Apolipoprotein-mediated Plasma Membrane Microsolubilization
ROLE OF LIPID AFFINITY AND MEMBRANE PENETRATION IN THE EFFLUX OF CELLULAR CHOLESTEROL AND PHOSPHOLIPID*

Kristin L. GillotteDagger §, Mohamed Zaiou§, Sissel Lund-Katz, G. M. Anantharamaiah, Paul Holvoetparallel , Ann Dhoestparallel , Mayakonda N. Palgunachari, Jere P. Segrest, Karl H. Weisgraber**, George H. Rothblat, and Michael C. PhillipsDagger Dagger

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 parallel  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

    ABSTRACT
Top
Abstract
Introduction
References

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 alpha -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 (Delta 222-243), apo A-I (Delta 190-243), apo E3 (Delta 192-299) and apo E4 (Delta 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 alpha -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

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 alpha -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 alpha -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.

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 alpha -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 (Delta 190-243) and apo A-I lacking residues 222-243 (Delta 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 (Delta 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 alpha -helical segments that can penetrate into the plasma membrane and induce the solubilization of lipids.

    EXPERIMENTAL PROCEDURES

Materials

Chloramphenicol, cholesterol, cholesteryl methyl ether, isopropyl-beta -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.

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 -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 (epsilon  = 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).

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 (epsilon  = 1.4 and 1.31 ml/mg·cm for total apo C and apo E4, respectively). Recombinant apo E3, apo E3 Delta 192-299, and apo E4 Delta 192-299 samples expressed in E. coli were prepared as described previously (35).

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 alpha -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).

Expression and Purification of apo A-I Deletion Mutant Proteins-- Construction of the cDNA for apo A-I (Delta 222-243) and apo A-I (Delta 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-beta -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).

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 (epsilon  = 1.28 and 1.41 ml/mg·cm for apo A-I (Delta 222-243) and apo A-I (Delta 190-243), respectively).

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 (pi 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 (Delta pi 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 Delta pi i values were plotted as a function of pi  and linear extrapolation to the point at which Delta pi i = 0 dyn/cm provided the monolayer exclusion pressure (pi e).

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-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

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 alpha -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 (black-triangle), apo C (black-square), apo E3 (black-down-triangle ), and apo E4 (black-diamond ). 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 alpha -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 alpha -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 alpha -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 alpha -helices), as well as peptide 209-241, representing the C terminus, are capable of solubilizing cellular lipids.

                              
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Table I
FC and PL efflux to peptide segments of apo A-I

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 (pi 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 pi e represents an increase in lipid binding affinity. It is clear that as the lipid binding affinity increases beyond a threshold pi e of approximately 30 dyn/cm, the peptides possess the ability to interact with the fibroblast plasma membrane and successfully remove FC. The pi e values for peptides that effectively stimulate membrane microsolubilization are listed in Table II; it is apparent that all pi 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 pi e of 28dyn/cm is slightly less than the value of 30dyn/cm apparently required for membrane microsolubilization.


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Fig. 3.   Correlation of peptide lipid binding affinity with the ability to remove cellular FC. The percentage FC released from the FC-enriched fibroblasts was determined as described in Table I. Monolayer exclusion pressures for each peptide and intact apo A-I, obtained as described under "Experimental Procedures," were plotted against the FC efflux values; each symbol represents a specific peptide. Data were best fitted by a Boltzmann sigmoidal curve (r2 = 0.87). Peptides that can solubilize DMPC multilamellar vesicles are indicated by asterisks.

                              
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Table II
Monolayer exclusion pressures of apo A-I peptide segments

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 (Delta 222-243) and apo A-I (Delta 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 (Delta 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 (Delta 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 alpha -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 alpha -helix (222-243) reduces pi 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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Fig. 4.   Membrane microsolubilization by intact apo A-I and various apo A-I deletion mutants. FC and PL efflux from human fibroblasts trace labeled with [3H]cholesterol or [3H]choline chloride and enriched 2-fold in FC content was measured as described for Figs. 1 and 2. The indicated apolipoproteins present at 50 µg/ml in MEM-HEPES were incubated with the monolayers for 4 h. Columns represent the mean release of triplicate samples from two separate experiments ± 1 S.D.; all values have been corrected for radioactivity detected in incubation medium consisting of MEM-HEPES alone. Intact apo A-I and apo A-I (Delta 190-243) displayed significantly more FC and PL efflux than apo A-I (Delta 222-243) as determined by Student's t test (for all comparisons, p values were <= 0.02). PL efflux was decreased upon deletion of residues 190-243 as compared with apo A-I (p = 0.02).


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Fig. 5.   Interaction of apo A-I, apo E, and protein variants with phospholipid monolayers. The surface balance technique utilized to determine the surface pressure at which proteins are no longer able to penetrate an egg PC monolayer was carried out as described under "Experimental Procedures." A plots the Delta pi i values for apo A-I (), apo A-I (Delta 190-243) (triangle ), and apo A-I (Delta 222-243) (down-triangle) as a function of the initial surface pressure, pi i; B displays the equivalent data for apo E3 (black-square), apo E4 (black-down-triangle ), apo E3 (Delta 192-299) (black-triangle), and apo E4(Delta 192-299) (black-diamond ). Symbols represent duplicate measurements, and lines were generated by the linear regression of the data points.

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 pi e from the 33dyn/cm measured with the full-length proteins to 28 and 27dyn/cm for apo E3 (Delta 192-299) and apo E4 (Delta 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 (Delta 192-299) and apo E4 (Delta 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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Table III
Membrane microsolubilization by apo E variants

The correlation between cellular PL efflux and the pi e of the various apolipoprotein molecules is plotted in Fig. 6. In contrast to the peptide results plotted in Fig. 3, reduction of pi 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 pi e values is significant, with an r2 value of 0.86 (Fig. 6).


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Fig. 6.   Correlation between the lipid binding affinity of apo A-I and its ability to remove cellular PL. The percentage of PL released from the FC-enriched fibroblasts was determined as described in Fig. 4. Monolayer exclusion pressures for full-length apolipoproteins and the apo E and apo A-I variants were obtained as described under "Experimental Procedures." Each symbol represents the protein species as indicated. Linear regression was performed; the resulting r2 value is 0.86.


    DISCUSSION

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 alpha -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 alpha -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 alpha -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 pi 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 alpha -helical domains in apo A-I exhibit different lipid affinities as reflected in their pi e values (26). The interaction of the weakly binding alpha -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 pi e apparent in Fig. 6. In contrast, for small peptides, the threshold pi e of ~30 dyn/cm occurs because presumably all the alpha -helices have the same lipid affinity (i.e. the same pi 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 pi 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 alpha -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 alpha -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 alpha -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 pi 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 alpha -helices among the lipids, and 2) the alpha -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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Fig. 7.   Model of the mechanism of membrane microsolubilization by apo A-I. The end two helices of apo A-I, exhibiting high affinity for lipid, penetrate into the phospholipid bilayer at specific FC-enriched sites of solubilization. The lipid binding affinity of these helices, as well as helix-helix interactions between the remaining helices of apo A-I induce the solubilization of a portion of the plasma membrane, forming a nascent HDL species. See under "Discussion" for further details. C, cholesterol.


    ACKNOWLEDGEMENTS

We thank Faye Baldwin, Sheila Benowitz, and Margaret Nickel for expert technical assistance.

    FOOTNOTES

* 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.

Dagger Current address: University of California, San Diego, Department of Medicine, La Jolla, CA 92093-0682.

§ These authors contributed equally.

Dagger Dagger 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 (Delta 190-243), human apo A-I lacking residues 190-243; apo A-I (Delta 222-243), human apo A-I lacking residues 222-243; apo E (Delta 192-299), human apo E lacking residues 192-299; DMPC, dimyristoylphosphatidylcholine; FC, free (unesterified) cholesterol; MEM, minimal essential medium; pi i, initial surface pressure; pi e, monolayer exclusion pressure; PBS, phosphate-buffered salt solution; PL, phospholipid.

2 K. L. Gillotte, unpublished results.

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