From the Section of Molecular Genetics, Whitaker
Cardiovascular Institute, Departments of Medicine and Biochemistry, and
the § Department of Physiology and Biophysics, Boston
University School of Medicine, Boston, Massachusetts 02118 and the
¶ Institute of Clinical Chemistry and Laboratory Medicine,
University of Munster, Albert-Schweitzer-Strasse 33, 48149 Munster, Germany
Received for publication, May 28, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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We have mapped the domains of lipid-free
apoA-I that promote cAMP-dependent and cAMP-independent
cholesterol and phospholipid efflux. The cAMP-dependent
lipid efflux in J774 mouse macrophages was decreased by ~80-92% by
apoA-I[ Apolipoprotein A-I (apoA-I)1 is the major protein
constituent of high density lipoproteins
(HDL) and plays crucial roles in the synthesis, structure, and function
of HDL (1). One important function of apoA-I and HDL is the induction
of cholesterol efflux from cells, a process that is crucial for
regulating cellular free cholesterol homeostasis (2, 3), receptor
endocytosis and recycling (4), and cell signaling (5). Four different kinds of interaction between extracellular acceptors have been proposed
to cause cholesterol efflux. (a) Natural or reconstituted HDL particles or other lipophilic acceptor particles can cause a
passive diffusion of cholesterol from cholesterol-poor membrane domains
through the aqueous phase (2, 6). (b) Interaction of
cellular proteins (e.g. scavenger receptor B-I) with HDL
facilitates the efflux of unesterified cholesterol to extracellular
cholesterol acceptors (7-9). Activation of the cholesterol-esterifying
enzyme lecithin:cholesterol acyltransferase by apoA-I also facilitates this process (10, 11). (c) Lipid-free apolipoproteins with a
high content of amphipathic The mechanism of interaction between ABCA1 and apoA-I is not fully
resolved. On one hand overexpression of ABCA1 was shown to enhance the
cellular binding and uptake of apoA-I (27-30). Because apoA-I can be
cross-linked with ABCA1, a direct physical interaction between apoA-I
and ABCA1 has been postulated as important for mediating the
ABCA1-dependent lipid efflux. On the other hand it has also
been suggested that ABCA1 does not interact physically with apoA-I but
changes the membrane lipid microenvironment so that apoA-I can
associate with the cell membrane and acquire phospholipids by binding
in the proximity of ABCA1 (20). Analysis of five naturally occurring
ABCA1 mutants, in the first and fourth extracellular domains, that are
defective in cholesterol efflux showed that four of the five mutants
failed to cross-link to apoA-I. The findings suggest that either direct
binding of apoA-I to ABCA1 or insertion of apoA-I in the plasma
membrane in close proximity to ABCA1 is necessary but not sufficient to
promote cholesterol efflux (31). It has also been shown that
ABCA1-mediated binding and lipid efflux are not specific for apoA-I but
may also occur with other apolipoproteins that contain multiple
amphipathic helical domains (27).
Previously, deletion mutants of apoA-I have been analyzed to unravel
the structural requirements of apoA-I for inducing cholesterol efflux.
Deletion of the carboxyl-terminal helices 8-10 in apoA-I (apoA-I[ In the present study we have analyzed the ability of various
recombinant apoA-I mutants lacking amino- and carboxyl-terminal domains
to promote efflux of cellular cholesterol and phospholipid from J774
mouse macrophages in the presence of a cAMP analog (i.e. ABCA1-catalyzed) or in the absence of a cAMP analog (i.e. at
least partially independent of ABCA1). We have performed similar
analysis in human embryonic kidney (HEK) 293 cells following transient transfection with an ABCA1 expression plasmid. Thereby we found evidence that the ABCA1-mediated cholesterol efflux by the full-length apoA-I requires a carboxyl-terminal domain in apoA-I, which encompasses amino acid residues 220-231. The analysis of apoA-I mutants containing deletions in the amino-terminal region, carboxyl-terminal region, or
both domains suggests that although there is ligand specificity of the
full-length apoA-I, the central helices of apoA-I alone are capable of
promoting ABCA1-mediated lipid efflux and the formation of discoidal
HDL in vitro and in vivo.
Materials
DNA-modifying enzymes were purchased from New England Biolabs,
Inc. (Beverly, MA). Oligonucleotides for PCR, DH10Bac competent cells,
cell culture reagents, recombinant Tobacco Etched Viral (rTEV)
protease, and LipofectAMINE-2000 were purchased from Invitrogen. 1,2-[3H]Cholesterol (1 mCi/ml, specific activity range
40-60 Ci/mmol), methyl[3H]choline chloride (1 mCi/ml,
specific activity range 60-90 Ci/mmol), and materials for PCR were
obtained from PerkinElmer Life Sciences. Nickel-nitrilotriacetic acid
resin was purchased from Qiagen, Inc. (Valencia, CA). HiTrap Q HP
columns, HiPrep Sephacryl S-200 column, Sepharose 6 PC column, and
Q-Sepharose high performance resin were from Amersham Biosciences.
8-(4-Chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP)
and bovine serum albumin (BSA) were from Sigma. All other reagents were
purchased from Sigma, Bio-Rad, Fisher, or other standard commercial sources.
Generation and Isolation of Recombinant Wild-type ApoA-I or
ApoA-I Mutants in the Baculovirus System
To generate baculoviruses expressing wild-type (WT) and mutant
apoA-I forms, a BamHI-SalI fragment containing
human apoA-I cDNA was cloned into the polylinker region of pFASTBAC
donor plasmid that contains the ampicillin and gentamycin resistance
genes (Invitrogen). This recombinant plasmid also contains a histidine
(His) tag and the TEV protease cleavage site. The apoA-I containing
plasmid was used to transform DH10Bac Escherichia coli
cells. These cells had been transformed previously with the baculovirus
genome containing the lacZ and kanamycin resistance genes,
along with a helper plasmid containing the tetracycline resistance gene
and the transposase genes. Transposition of the
apoA-I gene into the lacZ gene disrupts the expression of the lacZ and provides a recombinant
plasmid named bacmid.
The generation of the donor plasmid expressing WT apoA-I and of
plasmids expressing apoA-I[(185-243)], only by 15% by apoA-I[
(1-41)] or
apoA-I[
(1-59)], and was restored to 75-80% of the wild-type
apoA-I control value by double deletion mutants apoA-I[
(1-41)
(185-243)] and
apoA-I[
(1-59)
(185-243)]. Similar results were obtained in
HEK293 cells transfected with an ATP-binding cassette transporter A1
(ABCA1) expression plasmid. The double deletion mutant of apoA-I had
reduced thermal and chemical stability compared with wild-type apoA-I.
Sequential carboxyl-terminal deletions showed that
cAMP-dependent cholesterol efflux was diminished in all the
mutants tested, except the apoA-I[
(232-243)] which had normal
cholesterol efflux. In cAMP-untreated or in mock-transfected cells,
cholesterol efflux was not affected by the amino-terminal deletions,
but decreased by 30-40% and 50-65% by the carboxyl-terminal and
double deletions, respectively. After adenovirus-mediated gene transfer
in apoA-I-deficient mice, wild-type apoA-I and apoA-I[
(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[
(1-41)
(185-243)] formed discoidal HDL. The
findings suggest that although the central helices of apoA-I alone can
promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1
that are required for lipid efflux and HDL biogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices, such as apoA-I, can associate with multiple molecules of cellular phospholipids and free cholesterol by a process that was termed microsolubilization (12, 13). (d) Lipid-free apoA-I can also promote efflux of cellular
phospholipids and cholesterol by an ATP-dependent process,
which involves the Tangier disease gene product, i.e. the
ATP-binding cassette transporter A1 (ABCA1) (14-17). It has been
proposed that initial interactions of apoA-I and ABCA1 promote efflux
of membrane phospholipids, which results in the gradual extracellular
lipidation of apoA-I and in the formation of pre-
1-HDL.
This apoA-I-phospholipid complex promotes further efflux of
cellular cholesterol (14, 18-22) and the formation of discoidal
pre-
2-HDL and, after cholesterol esterification by
lecithin:cholesterol acyltransferase, spherical
-HDL particles (23-26).
(187-243)] or apoA-I[
(210-243)]) reduced the
ability of the mutant protein to bind to macrophages and to promote
efflux of cholesterol and phospholipid (32, 33). Likewise, deletion of
amino acid residues 211-243 and 222-243 impaired the ability of
apoA-I to promote cholesterol efflux from cultures of hepatocytes and
fibroblasts (13, 34), whereas deletion of several helices in apoA-I
(apoA-I[
(100-143)], apoA-I[
(122-165)],
apoA-I[
(144-186)], apoA-I[
(135-243)],
apoA-I[
(150-243)], and apoA-I[
(187-243)]) did not affect
cholesterol efflux from the same cells (13, 32, 34).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1-59)], apoA-I[
(185-243)], and apoA-I[
(1-59)
(185-243)] mutants was described previously (35, 36). The other mutant forms of apoA-I were produced in a similar manner. Briefly, the human apoA-I cDNA was mutagenized by PCR, using a set of flanking universal primers containing the restriction sites BamHI and SalI, using the pBluescript-AI
cDNA plasmid as template. The set of specific primers used in
mutagenesis, and the set of universal primers 5'(AIWC-5) and 3'(AIWC-3)
are shown in Table I. Specifically, we
used 5'(AIM1-5) and 3'(AIM1-3) primers for the generation of
apoA-I[
(1-41)], and 5'(AIM1-5) and 3'(AIM3-3) primers for the
generation of apoA-I[
(1-41)
(185-243)]. The DNA fragment
containing the mutation of interest was digested with BamHI
and SalI, and cloned into the corresponding sites of
pFASTBAC donor plasmid. Cells containing recombinant bacmids were
selected by kanamycin, tetracycline, and gentamycin resistance as white colonies due to the disruption of lacZ sequence in
the recombinant bacmid. Recombinant bacmid DNA was isolated from
minipreps and used to transfect a monolayer of Sf-9 insect cells
(38-40). Recombinant viruses were isolated, amplified, titrated, and
used to infect larger Sf-9 cell cultures grown in suspension at
27 °C. Sf-9 cells were pelleted and resuspended in a lysis buffer.
The supernatant was used for the purification of apoA-I fusion proteins
(His+-apoA-I) using a nickel-nitrilotriacetic acid affinity
column (41, 42). The pure apoA-I without the His tag
(His
-apoA-I), when needed, was obtained by cleavage with
rTEV protease and purified by using again the nickel-nitrilotriacetic
acid affinity chromatography.
Oligonucleotides used in PCR amplification and mutagenesis
Expression and Purification of Recombinant WT and Mutant Pro-apoA-I Isoforms in C127 Cells
Construction of plasmids expressing WT pro-apoA-I and
pro-apoA-I[(198-243)] has been described previously (44, 45). The pro-apoA-I[
(209-243)] deletion mutant was constructed in a
similar manner. Briefly, the fourth exon of the human apoA-I
gene was amplified and mutagenized by PCR, using a set of specific
mutagenic primers (209S and 209A) containing the mutation of interest
(a stop codon at nucleotide 209) and a set of flanking universal primers (AINotI and AIXhoI) containing the
restriction sites NotI and XhoI. The sequences of
the primers are shown in Table I. The pUCA-IN* vector,
which contains a NotI site in intron 3 and a XhoI
site in the 3'-end of the apoA-I gene, was used as a
template in the amplification reactions (43). The DNA fragment
containing the mutation of interest was digested with NotI
and SalI and cloned into the NotI and
XhoI sites of the pBMT3X-AI vector.
Permanent cell lines in mouse mammary tumor C127 cells expressing the
WT pro-apoA-I and the carboxyl-terminal truncation
pro-apoA-I[(198-243)] and pro-apoA-I[
(209-243)] were
generated as described previously (43). C127 cell clones overproducing
the WT and the variant apoA-I forms were grown in roller bottles on
collagen-coated lead microspheres (Verax Corp.), and the proteins were
purified from the serum-free medium as described previously (43) with
some modifications. Briefly, the medium collected from the roller
bottles was dialyzed against 0.01 M Tris, pH 8, and
filtered. Up to 350 ml of the medium were passed through a 5-ml HiTrap
Q HP column or up to 1200 ml were passed through a 25-ml Q-Sepharose
high performance column at a rate of 1 ml/min. Both columns had been equilibrated with the Tris buffer. The proteins were eluted with a
linear gradient of 1 M NH4HCO3 in
the Tris buffer (15 volumes of elution buffer for HiTrap Q column and 5 volumes for Q-Sepharose high performance column) at the same flow rate.
The fractions were analyzed by SDS-PAGE, and those containing the
apoA-I protein were pooled and further concentrated down to 2 ml, using
a Centriplus concentrator (Amicon) with a molecular weight cut-off of
10,000. The concentrated sample was applied to a gel filtration column HiPrep Sephacryl S-200 and eluted with 1 column volume (120 ml) of 0.15 M NH4HCO3 buffer at a rate of 0.4 ml/min. The purity of the apoA-I preparation was assessed by SDS-PAGE,
and fractions greater than 95% pure were recovered.
Expression of WT and Mutant Pro-apoA-I Isoforms in an Adenovirus System
The construction of recombinant adenoviruses carrying the
genomic sequence for the WT pro-apoA-I and pro-apoA-I[(220-243)] has been described before (45). The adenovirus expressing
pro-apoA-I[
(232-243)] was generated in a similar way. The DNA
fragments containing the mutations of interest were produced as
described in the previous paragraph, using the 220S and 220A primers
for the mutagenesis of pro-apoA-I[
(220-243)] and the 232S and
232A primers for pro-apoA-I[
(232-243)] (Table I). The recombinant
adenoviruses expressing pro-apoA-I[
(1-41)] and
pro-apoA-I[
(1-41)
(185-243)] were generated using cDNAs
for apoA-I deletion mutants. The human apoA-I cDNA was mutagenized by PCR, using a set of primers containing the restriction sites BamHI and SalI and using the pBluescript-AI
cDNA plasmid as template. The set of specific primers used in
mutagenesis and the set of primers containing the restriction sites
BamHI and SalI, designated AI(1-59)F and
AI(1-59)R, are shown in Table I. Specifically, we used AI(1-41)DF and
AI(1-41)DR primers for the generation of pro-apoA-I[
(1-41)] and
AI185F and AI185R primers for generating the
pro-apoA-I[
(185-243)] form. For the generation of
pro-apoA-I[
(1-41)
(185-243)], AI(1-41)DF and AI(1-41)DR
primers were used to further mutagenize the apoA-I cDNA using
pBluescript-AI[
(185-243)] as template. Following amplification
and mutagenesis, the DNA fragments were digested with BamHI
and SalI and subcloned into the pCA13 vector under the
control of the cytomegalovirus promoter (Microbix Biosystems Inc.).
Recombinant adenovirus constructs carrying this cDNA were prepared
following co-transfection of E1 transformed 911 cells with the plasmid
pJM17 (Microbix Biosystems Inc.) and the appropriate pCA13 plasmid
(45).
Human HTB13 cells (SW 1783, human astrocytoma grown to 80% confluence
in Leibovitz's L-15 medium containing 10% (v/v) fetal bovine serum
(FBS) in roller bottles) were infected with adenoviruses expressing WT
pro-apoA-I, pro-apoA-I[(220-243)], and
pro-apoA-I[
(232-243)] at a multiplicity of infection of 20. After
24 h of infection, cells were washed twice with serum-free medium
and preincubated in serum-free medium for 30 min, and fresh serum-free
medium was added. After 24 h the medium was harvested, and fresh
serum-free medium was added to the cells. The harvests were repeated
8-10 times.
The proteins were purified from the serum-free medium by ion exchange chromatography followed by gel filtration, as described above for C127 cells.
ABCA1 Expression Plasmid
The expression plasmid of human ABCA1 was a generous gift of Dr. M. W. Freeman and was constructed as described (46).
Cell Cultures and Lipids Efflux Assays
Studies in J774 Mouse Macrophages-- J774 mouse macrophages were grown in RPMI 1640 with 10% (v/v) FBS and antibiotics. J774 macrophages were plated in 12-well plates at density of 5 × 105 cells/well. The following day, cells were labeled with 1 ml of labeling medium (6 µCi/ml 1,2-[3H]cholesterol or 1 µCi of methyl[3H]choline chloride dissolved in 0.1% (v/v) ethanol as the final concentration and dispersed into RPMI 1640 supplemented with 0.2% (w/v) BSA). Following 24 h of labeling, cells were washed twice with serum-free medium and equilibrated for 24 h with or without 0.3 mM cpt-cAMP in 1 ml of RPMI 1640 supplemented with 0.2% (w/v) BSA. At the end of the treatment period with cpt-cAMP, cells were washed twice with serum-free medium and incubated with 1 ml of RPMI 1640, supplemented with 0.2% (w/v) BSA, with or without 1 µM WT apoA-I or mutant forms at 37 °C. For cholesterol efflux experiments, at different time points up to 6 h, 55 µl of medium were collected and clarified by centrifugation in a microcentrifuge for 2 min. The radioactivity in 40 µl of the supernatant was determined by liquid scintillation counting. For phospholipid efflux experiments, after 4 h of incubation, the media were collected and clarified by centrifugation in a microcentrifuge for 2 min. A 100-µl aliquot of the supernatant was first extracted with chloroform and methanol (47), and then the radioactivity of the chloroform phase was determined by liquid scintillation counting. At the end of the incubation, cells were lysed by 800 µl of lysis buffer (PBS containing 1% (v/v) Triton X-100) for 30 min at room temperature. For cholesterol efflux experiments, the radioactivity in 40 µl of each cell lysate was determined by scintillation counting. For phospholipid efflux experiments, the phospholipids in 100 µl of each lysate were extracted with chloroform and methanol (47), and the radioactivity was determined by scintillation counting. The percentage of secreted 3H-lipid was calculated by dividing the medium-derived counts by the sum of the total. To calculate the net cpt-cAMP-dependent efflux, the lipid efflux of the untreated cells was subtracted from the lipid efflux of the cells treated with cpt-cAMP.
Studies in HEK293 Cells following Transfection with an ABCA1 Expression Plasmid-- HEK293-EBNA cells were cultured in DMEM (high glucose) with 10% (v/v) FBS and antibiotics (31, 46). One day before transfection, the cells were plated on 24-well plates coated with poly-D-lysine at density of 2 × 105 cells/well in the same medium without antibiotics. The next day, cells at about 95% confluence were transfected with LipofectAMINE 2000 and ABCA1 or pcDNA1 (mock) (Invitrogen) plasmids for 16 h. The DNA-LipofectAMINE 2000 complexes were formed in serum-free Opti-MEM I. After this period of time, cells were labeled with 0.5 µCi/ml 1,2-[3H]cholesterol in DMEM (high glucose) supplemented with 10% (v/v) FBS. Following 24 h of labeling, cells were washed twice with PBS and incubated for 1 h with DMEM (high glucose) supplemented with 0.2% (w/v) fatty acid-free BSA. At the end of this period, cells were incubated with 0.5 ml of DMEM (high glucose) and supplemented with 0.2% (w/v) fatty acid-free BSA with or without 1 µM WT apoA-I or mutant forms at 37 °C for 4 h. The media were collected and clarified by centrifugation in a microcentrifuge for 2 min, and the radioactivity in 300 µl of the supernatant was determined by liquid scintillation counting. The cells were lysed with 0.1 M NaOH, and the radioactivity in a 300-µl aliquot of the cell lysate was determined by scintillation counting. [3H]Cholesterol efflux was expressed as the percentage of the radioactivity released in the medium relative to the total radioactivity in cells and medium. To calculate the net ABCA1-mediated efflux, the cholesterol efflux of the cells transfected with the control plasmid (mock) was subtracted from the cholesterol efflux of the cells transfected with the ABCA1 plasmid.
Animal Studies
Male apoA-I-deficient mice (apoA-I/
)
(ApoA1tm1Unc) C57BL/6J mice (48) were purchased from The
Jackson Laboratories (Bar Harbor, ME). The mice were maintained on a
12-h light/dark cycle and standard rodent chow. The mice were 6-8
weeks old at the time of injection of the adenoviruses. All procedures
performed on the mice were in accordance with National Institutes of
Health and institutional guidelines. The A-I
/
mice were
injected via the tail vein with 1 × 109
plaque-forming units of recombinant adenovirus per animal, and the
animals were sacrificed 4 days post-injection following a 4-h fast. The
plasma of the mice was adjusted to 1 mM
phenylmethylsulfonyl fluoride, 0.2% (w/v) aprotinin, 0.1% (w/v) EDTA,
and 0.002% (w/v) sodium azide prior to analysis of lipids,
lipoproteins, and apoA-I.
Cholesterol, Triglycerides, and Plasma ApoA-I Levels and Steady-state Hepatic Human ApoA mRNA Levels
The concentration of cholesterol and triglycerides in plasma was determined using the INFINITY cholesterol reagent (Sigma) and INFINITY triglyceride reagent (Sigma) according to the manufacturer's instructions. Cholesterol and triglyceride concentrations were determined spectrophotometrically at 490 nm. The concentration of human apoA-I in plasma was determined by sandwich ELISA (36). Mouse monoclonal anti-human apoA-I antibodies 5F6 (University of Ottawa Heart Institute) (the epitope is amino acids 118-141 of the human apoA-I) were used for coating microtiter plates, and sheep polyclonal anti-human apoA-I coupled to horseradish peroxidase (Biodesign International) was used as a secondary antibody. The immunoperoxidase procedure was employed for the colorimetric detection of apoA-I at 490 nm, using o-phenylenediamine dihydrochloride as substrate. The concentration was estimated based on standard curves constructed either with a human serum standard (Cardiolipid Level 1, Sigma) or with the purified lipid free WT and mutant forms of apoA-I. The concentration of human apoA-I in plasma was also estimated by Western blot analysis of the apoA-I samples. The membrane was probed with a goat polyclonal anti-human apoA-I antibody (Chemicon International). The proteins were visualized by chemiluminescence following incubation with rabbit anti-goat IgG (Biodesign International) coupled to horseradish peroxidase. ApoA-I was quantitated by scanning densitometry using different concentrations of purified WT apoA-I so that the intensities of the signal of plasma WT and mutant apoA-I forms were similar and in the linear range.
Total cellular RNA was isolated from the mouse liver using the Trizol reagent (Invitrogen) as recommended by the manufacturer. 15 µg of RNA were separated on 1.0% agarose-formaldehyde gels, transferred to Hybond-N+ nylon membrane (Amersham Biosciences), and cross-linked to the filter by UV irradiation (Stratalinker, Stratagene). The apoA-I probe for hybridization contained 290 bp of exon 4 of human apoA-I and 148 bp of the intergenic sequence between the apoA-I and apoCIII genes. The mouse glyceraldehyde-3-phosphate dehydrogenase probe (905 bp) was obtained from Ambion. The probe was labeled with 32P using the Multiprime DNA labeling system (Amersham Biosciences). Quantitation of the x-ray film was performed by a PhosphorImager (Amersham Biosciences) using the ImageQuant program. The apoA-I mRNA signal was normalized for the glyceraldehyde-3-phosphate dehydrogenase mRNA signal.
Fractionation of Plasma by Equilibrium Density Gradient Centrifugation and Electron Microscopy Analysis of the ApoA-I-containing Fractions
An aliquot of 250 µl of plasma was adjusted to 2 ml with PBS, and the lipoproteins were separated on 10-20% sodium bromide gradients (49). The tubes were spun for 66 h at 32,000 rpm on an SW-41 rotor (Beckman Instruments), and fractions (0.4 ml) were collected from top to bottom. The densities of the fractions were determined by comparing the readings measured with a refractometer (American Optical Corp.) to values obtained for solutions with known densities. The fractions were dialyzed against ammonium acetate and carbonate buffer (126 mM ammonium acetate, 2.6 mM ammonium carbonate, 0.26 mM EDTA, pH 7.4). Aliquots of the fractions were subjected to 13% SDS-PAGE, and the gels were transferred to nitrocellulose membrane for Western blot analysis. The membrane was probed with the mouse monoclonal anti-human apoA-I antibody 5F6 (University of Ottawa Heart Institute). The proteins were visualized by chemiluminescence following incubation with goat anti-mouse IgG1 (Roche Molecular Biochemicals) coupled to horseradish peroxidase. Fractions containing peak concentration of apoA-I were analyzed by electron microscopy.
Electron Microscopy
Aliquots of the fractions from equilibrium density gradient centrifugation after dialysis against ammonium acetate and carbonate buffer were stained with sodium phosphotungstate, visualized in the Phillips CM-120 electron microscope (Phillips Electron Optics, Eindhoven, Netherlands), and photographed as described previously (50). The photomicrographs were taken at ×75,000 magnification and enlarged 3 times.
Preparation and Electron Microscopy Analysis of rHDL Containing POPC:Cholesterol:ApoA-I
rHDL particles were prepared by the sodium cholate dialysis method (51), using POPC:cholesterol:apoA-I:sodium cholate in a molar ratio of 100:10:1:100, as described previously (52). The lipid-free apoA-I that was not incorporated in the lipid-protein complexes was removed by dialysis overnight against PBS buffer at 4 °C with at least 3 changes of 4 liters each, using dialysis tubing of 50,000 daltons molecular weight cut-off. The rHDL samples were adjusted either by dilution or by concentration to a final protein concentration of ~1 mg/ml. A 5-µl aliquot of each sample was used for electron microscopy as described above. The photomicrographs were taken at ×45,000 magnification and enlarged 2.5 times.
CD Spectroscopy
CD measurements were performed on an AVIV 62 DS
spectropolarimeter (AVIV Associates, Inc.), equipped with a
thermoelectric temperature control and calibrated with
d-10-camphorsulfonic acid in 5-, 2-, or 1-mm path length
quartz cuvettes, at protein concentration 20-100 µg/ml. Far-UV CD
spectra were recorded as described previously (44, 53). The spectra
were normalized to molar residue ellipticity using a mean residue
weight of 115.0 for WT apoA-I, 115.3 for A-I[(1-41)], and 116.7 for A-I[
(1-41)
(185-243)]. The complete superimposition of the
normalized spectra obtained at various protein concentrations for each
recombinant apoA-I is consistent with the absence of protein
self-association within the range of protein concentrations used in our
experiments. The
-helix content was estimated from the molar residue
ellipticity at 222 nm [
222] (54).
Thermal and Chemical Unfolding Monitored by CD
Monitoring of thermal and chemical unfolding of apoA-I by
ellipticity at 222 nm was performed at protein concentrations 25-80 µg/ml as described previously (44, 53). The midpoint (melting temperature), Tm, and van't Hoff enthalpy,
Hv, of the temperature-induced transitions were
determined from van't Hoff plots (55), ln Keq
versus 1/T, where Keq is
the apparent equilibrium constant, and T is temperature in
degrees Kelvin. The equilibrium constant was calculated as
Keq (T) = (
F
obs)/(
obs
U), where
obs is the observed ellipticity, and
F and
U are base lines for the protein folding and unfolding
states determined by linear extrapolations of the pre- and
post-transitional regions. In the transition region, the plots were
fitted by the linear function, ln Keq =
S/R
(
Hv/R) × 1/T, where
R = 1.98 cal/mol × K is the universal gas constant, and
S is the transition entropy.
For chemical unfolding, the free energy of denaturation (conformational
stability), GD0, the midpoint of
denaturation, D1/2, and m value that
reflects the steepness of the denaturation curve in the transition
region were determined using a linear extrapolation method (56). A plot
of Gibbs free energy
GD =
RT ln
Keq, where T = 25 °C = 298.15 K, versus denaturant concentration, [D],
was fitted by the linear function,
GD =
GD0
m [D].
The apparent equilibrium constant, Keq, was
calculated as shown above, except
obs ([D])
is the observed ellipticity at a given concentration of GdnHCl,
[D], and
F and
U are the ellipticity
in the folded and unfolded states.
F and
U were
taken as constants.
DMPC Clearance Kinetic Analysis
The initial rate of binding of WT and mutant proteins to DMPC
multilamellar liposomes was studied using a kinetic-turbidimetric method (57). Briefly, 250 µg of DMPC in chloroform:methanol (2:1)
solution were dried under nitrogen and resuspended in 900 µl of PBS
by vortexing to form liposomes. 100 µg of apoA-I in 100 µl of PBS
were added to the lipid emulsion to give DMPC:apoA-I ratio of 2.5:1
(w/w) and apoA-I concentration of 0.1 mg/ml. The DMPC clearance was
followed by monitoring the decrease in absorbance at 325 nm at 5-min
intervals at 24 °C. A plot of absorbance at 325 nm versus
time in min, A(t), was fitted by an exponential decay
function: A(t) = A + ae
t/k, where
A
, a, and k are fitting parameters.
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RESULTS |
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Production and Purification of WT and Mutant ApoA-I Forms Used in
Lipid Efflux Studies--
Fig. 1 shows
the apoA-I forms used in this study and the expression system used for
their production. The WT and the variant apoA-I forms were produced
using three different expression systems (baculovirus, adenovirus, and
permanent cell lines). SDS-PAGE analysis showed that all apoA-I forms
were apparently free of any contaminations (Fig. 1).
|
Effect of Amino-, Carboxyl-terminal and Double Deletion Mutants of ApoA-I on cAMP-dependent and cAMP-independent Efflux of Cellular Cholesterol from J774 Mouse Macrophages and the Influence of the His Tag-- To determine the potential domains of apoA-I that contribute to cholesterol efflux, J774 mouse macrophages were labeled with [3H]cholesterol for 24 h and then incubated in serum-free culture medium for 24 h in the presence or absence of 0.3 mM of the cAMP analog cpt-cAMP. cAMP analogs have been reported to enhance the expression of ABCA1 (18, 58, 59). Cholesterol efflux was monitored as a function of time in the presence of 1 µM lipid-free WT or mutant forms of apoA-I, as described under "Experimental Procedures." The apoA-I concentration used is higher than that required for saturable cholesterol or phospholipid efflux in untreated or cpt-cAMP-treated J774 macrophages (60) and in transiently or stably transfected cells expressing the ABCA1 transporter (27, 28).
The time course of cholesterol efflux from untreated (cAMP-independent
cholesterol efflux) and cpt-cAMP-treated (total cholesterol efflux)
cells, respectively, using apoA-I forms lacking the amino-terminal 1-41 or 1-59 segments or the carboxyl-terminal 185-243 segment or
both the amino- and carboxyl-terminal segments, was monitored over a
6-h period. The time course of the net cpt-cAMP-dependent cholesterol efflux was obtained by subtracting the efflux curves of the
untreated cells from the treated cells. This analysis indicated that
the efflux curves (total, cpt-cAMP-independent (data not shown) and
cpt-cAMP-dependent (Fig.
2A)) are almost linear over a
4-h period. To facilitate the analysis of the cholesterol efflux by the
different apoA-I forms, we tabulated total, cpt-cAMP-independent, and
cpt-cAMP-dependent efflux after 4 h of incubation with
1 µM WT apoA-I and mutants at 37 °C, setting the
efflux values for WT apoA-I to 100% (Fig. 2B). (Similar
values were obtained using the efflux results determined after 2 h
of incubation.)
|
The cpt-cAMP-independent efflux of cellular cholesterol was not
affected by amino-terminal deletions (apoA-I[(1-41)] and apoA-I[
(1-59)]) but was decreased by ~30% by the
carboxyl-terminal truncation of apoA-I (apoA-I[
(185-243)]).
Removal of both amino-terminal and carboxyl-terminal domains in apoA-I
(apoA-I[
(1-41)
(185-243)] and
apoA-I[
(1-59)
(185-243)]) decreased the cpt-cAMP-independent cholesterol efflux by 50-65% (Fig. 2B, white
bars). The cpt-cAMP-dependent cholesterol efflux was
reduced by 80% compared with WT apoA-I when the carboxyl-terminal
apoA-I[
(185-243)] deletion mutant was used as the cholesterol
acceptor. Much less pronounced impairments of cholesterol efflux were
seen in the presence of apoA-I variants with amino-terminal
deletions since removal of the amino terminus decreased cholesterol
efflux by 15%. Unexpectedly, cpt-cAMP-dependent cholesterol efflux was restored to 78% of WT apoA-I when the
cholesterol efflux assay was performed in the presence of double
deletion mutants lacking both the amino- and carboxyl-terminal domains (Fig. 2B, shaded bars). These findings indicate
that the various apoA-I mutants affect differently
cpt-cAMP-dependent and -independent cholesterol efflux.
The baculovirus system produces a fusion of apoA-I-(1-243) with a
28-residue amino-terminal segment with the sequence
MSYYHHHHHHDYDIPTTFNLYFQGAMGS. This segment contains 6 His residues, a
spacer region, and the TEV protease cleavage site. Removal of the His
tag by rTEV protease generates a His-apoA-I, which still
contains an additional 5 amino-terminal sequence (GAMGS). To assess the
potential interference of the His tag on the acceptor properties of
apoA-I, control cholesterol efflux experiments were performed for
4 h using 1 µM His
and
His+ apoA-I forms (WT, apoA-I[
(1-41)],
apoA-I[
(1-59)], apoA-I[
(185-243)], apoA-I[
(1-41)
(185-243)], and
apoA-I[
(1-59)
(185-243)]). This analysis showed that the His
tag does not affect the cpt-cAMP-dependent and
cpt-cAMP-independent cholesterol efflux properties of WT apoA-I and the
carboxyl-terminal deletion mutant (data not shown). However, the
presence of His tag reduced by 38-41 and 25-32% the
cpt-cAMP-dependent cholesterol efflux of the amino-terminal
deletion mutants and the double deletion mutants, respectively, as
compared with the corresponding His
-apoA-I forms (data
not shown). The presence of His tag also reduced 27-53% the
cpt-cAMP-independent efflux capacities of amino-terminal deletion
mutants but had no significant effect on the double mutants (data not
shown). These observations led us to utilize the
His
-apoA-I forms in the preceding and the following experiments.
Effect of Amino-, Carboxyl-terminal and Double Deletion Mutants of ApoA-I on ABCA1-mediated Efflux of Cellular Cholesterol in HEK293 Cells Transfected with an ABCA1 Expression Plasmid-- To confirm that cAMP-dependent cholesterol efflux of amino-, carboxyl-terminal and double deletion mutants of apoA-I was mediated by ABCA1, we compared the cAMP-dependent cholesterol efflux to the ABCA1-dependent efflux. HEK293-EBNA cells were transfected with empty vector or an ABCA1 expression plasmid for 16 h, as described under "Experimental Procedures." Following transfection, the cells were labeled with [3H]cholesterol for 24 h and then incubated with or without 1 µM lipid-free WT or mutant forms of apoA-I for 4 h. The efflux values obtained for WT apoA-I were set as 100%.
The cholesterol efflux pattern in HEK293-EBNA cells transfected with an
ABCA1 plasmid was similar to that of cAMP-dependent cholesterol efflux in J774 macrophages (compare Fig. 2, B
and C). Compared with WT-apoA-I, ABCA1-mediated cholesterol
efflux was 20% in the presence of apoA-I[(185-243)] and 72 and
107% in the presence of apoA-I[
(1-41)] and apoA-I[
(1-59)],
respectively. Similar to the cAMP-dependent cholesterol
efflux experiments, the deletion of both the amino-terminal and the
carboxyl-terminal regions restored the ABCA1-mediated cholesterol
efflux capacity of apoA-I to 76 and 97% of the WT control.
In mock-transfected cells, efflux of cellular cholesterol followed the
same pattern as in cAMP-untreated cells (compare Fig. 2, B
and C). Similar to the findings of Fig. 2B, the
control experiment of Fig. 2C showed that cholesterol efflux
was not affected by amino-terminal deletions (apoA-I[(1-41)] and
apoA-I[
(1-59)]), was decreased by ~40% by the
carboxyl-terminal truncation of apoA-I (apoA-I[
(185-243)]), and
was further decreased by 45-60% by the double amino- and
carboxyl-terminal deletion mutants (apoA-I[
(1-41)
(185-243)] and apoA-I[
(1-59)
(185-243)]) compared with the WT control.
Mapping the Carboxyl-terminal Domains Necessary for
cAMP-dependent Cholesterol Efflux by the Full-length ApoA-I
Molecule--
For a more detailed localization of the cholesterol
efflux promoting domain within the carboxyl-terminal domain of apoA-I, we studied pro-apoA-I[(198-243)], pro-apoA-I[
(209-243)],
pro-apoA-I[
(220-243)], and pro-apoA-I[
(232-243)] (Fig. 1)
for their cholesterol efflux capacity.
The cpt-cAMP-independent cholesterol efflux values for the WT pro-apoA-I produced by either the permanent cell lines or the adenovirus system were much lower than the efflux values observed for the WT apoA-I produced with the baculovirus system (compare Fig. 2, B and D) but were at the same levels as the efflux values obtained with human apoA-I isolated from plasma (data not shown). By contrast to cpt-cAMP-independent, the cpt-cAMP-dependent cholesterol efflux values for all four WT apoA-I were similar (Fig. 2, B and D).
The cpt-cAMP-independent cholesterol efflux was very low for the WT pro-apoA-I and barely detectable in the pro-apoA-I mutants with carboxyl-terminal truncations (Fig. 2D, white bars). The cpt-cAMP-dependent cholesterol efflux was 20, 33, 9, and 99% of the WT control value for the mutants lacking the carboxyl-terminal domains 198-243, 209-243, 220-243, and 232-243, respectively (Fig. 2D, shaded bars). These data suggest that amino acid residues 220-231 of apoA-I form a domain that is important for the ability of the intact apoA-I to induce cpt-cAMP-dependent cholesterol efflux.
Effect of Amino-, Carboxyl-, and Double Deletion Mutants of ApoA-I
on cAMP-dependent Efflux of Cellular Phospholipid from J774
Mouse Macrophages--
To explore further whether the amino- and
carboxyl-terminal domains of apoA-I affect the cholesterol and the
phospholipid efflux to a similar degree, we performed phospholipid
efflux studies in J774 mouse macrophages using WT apoA-I,
apoA-I[(1-41)], apoA-I[
(1-59)], apoA-I[
(185-243)],
apoA-I[
(1-41)
(185-243)], and
apoA-I[
(1-59)
(185-243)] as phospholipid acceptors. For these
studies the cells were labeled with [3H]choline chloride
for 24 h and then incubated in serum-free medium in the presence
or absence of 0.3 mM cpt-cAMP for 24 h, and the efflux
of 3H-phospholipid in the presence of 1 µM of the WT and the indicated apoA-I forms was
determined following a 4-h incubation period. The efflux values
obtained for WT apoA-I were set as 100%.
The analysis showed that the cpt-cAMP-dependent
phospholipid efflux followed similar patterns to those described for
cholesterol efflux. Compared with WT apoA-I,
cpt-cAMP-dependent phospholipid efflux was 8% in the
presence of apoA-I[(185-243)] and 72 and 102% in the presence of
apoA-I[
(1-41)] and apoA-I[
(1-59)], respectively. As in the
cholesterol efflux experiments, the deletion of both the amino- and the
carboxyl-terminal segments restored the phospholipid efflux capacity of
apoA-I to 75% (compare Fig. 2, B and E).
The cpt-cAMP-independent phospholipid efflux was low for WT apoA-I, was decreased approximately by about 50% for the two amino-terminal deletion mutants, and was barely detectable in the other mutants.
Physicochemical Analysis of the Amino-terminal Deletion
ApoA-I[(1-41)] and Double Deletion
ApoA-I[
(1-41)
(185-243)] Mutants--
The unexpected
observation that the double deletion mutants restored the lipid efflux
activity that was lost when only the carboxyl-terminal domain of apoA-I
was deleted prompted us to study the conformation and the thermal and
chemical stability of this unusual mutant.
The -helical content of the WT apoA-I, the amino-terminal deletion
apoA-I[
(1-41)], and the double deletion
apoA-I[
(1-41)
(185-243)] mutants was estimated from the
normalized far-UV CD spectra. It was found that compared with the WT
apoA-I, the 41-amino acid amino-terminal truncation
(1-41) of
apoA-I does not change the
-helical content significantly. In
contrast, the 100-amino acid double truncation
(1-41)
(185-243)
of apoA-I leads to an ~7% reduction in the
-helical content of
the mutant protein (Table II).
|
The thermal unfolding curves monitored by the ellipticity at 222 nm,
[222] (T), are shown in Fig.
3A; the corresponding van't Hoff plots, ln Keq (1/T), are shown
in the inset; and the values of the melting temperature,
Tm, and the effective enthalpy,
Hv, determined by the van't Hoff analysis are
given in Table II. This analysis did not show a significant effect of the
(1-41) truncation on the melting temperature
(Tm) but did show an ~9 °C reduction in
Tm as a result of the double truncation
(1-41)
(185-243), indicating a significant destabilization of
the double truncated mutant protein. Both mutations lead to a reduction
in
Hv, suggesting a lower cooperativity of the
thermal transitions for the mutants compared with WT apoA-I.
|
The chemical unfolding curves for WT apoA-I and the mutant proteins
tested, monitored by ellipticity at 222 nm, are shown in Fig.
3B, and the conformational stability,
GD0, and midpoint of chemical
denaturation, D1/2, and m values
determined from the curves are listed in Table II. The denaturation
curves of both the apoA-I[
(1-41)] and the
apoA-I[
(1-41)
(185-243)] mutants are shifted to lower
concentrations of denaturant compared with WT apoA-I in accordance with
a reduction in D1/2. Both mutations also lead to
~1.1 kcal/mol reduction in
GD0
indicating a large destabilizing effect as a result of the deletion of
the amino-terminal domain or both the amino- and carboxyl-terminal domains.
Kinetics of Binding of ApoA-I to DMPC Liposomes--
The kinetics
of association of lipid-free WT apoA-I, amino-terminal deletion and
double deletion mutants of apoA-I with DMPC multilamellar vesicles was
followed by the decrease in the turbidity at 325 nm, which reflects the
formation of DMPC-apoA-I complexes. These data for WT and each mutant
apoA-I are shown in Fig. 4A. It was found that both the apoA-I[(1-41)] mutant and the double deletion mutant apoA-I[
(1-41)
(185-243)] have a much lower
rate of association with DMPC liposomes as compared with the WT apoA-I (Fig. 4A).
|
Formation of Discoidal POPC-ApoA-I Particles by the WT ApoA-I and
the Truncated ApoA-I Form ApoA-I[(1-41)
(185-243)]--
The
sodium cholate dialysis method allowed the formation of discoidal
particles with the WT and the double-truncated apoA-I form (Fig. 4,
B and C). Under the negative staining conditions used, the electron micrographs showed that these particles form the
typical "rouleaux," indicating that they are discoidal and have the
thickness of a phospholipid bilayer. The number of rouleaux observed
depends on the concentration of the sample on the carbon grid. In less
concentrated samples, a large number of round particles that lie flat
on the grid were also observed. The findings indicate that the double
truncation of apoA-I did not affect the ability of the central helices
to form discoidal particles in vitro.
Analysis of the Ability of the Amino-terminal Deletion and Double
Deletion Mutant of ApoA-I to Form HDL in Vivo--
The ability of the
WT and truncated mutants of apoA-I to form HDL in vivo was
tested by adenovirus-mediated gene transfer in apoA-I/
mice. Four days post-infection, plasma was collected and analyzed for
plasma lipid and apoA-I levels and the presence of HDL particles. Livers were also collected and analyzed for human apoA-I mRNA levels to assess the expression of the apoA-I. Table
III shows the plasma cholesterol,
triglycerides, and apoA-I levels and the hepatic apoA-I mRNA levels
of the mice 4 days post-infection. The apoA-I concentration was
estimated by three different approaches as described under
"Experimental Procedures," and the results are shown in Table III.
These analyses showed that after normalization for the mRNA levels,
there is still an ~15-fold difference between the observed and
expected plasma concentration of apoA-I between the WT and the double
deletion mutant. A similar difference is also observed between the WT
and the amino-terminal deletion mutant. It appears that the low levels
of the double deletion mutant may be the result of both faster
catabolism and lower hepatic expression of the recombinant adenovirus.
On the other hand, the low plasma levels of apoA-I[
(1-41)] can
only be attributed to the faster catabolism because the hepatic apoA-I
mRNA levels of this mutant are high.
|
Analysis of the apoA-I distribution following equilibrium density
gradient centrifugation of plasma showed that the WT apoA-I and the
apoA-I[(1-41)] mutant were distributed predominantly in the
HDL2 and HDL3 region (Fig.
5A), whereas the double
deletion mutant apoA-I[
(1-41)
(185-243)] was shifted more
toward the HDL2 region (Fig. 5A). Similar
results were obtained by analysis of the apoA-I distribution in the gel
filtration fractions of plasma (data not shown).
|
Analysis of the nature of the lipoprotein particles formed by
electron microscopy showed that WT and apoA-I[(1-41)] mutant formed spherical particles, whereas apoA-I[
(1-41)
(185-243)] mutant formed discoidal particles, and apoA-I
/
mice had
few spherical particles (Fig. 5, B-E). Fig. 5,
A-E, indicates that WT apoA-I and apoA-I[
(1-41)] form
mature HDL. ApoA-I[
(1-41)
(185-243)] forms discoidal HDL
in vivo. The findings of Figs. 4C and
5D establish that the central helices 3-7 of apoA-I retain
their ability to form discoidal HDL particles in vitro and
in vivo.
![]() |
DISCUSSION |
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Lipid-free apoA-I has been shown to induce efflux of phospholipid and cholesterol from cellular plasma membranes by two processes, one independent of cellular proteins (i.e. "microsolubilization") and one dependent on ABCA1 (12-17). In this study we performed systematic deletions of helices of apoA-I to localize domains that are required for the fulfillment of these two lipid efflux functions. To differentiate ABCA1-mediated and ABCA1-independent cholesterol efflux mechanisms, we performed the experiments in the presence and absence of a cAMP analog that was shown previously to be a potent inducer of ABCA1 gene expression and activity (18, 58, 59, 61). To prove that cAMP-dependent lipid efflux represents ABCA1-mediated lipid efflux, we also investigated the ability of apoA-I variants to induce cholesterol efflux from cells that are transfected to overexpress ABCA1. For the interpretation of the data on ABCA1-independent lipid efflux it is, however, important to bear in mind that in the absence of cAMP, J774 cells have some ABCA1 activity. Thus cAMP-independent lipid efflux probably includes some ABCA1-mediated processes.
The present study shows that in cAMP-stimulated cells and
ABCA1-transfected cells, cholesterol and phospholipid efflux was strongly impaired in the presence of several apoA-I variants with a
carboxyl-terminal deletion, such as apoA-I[(185-243)] and
apoA-I[
(220-243)], but was either not affected or affected to a
lesser extent by amino-terminal deletions (apoA-I[
(1-41)] and
apoA-I[
(1-59)]). These findings are consistent with a previous
study, which also showed that truncation of a carboxyl-terminal
(apoA-I[
(210-243)]) but not of the amino-terminal region
(apoA-I[
(1-43)] and apoA-I[
(1-65)]) of apoA-I interferes
with cAMP-induced cholesterol efflux from macrophages (33). The
objective of the present study was to map the domain(s) of apoA-I that
are required for cholesterol efflux. For this purpose we analyzed
several additional apoA-I mutants containing deletions in the
carboxyl-terminal region. This analysis showed that the
cAMP-dependent cholesterol efflux capacity of several
apoA-I variants truncated in the carboxyl-terminal region remained
decreased for all the mutants that lacked the 220-231 amino acid
sequence of apoA-I. However, efflux was restored to normal levels when
the 232-243 region of apoA-I was deleted. This analysis indicated that
the region 220-231 of the intact apoA-I molecule is required for the
ABCA1-mediated cholesterol efflux. Interestingly, this is a domain
within apoA-I that shows a much higher degree of evolutionary
conservation than other domains of apoA-I (62). It has also been shown
that substitution of the hydrophobic amino acids L222K, F225K, and
F229K of apoA-I in this region diminishes the ability of apoA-I to
associate with HDL and phospholipids in vitro (43).
Furthermore, expression of this mutant in apoA-I-deficient mice by
adenovirus gene transfer prevents the maturation of HDL in
vivo and results in the accumulation of discoidal HDL particles in
plasma (45). Similarly, it has been shown that expression of a
carboxyl-terminal apoA-I[
(220-243)] mutant, which lacks the
220-231 region in apoA-I deficient mice, produced only small numbers
of discoidal HDL particles in vivo (45). These data are also
in agreement with the virtual HDL deficiency of a patient in whom a
frameshift mutation resulted in the synthesis and secretion of a
truncated apoA-I mutant with a changed sequence of amino acids 203-229
(63). Surprisingly however, the lipid efflux defect of apoA-I was
restored when, in addition to the carboxyl terminus, the amino terminus
of apoA-I was also deleted. A possible explanation of these data could
be that conformational constraints brought about by the deletion of the
carboxyl-terminal region diminish the functional interactions between
apoA-I and ABCA1. Further deletion of both the amino- and
carboxyl-terminal regions may relieve these conformational constraints
to allow the double deletion mutant of apoA-I to interact functionally
with the ABCA1 and thus promote cholesterol efflux from cell cultures.
To gain further insight into the relationship between the structure and
function of apoA-I, we studied the physicochemical properties of the
double deletion mutant of apoA-I and compared them with those of WT
apoA-I and the amino- or the carboxyl-terminal deletion mutants.
Structural analysis showed that neither deletions of amino-terminal
amino acid residues 1-41 performed in this study nor of the
carboxyl-terminal amino acid residues 185-243 studied previously (44)
changed significantly the -helical content. The carboxyl-terminal
truncation did not interfere with the stability of the mutant protein
(44). On the other hand, based on chemical denaturation data the
amino-terminal truncation destabilizes the protein.
The functional studies showed that the carboxyl-terminal deletion diminished greatly the ABCA1-mediated lipid efflux, as well as the ability to solubilize multilamellar DMPC phospholipid vesicles (43). In contrast, the amino-terminal deletion has only small effect on lipid efflux and solubilizes with a slower rate multilamellar DMPC phospholipid vesicles. These data indicate that the carboxyl-terminal domain of apoA-I is required to enable the WT apoA-I to induce cAMP- and hence ABCA1-dependent lipid efflux from cells and to solubilize multilamellar DMPC phospholipid vesicles, whereas the amino-terminal domain has a smaller effect on both processes.
The current study also showed that the
apoA-I[(1-41)
(185-243)] mutant which lacks both the amino-
and the carboxyl-terminal domain shows a ~7% reduction in its
-
helical content and has greatly reduced chemical and thermal stability
and cooperativity of unfolding as compared with WT apoA-I. When tested
in vitro and in vivo, the double deletion mutant
has the ability to form discoidal HDL particles. Physicochemical
analysis of rHDL particles containing the
apoA-I[
(1-41)
(185-243)] mutant showed that the lipid-bound
form of this mutant has 0.4 kcal/mol increased chemical stability
compared with the lipid-free form (data not shown). Thus the
lipid-bound form of this mutant appears to be thermodynamically more
favorable than the lipid-free form. This may explain the partial
ability of this mutant to solubilize multilamellar DMPC vesicles and to
promote ABCA1-mediated lipid efflux. Furthermore, the
-helical
content of the double deletion mutant apoA-I[
(1-41)
(185-243)] increases from 51% in the lipid-free form to 78% in the rHDL
particles, whereas the
-helical content of the WT apoA-I increases
from 58% in the lipid-free form to 67% in the rHDL particles (data not shown). This indicates that compared with WT apoA-I a greater percentage of amino acids of the double deletion mutant organizes into
-helical structure when this mutant associates with phospholipids. It is possible that similar conformational changes that increase the
-helical content of the apoA-I[
(1-41)
(185-243)] mutant may
occur as a result of functional interactions between the lipid-free form of the double deletion mutant and the ABCA1. Although we have no
evidence to support direct protein-protein interactions between the
double deletion mutant and ABCA1, it has to be noted that this mutant
lacks the carboxyl-terminal helices 8-10 which might promote the
association of apoA-I with lipid bilayers.
Previous studies (19, 22) have indicated that ABCA1 is not directly involved in the efflux of free cholesterol. Other groups have proposed that the interaction of apoA-I with ABCA1 or a partner protein stimulates translocation of intracellular cholesterol and phospholipids from the Golgi apparatus to the plasma membrane ABCA1, where it associates with apoA-I to form the precursors HDL particles, which subsequently mature to spherical HDL (61, 64). One study has suggested that apoA-I-ABCA1 complexes may recycle between plasma membrane and intracellular lipid pools where apoA-I may be lipidated by the action of ABCA1 and subsequently exocytosed (61, 64, 65). Our data show that the ABCA1-dependent cholesterol and phospholipid effluxes follow similar patterns, suggesting that the two processes are kinetically indistinguishable.
It was observed previously (13, 27, 66) that water-soluble
apolipoproteins as well as artificial peptides with amphipathic -helices induce cholesterol efflux from cells. Because the number of
amphipathic
-helices was correlated roughly with the efficacy of
cholesterol efflux, it was believed that apoA-I does not contain stringent structural domains that are important for this process. The
impaired ABCA1-mediated cholesterol efflux of apoA-I variants with
carboxyl-terminal deletions does not seem to be a consequence of
alterations in the structure of apoA-I. Thus, it is reasonable to
assume that stringent structure-function relationships between apoA-I
and ABCA1 are required to enable apoA-I to induce cAMP- and hence
ABCA1-dependent lipid efflux from cells. The present study
indicates that these specific functional interactions between the
full-length apoA-I and ABCA1 require the carboxyl-terminal residues
220-231 of apoA-I.
Carboxyl-terminal truncation interfered much less with the ability of
apoA-I to induce cAMP-independent efflux (Fig. 2B). Moreover, and in contrast to the cAMP-dependent lipid
efflux, further removal of the amino terminus did not restore the
defect in the double deletion mutant but rather reduced further the
cAMP-independent cholesterol efflux. This ABCA1-independent cholesterol
efflux pathway, i.e. microsolubilization, appears to be
largely a function of the apolipoprotein content of amphipathic
-helices rather than of a specific domain. Because the number of
amphipathic
-helices is a major determinant of the phospholipid
binding capacity of apoA-I, these data also indirectly support the
previous notion that physicochemical interactions between apoA-I and
plasma membrane phospholipids rather than biological ligand/receptor
interactions may underlie "microsolubilization" (12, 13).
Our in vivo data suggest that the specific, i.e.
cAMP- and ABCA1-dependent lipid efflux pathway, and the
nonspecific, i.e. cAMP- and ABCA1-independent lipid
"microsolubilization" pathway, have different impacts on HDL
biogenesis. WT apoA-I or apoA-I mutants with a preserved carboxyl
terminus such as apoA-I[(1-41)] maintain their capacity to induce
ABCA1-mediated cholesterol efflux and to form spherical HDL in
vivo. Previous studies also showed that the apoA-I[
(7-43)]
mutant forms spherical particles and few discoidal particles, whereas
the apoA-I[
(7-65)] mutant and various internal deletion mutants
form discoidal HDL particles (33, 67). By contrast, apoA-I variants
with carboxyl-terminal truncations such as apoA-I[
(220-243)],
which lacks the 220-231 domain, interfere with ABCA1-mediated
cholesterol efflux and, although not strongly interfering with
ABCA1-independent cholesterol efflux, do not form HDL in
vivo (45). The inability of the carboxyl-terminal truncated apoA-I
variants to form HDL most likely reflects their limited capacity to
promote cholesterol efflux and form pre-
-HDL precursors, as well as
their reduced affinity for phospholipids and pre-formed HDL particles.
Previous studies have shown that carboxyl-terminal truncated apoA-I
forms fail to associate with preformed HDL particles following density
gradient ultracentrifugation (43). Similarly, carboxyl-terminal
truncated apoA-I forms, produced by proteolysis of HDL by
metalloproteinases secreted by macrophage cultures, are found in
lipid-free fractions after gel filtration (68). Thus, the lipid-poor
particles or lipid-free mutant apoA-I forms generated by
adenovirus-mediated gene transfer most likely are catabolized rapidly
by the cubilin receptor or other mechanisms (69, 70). This may explain
our findings that indicate that the steady-state apoA-I mRNA levels
of the WT and mutant apoA-I forms do not correlate with the plasma
apoA-I levels. The combined deletion of the carboxyl and amino termini
partially restores the inability of the carboxyl-terminal deletion
variant to induce ABCA1-dependent cholesterol efflux and to
form lipidated discoidal HDL precursors, despite the fact that this
variant has an impaired ability to release cellular lipids by
cAMP-independent mechanisms. We therefore conclude that not
apolipoprotein-mediated lipid efflux per se, but rather the
mechanism of lipid efflux which involves functional interactions
between ABCA1 and apoA-I, is essential for the formation of HDL
precursors which can mature to HDL. This process has at least two
prerequisites, namely the cellular abundance of ABCA1 and the presence
of a full-length apoA-I with an intact carboxyl-terminal domain.
Overall, our findings suggest that the central region of apoA-I alone, which contains helices 3-7, has the capacity to promote ABCA1-mediated lipid efflux. Following acceptance of cholesterol and phospholipids, the lipidated particles containing the central apoA-I helices 3-7 may proceed to form discoidal particles in vivo.
When carboxyl-terminal segments that contain residues 220-231 are deleted, the apoA-I cannot function in ABCA1-mediated cholesterol efflux. This may lead to the formation of unstable HDL precursors that may be catabolized rapidly by the kidney.
It is possible that deletion of the carboxyl-terminal domain hinders the functional interactions of the mutant apoA-I with ABCA1, whereas this hindrance may be limited when both the amino- and carboxyl-terminal domains are deleted.
The 220-231 domain of apoA-I has a smaller effect on the ability of apoA-I to induce ABCA1-independent efflux, i.e. microsolubilization, of cellular lipids.
The restoration of ABCA1 lipid efflux in the apoA-I[(232-243)]
mutant, which contains the 220-231 region, as well as in the double
deletion mutant, which lacks both the amino- and the carboxyl-terminal domains, indicates that specific interactions of the 220-231 region with amino-terminal domain of apoA-I may be necessary to allow functional interactions between the full-length apoA-I and ABCA1 that
are required for cholesterol efflux and HDL biogenesis.
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ACKNOWLEDGEMENTS |
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We thank Gayle Forbes for technical assistance and Anne Plunkett for typing the manuscript. We also thank Dr. Michael Fitzgerald for technical assistance with the ABCA1 transfection assay, Dr. Mason Freeman for providing the ABCA1 expression plasmid, and Donald Gantz for performing the electron microscopy analysis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-33952 and HL-48739 and European Community Grant BMH4-CT98-3699.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.
Present address: Institute of Clinical Chemistry, University
Hospital of Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland.
** To whom correspondence should be addressed: Section of Molecular Genetics, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany St., W509, Boston, MA 02118. Tel.: 617-638-5085; Fax: 617-638-5141; E-mail: vzannis@bu.edu.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M205232200
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ABBREVIATIONS |
---|
The abbreviations used are:
apoA-I, apolipoprotein A-I;
ABCA1, ATP-binding cassette transporter A1;
apoA-I/
mice, apoA-I-deficient mice;
BSA, bovine serum
albumin;
cpt-cAMP, 8-(4-chlorophenylthio)adenosine 3':5'-cyclic
monophosphate;
FBS, fetal bovine serum;
HDL, high density lipoproteins;
HEK293 cells, human embryonic kidney 293 cells;
His+-apoA-I, apoA-I with a histidine tag;
His
-apoA-I, apoA-I without a histidine tag;
rTEV
protease, recombinant Tobacco Etched Viral protease;
WT, wild-type;
DMPC, dimyristoyl-L-
-phosphatidylcholine;
POPC,
-oleoyl-
-palmitoyl-L-
-phosphatidylcholine;
PBS, phosphate-buffered saline;
DMEM, Dulbecco's modified Eagle's medium;
ELISA, enzyme-linked immunosorbent assay.
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