Identification of Human Apolipoprotein E Variant Gene ...
Transcript of Identification of Human Apolipoprotein E Variant Gene ...
J. Biochem. 105, 51-54 (1989)
Identification of Human Apolipoprotein E Variant Gene:
Apolipoprotein E7 (Glu244 ,245•¨Lys244,245)1
Hideo Maeda,* Hiroyo Nakamura,* Shozo Kobori,** Mitsuko Okada,* Hirotada Mori,*** Hironori Niki,*** Tern Ogura,*** and Sota Hiraga****Faculty of Health and Living Sciences, Naruto University of Education, Naruto, Tokushima 772;** Department of Metabolic Medicine, Kumamoto University Medical School, Kumamoto, Kumamoto 860; and*** Department of Molecular Genetics, Institute for Medical Genetics, Kumamoto University Medical School, Kumamoto, Kumamoto 862
Received for publication, June 20, 1988
Apolipoprotein E (apoE) is one of the protein moieties of the human serum lipoproteins.
Three major isoforms of apoE (apoE2, apoE3, and apoE4) and minor variant isoforms
(apoE1, apoE5, and apoE7) have been detected by isoelectric focusing. In this study we have
cloned the apoE7 gene from a patient with the apoE3/E7 phenotype associated with
hypertriglyceridemia and diabetes mellitus. DNA sequencing revealed that the apoE7 gene
has two base substitutions (G•¨A) changing GIu244 ,245•¨LyS244,245, compared with the apoE3
gene. The replacement of the two amino acids is consistent with the result of isoelectric focusing of the apoE7 isoprotein, which shifts to four positively charged units compared
with the apoE3 isoprotein.
Apolipoprotein E (apoE) is one of the protein constituents of plasma lipoproteins (1, 2). Human apoE is synthesized in a variety of tissues as a preprotein of 317 amino acid residues (3, 4). The preprotein undergoes intracellular proteolysis, glycosylation, and extracellular desialylation, resulting in a single polypeptide chain of 299 amino acid residues with the molecular weight of 34,000 (5, 6). The human apoE gene has been isolated, characterized, and mapped to chromosome 19 (7, 8). The genes coding for apoC-I, apoC-II, and low density lipoprotein (LDL) receptor also have been described (9-11). ApoE mediates lipoprotein catabolism by binding to the LDL receptor (apoB, E-receptor), as well as to the apoE receptor (12), and is therefore important in determining triglyceride-rich lipoprotein catabolism. In plasma, three major apoE isoforms, E2, E3, and E4, differing in their isoelectric points, have been detected (13-15). These isoforms are determined by three alleles at a single genetic locus (14). The commonest apoE allele is apoE3, while apoE2 and apoE4 are less common in Western countries (16-20) and in Japan (21). It is known that these different isoforms have resulted from a series of amino acid substitutions occurring at several positions in the apoE isoprotein (22). It has been demonstrated that these different apoE phenotypes are associated with plasma lipoprotein disorders (23-27). Especially, apoE2 has been described as showing altered binding activity to lipoprotein receptors, which is consistent with this mutation, causing lipoprotein abnormalities in patients with type III hyperlipoproteinemia (28). In addition to these three isoforms, other isoforms (apoE5 and apoE7) associated with lipoprotein disorders have been reported by
Yamamura et al. (29, 30). It has been indicated that the incidence of these abnormal isoforms is low among healthy subjects, but is high among patients with hyperlipidemia and ischemic heart disease. A structural analyses of these mutants must be performed with respect to their association with atherosclerosis (30)
Recently we found a patient in Japan with an apoE3/E7 phenotype, associated with hypertriglyceridemia and diabetes mellitus. The patient is of a different family than the case reported by Yamamura et al. (30). In the present study, we have cloned and sequenced the genomic DNA segments of the apoE7 gene from a proband. We demonstrate that two base substitutions have occurred in two glutamic acid codons at positions 244 and 245 of apoE3. These base substitutions resulted in the replacement of two glutamic acid residues by two lysine residues, coinciding with a shift to a four positively charged unit from apoE3.
MATERIALS AND METHODS
Proband•\The 55-yr-old man with the apoE3/apoE7
phenotype had been well until 53 yr of age, when general
fatigue, polydipsia and polyurea developed slowly. He was
admitted to the hospital for 2 mo under a diagnosis of
diabetes mellitus, and hypertriglyceridemia was noted at
this time. He was referred to our lipid clinic. The serum
cholesterol, triglyceride, and high-density lipoprotein
(HDL)-cholesterol levels were 205, 483, and 49mg/dl,
respectively. The ECG and physical examination (xan
thoma, achilles tendon hypertrophy, and neurological
findings) revealed nothing remarkable.
Preparation of DNA-Approximately 15ml of peripheral blood was collected from the proband. Following purification of the white blood cells, genomic DNA was extracted according to the procedure of Kunkel et al. (31).
Separation of Lipoproteins•\Venous blood was obtained
after overnight fasting. Plasma very-low-density lipo-
1 This work was supported by Grants-in-Aid for Scientific Research
(Nos. 6244001 and 62570399) from the Ministry of Education, Science and Culture of Japan and a grant from Chiyoda Mutual Life Foundation.
Abbreviations: apo-, apolipoprotein-; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein.
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52 H. Maeda et al.
protein (VLDL) particles (d<1.006g/ml) were isolated in a Type 100.2 rotor using a Beckman TL-100 ultracentrifuge or in a Type 50.2Ti rotor using a Beckman L5-50B ultracentrifuge (32).
Isoelectric Focusing and Electrophoresis•\ApoE iso
forms in apoVLDL were analyzed by isoelectric focusing on
polyacrylamide gels. Analytical isoelectric focusing was
performed according to the method of Warnick et al. (33).
ApoVLDL (100-150ƒÊg) protein was solubilized in 10mM
Tris-HCl (pH 8.6) containing 8M urea and 10mM dithio
threitol. The apoproteins were subjected to isoelectric
focusing with 7.5% acrylamide gel rod containing 8M urea
and 2% ampholytes (LKB Inst., Sweden). Ampholine at pH
3.5-5, 5-8, and 3.5-10 were mixed in proportions of 2:2:
1 to create a pH gradient between 4-8. The cathlyte and
anolyte consisted of 1M NaOH and 1M H3PO4, respective
ly. The gels were focused with a current of 0.5mA per tube.
After a potential of 300V was attained, electrofocusing was
performed for 16h at 5•Ž. The gels were fixed and stained
for 3h in 3.5% perchloric acid containing 0.04% Coomassie
Brilliant Blue G (Sierra Chemical Co U.S.A)
Cloning of the Genomic ApoE DNA•\A library of
genomic DNA from the proband was constructed using the
EMBL 4 bacteriophage lambda cloning vector. Genomic
DNA was partially digested with Sau3AI and ligated with
BamHI-digested EMBL 4 phage DNA. The ligated DNA
was packaged in vitro ,into infectious particles using a
Gigapack kit (Stratagene, San Diego, Calif.). The library
was screened with the nick translated apoE cDNA segment
(6) according to previously described methodology (34).
The cloned DNA was digested with EcoRI and BamHI-
EcoRI, and DNA fragments were isolated after electro
phoresis in low melting point agarose gels (Nippon Gene
Co., Ltd., Toyama). A 2.3-kb BamHI-EcoRI fragment
containing exons 1, 2, and 3 of apoE was subcloned into the
pUC118 vector, and a 1.9-kb EcoRI fragment containing
Fig. 1. Isoelectric focusing patterns of apoVLDL. Lane A and lane B, apoVLDL from control subjects with the phenotype apoE3/E3 (lane A) and phenotype apoE3/E4 (lane B); lane C, apoVLDL from the proband with the phenotype apoE3/E7. The cathode is at the top and the anode at the bottom. The arrows represent apoE3 and apoE7 isoproteins (long arrows), and its sialylated derivatives (short arrows).
exon 4 of the apoE gene was subcloned into the bacteriophage M13mp10 vector (35).
DNA Sequence Analysis-DNA sequence was determined according to Sanger et al. (36). The sequencing reactions were carried out using a kit (Takara Shuzo Co., Kyoto). Oligonucleotide primers were synthesized using the solid-phase phosphotriester method and an Applied Biosystems DNA synthesizer (Foster, Calif.) and were used after desalting with a Sep-Pak column (Waters Associate, Inc., Milford, Mass.). The specific primers were used at 1pmol for each DNA sequencing reaction.
RESULTS AND DISCUSSION
Isoelectric Focusing•\Plasma VLDL particles were iso
lated from the serum of the proband and analyzed for apoE
isoforms by isoelectric focusing. As shown in Fig. 1 (lane
C), in apoVLDL of the proband, nine bands of apoE
isoproteins and their sialylated derivatives were observed
in addition to four bands of apoC proteins. The isoelectric
points (pI) of the two major bands were 5.72 and 6.74. One
of the major bands (pI 5.72) was focused on the position
corresponding to the apoE3 isoprotein. The other major
band (pI 6.74) was focused on three basic units from apoE4
and on four basic units from apoE3. The pI value of this
most basic apoE isoprotein was similar to that of apoE-
Suita (apoE7), described by Yamamura et al. (30). We also
conducted two-dimensional gel electrophoresis for this
apoVLDL from the proband, and it was blotted onto
nitrocellulose filters. The two major bands and three minor
bands all reacted with anti-human apoE antiserum (data
not shown). This most basic apoE protein (pI 6.74) was also
detected in some family members of this proband (data not
shown). From these results we diagnosed that the pheno
type of the apoE isoproteins of this proband was apoE3/E7
and that this apoE7 isoprotein was genetically inherited in
family members.
Cloning of the Variant ApoE Gene-We screened 6
•~ 105 recombinants from the genomic library from the
proband using the 32P-labeled cDNA of apoE as a probe. A
restriction map of the human DNA segments carried by one
Fig. 2. Restriction map of the genomic DNA segments contain-
ing the apoE gene. Solid blocks indicate coding regions . Hatched
regions indicate untranslated sequences. (A) The genomic DNA
segment carried by ă apoE-HK101. The numbers of exons in the
apoE gene are shown as E1, E2, E3, and E4 in the figure. (B) DNA
segments subcloned into pUC118 plasmid. (C) DNA segments sub
cloned into M13mp10 phage vector. Arrows indicate the direction and
extent of nucleotide sequencing determined by using the dideoxy
chain termination method. Relevant restriction sites are as follows:
BamHI (B), EcoRI (R), HindIII (H), and PstI (P).
J. Biochem .
Molecular Cloning of Human apoE7 Gene 53
Fig. 3. Nucleotide and amino acid sequences of the genomic DNA segment of the human apoE7 isoprotein gene. As compared with
the cDNA sequence of the most common apoE3 gene (6), two nucleotide substitutions (G-A) were found at nucleotide positions 730 and 733, resulting in changes of two GAG codons of glutamic acid to two AAG codons of lysine at amino acid residues 244 and 245.
TABLE I. T Human apolipoprotein E variants.8
a Differences in amino acid residues of apoE isoproteins (apoE2 and apoE4) and rare variant apoE isoproteins are commonest isoprotein, apoE3.
of the positive clones, named A apoE-HK101, which was found to carry the apoE7 gene as described below, is shown in Fig. 2. This clone contained the 13.9-kb DNA segment of the proband, including the entire coding sequence of the apoE gene which consists of four coding segments (exons) that were interrupted by three noncoding segments (introns).
Sequencing of the Cloned ApoE Gene•\The genomic
DNA segment of d. apoE-HK101 was subcloned into
pUC118 plasmid or M13mp10 bacteriophage. All four
exons of the apoE gene and their flanking regions were
analyzed by DNA sequencing. As shown in Fig. 3, it was
found that two transition mutations (G•¨A) occurred at
nucleotide positions 730 and 733. These mutations led to
changes in the two GAG codons of glutamic acid to two AAG
codons of lysine, resulting in the substitution of two lysine
residues for two glutamic acid residues at amino acid
positions 244 and 245. By comparison with the DNA sequence of the apoE3 cDNA (6), we confirmed that there
are no other mutations in the DNA segment, except for the
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54 H. Maeda et al.
two mutations described above. These substitutions of
lysine for glutamic acid should result in alteration of the
isoelectric point to four positive charges for the apoE7
isoprotein in isoelectric focusing, as compared with the
apoE3 isoform. This is consistent with the experimental
results of the isoelectric point for the apoE7 isoprotein .
These data suggest that the genomic DNA segment of ă
apoE-HK101 carries the ƒÃ 7 apoE allele, which encodes for
a kind of apoE7 isoprotein, hitherto the most basic iso
protein of apoE. DNA sequences of all exon-intron junc
tions were consistent with the results obtained for the
apoE4 genome (37).
Various apoE alleles have been described, as summarized in Table I. The apoE7 allele described in this paper codes for Cys112 and Arg158, as does the apoE3 allele. This suggests that the apoE7 allele was presumably derived from the apoE3 allele, not from the apoE2 and apoE4 alleles.
From the physiological point of view, the mutated residues of apoE7 isoprotein are located in one of the putative heparin binding domains (44), but not the receptor binding domain (45, 46). The two amino acid substitutions of the apoE7 isoprotein create a lysine-rich cluster (-Lys-Leu-Lys-Lys-) in residues 242-245. Although we have not determined the binding affinity of apoE7 isoprotein to the receptor and heparin, this lysine cluster with positive charge may have an impact on defective lipoprotein metabolism by some mechanism through the interaction of LDL receptors. heparin binding. and/or unknown factors.
Yamamura et al. (30) pointed out the prevalence of unusual apoE mutants, including both apoE5 and apoE7, which were as high as 5% among patients with atherosclerotic disease and hyperlipidemia. To date, another three independent subjects (45 to 60-yr-old men) with apoE7 isoprotein have been found by our laboratory. All three subjects were diagnosed as having diabetes mellitus or impaired glucose tolerance. In addition, two subjects among them showed hypertriglyceridemia (485 and 860mg/dl), and one showed hypercholesterolemia (330mg/dl) (data not shown). This high incidence of these clinical signs suggests that the apoE7 isoprotein affects the normal lipoprotein metabolism even in heterozygotes such as apoE7/apoE3. Further studies are necessary to learn the direct effect of the apoE7 isoprotein in vitro on lipoprotein metabolism.
We thank Dr. Jan L. Breslow, Rockefeller University, New York, for
sending the plasmid carrying cDNA of apoE. We wish to express our thanks to Drs. Haruo Uzawa and Yasuo Kishino for encouragement and advice. We thank Otsuka Pharmaceutical Co., Ltd. in Tokushima for the use of their facilities and equipment.
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