© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
The Arg123-Tyr166 Central Domain of Human ApoAI Is Critical for Lecithin:Cholesterol Acyltransferase–Induced Hyperalphalipoproteinemia and HDL Remodeling in Transgenic Mice
From the Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
Correspondence to Paul Holvoet, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail paul.holvoet{at}med.kuleuven.ac.be
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Abstract |
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Abstract—High density lipoprotein (HDL) metabolism and lecithin:cholesterol acyltransferase (LCAT)–induced HDL remodeling were investigated in transgenic mice expressing human apolipoprotein (apo) AI or an apoAI/apoAII chimera in which the Arg123-Tyr166 domain of apoAI was substituted with the Ser12-Ala75 domain of apoAII. Expression of apoAI and of the apoAI/apoAII chimera resulted in a respective 3.5-fold and 2.9-fold increase of HDL cholesterol. Human LCAT gene transfer into apoAI-transgenic mice resulted in a 5.1-fold increase of endogenous LCAT activity. This increase was associated with a 2.4-fold increase of the cholesterol ester–to–free cholesterol ratio of HDL, a shift from HDL3 to HDL2, and a 2.4-fold increase of HDL cholesterol levels. Agarose gel electrophoresis revealed that human LCAT gene transfer into human apoAI–transgenic mice resulted in an increase of pre-ß-HDL and of pre-
-HDL. In contrast, human LCAT
gene transfer did not affect cholesterol levels and HDL
distribution profile in mice expressing the apoAI/apoAII chimera. Mouse
LCAT did not "see" a difference between wild-type and mutant human
apoAI, whereas human LCAT did, thus localizing the species-specific
interaction in the central domain of apoAI. In conclusion, the
Arg123-Tyr166 central domain of apoAI is not critical for in vivo
lipoprotein association. It is, however, critical for LCAT-induced
hyperalphalipoproteinemia and HDL remodeling independent of the
lipid-binding properties of apoAI.
Key Words: transgenic mice • adenovirus • apoAI • LCAT • HDL remodeling
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Introduction |
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Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the metabolism of HDL cholesterol.1 2 Naturally occurring variants with point mutations in the central domain of apoAI, such as apoAIFin (Leu159
Arg),3
apoAIOita (Val 156Glu),4
apoAIOslo (Arg160Leu),5
apoAIPisa (Leu141 Arg),6 or with
the deleted Glu146Arg160 central domain
(apoAISeattle),7 are associated with
impaired LCAT activation and reduced HDL cholesterol
levels. Expression of human LCAT in mice8 9 10 11 12 13 or rabbits14 15 16 17 18 resulted in a significant increase of HDL cholesterol levels. Expression of human LCAT resulted in a 4-fold increase of HDL cholesterol levels in human apoAI–transgenic mice but only a 2-fold increase in transgenic mice expressing both human apoAI and apoAII. These data indicated that human LCAT has a significant preference for HDLs containing human apoAI.
Deletion of the Leu122-Pro165, the Leu144-Gly186, or the Glu146-Arg160 domain of apoAI resulted in impaired LCAT activation with in vitro reconstituted HDL.19 20 21 Furthermore, the hydrophobic face orientation of the Pro143-Ala164 helix domain of apoAI was found to be critical for LCAT activation.22 Finally, substitution of the Arg123-Tyr166 domain of human apoAI with the Ser12-Ala75 helical domain of apoAII resulted in a >20-fold reduction of the in vitro LCAT activity in reconstituted HDL. This decrease in LCAT activity was independent of the size and of the apolipoprotein, phospholipid, and cholesterol contents of the reconstituted HDL.23
In the present study, transgenic mice that expressed human apoAI or
the apoAI[(
Arg123-Tyr166);
apoAII(Ser12-Ala75)] chimera were
used to study the role of the Arg123-Tyr166 central domain of apoAI in
HDL metabolism both in the absence and presence of human
LCAT expressed with a recombinant human LCAT
adenovirus.23
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Methods |
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Preparation of Human ApoAI DNA Constructs for Zygote Injection
The isolation of a genomic clone containing the human apoAI gene24 has been previously reported. A 2.2-kb EcoRI-XbaI fragment was obtained by courtesy of E.M. Rubin, Lawrence Berkeley Laboratory, Berkeley, Calif. The 2.2-kb fragment was inserted in the pBlueScript (pBS) vector resulting in the pBS-AI vector. The pBS–AI(1–122)–AII(12–75) DNA fragment was amplified from the pBS-AI vector by polymerase chain reaction (PCR) using the 5'-dCTCGAAATTAACCCTCACTAAA forward primer and the 5'-dCTCCACCTTCTGGCGG reverse primer. The apoAI[
(123–166);AII(12–75)] DNA fragment was
amplified from the pMc-5–apoAI[(123–166);AII(12–75)]
vector14 by using the
5'-dGAAGTACTGAGACAGCGGCTCCACCTTCTGGCGG forward
primer and the 5'-dCACTTTGGAAAGTTTATTCTGAGC reverse primer that
contains an additional Psp site. The
apoAI(1–122)–apoAII(12–75)–apoAI(166–243) DNA fragment was
amplified from pBS–AI(1–122)–AII(12–75) and the
apoAI[(123–166);AII(12–75)] fragments by using the
5'-dCTCGAAATTAACCCTCACTAAA forward primer and the
5'-dCACTTTGGAAAGTTTATTCTGAGC reverse primer. The PCR product was
digested with Psp and XbaI and ligated in the
Psp and XbaI-treated pBS-AI vector. A 2.2-kb
EcoRI-XbaI fragment was excised and purified for
zygote injection. All DNA constructs were confirmed by DNA
sequencing.
Generation of Transgenic Mice
Transgenic mice expressing human apoAI or the apoAI/apoAII
chimera were generated by zygote injection into the C57BL/6 background
according to previously published procedures.25 26 All
experimental procedures in mice were performed in accordance with
protocols approved by the Institutional Animal Care and Research
Advisory Committee.
Production of Recombinant LCAT Adenovirus
The pUC19 LCAT plasmid, containing the entire human
LCAT cDNA, was a kind gift of Dr J. McLean (Department of
Cell Biology, Genentech Inc, South San Francisco, Calif). The
human LCAT cDNA fragment was obtained by digestion of pUC19
LCAT with EcoRI and HindIII and was subcloned by
using identical restriction sites in the shuttle plasmid pACCMVpLpA,
which contains the 760-bp cytomegalovirus (CMV) promoter/enhancer and
the simian virus splice/polyadenylation site. The recombinant human
LCAT virus was generated by cotransfection of pACCMVpLpA.LCAT and the
rescue plasmid pJM17 in 293 cells. The LCAT adenovirus was amplified
and purified as described previously.27
DNA Analysis
Tail tips were excised from 3- to 4-week-old mice and incubated
overnight at 55°C in lysis buffer (2 mmol/L EDTA, 1% sarcosyl,
0.2 mol/L Tris-HCl [pH 8.0], 0.4 mol/L NaCl, and 8 mol/L urea)
including proteinase K (3.6 mg/mL). The mixture was then incubated with
RNase (13 µg/mL) at 37°C for 2 hours. After
centrifugation, DNA was extracted with
phenol/chloroform (1:1, vol/vol) and precipitated in
isopropanol/ethanol. The DNA was resuspended in 700 µL of 10
mmol/L Tris-HCl, (pH 8.0), and 0.1 mmol/L EDTA. DNA (10 µg) was
digested with PstI, fractionated in 0.7% agarose,
transferred to a charge-modified nylon 66 binding matrix, and
hybridized in 50% formamide with nick-translated human apoAI genomic
DNA (specific activity 2 to 5x108 counts per
minute per microgram). Filters were washed with a stringency of 0.1x
NaCl/citrate (1x NaCl/citrate is 150 mmol/L NaCl and 15
mmol/L sodium citrate, pH 7.5) and 0.1% SDS at 65°C and
autoradiographed. Liver-specific human LCAT gene transfer
was demonstrated by the appearance of a 1.4-kb
EcoRI-HindIII fragment on Southern blots
developed with a human LCAT cDNA probe.
RNA Analysis
Total RNA was isolated from mouse liver by a single-step method
(Trizol reagent, Life Technologies) based on the guanidinium
isothiocyanate–acid–phenol RNA isolation method developed by
Chomczynski and Sacchi. Twenty micrograms of total RNA was separated on
a 1.25% agarose gel containing 0.21 mol/L formamide and 3.8 volume %
formaldehyde. RNA was blotted overnight in 20x SSC (20x SSC is
3 mol/L NaCl and 0.3 mol/L trisodium citrate ·
2H2O, pH 7.0). After UV cross-linking, the
membrane was prehybridized in Quickhyb (Stratagene) containing 800 µL
of herring sperm DNA for 6 hours. Human apoA-I cDNA and
human GAPDH cDNA probes were labeled with
[-32P]dCTP by using the Rediprime labeling
system (Amersham Life Sciences). Hybridization was performed for 18
hours by adding 3x107 cpm of denatured probe.
The membrane was washed 2 times for 20 minutes at room temperature in a
solution containing 2x SSC and 0.05% SDS and subsequently washed at
37°C for 20 minutes in a buffer containing 0.01% SSC and 0.1% SDS.
RNA signals were quantified in a PhosphorImager. The human apo A-I mRNA
signals were normalized for the GAPDH signal.
ApoAI Quantification by ELISA and Western Blotting
Blood was collected in heparinized microhematocrit capillary
tubes from the tail or from the retro-orbital plexus. After
centrifugation (2000g for 5 minutes), levels
of human apoAI and of the apoAI/apoAII chimera were measured in a
sandwich ELISA by using mouse monoclonal antibodies to human apoAI as
described previously.25 The antibodies used in this
assay had <0.01% cross-reactivity with mouse apoAI. Purified
proteins23 were used as internal standards. The interassay
variation coefficient of the ELISA is 12%. Alternatively, levels of
human apolipoproteins were determined by quantitative scanning of
Western blots of 10% to 15% gradient SDS-polyacrylamide gels
that had been developed with the monoclonal antibodies to human apoAI.
For quantification of mouse apoAI, Western blots were developed with
polyclonal anti-mouse apoAI antibodies prepared in cynomolgus monkeys
and generously supplied by Dr George Melchior (The Upjohn Co,
Kalamazoo, Mich). These antibodies had <0.01% cross-reactivity
with human apoAI. The interassay variation coefficient of the
immunoblot assay is 20%.
Plasma Lipid and Lipoprotein Analyses
At 
3 months of age, the mice were fasted overnight and blood
was collected. Plasma was obtained by centrifugation,
and lipoprotein fractions were separated by gel filtration on a
Superdex 200 HR column equilibrated with 20 mmol/L Tris-HCl
buffer, pH 8.1, containing 0.15 mol/L NaCl, 1 mmol/L EDTA, and
0.02 mg/mL NaN3 in a fast protein liquid
chromatography system (Waters Associates). Samples (200
µL) were applied. Human apoAI distribution in HDL was assessed by
ELISA. Mouse apoAI in HDL was assessed by Western
blotting.25 26 HDL particle size distribution was either
assessed by gel filtration after injection of radiolabeled purified
apolipoproteins28 or estimated by comparison of their
migration position on native 4% to 15% gradient
polyacrylamide gels with that of standard proteins:
thyroglobulin (Stokes’ radius of 8.5 nm), apoferritin (6.1 nm),
catalase (5.2 nm), and lactate dehydrogenase (4.1 nm) (Pharmacia).
Electrophoretic mobilities of plasma lipoproteins were analyzed
by agarose gel electrophoresis, and blots were analyzed by
quantitative scanning densitometry with the use of the NIH image
analysis program.
Endogenous LCAT Activity
The endogenous LCAT activity of the plasma of
transgenic mice was measured by determining the esterification rate of
cholesterol in human HDL3. Aliquots
containing 5 µg of cholesterol were incubated with 10
µL of plasma at 37°C for 30 minutes. Free cholesterol
(FC) and cholesterol esters (CEs) were quantified by
high-performance liquid chromatography. They
were eluted isocratically at 45°C with a mixture of
acetonitrile/isopropanol at ratios of 50:50 and 90:10 (vol/vol),
respectively.29 Endogenous LCAT activity,
expressed as nanomoles of CE per milliliter per hour, was
quantified in samples collected before and at 6 days after
intravenous injection of 109
plaque-forming units of the recombinant LCAT adenovirus.
In Vivo ApoAI Metabolism
Recombinant apoAI and
apoAI[(Arg123-Tyr166);apoAII(Ser12-Ala75]
were purified from the periplasmic fraction of Escherichia
coli cells as previously described.23 Proteins
were iodinated by the Iodo-Gen (Pierce Chemical Co) method.
The specific activities were 3 µCi/µg apolipoprotein. The clearance
rates (Cl) and the fractional catabolic rates (FCRs) of radiolabeled
apolipoproteins were determined in mice as described
previously.25 26 The endogenous
production rates (PRs) of the apolipoproteins were
calculated from the plasma steady-state concentrations (mg/dL), the
clearance rates (mL · h-1), and the body
weights (g) as described by Kleinberger et al.30
Statistical Analysis
The significance of differences in lipoprotein levels,
cholesterol-CE ratios, endogenous
apolipoprotein PRs, and FCRs were tested by the Mann-Whitney
nonparametric test. Human apoAI levels in mice before and
after human LCAT gene transfer were compared in a paired
t test. A P value of <0.05 was considered to be
statistically significant.
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Results |
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Effects of ApoAI Genotype on Apolipoproteins and Lipoproteins
Total cholesterol, non-HDL cholesterol, and HDL cholesterol levels of C57BL/6 mice were (mean±SEM, n=8) 45±4.0, 12±1.2, and 31±12.5 mg/dL, respectively. HDL cholesterol levels were 3.5-fold increased in apoAI-transgenic mice (n=8) and 2.9-fold increased in mice expressing the apoAI/apoAII chimera (n=8, Figure 1
).
The ratio of HDL to total cholesterol was 0.67±0.041 in
C57BL/6 mice, 0.91±0.077 (P<0.001) in apoAI-transgenic
mice, and 0.87±0.070 (P<0.001 versus C57BL/6 mice and
P=NS versus apoAI-transgenic mice) in mice expressing the
apoAI/apoAII chimera. Levels of human apoAI and of the apoAI/apoAII
chimera in transgenic mice were similar: 220±11 and 180±5.8 mg/dL,
respectively. Mouse apoAI levels were 70±6.7 mg/dL in C57BL/6 control
mice and <1 mg/dL in mice expressing human apoAI and in mice
expressing the apoAI/apoAII chimera (data not shown). Expression of
human apoAI or of the apoAI/apoAII chimera on the C57BL/6 background
did not change ß-VLDL cholesterol levels (Figure 1).
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Effect of Human LCAT Gene Transfer on LCAT Activity,
HDL Cholesterol Levels, and ApoAI Levels
Before human LCAT gene transfer, the
endogenous LCAT activity in apoAI-transgenic mice and in
mice expressing the chimera was very similar (Figure 2). The FC-CE ratios of HDL in apoAI- and
apoAI/apoAII-expressing mice were very similar (0.39±0.013 and
0.49±0.038, respectively). After human LCAT gene transfer,
the endogenous LCAT activity in apoAI-transgenic mice was
5.1-fold higher than in mice expressing the apoAI/apoAII chimera
(Figure 2). This higher LCAT activity was associated with a
2.4-fold increase of the CE-cholesterol ratio in the
HDLs of apoAI-transgenic mice (Figure 2). Human LCAT
gene transfer into transgenic mice expressing the apoAI/apoAII chimera
did not increase the endogenous LCAT activity and did not
alter the FC-CE ratio of their HDL (Figure 2). The differences
in LCAT activity in mice expressing apoAI or the apoAI/apoAII chimera
could not be attributed to differences in human LCAT levels.
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Adenovirus-mediated expression of human LCAT resulted in a 2.4-fold
further increase of HDL cholesterol levels in
apoAI-transgenic mice 6 days after intravenous injection of
the recombinant LCAT adenovirus (Figure 1
). In contrast, human
LCAT gene transfer did not induce a significant increase of
HDL cholesterol levels in C57BL/6 mice or in transgenic
mice expressing the apoAI/apoAII chimera (Figure 1). Whereas HDL
cholesterol levels in mice expressing human apoAI or the
apoAI/apoAII chimera were similar before human LCAT gene
transfer, HDL cholesterol levels were 2.8-fold higher in
apoAI-transgenic mice than in mice expressing the apoAI/apoAII chimera
after LCAT gene transfer (Figure 1). Human
LCAT gene transfer into apoAI-transgenic mice resulted in a
2.5-fold increase of non-HDL cholesterol levels, whereas
non-HDL cholesterol levels in C57BL/6 mice or in transgenic
mice expressing the apoAI/apoAII chimera were not affected by human
LCAT gene transfer (Figure 1).
Figure 3 shows that at day 0, plasma
levels of apoAI in the 2 groups of 8 apoAI transgenic mice were very
similar: 220±11 and 200±9.8 mg/dL. At day 6, plasma levels of apoAI
were very similar in the group that did not receive the human LCAT
adenovirus, whereas plasma levels of apoAI in mice treated with the
LCAT adenovirus were increased to 420±30 mg/dL. Human LCAT
gene transfer did not alter plasma levels of the apoAI/apoAII chimera
(Figure 3 and Table 1). Northern
blotting showed that human LCAT gene transfer in
apoAI-transgenic mice did not result in an increase of human apoAI mRNA
levels (data not shown).
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Endogenous Apolipoprotein Production and
Clearance
The plasma clearance of radiolabeled apolipoproteins was
analyzed in transgenic mice before and after human
LCAT gene transfer. The apolipoprotein steady-state plasma
concentration (Css), the FCR, the plasma
clearance rate (Clp), and the endogenous PR of apoAI and of
the apoAI/apoAII chimera were very similar (Table 1). Human
LCAT gene transfer did decrease the FCR of apoAI but not
that of the apoAI/apoAII chimera (Table 1). Human
LCAT gene transfer also resulted in an increased
endogenous PR of apoAI but not of the apoAI/apoAII chimera
(Table 1).
Lipoprotein Association of ApoAI and of the ApoAI/ApoAI Chimera
Before and After Human LCAT Gene Transfer
Figure 4 illustrates the
distribution of apoAI and of the apoAI/apoAII chimera in HDL fractions
isolated by gel filtration from the plasma of transgenic mice
expressing apoAI or the apoAI/apoAII chimera before and after
LCAT gene transfer. More than 85% of apoAI and of the
apoAI/apoAII chimera were recovered in HDL fractions (Table 2). As previously
demonstrated,17 HDL particles of apoAI-transgenic
mice were polydisperse, with major populations of particles with
diameters of 8.4, 9.2, and 9.6 nm, respectively, thus corresponding to
humanlike HDL3. Nondenaturing
polyacrylamide gel electrophoresis revealed a similar pattern
of HDL in transgenic mice expressing the apoAI/apoAII chimera. Human
LCAT gene transfer into apoAI-transgenic mice resulted in a
shift to larger HDL particles, with sizes ranging between 11 and 13 nm,
thus corresponding to humanlike HDL2. In
contrast, human LCAT gene transfer into transgenic mice
expressing the apoAI/apoAII chimera did not affect the size of the HDL
particles. Whereas human LCAT gene transfer into
apoAI-transgenic mice thus resulted in a shift from
HDL3 to HDL2, in agreement
with the enhanced LCAT activity and HDL cholesterol
esterification, HDL particles in LCAT adenovirus–treated
apoAI/apoAII-transgenic mice migrated primarily as
HDL3. In addition, human LCAT gene
transfer into apoAI-transgenic mice resulted in a 2-fold increase of
small, dense HDL (Table 2). In contrast, human LCAT
gene transfer into transgenic mice expressing the apoAI/apoAII chimera
did not result in an increase of small, dense HDL (Table 2).
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The cholesterol to apoAI, FC to apoAI, and CE to apoAI molar ratios of HDLs from apoAI-transgenic mice were 51±5.4, 15±1.5, and 39±3.0, respectively. After human LCAT gene transfer, these values were 64±5.5 (P=NS), 9.5±0.81 (P=0.0080), and 65±5.9 (P=0.0013), respectively.
Plasma lipoproteins were also separated by nondenaturing agarose gel
electrophoresis. As shown in Figure 5, 2
distinct HDL populations were observed in the plasma of
apoAI-transgenic mice: 1 migrated in the -position (90%) and 1
migrated in the pre-ß position (10%). After human LCAT
gene transfer, we observed 3 HDL populations: 1 in the pre-ß
position, 1 in the -position, and 1 in the pre- position. Human
LCAT gene transfer into apoAI-transgenic mice resulted in an
increase of pre-ß-HDL and of pre--HDL and a decrease of -HDL
(Figure 5). In contrast, human LCAT gene transfer
into transgenic mice expressing the apoAI/apoAII chimera did not alter
the HDL distribution profile (Figure 5). Both before and after
LCAT gene transfer, HDL particles of transgenic mice
expressing the apoAI/apoAII chimera showed -electrophoretic
mobility.
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) and after (
) human
LCAT gene transfer. |
Discussion |
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The present study in transgenic mice demonstrates that (1) the Arg123-Tyr166 domain of human apoAI is not critical for its in vivo lipoprotein association, as evidenced by the similar increases of HDL cholesterol levels in transgenic mice expressing the central domain apoAI/apoAII chimera as in apoAI-transgenic mice; (2) human LCAT gene transfer into apoAI-transgenic mice is associated with hyperalphalipoproteinemia and HDL remodeling; and (3) the Arg123-Tyr166 domain of human apoAI is critical for in vivo LCAT activation and thereby for inducing hyperalphalipoproteinemia and HDL remodeling.
A first very important observation was that substitution of the Arg123-Tyr166 domain of apoAI with the apoAII segment did not affect in vivo apolipoprotein synthesis and lipoprotein association. Indeed, the in vivo PRs of human apoAI and of the apoAI/apoAII chimera were very similar, as well as the HDL cholesterol levels and the HDL distribution profiles of mice expressing apoAI or the apoAI/apoAII chimera. Thus, these transgenic mice were indeed suitable to investigate the role of LCAT activation in HDL metabolism independent of lipid binding and thus, independent of baseline levels of HDL cholesterol as well as of the baseline distribution profiles of HDL.
Adenovirus-mediated expression of human LCAT in apoAI-transgenic mice
was associated with an increase of HDL cholesterol levels
as has previously been shown in human apoAI/LCAT-transgenic
mice8 and in human apoAI-transgenic mice that were treated
with a similar human LCAT adenovirus.9 Increased LCAT
activity in LCAT adenovirus–treated apoAI-transgenic mice was
associated with an increase of both pre-ß-HDL and of pre--HDL.
Davidson et al31 demonstrated that the increased
electronegative charge on -HDL and pre--HDL, compared with that
on pre-ß-HDL, is mostly due to the presence of a neutral lipid ester
core and only in a small way to a direct effect of negatively charged
phospholipids. Because neutral lipids do not possess a net negative
charge, they cannot directly contribute a charge to a lipoprotein
particle. Therefore, the change in charge is most probably due to a
conformational change in the apolipoprotein molecules. It has been
suggested that this electronegative HDL fraction may be the
preferential substrate for cholesterol ester transfer
protein (CETP)–mediated lipid exchange. This electronegative HDL
fraction is similar to an HDL fraction in the plasma of CETP-deficient
patients.32 Thus, our results suggest that "recycling"
of the large HDL that is generated as a consequence of increased LCAT
activity is impaired in the absence of CETP and that the increase in
HDL cholesterol is due to impaired HDL
cholesterol flux. This hypothesis will be investigated in
mice that overexpress human apoAI, human LCAT, and human CETP.
At this moment, we can only speculate about the mechanisms that lead to
the increase of pre-ß-HDL in apoAI-transgenic mice, especially in
those mice treated with the LCAT adenovirus. Jiang et al33
have demonstrated that the phospholipid transfer protein (PLTP)
increases the influx of phospholipid and secondary
cholesterol into HDL, leading to an increase in potentially
antiatherogenic pre-ß particles. Increased PLTP activity and
PLTP-mediated HDL remodeling in human apoAI-transgenic mice may explain
to some extent the increase of pre-ß-HDL in human apoAI-transgenic
mice.34 Another molecule that may play a key role in HDL
remodeling is the scavenger receptor BI.35 This molecule
mediates the selective uptake of HDL CEs in the liver and may thus
contribute to the generation of pre-ß-HDL.35 It is thus
possible that by the action of human LCAT in LCAT adenovirus–treated
apoAI-transgenic mice, -HDL is converted into pre--HDL by the
accumulation of CEs and that the scavenger receptor BI–mediated
selective uptake of these CEs results in the generation of pre-ß-HDL.
Finally, the increase of pre-ß-HDL may partly be due to an increased
production of nascent HDL, as evidenced by the increased apoAI
PR in apoAI-transgenic mice after LCAT gene transfer. These
data are not necessarily in conflict with the lack of an increase in
human apoAI mRNA levels in these mice. Northern blotting may not be
sensitive enough to detect a 50% increase in apoAI
production.
Baseline levels of HDL cholesterol and baseline HDL distribution profiles of mice expressing the central domain chimera were very similar to these of apoAI-transgenic mice, demonstrating similar lipid-binding properties of apoAI and of the apoAI/apoAII chimera. Human LCAT gene transfer in mice expressing the central domain chimera did, however, not increase the endogenous LCAT activity and HDL cholesterol levels and did not induce HDL remodeling in these mice. We did not observe differences in size and lipid composition between HDLs of apoAI-transgenic mice and of mice expressing the apoAI/apoAII chimera before human LCAT gene transfer. It is therefore very unlikely that the observed differences in LCAT activity after LCAT gene transfer were due to differences in apolipoprotein/lipid content. Most likely differences in LCAT activity are due to the presence in apoAI, but not in the apoAI/apoAII chimera, of an amino acid sequence that may allow a conformation in apoAI that is critical for LCAT activation. Sparks et al36 demonstrated that LCAT can interact with lipid-poor apoAI, suggesting that LCAT does not need to bind to the lipid interface on an HDL particle but may directly interact with apoAI; thus, the conformation of apoAI may be critical for interaction with LCAT. Mouse LCAT did not see a difference between wild-type human apoAI and mutant apoAI, whereas human LCAT did so, thus localizing the species-specific interaction to the central domain of apoAI.
Serum cholesterol levels of carriers of the
apoAIFin mutation,3 a Leu159Arg
point mutation, are decreased to 20% to 30% of normal. Whereas the
ability of this mutant to activate LCAT is defective, its
phospholipid-binding and cholesterol efflux properties are
normal. Serum cholesterol levels of carriers of the
apoAIOita mutation,4 a Val156Glu
point mutation; of the apoAIOslo
mutation,5 an Arg160Leu point mutation; or of the
apoAISeattle mutation, a Glu146Arg160
deletion7 19 are <10% of normal. All of these mutations,
in contrast with the apoAIFin mutation, are
associated with both defective lipid binding and defective LCAT
activation. The latter mutants are thus not well suited to study the
effect of defective LCAT activity on HDL cholesterol levels
independently of defective phospholipid binding and lipoprotein
association. In the present study, an apoAI variant with normal
phospholipid binding properties but defective LCAT was used to study
the effect of reduced LCAT activity on HDL cholesterol
levels in the absence of reduced capacity to interact with
phospholipids and thus to produce HDL particles. In the presence of
human LCAT, HDL cholesterol levels in transgenic mice
expressing the apoAI/apoAII chimera were only 30% of those of mice
expressing wild-type human apoAI. As discussed above, the similar
levels of HDL cholesterol in mice expressing human apoAI or
the apoAI/apoAII chimera in the absence of human LCAT may be explained
by a lack of interaction between human apoAI-containing HDL and mouse
LCAT.
In conclusion, the present study demonstrates clear evidence for the critical role of the central domain of apoAI for in vivo LCAT activation and associated hyperalphalipoproteinemia and HDL remodeling, independently of its capacity to associate with lipids.
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Acknowledgments |
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This work was supported by the Interuniversitaire Attractiepolen Program (P4/34) and by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (program G.0110.98). Bart De Geest is a postdoctoral Fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. Sophie Van Linthout is a Research Assistant of the Instituut voor Wetenschappelijk Onderzoek-Vlaanderen. We thank Eddy Demarsin, Els Deridder, and Michèle Landeloos for excellent technical assistance.
Received March 5, 1999; accepted June 8, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Chen C, Applegate K, King WC, Glomset JA, Norum KR, Gjone E. A study of the small spherical high density lipoproteins of patients afflicted with familial lecithin: cholesterol acyltransferase deficiency. J Lipid Res. 1984;25:269–282.[Abstract]
- McCall M, Nichols AV, Blanche PJ, Shore VG, Forte TM. Lecithin:cholesterol transferase-induced transformation of HepG2 lipoproteins. J Lipid Res. 1989;30:1579–1589.[Abstract]
-
Miettinen HE, Jauhiainen M, Gylling H, Ehnholm S,
Palomaki A, Miettinen TA, Kontula K. Apolipoprotein
A-IFIN (Leu159Arg) mutation affects lecithin
cholesterol acyltransferase activation and subclass
distribution of HDL but not cholesterol efflux from
fibroblasts. Arterioscler Thromb Vasc Biol. 1997;17:3021–3032.
[Abstract/Free Full Text] -
Huang W, Sasaki J, Matsunaga A, Nanimatsu H, Moriyama
K, Han H, Kugi M, Koga T, Yamaguchi K, Arakawa K. A novel homozygous
missense mutation in the apoA-I gene with apoA-I deficiency.
Arterioscler Thromb Vasc Biol. 1998;18:389–396.
[Abstract/Free Full Text] -
Daum U, Leren TP, Langer C, Chirazi A, Cullen P,
Pritchard PH, Assmann G, von Eckardstein A. Multiple dysfunctions of
two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I(P165R),
that are associated with hypoalphalipoproteinemia in heterozygous
carriers. J Lipid Res. 1999;40:486–494.
[Abstract/Free Full Text] -
Miccoli R, Zhu Y, Daum U, Wessling J, Huang Y,
Navalesi R, Assmann G, von Eckardstein A. A natural apolipoprotein A-I
variant, apoA-I (L141R)Pisa, interferes with the formation of
-high density lipoproteins (HDL) but not with the formation of
pre-ß1-HDL and influences efflux of cholesterol into
plasma. J Lipid Res. 1997;38:1242–1253.[Abstract]
-
Deeb SS, Cheung MC, Peng RL, Wolf AC, Stern R, Albers
JJ, Knopp RH. A mutation in the apolipoprotein A-I gene: dominant
effect on the level and characteristics of plasma high density
lipoproteins. J Biol Chem. 1991;266:13654–13660.
[Abstract/Free Full Text] - Francone OL, Gong EL, Ng DS, Fielding CJ, Rubin EM. Expression of human lecithin-cholesterol acyltransferase in transgenic mice: effect of human apolipoprotein AI and human apolipoprotein AII on plasma lipoprotein cholesterol metabolism. J Clin Invest. 1995;96:1440–1448.
-
Séguret-Macé S, Latta-Mahieu M, Castro G,
Luc G, Fruchart JC, Rubin E, Denefle P, Duverger N. Potential gene
therapy for lecithin-cholesterol acyltransferase
(LCAT)-deficient and hypoalphalipoproteinemic patients with
adenovirus-mediated transfer of human LCAT gene.
Circulation. 1996;94:2177–2184.
[Abstract/Free Full Text] - Francone OL, Haghpassand M, Bennett JA, Royer L, McNeish J. Expression of human lecithin:cholesterol acyltransferase in transgenic mice: effects on cholesterol efflux, esterification, and transport. J Lipid Res. 1997;38:813–822.[Abstract]
- Berard AM, Foger B, Remaley A, Shamburek R, Vaisman BL, Talley G, Paigen B, Hoyt RF Jr, Marcovina S, Brewer HB Jr, Santamarina-Fojo S. High plasma HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin-cholesteryl acyltransferase. Nat Med. 1997;3:744–749.[Medline] [Order article via Infotrieve]
- Dugi KA, Vaisman BL, Sakai N, Knapper CL, Meyn SM, Brewer HB Jr, Santamarina-Fojo S. Adenovirus-mediated expression of hepatic lipase in LCAT transgenic mice. J Lipid Res. 1997;38:1822–1832.[Abstract]
-
Vaisman BL, Klein HG, Rouis M, Berard AM, Kindt MR,
Talley GD, Meyn SM, Hoyt RF Jr, Marcovina SM, Albers JJ, Hoeg JM,
Brewer HB Jr, Santamarina-Fojo S. Overexpression of human lecithin
cholesterol acyltransferase leads to
hyperalphalipoproteinemia in transgenic mice. J Biol
Chem. 1995;270:12269–12275.
[Abstract/Free Full Text] -
Hoeg JM, Vaisman BL, Demosky SJ Jr, Meyn SM, Talley GD,
Hoyt RF Jr, Feldman S, Berard AM, Sakai N, Wood D, Brousseau ME,
Marcovina S, Brewer HB Jr, Santamarina-Fojo S. Lecithin:
cholesterol acyltransferase overexpression generates
hyperalphalipoproteinemia and a nonatherogenic lipoprotein pattern in
transgenic rabbits. J Biol Chem. 1996;271:4396–4402.
[Abstract/Free Full Text] - Brousseau ME, Santamarina-Fojo S, Zech LA, Berard AM, Vaisman BL, Meyn SM, Powell D, Brewer HB Jr, Hoeg JM. Hyperalphalipoproteinemia in human lecithin cholesterol acyltransferase transgenic rabbits: in vivo apolipoprotein A-I catabolism is delayed in a gene dose-dependent manner. J Clin Invest.. 1996;97:1844–1851.[Medline] [Order article via Infotrieve]
-
Hoeg JM, Santamarina-Fojo S, Berard AM, Cornhill JF,
Herderick EE, Feldman SH, Haudenschild CC, Vaisman BL, Hoyt RF Jr,
Demosky SJ Jr, Kauffman RD, Hazel CM, Marcovina SM, Brewer HB Jr.
Overexpression of lecithin:cholesterol acyltransferase in
transgenic rabbits prevents diet-induced
atherosclerosis. Proc Natl Acad Sci U S A. 1996;93:11448–11453.
[Abstract/Free Full Text] - Brousseau ME, Santamarina-Fojo S, Vaisman BL, Applebaum Bowden D, Berard AM, Talley GD, Brewer HB Jr, Hoeg JM. Overexpression of human lecithin:cholesterol acyltransferase in cholesterol-fed rabbits: LDL metabolism and HDL metabolism are affected in a gene dose-dependent manner. J Lipid Res. 1997;38:2537–2547.[Abstract]
-
Brousseau ME, Wang J, Demosky SJ Jr, Vaisman BL, Talley
GD, Santamarina-Fojo S, Brewer HB Jr, Hoeg JM. Correction of
hypoalphalipoproteinemia in LDL receptor-deficient rabbits by lecithin:
cholesterol acyltransferase. J Lipid Res. 1998;39:1558–1567.
[Abstract/Free Full Text] -
Frank PG, N'Guyen D, Franklin V, Neville T, Desforges
M, Rassart E, Sparks DL, Marcel YL. Importance of central
-helices of human apolipoprotein A-I in the maturation of
high-density lipoproteins. Biochemistry.. 1998;37:13902–13909.[Medline]
[Order article via Infotrieve]
-
Lindholm EM, Bielicki JK, Curtiss LK, Rubin EM, Forte
TM. Deletion of amino acids Glu146Arg 160 in human
apolipoprotein A-I (ApoA-ISeattle) alters
lecithin:cholesterol acyltransferase activity and
recruitment of cell phospholipid. Biochemistry. 1998;37:4863–4868.[Medline]
[Order article via Infotrieve]
-
Minnich A, Collet X, Roghani A, Cladaras C, Hamilton
RL, Fielding CJ, Zannis VI. Site-directed mutagenesis and
structure-function analysis of the human apolipoprotein A-I:
relation between lecithin-cholesterol acyltransferase
activation and lipid binding. J Biol Chem. 1992;267:16553–16560.
[Abstract/Free Full Text] -
Sorci-Thomas MG, Curtiss L, Parks JS, Thomas
MJ, Kearns MW, Landrum M. The hydrophobic face orientation of
apolipoprotein A-I amphipathic helix domain 143–164 regulates
lecithin: cholesterol acyltransferase activation.
J Biol Chem. 1998;273:11776–11782.
[Abstract/Free Full Text] -
Dhoest A, Zhao Z, De Geest B, Deridder E, Sillen A,
Engelborghs Y, Collen D, Holvoet P. Role of the Arg123-Tyr166 paired
helix of apolipoprotein A-I in lecithin:cholesterol
acyltransferase activation. J Biol Chem. 1997;272:15967–15972.
[Abstract/Free Full Text] -
Karathanasis SK, Zannis VI, Breslow JL. Isolation and
characterization of the human apolipoprotein A-I gene. Proc Natl
Acad Sci U S A. 1983;80:6147–6151.
[Abstract/Free Full Text] - Holvoet P, Danloy S, Collen D. Role of the carboxy-terminal domain of human apolipoprotein AI in high density lipoprotein metabolism. Eur J Biochem. 1997;245:642–647.[Medline] [Order article via Infotrieve]
- Holvoet P, Danloy S, Deridder E, Lox M, Bernar H, Dhoest A, Collen D. Substitution of the carboxyl-terminal domain of apo AI with apo AII sequences restores the potential of HDL to reduce the progression of atherosclerosis in apo E knockout mice. J Clin Invest. 1998;102:379–385.[Medline] [Order article via Infotrieve]
-
De Geest B, Zhao Z, Collen D, Holvoet P. Effects of
adenovirus-mediated human apo A-I gene transfer on
neointima formation after endothelial
denudation in apo E-deficient mice. Circulation.. 1997;96:4349–4356.
[Abstract/Free Full Text] -
Holvoet P, Zhao Z, Deridder E, Dhoest A, Collen D.
Effects of deletion of the carboxyl-terminal domain of apoA-I or of its
substitution with helices of apoA-II on in vitro and in vivo
lipoprotein association. J Biol Chem. 1996;271:19395–19401.
[Abstract/Free Full Text] - Vercaemst R, Union A, Rosseneu M, De Craene I, De Backer G, Kornitzer M. Quantitation of plasma free cholesterol and cholesteryl esters by high performance liquid chromatography: study of a normal population. Atherosclerosis. 1989;79:245–250.[Medline] [Order article via Infotrieve]
- Kleinberger G, Heinzel G, Druml W, Laggner A, Lenz K. Loading and maintenance dose for the determination of amino acid kinetics in plasma. Infusionsther Klin Ernahr. 1987;1:40–44.
-
Davidson WS, Sparks DL, Lundkatz S, Phillips MC. The
molecular basis for the difference in charge between pre-ß and
-migrating high density lipoproteins. J Biol Chem. 1994;269:8959–8965.
[Abstract/Free Full Text] - Duverger N, Rader D, Ikewaki K, Nishiwaki M, Sakamoto T, Ishikawa T, Nagano M, Nakamura H, Brewer HB. Characterization of high-density apolipoprotein particles A-I and A-I:A-II isolated from humans with cholesteryl ester transfer protein deficiency. Eur J Biochem. 1995;227:123–129.[Medline] [Order article via Infotrieve]
- Jiang X, Francone OL, Bruce C, Milne R, Mar J, Walsh A, Breslow JL, Tall AR. Increased pre-ß-high density lipoprotein, apolipoprotein AI, and phospholipid in mice expressing the human phospholipid transfer protein and human apolipoprotein AI transgenes. J Clin Invest. 1996;98:2373–2380.[Medline] [Order article via Infotrieve]
-
Ehnholm S, van Dijk KW, van’t Hof B, van der Zee A,
Olkkonen VM, Jauhiainen M, Hofker M, Haveks L, Ehnholm C. Adenovirus
mediated overexpression of human phospholipid transfer protein alters
plasma HDL levels in mice. J Lipid Res. 1998;39:1248–1253.
[Abstract/Free Full Text] - Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996;271:518–520.[Abstract]
-
Sparks DL, Frank PG, Braschi S, Neville TA, Marcel YL.
Effect of apolipoprotein A-I lipidation on the formation and function
of pre-ß and -migrating LpA-I particles.
Biochemistry. 1999;38:1727–1735.[Medline]
[Order article via Infotrieve]
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