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.

Vol. 105, No. 1, 1989 51

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

Vol. 105, No. 1, 1989

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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