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• W2153350589 abstract "Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP).Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors. Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors. Apolipoprotein E (apoE) is a 34 kDa protein that plays an important role in lipoprotein metabolism by association with lipoprotein particles and with members of the LDL receptor family (1Mahley R.W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology.Science. 1988; 240: 622-630Google Scholar, 2Weisgraber K.H. Apolipoprotein E: structure-function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar). ApoE contains a 22 kDa N-terminal domain (residues 1–191) that is recognized by receptors and a 10 kDa C-terminal domain (residues 222–299) that has high affinity for lipid and is responsible for the association of apoE with lipoproteins (3Wetterau J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains.J. Biol. Chem. 1988; 263: 6240-6248Google Scholar, 4Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain.Biochemistry. 2000; 39: 11657-11666Google Scholar). Three major isoforms of apoE exist in the population and differ by cysteine and arginine at residues 112 and 158. The most common isoform, apoE3, contains cysteine and arginine at these positions, respectively, whereas apoE2 contains cysteine at both positions and apoE4 contains arginine at both positions (5Weisgraber K.H. Rall Jr., S.C. Mahley R.W. Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms.J. Biol. Chem. 1981; 256: 9077-9083Google Scholar). These substitutions have important biological consequences. First, the various apoE isoforms are differentially recognized by the LDL receptor. Thus, apoE3 and apoE4 readily bind to the LDL receptor, whereas apoE2 binds poorly to the LDL receptor and is associated with type III hyperlipidemia (6Mahley R.W. Huang Y. C. Rall Jr, S. Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes.J. Lipid Res. 1999; 40: 1933-1949Google Scholar). Second, the APOE-ε4 allele is associated with the common late-onset familial and sporadic forms of Alzheimer's disease (AD) (7Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Apolipoprotein E: high avidity binding to B-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease.Proc. Natl. Acad. Sci. USA. 1993; 90: 1977-1981Google Scholar, 8Corder E.H. Saunders A.M. Strittmatter W.J. Schmechel D.E. Gaskell P.C. Small G.W. Roses A.D. Haines J.L. Pericak-Vance M.A. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families.Science. 1993; 261: 921-923Google Scholar). The biochemical mechanism by which the APOE-ε4 allele increases the risk of AD is unknown, but several possibilities have been proposed (9Nathan B.P. Bellosta S. Sanan D.A. Weisgraber K.H. Mahley R.W. Pitas R.E. Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro.Science. 1994; 264: 850-852Google Scholar, 10LaDu M.J. Falduto M.T. Manelli A.M. Reardon C.A. Getz G.S. Frail D.E. Isoform-specific binding of apolipoprotein E to B-amyloid.J. Biol. Chem. 1994; 269: 23403-23406Google Scholar, 11Evans K.C. Berger E.P. Cho C-G. Weisgraber K.H. T. Lansbury Jr, P. Apolipoprotein E is a kinetic but not a thermodynamic inhibitor of amyloid formation: implications for the pathogenesis and treatment of Alzheimer disease.Proc. Natl. Acad. Sci. USA. 1995; 92: 763-767Google Scholar), including differential functions of apoE isoforms upon interaction with members of the LDL receptor family (9Nathan B.P. Bellosta S. Sanan D.A. Weisgraber K.H. Mahley R.W. Pitas R.E. Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro.Science. 1994; 264: 850-852Google Scholar). The LDL receptor family includes the LDL receptor, the LDL receptor-related protein (LRP), LRP1b, megalin (or LRP-2), the VLDL receptor, and apoE receptor 2 (for review, see 12Strickland D.K. Gonias S.L. Argraves W.S. Diverse roles for the LDL receptor family.Trends Endocrinol. Metab. 2002; 13: 66-74Google Scholar). The LDL receptor recognizes apoE and apoB-100 and plays a critical role in cholesterol homeostasis (13Brown M.S. Goldstein J.L. Receptor-mediated control of cholesterol metabolism.Science. 1976; 191: 150-154Google Scholar), whereas the structurally related VLDL receptor recognizes apoE, but not apoB-100, and plays an important role in triglyceride metabolism (14Tacken P.J. Teusink B. Jong M.C. Harats D. Havekes L.M. Van Dijk K.W. Hofker M.H. LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice.J. Lipid Res. 2000; 41: 2055-2062Google Scholar). In addition, the VLDL receptor also participates in the reelin signaling pathway, which is important for correct cortical neuron migration during development (15Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and apoE receptor 2.Cell. 1999; 97: 689-701Google Scholar, 16Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Direct binding of reelin to VLDL receptor and apoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation.Neuron. 1999; 24: 481-489Google Scholar). LRP is widely expressed in a variety of tissues and cells and is a major neuronal apoE receptor that has been implicated in the development of AD by virtue of its ability to affect the metabolism of amyloid precursor protein (17Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. LDL receptor-related protein, a multifunctional apoE receptor, binds secreted B-amyloid precursor protein and mediates its degradation.Cell. 1995; 82: 331-340Google Scholar, 18Knauer M.F. Orlando R.A. Glabe C.G. Cell surface APP751 forms complexes with protease nexin 2 ligands and is internalized via the low density lipoprotein receptor-related protein (LRP).Brain Res. 1996; 740: 6-14Google Scholar, 19Ulery P.G. Beers J. Mikhailenko I. Tanzi R.E. Rebeck G.W. Hyman B.T. Strickland D.K. Modulation of beta-amyloid precursor protein processing by the low density lipoprotein receptor-related protein (LRP). Evidence that LRP contributes to the pathogenesis of Alzheimer's disease.J. Biol. Chem. 2000; 275: 7410-7415Google Scholar, 20Pietrzik C.U. Busse T. Merriam D.E. Weggen S. Koo E.H. The cytoplasmic domain of the LDL receptor-related protein regulates multiple steps in APP processing.EMBO J. 2002; 21: 5691-5700Google Scholar). In the liver, LRP plays an important role in chylomicron remnant metabolism (21Rohlmann A. Gotthardt M. Hammer R.E. Herz J. Inducible inactivation of hepatic LRP gene by Cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.J. Clin. Invest. 1998; 101: 689-695Google Scholar), whereas in the vasculature, LRP plays an atheroprotective role by suppressing platelet-derived growth factor signaling pathways, thereby inhibiting vascular smooth muscle cell proliferation and migration (22Boucher P. Gotthardt M. Li W.P. Anderson R.G. Herz J. LRP: role in vascular wall integrity and protection from atherosclerosis.Science. 2003; 300: 329-332Google Scholar). Like the VLDL receptor, LRP recognizes numerous ligands, many of which are proteinases and complexes of these proteinases with their target inhibitors. It is well established that association of apoE with lipid is required for its high-affinity binding to the LDL receptor (23Innerarity T.L. Pitas R.E. Mahley R.W. Binding of arginine-rich (E) apoprotein after recombination with phospholipid vesicles to the low density lipoprotein receptors of fibroblasts.J. Biol. Chem. 1979; 254: 4186-4190Google Scholar) and that the LDL receptor preferentially recognizes apoE3 and apoE4 isoforms. However, the binding of apoE to other receptor family members, such as LRP and the VLDL receptor, is not completely characterized. Data to date suggest that binding of apoE to these two receptors may differ from that of the LDL receptor. Thus, Takahashi et al. (24Takahashi S. Oida K. Ookubo M. Suzuki J. Kohno M. Murase T. Yamamoto T. Nakai T. Very low density lipoprotein receptor binds apolipoprotein E2/2 as well as apolipoprotein E3/3.FEBS Lett. 1996; 386: 197-200Google Scholar) found that the VLDL receptor readily recognizes apoE2 containing VLDL particles, whereas Narita et al. (25Narita M. Holtzman D.M. Fagan A.M. LaDu M.J. Yu L. Han X. Gross R.W. Bu G. Schwartz A.L. Cellular catabolism of lipid poor apolipoprotein E via cell surface LDL receptor-related protein.J. Biochem. (Tokyo). 2002; 132: 743-749Google Scholar) found that cells can catabolize lipid-poor apoE forms via an LRP-mediated process. In the current investigation, we sought to characterize the interactions between apoE, the VLDL receptor, and LRP to gain understanding of the role of these receptors in a variety of physiological processes. Our results indicate that the apoE binding properties of the VLDL receptor differ markedly from those of the LDL receptor. The soluble VLDL receptor fragment containing ligand binding repeats 1–8 (sVLDLr1–8) was prepared and characterized as described (26Hembrough T.A. Ruiz J.F. Papathanassiu A.E. Green S.J. Strickland D.K. Tissue factor pathway inhibitor inhibits endothelial cell proliferation via association with the very low density lipoprotein receptor.J. Biol. Chem. 2001; 276: 12241-12248Google Scholar). In some experiments, we used a soluble form of the human VLDL receptor termed sVLDLr that contains the entire ectodomain. This receptor was prepared using the Drosophila Expression System (Invitrogen) using the inducible/secreted kit according to the manufacturer's protocol. The secreted sVLDLr was purified by first removing Cu2+ ions from the media by passage over a Chelex-100 (Bio-Rad) column and then by affinity chromatography over receptor-associated protein (RAP)-Sepharose as described (26Hembrough T.A. Ruiz J.F. Papathanassiu A.E. Green S.J. Strickland D.K. Tissue factor pathway inhibitor inhibits endothelial cell proliferation via association with the very low density lipoprotein receptor.J. Biol. Chem. 2001; 276: 12241-12248Google Scholar). The apoE binding properties of the two soluble forms of the VLDL receptor were similar. Soluble forms of the LDL receptor were prepared in Escherichia coli (27Simmons T. Newhouse Y.M. Arnold K.S. Innerarity T.L. Weisgraber K.H. Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli.J. Biol. Chem. 1997; 272: 25531-25536Google Scholar). LRP was purified from human placenta (28Ashcom J.D. Tiller S.E. Dickerson K. Cravens J.L. Argraves W.S. Strickland D.K. The human A2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of A2-macroglobulin.J. Cell Biol. 1990; 110: 1041-1048Google Scholar), whereas RAP was expressed in E. coli and prepared as described (29Williams S.E. Ashcom J.D. Argraves W.S. Strickland D.K. A novel mechanism for controlling the activity of A2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.J. Biol. Chem. 1992; 267: 9035-9040Google Scholar). ApoE2, apoE3, and apoE4 were prepared as described (30Morrow J.A. Arnold K.S. Weisgraber K.H. Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.Protein Expr. Purif. 1999; 16: 224-230Google Scholar). Because of the presence of cysteine in apoE2 and apoE3, they are prone to form intermolecular disulfide-linked forms that were visualized by SDS-PAGE under nonreducing conditions. When present, the disulfide-linked aggregates were removed by dialyzing the protein into 20 mM HEPES, 150 mM NaCl, pH 7.4 (HBS buffer) containing 20 mM DTT for 1 h at room temperature, followed by dialysis overnight against nitrogenated HBS buffer. SDS-PAGE under nonreducing conditions and fast-protein liquid chromatography analysis confirmed that apoE2 and apoE3 preparations were free of disulfide-linked structures after treatment. ApoE monoclonal antibodies 3H1 (31Weisgraber K.H. Rall Jr., S.C. Mahley R.W. Milne R.W. Marcel Y.L. Sparrow J.T. Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3.J. Biol. Chem. 1986; 261: 2068-2076Google Scholar) and 1D7 (32Milne R.W. Douste-Blazy P. Marcel Y.L. Retegui L. Characterization of monoclonal antibodies against human apolipoprotein E.J. Clin. Invest. 1981; 68: 111-117Google Scholar) have been described. Mouse monoclonal anti-VLDL receptor antibodies 5F3, 1H5, and 1H10 were generated by immunizing VLDL receptor knockout mice with recombinant sVLDLr1–8 and prepared as described (33Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. Sequence identity between the A2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor.J. Biol. Chem. 1990; 265: 17401-17404Google Scholar). Screening was performed using microtiter wells coated with sVLDLr1–8. Antibodies were purified using protein G-Sepharose (Amersham Pharmacia Biotech). Purified mouse IgGs from Sigma-Aldrich, Inc. (St. Louis, MO), were used as controls for mouse anti-VLDL receptor antibodies. For assays involving cells, IgG samples were heat-inactivated for 30 min at 50°C before use. BSA was purchased from Sigma-Aldrich, Inc. 293 cells were cultured in Dulbecco's modified eagle's medium (DMEM), 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. 293 cells were transfected with a plasmid expressing the human VLDL receptor, and clones were selected by growing in EMEM, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml hygromycin. All cells were passaged at subconfluence using enzyme-free cell dissociation buffer (Sigma-Aldrich, Inc.). Tissue culture plates, including 6-well and 12-well plates, were from Fisher Scientific (Pittsburgh, PA). ApoE isoforms were immobilized on microtiter wells (IMMULON 2HB plates from Fisher Scientific) at a concentration of 4 μg/ml. The microtiter wells were then blocked with 3% BSA. LRP and sVLDLr1–8 were added at the concentrations indicated, and binding was allowed to occur for 16 h at 4°C. After binding, wells were washed three times. Bound LRP was detected with monoclonal antibody 11H4, and bound sVLDLr1–8 was detected with mouse polyclonal antibodies against sVLDLr1–8. To determine specificity, the binding of LRP and sVLDLr1–8 to BSA-coated wells was also measured. Bound monoclonal antibodies were detected with anti-mouse IgG-alkaline phosphatase-conjugated antibodies (Bio-Rad). After incubation with phosphatase substrate (Sigma number 104) in 0.1 M glycine, 1 mM MgCl2, and 1 mM ZnCl2, pH 10.4, the absorbance for each sample was measured at 405 nm. Data were analyzed by nonlinear regression analysis using SigmaPlot. To measure the binding of monoclonal antibodies to the VLDL receptor, sVLDLr1–8 was first immobilized onto microtiter wells. After blocking with BSA, increasing amounts of antibodies were added. After binding and washing, bound monoclonal antibodies were detected with anti-mouse IgG-alkaline phosphatase-conjugated antibodies (Bio-Rad). After incubation with phosphatase substrate (Sigma number 104) in 0.1M glycine, 1 mM MgCl2, and 1 mM ZnCl2, pH 10.4, the absorbance for each sample was measured at 405 nm. Data were analyzed using nonlinear regression analysis using SigmaPlot. To evaluate the affinity of lipid-free apoE isoforms for VLDL receptor and LRP, we used surface plasmon resonance (SPR) with a BIAcore 3000 biosensor (BIAcore AB, Uppsala, Sweden). Purified sVLDL1–8 and LRP were immobilized onto a CM5 sensor chip surface at densities of 3.5 fmol/mm2 [120 resonance units (RU)] and 5.8 fmol/mm2 (3,500 RU), respectively, by amine coupling in accordance with the manufacturer's instructions (BIAcore AB). One flow cell was activated and blocked with 1 M ethanolamine without any protein and was used as a control surface to normalize SPR signal from receptors immobilized with flow cells. Most of the binding experiments were conducted in standard HBS-P buffer, pH 7.4 (BIAcore AB), containing 0.005% Tween 20 at a flow rate of 30 μl/min and temperature of 25°C. Some direct binding experiments with the LRP and sVLDLr1–8 immobilized receptors were carried out in the presence of 2 mM CaCl2 in HBS-P buffer at a flow rate of 10 μl/min. Sensor chip surfaces were regenerated by 30 s pulses of 100 mM H3PO4. All injections used the Application Wizard in the automated method. Data were analyzed with BIA evaluation 3.0 software (BIAcore AB) using the equilibrium analysis model. The maximum change in response units (Rmax) from this analysis was replotted versus apoE concentration, and the data were fit to a single class of sites by nonlinear regression analysis using SigmaPlot 9.0 software. To test the binding of wild-type and mutant apoE3 with monoclonal antibodies 3H1 and 1D7, we used a capture assay in which monoclonal antibodies 3H1 and 1D7 (100 nM) were first captured on goat anti-mouse Fc-γ-specific IgG for 3 min in QUICKINJECT mode. After capture, 100 nM wild-type or mutant apoE3 or running buffer was injected using KINJECT mode. Because the concentration of goat anti-mouse Fc-γ-specific IgG::monoclonal antibody complexes was constant in each cycle, the changes in Rmax value reflect the differences in affinity for wild-type and mutant forms of apoE3. Chip surfaces were regenerated by a 1 min pulse of 10 mM glycine buffer, pH 1.5, or 20 mM HCl followed by a 2 min equilibration with running buffer. The absolute SPR responses for wild-type and mutant apoE were compared and expressed as relative values (percentages) to wild-type apo E. To measure the binding of apoE3 and mutant proteins to the VLDL receptor, 100 nM of each protein was injected directly over the CM5 chip surface in which sVLDLr was immobilized at a density of 3,000 RU. As a control for the experiment, a flow cell with immobilized ovalbumin (500 RU) was used. All injections were done in KINJECT mode, and Rmax reflected the SPR response of apoE3 or mutant protein binding to the VLDL receptor. Anti-VLDL receptor IgG 5F3 and anti-apoE IgG 3H1 were radiolabeled with 125I (Amersham Pharmacia Biotech, Piscataway, NJ) to a specific activity ranging from 2 to 10 μCi/μg protein using Iodogen (Pierce Chemical Co., Rockford, IL). For these assays, wild-type 293 cells, 293/VLDLR transfected cells, LRP-expressing mouse embryonic fibroblasts, or PEA-13 cells (LRP-deficient) were seeded onto 6-well plates (precoated with 0.1% gelatin) as indicated and grown overnight at 37°C in 5% CO2. Cell media were removed, and cells were washed once with assay medium (DMEM, 20 mM HEPES, pH 7.4, and 1.5% BSA) and incubated in this medium for 20–30 min. Cells were then incubated with assay medium containing radiolabeled proteins in the absence or presence of excess unlabeled competitors as indicated at 37°C. In those experiments measuring the internalization of apoE using radiolabeled antibody 3H1, we confirmed that the uptake of this monoclonal antibody was totally dependent upon the addition of exogenous apoE. After incubation, cell media were removed for the determination of degraded counts, and cells were detached with trypsin/proteinase K, then pelleted by centrifugation. Bound counts were measured by determining the counts in the supernatant of pelleted cells. Internalized counts were those associated with the cell pellet, whereas degraded counts were measured in the trichloroacetic acid-soluble fraction of the culture supernatant. Radioactivity was measured in a γ-counter. Soluble fragments of the VLDL receptor ligand binding domain were transiently expressed in Cos-1 cells using calcium phosphate precipitation as described (34Mikhailenko I. Considine W. Argraves K.M. Loukinov D. Hyman B.T. Strickland D.K. Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms.J. Cell Sci. 1999; 112: 3269-3281Google Scholar). Briefly, a 100 mm dish at ∼60% confluence was cotransfected with 10 μg of pSecTagB containing the cDNA for soluble human VLDL receptor fragments and 5 μg of pcDNA3RAP. Cells were washed 18 h after transfection and kept in serum-containing medium for another 24 h. Then, the medium was replaced with DMEM containing 1% Nutridoma. This medium was harvested after 48 h of incubation, subjected to immunoblot analysis using anti-myc antibody to detect recombinant proteins, and used in the binding assays. Normally, apoE only binds to the LDL receptor when incorporated into lipoprotein particles. However, Simmons et al. (27Simmons T. Newhouse Y.M. Arnold K.S. Innerarity T.L. Weisgraber K.H. Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli.J. Biol. Chem. 1997; 272: 25531-25536Google Scholar) demonstrated that the LDL receptor recognition site on apoE is exposed when lipid-free apoE is immobilized on microtiter wells. Using this assay, they demonstrated that a soluble LDL receptor bound poorly to immobilized apoE2 but bound with high affinity to immobilized apoE3 and apoE4, confirming the known specificity of the LDL receptor for apoE isoforms (27Simmons T. Newhouse Y.M. Arnold K.S. Innerarity T.L. Weisgraber K.H. Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli.J. Biol. Chem. 1997; 272: 25531-25536Google Scholar). To gain insight into the apoE binding properties of LRP and the VLDL receptor, we used this assay and measured the binding of purified LRP and VLDL receptor to various apoE isoforms immobilized on microtiter wells. The results of these experiments reveal that both the VLDL receptor (Fig. 1A–C)and LRP (Fig. 1D–F) bind to all isoforms of apoE when immobilized on microtiter wells. The apparent Kd values for the interaction of the VLDL receptor with immobilized apoE2, apoE3, and apoE4 are 86, 59, and 77 nM, respectively. The interaction of LRP with immobilized apoE was considerably stronger, with apparent Kd values of 1.6, 1.7, and 1.1 nM for the apoE2, apoE3, and apoE4 isoforms, respectively. Thus, the results from this solid-phase assay suggest that both the VLDL receptor and LRP do not discriminate between apoE isoforms. We next investigated the ability of the VLDL receptor and LRP to bind lipid-free forms of apoE. Previously, LRP has been immobilized on SPR surfaces and successfully used to investigate its binding of various ligands (35Neels J.G. van Den Berg B.M. Lookene A. Olivecrona G. Pannekoek H. Van Zonneveld A.J. The second and fourth cluster of class A cysteine-rich repeats of the low density lipoprotein receptor-related protein share ligand-binding properties.J. Biol. Chem. 1999; 274: 31305-31311Google Scholar, 36Neels J.G. van Den Berg B.M. Mertens K. Maat H. ter Pannekoek H. Van Zonneveld A.J. Lenting P.J. Activation of factor IX zymogen results in exposure of a binding site for low-density lipoprotein receptor-related protein.Blood. 2000; 96: 3459-3465Google Scholar, 37Loukinova E. Ranganathan S. Kuznetsov S. Gorlatov N. Migliorini M.M. Loukinov D. Ulery P.G. Mikhailenko I. Lawrence D.A. Strickland D.K. PDGF-induced tyrosine phosphorylation of the LDL receptor-related protein (LRP): evidence for integrated co-receptor function between LRP and the PDGF receptor.J. Biol. Chem. 2002; 277: 15499-15506Google Scholar). To determine if either LRP or the VLDL receptor is capable of also recognizing lipid-free forms of apoE in solution, we immobilized purified forms of these receptors on CM5 chips and monitored the real-time binding of different lipid-free apoE isoforms injected over the chip surfaces as analytes using biosensor BIAcore 3000. The data reveal that all apoE isoforms readily bound to the immobilized sVLDLr1–8 (Fig. 2A–C). Equilibrium analysis of observed sensorgrams revealed a high-affinity interaction between the VLDL receptor and all apoE isoforms, with no distinction between them (Table 1). In contrast, LRP failed to recognize lipid-free apoE2 and apoE3 and bound weakly to lipid-free apoE4 in solution (Fig. 2D–F). In some experiments, we noted weak binding of lipid-free apoE2 and apoE3 isoforms to LRP, and this was attributed to the presence of disulfide-linked oligomers that these proteins are known to form (38Weisgraber K.H. Shinto L.H. Identification of the disulfide-linked homodimer of apolipoprotein E3 in plasma. Impact on receptor binding activity.J. Biol. Chem. 1991; 266: 12029-12034Google Scholar). Upon removal of these oligomers, no binding was detected (data not shown). The weak interaction of LRP with lipid-free apoE4 might result from the tendency of this apoE isoform to undergo aggregation (39Chou C.Y. Lin Y.L. Huang Y.C. Sheu S.Y. Lin T.H. Tsay H.J. Chang G.G. Shiao M.S. Structural variation in human apolipoprotein E3 and E4: secondary structure, tertiary structure, and size distribution.Biophys. J. 2005; 88: 455-466Google Scholar). As expected, the LDL receptor did not bind lipid-free apoE isoforms in SPR experiments (data not shown). We conclude from these experiments that the VLDL receptor readily recognizes lipid-free forms of apoE in solution, whereas LRP does not recognize lipid-free forms of apoE2 and apoE3 and only weakly binds to lipid-free forms of apoE4.TABLE 1Binding constants measured for the binding of lipid-free apoE isoforms to the VLDL receptor and to LRPReceptorapoE2apoE3apoE4nMVLDL receptor20 ± 525 ± 1017 ± 9LRPnbnbweak bindingLDL receptornbnbnbapoE, apolipoprotein E; LRP, LDL receptor-related protein; nb, no binding detected. Kd values were determined using an equilibrium method and replotting maximal change in response units (Rmax) versus apoE concentration. The data were fit to a single binding site using SigmaPlot software. Open table in a new tab apoE, apolipoprotein E; LRP, LDL receptor-related protein; nb, no binding detected. Kd values were determined using an equilibrium method and replotting maximal change in response units (Rmax) versus apoE concentration. The data were fit to a single binding site using SigmaPlot software. We next determined whether the VLDL receptor is capable of mediating the cellular internalization of apoE isoforms when added to cells in a lipid-free state. Very likely, this apoE remains in a lipid-poor state, as Narita et al. (25Narita M. Holtzman D.M. Fagan A.M. LaDu M.J. Yu L. Han X. Gross R.W. Bu G. Schwartz A.L. Cellular catabolism of lipid poor apolipoprotein E via cell surface LDL receptor-related protein.J. Biochem. (Tokyo). 2002; 132: 743-749Google Scholar) found no detectable" @default.
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• W2153350589 date "2005-08-01" @default.
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• W2153350589 title "The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor" @default.
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