© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
A Novel Mutant, ApoA-I Nichinan (Glu235
0), Is Associated With Low HDL Cholesterol Levels and Decreased Cholesterol Efflux From Cells
From the Department of Internal Medicine, School of Medicine, Fukuoka University, Fukuoka (H. Han, J.S., A.M., W.H., T.K., K.A.); the Department of Internal Medicine, Miyazaki Prefectural Nichinan Hospital, Nichinan (M.A., T.T.); and the Department of Biochemistry, Kumamoto University, School of Medicine, Kumamoto (H. Hakamata, S.H.), Japan.
Correspondence to Jun Sasaki, MD, Department of Internal Medicine, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. E-mail mm03455{at}msat.fukuoka-u.ac.jp
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Abstract |
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Abstract—A novel variant of apolipoprotein (apo) A-I associated with low high density lipoprotein (HDL) cholesterolemia has been identified in a Japanese family during screening for apoA-I variants by isoelectric focusing (IEF) gel analysis. ApoA-I (Glu235
0) Nichinan was caused by
a 3-bp deletion of nucleotides 1998 through 2000 in exon 4
of the apoA-I gene. Four subjects in the family were heterozygous
carriers for this mutation; the mean plasma concentrations of apoA-I
and HDL cholesterol of affected family members were 30%
and 32% lower, respectively, than those of unaffected family members.
There were no differences in the levels of very low density lipoprotein
and low density lipoprotein cholesterol,
triglycerides, and other apolipoproteins between the
carriers and the noncarrier family members. In the proband, plasma
lecithin:cholesterol acyltransferase activity was normal.
Functional consequences of the mutation were examined by expressing the
mutated and wild-type proapoA-I cDNAs in Escherichia
coli. Cholesterol efflux to recombinant
proapoA-I Nichinan from mouse peritoneal macrophages loaded
with [3H]cholesterol-labeled
acetylated low density lipoprotein was decreased by 54% when
compared that of normal recombinant proapoA-I. In vivo turnover studies
in normal rabbits demonstrated that the recombinant proapoA-I Nichinan
was rapidly cleared (22% faster) compared with normal
recombinant proapoA-I. We conclude that apoA-I (Glu2350) Nichinan
induced a critical structural change in the carboxyl-terminal domain of
apoA-I for cellular cholesterol efflux and increased the
catabolism of apoA-I, resulting in low HDL cholesterol levels.
Key Words: HDL cholesterol • apoA-I variant • cholesterol efflux • kinetics
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Introduction |
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Apolipoprotein (apo) A-I, the major protein constituent of plasma HDLs, plays a leading role in cholesterol efflux from peripheral cells and acts as a cofactor for lecithin:cholesterol acyltransferase (LCAT) activation.1 Human apoA-I is synthesized in the liver and small intestine as a 267–amino acid–long preproapolipoprotein of known sequence.2 After cotranslational cleavage of the 18–amino acid residue–long presegment, proapoA-I is secreted into the plasma and lymph, where it is processed to the 243 amino acid residues encompassing mature apoA-I.3 The entire sequence and structure of the apoA-I gene located on chromosome 11q13 have been determined.4 The gene encompasses some 2 kb of DNA and is composed of 4 exons. The fourth exon contains homologous sequences that code for ten 11- or 22-amino acid repeats to form amphipathic helixes that mediate biological functions.5 However, the domains of apoA-I necessary to form distinct HDL subclasses,6 activate LCAT,7 bind to cell-surface receptors,8 and promote cholesterol efflux from cells9 are not clearly understood. Monoclonal antibodies with epitopes recognizing discontinuous groups of amino acids in residues 143 to 164 were demonstrated to inhibit LCAT activation and cellular cholesterol efflux.1 Mutagenized apoA-I with a deletion of residues 143 to 164 completely abolishes LCAT activation.10 The majority of reported apoA-I variants with a dominant effect on HDL metabolism are positioned in this region. On the other hand, the carboxyl-terminal region (residues 209 to 243) of apoA-I has been proposed to be important in cell binding,11 lipid binding, promoting cholesterol efflux from cells,12 13 LCAT activation,14 and in vivo metabolism of HDL.15
To date, >40 genetically determined structural variants of apoA-I have been identified and have helped to characterize the structure-function relationships of apoA-I. In our laboratory, we have characterized 10 apoA-I variants, including apoA-I (Val156Glu) Oita.16 17 18 19 20 21 Only a minority of these variants were associated with altered HDL cholesterol levels or increased risk for cardiovascular disease.22 23 24 25 26 27 28
Here, we report a novel mutation in the human apoA-I gene, apoA-I Nichinan, that is associated with low plasma concentrations of apoA-I and HDL cholesterol. To gain insight into the functional features of apoA-I Nichinan, we produced recombinant proapo (r-proapo) A-I Nichinan and investigated the cellular cholesterol efflux and catabolism of the variant apoA-I.
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Methods |
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Subjects
The patient was a 45-year-old Japanese woman who came to Nichinan Hospital because of ankylosis of the hip joint. She was found to have the apoA-I Nichinan mutation during the course of screening for apoA-I variants in 33 000 individuals by isoelectric focusing (IEF) gel electrophoresis in western Japan. Physical examination revealed a normally developed Japanese woman (height, 157 cm; weight, 52 kg; blood pressure, 134/80 mm Hg; and cardio-thoracic ratio, 48%). Her plasma HDL cholesterol and apoA-I levels were 40 and 118 mg/dL, respectively (Tables 1
and 2). Laboratory tests
showed plasma aspartate aminotransferase and alanine aminotransferase
levels of 14 and 14 IU/L, respectively. On physical examination, her
skin appeared normal, with no xanthelasmas. She was not
symptomatic for coronary artery disease (CAD), did
not have any clinical signs of CAD, and had a normal resting
ECG. Family history was not indicated for any increased prevalence of
CAD. A pedigree of the family is presented in Figure 1. The 4 affected subjects and 4
unaffected members of the apoA-I Nichinan family did not present
any clinical signs of CAD. The proband and family members gave
their informed consent prior to their participation in this study.
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Plasma Lipoprotein and LCAT Analyses
Blood samples were collected into tubes containing
Na2EDTA after a 12-hour fast. Plasma lipoproteins
were isolated by sequential
ultracentrifugation.29 The following
density fractions were obtained: VLDL (d<1.019 g/mL), LDL
(d=1.019 to 1.063 g/mL), HDL2
(d=1.063 to 1.125 g/mL), and HDL3
(d=1.125 to 1.21 g/mL). Acetylated LDL (acetyl-LDL)
was prepared by chemical modification of LDL with acetic
anhydride30 and labeled with
[3H]cholesteryl oleate as
described.31 The specific radioactivity of
[3H]cholesteryl oleate–labeled acetyl-LDL was
3900 disintegrations per minute per microgram of protein. Plasma
concentrations of cholesterol and triglyceride
were determined by an enzymatic method. Apolipoprotein concentrations
were measured by single radial immunodiffusion. LCAT activity in plasma
was determined by the dimyristoyl phosphatidylcholine
method.32 Control samples for comparing plasma
concentrations of lipids, lipoproteins, and LCAT activity were obtained
from healthy subjects matched for age and sex.
Electrophoresis and Immunoblotting
IEF gel electrophoresis on a pH gradient from 4 to 6 with plasma
samples from the proband was performed as described
previously.16 In brief, 1 µL of plasma was incubated for
1 hour at room temperature with 30 µL of 10 mmol/L Tris-HCl (pH
8.2) containing 1% SDS, 2% ampholyte (pH 4 to 6), and 10%
ß-mercaptoethanol. The mini-slab gel system (Biometra-Multigel
G-44) with a 7.2% polyacrylamide gel containing 5 mol/L
urea and 2% ampholyte (pH 4 to 6) was used for IEF. The apoA-I bands
were detected by immunoblotting with polyclonal rabbit
anti-human apoA-I antiserum.33
Nondenaturing Gradient Polyacrylamide Gel
Electrophoresis (PAGE)
HDL particle sizes were determined by nondenaturing gradient
PAGE by using 4% to 30% gels (Pharmacia LKB
Biotechnology).34 Plasma with a density <1.21 g/mL was
obtained by ultracentrifugation at 100 000 rpm for 2
hours (Beckman TL-100 ultracentrifuge). This step was followed
by the addition of 20 µg protein to the gel. Protein was visualized
with 0.05% Coomassie Brilliant Blue G-250, and then the gel was
scanned with a personal densitometer SI (Molecular Dynamics, Inc). The
gel was calibrated with a standard protein mixture
(high-molecular-weight calibration kit, Pharmacia LKB Biotechnology).
HDL particle size was calculated from the calibration curve of the peak
migration distance versus that of the standard.
DNA Amplification by Polymerase Chain Reaction (PCR)
Genomic DNA was isolated from 120 µL of peripheral
blood sample obtained from the proband.
Oligodeoxynucleotide primers used in amplification and
sequencing were chosen according to the published apoA-I gene
structure.35 The sequences and positions of these primers
in the apoA-I sequence are shown in Table 3. Two DNA segments carrying exon 3 and
exon 4 of apoA-I of the subject were amplified by PCR with genomic DNA
as a template. The PCR was performed in a reaction volume of 100 µL
containing 2.5 U Taq polymerase (Perkin-Elmer Cetus), 150 ng
genomic DNA, 20 pmol of each primer, and 200 µmol of each dNTP
in 10x reaction buffer containing 0.1 mol/L Tris-HCl (pH 8.3), 0.5
mol/L KCl, 15 mmol/L MgCl2, and 0.1%
(wt/vol) gelatin, as described previously.16
Amplification was performed by initial denaturation at 96°C for 10
minutes, followed by 30 cycles at 96°C for 30 seconds, 60°C for 30
seconds, and 72°C for 1 minute, and a final extension of 7 minutes at
72°C.
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DNA Sequence Analysis
The PCR-amplified DNA products were purified with the
Geneclean kit (Bio101), and purified DNA fragments were ligated to
pT7Blue T vector (Novagen). After subcloning, the single-stranded DNA
was sequenced with the dideoxy chain-termination reaction method and a
sequence kit (US Biochemical).36
Amplification-Refractory Mutation System (ARMS)
The presence of the apoA-I gene mutation was also
analyzed by ARMS.37 For this analysis, a
fragment of apoA-I gene was amplified by the use of primers 5 and 6 in
Table 1
. PCR was performed for 30 cycles of 30 seconds at
96°C, 30 seconds at 65°C, and 1 minute at 72°C. The PCR
products were determined by 5% PAGE. DNA was stained with ethidium
bromide and visualized by UV transillumination.
Construction of the Expression Vector pGEX/A-I
PCR was utilized to introduce appropriate DNA sequences
for constructing a fusion protein of apoA-I to glutathione
S-transferase (GST). The following 5' and 3' primers
were used in this study:
5'-CTCGGATCCACGATCGAAGGTCGTCGG-CATTTCTGGCAGCAAGA-3'
and
5'-CTTTGGAAACGTGA-ATTCTGGGATCCGGGAAGGG-3',
respectively. The 5' primer contained a BamHI site and
factor Xa cleavage site (underlined), which was immediately followed by
the first 6 amino acids of proapoA-I as described.14
The 3' primer has an EcoRI site and a BamHI site.
The pL–A-I plasmid (a gift from Mitsubishi Chemical Co, Yokohama,
Japan), which contains a full-length cDNA sequence of apoA-I, was used
as a template for PCR. The PCR fragment was ligated into the pT7Blue T
vector and subcloned. The pT7Blue T–apoA-I vector was digested with
BamHI and ligated into the BamHI site of the
pGEX-2T plasmid (Pharmacia Biotech, Inc). Mutant versions of apoA-I
cDNA were prepared by using the site-directed mutagenesis system
described by Kunkel.38
Expression and Purification of Human r-ProapoA-I
Human wild-type and variant proapoA-I were expressed according
to GST (gene fusion system, Pharmacia). Escherichia coli,
JM109 transformed with pGEX/apoA-I constructs, was cultured in LB broth
containing 50 mg/mL ampicillin at 37°C to an optical density of 0.5
to 0.6 (550 nm). Expression was induced with 0.5 mmol/L
isopropyl-1-thio-ß-D-galactopyranoside, after which the culture was
grown for another 1 hour at 30°C. Cells were pelleted
(4000g, 15 minutes), resuspended in PBS, and sonicated. Cell
debris was removed by centrifugation and the soluble
fraction loaded onto a glutathione–Sepharose 4B column (Pharmacia).
Fusion protein bound to the glutathione–Sepharose 4B was eluted by the
addition of reduced glutathione (10 mmol/L in 50 mmol/L
Tris-HCl, pH 8.0), and to obtain proapoA-I free of the carrier GST, the
fusion protein was cleaved by the addition of factor Xa (1:400, wt/wt)
at 4°C for 24 hours. The solution was dialyzed, and free proapoA-I
was separated from the GST carrier by repeating the glutathione-agarose
affinity chromatography as described above. The
r-proapoA-I was further purified by immunoaffinity
chromatography with a polyclonal anti-human apoA-I
antibody (Daiichi Pure Chemicals Co, Tokyo, Japan), after Sephacryl
S200 chromatography (Pharmacia). The final product
was checked by 12% SDS-PAGE. The amino acid sequence analysis
of the r-proapoA-I was performed as described
previously.16 In brief, r-proapoA-I was digested by
lysyl endopeptidase (enzyme-to-substrate ratio of
1:100, wt/wt) for 6 hours at 30°C in 1 mL of 10 mmol/L Tris-HCl
buffer (pH 9.0). The digested peptide fragments were separated by
high-performance liquid chromatography on a
Wakosil 5C18-200 column (4.0x250 mm). The
NH2-terminal amino acid sequences of r-proapoA-I
and the peptide fragments were analyzed by an Applied
Biosystems model 491 protein sequencer.
Iodination of ApoA-I
Labeling of normal r-proapoA-I and r-proapoA-I Nichinan with
Na[125I] (Du Pont–New England Nuclear) was
carried out according to the ICl method of McFarlane.39
The labeled apoA-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 apoA-I ranged from 350 to 450 counts per minute per
nanogram protein, and 99% of the radioactivity of labeled apoA-I was
precipitated by trichloroacetic acid. Before the turnover studies,
labeled r-proapoA-I was passed through a 0.45-µm filter with the
addition of bovine albumin (fatty acid–free, Sigma) to a final
concentration of 10 mg/mL and stored for 1 to 2 days at 4°C.
In Vivo Turnover Study of ApoA-I
Male Japanese White rabbits (body weight, 2.5 to 2.6 kg) were
purchased from Kyudo Co, Fukuoka, Japan. The animals were housed
individually in a 12:12-hour light/dark cycle environment and provided
with a commercial rabbit diet and water ad libitum. The experimental
protocol was approved by the Ethics Review Committee for Animal
Experimentation of Fukuoka University. The in vivo turnover studies
were performed essentially as described previously.40 In
brief, 2 days before and throughout the entire turnover study, 5 drops
of saturated KI were added each day to the drinking water. Blood
samples were collected via the marginal ear vein at 10 minutes; 3, 6,
9, and 24 hours; and then once a day for 4 days after injection
(25 µCi of 125I-labeled apoA-I in <1 mL saline). The
fractional catabolic rate (FCR) was calculated as described by
Matthews.41 The clearance of each labeled apoA-I was
measured in 4 different rabbits.
Cholesterol Efflux From Macrophage Foam
Cells
Mice were purchased from Kyudo Co, Fukuoka, Japan. Mouse
peritoneal macrophages were collected from nonstimulated male
DDY mice with 8 mL ice-cold PBS, centrifuged at 200g
for 5 minutes at 4°C, and suspended in Dulbecco's modified Eagle's
medium containing 3% BSA, 0.1 mg/mL streptomycin, and 100 U/mL
penicillin (medium A).42 One milliliter of cell
suspension (2x106 cells) was seeded onto each
plastic culture dish (35-mm diameter, Falcon) and incubated at 37°C
for 2 hours. Cells were washed once with 1 mL of PBS containing 0.3%
BSA and twice with 1 mL of PBS, and the monolayers thus formed were
used in the following cell experiments.42
Macrophages were converted to foam cells by incubation for 18 hours with medium A containing 50 mg/mL [3H]cholesteryl oleate–labeled acetyl-LDL. The cells were washed 3 times with PBS and incubated for an additional 18 hours with Dulbecco's modified Eagle's medium containing 0.2% BSA, 0.1 mg/mL streptomycin, and 100 U/mL penicillin (medium B) in the presence of 20 µg/mL native apoA-I, r-proapoA-I, or r-proapoA-I Nichinan. The incubation medium was collected and centrifuged to remove detached cells, and the radioactivity released into the medium from cells was determined by liquid scintillation spectrometry.31 The cellular lipids were extracted and run on a thin-layer chromatography system, followed by determination of the radioactivity in [3H]cholesterol and [3H]cholesterol esters as described previously.31 Native human apoA-I was purified by Sephacryl S-300 gel chromatography from delipidated HDL as described previously.31
Statistical Analyses
Data are expressed as mean±SD. Differences in FCR in the
turnover study and cholesterol efflux in vitro were
evaluated for statistical significance by Student's t test.
A value of P<0.05 denoted the presence of statistical
significance.
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Results |
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DNA Sequence Analysis
The sequence of the protein-coding regions of the apoA-I gene revealed that the proband (Figure 2
) was
heterozygous for a 3-bp deletion in exon 4 from nucleotides
1998 through 2000, removing Glu (GAG) at codon 234 or 235 of the mature
apoA-I. Because this deletion had not been previously reported, we
named this apoA-I variant (Glu 2350) apoA-I Nichinan, for the
proband's place of residence. The rest of the apoA-I gene sequence was
normal.
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We identified 3 other affected members in the proband's family who are
heterozygous for apoA-I Nichinan by using ARMS (Figure 3). PCR products from 4 heterozygotes
with apoA-I Nichinan presented a 297-bp band with the mutant
primers, whereas the apoA-I from other family members was not amplified
with these primers.
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Plasma Lipoprotein Analysis and LCAT Activity
IEF gel electrophoresis and immunoblotting of the
proband's plasma revealed that apoA-I Nichinan had a relative charge
of +1 compared with normal apoA-I4 (Figure 4). The concentrations of plasma
lipoproteins in the apoA-I Nichinan family members are shown in Tables 2 and 3. The mean concentration of HDL
cholesterol in the affected family members was reduced by
32% compared with that of the unaffected family members
(P<0.01). The concentrations of total
cholesterol, LDL cholesterol, and
triglyceride in apoA-I Nichinan carriers were similar to
those of unaffected subjects. The mean plasma concentrations of apoA-I
and apoA-II in the carriers were reduced by 30% (P<0.05)
and 15% (P=0.3), respectively, compared with those of
unaffected family members. The mean apoC-II and apoC-III concentrations
of the carriers were similar to those of unaffected family members.
Although cholesterol concentrations in both
HDL2 and HDL3 subfractions
were reduced, the degree of reduction in the HDL2
subfraction was greater than that in HDL3.
The nondenaturing PAGE pattern of HDL particles followed by scanning is
shown in Figure 5. The HDL particle size
profile of the proband was characterized by a predominance of the
HDL3 subclass and demonstrated that the
heterozygote had a decrease in HDL2. In the
proband, plasma LCAT activity as measured by exogenous assay was not
different from normal controls (Table 3).
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Characterization of r-ProapoA-I
The purified recombinant normal and mutant proapoA-Is were
characterized by 12% SDS-PAGE, IEF, and amino acid sequence
analysis. Normal r-proapoA-I and r-proapoA-I Nichinan formed a
single band on PAGE, with a similar molecular weight for apoA-I. IEF
gel electrophoresis and immunoblotting of r-proapoA-I
Nichinan was consistent with its having +1 charge units more
basic than normal r-proapoA-I. The amino acid sequence analysis
showed a Glu 235 deletion in r-proapoA-I Nichinan.
N-Terminal sequencing of r-proapoA-I and the digested
peptide fragments confirmed the expected proapoA-I sequence (data not
shown).
In Vivo ApoA-I Kinetic Study
Plasma decay curves of 125I-labeled normal
and variant r-proapoA-I in normal rabbits were compared. Table 4 shows that the mean FCR of
125I-labeled proapoA-I Nichinan (0.98±0.01
· d-1) was 22.2% higher than that of
125I-labeled normal r-proapoA-I (0.76±0.09
· d-1).
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Cholesterol Efflux by r-ProapoA-I From
Macrophage Foam Cells
In the next step, we examined the effect of r-proapoA-I Nichinan
on cholesterol efflux by using mouse peritoneal
macrophages. For this purpose, macrophages were
converted to foam cells with [3H]cholesteryl
oleate–labeled acetyl-LDL and then reacted with native apoA-I,
r-proapoA-I, or r-proapoA-I Nichinan. As shown in Figure 6, significant amounts of radioactivity
were released to the medium by native apoA-I. Thin-layer
chromatographic analyses of the lipids extracted
from the medium showed that all of the radioactivity had been derived
from [3H]cholesterol. Normal
r-proapoA-I also induced
[3H]cholesterol release into the
medium. Similarly, on incubation with r-proapoA-I Nichinan, significant
amounts of [3H]cholesterol were
released into the medium from macrophage foam cells. However,
the amount of [3H]cholesterol
released by r-proapoA-I Nichinan was 46% compared with that of normal
r-proapoA-I (Figure 6). Consistent with these results,
the capacity of r-proapoA-I Nichinan to reduce cellular cholesteryl
[3H]oleate (38% reduction from control) was
significantly weak when compared with that of normal r-proapoA-I (52%
reduction from control; data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We identified a heterozygote for a 3-bp deletion of nucleotides 1998 through 2000 in exon 4 of the apoA-I gene, resulting in a Glu 235 deletion. ApoA-I (Glu235
0) Nichinan was
associated with low levels of apoA-I and HDL cholesterol.
The HDL particle size distribution of the heterozygous apoA-I Nichinan
proband showed a low HDL2 subfraction. Plasma
LCAT activity in the proband was normal, and none of the apoA-I
Nichinan carriers presented with premature CAD. To examine the
functional role of apoA-I Nichinan, we produced r-proapoA-I Nichinan in
an E coli system. We characterized r-proapoA-I and
r-proapoA-I Nichinan by SDS-PAGE and amino acid analysis. It
has been reported that human apoA-I and r-proapoA-I show similar LCAT
activation properties, and the 
-helical contents of DMPC-associated
forms of apoA-I and r-proapoA-I were comparable.43 44
However, it has also been reported that cholesterol efflux
to lipid-free apoA-I was twice as much as that to
r-proapoA-I.45 In this study, the cholesterol
efflux to r-proapoA-I was 
60% of that to apoA-I. The
cholesterol efflux to r-proapoA-I Nichinan from mouse
peritoneal macrophages was decreased 54% compared with that of
r-proapoA-I. Studies on r-proapoA-I kinetics demonstrated accelerated
catabolism of r-proapoA-I Nichinan relative to normal r-proapoA-I in
rabbits. Many studies have shown that HDL stimulates cholesterol efflux from cultured cells. Aqueous diffusion and apolipoprotein binding models have been proposed as mechanisms by which HDL removes cellular cholesterol.46 The cell culture studies also imply that apolipoproteins containing very little or no lipid are the precursors for the lipid pathway of HDL in vivo.47 The best characterized of the lipid-poor forms of apoA-I is pre-ß1 HDL, a small particle containing only apoA-I and some phospholipid.1 46 Fielding et al6 have reported that apoA-I residues 137 to 144 are adjacent to or part of a structural site in pre-ß1 HDL that is active in promoting the efflux of cellular cholesterol. Another study has attempted to demonstrate that monoclonal antibodies specific to apoA-I residues 140 to 150 are able to inhibit cellular cholesterol efflux from intracellular or plasma membrane pools of cholesterol.9 Miccoli et al48 have reported that the cholesterol efflux capacity in plasma from apoA-I (Leu141Arg) Pisa hemizygotes with a nonsense mutation was significantly lower than that in normal plasma. von Eckardstein et al8 reported that cholesterol efflux from adipocytes obtained with a DMPC complex of apoA-I (Pro165Arg) was decreased by 30% and demonstrated that the substitution of proline at residue 165 interfered with the formation of a structural domain in apoA-I that is crucial for cellular cholesterol efflux stimulation.
Structural analysis revealed that the carboxyl-terminal region
of apoA-I also plays an important role in cholesterol
efflux, lipid binding,49 and the interaction of HDL with
cell membranes.50 The carboxyl-terminal region (residues
190 to 243) of apoA-I has been shown to be important for lipid and HDL
binding as well as for binding to cell membranes and thus, more rapid
clearance.15 Allan et al11 indicated that Fab
fragments from anti–A-I 205–220 and anti–A-I 230–243 antibodies
inhibited the binding of
125I-HDL3 to the liver
membrane. Holvoet et al51 suggested
that the carboxyl-terminal pair of helixes of apoA-I was involved in
the initial rapid binding of apoA-I to the lipid surface of HDL. ApoA-I
223–239 may affect cooperative interactions with the middle
amphipathic helixes of apoA-I that are critical for its specific
distribution over the different HDL species.
Sviridov et al45 have demonstrated
that truncation of carboxyl-terminal residues to 223 caused a
significant reduction in the ability of apoA-I to promote efflux of
cellular lipids, suggesting that the 223 to 243 region is most likely
involved in the formation of apoA-I–lipid complexes.
The mutation in apoA-I Nichinan is located within
the epitope, which inhibits the ability of apoA-I to promote
cholesterol efflux from cells. Therefore, we examined the
influence of apoA-I Nichinan on cholesterol efflux from
mouse peritoneal macrophages by using r-proapoA-I. There were
decreases by 54% in cholesterol efflux to r-proapoA-I
Nichinan from cholesterol-enriched mouse
macrophages when compared with normal r-proapoA-I. One possible
explanation may be that a critical structural change in the
carboxyl-terminal domain of apoA-I is induced by the Glu2350
mutation, leading to a decrease of helicity on lipid binding. By using
a computer model of the apoA-I secondary structure, apoA-I has been
found to be composed of 10 -helixes, which are composed of 11 or 22
amino acids.5 The hydrophobic moments calculated by the
method of Kyte and Doolittle52 are 1.05 for the residue
220 to 241 helix. Deletion of polar residues in the case of apoA-I
(Glu2350) Nichinan changes the hydrophobic moment of the helix to
0.93 and thus, also changes the orientation of the hydrophobic face in
the terminal amphipathic -helical domain of the mutant apoA-I. Thus,
this change of orientation in the -helical domain of residues 220 to
241 could affect to a certain extent the lipid-binding properties of
the helix.
The variability in HDL cholesterol levels was largely
determined by differences in the FCR of apoA-I, which was inversely
correlated with HDL particle size.15 The apoA-I (Arg26Gly)
Iowa mutation increased apoA-I catabolic rate and shifted it to a
higher density HDL fraction (HDL3 and the
d>1.21 g/mL fraction), resulting in decreased HDL
cholesterol levels.25 In addition to
apoA-I Iowa, apoA-I (Lys1070), apoA-I (Leu141Arg) Pisa, apoA-I
(Arg151Cys) Paris, apoA-I (Leu159Arg) Fin, apoA-I (Arg160Leu) Oslo,
apoA-I (Pro165Arg), and apoA-I (Arg173Cys) Milano provide other
examples of a mutation in apoA-I that reduces HDL
cholesterol levels in heterozygous carriers and that have
also been reported to have a more rapid catabolic
rate.8 23 35 53 54 55 56 In apoA-I (Leu159Arg) Fin, it was
speculated that moderate
hypertriglyceridemia in affected subjects
may contribute to the increased apoA-I FCR and markedly reduced
concentration of serum HDL cholesterol. Dimerization of
apoA-I (Arg173Cys) Milano accounts for the faster catabolism of normal
and variant apoA-I in heterozygous carriers.57 It was
reported that apoA-I (Leu141Arg) Pisa has a reduced ability to
activate LCAT.48 Recently, in our laboratory, a
turnover study demonstrated that apoA-I (Val156Glu) Oita had an
accelerated catabolic rate in rabbits. The shift of apoA-I to a higher
density HDL fraction and the reduced LCAT activity in apoA-I Oita
may be related to faster catabolism of apoA-I, which may contribute to
apoA-I deficiency and a low HDL cholesterol
level.21 A frameshift mutation in residue 202 of the
carboxyl terminal of human apoA-I was reported to be the causative
defect in HDL deficiency with corneal opacities.58
Moreover, truncation to residues 227 to 243 of apoA-I, which disrupts
the last amphipathic -helical domain, decreased the ability of
apoA-I to associate with HDL and also produced a marked increase in the
clearance of apoA-I.14
In this study, we obtained r-proapoA-I Nichinan and studied its catabolic rate in rabbits. The turnover study demonstrated that r-proapoA-I Nichinan has an accelerated catabolic rate in rabbits compared with normal r-proapoA-I. Because plasma LCAT activity, as measured by exogenous assay, which reflect the mass or concentration of LCAT, was not decreased, low concentrations of plasma apoA-I and HDL cholesterol in the carriers of apoA-I Nichinan may not be directly related to in vivo LCAT activity. Considering the role of apoA-I in HDL metabolism, a decrease in the capacity of apoA-I to promote cholesterol efflux from cells in apoA-I Nichinan is likely to be the consequence of the abnormal apoA-I due to the mutation in the apoA-I gene.
An association between apoA-I mutations and premature CAD is still a matter of debate. Most patients with apoA-I mutations do not show an increased susceptibility to the development of CAD, despite previous epidemiological evidence that plasma HDL concentration is inversely correlated with the risk of CAD.59 Evaluation of the relation between apoA-I mutations and CAD risk is hampered by the fact that these mutations are genetically heterogeneous and very rare in the population. In 4 different families, homozygous patients for different apoA-I null alleles suffered from CAD.60 61 62 63 Compound heterozygous apoA-I (Leu141Arg) Pisa23 was also identified to be associated with CAD. In addition to the differences in basic defects, there is also variation in sex, age, ethnic origin, and individual habits among homozygous proband patients. These factors are known to significantly modulate CAD risk. Because the proband's family members were living in a rural area of western Japan, all carriers of apoA-I Nichinan characterized so far are heterogeneous, and clinical signs of CAD have not been presented despite the low plasma HDL cholesterol level. The CAD risk cannot, however, be ruled out, owing to the relatively small sample size and young age of 2 affected family members. Thus, many important questions, such as the prevalence of the apoA-I Nichinan mutation in different populations, the exact functional role of apoA-I Nichinan in reverse cholesterol transport, and its possible influence on the development of CAD, remain to be answered.
In conclusion, heterozygosity for apoA-I Nichinan results in a reduction of plasma HDL cholesterol concentration, a decrease in the number of HDL2 size particles, and enhanced catabolism of apoA-I. In vitro studies with recombinant lipid-free apoA-I showed a decreased ability to promote cholesterol efflux from mouse peritoneal macrophages. Elucidation of the atherogenicity, if any, of apo-I Nichinan still requires further large-scale studies in additional affected kindreds.
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Acknowledgments |
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This work was partly supported by grants from the Japanese Ministry of Education, Science, Sports and Culture (No. 08671195 to J.S., No. 08671196 to A.M.). We wish to thank Dr Yoshio Mismi and Dr Fumiko Arakawa in the Department of Biochemistry, School of Medicine, Fukuoka University, for their technical advice; Tomoko Shinkawa and Akiyo Hamachi for their excellent technical assistance; and Akiko Kato for the preparation of the manuscript.
Received July 9, 1998; accepted September 29, 1998.
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