© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
A Single Amino Acid Deletion in the Carboxy Terminal of Apolipoprotein A-I Impairs Lipid Binding and Cellular Interaction
From the Department of Internal Medicine (W.H., J.S., A.M., H.H., W.L., T.K., M.K., K.A.), School of Medicine; and the Department of Chemistry (S.A.), Fukuoka University, Fukuoka, Japan.
Correspondence to Jun Sasaki, MD, Department of Internal Medicine, School of Medicine, Fukuoka University, 45-1, 7-chome Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. E-mail mm034515{at}msat.fukuoka-u.ac.jp
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Abstract |
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Abstract—The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding. However, the precise functional domain of the C-terminal remains to be defined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences. A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding. On preincubation with human plasma lipoprotein fractions (d<1.225 g/mL) at 37°C for 1 hour, 125I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas 125I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity). Circular dichroism measurements showed that the
-helix content of
lipid-bound r-proapoA-I Nichinan was reduced, being 62% (versus 73%)
of normal r-proapoA-I. Nondenaturing gradient gel electrophoresis of
reconstituted HDL particles assembled with r-proapoA-I Nichinan and
normal r-proapoA-I showed similar particle size. To study
cholesterol efflux, human skin fibroblasts were labeled
with [3H]cholesterol, followed by incubation
with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex.
Fractional cholesterol efflux from
[3H]cholesterol-labeled fibroblasts to
lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan complexes
was significantly reduced relative to that of normal r-proapoA-I or
DMPC/r-proapoA-I during the 6-hour incubation. Binding
assays of human skin fibroblasts by lipid-free r-proapoA-I showed that
r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal
r-proapoA-I. Our data demonstrate that the deletion of glutamic acid
235 at the C-terminus substantially reduces the
lipid-binding properties of r-proapoA-I Nichinan, which may cause a
reduction in its capacity to interact with plasma membranes as well as
to promote cholesterol efflux from cultured
fibroblasts.
Key Words: apo A-I variants • lipid binding • cellular binding • secondary structure
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Introduction |
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ApoA-I, the major protein moiety of HDL, is a 243–amino acid polypeptide and contains 22- or 11-residue repeat units, folding into 6 or 8 putative amphipathic
-helixes, presumably
for lipid binding, enhancing cholesterol efflux from
peripheral tissue, and activating the plasma enzyme
lecithin: cholesterol acyltransferase (LCAT).1
ApoA-I is also thought to be an important ligand for receptor-dependent
and receptor-independent binding to the cell
surface.2 3
The functional domains of apoA-I have been recently studied with
synthetic peptides, mutagenized apoA-I, limited proteolysis, and
monoclonal anti–apoA-I antibodies. These studies have suggested that
residues 44 to 126 in the N-terminus are responsible for
maintaining the -helix structure of lipid-free
apoA-I,4 5 while residues 137 to 186 in the central
-helical region are involved in LCAT activation, cellular
cholesterol efflux, and interaction with cell-surface
binding sites.6 7 8 On the other hand, the
C-terminus (residues 193 to 243) of the -helical domain
of apoA-I exhibits a significantly higher lipid affinity than do other
helixes.9 10 Residues 190 to 243 or 222 to 243
in the C-terminus are actively involved in protein-lipid
interactions, which are critical in the initial rapid binding to HDL,
cholesterol efflux from the plasma membrane, and in vivo
HDL catabolism.11 12 13 14 In addition, the
C-terminus also binds the putative HDL
receptor.15 However, mapping of apoA-I domains that
are functionally important requires a more precise point mutagenesis
that produces minimal disturbance of the 3-dimensional
structure of apoA-I.16 In this regard, naturally occurring
point mutations that may subtly alter the conformation of apoA-I and
that may be associated with low HDL cholesterol levels can
be useful for structure-function studies of apoA-I functional
domains.
We have recently reported a novel mutant, apoA-I Nichinan (Glu235del), that is associated with low plasma HDL cholesterol levels and reduced cholesterol efflux from macrophages.17 In the present study, recombinant (r-) proapoA-I Nichinan was used to examine the effect of this mutation on the lipid-binding properties of apoA-I and related consequences.
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Methods |
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Materials
[1,2,3H]cholesterol and 125I were obtained from NEN Life Science Products. The Sequenase sequencing kit was purchased from US Biochemical Corp. Reagents for the polymerase chain reaction were purchased from Perkin-Elmer. The kit for mutagenesis was from Takara Biochemicals. pT-7 Blue T vector was from Novagen. Materials used for chromatography were from Bio-Rad Laboratories. Strains of Escherichia coli used for transfection were obtained from Takara Biochemicals. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were from GIBCO. BSA was from Sigma Chemical Co. Culture plates were purchased from Falcon (Becton-Dickinson). Other chemical reagents were obtained from Sigma and Wako Pure Chemical Industries.
Construction of cDNA for Expression of r-ProapoA-I
The expression of human mature apoA-I in the E coli
system is difficult owing to intracellular degradation of the protein
during bacterial growth.18 19 To overcome this
problem, a pro- segment was used to optimize the efficiency of protein
expression in the E coli system.20 21 On
the other hand, proapoA-I has been shown to have
physiological properties similar to those of plasma
apoA-I.22 23 Therefore, we used proapoA-I in the
present study. cDNAs for expression of wild-type human proapoA-I
and proapoA-I Nichinan in E coli were obtained as described
previously.17 Variant versions of proapoA-I cDNA were
prepared by using the site-directed mutagenesis system described by
Kunkel et al.24 All plasmids from clones selected for the
expression of r-proapoA-I were subsequently sequenced.
Expression and Purification of Human r-ProapoA-I
Human r-proapoA-I (wild-type and variant) was expressed as a
fusion protein in the E coli/pGEX vector expression system
as described previously.17 In brief, proapoA-I
fusion protein was purified by glutathione-agarose affinity
chromatography, cleaved by factor Xa, and repurified by
repeating the glutathione-agarose chromatography and
performing chromatography on anti–apoA-I affinity and
Sephacryl S300 columns. The final product was checked with a 12%
SDS-polyacrylamide gel and NH2-terminal
protein sequencing25 and was confirmed to be purified
proapoA-I (wild-type and variant).
Binding of r-ProapoA-I to DMPC
The association of normal r-proapoA-I and r-proapoA-I Nichinan
with dimyristoyl phosphatidylcholine (DMPC) multilamellar liposomes was
determined by the kinetic turbidimetric method.26 Weighed
amounts of DMPC, dissolved with chloroform in a glass tube, were dried
under N2 gas. DMPC was then dispersed in a buffer
composed of 8.5% KBr, 10 mmol/L Tris, 0.01% EDTA, and 0.01%
NaN3 (pH 7.4). DMPC was mixed with r-proapoA-I at
a 3.5:1 weight ratio to a final protein concentration of 0.1 mg/mL in
quartz spectrophotometer cells (1-cm path length). The rate of
disappearance of liposomal turbidity was measured at 325 nm in a
Beckman DU series 60 spectrophotometer equipped with a thermostatically
controlled cuvette holder that was maintained at 24±0.10°C.
Preparation of Reconstituted HDL
Reconstituted HDL containing normal r-proapoA-I or r-proapoA-I
Nichinan was prepared by the sodium cholate dialysis method as
described by Jonas.27 The molar ratio of DMPC to
r-proapoA-I in the initial preparation was 150:1. r-ProapoA-I, DMPC,
and cholate mixtures were equilibrated for 24 hours at 24°C, followed
by removal of cholate by extensive dialysis. The reconstituted HDL was
used for assessment of -helical structure by circular dichroism (CD)
spectrometry and cholesterol efflux. In addition,
electrophoretic mobility of reconstituted HDL was analyzed by
agarose gel electrophoresis. The size of reconstituted HDL was also
determined by nondenaturing gradient polyacrylamide gradient
gel electrophoresis on 4% to 30% gels (Pharmacia LKB Biotechnology).
After protein staining with Coomassie Blue G-250, the gel was
analyzed by laser densitometric scanning with
high-molecular-weight standards (Pharmacia Biotech).28
CD Spectrometry
The average -helix content of normal r-proapoA-I and
r-proapoA-I Nichinan in lipid-free and lipid-bound form was estimated
by CD spectroscopy by using a Jasco J-600 spectropolarimeter (Japan
Spectroscopy) at 222 nm. Spectra were recorded at 25°C in a
0.1-cm quartz cell at wavelengths between 195 and 260 nm. The final
concentration of the sample was 0.10 mg/mL. PBS was used as the buffer
throughout the measurement. Four scans for each sample were
recorded and averaged. The background of the buffer was subtracted.
The mean molar residue ellipticity (
) was expressed as
degrees · cm2 ·
dmol-1, which was calculated from the equation
[]=obs115/10lc,
where obs is the observed ellipticity at
222 nm in degrees, 115 is the mean residue molecular weight of the
protein, l is the optical path length in centimeters, and
c is the protein concentration in g/mL.21
The -helix content was calculated from the formula
[]222=-30 300fH-2340,
where fH is the fraction of -helical
structure.29
Cultivation of Human Skin Fibroblasts
Normal human skin fibroblasts, seeded in 35-mm tissue-culture
plates at a density of 105 cells per plate, were
maintained in bicarbonate-buffered DMEM supplemented with 10% fetal
bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at
37°C and cultured to subconfluence. Cells used in the study were
between the 10th and 20th passage.
Cholesterol Efflux Assay
The protocols used for labeling fibroblasts and measuring the
efflux of plasma membrane cholesterol were essentially
based on the method described previously by Rothblat et
al.30 In brief, before being labeled, fibroblasts were
washed twice with PBS containing 0.2% BSA and twice with PBS alone.
They were then labeled in the presence of 10% FBS with 20 µCi of
[3H]cholesterol for 48 hours before
the efflux experiment.
[3H]Cholesterol-labeled fibroblasts
were washed twice with DMEM containing 2 mg/mL BSA and twice with DMEM
alone. Washed cells were incubated with DMEM containing 3 mg/mL BSA for
1 hour at 37°C, transferred to DMEM or to DMEM containing different
cholesterol acceptors, and then incubated for 6 hours at
37°C. Both lipid-free r-proapoA-I and reconstituted HDL particles
assembled with DMPC and r-proapoA-I were used in the efflux
experiments. The final concentration of r-proapoA-I was 30 µg/mL. The
molar ratio of DMPC to r-proapoA-I in the initial preparation was
150:1. Thereafter, the medium was transferred to a tube at the
indicated time and centrifuged at 15 000 rpm for 10 minutes at
4°C. Cell monolayers were washed 4 times with fresh medium. Cellular
lipids were extracted by incubation with hexane-isopropanol (3:2
vol/vol). Aliquots of the supernatant medium and cellular lipid
extracts were then counted by LKB liquid scintillation spectrometry.
Fractional cholesterol efflux was expressed as counts per
minutemedium/(cpmmedium+cpmcells)x100
as previously described.31
Iodination of r-ProapoA-I
Labeling of normal r-proapoA-I and r-proapoA-I Nichinan with
Na125I was carried out according to the iodine
monochloride method of McFarlane.32 Labeled r-proapoA-I
was dialyzed at 4°C for 40 hours against 0.15 mol/L NaCl containing
0.3 mmol/L EDTA, pH 7.4. The specific activity of labeled
r-proapoA-I ranged from 250 to 300 cpm/ng protein, and 99% of the
radiolabeled r-proapoA-I could be precipitated by trichloroacetic acid.
Before the experiment, labeled r-proapoA-I was stored at 4°C for 1 or
2 days.
Gel Filtration Chromatography
r-ProapoA-I distribution among human plasma lipoprotein
fractions was analyzed by agarose gel filtration. Separation of
human plasma lipoprotein fractions was performed as described by Rudel
et al.33 Lipoproteins fractions (d<1.225 g/mL)
from healthy donors were isolated by
ultracentrifugation for 40 hours at 40 000 rpm at
4°C (Hitachi 70p-72, RP50T-2 rotor). In the next step, 1 mL of each
lipoprotein fraction (containing 235 mg of cholesterol) was
incubated with 10 µg of 125I-labeled
r-proapoA-I at 37°C for 60 minutes. The mixture was loaded onto a
Bio-Gel A5 m agarose gel column (1.5x100 cm, Bio-Rad Laboratories) and
eluted at a flow rate of 10 mL/h with a buffer containing 0.9% NaCl,
0.01% EDTA, and 0.01% NaN3 (pH 7.4) and 2 mL of
each fraction. Fractions were counted for radioactivity in a gamma
counter and checked by absorbance at 280 nm. The concentrations of
triglycerides and cholesterol were determined
by standard enzymatic assays according to the instructions provided by
the manufacturer (Wako).
Binding Assay
Binding of radiolabeled r-proapoA-I to human fibroblasts was
performed at 4°C for 2 hours. Before the experiment, fibroblasts were
loaded with cholesterol at 50 µg/mL in each 30-mm plate
for 24 hours. The protocol for the binding assay was essentially based
on the method previously described by Oram.34 In brief,
confluent fibroblasts were washed with PBS containing 1 mg/mL BSA and
then incubated with DMEM containing 2 mg/mL BSA for 1 hour at 37°C.
After being washed with PBS containing 1 mg/mL BSA, cells were
incubated with 1.25, 2.5, 5.0, 7.5, 10, or 20 µg/mL
125I-labeled r-proapoA-I in 2 mg/mL
BSA–containing DMEM buffered with 20 mmol/L HEPES. After a 2-hour
incubation on ice, cells were extensively washed with cold PBS
containing 2 mg/mL BSA and PBS alone, allowed to warm to room
temperature, and then dissolved in 0.1N NaOH for 30 minutes. Aliquots
were assayed for 125I radioactivity in a gamma
counter and for protein concentration. Nonspecific binding was
determined in the presence of a 100-fold excess of unlabeled HDL.
Analytical Methods
Protein concentrations of cells and r-proapoA-I were determined
by the bicinchoninic acid protein assay. All experiments were
carried out in triplicate or quadruplicate and repeated at least twice.
All data are expressed as mean±SEM. Differences between groups were
examined for statistical significance by using Student’s 2-tailed
t test. A value of P<0.05 denoted a
statistically significant difference.
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Results |
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Interaction With DMPC
The kinetic interaction of r-proapoA-I with DMPC was monitored by changes in the clearance of liposomal turbidity as a function of time at 24°C, ie, the transition temperature of the lipid. Figure 1
shows the kinetic interaction of
r-proapoA-I with DMPC recorded during the initial 60 minutes of
incubation. Normal r-proapoA-I interacted rapidly with DMPC, with a
decrease in the rate of turbidity (Figure 1 and Table 1), a finding compatible with published
data.11 35 In contrast, r-proapoA-I Nichinan and DMPC
interacted slowly, with a rate constant 8 times slower than that of
normal r-proapoA-I (Figure 1 and Table 1).
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Binding of r-ProapoA-I to HDL
To compare the lipid-binding properties of r-proapoA-I Nichinan
with endogenous plasma apolipoprotein, we analyzed
the relationship between r-proapoA-I Nichinan and human lipoprotein
fractions (d<1.225 g/mL) by agarose gel
chromatography. As shown in Figure 2, on preincubation with lipoprotein
fractions at 37°C for 1 hour, normal r-proapoA-I was
chromatographed as a single peak at the HDL fraction (Figure 2, middle panel), followed by a negligible small shoulder peak
corresponding to the free r-proapoA-I fraction. The
chromatographic pattern of r-proapoA-I Nichinan was
similar: 23% of the radioactivity was accounted for by the latter peak
(Figure 2, lower panel), indicating that r-proapoA-I Nichinan
might be dissociated from the HDL fraction.
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) was detected by absorbance at 280 nm, and cholesterol
(
) was determined by enzymatic assay. Top, Elution position of
VLDL/LDL and HDL fractions determined by the mass of proteins and
cholesterol; middle, elution pattern of normal r-proapoA-I;
bottom, elution pattern of r-proapoA-I Nichinan. OD indicates optical
density.
Secondary Structure of r-ProapoA-I Nichinan Assessed by CD
We also examined the effect of the Glu235 deletion on the
secondary structure of r-proapoA-I Nichinan by using CD spectroscopy.
As indicated in Table 2, lipid-free
normal r-proapoA-I and r-proapoA-I Nichinan had similar -helical
contents of 41% to 45%, but these contents in normal r-proapoA-I and
r-proapoA-I Nichinan were markedly increased when DMPC was complexed
with r-proapoA-I. However, r-proapoA-I Nichinan exhibited significantly
less helical content on lipid binding: 62% versus 73% of normal
r-proapoA-I (Table 2), as confirmed by the ellipticity value at
222 nm, which was lower for r-proapoA-I Nichinan (Figure 3). The average -helical contents of
lipid-free and lipid-bound r-proapoA-I are indicated in Table 2.
The number of -helixes per reconstituted HDL molecule was estimated
from the -helical content and protein length, after assuming a
length of 16 amino acids for amphipathic helixes and of 6 amino acids
for the adjacent ß-strands.11 36 r-ProapoA-I Nichinan
had 7 -helixes per apoA-I molecule compared with 8 -helixes for
normal r-proapoA-I (Table 2).
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Size of Reconstituted HDL Particles Assessed by GGE
Because the Glu235 deletion affected the arrangement of the
secondary structure of r-proapoA-I Nichinan in its lipid-bound state,
we used gradient gel electrophoresis (GGE) to examine reconstituted HDL
particles assembled with r-proapoA-I Nichinan to determine whether the
Glu235 deletion affected particle size. When r-proapoA-I was complexed
with DMPC, normal r-proapoA-I and r-proapoA-I Nichinan formed doublet
particles of a size similar to those already reported under similar
conditions (Figure 4).6 37 38
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Cholesterol Efflux From
[3H]Cholesterol-Labeled Fibroblasts
ApoA-I–mediated efflux of cellular cholesterol may
require the interaction of the cell plasma membrane with either
lipid-free apoA-I or lipidated apoA-I.1 Our previous study
indicated that lipid-free r-proapoA-I Nichinan was defective in
promoting cholesterol efflux from
macrophages.17 Because the Glu235 deletion
affected the lipid binding of apoA-I, we examined
cholesterol efflux by r-proapoA-I in its lipid-free and
lipid-bound forms in
[3H]cholesterol-labeled fibroblasts
within 6 hours of incubation. As shown in Table 3, the cholesterol released
from [3H]cholesterol-labeled
fibroblasts by both normal r-proapoA-I and r-proapoA-I Nichinan
increased proportionally with incubation time. At all time points,
however, the rate of release of
[3H]cholesterol from fibroblasts by
r-proapoA-I Nichinan was significantly lower than that from normal
r-proapoA-I (reductions by 34%, 33%, and 28% at 1, 3, and 6 hours,
respectively). On the other hand, conjugation of proapoA-I with DMPC
resulted in marked enhancement of cholesterol efflux by
DMPC/r-proapoA-I complexes, being 4 to 6 times higher than that of
lipid-free r-proapoA-I for both normal r-proapoA-I and r-proapoA-I
Nichinan (Table 3), compatible with the results of previous
studies.3 39 However, the capacity to release
[3H]cholesterol from fibroblasts by
DMPC/r-proapoA-I Nichinan complexes was also significantly reduced
compared with that of DMPC/normal r-proapoA-I complexes (reductions by
24.4%, 23%, and 23.6% at 1, 3, and 6 hours, respectively; Table 3).
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Binding of r-ProapoA-I to Cholesterol-Loaded Fibroblasts
Lipid-free apoA-I is thought to interact with specific cellular
factor(s) or lipid domains on the cell surface for subsequent
acceptance of cholesterol efflux.3 40
To test whether reduced cholesterol efflux from fibroblasts
was due to a change in the ability of r-proapoA-I Nichinan to interact
with the cell surface, we performed binding assays by incubating
125I-labeled r-proapoA-I with
cholesterol-loaded fibroblasts in a dose-dependent manner.
Specific binding reached saturation at 
10 µg/mL for both normal
r-proapoA-I and r-proapoA-I Nichinan (Figure 5). However, the cell-binding capacity of
r-proapoA-I Nichinan was reduced by 35% at saturation relative to that
of normal r-proapoA-I (11.14±0.62 versus 17.40±0.71 ng/mg cell
protein, respectively, P<0.05).
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) or r-proapoA-I
Nichinan (•). Nonspecific binding was measured in the presence of a
100-fold excess of unlabeled HDL. After incubation, cells were digested
with 0.1N NaOH and then counted. |
Discussion |
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In the present study, we found that deletion of glutamic acid at codon 235 in the C-terminus of apoA-I resulted in a significantly slow initial interaction of r-proapoA-I Nichinan with DMPC. Furthermore, the in vitro distribution of r-proapoA-I Nichinan in human plasma HDL fraction was altered, with 23% of r-proapoA-I Nichinan being associated in the free r-proapoA-I fraction. These results may explain the previous observation that r-proapoA-I Nichinan was rapidly catabolized relative to normal r-proapoA-I when injected into normal rabbits.17 Structural analysis by CD scanning suggested that the Glu235 deletion affected the arrangement of the secondary structure of apoA-I Nichinan in reconstituted HDL, consistent with the data from the predicted helical wheel presentation of the presumed
-helical structure of
segments 220 to 240 of apoA-I Nichinan.17 On the other
hand, the extent of lipid binding of normal r-proapoA-I was similar to
that of r-proapoA-I Nichinan, since we observed a >90% clearance of
liposomal turbidity for r-proapoA-I Nichinan after 24 hours of
incubation (data not shown). Consistent with this observation,
reconstituted HDL particles assembled with normal r-proapoA-I or
r-proapoA-I Nichinan showed a similar particle size, suggesting that
r-proapoA-I Nichinan underwent a structural conformation arrangement to
allow for some configurational adaptation to bind lipids. Thus, our
present findings provide further evidence to suggest that residues
220 to 243 of apoA-I are important for lipid binding and for binding to
HDL, consistent with earlier findings by mutagenesis, synthetic
peptide, and limited proteolysis studies on lipid binding of the
C-terminus.9 11 12 13 Considered together,
our data suggest that deletion of Glu235 impairs lipid binding of
apoA-I Nichinan.
It has been proposed that residues 187 to 241 of apoA-I bind lipids in
the initial stage of lipid binding and that residues near 230 may
penetrate deeper into the HDL molecule, which may be important for
anchoring apoA-I to the surface of HDL particles.13 41 Our
results generally agree with these schemes. In addition, the
apoA-I(Lys107del) variant results in abnormal activation of LCAT,
probably due to a disruption in the secondary structure of the mutant
protein.42 Although replacement of various amino acids in
the C-terminus demonstrated that substitution of
specifically charged residues between 195 and 238 did not affect the
binding of mutant protein to HDL,16 penetration of
exchangeable apolipoproteins in the phospholipid layer involves both
electrostatic and hydrophobic interactions.43 Deletion of
polar residue Glu235 affects the secondary structure of apoA-I
Nichinan, and it may also influence penetration of the extreme
C-terminus into HDL particles and the ionic
interactions between antiparallel helixes in lipid-bound apoA-I
Nichinan. Thus, the change in amphipathic character of the -helical
structure of segment 220 to 240 due to deletion of Glu235 may be
critical to the observed impaired lipid binding of apoA-I Nichinan.
The molecular mechanisms of apoA-I–mediated cellular cholesterol efflux may involve the interaction of the cellular plasma membrane with either lipid-free apoA-I or lipidated apoA-I.1 Recently, the "shuttle-and-sink" model has been proposed for the function of various HDL subclasses in cholesterol efflux.44 45 In this model, small particles, such as pre-ß-HDL and lipid-free apolipoproteins, function as high-efficiency initial cholesterol acceptors (ie, shuttles) to move cholesterol from the plasma membrane to larger HDL particles that serve as reservoirs (sinks). Recent studies have shown that the interaction of lipid-free apoA-I with cultured cells causes efflux of both cellular phospholipid and free cholesterol.3 46 This process may involve either specific cellular factor(s) or plasma membrane domains and occurs in many cell types, including fibroblasts.3 40 In another study, Sviridov et al14 demonstrated that truncation to residue 222 significantly affected the ability of apoA-I to interact with cholesterol and phospholipid, a process related to the transfer of cholesterol from the plasma membrane to extracellular acceptors. We previously indicated that r-proapoA-I Nichinan was associated with diminished cholesterol efflux from macrophages/foam cells.17 In the present study, we demonstrated that reduced fractional cholesterol efflux from [3H]cholesterol-labeled fibroblasts by r-proapoA-I Nichinan occurred during a 6-hour incubation. Moreover, the change in fractional cholesterol efflux from these cells seemed to be correlated with reduced binding of r-proapoA-I Nichinan to fibroblasts. These data suggest that reduced cholesterol efflux from fibroblasts by r-proapoA-I Nichinan may be due to a change in the ability of r-proapoA-I Nichinan to interact with cellular plasma membranes. On the other hand, we also found a defective promotion of cholesterol efflux from [3H]cholesterol-labeled fibroblasts by DMPC/r-proapoA-I Nichinan. Because r-proapoA-I Nichinan showed a slow interaction with DMPC phospholiposomes, as well as the ability to form reconstituted HDL particles, perhaps the reduced cholesterol efflux by DMPC/r-proapoA-I Nichinan may be related to the reduced initial association of r-proapoA-I Nichinan with lipids. In addition, the amount of DMPC in DMPC/r-proapoA-I was similar to that in DMPC/r-proapoA-I Nichinan complexes, suggesting that the difference in cholesterol efflux between DMPC/normal r-proapoA-I and DMPC/r-proapoA-I Nichinan was not related to DMPC. The nature of the apoA-I domain involved in cellular surface binding remains unknown. The C-terminus is thought to be involved in interaction with HDL binding sites.15 However, previous studies showed that the last 20 to 24 residues of apoA-I were not essential for binding to the putative HDL receptor on the liver, and binding of truncated apoA-I (deletion of the last 21 residues) to HepG2 was similar to that of full-length apoA-I.14 47 Perhaps truncated apoA-I (deletion of the last 21 or 24 residues) can interact selectively with the cell-surface binding site, a process probably associated with mobilization of intracellular cholesterol. The central region (residues 121 to 186) with a weaker lipid affinity has been implicated in protein-protein interaction.10 Indeed, a limited proteolysis study showed that cleavage of 70 amino acids from the C-terminus resulted in disruption of the HDL receptor–binding domain of apoA-I.47 Other studies demonstrated that the central region was involved in LCAT activation and intracellular cholesterol efflux, probably through selective cell membrane interaction and biochemical signaling.6 7 8 Because the extent of cholesterol efflux corresponds to the lipid-binding affinity of acceptors and the unstable C-terminus can be stabilized by lipid binding,4 we speculate that the interaction of the extreme C-terminus (residues 220 to 243) with cell plasma membranes may be an important step for cholesterol efflux from cells.
In conclusion, we analyzed in the present study the lipid-binding properties of r-proapoA-I Nichinan. Our data suggest that deletion of Glu235 from the C-terminus affects lipid binding of apoA-I Nichinan. This change is correlated with a reduced binding of the variant protein to plasma HDL particles, which may cause a reduced ability of the variant protein to interact with cells and to promote cholesterol efflux from cultured fibroblasts. Interaction of the extreme C-terminus of apoA-I with the cell plasma membrane may be an important step for cholesterol efflux from cells.
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Acknowledgments |
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This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, and Culture (No. 08671195 to J.S. and No. 11671064 to A.M.) and the Science Research Promotion Fund from the Japan Private School Promotion Foundation. We thank Prof Seikoh Horiuchi (Department of Biochemistry, Kumamoto University, Kumamoto, Japan) for the critical reading and helpful discussion of our manuscript. We also thank Tomoko Shinkawa for her excellent technical assistance and Kayo Nishi for secretarial assistance in the preparation of this manuscript.
Received February 26, 1999; accepted July 20, 1999.
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References |
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1. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res. 1995;36:211–228.[Abstract]
2.
Oram JF, Mendez AJ, Slotte JP, Johnson TF. High
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