Apolipoprotein B-48 or Its Apolipoprotein B-100 Equivalent Mediates the Binding of Triglyceride-Rich Lipoproteins to Their Unique Human Monocyte-Macrophage Receptor

From the University of Alabama at Birmingham, Department of Medicine, Division of Gerontology and Geriatrics (S.H.G., R.S., M.L.B., W.A.B.), Birmingham, Ala; and the Department of Medicine, University of California at San Diego (M.P.R.).

Correspondence to Sandra H. Gianturco, PhD, and William A. Bradley, PhD, 690 Diabetes Research and Education Building, University of Alabama at Birmingham, 1808 Seventh Ave S, Birmingham, AL 35294-0012.

	Abstract

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Abstract—Studies in animals and humans have demonstrated uptake of plasma chylomicrons (triglyceride-rich lipoprotein [TGRLP] of S_f>400) by accessible macrophages in vivo. One potential mechanism is via a unique receptor pathway we previously identified in human blood and THP-1 monocytes and macrophages for the lipoprotein lipase (LpL)– and apolipoprotein (apo) E–independent, high-affinity, specific binding of plasma chylomicrons and hypertriglyceridemic VLDL (HTG-VLDL) to cell-surface membrane-binding proteins (MBP 200, 235; apparent M_r 200, 235 kD on SDS-PAGE) that leads to lipid accumulation in vitro. Competitive binding studies reported here demonstrate that anti-apoB antibodies specifically block the high-affinity binding of TGRLP to this receptor on THP-1 cells and on ligand blots. LpL, which binds to an N-terminal domain of apoB, also inhibits TGRLP binding both to this site on THP-1s and to MBP 200, 235 by binding to apoB. Chylomicrons of S_f>1100 that contain apoB-48, but not apoB-100, bind specifically to MBP 200, 235, and this binding is blocked by anti-apoB IgG. In contrast, lactoferrin and heparin do not inhibit TGRLP binding. We conclude that the receptor-binding domain is within apoB-48 (or an equivalent in apoB-100) near the LpL-binding domain, but not a heparin-binding domain. Uptake of TGRLP by this mechanism could provide essential nutrients or, in HTG, cause excess lipid accumulation and foam cell formation.

Key Words: foam cells • atherosclerosis • hypertriglyceridemia

	Introduction

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The majority of chylomicron remnants are taken up by the liver¹ after sequestration in the space of Disse,² which is enriched with apoE,³ the mediator of hepatic remnant uptake by the LDL receptor or LRP. Human and animal studies indicate that peripheral macrophages also take up intestinally derived plasma chylomicrons (ie, partially lipolyzed plasma TGRLP of S_f>400), which contain apoB-48 as the only apoB species. Ross and Zilversmit⁴ demonstrated extrahepatic uptake of 40% of plasma chylomicrons in rabbits that was decreased by inhibition of the reticuloendothelial system,⁵ which implicated monocyte-macrophages in this extrahepatic uptake. Studies in marmosets (a primate) and rabbits demonstrated substantial (20% to 40% of total) uptake of chylomicrons in vivo by accessible peripheral macrophages, particularly in bone marrow (both animals) and spleen (marmosets and several nonprimate animals).^{6 7} Studies in humans demonstrate that dietary lipoproteins deliver retinyl esters to blood cells, as well as to the liver,⁸ suggesting that plasma chylomicrons serve as a nonmodified, native source of lipid and lipid-soluble vitamins for monocyte-macrophage nutrition in the normal state. In pathological states in which chylomicrons persist in the fasting state, TGRLP (including chylomicrons and their remnants) appear to be involved in the conversion of monocyte-macrophages to foam cells. For example, foam cells are found in the skin, spleen, atherosclerotic lesions, and bone marrow of nondiabetic subjects with fasting chylomicrons (hyperlipoproteinemia types 1, 3, and 5).⁹ Diabetic subjects with fasting chylomicronemia also develop monocyte-macrophage–derived foam cells in eruptive xanthomas that are filled with triglyceride and cholesteryl ester due to uptake of chylomicron-sized lipoproteins by monocyte-macrophages, as demonstrated by chemical analysis and electron microscopy.¹⁰

The in vivo rate and magnitude of TG-rich chylomicron uptake by bone marrow monocyte-macrophages in rabbits and marmosets (20% to 40% of chylomicrons cleared from the plasma by 20 minutes)⁶ suggest this uptake is at least in part receptor mediated, since a bone marrow equivalent to hepatic-like sequestration of chylomicrons has not been reported. This rapid uptake of TG-rich chylomicrons in vivo by accessible bone marrow and spleen macrophages was not, however, accelerated by infusion of apoE,⁶ a surprising finding because apoE is required for the uptake of large (S_f>60) TGRLP by members of the LDL receptor gene family^{11 12 13 14 15 16 17 18} and for hepatic uptake of chylomicron remnants and ß-VLDL.¹⁹ Moreover, in rabbits,⁶ infused apoE diverted much of the uptake from the peripheral macrophages to the liver, suggesting that the observed preinfusion chylomicron uptake by peripheral macrophages was not mediated by apoE and that these macrophages therefore have an alternative apoE-independent uptake mechanism for these TGRLP.

We have demonstrated in human and murine macrophages a receptor that has the ligand-binding characteristics suggested by the above in vivo studies. It is an apoE- and LpL-independent TGRLP receptor pathway that differs from the LDL receptor family and the scavenger receptor family pathways in its properties, including: (1) constitutive expression during differentiation; (2) retarded intracellular ligand degradation; (3) ligand specificity; (4) apparent mass of the candidate receptor proteins (M_r 200, 235 kD); and (5) cellular distribution.^{20 21} Uptake of the high-affinity ligands plasma chylomicrons (S_f>400), HTG-VLDL (S_f 100 to 400), and tryp-VLDL (S_f 100 to 400) immunochemically devoid of apoE, which was used as a model lipoprotein to study apoE-independent mechanisms, via this pathway resulted in rapid (4 hours), massive lipid accumulation and foam cell morphology of these cells in vitro.^{20 21} In contrast, normal VLDL and LDL did not compete for this site and did not cause lipid accumulation. Acetyl LDL also did not compete for this site. Two major TGRLP membrane-binding activities with apparent M_r on SDS-PAGE of 200 and 235 kD (MBP 200, 235) were identified as likely receptor candidates in normal human blood-borne, THP-1, and U937 monocyte-macrophages that have the identical ligand specificities as the cellular site.^{20 21} Limited proteolysis of THP-1 monocyte-macrophages, but not heparinase or heparitinase treatment, abolished both MBP 200, 235 and the high-affinity cell-binding site; both activities recovered in parallel when proteolysis was quenched, strongly implicating MBP 200, 235 as the cellular receptor.²² MBP 200 and 235 are cell-surface proteins with a common protein subunit (MBP 200) that contains the ligand-binding domain.²² The MBPs exhibit the same high-affinity (nanomolar K_d), saturable, specific ligand-binding characteristics of the cellular pathway.^{21 22} MBP 200 has been purified,²³ and the molecular cloning and sequencing of its cDNA are currently in progress. The approximately 3.8 kb so far sequenced (encoding the carboxy-terminal 115 kD of the macrophage protein, including a potential transmembrane domain, and the 3' untranslated region) is unique, with no matches or homologies in GenBank (S.H. Gianturco, unpublished data, 1998).

The current studies were undertaken to identify the major binding determinant present in chylomicrons and HTG-VLDL for MBP 200, 235, the most likely candidates^{20 21 22} for the human apoE- and LpL-independent monocyte-macrophage receptor for TGRLP.

	Methods

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Materials
Heparan sulfate, bovine LpL, bovine serum albumin, biotin hydrazide, and nonimmune IgG were from Sigma Chemical Company. Streptavidin–alkaline phosphatase was from Boehringer Mannheim and Southern Biotechnology Associates.

Lipoproteins
HTG-VLDL (S_f 100 to 400 and 60 to 100) was isolated from plasma of fasting subjects with type 4 lipoprotein profiles or in some cases from lipemic plasma obtained from the Red Cross. To obtain chylomicrons and remnants enriched in apoB-48, normal and hypertriglyceridemic volunteers consumed a standardized solid, high-fat meal^{24 25} at 1000 calories per meter squared. Blood for the isolation of lipoproteins was drawn 4 hours after the meal was consumed. Total chylomicrons S_f>400 were isolated directly from plasma by flotation for 20 minutes at 20 000 RPM in an SW27 rotor at 23°C.

Chylomicron subfractions (S_f>3200, S_f 1100 to 3200, and S_f 400 to 1100) and HTG-VLDL S_f 100 to 400 were isolated from total chylomicrons or the d<1.006 fraction, respectively, by cumulative flotation through a discontinuous NaCl gradient from d=1.063 to 1.006 g/mL²⁶ and as detailed previously for VLDL subspecies.²⁷ Protein concentrations of the lipoproteins were obtained by a modified Lowry procedure.^{28 29} Tryp-VLDL, reisolated and devoid of immunochemically detectable apoE, was prepared as previously described.^{12 13 27} Functional loss of apoE was demonstrated by lack of binding of tryp-VLDL to partially purified bovine LDL receptors on ligand blots.^{12 13 20 21 22 30} Although tryp-VLDL is devoid of immunochemically detectable apoE^{12 13 27} and apoCIII (W.A. Bradley, unpublished data, 1998), it retains essentially all immunochemically detectable apoB as fragments of 100 kD and less, as determined by SDS-PAGE (size) and radioimmunoassay^{13 27} or by SDS-PAGE and quantitative dot-blot analysis of parent VLDL and tryp-VLDL.²² For cell-binding studies, lipoproteins were radioiodinated by the iodine monochloride method as described³¹ and as we used previously.^{11 12 13 20 21 27} Specific activities ranged from 100 to 200 cpm/ng protein. Less than 10% of the label was extracted into organic solvent. For competitive ligand blots, TGRLP was biotinylated³² as described and dialyzed extensively before use.

Cells and Cell Culture
THP-1 cells (a human monocytic leukemia cell line) were purchased from the American Type Culture Collection and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 µg/mL penicillin, 100 U/mL streptomycin, and 5x10^–5 mol/L 2-mercaptoethanol. Cells were maintained in tissue-culture flasks at 37°C in a humidified atmosphere of 5% CO₂ and 95% air at <1.0x10⁶mL. For differentiation, cells (1.5x10⁶ cells per well of 6-well plates) were seeded in complete media; phorbol 12-myristate, 13-acetate was then added (10^-7 molL final concentration), as described.²¹ Human skin fibroblasts were early-passage cells derived in our laboratory from newborn foreskin and maintained as previously described.^{33 34}

Cell-Binding Studies
Lipoprotein binding studies were carried out essentially as initially described by Goldstein and Brown.³⁵ THP-1 monocytes were seeded in 6-well tissue-culture plates (1.5x10⁶ cells per well), phorbol ester (10^–7 mol/L) was added to induce adherence, and then they were used for experiments after 24 hours.^{21 22} As controls, cultured human skin fibroblasts were subcultured and grown to 75% confluence (3 to 4 days after subculture at a 1-to-4 split ratio) in complete medium (DMEM containing 10% fetal bovine serum, 2 mmol/L glutamine, 100 µg streptomycin per milliliter, and 100 U penicillin per milliliter), washed with sterile saline, and preincubated in DMEM containing 5% lipoprotein-deficient serum for 36 hours to induce the LDL receptor.^{11 12 13 27} Cells were then preincubated for 30 minutes at 4°C to cool the cells. Cells were then incubated with RPMI-1640 (THP-1 cells) or DMEM (fibroblasts) containing 10 mmol/L HEPES, pH 7.4, 2 mg BSA per milliliter, and indicated amounts of ¹²⁵I–HTG-VLDL or ¹²⁵I–tryp-VLDL alone and in the presence of 200 µg/mL unlabeled VLDL or other potential competitors for 1.5 hours at 4°C before extensive washing with cold buffered saline containing 2 mg BSA per milliliter³⁵ as previously described.^{11 12 13 20 21 22 27} Cells were dissolved in 0.1 N NaOH prior to the measurements of cell-associated radioactivity and cell protein. Dishes with no cells were used to correct for the amount of nonspecific binding to the plastic wells, as described.²⁷

Antibodies
Sheep anti-human apoB IgG (1001400, Boehringer Mannheim Biochemicals) was purified by affinity chromatography using an LDL-conjugated Sepharose column, prepared as previously described.³⁶ Immunoaffinity-purified rabbit anti-sheep IgG conjugated to alkaline phosphatase and sheep -globulin were purchased from Jackson Laboratories. Rabbit anti-human apoB antibodies were isolated by ammonium sulfate precipitation of serum from rabbits immunized intradermally with human LDL, isolated at d=1.03 to 1.05 g/mL, and emulsified in adjuvant. The anti–apoB-100 antibodies generated and/or affinity purified in our laboratory were monospecific for apoB and did not recognize apoE, apoCs, or apoHDL. Anti-apoE was generated in rabbits using human apoE purified in our laboratory and was monospecific for apoE. Affinity-purified goat anti-apoCIII and anti-apoCII were generous gifts from Dr Ronald Krauss and Dr G.M. Anantharamaiah, respectively.

Preparation of Cell Extracts
THP-1 monocytes (1.5x10⁸) were harvested and washed twice with 50 mL of buffer A (0.15 mmol/L NaCl containing 50 U aprotinin per milliliter, 5 mmol/L benzamidine, and 0.1 mmol/L PMSF) and resuspended in 2 mL of 20 mmol/L Tris, pH 8.0, 50 mmol/L NaCl, 0.1 mmol/L EDTA, containing the protease inhibitor mix of buffer A plus leupeptin and D-phenylalanyl-1-propyl-L-arginine chloromethylketone (PPACK) and solubilized with 1% Triton X-114 for 15 minutes on ice. Aqueous-phase extracts were prepared as previously described^{20 21 22} by the method of Bordier³⁷ and immediately frozen in liquid nitrogen after the addition of glycerol to a final concentration of 10% (vol/vol). Protein content was estimated by the Bradford method using the Bio-Rad protein assay reagent.³⁸

Ligand Blotting
The ligand-blotting assay was performed essentially as described earlier^{20 21 22} with minor modifications. Aliquots of the detergent extracts were electrophoresed on 5% polyacrylamide gels containing 0.1% SDS³⁹ under nonreducing conditions in a Bio-Rad minigel apparatus and electrotransferred to nitrocellulose. After blocking for 1 hour with 5% Carnation nonfat dry milk in ligand buffer (50 mmol/L Tris-HCl, pH 8, 90 mmol/L NaCl, and 2 mmol/L CaCl₂), the blots were rinsed with 0.5% milk in ligand buffer before incubation with lipoproteins in ligand buffer containing 0.05% milk.^{20 21 22} Biotin-labeled lipoproteins, with and without antisera, IgGs (the 50% [NH₄]₂ SO₄ precipitate of antisera), or other potential competitors, were preincubated for 30 minutes at 4°C and then incubated with the nitrocellulose strips for 1.5 to 3 hours as indicated. After extensive washing, bound lipoprotein was detected by incubation with streptavidin linked to alkaline phosphatase, followed by the substrates BCIP and NBT (Bio-Rad). In some experiments without antibodies as potential competitors, native, unlabeled TGRLP was used as the ligand and bound TGRLP was detected with anti-apoB followed by alkaline phosphatase-linked secondary antibody. Ligand blots were scanned on an optical scanner (Hewlett Packard), and binding activity was quantified using the Image Quant software (Molecular Dynamics densitometer) as previously described.^{21 22}

	Results

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Anti-ApoB Antibodies Inhibit the Binding of HTG-VLDL S_f 100 to 400 to MBP 200, 235
Previous experiments in human blood-borne and THP-1 monocytes and macrophages^{21 22} with the surrogate ligand tryp-VLDL, which retained essentially all apoB immunoreactivity (in fragments of 100 kD) but was devoid of immunochemically detectable apoE^{12 13 22 27} and apoCIII (S.H. Gianturco, unpublished data, 1998) and failed to bind to the LDL receptor in cells^{12 13} and in ligand blots,^{20 21 22 27 30} suggested that apoB may be the ligand for this monocyte-macrophage cell site and corresponding MBPs. Thus, competitive ligand-blotting experiments were done with several polyclonal anti-apoB–specific antibodies to determine whether they were capable of specifically blocking binding of HTG-VLDL to the putative TGRLP receptor proteins MBP 200, 235. In the representative experiment shown in Figure 1, THP-1 monocyte extracts were electrophoresed and transferred to nitrocellulose, blocked, and incubated with biotinylated VLDL in the absence (lane 1) and presence of 2 levels of a monospecific rabbit anti-apoB IgG (40 µg/mL, lane 2; 400 µg/mL, lane 3), or the corresponding preimmune IgG (90 µg/mL, lane 4; 400 µg/mL, lane 5). Binding of biotinylated HTG-VLDL S_f 100 to 400 was visualized with streptavidin-linked alkaline phosphatase and the image digitized (Figure 1a) and quantified by scanning densitometry (Figure 1b). The lower level (40 µg/mL, lane 2) of anti-apoB IgG blocked 50% of binding, and the higher level (400 µg/mL, lane 3) blocked all visibly detectable (80% by densitometry) HTG-VLDL binding to MBP 200, 235. In contrast, the preimmune IgG at 90 µg/mL blocked none of the binding (lane 4) and at 400 µg/mL blocked 25% (lane 5). In a separate ligand-blotting experiment, this apoB antibody did not inhibit the binding of the same biotinylated VLDL to the bovine LDL receptor (data not shown). The experiment shown in Figure 1 is representative of 12 separate experiments with 4 different anti-apoB antibodies; in all cases, anti-apoB antibodies specifically inhibited binding of HTG-VLDL, chylomicrons, or tryp-VLDL to MBP 200, 235. These experiments suggest that apoB is involved in the binding of HTG-VLDL to the MBPs. These results are consistent with studies demonstrating specific, high-affinity binding of tryp-VLDL^{20 21 22 23} and implicate apoB in the binding of TGRLP to MBP 200, 235. Alternatively, the anti-apoB antibodies could sterically hinder another component that is the ligand for MBP 200, 235.

View larger version (33K): [in this window] [in a new window]   Figure 1. Inhibition by monospecific rabbit anti-human apoB IgG of binding of HTG-VLDL to the monocyte TGRLP receptor (MBP 200, 235). THP-1 monocyte aqueous-phase extracts were electrophoresed and transferred to nitrocellulose (100 µg per lane). Biotinylated HTG-VLDL Sf 100 to 400 (0.5 µg/mL) was preincubated with buffer (lane 1) or with 2 levels of anti-apoB (rabbit 1325): 40 µg/mL (lane 2), 400 µg/mL (lane 3); or 2 levels of preimmune (rabbit 1325) IgG: 90 µg/mL (lane 4), 400 µg/mL, (lane 5). Lipoproteins and IgGs were preincubated for 30 minutes and then incubated with the nitrocellulose strips for 3 hours at 4°C. After extensive washing, bound lipoprotein was detected with streptavidin linked to alkaline phosphatase followed by colorimetric substrates (the digitized image is shown in A) and quantified by scanning densitometry (B) using two-dimension area integration and illustrated as VLDL binding in densitometric units (pixels).

Antibodies Against Other Apoproteins of HTG-VLDL Fail to Inhibit Its Binding to MBP 200, 235
ApoB is only 30% of the total protein mass in HTG-VLDL S_f 100 to 400; apoE is 6% to 8%, and apoCs are 63%.^{12 13 34} On a molar basis, HTG-VLDL S_f 100 to 400 contains 1 mol apoB, approximately 3 to 6 mol apoE, and 150 mol apoCs (primarily apoCIII) per mole VLDL. To directly determine whether any of these other apoproteins are the ligand, sterically hindered by the anti-apoB antibodies in the experiments represented by Figure 1, or whether they contribute to the binding of HTG-VLDL to MBP 200 and 235, we did a series of competitive ligand-blotting experiments with polyclonal antibodies against the other major apoproteins of HTG-VLDL. All antibodies used recognized their antigens in native VLDL and the anti-apoCIII antibody is used to isolate apoCIII-rich VLDLs (Dr Ronald Krauss, personal communication, April 1996). One experiment is shown in Figure 2. In this ligand blot, as in many but not all, MBP 200, 235 activities (either or both) appear as a complex of 2 or more bands due to the existence of several permissible oxidation states and/or disulfide isomers, as previously published.²² In the experiment shown in Figure 2A, biotinylated HTG-VLDL S_f 100 to 400 incubated with buffer (lane 1) or with nonimmune IgG (lane 3) binds to MBP 200 and 235 to similar extents. Neither anti-apoE (lane 4) nor anti-apoCIII (lane 5) diminishes the binding of HTG-VLDL to MBP 200, 235 but anti-apoB again effectively blocks >90% of the binding of HTG-VLDL (lane 2 and Figure 2B). The finding that the anti-CIII antibody failed to block binding of HTG-VLDL to MBP 200, 235, even though the total apoCIII mass is 2 times the mass of apoB in HTG-VLDL S_f 100 to 400, argues against the alternative explanation offered above that the anti-apoB antibodies blocked by sterically hindering another apoprotein's interaction with the MBPs. In other ligand-blotting experiments, we determined that the concentration of anti-apoE IgG used here blocked all binding of HTG-VLDL to the LDL receptor. Additional competitive ligand-blotting studies with anti-apoCII IgGs demonstrate that these antibodies do not inhibit binding (data not shown). Taken together, the competitive ligand-blotting studies strongly suggest that apoB, but not apoE, apoCIII, or apoCII, mediates the binding of HTG-VLDL to MBP 200, 235.

View larger version (35K): [in this window] [in a new window]   Figure 2. Inhibition by anti-apoB, but not anti-apoE, anti-apoCIII, or nonimmune IgG, of binding of HTG-VLDL Sf 100 to 400 to MBP 200, 235. THP-1 monocyte aqueous-phase extracts were electrophoresed and transferred to nitrocellulose, blocked, and incubated with biotinylated HTG-VLDL and the indicated antibodies at 4°C. Lane 1, buffer; lane 2, 3 mg/mL anti-apoB (1325; apoB); lane 3, 2.4 mg/mL nonimmune IgG (nonimm); lane 4, 2.3 mg/mL anti-apoE (apoE); and lane 5, 2 mg/mL anti-apoCIII (apoCIII). The IgGs were preincubated with biotinylated HTG-VLDL (2.5 µg/mL) for 30 minutes at 4°C and then were incubated with the nitrocellulose strips for 3 hours at 4°C. Lipoprotein binding was visualized (A) and quantified by densitometry (B) as described in Figure 1. Ab indicates antibody.

Anti-ApoB Antibodies Inhibit the Binding of TGRLP to the TGRLP Receptor of THP-1 Monocytes, but Not to the LDL Receptor of Fibroblasts
To confirm that apoB mediates the binding of TGRLP to the LpL- and apoE-independent TGRLP cellular receptor, competitive cell-binding studies with THP-1 monocyte-macrophages were conducted under experimental conditions that minimize the expression of the LDL receptor, the LRP, LpL, and apoE (1 day after adherence was induced by phorbol 12-myristate, 13-acetate) as previously described.²¹ As a control, competitive binding studies were also done simultaneously with cultured human skin fibroblasts with upregulated LDL receptors, since HTG-VLDL S_f 100 to 400 binds to the LDL receptor via apoE and not via apoB.^{12 13 14 15} Consistent with the ligand-blotting studies (Figure 1) and shown in Figure 3A, the high-affinity, specific binding of ¹²⁵I–HTG-VLDL to THP-1 cells was inhibited by antibodies to apoB, but not by the equivalent level of nonimmune IgGs. In contrast, and indicating the specificity of the blocking experiments in THP-1 cells, the same anti-apoB antibody did not inhibit the LDL receptor–specific binding of ¹²⁵I–HTG-VLDL to the fibroblasts (Figure 3B), consistent with previously published studies.^{12 13 14 15} This representative experiment shows that the inhibition of ¹²⁵I–HTG-VLDL binding to THP-1 by anti-apoB antibodies was not significantly different from the inhibition by homologous, unlabeled HTG-VLDL (self). This finding indicates that apoB is the component of TGRLP responsible for its high-affinity, specific binding to THP-1 cells when the LDL receptor, LRP, LpL, and apoE are suppressed.

View larger version (14K): [in this window] [in a new window]   Figure 3. Inhibition by anti-apoB IgG, but not nonimmune IgG, of binding of 125I–HTG-VLDL to THP-1 macrophages, but not to human fibroblasts with upregulated LDL receptors. A, THP-1 monocyte-macrophages 1 day after adherence were grown as described in "Methods." Duplicate dishes of cells and no cells for controls were incubated with 125I–HTG-VLDL Sf 100 to 400, 5 µg/mL, alone (none) or in the presence of a 30-fold excess of unlabeled HTG-VLDL (self), in the presence of affinity-purified sheep anti-apoB IgG (anti-apoB), or the equivalent level of sheep nonimmune IgG (NonImm) at 4°C for 16 hours and then incubated with precooled, washed cells for 1.5 hours at 4°C. After extensive washing, the cells were dissolved in 0.1 NaOH for determination of bound 125I–HTG-VLDL, as described in "Methods." Values represent the average of duplicate dishes corrected for background by subtracting the averages of the no-cell controls and are expressed in terms of percent of control, ie, percent of the uninhibited activity (100%). Specific binding activity for 125I–HTG-VLDL, 5 µg/mL, was 24 ng/mg cell protein, which represented 100% uninhibited activity. B, Human skin fibroblasts were grown to 75% confluence and preincubated with DMEM containing 5% lipoprotein-deficient serum for 36 hours to induce the LDL receptor as described in "Methods." The binding and competition protocols were identical to those for the THP-1 cells as described in A above. Specific binding activity for 125I–HTG-VLDL, 5 µg/mL, was 237 ng/mg cell protein, which represented 100% uninhibited activity.

Effects of Lactoferrin, Heparin, and LpL on Binding of HTG-VLDL S_f 100 to 400 to MBP 200, 235 and to THP-1 Monocyte-Macrophages
A series of competitive ligand-blotting studies were carried out to further distinguish MBP 200 and 235 from receptors of the LDL receptor family and to further delineate the binding domains in apoB for this distinct receptor. As shown in Figure 4, neither lactoferrin nor heparin is an effective inhibitor of the binding of HTG-VLDL to MBP 200 and 235. In this representative experiment, nitrocellulose strips containing MBP 200, 235 were incubated with 0.5 µg of biotinylated HTG-VLDL per milliliter in the absence (lane 1) or presence of lactoferrin at 50 µg protein per milliliter (lane 2) or 500 µg protein per milliliter (lane 3); heparin at 10 U/mL (lane 4) and 100 U/mL (lane 5); or HTG-VLDL at 25 µg/mL (lane 6) or 5 µg/mL (lane 7). Binding of biotinylated VLDL was visualized with streptavidin-linked alkaline phosphatase (Figure 4A) and quantified by scanning densitometry (Figure 4B). Only unlabeled HTG-VLDL effectively competed with biotinylated HTG-VLDL for binding to MBP 200, 235. Lack of inhibition of TGRLP binding to MBP 200, 235 by heparin at levels that are known to displace LpL from cells indicates that binding is not mediated by LpL potentially bound to the VLDL. It also suggests that the apoB domain involved in binding to MBP 200, 235 is different from the apoB domain involved in the binding of LDL to the LDL receptor, because the apoB-LDL receptor interaction is disrupted by heparin.³⁵ Further, it suggests that the domain in apoB that binds to MBP 200, 235 is not in a heparin-binding domain of apoB. Lack of inhibition of binding by lactoferrin indicates that MBP 200 and 235 are distinct from the putative hepatic remnant receptors.⁴⁰

View larger version (42K): [in this window] [in a new window]   Figure 4. Effects of lactoferrin and heparin on binding of HTG-VLDL to MBP 200, 235. THP-1 monocyte aqueous-phase extracts were electrophoresed and transferred to nitrocellulose. The nitrocellulose strips were incubated for 4 hours at 4°C with 0.5 µg biotinylated HTG-VLDL per milliliter in the absence (lane 1) or presence of lactoferrin at 50 µg protein per milliliter (lane 2) or 500 µg protein per milliliter (lane 3); heparin at 10 U/mL (lane 4) and 100 U/mL (lane 5); or unlabeled HTG-VLDL at 25 µg/mL (lane 6) or 5 µg/mL (lane 7). Biotinylated HTG-VLDL binding was detected with streptavidin-linked alkaline phosphatase (digitized image, A) and quantified by densitometry (B). Lacto indicates lactoferrin; Hep, heparin.

To further characterize the receptor-binding domain in apoB of HTG-VLDL and distinguish its binding to the monocyte TGRLP receptor from binding to the LDL receptor or related receptors, competitive ligand-binding studies were carried out with levels of LpL reported to enhance binding of lipoproteins to LDL receptor family members or to HSPG on cells. In the representative experiment shown in Figure 5, THP-1 monocyte extracts were electrophoresed and transferred to nitrocellulose. Biotinylated HTG-VLDL (3 µg of protein per milliliter) was preincubated for 30 minutes at 4°C (to inhibit potential lipolysis by LpL) and then with the nitrocellulose strips for 3 hours at 4°C with buffer (lane 1) or LpL (at 0.2, 2.0, and 20 µg/mL; lanes 2 through 4) or with the same levels of bovine serum albumin as controls (lanes 5 through 7). Surprisingly, LpL, at levels that enhance binding to LRP,⁴¹ blocks the binding of HTG-VLDL to MBP 200, 235 in a concentration-dependent manner (Figure 5B). In contrast, albumin has minimal effects on HTG-VLDL binding. Thus, LpL does not mediate the interaction of VLDL with this monocyte-macrophage receptor; rather, it inhibits binding. Inhibition of binding by LpL is likely due to its binding to the N-terminal domain of apoB,⁴² since preincubation of the receptors on nitrocellulose strips with LpL failed to inhibit the binding of TGRLP subsequently incubated with the strips (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 5. Inhibition by LpL of binding of HTG-VLDL to MBP 200, 235. THP-1 monocyte aqueous-phase extracts were electrophoresed and transferred to nitrocellulose, blocked, and incubated at 4°C with biotinylated HTG-VLDL Sf 100 to 400 (3 µg of protein per milliliter) in the absence (lane 1) or presence of LpL (0.2 µg/mL, lane 2; 2.0 µg/mL, lane 3; 20 µg/mL, lane 4) or in the presence of bovine serum albumin (0.2 µg/mL, lane 5; 2.0 µg/mL, lane 6; 20 µg/mL, lane 7). Binding was detected by incubation with streptavidin/alkaline phosphatase and the image was digitized (A) and quantified by densitometry (B).

Competitive cell-binding studies also demonstrate that lactoferrin fails to inhibit significantly (<5%) the binding of tryp-VLDL or HTG-VLDL to monocyte-macrophages, and high levels of heparin (10 mg/mL) have only a small (18%) effect (Table). Other studies with heparin at lower levels (1 mg/mL) show little to no inhibition of TGRLP binding. These cell studies also indicate that LpL at levels shown by others to enhance binding of lipoproteins to cellular HSPG,^{43 44} LRP,^{41 45} and the LDL receptor⁴⁶ (1 to 2 µg/mL) does not enhance uptake of tryp-VLDL; rather it partially inhibits binding (to 26% at 1.6 µg LpL per milliliter). That the inhibition of binding of TGRLP to cells by LpL is less than the inhibition of TGRLP binding to MBP 200, 235 is likely due to the competing enhancement of lipoprotein binding to cellular HSPGs by LpL, which is not a confounder in ligand blots. Thus, the results of cell-binding studies are similar to the results of ligand-blotting studies, with inhibition by anti-apoB and LpL, but not by preimmune or nonimmune IgG, lactoferrin, or heparin.

Chylomicrons S_f>1100 Containing ApoB-48, but Not ApoB-100, Bind to MBP 200, 235
The specific inhibition of HTG-VLDL and tryp-VLDL binding to cells and to MBP 200, 235 on ligand blots by antibodies to apoB indicates that this apoprotein is necessary for the binding of HTG-VLDL to this receptor. The inhibition by LpL in cells and in ligand blots implicates the N-terminal domain of apoB. Previous studies showed that HTG-VLDL, but not normal VLDL S_f>60, binds with high affinity to cells, causes lipid accumulation, and binds to MBP 200 and 235.²¹ HTG-VLDL subfractions from subjects with elevated plasma triglyceride (>150 mg/dL) contain more apoB-48 than normal VLDL subfractions from subjects with normal plasma triglycerides (<150 mg/dL) after purification by cumulative flotation because of delayed chylomicron remnant clearance.¹³ Taken together, these results suggest that apoB-48 may be a preferred ligand, or at least contain a preferred conformational domain of apoB that enhances binding to this receptor. Thus, we studied chylomicron subfractions isolated 4 hours after a standardized fat load.^{24 25} Chylomicrons, ie, TGRLP of S_f>400, were purified further by cumulative flotation into more homogeneous subfractions of S_f>3200 (CM I), S_f 1100 to 3200 (CM II), and S_f 400 to 1100 (CM III).²⁶ The largest 2 chylomicron fractions (CM I and II) contained apoB-48 as the only detectable apoB species (Figure 6, lanes 1 to 4), whereas the smallest fraction (CM III; lanes 5 and 6) contained both apoB-48 and apoB-100, as determined by immunochemical blotting (Figure 6), which allowed us to estimate that <0.1%, or <1 in 1000 particles, contains apoB-100 in the S_f>1100 subfractions. Lane 7 contains a typical fasting HTG-VLDL S_f 100 to 400 with apoB-48, as well as apoB-100 and apoE. All chylomicron subfractions contained immunochemically detectable apoE (Figure 6), as well as apoCs (not shown).

View larger version (54K): [in this window] [in a new window]   Figure 6. Immunoblot evidence that plasma chylomicrons of Sf>1100 contain apoB-48, but not apoB-100. Plasma was isolated from a hypertriglyceridemic subject 4 hours after a standardized test fat meal. Total chylomicrons (CM) Sf>400 were subfractionated through a salt gradient into 3 subclasses: Sf>3200 (CM I), Sf 1100 to 3200 (CM II), and Sf 400 to 1100 (CM III). Samples of each were electrophoresed at 2 levels (1 and 2 µg total apoprotein per lane) on a 4% to 20% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed for apoB (above the line) and apoE (below the line). CM I, lanes 1 and 2; CM II, lanes 3 and 4; CM III, lanes 5 and 6; and control hypertriglyceridemic VLDL Sf 100 to 400, containing apoB-100, apoB-48, and apoE, lane 7; lane M shows the prestained protein molecular-weight markers.

The 3 chylomicron subfractions were then tested for binding to MBP 200, 235 and to the partially purified bovine LDL receptor by ligand-blotting analysis; a representative experiment is shown in Figure 7. All of the chylomicron subfractions, added at equivalent concentrations, bound with high affinity to MBP 200, 235 (lanes 1, 3, and 5), as well as to the LDL receptor (lanes 2, 4, and 6). Since apoB-48 is the only apoB species immunochemically detectable in the largest 2 chylomicron subfractions (CM I and CM II; Figure 6), apoB-48 or an apoB-48 domain is strongly implicated as the primary apoprotein binding determinant in postprandial TGRLP for the distinct human apoE- and LpL-independent monocyte-macrophage receptor for TGRLP and its candidate receptor proteins MBP 200 and 235. Indeed, the binding of CM II to MBP 200, 235 was specifically inhibited by anti-apoB IgG by 90%, but not by nonimmune IgG (Figure 8).

View larger version (51K): [in this window] [in a new window]   Figure 7. Binding of chylomicron subspecies to MBP 200, 235 (odd lanes) or to the partially purified LDL receptor (even lanes). THP-1 monocyte aqueous-phase extracts and the partially purified bovine LDL receptor were electrophoresed in alternating lanes, transferred to nitrocellulose, blocked, and incubated with chylomicron (CM) subfractions (10 µg/mL). CM I (Sf>3200) was incubated with lanes 1 and 2; CM II (Sf 1100 to 3200) with lanes 3 and 4; and CM III (Sf 400 to 1100) with lanes 5 and 6. Binding was detected with a polyclonal anti-apoB antibody followed by a second antibody linked to alkaline phosphatase. The sharp band in lanes with THP-1 extracts that migrates between MBP 200 and the LDL receptor is a nonspecific lipoprotein-binding protein apparent in some, but not all, ligand blots.

View larger version (30K): [in this window] [in a new window]   Figure 8. Blocking by anti-apoB antibodies of binding of chylomicrons Sf 1100 to 3200 (CM II) that contain apoB-48 as the only apoB species to MBP 200, 235. THP-1 aqueous-phase extracts were electrophoresed and transferred to nitrocellulose. Prior to incubation with the nitrocellulose strips for 3 hours at 4°C, biotinylated CM II was preincubated at 4°C for 30 minutes with buffer (lane 1), anti-apoB (rabbit 1325; lane 2), or an equivalent level of nonimmune IgG (lane 3). Chylomicron binding was visualized after incubation with streptavidin-linked alkaline phosphatase, digitized (A), and quantified by scanning densitometry (B).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our earlier reported studies with tryp-VLDL, in which apoE and apoCs were removed from TGRLP by trypsinolysis and reflotation, leaving apoB fragments as the only apparent apoproteins associated with the particle, suggested apoB mediates TGRLP binding to the apoE- and LpL-independent monocyte-macrophage receptor for TGRLP and to the cell-surface receptor candidate proteins MBP 200 and 235.^{20 21 22} The tryp-VLDL bound with high affinity and specificity to this macrophage receptor and MBP 200, 235 and failed to bind to the LDL receptor.^{20 21}

Herein we present several additional lines of evidence that apoB is the ligand for this receptor. First, anti-apoB antibodies specifically inhibit the high-affinity, specific binding of TGRLP S_f>100 both to this receptor on THP-1 cells and to the MBP on ligand blots. In contrast, the same anti-apoB antibodies failed to inhibit the binding of HTG-VLDL S_f 100 to 400 to the LDL receptor, which is known to be mediated by apoE,^{12 13 14 15 30} indicating that the blocking in THP-1 was a specific effect on apoB and not due to nonspecific effects on other TGRLP apoproteins. Second, antibodies that bind to the other major apoproteins that make up 70% of the total protein mass of native TGRLP S_f 100 to 400 (apoE, apoCIII, and apoCII) do not inhibit binding, further emphasizing the specificity of the anti-apoB inhibition. Because anti-apoB IgG alone is as effective as homologous unlabeled TGRLP in competition for this macrophage receptor (ie, anti-apoB IgG can block essentially all of the high-affinity, specific binding of TGRLP S_f>100 to this receptor, but not to the LDL receptor), apoB appears to be the essential TGRLP component directly involved in binding to this receptor. Third, LpL, which binds to an N-terminal domain of apoB,⁴² also inhibits the binding of TGRLP both to the cellular site and to MBP 200, 235. Inhibition by LpL is highly specific and effective, occurring at low levels (<2 µg/mL) and in a concentration-dependent manner. The mechanism by which LpL inhibits the TGRLP-receptor interaction appears to result from its binding to the N-terminal portion of apoB,⁴² since LpL preincubated with the receptor on nitrocellulose does not inhibit the binding of TGRLP subsequently added. In contrast to the effective inhibition of TGRLP binding by LpL, neither lactoferrin nor heparin, used at much higher concentrations, inhibited binding, emphasizing the specificity of inhibition by LpL and the differences of this binding site's characteristics from those of the LDL receptor family. Fourth, plasma chylomicron subfractions that contain apoB-48 as the only apoB species (S_f>3200 and S_f 1100 to 3200 isolated 4 hours postprandially) bind to MBP 200, 235, and this binding is also specifically inhibited by anti-apoB IgG. Together, these results implicate an apoB-48 domain (or an equivalent in apoB-100) as the receptor-binding determinant in TGRLP for its specific, high-affinity binding to MBP 200, 235 and to the cellular apoE- and LpL-independent monocyte-macrophage TGRLP receptor.

This TGRLP/apoB 48/100 receptor-binding domain that is present in plasma chylomicrons, HTG-VLDL, and tryp-VLDL apparently is relatively inaccessible in normal VLDL and LDL, which do not exhibit high affinity for the cellular TGRLP receptor or for MBP 200, 235, as previously demonstrated by direct and competitive binding studies both in cells and on ligand blots.^{20 21} Our previous studies demonstrated, however, that trypsinization followed by reflotation of normal VLDL S_f>60 exposes the receptor-binding domain, since trypsinized normal VLDL S_f 100 to 400, but not native normal VLDL, binds with high affinity to the MBPs on ligand blots.²⁰ In contrast, trypsinization of LDL does not induce high-affinity binding to this site on macrophages or to MBP 200, 235 on blots (data not shown), and it has little to no effect on its binding to the LDL receptor in cells or on ligand blots, as previously documented.^{12 13 27} These data parallel, but in the reverse sense, a characteristic of apoB that we and others have demonstrated in the past that the exposure of metabolically important domains of apoB are determined in part by the size of the lipoprotein particle in which they are found. For example, the binding domain in apoB in LDL, IDL, and VLDL S_f 20 to 60 for the LDL receptor is not accessible in TGRLP S_f>60.^{12 13 14 15}

The studies reported here also add to the list of properties that distinguish this TGRLP/apoB receptor from other lipoprotein receptor families. ApoB-mediated binding to the LDL receptor⁴⁷ or LpL-mediated binding to members of the LDL receptor family or to cell-surface HSPGs can be inhibited by heparin.⁴⁴ In contrast, heparin has little effect on the binding of TGRLP to this distinct cellular receptor or the MBPs (present study) or on macrophage lipid accumulation under conditions in which this receptor, but not the LDL receptor, is fully expressed.⁴⁸ Lactoferrin, which inhibits the uptake of chylomicron remnants by the liver,⁴⁰ also fails to inhibit the interaction of TGRLP with THP-1 monocyte-macrophages and with MBP 200, 235, indicating they are not related to putative hepatic remnant receptors. The inhibition by LpL of TGRLP binding both (1) to THP-1 cells grown under conditions in which this receptor, but not the LDL receptor or LRP, is expressed and (2) to MBP 200, 235 also distinguishes this receptor from members of the LDL receptor family, because LpL can augment binding of lipoproteins to these receptors.^{41 43} These competitive binding studies indicate that the receptor-binding domain in TGRLP is within apoB-48 (or a corresponding domain in apoB-100) at or near the LpL-binding domain and not in a heparin-binding domain.

In addition, the specific and parallel inhibition of TGRLP binding to the cellular site and to MBP 200, 235 by anti-apoB and LpL provides further evidence that corroborates the previously published data supporting their role as the high-affinity monocyte-macrophage LpL- and apoE-independent TGRLP receptors.^{21 22} Finally, since chylomicrons that contain apoB-48 as the only apoB species bind to this receptor and this binding is inhibited by anti-apoB antibodies, we conclude that an apoB-48 domain is sufficient to mediate the binding of TGRLP to the receptor and suggest that this receptor functions as a TGRLP/apoB-48 monocyte-macrophage receptor.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein DMEM = Dulbecco's modified Eagle's medium HSPG = heparan sulfate proteoglycan HTG-VLDL = hypertriglyceridemic VLDL LpL = lipoprotein lipase LRP = LDL receptor-related protein/2 macroglobulin receptor MBP = membrane-binding protein PAGE = polyacrylamide gel electrophoresis TGRLP = triglyceride-rich lipoproteins tryp = trypsinized

	Acknowledgments

 
This research was supported by grants HL44480 and HL46304 from the National Institutes of Health. M.L.B. was supported by the National Institutes Research Service award T32 HL-07703 during the course of this study. Our patient volunteer protocol for blood donation was conducted through the General Clinical Research Center of the University of Alabama at Birmingham and supported by the NIH, Division of Research Resources grant RR00032. Animals for antibody production were under the expert supervision of the professional staff of the UAB Animal Resources Program. Affinity-purified goat anti-apoCIII and anti-apoCII were generous gifts from Dr Ronald Krauss and Dr G.M. Anantharamaiah, respectively. We thank the nurses at the UAB GCRC for their assistance with patients and Betty Darnell for her expertise in nutritional calculations for our patients. We thank Caryl Reese and Dana Stinson for their invaluable technical assistance and Marilyn Robinson for editorial assistance and preparation of the manuscript.

	Footnotes

 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13–16, 1995, and published in abstract form (Circulation. 1995;92[pt 2]:I-690).

Received May 27, 1997; accepted January 13, 1998.

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