From the Department of Biochemistry, MCP Hahnemann University,
Philadelphia, Pennsylvania 19129, the ¶ Departments of Medicine,
Biochemistry, and Molecular Genetics and the Atherosclerosis Research
Unit, University of Alabama at Birmingham Medical Center, Birmingham,
Alabama 35294, the Center for Molecular and Vascular Biology,
University of Leuven, B-3000 Leuven, Belgium, and the ** Gladstone
Foundation Laboratories for Cardiovascular Disease, Department of
Pathology, Cardiovascular Research Institute, University of California,
San Francisco, California 94140
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ABSTRACT |
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Lipid-free apolipoprotein (apo) A-I
contributes to the reverse transport of cholesterol from the periphery
to the liver by solubilizing plasma membrane phospholipid and
cholesterol. The features of the apolipoprotein required for this
process are not understood and are addressed in the current study.
Membrane microsolubilization of human fibroblasts is not specific for
apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast
monolayers at a saturating concentration of 50 µg/ml are all able to
release cholesterol and phospholipid similarly. To determine the
properties of the apolipoprotein that drive the process, apo A-I
peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic High density lipoprotein
(HDL)1 and its major protein
component, apolipoprotein (apo) A-I, have been established to be
anti-atherogenic in nature (1, 2). This is presumably due to their
central role in mediating the transport of cholesterol from peripheral cells to locations of catabolism, a process termed reverse cholesterol transport (3-5). It is well established that the initial efflux of
unesterified (free) cholesterol (FC) to HDL in this process occurs by
one of two distinct mechanisms (6-8), depending on the degree of
lipidation of apo A-I (9). When apo A-I is present as a structural
component of fully lipidated HDL particles, cellular FC is incorporated
by the well defined aqueous diffusion mechanism (10). Alternatively,
under conditions that cause the apolipoprotein to dissociate from HDL,
creating a pool of lipid-free (poor) apo A-I, cellular FC associates
with the apolipoprotein by a membrane microsolubilization mechanism. In
this process, apo A-I associates with the plasma membrane and
stimulates FC efflux by simultaneously solubilizing the cellular FC and
phospholipid (PL) (9, 11). The details of this mechanism are not well
understood at this point; the current paper focuses on understanding
the structural characteristics of apo A-I that drive this process.
The exchangeable apolipoproteins of HDL (apo A-I, apo A-II, apo A-IV,
apo C, apo E) are all able to access cellular FC and PL (6, 12-15).
This suggests that FC efflux mediated by lipid-free apolipoproteins is
dependent on a shared structural motif. In fact, each of these
apolipoproteins contains several Several groups have reported that the C-terminal domain of the apo A-I
molecule is particularly important for the lipid-associating properties
of the protein (22-25). Consistent with this, recent studies of
amphipathic peptides representing the Materials
Chloramphenicol, cholesterol, cholesteryl methyl ether,
isopropyl- Methods
Preparation of Lipoproteins--
HDL and low density lipoprotein
were isolated from fresh plasma obtained from normolipidemic donors by
sequential ultracentrifugation as described previously (30), and each
appeared as a single band on agarose gel electrophoresis. Prior to use
the isolated lipoproteins were dialyzed extensively against Tris buffer
(10 mM Tris, 150 mM NaCl, 1.0 mM
EDTA; pH 8.2).
Purification of Apo A-I and Apo A-II--
Human HDL was
delipidated in ethanol/diethyl ether (31), and apo A-I and apo A-II
were isolated by anion exchange chromatography on Q-Sepharose (32). The
proteins were stored in lyophilized form at Purification of Apo C and Apo E4--
Human very low density
lipoprotein isolated from the fresh plasma of normolipidemic subjects
was delipidated in chloroform:methanol, and the total apo C fraction (a
mixture of apo CI, CII, CIII1, and CIII2) was
isolated by gel filtration on Sephacryl S-300 followed by anion
exchange chromatography on Q-Sepharose (34). Apo E4 was isolated in a
similar fashion from very low density lipoprotein obtained by
plasmaphoresis of an individual with hyperlipidemia and phenotyped as
apo E4/4; the very low density lipoprotein was kindly provided by Dr.
Daniel Rader (University of Pennsylvania, Philadelphia, PA). Prior to
use the purified apolipoproteins were resolubilized in 6 M
guanidine HCl and dialyzed extensively against the above Tris buffer.
Concentration was determined by absorbance at 280 nm ( Preparation of Apo A-I Peptides--
Pure peptides representing
segments of apo A-I were synthesized using an automated solid phase
peptide synthesizer as described previously (26, 36, 37). To promote
the Expression and Purification of apo A-I Deletion Mutant
Proteins--
Construction of the cDNA for apo A-I (
The apo A-I mutants were purified from the bacterial proteins through
hydrophobic and ion exchange chromatography on phenyl-Sepharose and
Q-Sepharose columns using a FPLC system (Amersham Pharmacia Biotech).
Purified proteins were confirmed as apo A-I variants through Western
blot analysis utilizing a polyclonal rabbit anti-human apo A-I antibody
(Accurate) and secondary antibody with a specific conjugate for
alkaline phosphatase (Pierce). The concentration of the purified
proteins was determined by their absorbance at 280 nm after calculating
the extinction coefficient for each of the apo A-I variants ( Determination of the Lipid Binding Affinities of Apolipoproteins
and Apo A-I Peptides--
The relative affinities of the proteins for
a lipid-water interface were determined as described in detail
previously (26). The procedure utilizes a surface balance technique to
measure the surface pressure at which the proteins are no longer able to penetrate an egg PC monolayer. Egg PC was spread at the air-water interface in a Teflon dish containing 80 ml of PBS to provide an
initial surface pressure (
Association of the peptides with dimyristoylphosphatidylcholine (DMPC)
multilamellar vesicles was performed as described in detail previously
(26). Briefly, peptides were mixed with multilamellar vesicles at a 1:1
(w:w) ratio. The ability of the peptide to solubilize the DMPC and
clarify the turbid solution was determined by the scattered light
intensity measured at 400 nm on a SLM 8100 photon-counting spectrofluorometer.
Preparation of Cell Monolayers--
Normal human skin GM3468A
fibroblasts (passages < 30) were plated in 22-mm, 12-well plates
and grown to confluence in minimal essential medium (MEM)/bicarbonate
supplemented with 10% fetal calf serum in a 37 °C humidified
incubator (95% air, 5% CO2). Upon reaching confluence,
the cells were labeled with either 10-20 µCi/ml
[3H]cholesterol diluted in MEM supplemented with 10%
lipoprotein-deficient serum (40) or 15 µCi/ml
[3H]choline chloride diluted in MEM with 10%
lipoprotein-deficient serum for 48-72 h. In these preparations,
radioactivity present in ethanol was dried under nitrogen and
resolubilized in 50 µl ethanol before adding the media to ensure that
the ethanol concentration was always less than 1% (v/v). For
experiments utilizing unesterified cholesterol (FC)-enriched
monolayers, the radiolabeled fibroblast monolayers were incubated an
additional 24 h with a FC-loading medium (15). This medium
contained 2 µg/ml Sandoz compound 58035 to inhibit FC esterification
(41), 100 µg of FC/ml of FC:PL dispersions (>2:1, mol:mol) (42), 50 µg of protein/ml of human low density lipoprotein, 2 mg/ml fatty
acid-free bovine serum albumin, and either 20 µCi/ml
[3H]cholesterol or 20 µCi/ml [3H]choline
chloride. The [3H]choline label was used to follow the
movement of PL as it has been shown previously that 90% of the PL
released from cholesterol-enriched fibroblasts to lipid-free apo A-I is
choline-containing (15). The above procedure results in a 2-3-fold
increase in cellular FC.
Efflux of Cellular Cholesterol--
(43). After washing the cell
monolayers with 1 ml of MEM-HEPES (four times), FC efflux measurements
were initiated by the application of 1.0 ml/well of the test medium,
consisting of acceptor diluted to the desired protein concentration in
MEM-HEPES. The experiments were conducted in a covered 37 °C water
bath, and all media were supplemented with HEPES. The radioactivity in
an aliquot of the medium was determined by liquid scintillation
counting at specific time intervals to estimate the fraction of FC
released into the medium; any cellular material was removed prior to
counting by filtration of the medium through a 0.45 µm filter. Upon
completion of the time course, all cell wells were washed with PBS
(BioWhittaker) three times, and the cellular lipids were extracted with
isopropanol (44). From the extracted lipids, the total amount of
radioactive FC per well was measured by liquid scintillation counting,
and FC mass was also assayed. Total cellular FC present in the
monolayers at zero time was typically 40-60 µg/mg cell protein.
Efflux of Cellular Phospholipid--
After washing the
monolayers with MEM-HEPES (three times), PL efflux measurements were
initiated as described for FC efflux. An aliquot of the medium was
removed at specific time intervals and filtered through a 0.45 µm
filter; 10 µg of butylated hydroxytoluene was then added to prevent
the oxidation of PL. The lipids were extracted from the sample by
treatment with 1:1 (v/v) chloroform/methanol (45), and then the aqueous
phase was aspirated, and the chloroform phase was washed three times
with 10/9 (v/v) methanol/water. The chloroform phase was dried under
nitrogen in liquid scintillation vials and radioactivity was
quantitated by liquid scintillation counting. Upon completion of the
time course, all cell wells were washed with PBS three times, and the
cellular lipids were extracted with isopropanol containing 10 µg of
butylated hydroxytoluene/ml. The extracts were dried under nitrogen,
and the free [3H]choline was removed as described above.
From the extraction, the total amount of radioactive,
choline-containing PL per well was determined. The mass of
choline-containing PL was determined by an enzymatic assay (Wako).
Total cellular choline-containing PL present in the monolayers at zero
time was typically 120-140 µg/mg cell protein.
Data Analysis--
The fractional release of FC or PL was
determined experimentally and analyzed as described previously for this
system (43). The kinetic analysis was based on the assumptions that the
system is closed and that all lipid therefore exists in either the
cellular lipid pool or the acceptor pool. The cellular cholesterol pool exists primarily in the FC state as cholesterol esterification is
negligible in the human fibroblast cells and compound 58035, an
acyl-CoA:cholesterol O-acyltransferase inhibitor, was
included in all manipulations. The equilibration of FC or PL between
the lipid and acceptor pools was fitted to the mono-exponential rate equation Y = Ae It is well established that apo A-I, as well as the other HDL
apolipoproteins, has the ability to solubilize FC and PL from the
plasma membrane of cells (6). To evaluate the requirements of the FC
acceptor protein in the membrane microsolubilization process, the
abilities of lipid-free apo A-I, apo A-II, apo C, apo E3, and apo E4 to
remove cellular lipids were compared. These HDL proteins are all
composed of several amphipathic -helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N-and
C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol. Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%,
respectively, of that to intact apo A-I. Deletion mutants of apo E and
apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (
222-243), apo A-I (
190-243), apo E3 (
192-299) and apo E4 (
192-299)
all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of
these proteins to penetrate into a phospholipid monomolecular film.
Overall, the results indicate that insertion of amphipathic
-helices
between the plasma membrane phospholipid molecules is a required step
in the mechanism of apolipoprotein-mediated cellular lipid efflux.
Therefore the lipid binding ability of the apolipoprotein is critical
for efficient membrane microsolubilization.
INTRODUCTION
Top
Abstract
Introduction
References
-helical segments that are
amphipathic in nature; these domains provide the apolipoprotein
molecules with the ability to associate with lipids and act as
structural components of HDL (16, 17). Studies investigating lipid-free
apolipoprotein-mediated FC efflux have focused on apo A-I as an
acceptor, as this component of HDL readily dissociates from the fully
lipidated particle to a lipid-free (poor) form (18-21). The lipid
affinities of the amphipathic
-helices of apo A-I are likely to be
significant for the apo A-I-mediated solubilization of plasma membrane
PL and FC, although this issue has not been examined in detail.
-helical domains of apo A-I
have indicated that the first and last helices of the intact protein
have the greatest lipid affinities (26). In the current work, we
address the importance of these regions in apo A-I-mediated membrane
microsolubilization through the use of synthetic apo A-I peptides. The
proposed structural organization of residues 44-243 includes eight
22-mer and two 11-mer amphipathic helical domains. The abilities of
peptides that represent these helical domains alone as well as in
combination to solubilize FC and PL from FC-enriched human fibroblast
cells were investigated; the peptides systematically address the entire
apo A-I sequence. To further investigate the role of domains of high
lipid binding affinity in FC efflux, apo A-I deletion mutants were
constructed. Apo A-I molecules lacking residues 190-243 (
190-243)
and apo A-I lacking residues 222-243 (
222-243) bind PL less well
than wild-type apo A-I. The C-terminal domain of human apo E is also responsible for its lipid binding (27-29). To check whether any correlation between lipid binding affinity and ability to remove cellular lipids is peculiar to apo A-I, apo E variants lacking the
lipid binding domain were also utilized. Apo E3 and apo E4 lacking
residues 192-299 (
192-299) display reduced association with lipids
as compared with intact apo E3 and apo E4 molecules (29). The results
of experiments with synthetic peptides and engineered apo A-I and apo E
molecules demonstrate that there is a correlation between the lipid
binding affinities of apolipoproteins and their abilities to
participate in membrane microsolubilization of cellular PL and FC. The
mechanism of apolipoprotein-mediated lipid efflux requires the presence
of amphipathic
-helical segments that can penetrate into the plasma
membrane and induce the solubilization of lipids.
EXPERIMENTAL PROCEDURES
-D-thiogalactopyranoside, lysozyme, and yeast
growth media were purchased from Sigma.
[1,2-3H]cholesterol (43.5 Ci/mmol) and
[methyl-3H]choline chloride (81 Ci/mmol) were
obtained from NEN Life Science Products. Minimal essential medium and
phosphate-buffered saline (PBS) were purchased from BioWhittaker
(Walkersville, MD). Bovine calf and fetal serum were supplied by Life
Technologies, Inc. All media were supplemented with 50 µg/ml
gentamycin (Sigma). Anti-human apo A-I (rabbit) polyclonal antibody was
purchased from Accurate (Westbury, NY) and the anti-rabbit IgG (goat),
alkaline phosphatase conjugated antibody was from Pierce. All other
reagents were analytical grade. FPLC columns and supplies were
purchased from Amersham Pharmacia Biotech.
70 °C. Prior to use,
the purified apolipoprotein was resolubilized in 6 M
guanidine HCl and dialyzed extensively against the Tris buffer
mentioned above. Concentration was determined by absorbance at 280 nm
(
= 1.23 and 0.87 ml/mg·cm for apo A-I and apo A-II, respectively)
or by a modification of the Lowry method (33).
= 1.4 and
1.31 ml/mg·cm for total apo C and apo E4, respectively). Recombinant
apo E3, apo E3
192-299, and apo E4
192-299 samples expressed in
E. coli were prepared as described previously (35).
-helical stability of the peptide molecules, the N and C termini
were acetylated and amidated (except for peptide 1-33), respectively
(38). Peptide solutions were prepared by weighing known amounts of the
peptides into tubes and solubilizing them in Tris buffer, assuming that 90% of the dry weight was pure peptide (determined by dissolving known
weights of the lyophilized peptides and measuring either the absorbance
at 280 nm for peptides containing Tyr and/or Trp or the concentration
by quantitative amino acid analysis).
222-243)
and apo A-I (
190-243) was carried out by Holvoet et al.
(23) as formerly described. Wild-type apo A-I and the apo A-I mutants
were expressed in the periplasmic space of Escherichia coli
WK6 host cells (23). Briefly, the cells were grown in 2YT medium at
37 °C until the absorbance at 600 nm reached 0.5. Isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 0.4 mM, and the induction was carried out
for 2.5 h at 37 °C. The cells were centrifuged at 10,000 × g for 10 min; periplasmic cell fractions were prepared from the pellet as described previously (39).
= 1.28 and 1.41 ml/mg·cm for apo A-I (
222-243) and apo A-I
(
190-243), respectively).
i) ranging from 5 to 35 dyn/cm. Apo A-I, apo E, or apo A-I peptides in buffer containing 1.5 M guanidine HCl were injected into the subphase at an
initial concentration of 50 µg/dl, which was diluted by the PBS to
result in a final guanidine HCl concentration of
1 mmol/liter. The
change in surface pressure (
i) was recorded by a mica
plate connected to a Cahn RTL recording electrobalance until a steady
state was achieved; the solution was stirred continuously with a
magnetic stirrer throughout the measurement. The equilibrium
i values were plotted as a function of
and linear
extrapolation to the point at which
i = 0 dyn/cm
provided the monolayer exclusion pressure (
e).
Bt + E, where Y represents the fraction of
radiolabeled lipid remaining in the cells, t is the
incubation time, A is a pre-exponential term that reflects
the fraction of lipid that exists in the medium at equilibrium,
B is a time constant characteristic of the release of FC or
PL, and E is a constant that represents the fraction of
labeled lipid that remains associated with the cells at equilibrium. These variables were derived by fitting the experimental data to the
equation by nonlinear regression (GraphPad Prism, GraphPad Software
Inc.). The apparent rate constant for efflux (ke) was derived from these parameters. The half-time of efflux value in
hours was then calculated as follows: t1/2 = ln2/ke. The computed half time values were
statistically compared by Student's t test (GraphPad Prism).
RESULTS
-helical segments, and they range in
molecular mass from approximately 6 to 34 kDa. The unlipidated
apolipoproteins were incubated at 50 µg/ml, a concentration well
above that which is required for saturable FC or PL efflux (15) with
FC-enriched human fibroblasts. From the 8-h time course displayed in
Fig. 1, it is clear that except for apo
A-II, the apolipoproteins all have similar abilities to stimulate FC
efflux. Apo A-II removes approximately 2% of the cellular FC in 8 h, whereas the other apolipoproteins solubilize 4.5-5%. These results
are not explained by variations in the initial rate at which FC is
obtained by the different proteins because the ke,
or efflux rate constant, determined from the first 1 h of the time
course was 1.1 ± 0.1, 1.2 ± 0.1, 1.1 ± 0.2, 0.9 ± 0.1 and 0.9 ± 0.1%/h for apo A-I, apo A-II, apo C, apo E3, and apo E4, respectively. The apparent inefficiency of apo A-II in the
membrane microsolubilization process was not observed when the PL
efflux was determined, as shown in Fig.
2. In this case, all of the
apolipoproteins initiated at least as much choline-containing PL efflux
as apo A-I, and apo C removed significantly more. Mouse apo A-I and
subspecies of apo C, apo CII, and apo CIII demonstrate efficiencies of
initiating cellular FC and PL efflux similar to that of apo A-I (data
not shown).
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Fig. 1.
Cholesterol efflux from FC-enriched
fibroblasts to various HDL apolipoproteins. Human fibroblasts
trace-labeled with [3H]FC were enriched 2-fold in FC
content through a 24-h incubation in the presence of low density
lipoprotein, FC/PL dispersions, and an acyl-CoA:cholesterol
acyltransferase inhibitor as described under "Experimental
Procedures." This procedure resulted in cellular FC levels of 39 ± 5 µg of FC/mg of cell protein. Apolipoproteins present at 50 µg/ml in MEM-HEPES were incubated with the monolayers for periods up
to 8 h. Aliquots were removed from the media at each time point
and filtered, and radioactivity was determined by liquid scintillation
counting. Symbols represent the mean (±1 S.D.) of triplicate
determinations of the percentage of FC released from the monolayers to
apo A-I ( ), apo A-II (
), apo C (
), apo E3 (
), and apo E4
(
). Curves were generated through fitting to a mono-exponential rate
equation. Values have been corrected for release to MEM-HEPES alone; FC
efflux to media alone was typically 15% of that observed in the
presence of apo A-I.
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Fig. 2.
Phospholipid efflux from FC-enriched
fibroblasts to various HDL apolipoproteins. Human fibroblasts
trace-labeled with [3H]choline chloride were enriched
2-fold in FC content as summarized in the legend to Fig. 1, and PL
efflux was measured. The indicated apolipoproteins present at 50 µg/ml in MEM-HEPES were incubated with the monolayers for 4 h.
Columns represent the mean (±1 S.D.) of triplicate
determinations of the relative percentage of PL released from the
monolayers, as compared with the efflux measured to apo A-I; all values
have been corrected for efflux to MEM-HEPES in the absence of
apolipoprotein. PL efflux to media alone was typically 10% of that
observed in the presence of apo A-I. The variability between apo A-I
and apo C (p = 0.006) is significant as determined by
Student's t test.
The ability of various -helical apolipoproteins to stimulate the
efflux of FC and PL indicates that a specific amino acid sequence is
not required for membrane microsolubilization (46, 47). Prior work with
helical peptides characteristic of exchangeable apolipoproteins
suggested that the
-helices must be amphipathic in nature to access
cellular lipids (46). To better understand how the amphipathicity of
the molecule affects the efficiency of this process, peptides
representing each of the 11- and 22-residue (11- and 22-mers)
amphipathic
-helical segments of apo A-I were constructed; tandem
dimer combinations of adjacent helical segments were also synthesized
as 33- and 44-mer peptides. The 20 peptides generated systematically
represent every region of the apo A-I molecule. These synthetic
peptides were incubated with the FC-enriched monolayers, and FC and PL
efflux was determined. From the results in Table
I, it is clear that none of the peptides
are as efficient as the full-length protein at removing cellular
lipids; the majority of the peptides did not stimulate any detectable
FC or PL efflux. However, peptides containing the first N-terminal
22-mer helix of apo A-I, 44-65 (amino acids 1-43 are not organized
into amphipathic
-helices), as well as peptide 209-241,
representing the C terminus, are capable of solubilizing cellular
lipids.
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These results are particularly intriguing as the two domains
represented by the active peptides have been proposed to be critical for the lipid binding ability of the apo A-I molecule (23, 25, 26, 37).
Therefore, the lipid monolayer exclusion pressures (e),
determined for 15 of the peptides and intact apo A-I, were plotted
against the abilities of the segments to stimulate FC efflux; the
resulting graph is presented in Fig. 3.
An increase in
e represents an increase in lipid binding
affinity. It is clear that as the lipid binding affinity increases
beyond a threshold
e of approximately 30 dyn/cm, the
peptides possess the ability to interact with the fibroblast plasma
membrane and successfully remove FC. The
e values for
peptides that effectively stimulate membrane microsolubilization are
listed in Table II; it is apparent that
all
e values are
30 dyn/cm. These results demonstrate a
strong relationship between the lipid binding properties of the
proteins and their effectiveness in membrane microsolubilization. The
capabilities of the peptides to stimulate FC efflux also
correlate well with their abilities to solubilize DMPC vesicles, as
indicated by Table I and Fig. 3, asterisks. The segment
representing amino acids 220-241 is the only peptide that can
solubilize DMPC vesicles but does not remove cellular FC; its
e of 28dyn/cm is slightly less than the value of 30dyn/cm
apparently required for membrane microsolubilization.
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To examine whether solubilization of cellular lipids by the apo A-I
molecule also is driven by the lipid binding affinity, apo A-I deletion
mutants were expressed and purified. As the C-terminal peptide 209-241
is able to efficiently stimulate FC and PL efflux and has a very high
monolayer exclusion pressure of 36 dyn/cm, we focused the mutations on
this region of the apo A-I molecule. Either a single helix or the last
two C-terminal helices were eliminated to give apo A-I (222-243)
and apo A-I (
190-243), respectively. The purified deletion mutants
and intact apo A-I were incubated with FC-enriched human fibroblasts
for the measurement of FC and PL efflux (Fig.
4). The elimination of the C-terminal helical segments clearly affects the ability of apo A-I to stimulate lipid efflux. Removal of this domain from apo A-I to create apo A-I
(
222-243) resulted in a significant reduction in both FC and PL
efflux; FC efflux to the deletion mutant was 55% and PL efflux 25% of
that measured to the intact apo A-I protein. Unexpectedly, removal of
an additional helix to form apo A-I (
190-243) restored much of the
ability to efficiently initiate membrane microsolubilization. FC and PL
efflux stimulated by this deletion mutant was 85 and 70%,
respectively, of that observed with the full-length protein. Similar
results have been reported previously for HepG2 cells in that apo A-I
with a deletion from residue 150-243 restores the loss of efflux
observed with a deletion from 222-243 (48). The deletion of C-terminal
-helical segments from apo A-I leads to parallel changes in membrane
microsolubilization and the lipid binding affinities of the variants.
The interaction of intact apo A-I and the variants with an egg PC
monolayer is shown in Fig. 5A.
Removal of a single
-helix (222-243) reduces
e from 34 to 25dyn/cm; elimination of the last two helical segments (190-243)
diminishes the exclusion pressure to only 29 dyn/cm. Therefore, there
is a distinct correlation between lipid binding affinity and the
solubilization of fibroblast plasma membrane lipids.
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To further investigate the role of lipid affinity in stimulating
cellular FC and PL efflux to lipid-free apolipoproteins, efflux to apo
E molecules with altered lipid affinities was monitored. The N-terminal
22-kDa segments of apo E3 and apo E4 were utilized; these proteins lack
residues 192-299, the lipid binding domain of the intact apo E
molecules (29). These apo E deletion mutants display a reduction in
e from the 33dyn/cm measured with the full-length proteins
to 28 and 27dyn/cm for apo E3 (
192-299) and apo E4 (
192-299),
respectively (Fig. 5B). A decreased ability to stimulate FC
and PL efflux is observed with these deletion mutants (Table
III). Both apo E3 (
192-299) and apo
E4 (
192-299) remove significantly less FC than intact apo E3 or apo
E4; approximately 50-60% of the FC accessed by the full-length
protein is released to the deletion mutants. Similarly, the proteins
lacking the lipid-binding region of apo E displayed 50% of the ability
of intact apo E to stimulate PL release.
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The correlation between cellular PL efflux and the e of the
various apolipoprotein molecules is plotted in Fig. 6. In contrast to the peptide results
plotted in Fig. 3, reduction of
e below the threshold value
of 30 dyn/cm does not completely eliminate the ability of the deletion
mutants to stimulate FC and PL efflux. Thus, although a major factor,
the lipid binding affinity of the near-full-length proteins seems not
to completely determine the efficiency of membrane microsolubilization.
However, the correlation between the ability of the apolipoproteins and variants to stimulate PL efflux and their respective
e values is significant, with an r2 value of 0.86 (Fig. 6).
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DISCUSSION |
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Lipid-free apo A-I mediates cellular FC and PL efflux through a membrane microsolubilization process (11). To understand the mechanism by which the apolipoprotein-lipid association occurs, the structural features and domains of the apo A-I molecule required for this process need to be clarified. Here we delineate some of the properties of apo A-I, as well as the other HDL apolipoproteins, that enable the unlipidated forms to efficiently solubilize human fibroblast FC and PL.
Requirements of the Protein-- To readily associate with plasma membrane lipids, specific structural features of apo A-I are presumably required. The amino acid sequence, molecular weight, and amphipathic nature of the protein are all potential determinants of the efficiency of this process. The results of this study clarify the contributions of these structural features to membrane microsolubilization.
Specific amino acid sequences could theoretically be necessary, particularly for association with cell surface receptors that mediate the microsolubilization process. However, despite differences in their primary structures, all of the apolipoproteins utilized as acceptors of FC are able to initiate FC and PL efflux (Figs. 1 and 2). This is consistent with previous findings with both full-length apolipoproteins (14, 15) and class A amphipathic peptides (46, 47). Furthermore, trypsin treatment of the fibroblast cell surface apparently has no inhibitory effect on membrane microsolubilization,2 supporting the idea of an interaction that does not involve a cell surface protein. Other cell types, such as macrophages, allow several of the HDL apolipoproteins to solubilize FC and PL yet have a trypsin-sensitive component (14, 49); the possible protein-protein interaction in this case must require structural aspects of the apolipoprotein other than a particular amino acid sequence.
The length of the apo A-I molecule may also contribute to its ability
to stimulate lipid solubilization as cooperativity between -helices
may drive the molecular solubilization of membrane PL and FC. Apo
Ctotal, apo CII, and apo CIII, the shortest apolipoproteins used, display a greater or equal ability to remove FC and PL as proteins five times as large when added on an equal mass basis. Of
course, the larger proteins are more effective on a molar basis. Furthermore, a single
-helix (44-65) of 22 residues in length and a
segment 33 residues (209-241) in length are able to initiate FC and PL
efflux (Table I). Increasing the length of segment 44-65 to a
double-helix structure of residues 44-87 does not enhance the FC and
PL release. Despite the ability of these peptides to access cellular
lipids, the extent of the removal is not as great as that observed with
the full-length proteins; peptides 44-65 and 209-241 remove
approximately 40 and 70%, respectively, of the lipids accessed by
intact apo A-I. These results indicate that simply increasing the
length of the protein molecule does not increase the ability to
stimulate FC and PL efflux from human fibroblast cells.
The lipid binding affinity of the proteins, arising from the
amphipathic nature of the -helical segments, appears to be the most
likely determinant of their effectiveness in membrane
microsolubilization because the ability of the proteins to associate
with lipids correlates with their capability to solubilize FC and PL.
The peptides must be able to exert a critical
e of
approximately 30 dyn/cm to support the efflux of FC and PL (Fig. 3);
this threshold value is not observed with the apolipoprotein deletion
mutants or full-length forms (Fig. 6). The various
-helical domains
in apo A-I exhibit different lipid affinities as reflected in their
e values (26). The interaction of the weakly binding
-helices is presumably facilitated by cooperative, intramolecular
interactions with the strongly binding terminal helical segments. For
this reason, there is not a defined threshold value for
e
apparent in Fig. 6. In contrast, for small peptides, the threshold
e of ~30 dyn/cm occurs because presumably all the
-helices have the same lipid affinity (i.e. the same
e), and there is an all-or-none effect with regard to
penetration into a PL monolayer.
Efflux of PL mediated by apo A-II was as great as that observed to apo
A-I and correlates well with the lipid binding affinity of the protein
(Fig. 6). However, apo A-II does not have as great a capacity for FC
efflux (Fig. 1) (50) as is expected from the measured e
value of 35 dyn/cm. The reasons for this are not clear, but perhaps the
dimeric structure of apo A-II alters the flexibility of the protein in
a manner that causes apo A-II to access plasma membrane lipid domains
that are not FC-enriched.
Model of Apo A-I Cellular Association--
The above results from
experiments studying apo A-I peptide-mediated FC and PL efflux are
consistent with a previously proposed model of the association of apo
A-I with lipid (26). In this model, the two helices nearest the ends of
the molecule have the greatest lipid affinity and drive the interaction
of apo A-I with the plasma membrane, initiating the solubilization of
cellular FC and PL. These -helices are best able to insert between
the molecules in a PL monolayer; this ability to penetrate into the surface of the PL bilayer is an essential step in the membrane microsolubilization process (Fig. 7). The
insertion into the lipids is expected to occur at sites of
solubilization, such as domain boundaries, where the plasma membrane
exhibits high lateral compressibility and the packing of the lipid
molecules is relatively loose (51). Cooperative interactions between
several
-helical segments and the lipid surface completes the
process, by creating an apolipoprotein/lipid structure that can
dissociate from the plasma membrane. These interactions are presumably
intermolecular for single-helix peptides and intramolecular for the
near full-length apolipoprotein molecules. The
-helical segments
that are successful in stimulating the solubilization of membrane
lipids are defined by the extent of their lipid binding affinity rather
than a specific amino acid sequence. In agreement with these ideas,
removal of the C-terminal helix of apo A-I that has the highest
e (Table II) decreases the ability to remove cellular
lipids (Fig. 4). Further support for a key role for lipid binding is
provided by previous findings that the C terminus of apo A-I dictates
the degree of association with lipid (22, 23, 25, 52). Similarly,
removal of the C-terminal lipid binding domain of apo E reduces the FC
and PL efflux to this protein (Table III). The mechanism of apo
A-I-mediated FC efflux involves two essential features: 1) the membrane
lipids must be organized in a manner that allows insertion of
-helices among the lipids, and 2) the
-helices must exhibit
sufficient binding affinity to induce the apolipoprotein-lipid
association. Thus, the domains of the apolipoprotein molecule that can
best penetrate between phospholipid molecules in a monolayer or bilayer (i.e. those in the C-terminal regions of apo A-I and apo E)
drive the association of the protein with the fibroblast plasma
membrane, thereby initiating membrane microsolubilization.
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ACKNOWLEDGEMENTS |
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We thank Faye Baldwin, Sheila Benowitz, and Margaret Nickel for expert technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL22633, HL56083, HL07443, and HL34343 and a predoctoral fellowship from the American Heart Association, Southeastern Pennsylvania Affiliate (to K. L. G.).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.
Current address: University of California, San Diego, Department
of Medicine, La Jolla, CA 92093-0682.
§ These authors contributed equally.
To whom correspondence should be addressed: Dept. of
Biochemistry, MCP Hahnemann University, 2900 Queen Ln., Philadelphia, Pennsylvania 19129.
The abbreviations used are:
HDL, high density
lipoprotein; apo, apolipoprotein; apo A-I (190-243), human apo A-I
lacking residues 190-243; apo A-I (
222-243), human apo A-I lacking
residues 222-243; apo E (
192-299), human apo E lacking residues
192-299; DMPC, dimyristoylphosphatidylcholine; FC, free (unesterified)
cholesterol; MEM, minimal essential medium;
i, initial
surface pressure;
e, monolayer exclusion pressure; PBS, phosphate-buffered salt solution; PL, phospholipid.
2 K. L. Gillotte, unpublished results.
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REFERENCES |
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