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Journal of Lipid Research, Vol. 42, 959-966, June 2001
Copyright © 2001 by Lipid Research, Inc.

Cationic domain 141-150 of apoE covalently linked to a class A amphipathic helix enhances atherogenic lipoprotein metabolism in vitro and in vivo

Correspondence to: G. M. Anantharamaiah, at the Department of Medicine, UAB Medical Center, 1530 3rd Ave. S., Birmingham, AL 35294., ananth{at}uab.edu (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We previously showed (1) that a peptide, Ac-hE18A-NH₂, in which the arginine-rich heparin-binding domain of apolipoprotein E (apoE) [residues 141;–150] (LRKLRKRLLR), covalently linked to 18A (DWLKAFYDKVAEKLKEAF; a class A amphipathic helix with high lipid affinity), enhanced LDL uptake and clearance. Because VLDL and remnants contain more cholesterol per particle than LDL, enhanced hepatic clearance of VLDL could lead to an effective lowering of plasma cholesterol. Therefore, in the present article we compared the ability of this peptide to mediate/facilitate the uptake and degradation of LDL and VLDL in HepG2 cells. The peptide Ac-hE18A-NH₂, but not Ac-18A-NH₂, enhanced the uptake of LDL by HepG2 cells 5-fold and its degradation 2-fold. The association of the peptides with VLDL resulted in the displacement of native apoE; however, only Ac-hE18A-NH₂ but not Ac-18A-NH₂ caused markedly enhanced uptake (6-fold) and degradation (3-fold) of VLDL. Ac-hE18A-NH₂ also enhanced the uptake (15-fold) and degradation (2-fold) of trypsinized VLDL Sf 100;–400 (containing no immunodetectable apoE), indicating that the peptide restored the cellular interaction of VLDL in the absence of its essential native ligand (apoE). Pretreatment of HepG2s with heparinase and heparitinase abrogated all peptide-mediated enhanced cellular activity, implicating a role for cell-surface heparan sulfate proteoglycans (HSPG). Intravenous administration of Ac-hE18A-NH₂ into apoE gene knockout mice reduced plasma cholesterol by 88% at 6 h and 30% at 24 h after injection.

We conclude that this dual-domain peptide associates with LDL and VLDL and results in rapid hepatic uptake via a HSPG-facilitated pathway.—Datta, G., D. W. Garber, B. H. Chung, M. Chaddha, N. Dashti, W. A. Bradley, S. H. Gianturco, and G. M. Anantharamaiah. Cationic domain 141-150 of apoE covalently linked to a class A amphipathic helix enhances atherogenic lipoprotein metabolism in vitro and in vivo. J. Lipid Res. 2001. 42: 959–966.

Supplementary key words: cationic peptides, enhanced uptake, degradation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Receptor interaction of atherogenic apolipoprotein B (apoB)-containing lipoproteins is essential for hepatic clearance (2) (3) (4), the major site of cholesterol catabolism to bile for elimination. Although LDL is cleared primarily by its interaction with the LDL-receptor (LDLR), VLDL and lipase-produced VLDL remnants are cleared, in large part, via the interaction of an appropriate conformation of apoE with several cell surface receptors. These include LDLR, the LDL-receptor related protein (LRP), and direct binding of apoE-containing VLDL and remnants to HSPG in the space of Disse (2) (3) (4). ApoE plays a critical role in lipoprotein metabolism. Its importance in inhibiting atherosclerosis in rabbits and mice has been established by numerous studies (5) (6) (7) (8) (9) (10). For example, overexpression of human apoE in New Zealand White rabbits enhances the clearance of diet-induced lipoprotein remnants when compared with non-transgenic animals (5); intravenous injection of human apoE into cholesterol-fed Watanabe rabbits protects these animals from atherosclerosis (6). Mice overexpressing rat apoE have lower plasma cholesterol (7). Furthermore, loss of the apoE gene elicits spontaneous atherosclerosis (8) (9) that is ameliorated when macrophage-specific apoE expression is restored in apoE-deficient mice (10) (11). Finally, direct addition of apoE to the VLDL surface enhances the binding and uptake of VLDL (12) (13) (14), which carries more cholesterol per particle compared with LDL.

ApoE is a 299 amino acid residue protein that has been shown, initially by proteolysis studies (15) (16), to have two distinct domains: a receptor binding domain in the N-terminal region [residues 1;–191] and a lipid binding domain at the C terminus [residues 192;–299]. Subsequent studies with synthetic peptides and monoclonal antibodies (17) (18) (19) (20) (21) support this observation. Wilson et al. (22) crystallized the amino terminal domain of apoE [residues 1;–191] containing the LDL receptor-binding domain, which demonstrated its globular structure. Using 1,2-dimyristoyl-sn-glycero-3-phosphocholine complexes of the N-terminal 22-kDa thrombin fragment mutants of apoE, it has been shown (23) that conserved lysines and arginines within the 140;–150 region of apoE are crucial for the interaction between apoE and the LDLR. The positive charge cluster appears to be involved in ionic interactions with negative charges of the LDLR and/or proteoglycans. We recently showed (1) that the dual-domain peptide Ac-hE18A-NH₂ with the sequence Ac-LRKLRKRLLRDWLKAFYDKVAEK LKEAF-NH₂, containing the arginine-rich cationic domain of apoE (LRKLRKRLLR), covalently linked to a class A amphipathic peptide 18A (DWLKAFYDKVAEKLKEAF), binds to LDL, and enhances its uptake and degradation in fibroblasts via the HSPG pathway. Previous studies from our laboratory (24) showed that class A amphipathic peptides interact with VLDL and displace apoE. We reasoned that an efficient removal of VLDL might result in a significant reduction of plasma cholesterol. Therefore we studied, and report here, the peptide-mediated uptake and degradation of LDL and VLDL by hepatocytes and the effect of acute intravenous administration of Ac-hE18A-NH₂ on plasma cholesterol levels in the apoE gene knockout mouse model.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Synthesis of the peptides
Peptides Ac-18A-NH₂ and Ac-hE18A-NH₂ were synthesized by Fmoc chemistry, using an automatic peptide synthesizer (PE Biosystems, Foster City, CA) according to the procedure described by us earlier (1). Purity of peptides was determined by C-18 analytical reversed-phase HPLC and confirmed by mass spectral analysis.

Preparation of VLDL, LDL, and lipoprotein-deficient serum
Plasma VLDL and LDL were isolated from human plasma obtained from the American Red Cross by sequential centrifugation as previously described (25) (26). The VLDL (d < 1.006 g/ml) was isolated by centrifugation of plasma at 50,000 rpm at 4°C for 24 h and washed once by reisolation under identical conditions. The LDL (1.006 < d < 1.063g/ml) was also isolated by centrifugation at 50,000 rpm at 4°C for 24 h and washed by overlayering with 150 mM NaCl and recentrifuged and dialyzed extensively against 150 mM NaCl containing 0.24 mM EDTA. The purity of the isolated lipoproteins was determined by Superose 6 (Pharmacia) column chromatography and by agarose electrophoresis. For preparation of lipoprotein-deficient serum (LPDS), the density of plasma was adjusted to 1.21 g/ml with potassium bromide and centrifuged at 50,000 rpm at 4°C. The top fraction consisting of lipoproteins was removed. The lower fraction, which is devoid of lipoproteins, was dialyzed extensively against 150 mM NaCl at 4°C for 60 h. The lipoprotein-deficient plasma was converted to LPDS by incubation with thrombin (10 U/ml) at 4°C for 24 h and removing the resulting clot by centrifugation.

Manual cholesterol analysis
Cholesterol was determined by adding 10-µl standard or plasma to 1-ml cholesterol reagent (4-g "Cholesterol 1000," Sigma Chemical Co., St. Louis, MO) dissolved in 100-ml deionized H₂O) and mixing. Absorbance at 500 nm was read in a spectrophotometer after 15 min of incubation at room temperature. Standards were Sigma Cholesterol Calibrator at 100, 200, and 400 mg/dl.

Trypsinolysis of VLDL
Trypsinolysis was carried out on VLDL S_f 100;–400, essentially as described by Gianturco et al. (27). Briefly, the VLDL S_f 100;–400 was treated with trypsin (1% by weight) in Tris buffer (pH 7.4) for 2 h at 37°C and passed over a benzamidine-sepharose column to remove active trypsin, and the protease-treated VLDL was reisolated by cumulative ultracentrifugation.

¹²⁵I labeling of LDL, VLDL, and peptides
LDL, VLDL, and peptides were labeled with ¹²⁵I using the method of Bilheimer, Eisenberg, and Levy (28). The ¹²⁵I-labeled material was separated from free ¹²⁵I by a desalting column (Biorad, Econopak 10DG). Fractions containing labeled material were pooled and specific activity determined. In the case of peptides, 90% of the peptide was labeled, whereas in LDL and VLDL, 80% and 70%, respectively, of the protein was labeled as determined by delipidation. The labeled LDL/VLDL was dialyzed extensively against PBS and was analyzed for protein and cholesterol content. ¹²⁵I-specific activities ranged from 250 to 400 dpm/ng of protein.

Interaction of the peptide with VLDL
The peptides Ac-hE18A-NH₂ and Ac-18A-NH₂ were added to VLDL at various protein/peptide (w/w) ratios and incubated at room temperature for 1 h. VLDL-peptide mixtures were subjected to ultracentrifugation to separate VLDL-peptide complexes from unbound peptides as previously described (24). The protein content of the isolated VLDL fraction was determined by the procedure of Lowry et al. (29). SDS-PAGE was carried out using the method of Laemmli (30). Pre-made Novex gels were used and the gel was stained with Coomassie blue to identify the protein bands. The bands were quantitated by densitometry using the gel documentation system from UVP (Upland, CA).

Binding of the peptides to VLDL was also examined by using ¹²⁵I-labeled peptides. Both Ac-hE18A-NH₂ and Ac-18A-NH₂ have a single Tyr residue that can be iodinated. VLDL (1-mg apoprotein) was incubated with 1 mg of labeled peptide, the VLDL-peptide complex was reisolated, and the bound peptide was separated from free peptide using density-gradient ultracentrifugation. The amount of peptide bound to VLDL was calculated using the specific activity of the peptide, which ranged from 250 to 300 dpm/ng of peptide.

Binding, internalization, and degradation of LDL and VLDL
The binding of lipoprotein (LDL or VLDL) to cells and its internalization and degradation were measured as described by Goldstein, Basu, and Brown (26). HepG2 cells were grown in DMEM containing 10% fetal calf serum (FCS) and penicillin-streptomycin-amphotericin in 6-well plates and used after reaching 75;–90% confluency (2;–3 days). The seeding density of cells used was 1.5 x 10⁵ to 3.0 x 10⁵ cells/ml medium. Cells were incubated with medium containing LPDS for 24 h prior to use. For binding experiments, the cells were incubated with peptide-treated and control ¹²⁵I-LDL or ¹²⁵I-VLDL at 4°C for 2 h and, for uptake (or internalization), they were incubated at 37°C for 2 h. After washing four times with ice cold PBS containing 2-mg/ml BSA to remove nonspecifically bound lipoprotein, the cells were incubated with dextran sulfate (4 mg/ml, Pharmacia, M_r 500,000) or heparin (10 mg/ml, Sigma) for 1 h to release specifically bound ¹²⁵I-LDL/ ¹²⁵I-VLDL. The counts in the dextran sulfate/ heparin wash reflected the amount of LDL/VLDL specifically bound to cells. The cells were washed with cold PBS, dissolved in 1-ml 0.1N NaOH, and a 0.5-ml aliquot was counted. These counts reflect the amount of LDL/VLDL internalized. Protein was estimated by the method of Lowry et al. (29).

Degradation of LDL/VLDL was measured using the protocol described above for binding, except that the cells were incubated at 37°C for 5 h. Degradation was determined by precipitating ¹²⁵I-LDL or ¹²⁵I-VLDL from the medium by incubation at 4°C for 30 min with 16.7% TCA (0.5 ml of 50% TCA was added to 1 ml of medium) (25). The resulting precipitate was removed by centrifugation. The supernatant was treated with 10 µl of 40% potassium iodide and 40 µl of 30% hydrogen peroxide. The free ¹²⁵I liberated was extracted with 2 ml of chloroform. The upper aqueous layer (0.5 ml) was then counted. This represents the degradation of apolipoprotein in LDL/VLDL.

Cell-surface HSPG was removed by incubating the cells with 3 U/ml heparinase/heparitinase (Sigma) at 37°C for 2 h as previously reported (1). In all the cell experiments, mean values from replicate measurements of three independent experiments were used.

Effect of peptide in intravenous administration in apoE(-/-) mice
Mice were purchased from Jackson Laboratories (Bar Harbor, ME). Following an overnight fast, female mice were injected intravenously with 100 µg of Ac-hE18A-NH₂ or Ac-18A-NH₂ in 100 µl PBS through the tail vein; control mice were injected with 100 µl PBS. Blood was collected from the retro-orbital sinus under anesthesia immediately before injection and at 6 and 24 h following injection, and cholesterol was measured manually on all samples. Food was provided to the mice following the 6-h sample. Lipoprotein cholesterol profiles were determined using the CLiP method (31).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have shown (1) that the dual-domain peptide, Ac-hE18A-NH₂, binds to LDL and enhances its uptake in fibroblasts. Given the importance of the liver in the clearance of LDL and VLDL, we determined the effect of this peptide on the uptake and degradation of LDL and VLDL in model hepatocytes. Ac-hE18A-NH₂ enhanced the uptake of LDL in HepG2 cells 5-fold and degradation 2-fold as seen in Fig 1. Heparinase and heparitinase pretreatment of cells completely eliminated this effect (Fig 1), suggesting that the LDL-Ac-hE18A-NH₂ is taken up via the HSPG pathway in hepatocytes as in fibroblasts (1). The level of enhanced uptake of LDL was dependent on the amount of Ac-hE18A-NH₂ incorporated into the LDL. Importantly, the control peptide Ac-18A-NH₂ not containing the cationic domain did not enhance the uptake of LDL in HepG2 cells at any concentration (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 1. Uptake and degradation of 125I-LDL alone and 125I-LDL-Ac-hE18A-NH2 complex by Hep G2 cells. 125I-LDL was incubated with Ac-hE18A-NH2 (1:1 w/w) and the complex reisolated by density-gradient ultracentrifugation. The 125I-LDL-peptide complex was used to determine the effect of the bound peptide on the uptake and degradation of 125I-LDL. In each case, particles containing 50 µg of cholesterol were added and incubated with cells at 37°C for 2 h for uptake and for 5 h for degradation. Cell-surface bound LDL was removed in two different ways; in one experiment heparin was used (main figure) and in another, dextran sulfate (inset). The data represent a mean of triplicate values. The solid bars represent native 125I-LDL, and the open bars represent 125I-LDL-Ac-hE18A-NH2. +H/H represents the uptake of LDL in cells pretreated with heparinase/heparitinase (3 U/ml).

Peptides associate with VLDL surface and displace apo E
Peptide binding to VLDL was studied by incubating VLDL with increasing amounts of Ac-hE18A-NH₂ at the weight ratios of 1:0.15, 1:0.3, and 1: 0.6 as indicated in the legend of Fig 2a. The peptide-VLDL mixtures were subjected to density gradient ultracentrifugation to separate VLDL-peptide complexes from unassociated peptide. The apolipoprotein distribution of the VLDL-peptide complexes re-isolated by density gradient ultracentrifugation was examined by SDS-PAGE and is shown in Fig 2a. Since addition of peptide to VLDL could interfere with the quantitation of proteins on the lipoprotein, VLDL particle concentration was determined by cholesterol content. At equal VLDL particle concentration (based on cholesterol content), the ratio of apoE to apoB was determined at each peptide concentration by quantitating the intensity of the bands and is shown in Table 1. The apoE/apoB ratio (relative intensities) of each complex (lanes 2, 4, and 5 in Fig 2a) was compared with that of VLDL alone (lane 1, Fig 2a). The apoE/apoB ratio was lower in Ac-hE18A-NH₂-treated VLDL (Fig 2a, lanes 2, 4, and 5) than in VLDL alone (Fig 2a, lane 1). The apoE/apoB ratio decreases from 0.2 to 0.06 with increasing concentration of Ac-hE18A-NH₂ (Table 1). Negligible amounts of apoE were detectable in VLDL at the VLDL: peptide ratio of 1.0:0.6 (Fig 2a, lane 5). The decrease in apoE content with increasing amount of peptide on VLDL indicated that apoE was displaced from the VLDL surface by Ac-hE18A-NH₂ in a concentration-dependent manner, whereas the increase in intensity of the band corresponding to the peptide demonstrated a concentration-dependent association of peptide with VLDL.

View larger version (81K): [in this window] [in a new window]   Figure 2. A: Effect of increasing amount Ac-hE18A-NH2 on its binding to VLDL. Apolipoproteins in VLDL and VLDL-Ac-hE18A-NH2 complexes reisolated by density-gradient ultracentrifugation were separated on SDS-PAGE (10;–20%). The gel is stained with Coomassie Brilliant blue. Lanes 1, VLDL alone; 2, VLDL-Ac-hE18A-NH2 (1:0.15 w/w); 3, molecular weight standards; 4, VLDL-Ac-hE18A-NH2 (1:0.3 w/w); and 5, VLDL-Ac-hE18A-NH2 (1:0.6 w/w). The positions of the apolipoproteins and the peptide are indicated. The band at <14.4 kDa in lane 1 represents apoCs present in VLDL. Each of the lanes contains equivalent VLDL based on cholesterol content (50 µg). The decrease in the content of apoE per 50 µg of VLDL cholesterol with increasing addition of Ac-hE18A-NH2 is evident in lanes 4 and 5. Increasing amounts of Ac-hE18A-NH2 bound to the VLDL can be seen in lanes 2,4, and 5 indicated by its marker on the right side of the gel. B: Comparison of the effect of Ac-hE18A-NH2 and Ac-18A-NH2 on VLDL. SDS-PAGE (4;–20%) of apolipoproteins in VLDL and VLDL-Ac-hE18A-NH2 (1:1) and VLDL-Ac-18A-NH2 (1:1) complexes reisolated by density-gradient centrifugation. Each of the lanes contains the same amount of VLDL based on cholesterol content (50 µg). Lanes 1, molecular weight standard; 2, control VLDL; 3, VLDL-Ac-hE18A-NH2 (1:1 w/w); 4, VLDL-Ac-18A-NH2 (1:1 w/w); and 5, Ac-hE18A-NH2 alone (6 µg). C: Demonstration of the difference in the chromogenicity of Ac-hE18A-NH2 and Ac-18A-NH2. Different concentrations (1;–10 µg) of Ac-hE18A-NH2 and Ac-18A-NH2 were loaded on a 10;–20% SDS gel and electrophoresed. These gels were stained with colloidal blue, and the integrated band intensity was measured using the gel documentation system. The integrated band intensity was plotted against the concentration of the peptide. Ac-hE18A-NH2 (open triangle) shows greater chromogenicity than Ac-18A-NH2 (solid circle).

To compare the interaction of VLDL and Ac-hE18A-NH₂ with that of VLDL and the control peptide, Ac-18A-NH₂, complexes (1:1 w/w) of the two peptides were made with VLDL, isolated, and compared on the same SDS gel, Fig 2b. Ac-18A-NH₂ also displaced apoE from VLDL (Fig 2b, lane 4) as had been reported earlier (24), however, it displaced less apoE than Ac-hE18A-NH₂ (Fig 2b, lane 3) at the same VLDL/peptide (w/w) ratio. Fig 2b, lane 5, shows the band due to the peptide Ac-hE18A-NH₂ alone. The apoE/apoB ratios (relative intensity) were determined as earlier and are found in Table 2. The apoE/apoB ratio decreased from 0.2 in VLDL alone (without peptide, Fig 2b, lane 2) to 0.04 with Ac-hE18A-NH₂ (Fig 2b, lane 3), and to 0.1 with Ac-18A-NH₂ (Fig 2b, lane 4), respectively. A faint band corresponding to Ac-18A-NH₂ can also be seen in Fig 2b, lane 4.

Using radiolabeled peptide (both Ac-hE18A-NH₂ and Ac-18A-NH₂), the binding of the peptide to VLDL was studied. It was determined that the amount of peptide bound to VLDL was the same in both cases (67 ± 5 nmol of Ac-hE18A-NH₂ and 73 ± 7 nmol of Ac-18A-NH₂ per mg of VLDL). Although the concentration of the two peptides in their respective complexes was the same, Ac-hE18A-NH₂ stained much more than Ac-18A-NH₂, possibly because the former contains a greater number of Arg and Lys residues. The difference in chromogenicity of the two peptides was also examined by staining SDS gels containing different amounts of peptides. Fig 2c shows the difference in chromogenicity, indicating increased chromogenicity of Ac-hE18A-NH₂ compared with that of Ac-18A-NH₂. The above results indicate that both peptides, Ac-hE18A-NH₂ and Ac-18A-NH₂, displaced apoE from the VLDL surface and that they bind to VLDL surface to produce an apoE-deficient, VLDL-peptide complex.

The dual-domain peptide, but not the class A amphipathic helical peptide, enhances VLDL binding and degradation in HepG2 cells
To determine the effect of the dual-domain peptide on the binding (4°C, 2 h), uptake (37°C, 2 h), and degradation (37°C, 5 h) of ¹²⁵I-VLDL, HepG2 cells were incubated with ¹²⁵I-VLDL-Ac-hE18A-NH₂ and with ¹²⁵I-VLDL-Ac-18A-NH₂ complexes under the conditions described above and in Materials and Methods. Although apoE was displaced by both peptides, the presence of Ac-hE18A-NH₂ on VLDL markedly enhanced the binding, uptake, and degradation of ¹²⁵I-VLDL by HepG2, whereas that of Ac-18A-NH₂ did not. Binding of ¹²⁵I-VLDL at 4°C (3 µg of apoprotein/mg of cell protein ± 0.008) was enhanced in the presence of Ac-hE18A-NH₂ to 16.5 µg of apoprotein/mg of cell protein ± 0.14 (data not shown). These experiments were done in triplicate. Uptake (internalization) was measured by determining the amount of radioactivity in the cells after incubation at 37°C for 2 h, washing surface-bound VLDL with dextran sulfate. Compared with VLDL alone, the uptake and degradation of VLDL-Ac-hE18A-NH₂ were stimulated by 6-fold and 3-fold, respectively ( Fig 3a, inset). To confirm that the observed enhancement was real and not due to inefficient removal of surface-bound VLDL, separate experiments were carried out using heparin to remove surface-bound VLDL. As can be seen, both dextran sulfate (Fig 3a, inset) and heparin (Fig 3a) yielded similar results. In stark contrast, Ac-18A-NH₂ did not increase the uptake of VLDL; rather, the uptake was less than that of VLDL alone, in agreement with the observation that Ac-18A-NH₂ displaced apoE from VLDL and produced apoE-deficient VLDL. Both peptides associated with VLDL to the same extent as indicated above and both displaced the endogenous ligand apoE. However, only Ac-hE18A-NH₂ containing the cationic domain increased uptake and degradation of VLDL in HepG2 cells.

View larger version (39K): [in this window] [in a new window]   Figure 3. A: Uptake and degradation of 125I-VLDL alone and 125I-VLDL-Ac-hE18A-NH2 complex by Hep G2 cells. Ac-18A-NH2 was used as a control. 125I-VLDL was incubated with the peptide (1:1 w/w) and reisolated by density centrifugation. The peptide bound to 125I-VLDL floated to the top and the unbound peptide was at the bottom. The 125I-VLDL-peptide complex was used to determine the effect of the peptide on the uptake and degradation of 125I-VLDL. In each case particles containing 50 µg of cholesterol were added and the cells incubated at 37°C for 2 h for uptake and 5 h for degradation. Cell-surface bound VLDL was removed in two different ways; in one experiment heparin was used (main figure) and in another, dextran sulfate (inset). 125I-VLDL alone (open bar); 125I-VLDL-Ac-hE18A-NH2 (1:1 w/w) (hatched bar); and 125I-VLDL-Ac-18A-NH2 (1:1 w/w) (solid bar). +H/H indicates the uptake of 125I-VLDL and 125I-VLDL-Ac-hE18A-NH2 in cells pretreated with heparinase/heparitinase (3 U/ml). The data represent a mean of triplicate values. B: Uptake and degradation of 125I-trypsinized VLDL Sf 100–400 alone and 125I-trypsinized VLDL-Ac-hE18A-NH2 complex by Hep G2 cells. Ac-18A-NH2 was used as a control. 125I-trypsinized VLDL was incubated with the peptide (1:1 w/w) and reisolated by density centrifugation. The peptide bound to 125I-trypsinized VLDL floated to the top and the unbound peptide was at the bottom. The 125I-trypsinized VLDL-peptide complex was used to determine the effect of the peptide on the uptake and degradation of 125I-trypsinized VLDL. In each case particles containing 50 µg of cholesterol were added. Conditions for uptake and degradation were as mentioned in Fig 3a. Cell-surface bound VLDL was removed in two different ways; in one experiment heparin was used (main figure) and in another, dextran sulfate (inset). 125I-trypsinized VLDL alone (open bars); 125I-trypsinized VLDL-Ac-hE18A-NH2 (1:1 w/w) (hatched bars); and 125I-trypsinized VLDL-Ac-18A-NH2 (1:1 w/w) (solid bars). +H/H indicates the uptake in presence of heparinase/heparitinase (3 U/ml), as in Fig 3a. The data represent a mean of triplicate values.

Heparinase and heparitinase remove HSPG from the surface of cells. We used this to determine whether HSPG had a role in Ac-hE18A-NH₂-mediated uptake of VLDL. Cells were pretreated with these enzymes as detailed in Materials and Methods. As can be seen in Fig 3a, treatment with heparinase and heparitinase eliminated the enhancement of VLDL uptake by Ac-hE18A-NH₂, implicating HSPG in this peptide-mediated enhanced uptake of VLDL, similar to our previous observation of LDL-peptide complex uptake and degradation.

Ac-hE18A-NH₂ binds to trypsinized VLDL and enhances its uptake by HepG2 cells
Trypsinization of VLDL removes immuno-detectable apoE on VLDL S_f 100;–400 (16). This VLDL subfraction is rendered LDL-receptor-inactive with the loss of its apoE receptor-binding domain on a particle in which the apoB is incompetent in binding to the LDLR (14) (15) (32). Both peptides studied here, Ac-hE18A-NH₂ and Ac-18A-NH₂, bound to trypsinized VLDL as determined by SDS electrophoresis (data not shown). It was therefore of interest to determine whether Ac-hE18A-NH₂ and/or Ac-18A-NH₂, when bound to trypsinized VLDL S_f 100-400, could facilitate its uptake by HepG2 cells. As with intact total VLDL, only Ac-hE18A-NH₂ enhanced the uptake of trypsinized VLDL S_f 100;–400. The uptake increased by 15-fold over the control trypsinized VLDL, whereas Ac-18A-NH₂ had no effect (Fig 3b). Degradation of trypsinized VLDL was also enhanced by Ac-hE18A-NH₂; again, Ac-18A-NH₂ had no effect. Although the Ac-hE18A-NH₂-mediated uptake was enhanced 15-fold, the degradation was only 2-fold greater than with trypsinized VLDL alone. To determine whether the HSPG pathway was involved in uptake and degradation in this case also, the cells were again pretreated with heparinase and heparitinase. The observed enhancement was nullified by pretreatment of the cells with heparinase and heparitinase. These data suggest that HSPG pathway plays a significant role in the enhanced uptake and degradation of Ac-hE18A-NH₂-mediated VLDL and trypsinized VLDL. The peptide mimics at least one of the functions of apoE; i.e., binding to HSPG.

Effect of acute administration of the peptide in apoE gene knockout mice
Intravenous administration of 100 µg Ac-hE18A-NH₂ in 100 µl PBS into fasting apoE gene knockout mice (n = 8) reduced plasma cholesterol from 394 ± 12 mg/dl (zero time) to 47.7 ± 10.3 mg/dl (88% reduction) 6 h after injection (P < 0.001 compared with zero time by one-way ANOVA; Fig 4a). Food was provided after the 6-h blood sample. At 24 h, cholesterol levels had increased, but remained significantly less than the initial levels (282 ± 18 mg/dl; P < 0.001 compared with zero time by one-way ANOVA; Fig 4a). Injection of PBS (n = 7) did result in significant reduction of plasma cholesterol at 6 h compared with initial levels (P < 0.001), but the reduction was significantly less than in the Ac-hE18A-NH₂ mice (Fig 4a). The reduction in plasma cholesterol in Ac-hE18A-NH₂-injected mice was primarily in the VLDL and IDL/LDL regions of the profile; a representative profile from an individual animal is presented in Fig 4b. Injection of Ac-18A-NH₂ into fasting apoE gene knockout mice did not result in significant reductions of plasma cholesterol at 4 h compared with PBS. Total cholesterol levels at 4 h in Ac-18A-NH₂-injected mice were reduced 22.7 ± 3.2% (n = 4) compared with initial levels, whereas those in PBS-injected mice were reduced 22.2 ± 2.7% (n = 4) compared with initial levels. These reductions were comparable to those seen in PBS-injected mice in Fig 4a (29.1 ± 1.4%).

View larger version (29K): [in this window] [in a new window]   Figure 4. Clearance of plasma cholesterol following intravenous injection of Ac-hE18A-NH2 in apoE gene knockout mice. Fasting female apoE gene knockout mice were injected intravenously through the tail vein with either PBS (100 µl; n = 7) or Ac-hE18A-NH2 (100 µg in 100 µl PBS; n = 8). Blood samples were taken under anesthesia immediately before injection (zero time) and at 6 and 24 h. Plasma was collected and analyzed for total cholesterol and for plasma cholesterol profiles by the CLiP method (31). A: Total cholesterol levels in PBS (solid bars) or peptide-injected (open bars) mice are shown as mean ± SEM. * P < 0.05 versus PBS at the same time point by two-tailed t-test; ** P < 0.0001 versus PBS at the same time point by two-tailed t-test. B: Representative plasma cholesterol profiles at zero time (solid line) and 6 h (dashed line) in a single peptide-injected mouse.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present data show that Ac-hE18A-NH₂ and Ac-18A-NH₂ associate with both LDL and VLDL, but only the dual-domain peptide, Ac-hE18A-NH₂, enhanced the uptake and degradation of VLDL and LDL by HepG2 cells. ApoE is required for the LDLR-mediated uptake of VLDL S_f >60 (2) (14). Both Ac-hE18A-NH₂ and Ac-18A-NH₂ displaced apoE from VLDL. Therefore, these apoE-deficient VLDL would be expected to be internalized by apoE-mediated cellular pathways to a lesser degree than the control VLDL. This was observed for Ac-18A-NH₂-treated and trypsinized VLDL; however, Ac-hE18A-NH₂-treated VLDL associated with HepG2 cells more efficiently (6-fold) than the control VLDL. The degradation was also enhanced 3-fold over the control. Thus the increased cell association of apoE-deficient VLDL-Ac-hE18A-NH₂ complex implies that Ac-hE18A-NH₂ is able to mimic apoE functionally for the enhancement of uptake and degradation of VLDL. The uptake of the VLDL-dual-domain peptide complex may be greater than VLDL alone because the number of Ac-hE18A-NH₂ molecules (potential ligand domains) bound per VLDL is greater than the number of apoE molecules in the native control VLDL. The relative enhanced uptake observed with trypsinized VLDL, despite the fact that all immunochemically detectable apoE was lost due to trypsinization, may be due to the same reason just stated or, perhaps, to the lower uptake of the trypsinized VLDL relative to the complex. Although the enhancement of VLDL internalization was 6-fold in presence of the peptide, degradation was not enhanced to the same extent (only 3-fold). This suggests the possibility that the VLDL-peptide complex internalization may occur through both receptor-dependent and receptor-independent pathways and/or that the degradation is at its maximum. It is also possible that the degradation pathway is inhibited by the peptide. These issues will be the subject of further investigation. Although HSPG is implicated in the internalization process, other receptor-dependent pathways may also be contributing to the transfer of VLDL from HSPG to receptor for endocytic degradation. All these parameters might reflect a greater internalization relative to the measured endocytic degradation.

The effect of Ac-hE18A-NH₂ on the uptake of LDL and VLDL in HepG2 cells (Fig 1 and Fig 3a) was examined using ¹²⁵I-VLDL treated with peptide at a 1:1 ratio and re-isolating the lipoprotein-peptide complex. When the peptide-mediated LDL and VLDL uptake and degradation were compared on the basis of the amount of cholesterol to which the cells were exposed, the following results were obtained. Assuming as a first approximation and based on the protein-to-total cholesterol ratio per lipoprotein, it can be seen from the apoprotein uptake in Fig 1 that 1.8 µg of LDL cholesterol/mg of cell protein are taken up. However, in the presence of peptide, 9.0 µg of cholesterol/mg of cell protein was taken up. The results with VLDL suggest that approximately 3.2;–5 µg of VLDL cholesterol/mg of cell protein are internalized in the absence of Ac-hE18A-NH₂ and 20;–32 µg of VLDL cholesterol/mg of cell protein in the presence of the dual-domain peptide (Fig 3a), based on the calculation. Because VLDL is a larger particle than LDL, it contains more total cholesterol per particle. Thus peptide-mediated enhanced uptake of VLDL could result in internalization of greater amounts of cholesterol per particle. In the range of the peptide concentration studied (1:0.15, 1:0.3, 1:0.6, and 1:1), the uptake of both lipoproteins was dependent on the concentration of the peptide. The greater the number of Ac-hE18A-NH₂ molecules bound to the lipoprotein, the greater the internalization of the lipoprotein. Thus it appears that due to the greater surface area of VLDL, less surface pressure on VLDL, or both, more peptide can bind per VLDL particle as compared with LDL. Furthermore, displacement of apoE from the VLDL surface by the peptide perhaps exposes more lipid surface for peptide to associate. A greater number of peptide molecules compared with apoE (increased ligand-binding domains relative to one apoE) could bind VLDL; thus more Ac-hE18A-NH₂-VLDL is taken up than VLDL itself. Although both Ac-hE18A-NH₂ and Ac-18A-NH₂ bind VLDL to the same extent on a molar basis, only Ac-hE18A-NH₂ facilitates an increased binding and uptake of VLDL, suggesting that the net positive charge plays an important role in the cell association to HepG2 cells and fibroblasts (1).

ApoE is involved normally in the clearance of remnant atherogenic lipoproteins via not only the LDLR and LDLR-related proteins but also the HSPG-mediated pathway (2). It is possible that Ac-hE18A-NH₂ on the basis of our in vitro results might also assist in the rapid clearance of atherogenic lipoproteins in vivo. In experiments presented here, intravenous administration of the Ac-hE18A-NH₂ in apoE knockout mice reduced plasma cholesterol by 88% within 6 h and reduced levels were maintained at 24 h even with only a single injection (Fig 4a). However, administration of the control peptide Ac-18A-NH₂ reduced plasma cholesterol levels only by 22% as did PBS. These results were similar to those reported by us (33) with intraperitoneal administration of Ac-18A-NH₂ in C57BL/6J mice fed an atherogenic diet.

HSPG mediate the internalization of apoE-containing lipoproteins either via the receptor-facilitated mechanism or independently. The Arg-rich heparin-binding region [141;–150] of apoE, LRKLRKRLLR, would be expected to have a high affinity for cell-surface proteoglycans (34); our data support this observation. The portion of the sequence in bold corresponds to the consensus sequence of heparin-binding proteins, which bind glycosaminoglycans (GAG) (34). It appears that the amphipathic lipid-binding domain of Ac-hE18A-NH₂ binds to the lipoprotein surface while the positively charged domain directs the lipoprotein-peptide complex to the negatively charged proteoglycan for enhanced clearance via several potential pathways.

In summary, Ac-hE18A-NH₂ significantly enhances the uptake of atherogenic lipoproteins LDL and VLDL by HepG2 cells, whereas the peptide Ac-18A-NH₂, which does not possess the Arg-rich sequence LRKLRKRLLR does not. Enhanced VLDL uptake and degradation was observed despite the loss of apoE from the VLDL surface. A rapid and enhanced clearance of VLDL/LDL cholesterol in apoE knockout mice suggests that the peptide has the potential to lessen lesion formation by lowering plasma levels of atherogenic apoB-containing lipoproteins. Studies are in progress to determine whether lesion formation is decreased and the mechanism and site of this enhanced uptake of the apoB-containing lipoproteins and their fate after internalization.

	ACKNOWLEDGMENTS

We thank Dr. Steve Barnes and Marion Kirk for the mass spectral analyses of peptides. This work was supported in part by NIH grants HL34343 (G.M.A.), HL44480 (S.H.G.), and HL64156 (W.A.B.).

Manuscript received November 29, 2000; and in revised form February 2, 2001

Abbreviations: Ac-, acetyl; apoB, apolipoprotein B; HSPG, heparan sulfate proteoglycan; LPDS, lipoprotein deficient serum; Mtr, 4-methoxy-2,3,6-trimethylbenzenesulfonyl; TFA, trifluoroacetic acid

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1. Datta, G., Chaddha, M., Garber, D. W., B-H. Chung, E. M., Tytler, N., Dashti, W. A., Bradley, S. H., Gianturco,, Anantharamaiah, G. M. 2000. The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts. Biochemistry. 39:213-220[Medline].

2. Mahley, R. W., S Ji, Z. 1999. Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J. Lipid Res. 40:1-16[Abstract/Free Full Text].

3. Mahley, R. W. 1988. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 240:622-630[Abstract/Free Full Text].

4. Zeng, B-I, B-C. Mortimer, I. J., Martins, U., Seydel,, Redgrave, T. G. 1998. Chylomicron remnant uptake is regulated by the expression and function of heparan sulfate proteoglycan in hepatocytes. J. Lipid Res. 39:845-860[Abstract/Free Full Text].

5. Fan, J., Ji, Z-S., Huang, Y., De Sliva, H., Sanan, D., Mahley, R. W., Innerarity, T. L., Taylor, J. M. 1998. Increased expression of apolipoprotein E in transgenic rabbits results in reduced levels of very low density lipoproteins and an accumulation of low density lipoproteins in plasma. J. Clin. Invest. 101:2151-2164[Medline].

6. Yamada, N., Shimano, H., Mokuno, H., Ishibashi, S., Gotoda, M., Kawakami, M., Watanabe, Y., Akanuma, Y., Murase, T., Takaku, F. 1989. Increased clearance of plasma cholesterol after injection of apolipoprotein E into Watanbe heritable hyperlipidemic rabbits. Proc. Natl. Acad. Sci. USA. 86:665-669[Abstract/Free Full Text].

7. Shimano, H., Yamada, N., Katsuki, M., Yamamoto, K., Gotoda, T., Harada, K., Shimada, M., Yazaki, Y. 1992. Plasma lipoprotein metabolism in transgenic mice overexpressing apolipoprotein E. Accelerated clearance of lipoproteins containing apolipoprotein B. J. Clin. Invest. 90:2084-2091.

8. Zhang, S. H., Reddick, R. L., Piedrahita, J. A., Maeda, N. 1992. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 258:468-471[Abstract/Free Full Text].

9. Plump, A. S., Smith, J. D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J. G., Rubin, E. M., Breslow, J. L. 1992. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 71:343-353[Medline].

10. Spangenberg, J., Curtis, L. K. 1997. Influence of macrophage-derived apolipoprotein E on plasma lipoprotein distribution of apolipoprotein A-I in apolipoprotein E-deficient mice. Biochim. Biophys. Acta. 1349:109-121[Medline].

11. Fazio, S., Babaev, V. R., Murray, A. B., Hasty, A. H., Carter, K. J., Gleaves, L. A., Atkinson, J. B., Linton, M. F. 1997. Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages. Proc. Natl. Acad. Sci. USA. 94:4647-4652[Abstract/Free Full Text].

12. Sehayek, E., Eisenberg, S. 1990. Abnormal composition of hypertriglyceridemic VLDL determines abnormal cell metabolism. Arteriosclerosis. 10:1088-1096[Abstract/Free Full Text].

13. Gianturco, S. H., Brown, F. B., Gotto, A. M., Jr., Bradley, W. A. 1982. Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts. J. Lipid Res. 23:984-993[Abstract].

14. Bradley, W. A., Gianturco, S. H. 1986. Apo E is necessary and sufficient for the binding of large triglyceride-rich lipoproteins to the LDL receptor; apo B is unnecessary. J. Lipid Res. 27:40-48[Abstract].

15. Bradley, W. A., Hwang, S. L., Karlin, J. B., Lin, A. H., Prasad, S. C., Gotto, A. M., Jr., Gianturco, S. H. 1984. Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglysceridemic very-low-density lipoprotein to low density lipoproteins. J. Biol. Chem. 259:14728-14735[Abstract/Free Full Text].

16. Gianturco, S. H., Gotto, A. M., Jr., C, S-L., Huang, J. B., Karlin, A. H. Y., Lin, S. C., Prasad,, Bradley, W. A. 1983. Apolipoprotein E mediates uptake of Sf 100;–400 hypertriglyceridemic very low density lipoproteins by the low density lipoprotein receptor pathway in normal human fibroblasts. J. Biol. Chem. 258:4526-4533[Free Full Text].

17. Sparrow, J. T., Sparrow, D. A., Fernando, G., Culwell, A. R., Kovar, M., Gotto, A. M., Jr. 1992. Apolipoprotein E: Phospholipid binding studies with synthetic peptides from the carboxyl terminus. Biochem. 31:1065-1068[Medline].

18. Rall, S. C., Jr., Weisgraber, K. H., Innerarity, T. L., Mahley, R. W. 1982. Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects. Proc. Natl. Acad. Sci. USA. 79:4696-4700[Abstract/Free Full Text].

19. Lalazar, A., Weisgraber, K. H., Rall, S., Giladi, H., Innerarity, T. L., Levanon, A. Z., Boyles, J. K., Amit, B., Gorecki, M., Mahley, R. W., Vogel, T. 1988. Site-specific mutagenesis of human apolipoprotein E. Receptor binding activity of variants with single amino acid substitutions. J. Biol. Chem. 263:3542-3545[Abstract/Free Full Text].

20. Dyer, C. A., Curtiss, L. K. 1991. A synthetic peptide mimic of plasma apolipoprotein E that binds the LDL receptor. J. Biol. Chem. 296:22803-22806.

21. Dyer, C. A., Smith, R. S., Curtiss, L. K. 1991. Only multimers of a synthetic peptide of human apolipoprotein E are biologically active. J. Biol. Chem. 266:15009-15015[Abstract/Free Full Text].

22. Wilson, C., Wardell, M. R., Weisgraber, K. H., Mahley, R. W., Agard, D. A. 1991. Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E. Science. 252:1817-1822[Abstract/Free Full Text].

23. Zaiou, M., Arnold, K. S., Newhouse, Y. M., Innerarity, T. L., Weisgraber, K. H., Segall, M. L., Phillips, M. C., Lund-Katz, S. 2000. Apolipoprotein E-low density lipoprotein receptor interaction: Influences of basic residue and amphipathic -helix organization in the ligand. J. Lipid Res. 41:1087-1095[Abstract/Free Full Text].

24. Chung, B. H., Palgunachari, M. N., Mishra, V. K., Chang, C. H., Segrest, J. P., Anantharamaiah, G. M. 1996. Probing the structure and function of VLDL by synthetic amphipathic helical peptides. J. Lipid Res. 37:1099-1112[Abstract].

25. Schumaker, V. N., Puppione, D. L. 1986. Sequential flotation ultracentrifugation. Methods Enzymol. 128:155-170[Medline].

26. Goldstein, J. L., Basu, S. K., Brown, M. S. 1983. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 98:241-260[Medline].

27. Gianturco, S. H., Gotto, A. M., Jr., Huang, L. C., Karlin, J. B., Lin, A. H. Y., Prasad, S. C., Bradley, W. A. 1983. Apolipoprotein E mediates uptake of Sf 100;–400 hypertriglyceridemic very low density lipoproteins by the low density lipoprotein receptor pathway in normal human fibroblasts. J. Biol. Chem. 258:4526-4533.

28. Bilheimer, D. W., Eisenberg, S., Levy, R. I. 1972. The metabolism of very low density lipoprotein proteins. I. Preliminary in vitro and in vivo observations. Biochim. Biophys. Acta. 260:212-221[Medline].

29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275[Free Full Text].

30. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-685[Medline].

31. Garber, D. W., Kulkarni, K. R., Anantharamaiah, G. M. 2000. A convenient and sensitive method for lipoprotein analysis of individual mouse plasma samples. J. Lipid Res. 41:1020-1026[Abstract/Free Full Text].

32. Bradley, W. A., Gotto, A. M., Jr., Gianturco, S. H. 1985. Expression of LDL receptor binding determinants in very low density lipoproteins. Ann. N.Y. Acad. Sci. 454:239-247[Medline].

33. Garber, D. W., Datta, G., Chaddha, M., Palgunachari, M. N., Hama, S. Y., Navab, M., Fogelman, A. M., Segrest, J. P., Anantharamaiah, G. M. 2001. A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis. J. Lipid Res. 42:545-552[Abstract/Free Full Text].

34. Verrecchio, A. M., Germann, W., Schick, B. P., Kung, B., Twardowski, T., San Antonio, J. D. 2000. Design of peptides with high affinities for heparin and endothelial cell proteoglycans. J. Biol. Chem. 275:7701-7707[Abstract/Free Full Text].

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