech)
and staining with Coomassie Blue.
Isolation of Lecithin:Cholesterol
Acyltransferase--
Lecithin:cholesterol acyltransferase was isolated
from samples of pooled, human plasma as described (18, 19). Activity was assessed using 1-palmitoyl-2-oleoyl phosphatidylcholine
(POPC)/unesterified cholesterol (UC)/apoA-I discoidal rHDL labeled with
[1 Isolation of CETP--
CETP was prepared as described (21).
Transfer activity was assessed as the transfer of [3H]CE
from [3H]CE-HDL3 to low density lipoproteins
(22, 23). The assay was linear when less than 30% of the
[3H]CE transferred from HDL3 to low density
lipoproteins. Activity is expressed in units/ml, with 1 unit being the
transfer activity of 1 ml of a preparation of pooled, human
lipoprotein-deficient plasma. The CETP used in this study had 22 units
of activity/ml.
Isolation of PLTP--
PLTP was isolated from samples of pooled
human plasma by ammonium sulfate precipitation, ultracentrifugation,
Macroprep Hydrophobic Interaction chromatography (Bio-Rad),
DEAE-Sepharose Fast Flow chromatography (Amersham Pharmacia Biotech),
and heparin-Sepharose Fast Flow chromatography (Amersham Pharmacia
Biotech) (9). These procedures were carried out at room temperature on
an FPLC system. PLTP activity was determined as the transfer of
1,2-di-[1-14C]palmitoyl
L-3-phosphatidylcholine (Amersham Pharmacia Biotech) from
1,2-di-[1-14C]palmitoyl
L-3-phosphatidylcholine-labeled small unilamellar egg PC
vesicles to HDL (24). The activities of the PLTP preparations used in
this study ranged from 1.7 to 3.1 µmol of phospholipid transferred/ml
of PLTP/h. Activities in individual incubations are presented in the
figure legends. The PLTP preparations were free of CETP and
lecithin:cholesterol acyltransferase activities and appeared as single
bands after electrophoresis on a 20% SDS Phast gel (Amersham Pharmacia
Biotech) and silver staining.
Preparation of rHDL--
Discoidal rHDL containing POPC, UC, and
apoA-I were prepared by the cholate dialysis method (25) and converted
into spherical CE-rHDL by incubation at 37 °C for 24 h with low
density lipoproteins and lecithin:cholesterol acyltransferase (26). The
spherical CE-rHDL were isolated by ultracentrifugation at 4 °C in
the 1.07 < d < 1.21 g/ml density range, with two
24-h spins at the lower density and a single 16-h spin at the upper
density (15). The 1.07 g/ml spins were carried out at 55,000 rpm using
a Beckman Ti 55.2 rotor in a Beckman L8-M ultracentrifuge. The 1.21 g/ml spin was carried out at 100,000 rpm using a Beckman TLA 100.4 rotor in a Beckman TL-100 Tabletop ultracentrifuge. The CE-rHDL were
dialyzed extensively against Tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 1 mM
EDTA-Na2 and 0.01% (w/v) NaN3 (TBS) before use.
Spherical CE-rHDL were enriched with TG by incubation at 37 °C for
either 2 or 20 min with the phospholipid/triglyceride emulsion, Intralipid (KabiVitrum AB, Stockholm, Sweden) and CETP (9). Under these
conditions, 12 and 32%, respectively, of the rHDL CE were replaced by
TG. The TG-rHDL were isolated by ultracentrifugation as described for
CE-rHDL and dialyzed against TBS before use.
Remodeling of rHDL by PLTP--
All incubations were carried out
in stoppered plastic tubes in a shaking water bath maintained at
37 °C. Details of the individual incubations are presented in the
figure legends. When the incubations were complete, the rHDL were
isolated by ultracentrifugation in the 1.063 < d < 1.25 g/ml density range with a single spin at the upper and lower
densities. The spins were conducted at 100,000 rpm for 16 h at
4 °C using a TLA 100.4 rotor in a Beckman TL-100 Tabletop
ultracentrifuge. The rHDL were concentrated in a CF25 membrane cone
(Amicon®) and then applied to a prepacked HR 10/30 Superose-6 column
(Amersham Pharmacia Biotech) attached to an FPLC system. The rHDL were
eluted from the column at a flow rate of 0.3 ml/min. Fractions were
collected at 1-min intervals.
Time Course of the Remodeling of rHDL by PLTP--
CE-rHDL and
TG-rHDL were either maintained at 4 °C, incubated at 37 °C for
24 h in the absence of PLTP, or incubated at 37 °C for 1, 3, 6, 12, and 24 h in the presence of PLTP. Aliquots of the unprocessed
incubation mixtures were subjected to nondenaturing 3-40%
polyacrylamide gradient gel electrophoresis, transferred electrophoretically to nitrocellulose membranes, and immunoblotted with
polyclonal sheep anti-human apoA-I antiserum (Roche Molecular Biochemicals). The transferred bands were detected by ECL (Amersham Pharmacia Biotech) (27).
Phospholipid Transfer Studies--
To study phospholipid
transfers from vesicles to rHDL, [14C]POPC-labeled small
unilamellar POPC vesicles were mixed with unlabeled CE-rHDL or
unlabeled TG-rHDL and incubated at 37 °C for 1, 3, 5, 10, and 20 min
in the presence of PLTP (24). The vesicles were precipitated with a
MnCl2/heparin solution (24, 28), and the
[14C]POPC content of the rHDL in the supernatant was
determined by liquid scintillation counting (Beckman LS 6000 TA Liquid
Scintillation Systems, Beckman Instruments, Inc., Fullerton, CA).
Precipitation of the vesicles with MnCl2/heparin was
quantitative, while more than 95% of the rHDL remained in solution.
To study phospholipid transfers from rHDL to vesicles,
[14C]POPC-labeled CE-rHDL and
[14C]POPC-labeled TG-rHDL were incubated with unlabeled
small unilamellar POPC vesicles and PLTP. The
[14C]POPC-labeled CE-rHDL were prepared by incubating
unlabeled CE-rHDL (6.6 µmol of PL) and
[14C]POPC-labeled small unilamellar POPC vesicles (0.66 µmol of PL) with PLTP at 37 °C for 3 h. The radiolabeled
CE-rHDL were isolated by ultracentrifugation and then either incubated
at 37 °C for 20 min with Intralipid and TBS, or enriched with TG by
incubation at 37 °C for 20 min with Intralipid and CETP. The
resulting [14C]POPC-labeled CE-rHDL and
[14C]POPC-labeled TG-rHDL were isolated by
ultracentrifugation. PLTP-mediated phospholipid transfers from the
radiolabeled rHDL to unlabeled, small unilamellar POPC vesicles were
determined by incubation at 37 °C for 1, 3, 5, 10, and 20 min
(24).
Transfer rates were calculated as the slope of the initial, linear
section of plots of the percentage of phospholipid transferred between
the rHDL and vesicles as a function of time.
Interaction of PLTP with rHDL--
The binding of PLTP to
CE-rHDL and TG-rHDL was studied using surface plasmon resonance
analysis on a BIAcore 2000 system. Rabbit anti-mouse Fc (RAMFc) was
covalently attached to CM5 research grade sensor chips as described
(29). Purified monoclonal antibodies were injected individually at 10 µl/min and captured at 25 °C by the RAMFc. The human
apoA-I-specific antibodies AI-1.2 (120 µg/ml), AI-11 (5 µg/ml), and
AI-137.1 (13.5 µg/ml) were used individually to capture CE-rHDL and
TG-rHDL. The human PLTP-specific antibody, mAb 66 (100 µg/ml)
was used to capture the PLTP. The analyte PLTP or rHDL was exposed to
the immobilized rHDL or PLTP, respectively, at 25 °C with a buffer
flow rate of 10 µl/min.
For the apoA-I-specific mAb studies, a typical experiment consisted of
the generation of a single chip of immobilized RAMFc followed
sequentially by exposure of each flow cell to the same concentration of
antibody and rHDL (25 nM). Then, 40 µl of PLTP analyte
(125-1000 nM) was injected, with each flow cell receiving a different analyte concentration.
For the PLTP-specific mAb studies, the RAMFc chip was exposed
sequentially to 100 µg/ml of mAb 66 and 1 µM PLTP
followed by CE-rHDL or TG-rHDL at 100-6000 nM.
Data were analyzed with the BIAevaluation version 3.0 software, and the
Langmuir model for 1:1 binding was used to calculate ka and kd rate constants.
Epitope Mapping of Anti-PLTP mAb 66--
Epitope mapping was
performed using a Peptide-Spot filter containing 140 peptides, each 13 amino acid residues long, with 11 amino acid overlaps (Jerini Bio
Tools, GmbH, Berlin, Germany). The filter was first soaked in methanol
for 10 min at room temperature and further treated with monoclonal
antibody mAb 66 as described (30). The bound antibodies were detected
by ECL (30). According to this analysis, mAb 66 reacts with the region
of amino acids 225-235 of PLTP, ASTSNLDMDFR. The molecular model of
PLTP (31) predicts that this peptide region, especially the sequence
STSN, is in a turn on the surface of PLTP and well exposed for
antigenic reactions.2
Structural Studies--
Phospholipid acyl chain and head group
packing order was determined by labeling rHDL with
1,6-diphenyl-1,3,5-hexatriene (DPH) and
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene
p-toluenesulfonate (TMA-DPH) (32, 33), respectively. Steady
state fluorescence polarization of the DPH- and TMA-DPH-labeled rHDL
was measured at 5 °C intervals from 5 to 50 °C using an
excitation wavelength of 366 nm. The molar ratio of rHDL
phospholipid/probe was 500:1, and the final phospholipid concentration
was 0.5 mM.
Lipid-water interfacial hydration was assessed by labeling the rHDL
with 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN) (34).
Uncorrected 390-600-nm emission spectra of the PRODAN-labeled rHDL
were recorded using an excitation wavelength of 366 nm and excitation
and emission band passes of 5 and 6 nm, respectively. The molar ratio
of rHDL phospholipid/probe was 500:1, and the final phospholipid
concentration was 0.5 mM. The ratio of the emission
intensities at 440 and 490 nm was measured at 5 °C intervals from 5 to 50 °C.
The unfolding of apoA-I in CE-rHDL and TG-rHDL was assessed by
incubation with 0-8 M guanidine hydrochloride (GdnHCl)
(21, 35). The rHDL were added to aliquots of 50 mM
Tris-HCl, pH 8.0, containing varying concentrations of GdnHCl. The
final apoA-I concentration was 20 µg/ml. Wavelengths of maximum
fluorescence were determined from 300-380-nm emission scans using an
excitation wavelength of 295 nm. The respective excitation and emission
band passes were 10 and 5 nm. Initial readings (t = 0 h) were made at 25 °C, immediately after the rHDL were added
to the GdnHCl. Subsequent measurements were made after 2, 5, 8, and
24 h of incubation at 25 °C.
Other Methods--
Nondenaturing 3-40% polyacrylamide
gradient gel electrophoresis was used to quantitate rHDL size (36). The
gels were stained with Coomassie Blue and scanned with a Sharp JX-610
scanner (Sharp, Japan). Imagemaster software was used to quantitate
particle size by reference to high molecular weight standards of known
diameter (Amersham Pharmacia Biotech). Agarose gel electrophoresis was used to determine rHDL surface charge (37).
A Cobas Fara centrifugal analyzer (Roche Diagnostics, Zurich,
Switzerland) was used for the compositional analyses. ApoA-I concentrations were determined either by the method of Lowry et al. (38), using bovine serum albumin as a standard, or by an immunoturbidometric assay (39). Enzymatic kits (Roche Molecular Biochemicals) were used to measure phospholipid, UC, and total cholesterol concentrations. CE concentrations were calculated as the
difference between the total cholesterol and UC concentrations. TG was
quantitated enzymatically (40).
The number of apoA-I molecules/particle was determined by covalently
cross-linking the rHDL with bis(sulfosuccinimidyl) suberate (41). The
cross-linked rHDL and a control sample of cross-linked, lipid-free
apoA-I were electrophoresed on nondenaturing polyacrylamide gradient
gels. The number of apoA-I molecules/particle was determined by
reference to the cross linked lipid-free apoA-I.
Statistical Analysis--
Analysis of variance, two-factor with
repeated measures, was used to assess differences between data sets.
Significance was determined as p < 0.05. The data
analysis package in Microsoft Excel 98 was used for these analyses.
Remodeling of CE-rHDL and TG-rHDL by PLTP (Tables
I and II, Figs.
1-3)--
Spherical CE-rHDL, prepared
as described under "Experimental Procedures," were enriched with TG
by incubation for 20 min with Intralipid and CETP. The respective
diameters of the CE-rHDL and TG-rHDL were 9.3 and 9.5 nm, and their
PL/UC/CE/TG/apoA-I molar ratios were 95.3/6.8/68.1/1.8/3.0 and
103.2/6.2/54.2/22.9/3.0 (Table I). The small amount of TG in the
CE-rHDL probably represents traces of Intralipid co-isolating with the
rHDL rather than spontaneous transfer of TG from Intralipid to the rHDL
(21). Since cross-linking with bis(sulfosuccinimidyl) suberate showed
that the CE-rHDL and TG-rHDL both contained three molecules of
apoA-I/particle, the stoichiometries are expressed relative to three
molecules of apoA-I. As judged by agarose gel electrophoresis, TG
enrichment did not affect rHDL surface charge, with the CE-rHDL and
TG-rHDL both having electrophoretic mobilities of
CE-rHDL and TG-rHDL size was not affected by incubation for 24 h
in the absence of PLTP (Fig. 1). After 24 h of incubation in the
presence of PLTP, ~76% of the original CE-rHDL were converted into
large (11.3-nm) and small (7.7-nm) particles. The remaining CE-rHDL
were unchanged in size. When the TG-rHDL were incubated for 24 h
with PLTP, they were completely converted into large (11.3-nm) and
small (7.7-nm) particles, thus confirming the earlier report that
TG-enrichment enhances the remodeling of HDL by PLTP (9).
The large and small conversion products were resolved by gel permeation
chromatography (Fig. 2). The lipid and
protein constituents in the rHDL that were either maintained at 4 °C
or incubated at 37 °C in the absence of PLTP eluted as a single peak
between fractions 12 and 27. This reflects the monodispersity of the
rHDL preparations. The large particles generated by incubation in the
presence of PLTP were enriched in CE, while the small conversion
products were enriched in apoA-I. Selected fractions were pooled as
indicated ( Time Course of the Remodeling of CE-rHDL and TG-rHDL by PLTP (Fig.
4)--
The time dependence of the
remodeling of CE-rHDL and TG-rHDL by PLTP was also examined (Fig. 4).
CE-rHDL and TG-rHDL were incubated for 0-24 h with PLTP. Aliquots of
the incubation mixtures were subjected to nondenaturing polyacrylamide
gradient gel electrophoresis and immunoblotted with an anti-apoA-I
polyclonal antibody. Tracks A and B
show rHDL that were either maintained at 4 °C or incubated at
37 °C for 24 h in the absence of PLTP. Tracks
C, D, E, F, and G represent rHDL that were incubated in the presence of PLTP
for 1, 3, 6, 12, and 24 h. Lipid-free apoA-I is shown in
track H. A proportion of the CE-rHDL were
converted into small particles during the first 6 h of incubation
with PLTP. Large CE-rHDL conversion products and dissociated apoA-I
were apparent after 12 h of incubation. In the case of
TG-rHDL, small particles were apparent at 1 h, while the
large conversion products and dissociated apoA-I appeared after 3 h of incubation with PLTP.
The finding that the small conversion products were formed before the
large conversion products provides convincing evidence that these
particles are generated by two independent processes. Furthermore, as
both the large and small TG-rHDL conversion products appeared more
rapidly than the CE-rHDL conversion products, it follows that TG
enrichment enhances both processes. The additional finding that the
dissociation of apoA-I coincided with the appearance of the large
conversion products suggests that their formation is related.
Influence of TG Enrichment on Phospholipid Transfers (Fig.
5)--
To determine whether transfers
of phospholipids from small unilamellar vesicles to TG-HDL were also
enhanced relative to CE-rHDL, the rHDL were incubated with
[14C]POPC-labeled small unilamellar POPC vesicles and
PLTP for 0-20 min. Transfers of [14C]POPC from the
vesicles to CE-rHDL (open squares) and TG-rHDL (closed squares) are shown in Fig. 5. The initial
rate of transfer from the vesicles to TG-rHDL was 7.1 µmol of POPC/ml
of PLTP/h, compared with 3.9 µmol of POPC/ml of PLTP/h for
CE-rHDL.
To determine whether the different rates of transfer could be explained
by modification of the vesicles by rHDL surface constituents, transfers
of phospholipids from rHDL to the vesicles were also examined.
[14C]POPC-labeled CE-rHDL and
[14C]POPC-labeled TG-rHDL were incubated for 0-20 min
with PLTP and unlabeled POPC vesicles. There was no detectable transfer
of POPC from the CE-rHDL to the vesicles (open
diamonds). Less than 5% of the TG-rHDL phospholipids
transferred to the vesicles (closed diamonds).
This indicates that the different rates of transfer of phospholipids
from the vesicles to the rHDL cannot be explained by rHDL phospholipids
altering the vesicle structure.
Binding of CE-rHDL and TG-rHDL to PLTP (Table
III)--
There was no difference
between the interaction of soluble PLTP with CE-rHDL and TG-rHDL that
were immobilized on the biosensor surface using either of three
separate apoA-I-specific monoclonal antibodies (AI-1.2, AI-11, or
AI-137.1). Likewise, no difference was observed between the interaction
of CE-rHDL and TG-rHDL with PLTP when the PLTP was immobilized on the
biosensor surface with the PLTP-specific antibody mAb 66 (Table
III).
The Influence of TG Enrichment on rHDL Structure (Table
IV, Fig.
6)--
Since the enhanced remodeling
of TG-rHDL compared with CE-rHDL could not be explained in terms of
differences in the binding to PLTP, the possibility that it was due to
structural differences between the particles was investigated.
Spherical CE-rHDL were enriched with increasing amounts of TG by
incubation with Intralipid and CETP for 2 or 20 min. Control incubations containing CE-rHDL and Intralipid but no CETP were also
carried out. The rHDL that were incubated with CETP contained TG as
12.2 and 32.4% of the total core lipids (Table IV). TG enrichment increased the size of the rHDL slightly from 9.2 to 9.5 nm.
Phospholipid acyl chain and head group packing order was determined
from the steady state fluorescence polarization of rHDL labeled with
DPH and TMA-DPH (Fig. 6). The DPH-labeled CE-rHDL that were either
maintained at 4 °C (data not shown) or incubated in the absence of
CETP (closed squares) had comparable polarization values. As the TG content of the rHDL increased, phospholipid acyl
chain packing order decreased as evidenced by the progressive reduction
in the polarization values for the rHDL with 12.2% TG (open
triangles) (p < 0.001 compared with all
other samples by analysis of variance) and 32.4% TG (open
circles) (p < 0.001 compared with all other
samples by analysis of variance).
The polarization values for the TMA-DPH-labeled CE-rHDL
(closed squares) and the TG-rHDL with 12.2% TG
(open triangles) and 32.4% TG (open
circles) were not significantly different, indicating that
TG enrichment did not affect rHDL phospholipid head group packing order.
The rHDL lipid-water interfacial hydration was assessed by labeling
with PRODAN and comparing the ratio of the intensities of the
fluorescence emission spectra at 440 and 490 nm as a function of
temperature. No differences were observed between the CE-rHDL and
TG-rHDL (results not shown). This indicates that TG enrichment has no
effect on rHDL lipid-water interfacial hydration.
The Influence of TG Enrichment on the Unfolding of apoA-I in rHDL
(Table IV, Figs. 7 and
8)--
The unfolding of the apoA-I
in spherical CE-rHDL and rHDL containing either 12.2 or 32.4% TG is
shown in Fig. 7. The rHDL were incubated with 0-8 M GdnHCl
for 0 (closed diamonds), 5 (open squares), and 24 h (closed
triangles). The results for the CE-rHDL are shown in Fig.
7A. Panels B and C
represent rHDL with 12.2 and 32.4% TG, respectively. The concentration
of GdnHCl required to achieve 50% unfolding of apoA-I is shown in
Table IV. These values were either calculated directly from Fig. 7, or
from a plot of the concentration of GdnHCl versus the free
energy of unfolding of apoA-I (35). The results show that the
concentration of GdnHCl required to unfold apoA-I decreases with
increasing rHDL TG content.
The kinetics of the unfolding of apoA-I in CE-rHDL
(closed diamonds) and rHDL with 12.2%
(open squares) and 32.4% (closed triangles) TG are shown in Fig. 8. These experiments were
carried out in the presence of 4.0 M GdnHCl The apoA-I in
the rHDL with 32.4% TG unfolded more rapidly than the apoA-I in the
rHDL with 12.2% TG or the apoA-I in CE-rHDL. When these results are
considered with the data in Table IV, it follows that enrichment with
TG destabilizes the apoA-I in rHDL.
The ability of PLTP to remodel HDL into large and small particles,
mediate the dissociation of apoA-I from HDL, and transfer phospholipids
between HDL and other lipoproteins is well documented (1-8). Earlier
work from this laboratory has also established that TG enrichment
enhances the remodeling of HDL by PLTP (9). While the PLTP-mediated
remodeling of HDL and the dissociation of apoA-I are both dependent on
efficient phospholipid transfers (42), the mechanism by which these
processes occur is not understood. The present study was carried out in
order to elucidate the mechanism of the remodeling of HDL by PLTP and
reasons why it is enhanced in TG-enriched particles.
These goals were achieved by (i) characterizing the large and small
particles that were formed when CE-rHDL and TG-rHDL were incubated with
PLTP, (ii) determining the time sequence for the formation of the large
and small particles and the dissociation of apoA-I, and (iii) defining
the structural changes that occur when rHDL are enriched with TG.
The relationship between the time sequence of the remodeling of rHDL
into large and small particles and the dissociation of apoA-I was
studied by incubating CE-rHDL and TG-rHDL with PLTP and monitoring rHDL
size changes as well as the dissociation of apoA-I (Fig. 4). These
results showed that the small conversion products were formed before
the large conversion products and that the appearance of the large
conversion products coincided with the dissociation of apoA-I.
Furthermore, both the large and small TG-rHDL conversion products were
generated more rapidly than the CE-rHDL conversion products.
In the case of CE-rHDL, small particles were formed during the first
6 h of incubation with PLTP, while the large conversion products
and dissociated apoA-I appeared after 12 h of incubation. These
time differences indicate that the large and small conversion products
are formed by independent processes. Furthermore, the finding that the
formation of the large particles coincided with the dissociation of
apoA-I suggested that both of these species were generated via a common pathway.
The data in Table II show that the large CE-rHDL and TG-rHDL conversion
products contain more surface and core lipid constituents/particle than
the original rHDL, indicating that they must have been formed by
particle fusion. Moreover, the data in Table III, showing that TG
enrichment does not affect the binding of PLTP to rHDL, suggests that
the rate at which the rHDL are remodeled is determined by processes
that occur after PLTP has mediated particle fusion. This
finding, together with the data in Table II and Fig. 4, indicate that
the PLTP-mediated remodeling of rHDL involves the following events
(Fig. 9). Interaction of PLTP with two
rHDL particles gives a large, unstable fusion product with six
molecules of apoA-I (Fig. 9i). The fusion product has one of
the following two fates: it rearranges into three small particles, each
of which has two molecules of apoA-I/particle, in a process that is not
accompanied by the dissociation of apoA-I (Fig. 9ii), or two
molecules of apoA-I dissociate from the fusion product, forming a more
stable, large conversion product with four molecules of apoA-I (Fig.
9iii).
The possibility that the small conversion products are further
remodeled into large conversion products was also considered. The data
in Table II indicate that this pathway requires interactions between
three small conversion products and the concomitant dissociation of two
molecules of apoA-I. Although the results in Fig. 4 indicate that
formation of large conversion products by this pathway is feasible, it
is likely that trimolecular collisions of this type are not
energetically favorable. As such, this pathway is unlikely to be a
major source of the large conversion products.
The results of the spectroscopic studies give an insight as to why the
TG-rHDL are remodeled by PLTP more rapidly than CE-rHDL. The data in
Fig. 8 show that the apoA-I in TG-rHDL unfolds more readily than the
apoA-I in CE-rHDL. This is in agreement with what has been reported by
other investigators (43) and is most likely caused by TG partitioning
from the core into the particle surface and preventing apoA-I
The apoA-I that dissociated from the CE-rHDL and TG-rHDL during the
incubations with PLTP appeared as three bands when subjected to
nondenaturing gradient gel electrophoresis and immunoblotting (Fig. 4).
One of the bands was comparable in size with lipid-free apoA-I. The
smallest band is possibly the 23-kDa fragment of apoA-I that is
generated when either lipid-free or lipid-associated apoA-I are
incubated with PLTP (45). The largest of the three bands may represent
apoA-I complexed with small amounts of rHDL lipids. This is consistent
with the reduced recovery of surface constituents relative to core
lipids in the large and small conversion products (Table II). It is
possible that the apoA-I that is associated with small amounts of
lipid may be comparable with the pre- Fig. 5 shows that PLTP-mediated phospholipid transfers from small
unilamellar vesicles to TG-rHDL are enhanced in TG-rHDL relative to
CE-rHDL. The additional finding that there is minimal transfer of
phospholipids in the reverse direction, from the rHDL to the vesicles,
excludes the possibility that this result can be explained by rHDL
phospholipids altering the vesicle surface. The increased transfer of
phospholipids from the vesicles to the TG-rHDL can be explained by an
enhanced ability of the TG-rHDL to accommodate additional
phospholipids. This is consistent with the results in Fig. 6, which
show that TG enrichment decreases the packing order of rHDL
phospholipid acyl chains. This is probably a result of TG partitioning
from the core into the rHDL surface and generating packing defects that
can be occupied by additional phospholipid molecules. These findings
are also in agreement with the observation that the TG content of HDL
correlates positively with the rate of PLTP-mediated phospholipid
transfers in plasma (47).
In conclusion, this study provides the first insight into the mechanism
by which PLTP mediates the remodeling of HDL. The results show that
PLTP acts as a fusogen when it interacts with rHDL and that the fusion
product is subsequently remodeled into large and small particles.
Evidence that the large and small conversion products are formed by two
independent processes is also presented. In addition, we show that the
apoA-I in TG-rHDL is destabilized compared with the apoA-I in CE-rHDL
and that this destabilization may be responsible for the enhanced
PLTP-mediated remodeling of TG-rHDL.
We thank Prof. P. J. Barter for advice
and R. Bright and D. J. Bonnett for technical assistance. Dr. Gerd
Wohlfahrt (Orion Corporation, Espoo, Finland) is gratefully
acknowledged for analysis of mAb 66 epitope location in the PLTP
molecular model.
*
This work was supported by the National Health and Medical
Research Council of Australia.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by a Royal Thai Government Scholarship.
§§
A Senior Career Research Fellow of the National Heart Foundation
of Australia. To whom correspondence should be addressed: Lipid
Research Laboratory, Level 1, Hanson Center, Frome Rd., Adelaide, South
Australia 5000, Australia. Tel.: 61 8 8222 3448; Fax: 61 8 8222 3154;
E-mail: karye@ozemail.com.au.
Published, JBC Papers in Press, April 26, 2001, DOI 10.1074/jbc.M010708200
2
G. Wohlfahrt, personal communication.
The abbreviations used are:
PLTP, phospholipid
transfer protein;
apoA-I, apolipoprotein A-I;
CE, cholesteryl esters;
CETP, cholesteryl ester transfer protein;
DPH, 1,6-diphenyl-1,3,5-hexatriene;
HDL, high density lipoprotein(s);
PL, phospholipid(s);
POPC, 1-palmitoyl-2-oleoyl
L-3-phosphatidylcholine;
PRODAN, 6-propionyl-2-(dimethylamino)-naphthalene;
rHDL, reconstituted HDL;
TG, triglyceride(s);
TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene
p-toluenesulfonate;
TBS, Tris-buffered saline;
UC, unesterified cholesterol;
GdnHCl, guanidine hydrochloride.
The Mechanism of the Remodeling of High Density Lipoproteins by
Phospholipid Transfer Protein*
§¶,§,
,§§
Department of
Medicine, The University of Adelaide, Royal Adelaide Hospital, Adelaide
5000, South Australia, Australia, The Scripps Research
Institute, La Jolla, California 92037, the ** Department of
Biochemistry, National Public Health Institute, Helsinki
FIN-00300, Finland, and the
Division of Cardiovascular Services, Royal
Adelaide Hospital,
Adelaide 5000, South Australia, Australia
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-migrating HDL, the initial acceptors of
cellular cholesterol in the first step of the reverse cholesterol pathway, and are also resistant to intracellular cholesterol
accumulation (10-12). Studies of PLTP knockout mice have shown that
PLTP is essential for maintaining normal HDL levels in plasma (13). Moreover, it has been reported recently that PLTP-mediated transfers of
phospholipids between HDL and other lipoprotein classes are not
interchangeable with the phospholipid transfers that are mediated by
cholesteryl ester transfer protein (CETP) (14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,2-3H]cholesterol ([3H]UC)
(Amersham Pharmacia Biotech) as the substrate (20). The assay was
linear when less than 30% of the [3H]UC was esterified.
The preparation used in this study generated 2.36 µmol of CE/ml
lecithin:cholesterol acyltransferase/h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.49
µm·s
1/V·cm1.
Physical properties of CE-rHDL and TG-rHDL
View larger version (18K):
[in a new window]
Fig. 1.
PLTP-mediated changes in rHDL size.
CE-rHDL (final apoA-I concentration 92.5 µg/ml) and TG-rHDL (final
apoA-I concentration 92.5 µg/ml; 28.0% TG) were either maintained at
4 °C or incubated at 37 °C for 24 h in the absence or
presence of PLTP (final activity 2.2 µmol of PL transferred/ml of
PLTP/h). The final volume of the incubation mixtures was 4.79 ml. When
the incubations were complete, the rHDL were isolated by
ultracentrifugation and subjected to nondenaturing polyacrylamide
gradient gel electrophoresis. The profiles represent scans of
Coomassie-stained gels. Particle diameters were calculated by reference
to known high molecular weight standards.
) and subjected to nondenaturing gradient gel
electrophoresis (Fig. 3).
Profiles A and B show, respectively,
the CE-rHDL and TG-rHDL, that were either maintained at 4 °C
or incubated at 37 °C for 24 h in the absence of PLTP.
Profiles C and D represent the large
(11.3-nm) and small (7.7-nm) conversion products, respectively. The
number of apoA-I molecules/particle in the pooled samples was
determined by cross-linking (Table II).
Whereas the original CE-rHDL and TG-rHDL contained three molecules of
apoA-I/particle, the large and small conversion products contained four
and two molecules of apoA-I/particle, respectively. The composition of the pooled samples and the recoveries of the individual rHDL
constituents are shown in Table II. The large particles contained
approximately twice as many phospholipid and core lipid molecules as
the original rHDL, indicating that they are derived from a fusion
product. Following incubation with PLTP, the recovery of rHDL core
lipids exceeded that of the surface constituents. This indicates that phospholipids and UC as well as apoA-I dissociated from the CE-rHDL and
TG-rHDL during the incubation.
View larger version (27K):
[in a new window]
Fig. 2.
Elution profiles of CE-rHDL and TG-rHDL.
CE-rHDL and TG-rHDL were either maintained at 4 °C or incubated at
37 °C for 24 h in the absence or presence of PLTP as described
in the legend to Fig. 1. When the incubations were complete, the rHDL
were isolated by ultracentrifugation, concentrated by ultrafiltration,
and applied to a HR 10/30 Superose-6 column. The concentrations of the
individual rHDL constituents in each fraction are shown. The values
represent the mean of triplicate determinations that varied by less
than 10%. Selected fractions were pooled as indicated ( ).
View larger version (18K):
[in a new window]
Fig. 3.
Size distribution of the pooled rHDL
fractions. Pooled samples from Fig. 2 were subjected to
nondenaturing polyacrylamide gradient gel electrophoresis. Scans of the
Coomassie-stained gels are shown. Profiles A and
B, respectively, show the pooled CE-rHDL and TG-rHDL
fractions that were either maintained at 4 °C or incubated at
37 °C for 24 h in the absence of PLTP. Profiles
C and D are the large and small conversion
products generated by incubation of CE-rHDL and TG-rHDL with PLTP at
37 °C for 24 h.
Physical properties of the pooled rHDL fractions
) in Fig. 2.
The composition, size, and number of apoA-I molecules/particle were
determined as described under "Experimental Procedures." The
stoichiometries represent the mean of triplicate determinations, which
varied by less than 10%.
View larger version (29K):
[in a new window]
Fig. 4.
Time course of the remodeling of rHDL by
PLTP. CE-rHDL (final apoA-I concentration 92.5 µg/ml) and
TG-rHDL (final apoA-I concentration 92.5 µg/ml) were either
maintained at 4 °C, incubated at 37 °C for 24 h in the
absence of PLTP (tracks A and B,
respectively), or incubated at 37 °C for 1, 3, 6, 12, and 24 h
in the presence of PLTP (final activity 5.1 µmol of PL transferred/ml
of PLTP/h) (tracks C, D, E,
F, and G, respectively). The final volume was 270 µl. An aliquot of each incubation mixture (0.25 µg apoA-I) was
subjected to nondenaturing polyacrylamide gradient gel electrophoresis.
Lipid-free apoA-I was also applied to the gels (track
H). The samples were transferred to nitrocellulose
membranes, immunoblotted with sheep anti-human apoA-I antiserum, and
detected by ECL.
View larger version (9K):
[in a new window]
Fig. 5.
Influence of TG enrichment on phospholipid
transfers. CE-rHDL ( 
; final apoA-I concentration 625.0 µg/ml)
and TG-rHDL (
; final apoA-I concentration 625.0 µg/ml; 31.1% TG)
were incubated with PLTP (final activity 0.16 µmol of PL
transferred/ml of PLTP/h) and [14C]POPC-labeled small
unilamellar POPC vesicles (final PL concentration 375.0 µg/ml) for 1, 3, 5, 10, and 20 min. The final incubation volume was 400 µl.
Identical conditions were used for incubations of
[14C]POPC-labeled CE-rHDL (
) and
[14C]POPC-labeled TG-rHDL (
) with unlabeled POPC
vesicles and PLTP. When the incubations were complete, the vesicles
were precipitated with a MnCl2/heparin solution, and the
[14C]POPC content of the rHDL was determined by liquid
scintillation counting. Data points represent the mean ± S.D. of
triplicate determinations.
Interaction of PLTP with CE-rHDL and TG-rHDL
Unfolding of apoA-I in CE-rHDL and TG-rHDL
View larger version (16K):
[in a new window]
Fig. 6.
Influence of TG enrichment on rHDL
structure. Spherical CE-rHDL were mixed with Intralipid and either
incubated at 37 °C for 20 min in the absence of CETP ( ) or
incubated at 37 °C for 2 (
) and 20 min (
) in the presence of
CETP as described in the legend to Table IV. The rHDL were isolated by
ultracentrifugation and labeled with DPH and TMA-DPH. Steady state
fluorescence polarization values for the DPH- and TMA-DPH-labeled rHDL
are shown. Values represent the mean ± S.D. of at least three
determinations. *, significantly different from all other samples by
analysis of variance, p < 0.001.
View larger version (22K):
[in a new window]
Fig. 7.
Influence of TG enrichment on the unfolding
of apoA-I in rHDL. Spherical CE-rHDL were mixed with Intralipid
and either incubated at 37 °C for 20 min in the absence of CETP
(A) or for 2 (B) and 20 min (C) in the
presence of CETP as described in the legend to Table IV. When the
incubations were complete, the rHDL were isolated by
ultracentrifugation then incubated with 0-8 M GdnHCl
for 0, 2, 5, 8, and 24 h at 25 °C. The data for 0 h ( ),
5 h (), and 24 h (
) are shown. Values represent the
mean of at least three determinations. Experimental errors for the
wavelength of maximum fluorescence are ±1.0 nm.
View larger version (11K):
[in a new window]
Fig. 8.
Influence of TG enrichment on the kinetics of
unfolding of apoA-I in rHDL. Spherical CE-rHDL were mixed with
Intralipid and incubated at 37 °C either for 20 min in the absence
of CETP ( ) or for 2 () and 20 () min in the presence of CETP
as described in the legend to Table IV. When the incubations were
complete, the rHDL were isolated by ultracentrifugation and then
incubated with 4.0 M GdnHCl for 0-24 h. Values for the
wavelength of maximum fluorescence represent the means of triplicate
determinations. Experimental errors are ±1.0 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 9.
Proposed mechanism for the remodeling of rHDL
by PLTP. i, PLTP mediates the fusion of two rHDL
particles, each of which contain three molecules of apoA-I, to give an
unstable particle with six apoA-I molecules. The fusion product either
(ii) rearranges into three small particles with two
molecules of apoA-I/particle or (iii) is converted into a
large particle with four molecules of apoA-I in a process that is
accompanied by the dissociation of two molecules of apoA-I.
-helices from intercalating between the rHDL phospholipid acyl
chains (44). This may be why apoA-I dissociates more rapidly from
TG-rHDL than from CE-rHDL and why the PLTP-mediated remodeling of HDL
is enhanced by TG enrichment.
1-migrating HDL in
human plasma that have been identified as the initial acceptors of
cellular cholesterol from peripheral tissues (12, 46).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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