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J Am Soc Nephrol 12:524-530, 2001
© 2001 American Society of Nephrology

Diminished LDL Receptor and High Heparin Binding of Apolipoprotein E2 Sendai Associated with Lipoprotein Glomerulopathy

MICHAEL M. HOFFMANN, HUBERT SCHARNAGL, ELEFTHERIA PANAGIOTOU, WERNER T. BANGHARD, HEINRICH WIELAND and WINFRIED MÄRZ

Division of Clinical Chemistry, Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany.

Correspondence to Dr. Michael M. Hoffmann, Division of Clinical Chemistry, Medical Clinic, Albert-Ludwigs-University, Hugstetter Strasse 55, 79106 Freiburg, Germany. Phone: 49-761-270-6357; Fax: 49-761-270-6358; E-mail: mhoff{at}medl.ukl.uni-freiburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. Variants of apolipoprotein E (apoE) have been linked to lipoprotein glomerulopathy, a new glomerular disease characterized by the deposition of lipoproteins in mesangial capillaries. One third of affected patients are heterozygous for apoE2 Sendai (Arg145 Pro). Variants of apoE can also produce type III hyperlipoproteinemia (HLP). Recessive type III HLP is caused by apoE2 (Arg158 Cys), a mutant with diminished low-density lipoprotein (LDL) receptor binding but halfnormal heparin binding. Dominant type III HLP is caused by mutations that markedly alter heparin binding but modestly reduce receptor binding. This study examined whether apoE2 Sendai (Arg145 Pro) was functionally different from type III HLP-producing apoE variants by expressing apoE3, apoE2 (Arg158 Cys), apoE1 (Arg146 Glu), a dominant apoE variant, and apoE2 Sendai (Arg145 Pro) in the baculovirus system. LDL receptor binding was studied using recombinant apoE complexed to phospholipid vesicles and to very lowdensity lipoprotein from a patient with familiar apoE deficiency. Compared with apoE3, receptor-binding activities of apoE2 (Arg158 Cys), apoE1 (Arg146 Glu), and apoE2 Sendai (Arg145 Pro) all were less than 5%. Heparin-binding activities were 53%, 23%, and 66%, respectively, of apoE3. The distribution of apoE2 Sendai among the major plasma lipoprotein fractions was similar to that of apoE3 and apoE2 (Arg158 Cys). ApoE2 Sendai (Arg145 Pro) represents the only known mutation within the heparin-binding domain of apoE (residues 142 through 147), revealing diminished receptor binding and almost normal heparin binding. These unique characteristics of apoE2 Sendai (Arg145 Pro) may relate to the development of lipoprotein glomerulopathy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lipoprotein glomerulopathy (LPG) is a newly recognized disease that is characterized mainly by the deposition of lipoprotein thrombi in glomerular capillaries (1). Recently, genetic variants of apolipoprotein E (apoE) have been implicated in the pathogenesis of LPG. ApoE is a glycoprotein of 34 kD (2,3). In plasma, it is associated with triglyceride-rich lipoproteins and high-density lipoproteins (HDL). ApoE is a ligand for members of the low-density lipoprotein (LDL) receptor family of membrane proteins. The best characterized function of apoE is to mediate the uptake of chylomicron and very low-density lipoprotein (VLDL) remnants into the liver. Furthermore, apoE has been implicated in the growth and differentiation of neurons (4,5), in the modulation of the immune response, and in the regulation of platelet aggregation (6).

In humans, there are three common variants of apoE. ApoE3 is the most frequent one. ApoE4 (Cys112 Arg), the second most frequent isoform, produces moderately elevated LDL concentrations and increases the risk of atherosclerosis and Alzheimer's disease (3,7). The third most prevalent allele is apoE2 (Arg158 Cys). This variant is defective in binding to lipoprotein receptors (8,9), although the replacement of cysteine for arginine at residue 158 resides outside the putative receptor-binding domain of apoE (residues 136 through 150). Homozygous carriers of apoE2 (Arg158 Cys) exhibit "normolipidemic" dysbetalipoproteinemia, which is characterized by slightly elevated concentrations of remnants (incompletely catabolized triglyceride-rich lipoproteins). In response to further genetic or environmental factors, these individuals may develop overt type III hyperlipoproteinemia (HLP), a condition characterized by the elevation of both cholesterol and triglycerides (2,10), accumulation of remnant particles, plamar xanthoma, and rapidly progressive atherosclerosis.

In rare cases, type III HLP has been found in individuals who are heterozygous rather than homozygous for apoE mutations. These dominant variants include apoE2 (Lys146 Gln), apoE3 (Cys112 Arg, Arg142 Cys), apoE4 (Glu13 Lys, Arg145 Cys), apoE2 (Arg145 Cys), apoE1 (Lys146 Glu), and apoE1 (Lys146 Asn, Arg147 Trp) (11). Clinically, these dominant variants show a high degree of penetrance. The common feature of the dominant mutations is that they involve substitutions of the basic residues at positions 142, 145, 146, and 147. These amino acids all are located within the receptor-binding domain (residues 136 through 150) of apoE and within the first heparin-binding domain (residues 142 through 147) of the molecule (12). The apoE variants that confer dominant type III HLP exhibit moderate reduction of LDL receptor binding along with markedly reduced binding to cell surface heparan sulfate proteoglycans (HSPG; 10% or less of normal) (10,13). This stands in contrast to the recessive apoE2 (Arg158 Cys), which possesses significant residual heparin binding (50% or more) but has less than 2% of normal LDL receptor-binding activity. Binding to heparin thus functionally distinguishes recessive (residual heparin binding) and dominant (defective in heparin binding) apoE variants.

ApoE concentrations are elevated in both LPG and type III HLP (10,14). This raises the possibility that the two entities are caused by similar molecular mechanisms. However, important differences between these diseases exist. Hyperlipidemia has not been observed consistently in LPG patients without nephrotic syndrome. Patients with LPG do not reveal the characteristic systemic manifestations of type III HLP (atherosclerosis or palmar xanthoma). Neither recessive nor dominant type III HLP appears to produce LPG. Finally, mesangial lesions are different. In LPG, lipoprotein deposition occurs within the lumen of the capillaries. In type III HLP, in contrast, lipids may accumulate in mesangial foam cells.

Among the genetic variants linked to LPG, apoE2 Sendai (Arg145 Pro) seems to be the most frequent one (15). None of the LPG patients bearing apoE2 Sendai (Arg145 Pro) was homozygous, suggesting that the variant dominantly causes LPG. Despite being located in the receptor- and heparin-binding domain of apoE, however, this mutation does not seem to lead to a predisposition for dominant type III HLP (14). To resolve this paradox and to approach the question of whether the molecular mechanisms that lead to LPG are distinct from those that underlie either dominant or recessive type III HLP, we obtained functional, recombinant apoE2 Sendai (Arg145 Pro) and characterized the protein with respect to LDL receptor binding and heparin binding, as well as its presence within the major plasma lipoprotein classes. Our results demonstrate that apoE2 Sendai (Arg145 Pro) showed dramatically impaired binding to the LDL receptor but only moderately diminished binding to heparin (66% of apoE3). Therefore, apoE2 Sendai (Arg145 Pro) is the first apoE variant that involves one of the positively charged amino acids of the first found heparin-binding domain that exhibits only moderately reduced heparin binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Site-Specific Mutagenesis of ApoE cDNA
To obtain apoE2 Arg145 Pro, we carried out site-directed mutagenesis of the apoE3 cDNA by using the splicing overlap extension method (16). The template for the PCR was pAc-E3 (kindly provided by Dr. Alan D. Attie, University of Madison, Madison, WI). The PCR product was subcloned into the pCRII vector (Invitrogen, Groningen, The Netherlands). pCRII harboring cDNA for apoE2 Arg145 Pro was double digested with NotI and FseI (New England Biolabs, Beverly, MA) to produce a 260-bp apoE cDNA fragment containing the mutant site. This fragment replaced the wild-type NotI-FseI fragment of apoE3 in a pGEM-apoE3 cDNA clone. After digestion of pGEM-apoE2 Arg145 Pro with BamHI, the mutant cDNA was inserted into the baculovirus transfer vector pAc-E3, replacing the apoE3 cDNA. The sequence of the mutant apoE cDNA constructs was verified by DNA sequencing. ApoE1 (Lys146 Glu), one of the dominant variants (17), was cloned using a similar strategy.

Expression and Purification of ApoE
Cotransfection of Bac3000 baculovirus DNA (Novagen, Madison, WI) and pAcYM1-apoE was performed using N[1-(2,3-Dioleoyloxy)propyl)]-N,N,N,trimethyl-ammonium propane (DOTAP; Roche, Diagnostics, Mannheim, Germany) as transfection reagent. Sf21 cells (3 x 106) were seeded in a 60-mm tissue culture plate. After 15 min, the medium was exchanged against 1 ml of fresh medium (Sf900II, Life Technologies, Eggenstein, Germany). Linearized baculovirus DNA (0.1 µg) and 0.5 µg recombinant plasmid DNA were diluted to a concentration of 0.1 µg/µl in 20 mmol/L HEPES buffer (pH 7.4). Fifteen µl of DOTAP were mixed with 35 µl of HEPES buffer; the DNA was added to the DOTAP solution and the mixture was incubated for 15 min at room temperature. The resulting transfection solution was added dropwise to the cells. After 6 h of incubation at 27°C, 3 ml of fresh Sf900II medium was added to the cells. At day 4 after transfection, the supernatant was collected and single virus clones were isolated. To produce recombinant apoE on a large scale, we seeded 2 x 106 Sf21 cells/ml in a spinner flask. The cells were infected with a high-titer stock solution of recombinant baculovirus. The multiplicity of infection was 10. Addition of leupeptin (0.5 mg/L) and pepstatin A (0.7 mg/L) to the culture medium 24 h postinfection improved the yield of apoE by approximately 20%. After incubation at 27°C for 72 to 96 h, the apoE-containing supernatant was collected by centrifugation and filtration. Before storage (-80°C) or purification, a protease inhibitor cocktail was added at final concentrations of 32 mg/L benzamidine-HCl, 20 mg/L aprotinin, 10 mg/L leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride.

Purification of Recombinant ApoE
In our initial protocol for the purification of recombinant apoE2, apoE3, apoE4, and apoE2 (Arg136 - >Cys) (18), we used a (NH4)2CO3 gradient to elute apoE from the column. Briefly, solid (NH 4)2CO3 was added to the cell culture supernatant to yield a final concentration of 25 mmol/L. Cell culture supernatant (250 ml) was circulated overnight on a heparin-Sepharose B6 column (C10/20) equilibrated with 25 mmol/L (NH4)2CO3. The column was washed with 15 vol of 25 mmol/L (NH4)CO3 and 10 vol of 300 mmol/L (NH4)2CO3. ApoE was eluted with 700 mmol/L (NH4)2CO3, dialyzed against 25 mmol/L (NH4)2CO3, and rechromatographed under identical conditions. Be - cause of the weak heparin-binding properties of apoE1 (Lys146 Glu), most of the protein eluted during the second wash. We therefore changed the second washing step to 10 vol of 150 mmol/L (NH4)2CO3 and eluted the protein at 300 mmol/L (NH 4)2CO3.

In the case of apoE2 Sendai (Arg145 Pro), the use of (NH4)2CO3 buffer resulted in the precipitation of the recombinant protein on the column. We therefore used KH2PO4 buffer and developed the column with a sodium chloride gradient. In detail, solid NaCl was added to the cell culture supernatant to yield a final concentration of 25 mmol/L. Cell culture supernatant (250 ml) was circulated overnight on a heparin-Sepharose 6B column (C10/20) equilibrated with 25 mmol/L NaCl and 20 mmol/L KH2PO4 (pH 6.5). The column was washed with 15 vol of 25 mmol/L NaCl and 20 mmol/L KH 2PO4 (pH 6.5) and was developed with a linear gradient of sodium chloride (25 mmol/L to 1 mol/L). To purify further the recombinant apoE variants, apoE-containing fractions were concentrated, using Centricon Plus-20 (Millipore, Bedford, MA) spin columns and applied to a Sephadex G-75 (Amersham Pharmacia Biotech, Uppsala, Sweden) column (2.6 x 50 cm). The elution buffer was chosen according to the requirement of the experiments to follow, e.g., 0.1 M NH4HCO3 (pH 8.1) was used when we prepared dimyristoylphosphatidyl-choline (DMPC) vesicles containing apoE.

Lipoproteins and Lipoprotein-Deficient Serum
VLDL (d < 1.006 kg/L) were isolated from the plasma of a patient with familial apoE deficiency and type III HLP (19) by preparative ultracentrifugation. VLDL were associated with recombinant apoE by incubation for 1 h at 37°C at a molar ratio of VLDL-apoB to recombinant apoE of 1:4. Human LDL (1.030 kg/L to 1.050 kg/L) were isolated from pooled plasma of normolipidemic donors. Lipoproteins were labeled with 125I using iodine-monochloride as oxidizing agent (20). Human lipoprotein-deficient serum (LPDS) was prepared by ultracentrifugation as described and stored at -20°C (21).

Preparation of ApoE-DMPC Complexes
Recombinant apoE isoforms were reconstituted with DMPC essentially as described (18,22). DMPC (15 mg) was suspended in 1 ml of 1 mmol/L Tris-HCl, 10 mmol/L ethylenediaminetetraacetate 160 Na2 (pH 7.6), and 150 mmol/L NaCl, incubated for 30 min at room temperature, and sonicated. Recombinant apoE isoforms (100 µg of each in 1.05 ml of 0.1 M NH4HCO3 [pH 8.1]) were incubated overnight with 50 µl of the DMPC suspension. The resulting apoE-DMPC complexes were isolated by preparative ultracentrifugation at a density of 1.25 kg/L.

Binding of 125 I-Labeled Lipoproteins
Human skin fibroblasts were from skin biopsies of healthy, normolipidemic individuals. The cells were grown in 24-well polystyrene plates containing Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum in a humidified incubator at 5% (vol/vol) CO2 and 37°C. Before the experiments, the cells were preincubated for 40 h in medium containing 10% (vol/vol) human LPDS to upregulate LDL receptors. Cellular binding of 125I-labeled VLDL and 125I-labeled LDL were measured by incubating the cells for 1 h at 4°C as described by Goldstein et al. (21) with slight modifications (18).

Heparin Binding of ApoE
Heparin binding was determined using the procedure of Mann et al. (13). ApoE-DMPC complexes were incubated with 20 mg of heparin-Sepharose or 20 mg Sepharose, respectively, for 4 h on an overhead shaker in 200 µl of Tris buffer (20 mmol/L Tris/HCl [pH 7.5], 50 mmol/L NaCl, 1% bovine serum albumin) containing 10 µg of protein. After centrifugation, the pellet was washed three times with Tris buffer, and the heparin-Sepharose-associated radioactivity was counted. Results were corrected for VLDL binding to Sepharose alone and expressed as nanograms of VLDL protein per milligram of heparin-Sepharose.

Distribution of ApoE among Plasma Lipoproteins
Iodinated apoE isoforms (0.5 µg, specific activity 60 to 80 cpm/ng protein) were incubated with plasma (500 µl) from a pool of normolipidemic, apoE3/3 homozygous donors for 1 h at 37°C. Samples (50 µl) of the incubation mixtures were applied to Superose 6 (Amersham Pharmacia Biotech) columns (300 mm) equilibrated with 100 mmol/L Na2HPO4 (pH 7.4) and 200 mmol/L NaCl. The eluent flow rate was 20 ml/h, and 0.7-ml fractions were collected (23). The radioactivity was determined in each fraction, and the relative distribution was calculated as a percentage of total radioactivity.

Statistical Analyses
Differences in cellular binding, uptake, and degradation as well as in heparin binding were examined for statistical significance using the unpaired t test. P values less than 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Receptor Binding of ApoE2 Sendai (Arg145 Pro)
Recombinant apoE was obtained using the baculovirus expression system (Figure 1 shows a sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the purification steps of recombinant apoE Sendai) (18). We compared the LDL receptor-binding activity of DMPC particles containing recombinant apoE3, apoE2 (Arg158 Cys), apoE1 (Lys146 Glu) (17), and apoE2 Sendai (Arg145 Pro) in cultured human fibroblasts. Relative binding activities were derived from the apoE concentrations required to displace one half of iodinated LDL from receptor binding. As expected, binding activity of apoE2 (Arg158 Cys) was significantly lower than that of apoE3 (Figure 2). The ability of apoE2 Sendai (Arg145 Pro) to displace iodinated LDL from receptor binding was similar to apoE2 (Arg158 Cys) and apoE1 (Lys146 Glu), the residual receptor binding of all three isoforms being less than 5% compared with apoE3 (Figure 2; P < 0.001).



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  		Figure 1. Purification steps of recombinant apolipoprotein E (apoE) Sendai. Coomassie blue stained 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant apoE was purified from 100 ml of supernatant of Sf21 cells transfected with recombinant apoE Sendai viruses (lane 1). In a first step, apoE was bound on a heparin-Sepharose column and eluted with a linear NaCl gradient as described in the Materials and Methods section (lane 2). For the final purification step, apoE Sendai was applied to a Sephadex G75 column.

 


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  		Figure 2. Receptor-binding activity of various apoE isoforms. Recombinant apoE3 ({blacksquare}), apoE2 (Arg158 Cys) ([UNK]), apoE-Sendai ({blacktriangleup}), and apoE1 (Lys146 Glu) ({diamondsuit}) were incorporated into dimyristoylphosphatidyl-choline (DMPC) particles. Human skin fibroblasts were grown in 24-well polystyrene plates and incubated for 40 h with medium containing 10% (vol/vol) lipoprotein-deficient serum (LPDS). The cells received 5 mg/l 125I-labeled low-density lipoprotein (LDL) and apoE-DMPC complexes at concentrations indicated for 1 h at 4°C. The average of control incubations without apoE-DMPC complexes was 68 ng of LDL protein/mg cell protein (100%). Each data point represents the average of two independent experiments, each performed in triplicate.

 

DMPC vesicles are artificial particles and may not reflect the conditions in vivo, especially because they do not contain any apolipoproteins other than apoE. Therefore, as a more physiologic model, we also used VLDL from an individual who was deficient in apoE as a result of a homozygous 10-bp deletion, which introduces a premature stop at codon 229 of the apoE mRNA (19). VLDL from this patient were loaded with recombinant apoE isoforms. Binding data obtained with these particles were in good agreement with the experiments using DMPC vesicles. Addition of recombinant apoE3 resulted in an approximately threefold increase in binding compared with apoE2 (Figure 3). There were no significant differences between the binding of VLDL complexed with apoE2 (Arg158 Cys), apoE1 (Lys146 Glu), and apoE2 Sendai (Arg145 Pro) (Figure 3).



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  		Figure 3. Binding of apoE-deficient very low-density lipoprotein (VLDL) enriched with apoE. Human skin fibroblasts were preincubated for 40 h with medium containing 10% (vol/vol) LPDS. The cells then received 125I-labeled VLDL supplemented with apoE3 ({blacksquare}), apoE2 (Arg158 Cys) ([UNK]), apoE-Sendai ({blacktriangleup}), and apoE1 (Lys146 Glu) ({diamondsuit}) for 1 h at the indicated concentrations. Cellular binding at 4°C was measured as described in the Materials and Methods section and was adjusted for nonspecific binding. Each data point represents the average of two independent experiments, each performed in triplicate.

 

Heparin Binding of ApoE2 Sendai (Arg145 Pro)
Variants of apoE associated with the dominant expression of type III HLP usually display higher receptor-binding activity than apoE2 (Arg158 Cys), but they bind less effectively to heparin and cell surface HSPG (10,13,18). Therefore, we measured the interaction of DMPC vesicles containing recombinant apoE isoforms with heparin-Sepharose in vitro. On the average of three different concentrations of apoE containing DMPC vesicles tested in three independent experiments (each in quadruplicate), the heparin-binding activities of apoE2 (Arg158 Cys), apoE1 (Lys146 Glu), and apoE2 Sendai (Arg145 Pro) were 53%, 23%, and 66% of normal, respectively (Figure 4). The differences between the three apoE variants and wild-type apoE as well as and the differences between each of the variants all were statistically significant (P < 0.001).



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  		Figure 4. Heparin binding of recombinant apoE variants. Recombinant 125I-labeled apoE3 ({blacksquare}), apoE2 (Arg158 Cys) ([UNK]) apoE-Sendai ([UNK]), and apoE1 (Lys146 Glu) ({square}) were incorporated into DMPC vesicles and incubated at a concentration of 5 mg/L with heparin-Sepharose or Sepharose, respectively. Each bar represents the average (±SD) of three independent experiments, each performed in quadruplicate. Results were corrected for binding to Sepharose alone.*, t test (P < 0.05).

 

Distribution of ApoE2 Sendai (Arg145 Pro) among Plasma Lipoproteins
To examine the distribution of apoE2 Sendai (Arg145 Pro) among plasma lipoproteins, we incubated iodinated apoE isoforms with plasma of normolipidemic apoE3/3 homozygous donors. Plasma lipoproteins were separated by gel chromatography, and the amount of radioactivity in each lipoprotein fraction was determined. ApoE2 Sendai (Arg145 Pro) preferentially associated with HDL particles, in a way similar to apoE2 and apoE3, whereas apoE4 mainly associated with VLDL particles (Figure 5). These data are in good agreement with the observation of Saito et al. (14) suggesting that most of apoE2 Sendai (Arg145 Pro) in plasma was found in the HDL fraction.



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  		Figure 5. Distribution of apoE variants among plasma lipoproteins. 125I-labeled apoE3 ({square}), apoE2 (Arg158 Cys) ({circ}), apoE4 (Cys112 Arg) ({diamond}), and apoE Sendai ({triangleup}; 0.5 µg) were incubated with plasma (500 µl) for 1 h at 37°C. The plasma was then applied to Superose 6 columns (300 mm). The eluent flow rate was 20 ml/h, and 0.7-ml fractions were collected. The radioactivity was determined in each fraction, and the relative distribution was calculated as a percentage of total radioactivity. Arrows indicate the elution position of VLDL, LDL, and HDL.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ApoE2 Sendai (Arg145 Pro) was first identified in a patient who had lipoprotein glomerulopathy (1,24). LPG is characterized by deposits of apoE- and apoB-containing lipoproteins in the lumen of mesangial capillaries. The clinical symptoms of LPG vary from mild proteinuria to nephrotic syndrome and chronic renal failure. LPG recurs in transplanted kidneys (25,26,27), implicating systemic rather than renal factors in the development of the disease. Until now, a total of 34 cases of LPG have been reported, predominantly in Asians: 27 in Japanese (24,25,28,29,30,31,32,33), 5 in Chinese (14,34,35), and 2 in French patients (36,37). At least 16 patients were heterozygous carriers of rare apoE variants. Among these, apoE2 Sendai (Arg145 Pro) was the most frequent one (11 patients) (15). Other variants found in patients with LPG are apoE2 Kyoto (Arg25 Cys) (31), apoE1 Tokyo (141 to 143 del) (30), apoE1 Maebashi (142 to 144 del) (33), apoE5 (Glu3 Lys) (25), and apoE1 (156 to 173 del) (32). Together, these findings suggest a role of apoE mutations in the development of LPG.

ApoE variants also produce dysbetalipoproteinemia or type III HLP. However, LPG and type III HLP are clinically dissimilar. Patients who have LPG but are not experiencing nephrotic syndrome may be normolipidemic. LPG has not been observed consistently in recessive or dominant type III HLP. Renal lesions in type III HLP contain mesangial foam cells rather than intracapillary thrombi. The molecular mechanisms that link LPG to apoE variants thus might differ from those that cause type III HLP.

This study provides the first comprehensive functional characterization of an apoE variant associated with LPG. In comparison to apoE3, apoE2 Sendai (Arg145 Pro) displayed impaired binding (<5%) to the LDL receptor, similar to apoE2 (Arg158 Cys) and apoE1 (Lys146 Glu), which both can be responsible for type III HLP. This is not surprising as Arg145 is part of the receptor-binding domain of apoE. The importance of residue 145 to LDL receptor binding is demonstrated by two other naturally occurring mutations at this position: apoE Kochi (Arg145 His) (38) and apoE2 (Arg145 Cys) (39), which is associated with dominant type III HLP (40). Whereas these two mutations lead only to the loss of one positive charge, the replacement of arginine 145 by proline may have a stronger impact on the structure of apoE because proline is known as a helix breaker in globular proteins (41). This is supported by Lalazar et al. (22), who designed an apoE variant in which proline was substituted for leucine 144. Similar to apoE2 Sendai (Arg145 Pro), this variant was functionally defective, exhibiting 13% of normal binding to the LDL receptor.

Differences in heparin binding functionally distinguish dominant from recessive apoE variants (10,13). Recently, we showed that the replacement of cysteine for arginine at position 136 of apoE, albeit located in the putative receptor-binding domain of the molecule, resulted in only moderate reduction of heparin binding (61% of normal), thus explaining that hyperlipidemic heterozygotes for apoE2 (Arg136 Cys) did not reveal type III HLP (18). Usually, dominant apoE variants display higher receptor-binding activity than apoE2 (Arg158 Cys), but they bind less effectively to heparin and cell surface HSPG (10,13). In comparison to apoE3, the heparin-binding activity of apoE2 Sendai (Arg145 Pro) was 66%, which is significantly more than the heparin-binding activity of the dominant variant apoE1 (Lys146 Glu) (23%) and even exceeds that of the recessive variants apoE2 (Arg158 Cys) (53%) and apoE2 (Arg136 Cys) (61%) (18). This finding was entirely unexpected. Residue 145 lies within the first heparin-binding domain of apoE (amino acids 142 and 147) (12). Therefore, it is not clear how the replacement of arginine 145 by proline results only in a moderate distortion of the heparin-binding activity of the molecule. One conceivable explanation for this paradox is that the introduction of the {alpha}-helix—breaking proline residue induces a conformational rearrangement profound enough to generate a novel heparin domain. However, regardless of the underlying mechanism responsible for the unforeseen high heparin binding of apoE2 Sendai (Arg145 Pro), the results of our in vitro studies are completely consistent with the quasirecessive rather than dominant inheritance of hyperlipoproteinemia encountered clinically in apoE2 Sendai (Arg145 Pro) families (27,42).

Our results indicate that apoE2 Sendai (Arg145 Pro) behaves distinctly from normal apoE and from mutants that produce recessive or dominant type III HLP. Recessive apoE2 (Arg158 Cys) displays a marked reduction of receptor binding and half-normal heparin binding. Dominant apoE mutants are defective in binding to both LDL receptors and heparin. LDL receptor binding of apoE2 Sendai (Arg145 Pro) is diminished as well, but it binds significantly more strongly to heparin than apoE2 (Arg158 Cys), despite the loss of a positive charge within a heparin-binding domain. These unique functional properties may explain why apoE2 Sendai (Arg145 Pro) specifically predisposes to LPG.

The question of whether other apoE mutants that are implicated in the causation of LPG share these characteristics is difficult to answer because none of these variants has been examined with regard to receptor and heparin binding. It is intriguing that apoE mutations associated with LPG seem to cluster within or close to the receptor binding site. There are three exceptions to this, namely apoE2 Kyoto (Arg25 Cys), apoE5 (Glu3 Lys), and apoE1 (156 to 173del) (25,31,32). At least apoE2 Kyoto has been shown to be defective in binding to the LDL receptor (31) as well, possibly because of the removal of an interhelical salt bridge between Arg25 (helix 1) and Glu70 (helix 2) (31). Whether the heparin-binding activities of apoE2 Kyoto (Arg25 Cys) or one of the other apoE mutants are similar to those of apoE2 Sendai (Arg145 Pro) remains an open question.

ApoE mRNA has been found to be expressed in the normal kidney (43). Saito and colleagues (14,44) proposed that the glomerular synthesis of apoE contributes to the development of LPG. It is not yet known whether LPG is accompanied by quantitative changes in the renal expression of apoE. However, renal production of normal quantities of mutant apoE still could account for the development of LPG. In this context, impaired receptor-mediated catabolism paired with high heparin binding might decrease the clearance and enhance the retention of apoE2 Sendai (Arg145 Pro) within the glomerular microenvironment.

In conclusion, the present study demonstrates that apoE2 Sendai (Arg145 Pro) has functional characteristics distinct from apoE mutants that are linked to type III HLP. Our findings may provide an explanation for the specific pathology of LPG. Further detailed investigation is now needed to examine whether apoE2 Sendai (Arg145 Pro) and other apoE variants found in patients with LPG have something in common and how these variants cause the pathologic features that define LPG.


   Acknowledgments
 
This study was supported by the Center of Clinical Research II, project D5, Albert-Ludwigs-University, Freiburg, Germany.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Saito T, Sato H, Kudo KI, Oikawa SI, Shibata T, Hara Y, Yoshinaga K, Sakaguchi H: Lipoprotein glomerulopathy: Glomerular lipoprotein thrombi in a patient with hyperlipoproteinemia. Am J Kidney Dis13 : 148-153,1989[Medline]
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Received for publication June 6, 2000. Accepted for publication August 16, 2000.




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