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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1240-1247.)
© 1995 American Heart Association, Inc.

Articles

Apolipoprotein B and E Basic Amino Acid Clusters Influence Low-Density Lipoprotein Association with Lipoprotein Lipase Anchored to the Subendothelial Matrix

Uday Saxena; Bruce J. Auerbach; Erika Ferguson; Joachim Wölle; Yves L. Marcel; Karl H. Weisgraber; Robert A. Hegele; Charles L. Bisgaier

From the Department of Atherosclerosis Therapeutics, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co, Ann Arbor, Mich (U.S., B.J.A., E.F., J.W., C.L.B.); University of Ottawa (Ontario, Canada) Heart Institute (Y.L.M.); Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, Department of Pathology, University of California–San Francisco (K.H.W.); and St Michael's Hospital, Division of Endocrinology and Metabolism, Toronto, Ontario, Canada (R.A.H.).

Correspondence to Uday Saxena, PhD, Atherosclerosis Therapeutics, Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd, Ann Arbor, MI 48105.

	Abstract

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Abstract Lipoprotein accumulation in the subendothelial matrix is an important step in atherogenesis. We have previously shown that addition of lipoprotein lipase (LPL) markedly increased binding of apolipoprotein B (apoB)–containing lipoproteins to an endothelial cell–derived matrix, and this enhanced lipoprotein binding was inhibited by apoE. In the present studies we examined the role of various regions of apoB in the binding of LDL to LPL-containing endothelial cell matrix and the ability of various apoE domains to decrease lipoprotein retention. We studied three apoB epitope-specific monoclonal antibodies for their ability to block the binding of ¹²⁵I-LDL to LPL-containing matrix. Of these, monoclonal antibody 4G3, which recognizes an arginine-containing epitope in apoB, was the most effective in reducing LDL binding. Chemical modification of LDL apoB lysines or arginines markedly reduced the ability of the lipoprotein to block the binding of ¹²⁵I-LDL to LPL-containing matrix, suggesting that apoB positively charged amino acids are involved in the interaction. Furthermore, polyarginine or polylysine markedly decreased ¹²⁵I-LDL binding to LPL-containing matrix, whereas polyleucine was ineffective. These data suggest that apoB positively charged regions are important in LDL binding. To explore the role of charge modifications on apoE by single arginine-cysteine interchanges, we examined the effects of the three major human apoE isoforms (apoE2, apoE3, and apoE4). ApoE3 was the most effective in decreasing ¹²⁵I-LDL retention, followed by apoE4; apoE2 was the least effective. Similarly, apoE2-containing HDL was much less effective than apoE3-containing HDL in decreasing ¹²⁵I-LDL retention. Therefore, both cysteine for arginine substitutions at amino acids 112 and 158, known to markedly reduce apoE binding to the LDL receptors, also had significant effects on the ability of this apoE isoform to displace LDL bound to LPL. Two peptides generated by thrombin cleavage of apoE3 both were able to decrease ¹²⁵I-LDL binding, indicating the presence of multiple sites within apoE that could participate in the inhibitory effect. We conclude that positively charged regions on apoB are responsible for the binding of LDL to LPL-containing matrix and that similar regions of positive charge in apoE allow it to compete and decrease the retention of LDL.

Key Words: lipoprotein lipase • LDL • subendothelial matrix • apo E • lipoprotein retention

	Introduction

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Atherosclerosis is a progressive disease involving both the vessel wall and plasma components. Developmentally, this process is complex and includes (1) infiltration of apoB-containing lipoproteins into the subendothelium and their matrix binding, (2) oxidative modifications of apoB-containing lipoproteins, resulting in their uptake by scavenger receptors on smooth muscle cells and macrophages transforming them to foam cells, (3) adhesion and transmigration of blood-borne monocytes through the arterial endothelium and their differentiation to macrophages, (4) cytokine-induced proliferation of smooth muscle cells, and (5) the eventual subendothelial accumulation of extracellular crystalline cholesterol and calcium deposits, presumably resulting from foam cell necrosis.^{1 2 3} This process eventually leads to raised lesions that may rupture, initiating a thrombogenic event.

An early stage of this process may include apoB-containing lipoprotein (VLDL, ß-VLDL, IDL, and LDL) binding to LPL, which is anchored to specific sulfate proteoglycan components (ie, heparan sulfate and dermatan sulfate) of the subendothelial matrix.⁴ Access to these matrix components by larger apoB-containing lipoproteins (VLDL and ß-VLDL) is likely facilitated under conditions in which the endothelium is compromised, such as at the edges of progressing lesions or those lesions induced procedurally (eg, balloon angioplasty). The trapping of apoB-containing lipoproteins within the subendothelial microenvironment may predispose the lipoproteins to oxidative modification.⁵

The inhibitory role that apoE and apoE-rich HDL play in atherosclerosis development has been demonstrated in a variety of models. Badimon et al⁶ have shown that intravenous injections of HDL that contained apoE caused regression of preestablished atherosclerotic lesions in cholesterol-fed rabbits. Yamada et al⁷ have shown that atherosclerotic lesion progression proceeds at a significantly reduced rate on apoE infusion in Watanabe heritable hyperlipidemic rabbits despite the absence of a change in plasma lipid levels. More recently, Shimano et al⁸ have shown that diet-induced atherosclerosis is inhibited in transgenic mice in which there is high expression of apoE localized within the artery wall. These data suggest a direct effect of apoE at the lesion site. Species such as rats, which have high amounts of apoE-rich HDL,⁹ are resistant to diet-induced atherosclerosis.¹⁰ Likewise, conditions such as human cholesteryl ester transfer protein deficiency are characterized by elevations in plasma HDL, apoE, and apoE-rich HDL and are also accompanied by an absence of coronary artery disease.^{9 11 12 13 14 15} Epidemiological studies have also suggested an inverse correlation between plasma levels of HDL₂, a subclass that contains apoE, and the severity of coronary and peripheral atherosclerosis.¹⁶ Mice made deficient in apoE by gene targeting develop severe atherosclerosis.^{17 18} In these mice prominent monocyte adhesion to lesion sites and high titers of mouse plasma antibodies to oxidized lipoprotein were demonstrable.¹⁹ Other studies have suggested that elevation of apoA-I in apoE knockout mice prevents or retards atherosclerosis, possibly through mechanisms that prevent the oxidation of LDL.^{20 21} This antiatherosclerotic effect occurs despite a lack of competition with apoE for apoB-containing lipoprotein binding to matrix LPL, which suggests that the antiatherosclerotic effects of HDL are not restricted to apoE-containing HDL fractions. Possibly, the rise in apoA-I in these mice results in an enhanced antioxidant activity associated with elevated HDL.²² Several epidemiological studies have suggested that the plasma level of apoE-poor HDL (HDL₃) is inversely related to the frequency of coronary heart disease.^{23 24 25} Perhaps the antiatherosclerotic effects of each of the major HDL subfractions affect different aspects of atherosclerosis. ApoE-poor HDL may largely contribute to reverse cholesterol transport,^{26 27} and apoE-rich HDL may affect lipoprotein retention.²⁸ Our previous in vitro data showed that binding of apoB-containing lipoproteins to LPL anchored to the subendothelium is inhibited by apoE and apoE-rich HDL.²⁸ Although the mechanism leading to increased lipoprotein oxidation in apoE-deficient mice is not clear, extrapolation of our previous in vitro data to an in vivo situation raises the possibility that trapping of apoB-containing lipoproteins is unchallenged and could result in enhanced lipoprotein oxidation.

Previous results have also shown that sulfated polysaccharides and proteoglycans can bind apoB-containing lipoproteins in the presence of divalent cations.^{29 30} The binding of the lipoproteins is mediated by positively charged regions of lipoproteins with the negatively charged carboxyl and sulfate moiety of polysaccharides.³⁰ Thus, the interactions between proteoglycans and lipoproteins are believed to be ionic in nature.

The nature of the chemical interactions between apoB, apoE, and LPL that results in modulating LDL binding to LPL in the subendothelial cell matrix is not known. Therefore, in the current study we focused on identifying the chemical basis for these interactions. Using epitope-specific Mabs to apoB; apoB chemical modification; synthetic peptides; purified apoE2, E3, or E4; thrombin-cleaved apoE3 peptides; and apoE2- or apoE3-containing HDL, we have identified the chemical basis for the interaction between apoB and apoE with subendothelial cell matrix–bound LPL.

	Methods

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Materials
Polylysine, polyleucine, and polyarginine (30– to 40–amino acid peptides) were obtained from Sigma Chemical Co. Tissue culture media, media supplements, and phosphate-buffered saline were purchased from GIBCO Laboratories. Fetal calf serum was purchased from HyClone Laboratories, Inc.

Plasma Lipoprotein Isolation and Radioiodination
LDL (d=1.019 to 1.063 g/mL) and HDL (d=1.063 to 1.21 g/mL) were isolated by sequential isopycnic centrifugation of plasma obtained from healthy human volunteers.³¹ LDL was radioiodinated by the iodine monochloride technique.^{32 33} Radioiodinated LDL (¹²⁵I-LDL) was separated from unincorporated ¹²⁵I by PD-10 gel filtration (Pharmacia Fine Chemicals Inc). The specific radioactivity of ¹²⁵I-LDL preparations ranged from 450 to 600 cpm/ng protein. More than 90% of the radioactivity associated with LDL was precipitable with 20% trichloroacetic acid, and less than 5% of the radioactivity was lipid associated (ie, chloroform extractable).

ApoE-Containing HDL Isolation
HDL from humans homozygous for apoE2 or apoE3 was used to isolate apoE-rich and apoE-poor HDL by Affi-Gel Heparin chromatography (Bio-Rad Laboratories) as described previously.^{28 34} Briefly, total HDL was dialyzed against 2 mmol/L sodium phosphate, pH 7.4, and applied to an Affi-Gel Heparin column equilibrated with the same buffer. Fractions were sequentially eluted with 2 mmol/L sodium phosphate, pH 7.4 (unbound); 50 mmol/L NaCl in 2 mmol/L sodium phosphate, pH 7.4 (apoE-poor HDL); and finally 500 mmol/L NaCl in 2 mmol/L sodium phosphate, pH 7.4 (apoE-rich HDL). Fractions were dialyzed and concentrated in 2 mmol/L sodium phosphate, pH 7.4, with the use of Centriflo CF25 ultrafiltration membrane cones (Amicon Division, WR Grace & Co). ApoE-rich HDL (apoE3) radioiodination (¹²⁵I-apoE–rich HDL) was performed as described previously.²⁸

Bovine Milk LPL Purification
Bovine milk LPL was purified from fresh unpasteurized milk with Affi-Gel Heparin chromatography (Bio-Rad Laboratories) as previously described.³⁵ With the use of triolein emulsions as substrate, lipase preparations typically had activities of 25 to 40 mmol free fatty acid released per hour per milligram of protein. LPL radioiodination was performed exactly as described before.³⁵

Protein Determination
Protein was determined with the method of Lowry et al³⁶ with the use of BSA as a standard.

Aortic Endothelial Cell Culture and LDL Retention Studies
Porcine aortic endothelial cells were isolated and cultured to confluence, and subendothelial cell matrix was prepared as previously described.³⁵ The matrix was washed four times with DMEM containing 3% BSA (DMEM-BSA). LPL (8 µg/mL) was then added in DMEM-BSA and the matrix incubated at 4°C for 2 hours. After this incubation the matrix was washed three times with DMEM-BSA to remove any unbound LPL. Next, ¹²⁵I-LDL (2.5 or 10 µg/mL LDL protein) or ¹²⁵I-apoE–rich HDL (12.5 or 100 µg/mL total HDL protein) in DMEM-BSA was added to the LPL-containing matrix alone or in the presence of apolipoproteins, polypeptides, or lipoproteins. The matrix was incubated for 1 hour at 37°C, the media removed, and the matrix washed three times with DMEM-BSA. Finally, the amount of ¹²⁵I-LDL or ¹²⁵I-apoE–rich HDL retained by the matrix was determined by the addition of 50 U heparin to the matrix as described previously.²⁸

Apolipoprotein Purification
ApoE isoforms were purified as described.³⁷ Thrombin digestion of apoE3 and isolation of the 10- and 22-kD peptide products were performed as previously described.³⁸

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis
LPL and apolipoprotein purity and protein composition of lipoproteins were assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.³⁹ The apoE content of HDL preparations was estimated by densitometric scanning of the gels.

LDL Chemical Modifications
1,2-Cyclohexanedione modification of apoB arginine residues and reversal and acetic anhydride modification of apoB lysine residues were performed as described previously.^{40 41} Reductive methylation of LDL was also performed as described previously.⁴⁰ Modified lipoproteins were used within 2 days of preparation. Agarose gel electrophoresis of LDL preparations was performed with Titan gels (Helena Laboratories).

Mabs to LDL
The production and characterization of the Mabs to apoB—4G3, 2D8, and 5E11—have been described previously.^{42 43} The IgG subclass containing the anti-LDL antibodies was isolated from the ascites fluid with a protein-A Sepharose column and used in the experiments. ¹²⁵I-LDL (2.5 µg/mL) was incubated with the Mabs at either 1 or 10 µg/mL concentration overnight at 4°C and then added to LPL-containing matrix for study of the retention of the lipoprotein.

	Results

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Mabs to ApoB Block LDL Retention by LPL-Treated Subendothelial Cell Matrix
Our prior studies demonstrated that bovine LPL treatment of porcine subendothelial matrix markedly enhanced ¹²⁵I-LDL binding.²⁸ With the use of a fixed LPL concentration, ¹²⁵I-LDL binding was shown to be saturable and displaceable by nonradioactive LDL, free apoE, liposome-bound apoE, and apoE-rich HDL. With the use of this same system, apoB epitope–specific Mabs (5E11, 4G3, or 2D8) were preincubated with ¹²⁵I-LDL, and retention by LPL-treated matrix was determined (Fig 1). In the absence of Mab treatment, 39 ng ¹²⁵I-LDL was bound per well, whereas heparin treatment reduced ¹²⁵I-LDL retention by greater than 97%. To varying degrees, all three Mabs tested diminished ¹²⁵I-LDL binding to LPL. Mab 5E11 minimally decreased ¹²⁵I-LDL binding by 15% at 1 µg/mL; no further decrease was observed with 10 µg/mL (13% decrease). Mab 4G3 showed the greatest decrease in ¹²⁵I-LDL binding to LPL-treated matrix by 29% and 42% at 1 and 10 µg/mL, respectively. Mab 2D8 interfered moderately (23% decrease) with ¹²⁵I-LDL binding only at the higher concentration tested (10 µg/mL). A control Mab (ie, Mab 6C5 to human apoE) that does not recognize apoB (10 µg/mL) had no effect on LDL retention. Overall, these data suggested that Mab 4G3 is the most effective Mab in blocking LDL retention by the matrix.

View larger version (27K): [in this window] [in a new window]   Figure 1. Bar graph shows that Mabs to apoB block LDL retention by LPL-treated subendothelial cell matrix. Porcine aortic endothelium cell–derived matrix was treated with LPL (8 µg/mL), washed, and exposed to human 125I-LDL (2.5 µg/mL) preincubated alone (control) or with protein A affinity-purified Mabs to human apoB. Plates were washed, and radioactivity remaining with matrix was determined as described in "Methods." 125I-LDL retention in the presence of heparin (50 U) was also determined. Data are mean±SEM of a representative experiment performed in triplicate. Statistical significance of the effect of Mabs relative to control was calculated with the two-sided unpaired t test.

LDL ApoB Cyclohexanedione Modification and Acetylation Decrease Its Ability to Block ¹²⁵I-LDL Retention
Cyclohexanedione modification of arginine residues in apoB has previously been shown to abolish its reactivity with Mabs 4G3 and 2D8.⁴² Since these two Mabs in the above experiment caused the largest block in the retention of LDL, it is possible that arginine residues in apoB play a role in LDL retention. In these experiments we chemically modified LDL basic amino acids by either acetylation (which introduces a negative charge on lysine residues) or cyclohexanedione treatment (which introduces a negative charge on arginine residues). The electrophoretic mobility of acetylated LDL and cyclohexanedione-modified LDL on agarose showed the expected increased anodal migration of LDL caused by the loss of positively charged amino acids on native LDL (Fig 2). Cyclohexanedione treatment followed by reversal slowed the increased LDL mobility, suggesting a partial restoration of positive charge.

View larger version (44K): [in this window] [in a new window]   Figure 2. Bar graph and blot show that cyclohexanedione modification and acetylation of LDL apoB decrease LDL competition. Aortic endothelial cell–derived matrix was treated with LPL (8 µg/mL) and washed, and human 125I-LDL (2.5 µg/mL) retention was determined in the absence (control) or presence of a 10- or 50-fold excess of unmodified and nonradioactive LDL, acetylated LDL (AcLDL), cyclohexanedione-treated LDL (CycLDL), or cyclohexanedione-treated then reversed LDL (Rev cycLDL). Nonspecific human 125I-LDL retention was determined in wells containing heparin (50 U). Plates were washed with buffer, and heparin-releasable radioactivity was determined as described in "Methods." Top, Lipoprotein electrophoresis pattern of charge-modified LDLs. Data are mean±SEM of a representative experiment performed in triplicate. Statistical significance of the effect of chemically modified lipoproteins relative to control was calculated with the two-sided unpaired t test.

These modified lipoproteins were tested for their ability to block the binding of radiolabeled native LDL (2.5 µg/mL) at 10- and 50-fold excess concentrations. Ten- and 50-fold excess native LDL blocked ¹²⁵I-LDL binding by 71% and 88%, respectively. LDL basic amino acid modification diminished the ability of LDL to compete with ¹²⁵I-LDL for binding to matrix-bound LPL. Acetylation, which mainly modifies lysine residues, made LDL less competitive than native LDL, displacing only 51% and 64% bound ¹²⁵I-LDL at 10- and 50-fold excess concentrations, respectively. Cyclohexanedione modification of LDL arginines further diminished the ability of LDL to displace bound ¹²⁵I-LDL; at 10- and 50-fold excess concentrations, bound ¹²⁵I-LDL was only displaced by 22% and 55%, respectively. Reversal of the cyclohexanedione modification of LDL restored some inhibitory property; at 10- and 50-fold excess concentrations ¹²⁵I-LDL binding was 35% and 73% of control, respectively. In contrast, methylation of LDL apoB, which does not change the charge of the lipoprotein yet blocks its interaction with the LDL receptor, had no effect on the ability of LDL to displace ¹²⁵I-LDL (data not shown). These data suggest that arginine and lysine residues in apoB play a significant role in the retention of LDL by LPL-containing matrix. Overall, chemical modification of arginine and lysine showed better effects than the inhibitory effects of the Mabs. One explanation for this may be that the chemical modifications may affect several domains on apoB, whereas the Mab should affect only specific epitopes on apoB.

Polyarginine and Polylysine Peptides Compete With LDL for Retention by LPL-Containing Matrix
To determine whether positively charged residues in LDL function to bind LPL, which is anchored to the subendothelial matrix, we assessed competition with positively charged peptides. Equivalent concentrations (200 µg/mL) of peptides of polyarginine and polylysine reduced ¹²⁵I-LDL binding by 97% and 75%, respectively (Fig 3A). Polyleucine at similar concentrations had no effect on decreasing the binding of ¹²⁵I-LDL (Fig 3A). In a separate study that determined the relative efficacy of these peptides in 50% displacement of ¹²⁵I-LDL from matrix-bound LPL, polyarginine was shown to be approximately 10 times more effective than polylysine (Fig 3B). However, unlike polyarginine, arginine alone up to 175 µg/mL had no effect on LDL retention (data not shown). Overall, these data suggest that clusters of positive charges are required for decreasing LDL retention to matrix.

View larger version (18K): [in this window] [in a new window]   Figure 3. Graphs show that polyarginine and polylysine peptides compete with LDL for retention by LPL. A, Aortic endothelial cell–derived matrix was treated with LPL (8 µg/mL) and washed, and human 125I-LDL (2.5 µg/mL) retention was determined in the absence (control) or presence of 200 µg/mL polyarginine, polylysine, or polyleucine. Data are mean±SEM of a representative experiment performed in triplicate. Statistical significance of the effect of the peptides was calculated with the two-sided unpaired t test. B, In a separate experiment human 125I-LDL retention was determined at the indicated concentrations of polyarginine and polylysine. Plates were washed with buffer, and heparin-releasable radioactivity was determined as described in "Methods." Data are the mean of a representative experiment performed in duplicate.

We explored the possibility that these peptides may release LPL from the matrix and thereby decrease LDL retention in experiments using radioiodinated LPL (8 µg/mL). Neither peptide (at 10 or 100 µg/mL) decreased the amount of radiolabeled LPL bound to the matrix.

ApoE Isoforms Differ in Their Ability to Reduce LDL Retention by Subendothelial Cell Matrix–Bound LPL
Results obtained in the above experiments suggested that positively charged domains of a molecule are important in its ability to inhibit LDL retention. ApoE is a 299–amino acid arginine-rich protein with three major isoforms—apoE2, apoE3, and apoE4—that vary by single arginine-cysteine interchanges at positions 112 and 158.⁴⁴ ApoE2, the rare isoform, contains its only two cysteines at both sites; apoE3, the most common isoform, contains an arginine substitution at position 158; and apoE4 has arginine substitutions at both sites. The major apoE isotypes have been shown to have major differences in ligand binding. For example, apoE3 and apoE4 bind the LDL receptor with approximately 100 times the affinity of apoE2.⁴⁴ We examined the effects of these isoforms on LDL retention. As shown in Fig 4, apoE3 appeared to be most effective, followed by apoE4 and apoE2, which was the least effective. These data suggest some selectivity of the apoE isoforms on the retention of LDL by the matrix. In these experiments we also assessed the effects of the 22- and 10-kD thrombin fragments of apoE3 on LDL retention. Each of these fragments contain heparin binding domains⁴⁵ and as shown in Fig 4 displaced ¹²⁵I-LDL. These data suggest a duplicity of functional domains within the apoE molecule that compete with LDL for subendothelial cell matrix–bound lipase.

View larger version (25K): [in this window] [in a new window]   Figure 4. Bar graph shows that apoE isoforms are dissimilar in reducing LDL retention by subendothelial cell matrix–bound LPL. Aortic endothelial cell–derived matrix was treated with LPL (8 µg/mL) and washed, and human 125I-LDL (2.5 µg/mL) retention was determined in the absence (control) or presence of a 50-fold excess of unmodified and nonradioactive LDL, apoE3, apoE4, apoE2, or the 10- or 22-kD thrombolytic fragments of apoE3 (all at 2.5 µg/mL). Nonspecific human 125I-LDL retention was determined in wells containing heparin (50 U). Plates were washed with buffer, and heparin-releasable radioactivity was determined as described in "Methods." Data are mean±SEM of a representative experiment performed in triplicate. Statistical significance of the effect of the isoforms and peptides relative to control was calculated with the two-sided unpaired t test.

ApoE3-Containing HDL Is More Effective Than ApoE2-Containing HDL in Inhibiting LDL Retention
Since differential effects of purified apoE2 and apoE3 were observed, we examined whether apoE3-containing HDL differed from apoE2-containing HDL in blocking LDL retention. The apoE content of apoE2-containing HDL preparations used in this study was atypical in that it was much lower (2.11 ng/µg HDL protein) than previously obtained.²⁸ To keep the apoE content comparable, we deliberately tested an apoE3-containing HDL preparation that was similar (1.24 ng apoE3/µg HDL). The data were normalized for apoE content and are shown in Fig 5. Addition of apoE2-containing HDL had no significant effect at lower concentrations; however, at the highest concentration used some inhibition was observed (Fig 5). In contrast, apoE3-containing HDL showed inhibitory effects at every concentration tested (Fig 5). Addition of either apoE2- or apoE3-poor HDL up to 400 µg (total protein) showed no effect. These data suggest that apoE3-containing HDL is far more effective than apoE2-containing HDL in displacing ¹²⁵I-LDL from LPL-bound matrix. These observations are consistent with the differential effects observed with purified apoE2 and apoE3 shown in Fig 4.

View larger version (18K): [in this window] [in a new window]   Figure 5. Line graph shows that apoE2-containing HDL is less effective than apoE3-containing HDL in its ability to displace 125I-LDL from subendothelial matrix–bound LPL. Porcine aortic endothelial cell–derived matrix was treated with LPL (8 µg/mL) and washed, and 125I-LDL (10 µg/mL) retention was determined in the absence or presence of apoE2-containing or apoE3-containing HDL at various concentrations. Plates were washed with buffer, and heparin-releasable radioactivity was determined as described in "Methods." Data are shown as nanograms 125I-LDL bound at various concentrations of the two HDL preparations as a function of apoE content. Data are mean±SEM of a representative experiment performed in triplicate. The inhibitory effect of the apoE3-containing HDL was statistically significant (*P=.002), calculated with the two-sided unpaired t test relative to control retention.

The ability of apoE-rich HDL to diminish LDL retention could have resulted from its direct binding to the matrix and blocking of LDL retention. We examined this possibility by incubating ¹²⁵I-apoE–rich HDL with matrix alone or matrix that contained LPL (8 µg/mL). Addition of 12.5 or 100 µg ¹²⁵I-apoE–rich HDL resulted in the binding of 2.1 and 9.6 ng, respectively, of HDL to the matrix alone, whereas 14.5 and 25.4 ng of HDL bound to matrix that contained LPL. These results demonstrate that some apoE3-containing HDL does bind to the matrix directly and LPL enhances this binding. Thus apoE-rich HDL may decrease LDL retention by binding to either matrix or LPL or both.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In addition to the well-known enzymatic function of LPL, this enzyme also possesses structural motifs important in the binding of apoB- and apoE-containing lipoproteins.^{3 28 46} For example, postheparin human plasma gel filtration studies of Goldberg et al⁴⁷ have shown LPL activity associated with apoB/E-containing chylomicron and VLDL remnants. Similarly, studies of Vilella et al⁴⁸ have shown LDL and apoE-containing HDL₂ associated with LPL. LPL has also been shown to facilitate the cell surface proteoglycan binding of VLDL and LDL and subsequent uptake by various cell types.^{49 50 51 52 53 54} In the studies presented here we examined the interactions of matrix-bound LPL with LDL and HDL. We have previously shown that apoE modulates apoB-containing lipoprotein retention by LPL anchored to the subendothelial matrix.²⁸ The studies demonstrated specificity of heparan and dermatan sulfates in LPL binding to the subendothelial matrix. The bound LPL serves as an anchor for the retention of apoB-containing lipoproteins. In those studies a specific role of apoE in competing for retained apoB bound to LPL and not for LPL bound to the glycosaminoglycans was demonstrated. In the current study we investigated the functional domains present on apoB and apoE that participate in the interactions between LDL and LPL-containing matrix. To this end, we used Mabs to apoB, chemical modification of apoB, apoE isoforms and apoE-containing HDL, apoE thrombin generated, and synthetic peptides to test their effects on LDL retention.

In our first series of studies, we investigated apoB domains that could potentially function in LPL binding. Both Mab (Fig 1) and arginine/lysine chemical modification (Fig 2) studies suggested that basic amino acids of apoB might play a role in the interaction with LPL. Using Mabs to human apoB we determined whether a reduction in ¹²⁵I-LDL binding to LPL-bound matrix could be observed. Mab 4G3 was the most effective of the three apoB-specific Mabs tested. Mab 4G3 has been previously mapped to arginine-containing sites, suggesting that arginine residues in apoB may mediate the binding of LDL to matrix. In addition, both Mab 4G3 and Mab 5E11 are known to block LDL binding to the LDL receptor.⁴³ Since Mab 4G3 only partially blocked ¹²⁵I-LDL retention, other sites on apoB might participate in its retention. Mab 2D8, which binds apoB but does not interfere with LDL receptor binding, had a small effect on matrix retention of LDL. It is of interest to note that the epitope for this Mab has also been mapped to an arginine-containing region on apoB. Thus, the most effective Mabs, 4G3 and 2D8, both map to arginine-containing residues. Overall, these Mab studies suggested that although sites involved in the binding of apoB to the LDL receptor may be involved in LPL matrix retention, additional sites on apoB might function in LPL retention. To further investigate apoB functional domains involved in LPL binding, we chemically modified both lysine and arginine residues (Fig 2). Lysine (acetylation) or arginine (cyclohexanedione treatment) modification resulted in a reduction in LDL basic charge and partial reduction in the ability of LDL to compete for matrix-retained LDL. The effects observed with cyclohexanedione-treated LDL were partially reversible (Fig 2). Thus, basic amino acid regions of apoB appear to participate in the binding to lipase.

Since apoE is arginine rich and blocks LDL binding to LPL, the possibility was raised that apoE basic amino acid residues also participate in LPL binding. In the current study we found that apoE3 blocked LDL retention more efficiently than apoE4. Although a single arginine substitution in apoE4 (Cys112 to Arg112) would cause a slight increase in the positive charge of this protein, a marked conformational change in the protein has also been observed.⁵⁵ For example, the substitution at position 112 alone can affect the lipoprotein association preference of apoE.⁵⁵ Thus, it is not surprising that a single substitution may also affect retention inhibition, again suggesting that apoE conformation may also direct its retention inhibitory properties. Examination of the effects of purified apoE isoforms showed that apoE2 and apoE3 were dissimilar in inhibiting LDL retention. Differences in the effects of apoE3 and apoE2 were also observed when examined in their natural form associated with HDL, although these differences were not as dramatic as those found in LDL receptor binding activity of these isoforms. With LDL receptor binding, the interaction of apoE2 is not detectable,⁴⁴ but in our assays apoE2 did have an inhibitory effect at high concentrations. These data indirectly suggest that other sites on apoE besides the LDL receptor binding domains compete for LPL retention of LDL. This idea is supported by the findings that both apoE 10- and 22-kD thrombin fragments retained the ability to displace LDL (Fig 4) even though the LDL receptor binding domain is contained in the 22-kD peptide.³⁸ Similarly, clusters of charged amino acids, such as those contained in polyarginine and polylysine, were effective competitors with LDL for retention (Fig 3). Thus, apoE appears to block LDL binding to lipase-containing matrix by virtue of possessing similar positively charged domains and thus competing with apoB for binding to lipase-containing matrix. The demonstration of apoE3-containing HDL binding to the matrix alone and LPL-containing matrix supports the possibility that apoE-rich HDL may inhibit LDL retention by either direct competition or steric hindrance. Previous studies have shown that apoE3 binds to cell surface and matrix heparan sulfate proteoglycans more efficiently than apoE2.⁵⁶ Thus, it is possible in our studies that the greater effectiveness of apoE3-containing HDL relative to apoE2-containing HDL is related to differential binding of these apoE isoforms to the matrix and/or LPL bound to the matrix.

Overall, these data suggest the importance of positively charged clusters as a component of LDL/apoB binding to lipase-containing matrix and as a constituent involved in the inhibitory effects of apoE on LDL retention. The availability of multiple positively charged regions in apoB and apoE can arise as a result of a primary sequence containing close clusters of lysine and arginine or by interaction between distant regions in the molecule to generate a high density of positive charges. Camejo et al⁵⁷ and Olsson et al⁵⁸ have proposed that at least 10 surface regions on apoB have three or more positive charges within fewer than 15 residues. Furthermore, Weisgraber and Rall⁴⁵ have isolated seven heparin-binding positively charged peptides from proteolytic digests of apoB. Future experiments with shorter apoB/E peptides and model peptides will establish the minimal apolipoprotein structure required for LPL-lipoprotein-matrix interactions. We hypothesize that both charge and conformation of apoB and apoE play a role in LPL recognition because (1) both polylysine and polyarginine are positively charged, but polyarginine was much more efficacious in inhibiting LDL retention; and (2) apoE2 and apoE3 showed differential effects on LDL retention despite only a single arginine substitution in this arginine-rich protein.

Arterial subendothelial matrix trapping of apoB-containing lipoproteins may have a number of important physiological implications, including those related to early steps in lipoprotein oxidative modification. The localized production of apoE by macrophages in the lesions and plasma apoE-containing HDL may have a dual function. First, apoE may play a role in the egress of cholesterol within lesions.⁴⁴ Second, the apoE may play a protective role in modulating LDL retention²⁸ and thereby prevent the oxidation of apoB-containing lipoproteins within the subendothelial space. In this regard, recent preliminary monocyte transmigration studies using cocultures of apoE-transfected human aortic endothelial cells with smooth muscle cells markedly reduced migration of monocytes through the endothelial layer, presumably by preventing LDL trapping and resulting oxidation.⁵⁹ The greater efficacy of apoE3 relative to apoE2 assumes great physiological importance if indeed such mechanisms are operative in vivo. Based on the data presented here, we propose that the predisposition for atherosclerosis observed in patients with apoE2^{44 56 60} may in part be related to inefficient inhibition of subendothelial LDL accumulation, possibly confounded by elevated levels of apoB-containing lipoproteins.

In conclusion, our studies have shown that arginine-lysine–containing regions on apoB and apoE play a critical role in LDL retention and its modulation by apoE. This information will provide the basis for the design of specific molecules that can block the extracellular accumulation of lipoproteins during atherosclerosis.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BSA = bovine serum albumin DMEM = Dulbecco's modified Eagle's medium LPL = lipoprotein lipase mAb = monoclonal antibody

	Acknowledgments

 
These studies were supported in part by National Institutes of Health Program Project Grant HL-41633 (K. Weisgraber), a Medical Research Council of Canada Group Grant, the Heart and Stroke Foundation of Ontario (Y. Marcel), and the Medical Research Council of Canada (MT 1162, R. Hegele).

Received November 20, 1994; accepted May 17, 1995.

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