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Annals of Clinical & Laboratory Science 31:163-170 (2001)
© 2001 Association of Clinical Scientists

Characterization of an Apolipoprotein E3 Variant (Arg 145 -> His) Associated with Mild Hypertriglyceridemia

Hiroya Hidaka1, Minoru Tozuka1, Eiko Hidaka1, Kazuyoshi Yamauchi1, Hiroyoshi Ota1, Takayuki Honda2 and Tsutomu Katsuyama2
1 Central Clinical Laboratories, Shinshu University Hospital, Matsumoto, Japan
2 Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan

Address correspondence to Minoru Tozuka, Ph.D., Central Clinical Laboratories, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan; tel 81 263 37 2805; fax 81 263 34 5316; e-mail mtozuka{at}hsp.md.shinshu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a proband (21-yr-old female), we previously identified an apolipoprotein (apo) E variant, apoE3 (Arg 145 -> His), with an isoelectric point midway between apoE3 and apoE2. ApoE gene analysis of 4 of the proband’s kin indicated that 3 possess the same variant. All 4 had a high concentration of apoE in plasma, while 3 of 4 had hypertriglyceridemia. In the proband (who had no hypertriglyceridemia), most apoE was distributed in slow-alpha lipoproteins (predominantly in the form of apoE-AII heterodimer) and in larger molecules with apparent molecular weights of 80 and 100 kDa. In the proband’s brother (with hypertriglyceridemia), however, most apoE was distributed in slow pre-ß lipoproteins, predominantly in the form of monomeric apoE. In each subject, the concentration of apoE3 variant was significantly higher than that of normal apoE3 in the predominant apoE-rich lipoprotein. The apoE3 variant, which displayed a slightly reduced binding ability to LDL-receptor and heparin, may induce an accumulation of apoE-rich lipoproteins. These observations suggest that the difference in distribution of apoE3 variant in plasma lipoproteins between the proband and her brother (combined with its reduced affinity for the LDL receptor) may provide key insights into the pathogenesis of hypertriglyceridemia.

(received 18 January 2001; accepted 6 February 2001)

Keywords: apolipoprotein E, isoform, variant, polymorphism, hypertriglyceridemia, heparin, LDL-receptor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Apolipoprotein E (apoE), a 34.2 kDa plasma protein composed of 299 amino acid residues [1], is a polymorphic protein with isoforms and disulfide-linked complexes. The major function of apoE is to regulate plasma lipid and lipoprotein levels by associating with lipoprotein subclasses, such as triglyceride-rich lipoproteins, remnants of VLDL and chylomicrons, and apoE-containing HDL, and by acting as a ligand to mediate high-affinity binding to heparan sulfate proteoglycans (HSPG) and lipoprotein receptors on the cell-surface [2–7].

Apo E has three common isoforms, designated apoE2 (Cys-112, Cys-158), apo E3 (Cys-112, Arg-158), and apoE4 (Arg-112, Arg-158), with different binding activities to lipoprotein receptors; apoE3 and apoE4 have almost equal binding abilities, while apoE2 is defective [8–10]. ApoE3 exists not only in monomeric form, but also as the apoE-AII heterodimer and in a higher molecular weight form (approximately 100 kDa) identified as a homodimer [11,12], while only the monomeric form exists for apoE4. In addition, an apo(AII-E2-AII) complex is found in subjects with an apoE phenotype including apoE2 [13]. The apoE-AII complex and the homodimer [and possibly the apo(AII-E2-AII) complex] are each known to have poor ability to bind to the lipoprotein receptor [12,14]. This may explain why plasma lipid levels are influenced by apoE isoforms [15].

A new apoE variant, apoE3 (Arg 145 -> His) named apoE-Kochi, has been reported previously [16]. We have identified the same apoE variant in a family whose members either had normal lipid levels or showed hypertriglyceridemia [17]. All of the family members with the variant had high concentrations of apoE in their plasma. However, an apoE-rich lipoprotein fraction with abnormal electrophoretic mobility, between ß and pre-ß, was observed in only three subjects, all of whom had mild hypertriglyceridemia. The remaining subject (with a normal lipid level) had apoE-rich lipoprotein in the slow-alpha fraction, despite having a high level of plasma apoE like the other members [17]. To investigate the effect of this apoE3 variant (Arg 145 -> His) on the pathogenesis of hypertriglyceridemia, we analyzed its distribution among plasma lipoproteins, its polymorphism, and its heparin- and LDL-receptor-binding abilities.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials.  Anti-apoE, -apoAI, and -apoB sera (goat) were obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). Peroxidase-conjugated anti-goat IgG antibody (rabbit) was obtained from MBL Co. (Tokyo, Japan). Agarose gel ("Titan Gel Lipoprotein," Cat. No. J3045), barbital buffer, pH 8.6, for electrophoresis, and cholesterol- and triglyceride-staining kits were obtained from Helena Laboratories (Beaumont, TX). Polyacrylamide gradient gel (8–16%, thickness 1 mm) containing sodium dodecyl sulfate was obtained from Tefco Co. (Tokyo, Japan). Neuraminidase, FITC, heparin-Sepharose gel, and dimyristoylphosphatidylcholine (DMPC) were obtained from Nacalai Tesque Co. (Kyoto, Japan), Wako Chemical Co. (Osaka, Japan), Pharmacia Biotech (Uppsala, Sweden), and Sigma Chemical Co. (St. Louis, MO), respectively. Sera from two subjects with the apoE3 variant, the proband and her brother, were investigated. Sera from normolipidemic subjects were also studied as controls.

Assays for serum lipids and apolipoproteins.  Total cholesterol, triglyceride (TG), and HDL-cholesterol were asayed by automated enzymatic methods, using an automatic analyzer. Apolipoproteins (apoAI, apoB, and apoE) were assayed by an automated immunoturbidimetric method (Daiichi Pure Chemicals Co.).

Preparation of lipoproteins.  Whole lipoprotein (d 1.006–1.2 g/ml) and low-density lipoprotein (LDL: d 1.006–1.063 g/ml) were isolated from sera by ultracentrifugation, by the method of Havel et al [18]. Apolipoproteins were obtained from the lipoprotein fractions by delipidation using ethanol:ether (3:1,v:v).

ApoE phenotyping.  ApoE phenotype was determined by isoelectric focusing (IEF) followed by immunoblotting with anti-apoE antibody [15]. Briefly, serum (5 µl) was incubated with 10 µl of neuraminidase solution (5 U/L in 0.1 mole/L citrate buffer, pH 6.0, including 0.2% Tween 20) in a polypropylene microcentrifuge tube for 4 hr at 37°C, followed by treatment with 10 µl of 75 mmol/L dithiothreitol including 0.2% Tween 20 for 30 min at room temperature. The mixture was then analyzed by isoelectric focusing, which was carried out using 4.8% polyacrylamide gel containing 8 mol/L urea and 2% Ampholine (mixture of pH 5–8, pH 3.5–10, and pH 7–9, 3:1:1) at 500V for 1 hr. The separated proteins were electrophoretically transferred onto nitrocellulose membranes, and the bands representing apoE isoforms were visualized using anti-apoE polyclonal antibody (goat), horseradish peroxidase-conjugated anti-goat IgG (rabbit), 3,3'-diaminobenzidine tetrahydrochloride (Dojin Chemical Co., Osaka, Japan), and hydrogen peroxide (Wako Pure Chemicals).

Lipoprotein electrophoresis.  Agarose gel electrophoresis for fractionation of lipoproteins was performed according to the manufacturer’s instructions. Serum (2 µl) was applied to agarose gel using a template, and electrophoresed for 25 min at 90V. Separated lipoproteins were stained with cholesterol- and triglyceride-staining kits. Visualization of apolipoproteins on the agarose gel was performed by an immunofixation method using specific antibodies. Briefly, after electrophoresis, cellulose acetate membrane strips (each soaked in one of the antibodies) were put on the agarose gel and incubated for 15 min at 37°C. The gel was exhaustively washed with saline followed by water, and then dried in a dryer box. The fixed apolipoproteins were stained with Coomassie Brilliant Blue G-250.

Apolipoprotein electrophoresis.  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the Tefco system (Tefco Co.). Samples (10 µl) obtained from the FPLC system were mixed with 5 µl of sample buffer (0.5 mol/L Tris-HCl, pH 6.8, containing 25% glycerol, 10% SDS). The mixtures were applied to gels and electrophoresed using SDS-PAGE buffer (0.025 mol/L Tris-0.192 mol/L glycine, pH 8.3, containing 0.1% SDS) at 10 mA/gel for 2 hr. After electrophoresis, apolipoproteins were transferred to a nitrocellulose membrane and visualized by immunoblotting, as described above.

Heparin-Sepharose affinity chromatography.  A heparin-Sepharose column (0.5 x 20mm) was equilibrated with 5 mmol/L Tris-HCl buffer, pH 7.4, containing 8 mol/L urea (Buffer A). Whole apolipoproteins were applied to the column, and the bound apolipoproteins were eluted with the same buffer containing 0–0.2 mol/L NaCl, using a stepwise method. The complexes containing apoE in each fraction were separated by SDS-PAGE or IEF, and then visualized by immunoblotting using anti-apoE antibody, as described above.

Binding assay for LDL-receptor.  ApoE-DMPC complexes were prepared as described by Rall et al [19]. FITC-labeled LDL (FITC-LDL) was prepared by the method of Riggs et al [20]. The binding ability of apoE-DMPC to the LDL-receptor on the surface of normal lymphocytes was estimated by a flow-cytometric method using a competitive binding assay between apoE-DMPC and FITC-LDL (at 4°C on ice) [21].


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Isoelectric focusing of the apoE variant.  ApoE phenotyping of the two subjects with the apoE variant revealed the presence of two isoproteins, one with a normal apoE3 mobility and the other with isoelectric point midway between apoE3 and apoE2 (Fig. 1Go). This variant was found by genetic analysis to contain a substitution of Arg145 for His in normal apoE3 (data not shown). In both subjects, the relative concentration of the apoE3 variant was significantly higher than that of normal apoE3. Both subjects showed higher levels of plasma apoE than subjects with the apoE3/E3 phenotype; however, the plasma triglyceride level was normal for one (the proband) and elevated for the others (her mother and brothers).



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  		Fig. 1. Identification of apoE variant and analysis of serum lipids and apoE. Panel A: Analytical isoelectric focusing on a polyacrylamide gel (pH 5–8) of sera from subjects with apoE3/E variant (lanes 1 and 2), apoE4/E2 (lane 3), apoE3/E3 (lane 4), and apoE3/E2 (lane 5). Sera (5 µl) treated with neuraminidase and dithiothreitol in 1% Tween 20 solution were applied to gel for isoelectric focusing. After electrophoresis, the apoE bands were detected by immunoblotting with anti-apoE serum. The arrow indicates the apoE variant. The focusing positions of apoE4, apoE3, and apoE2 are indicated. Panel B: Table shows concentrations of lipids and apoE in sera from subjects with apoE variant (subject 1: proband; subject 2: proband’s brother); those of control subjects with apoE3/E3 (n=144) are also indicated, with mean ± SD, for comparison.

 
Distribution of apoE in plasma lipoprotein classes.  The distribution of apoE in plasma lipoproteins was determined by agarose gel electrophoresis. Lipoprotein fractions and the distribution of apoE, apoAI, and apoB were identified by enzymatic staining of cholesterol and triglyceride and immunoblotting, respectively (Fig. 2Go). In the proband with the apoE3/E variant phenotype (whose plasma apoE was high, but whose plasma triglyceride was not), apoE was preferentially distributed in slow-alpha lipoprotein. In her brother, with the same apoE phenotype, both the apoE and the triglyceride in plasma were elevated, and the distribution of apoE revealed a broad ß-pattern.



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  		Fig. 2. Agarose gel electrophoresis of sera from subjects with apoE variant. After agarose gel electrophoresis, lipoproteins were detected by the various staining methods described in Methods. Lanes 1–3: cholesterol staining; lanes 4–6: triglyceride staining; lanes 7–9: apoA-I immunostaining; lanes 10–12: apoB immunostaining; lanes 13–15: apoE immunostaining. Lanes 1, 4, 7, 10, and 13: control serum with normal lipid concentration (apoE3/E3 phenotype); lanes 2, 5, 8, 11, and 14: the proband (apoE3/E variant phenotype); lanes 3, 6, 9, 12, and 15: the proband’s brother (with hypertriglyceridemia) (apoE3/E variant phenotype).

 
Two-dimensional electrophoresis.  A two-dimensional electrophoresis, using agarose gel for the first and SDS-polyacrylamide gel for the second dimension, revealed polymorphism in terms of molecular weight of the complexes containing apoE, apoE-AII heterodimer, and two higher molecular-mass forms (approximately 80 kDa and 100 kDa) (Fig. 3Go). In the proband, the apoE-AII heterodimer and the higher molecular-mass forms were predominantly observed in the position representing slow-alpha lipoprotein, whereas monomeric apoE was distributed in the slow pre-beta lipoprotein position. In the proband’s brother, most of the monomeric apoE and higher molecular-mass forms were distributed in the slow pre-beta lipoprotein position, but there was only a small amount of the apoE-AII heterodimer in this position.



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  		Fig. 3. Two-dimensional polyacrylamide gel electrophoresis of apoE from subjects with apoE variant Agarose gel electrophoresis was performed for sera of the proband (apoE3/E variant phenotype, A) and her brother (with hypertriglyceridemia) (apoE3/E variant phenotype, B) as the first dimension. The gels were incubated in SDS-PAGE buffer and applied to the top of acrylamide slab gels (8–16%) with 0.1% SDS. After electrophoresis, apoE was visualized by immunoblotting. Molecular mass standards (kDa) are indicated at the left side.

 
Isoelectric focusing of the lipoprotein fractions.  To determine the distribution of the apoE-variant among the various lipoprotein fractions, sera were applied to agarose gel electrophoresis. After the electrophoresis, the gel was sliced at intervals of 5 mm, and the separated lipoproteins were eluted from each piece of gel using 0.15 M NaCl followed by isoelectric focusing. In our two subjects, the apoE variant was widely distributed in all of the lipoprotein fractions, as was normal apoE3. In both subjects, the relative concentration of the apoE variant was significantly higher than that of normal apoE3 in each of the lipoprotein fractions predominantly containing apoE (Fig. 4Go).



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  		Fig. 4. ApoE isoforms among plasma lipoproteins separated by agarose electrophoresis. Electrophoresis was performed as in Fig. 3Go. After electrophoresis, the strips of agarose gel were sliced at intervals of 5 mm. Proteins were eluted in 0.15 M NaCl solution from each slice, and applied to an isoelectric focusing gel. After focusing, apoE was detected by immunoblotting using anti-apoE antiserum. Black and white arrows indicate the variant and normal apoE, respectively. Panel A shows the isoelectric focusing pattern for the proband, and panel B shows that for her brother. Each lane corresponds to the lipoprotein fraction shown under the patterns.

 
Heparin-Sepharose affinity chromatography.  To determine the affinities of the apoE variant and apoE3 for heparin, the proband’s whole apolipoproteins were applied to a heparin-Sepharose column followed by exhaustive washing with Buffer A. The bound apolipoproteins were eluted by an increasing NaCl concentration in Buffer A. In each fraction, the molecular size of the apoE complexes was analyzed by SDS-PAGE and the isoforms of apoE were analyzed by isoelectric focusing. The elution of the apoE-AII complex (43 kDa) started at approximately 42 mmol/L NaCl followed by elution of the apoE monomer (33 kDa) and homodimer (100 kDa) at approximately 45 and 50 mmol/L NaCl, respectively (Fig. 5Go). By isoelectric focusing, the elution of the apoE variant started at approximately 42 mmol/L NaCl, then both the apoE variant and apoE3 followed at approximately 45 mmol/L NaCl (Fig. 5Go).



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  		Fig. 5. Electrophoretic analysis of apoE for the fractions separated by heparin-Sepharose affinity chromatography. Apolipoproteins, prepared from the proband’s whole lipoproteins (d 1.006–1.21 g/ml) separated by ultracentrifugation, were resolved in 5 mmol/L Tris-HCl buffer (pH 7.4) containing 8 mol/L urea, and applied to the heparin-Sepharose column. The unbound fraction was eluted with resolution buffer, and then the bound fractions were eluted with resolution buffer containing various concentrations of NaCl (dotted line). The bound fractions (18 fractions) were analyzed using SDS-polyacrylamide gel electrophoresis (A) and isoelectric focusing (B). Lanes 1–5 of panel B correspond to lanes 5–9 of panel A. Positions of known molecular mass (kDa) obtained using pre-stained standards (lane 19) are indicated.

 
Binding assay.  The ability of the apoE variant-DMPC complex to bind to the LDL receptor on peripheral lymphocytes was approximately 80% of the ability of the normal apoE3-DMPC complex (Fig. 6Go).



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  		Fig. 6. Binding of apoE-DMPC complexes to LDL receptor on normal lymphocytes. Lymphocytes were incubated in a medium containing 1 µg protein/ml of FITC-LDL and the indicated concentration of apoE-DMPC (open circle) or apoE variant-DMPC (closed circle) complexes for 2 h at 4°C on ice. After washing the cells, the FITC-LDL bound to the cells was measured by flow-cytometry. Each value, the average of duplicate tests, is expressed as a percentage of the binding of FITC-LDL to cells without apoE-DMPC and apoE variant-DMPC complexes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We previously identified the present apoE3 variant (Arg 145-> His) in 4 subjects among 5 members of a family (all heterozygotes), and reported that all 4 subjects had high plasma apoE concentration [17]. In the present study, we characterized this apoE3 variant in two subjects (the proband and her brother) with respect to the existing form, its distribution in plasma lipoproteins, and its ability to bind to heparin and the LDL receptor.

The cause of the high plasma apoE concentration may possibly be related to the reduced ability of the apoE3 variant to bind to heparin and the LDL receptor (compared to that of the common apoE3), resulting in slower rates of catabolism of lipoproteins containing apoE. This notion is supported by the finding that the apparent concentration of the apoE3 variant (as obtained from IEF followed by immunoblotting) was significantly higher than that of normal apoE3 in both subjects. The predominant lipoprotein class containing apoE was, however, different in the two subjects: most apoE was in the slow-alpha lipoprotein fraction in the proband, but in the slow-pre-ß in her brother. The latter lipoprotein fraction could correspond to the IDL or ß-VLDL observed upon agarose gel electrophoresis in other apoE3 mutants [22,23].

Previous studies have shown a correlation between the serum total apoE level and the levels of triglyceride or HDL cholesterol [24]. Furthermore, the apoE in pre-beta lipoproteins is transferred from alpha lipoproteins with apoE [25–28]. This suggests that the low concentration of TG in the proband’s serum could be the result of a reduced transfer of apoE from slow-alpha lipoprotein to slow-pre-ß lipoprotein.

Mild hypertriglyceridemia was observed in the proband’s brother, but not in the proband. It is not obvious why this difference existed between the two subjects. Type III hyperlipoproteinemia is usually associated with homozygosity for apoE2 (Arg158 -> Cys); however, homozygosity for apoE2 is not always associated with type III hyperlipoproteinemia. Similarly, the apoE3 variant described here could sometimes, but not always, be one of the factors that induces mild type III hyperlipoproteinemia.

In recent years, several variants that induce type III hyperlipoproteinemia have been reported, including apoE variants: Arg136-> Cys [29], Arg136-> Ser [30], Arg142-> Leu [31], Arg142-> Cys [32], and Arg145-> Cys [33]. Interestingly, the apoE variant (Arg136-> His) is known to induce mild type III hyperlipoproteinemia [34]. This implies that the substitution of a weak positive charge (His) for a positive charge (Arg) has a mild effect compared to the substitution of a neutral charge (Cys) for a positive charge (Arg).

The apoE3 variant, especially the apoE3 variant-AII heterodimer, was found to bind to heparin and the LDL receptor with lower affinity than normal apoE3. This indicates that the Arg145 residue of apoE3 is an important site in the metabolism of lipoproteins. ApoE contains two binding domains for heparin, residues 142–147 and 243–272 [35,36]. When apoE forms complexes with phospholipids or materials on the surface of a lipoprotein particle, residues 142–147 appear on the surface [35]. This binding domain is consistent with the binding domain of apoE for the LDL receptor, residues 134–150. The binding of apoE to the LDL receptor and the remnant receptor is thought to be mediated by basic residues such as arginine and lysine in residues 134–150 of the apoE molecule [37]. Actually, there are 9 basic residues out of 17 among residues 134–150. Thus, the arginine at residue 145 may play a part in the binding of apoE to both heparin and the LDL receptor.

In the proband, most apoE3, including its variant, exists in the slow-alpha lipoprotein fraction in the form of disulfide-linked complexes, such as the apo(E-AII) complex and the apoE homodimer. On the other hand, in the proband’s brother, most apoE3 exists in the slow-pre-beta lipoprotein fraction as the mono-meric form. It is not clear whether this difference is the result of their different plasma triglyceride levels or the consequence of them. The increase in the slow-alpha lipoprotein fraction could reflect its role as storehouse for excess apoE in the proband (who does not seem to have a predisposition for hypertriglyceridemia). In contrast, excess apoE would accumulate in the slow-pre-beta lipoprotein fraction as TG-rich lipoprotein in her brother, who seems to have such a predisposition.

In conclusion, apoE 3 (Arg 145-> His), with slightly defective binding to heparin and LDL receptor, appears to play a significant role in the accumulation of apoE and in the expression of mild hypertriglyceridemia. However, this is not seen in every case, since hypertriglyceridemia was present in only 3 of the 4 subjects with the apoE mutation.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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H. Hidaka, M. Tozuka, B. Meyer, K. Yamauchi, M. Sugano, T. Nakabayashi, and T. Katsuyama
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