© 1997 American Heart Association, Inc.
Articles |
Possible Functional Interactions of Apolipoprotein B-100 Segments That Associate With Cell Proteoglycans and the ApoB/E Receptor
the Wallenberg Laboratory for Cardiovascular Research (U.O, G.C., E.H.-C., K.E., O.W., G.B.), Heart and Lung Department, Goteborg University, Goteborg, and the Preclinical Research Laboratories (G.C.), AB Astra Hassle, Molndal, Sweden.
Correspondence to German Camejo, Preclinical Research Laboratories, AB Astra Hassle, S-431 83, Molndal, Sweden. E-mail german.camejo@hassle.se.astra.com.
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Abstract |
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The interaction of apoE lipoproteins with cells appears to be mediated by an association with basic sequences of proteoglycans and the apoB/E receptor. ApoB-100 has basic sequences, homologous with those of apoE, that form part of the apoB/E receptor–binding domain. These sequences of apoB-100 also interact with proteoglycans. We investigated whether such segments, in analogy with apoE, could act cooperatively on LDL interactions with proteoglycans and the receptor. As a model we used the two most basic regions of apoB-100, 3147 through 3157 and 3359 through 3367, connected by three glycines (3145-3157–GGG–3359-3367). Such segments may be proximal in LDL by the presence of a disulfide bridge between Cys(3167) and Cys(3297). The apoB heterodimer but not the separated monomers inhibited 125I-LDL degradation in fibroblasts and THP-1 cells by 50% at
11 µmol/L. The heterodimer affinity with arterial proteoglycans was closer to that of LDL and higher than that of the individual peptides. The heterodimer appears to bind specifically to THP-1 cells, with a Kd of 6.2x10-8 mol/L and a Bmax of 1.3x106 molecules/cell. Monoclonal antibody C-7, which recognizes the apoB receptor, inhibited the binding to cells. Treatment of fibroblasts with chondroitinase ABC or chlorate decreased 125I-LDL degradation markedly. Hydrolysis of pericellular proteoglycans of fibroblasts by chondroitinases reduced mostly the low-affinity, high-capacity component of LDL binding. This compartment appears to hold 70% of the cell-associated LDL when internalization is inhibited at 4°C. Therefore, cell-surface chondroitin sulfate/dermatan sulfate proteoglycans appear to modulate binding and receptor-mediated internalization of LDL. This may be caused, at least in part, by the association of proteoglycans with the apoB-100 segments 3145 through 3157 and 3359 through 3367.
Key Words: apoB-100 • cell-surface proteoglycans • apoB/E receptor • LDL degradation • LDL cell binding
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Introduction |
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Brown et al1 in 1978 found that certain basic proteins or peptides inhibit the binding of LDL to its receptor. Because the receptor-active proteins bind heparin, they suggested that inhibition was caused by polycations interacting with the receptor, with the sulfated GAGs, or with both apoE-containing lipoproteins also binding the apoB/E receptor and sulfated GAGs. A synthetic dimeric repeat of amino acid residues 141 through 155 of apoE appears responsible for this.2 LDLs associate with the negatively charged LDL receptor and GAGs at a neutral pH even though the net charge of the lipoprotein is also negative, with an isoelectric point of 5.4 to 5.9.3 This suggests that the net charge of the LDL is mediated by localized positively charged regions of apoB-100. The apoE segment 142 through 150 is homologous (56% total identity and 67% functional identity) to one segment on apoB-100, amino acid residues 3359 through 3367. Two basic regions of similar size in apoB, segments 3147 through 3157 and 3359 through 3367, belong to the LDL receptor–binding domain.4 5 These segments are also important for the association of LDL with PGs and GAGs.6 7 8 9 10 11 A disulfide bond between cysteine residues 3167 and 3297 may approximate these regions that would form an extended positive domain recognized by the negatively charged LDL receptor, as suggested by Milne et al,4 Lawn and Scott,5 and Chan.12 This structure may also interact with PGs and GAGs. We have shown that other positive regions of apoB-100, such as 4230 through 4253 and 2106 through 2121, have a lower affinity for GAGs than sequences 3147 through 3157 and 3359 through 3367.10 Point mutations, in which arginine is substituted by uncharged amino acids at positions 3500 and 3531, can reduce receptor binding. Thus, positive charges situated downstream of segments 3147 through 3157 and 3359 through 3367 also appear to be involved.13 The LDL receptor–associating region is not accessible in the apoB-100 of VLDL.4 The acquisitions of such properties and increase in affinity with PGs displayed by LDL may be attributed to conformational changes in apoB-100. This may follow the shrinkage in surface area and the conversion of the particle from a triglyceride-rich to a cholesteryl ester–rich lipoprotein.11 Affinity for GAGs also increases with diminished surface area in VLDL subfractions.11 A conformation that could bring together two receptor-binding regions, analogous to an apoE dimer, may cause a high enough charge density for increased recognition by the LDL receptor. This may also explain why small LDL particles have a higher affinity for PGs and GAGs than buoyant ones.14 These two phenomena could regulate how and which lipoproteins should be taken up by cells and extracellular matrix and which lipoproteins should be left in circulation for additional processing.14 15 ApoB and apoE share the properties of binding to the LDL receptor and to sulfated GAGs,6 11 16 17 and Ji et al18 19 report that the interaction of apoB/E lipoproteins with hepatocytes is mediated by a cooperative process in which pericellular PGs and specific receptors in the liver are involved. Recently Saxena et al20 have shown that the basic amino acid clusters of apoB are responsible for the binding of LDL to endothelial cell pericellular matrix containing lipoprotein lipase. They also showed that apoE isoforms with analogous segments compete for this binding. In addition, pericellular PGs in smooth muscle cells and macrophages seem to regulate the metabolism of apoB lipoproteins by interactions with both lipases and apoB lipoproteins.21 22
In analogy with the experiments of Dyer and Curtiss,2 we explored the possibility of a dimer motif, a heterodimer in the case of apoB-100, in the interaction of LDL with PGs and the apoB/E receptor. We used as a model a synthetic peptide composed of the two most basic regions of apoB-100, 3147 through 3157 and 3359 through 3367, which are sequential and separated by three glycines (3145-3157–GGG–3359-3367). These two segments were used to map PG-binding regions on LDL.10 11 The data suggest that the apoB heterodimer binds to the LDL receptor and also binds GAGs with an affinity similar to that between LDL and GAGs. The synthetic apoB heterodimer could be a better model of LDL receptor– and GAG-binding regions of apoB-100 than the individual segments. In addition, the results suggest a role of cell-surface PGs in the binding and internalization of LDL.
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Materials
Chondroitin sulfate C (80% C6S and 20% chondroitin-4-sulfate) from shark cartilage (sodium salt; catalogue No. 400675) was purchased from Seikagaku Kogyo Co, and human aortic chondroitin sulfate PGs were prepared.14 Chondroitinase ABC (catalogue No. C-2905, EC 4.2.2.4), heparinase III (heparitinase) (H-8891, EC 4.2.2.8), and BSA (grade V) were from Sigma Chemical Co. Sodium [35S]sulfate was from Amersham. Disposable PD10 columns (Sephadex G-25) were from Pharmacia. Affigel-Hz coupling gel and reagents were from Bio-Rad. Cell culture media, fetal calf serum, and HEPES were from Flow Laboratories. Human blood serum was from young healthy donors. mAb C-7, from the medium of a hybridoma cell line obtained from the American Type Culture Collection, was isolated by using affinity chromatography on protein A sepharose (Pharmacia). A control mAb directed against an irrelevant antigen (Aspergillus niger glucose oxidase) was purchased from Dako. Sodium chlorate was from Fluka. All other chemicals were from Merck.
Lipoproteins
LDL (d=1.019 to 1.063 g/mL) was isolated from fresh human plasma by using differential ultracentrifugation, stored, checked for integrity,11 and labeled with [125I]iodide.14 LDL subfractions were isolated from plasma obtained from a donor with two distinct lipoprotein components (d=1.019 to 1.063 g/mL) by using a continuous deuterium oxide–water density gradient.23 The sizes of the LDL subfractions were determined by using nondenaturing 2% to 16% polyacrylamide gel electrophoresis with LDL of sizes determined by negative staining and electron microscopy as standards.
Peptide Synthesis and Purification
Synthetic apolipoprotein segments and model peptides were synthesized by using solid-phase procedures with the fluorenylmethoxycarbonyl method.24 The peptides were synthesized, purified, and sequenced.11 The final purity of the peptides was 
95% as assessed by analytical reversed-phase chromatography. Labeling of the apoB heterodimer with [125I]iodide was performed by using an Enzymobead radioiodination reagent kit.25 Peptide concentration in solution was evaluated by using amino acid analysis with analytical high-performance liquid chromatography after incubation for 24 hours at 100°C in 6 mol/L HCl. Tables 1
and 2 show the different apolipoprotein segments and synthetic peptides used. The first samples of the apoE dimer were generously provided by Cheryl A. Dyer and Linda K. Curtiss from The Scripps Research Institute, Calif.
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Cell Culture
Human fibroblasts were cultured in Dulbecco's minimal essential Eagle's medium supplemented with 10% fetal calf serum. Fibroblasts were cultured for 4 to 6 days before transfer to 10% lipoprotein-depleted serum for overnight upregulation of LDL receptors. THP-1 cells (American Type Culture Collection) were cultured in RPMI 1640 medium with 10% fetal calf serum. THP-1 cells were also cultured for 6 days before transfer to 10% lipoprotein-depleted serum for overnight upregulation of LDL receptors. Experiments with THP-1 cells were performed with the cells in suspension with gentle stirring. For subsequent experimental incubations, cells were changed to the corresponding medium supplemented with 0.2% BSA. For biolabeling of PGs, the medium was changed to Eagle's diploid (sulfate-depleted) medium with 105 IU/L penicillin, 0.1 g/L streptomycin, 1 mmol/L sodium pyruvate, and 4 mmol/L glutamine, and the cells were incubated for 72 hours. Labeled PGs were obtained by exposing cultured cells to 1 mL fresh Eagle's diploid medium containing sodium [35S]sulfate (20 mCi/mL) for 48 to 72 hours. We used heparitinase or chondroitinase ABC to degrade the cell GAGs.26 We chose 90 minutes at 37°C in the presence of 1.0 U/mL of enzymes and 0.03 mol/L sodium acetate. As cell-surface PGs may regenerate once the enzyme is removed, the concentration of enzymes at 1.0 U/mL was maintained during the subsequent experimental incubations of cells with 125I-labeled lipoproteins. Lyase activity was quantified by scintillation counting of 35S radioactivity in a 100-mL aliquot of the cell medium (LKB 1214 Rackbeta, Pharmacia-LKB).
Binding of 125I-labeled peptide to the THP-1 cells was measured by incubation with increasing amounts of 125I-labeled peptide for 4 hours at 4°C with or without cold peptide. The medium was HEPES buffer, pH 7.4, containing 1% BSA and 2 mmol/L CaCl2x2H2O (binding buffer) in 11x70-mm minisorb immunotubes (Nunc). After centrifugation at 1000 rpm for 5 minutes, the binding medium was aspirated, and the cells were washed twice by centrifugation with binding buffer at 4°C to remove any unbound radioactivity. The cells were washed twice with phosphate-buffered saline before adding 0.2 mol/L NaOH, and the protein was measured. The amounts of 125I-labeled lipoprotein bound to the cell surface and cell associated were determined, and the degradation of 125I-LDL was determined by the appearance of trichloroacetic acid–soluble radioactivity in the medium.14
The procedure described by Moscatelli27 was used for measuring the high-affinity, receptor-mediated and low-affinity, PG-mediated cell association of LDL with fibroblasts. The cells were incubated for four hours at 4°C in DPBS containing increasing amounts of 125I-LDL and 0.2% BSA. After being washed once with DPBS, bound LDL was dissociated from the cells by treatment with 2 mol/L NaCL and 20 mmol/L HEPES, pH 7.4. These washings were pooled and counted; they contained the LDL associated with the cells via low-affinity, pH-insensitive association, possibly pericellular PGs. The same cells were subsequently washed twice with 2 mmol/L NaCl and 20 mmol/L sodium acetate, pH 4.0, and the washings were pooled and counted. These last washings should contain mainly the LDL associated with pH-sensitive, high-affinity receptors. Protein content was estimated as described.22 Incubation with NaClO3 was used to block sulfation of the GAGs synthesized by the cultured fibroblasts.28 Binding to the apoB/E receptor was measured in detergent-solubilized membrane fractions prepared from fibroblasts.29 mAb C-7, which binds to the apoB/E receptor in human cells, was used for binding competition studies.30
Experiments With GAGs
The conditions for the evaluation of the affinity of LDL for GAGs by competition assay11 and for frontal affinity chromatography and coupling of GAGs to Affigel-Hz and to obtain the Kd as well as the methods used for preparing human arterial CSPGs6 14 have been described. Displacement experiments by peptides of LDL bound to C6S immobilized in Affigel-Hz were performed in a microtiter plate version. Fifty microliters of a 1:5 (vol/vol) suspension of C6S/Affigel-Hz in HEPES buffer (20 mmol/L NaCl and 0.1% ovalbumin) was added to each well, after which 50 µL LDL solution with or without competitor was added. The wells were covered, and after a 1-hour incubation on a shaking board at room temperature the plates were centrifuged at 2000 rpm for 5 minutes. The supernatant was carefully aspirated, and the wells were washed once with HEPES buffer. The LDL cholesterol associated with the beads (Bio-Rad) was determined directly in the wells by using an enzymatic microassay.31
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Results |
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Role of PGs in LDL Cell Association
The synthetic apoB-100 heterodimer inhibited LDL degradation by fibroblasts but not the monomeric segments (Fig 1
). Fibroblast degradation of 125I-LDL was inhibited 50% by the apoB heterodimer at 11 mmol/L and by the apoE dimer (dimeric repeat 141 through 155) at 2 mmol/L (Fig 1). One experiment with the monocytic cell line THP-1 gave similar results (not shown), indicating that the dimeric peptides also inhibited LDL degradation in other cell types. To investigate further the structural requirements for inhibition of 125I-LDL degradation, we tested a set of simplified model peptides using the apoB heterodimer as a pattern. The model peptides are listed in Table 2; their effect is shown as the concentration in micromoles per liter required to induce 50% inhibition (IC50) compared with the control. Inhibition with cold LDL is included for comparison (Table 2). All positively charged amino acids were exchanged for lysine (K), and all intervening amino acids were exchanged for glycine (G) (no side chain). Net charge seemed to be the most important factor associated with the inhibition of 125 I-LDL degradation as the decalysine, model peptide I, inhibited 125I-LDL degradation as efficiently as the apoB heterodimer. Spreading the 10 positive charges did not eliminate the inhibiting capacity but markedly increased the IC50 (model peptide II). Decreasing the number of charged lysines markedly decreased the inhibition in activity (peptides III through V). None of the peptides at the concentrations used diminished cell protein content.
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), apoB segment 3145 through 3157 (
), apoB segment 3359 through 3367 (
), apoB heterodimer (Het. Dimer) (
), and apoE dimer (
). Competitors were added to 5x104 fibroblast cells and incubated at 37°C for 5 hours with 5 mg/mL 125I-LDL. Each point is the average of duplicate samples in two separate experiments. SD is indicated when larger than symbol. Cell association data were similar (not shown).
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To investigate if cell-surface PGs were involved in LDL binding, we treated fibroblasts with heparitinase and chondroitinase ABC. This treatment should remove heparan and chondroitin/dermatan sulfate GAGs at the cell surface. Preincubation with chondroitinase ABC but not heparitinase decreased the 125I-LDL degradation by 52±7% when the cells were incubated with 25, 50, or 200 µg/mL 125I-LDL (P<.01). The binding curve at 4°C changed its characteristics after preincubation with lyases (Fig 2). The results were analyzed by using nonlinear regression (GraphPad Prism) and fitted to a saturation equation that assumed one class of binding sites. The binding isotherms of the controls consistently showed a higher Bmax and lower affinity (ie, higher Kd) than the treated cells. Clearly, partial elimination of the surface GAGs with chondroitinase ABC eliminated a contributor to the binding that has a lower affinity and a higher capacity for the lipoprotein. An alternative approach was also applied to investigate the potential involvement of PGs in the binding of LDL to cells. Sodium chlorate, an inhibitor of sulfate adenyltransferase, reduces protein and carbohydrate sulfation but not chain polymerization. We found that preincubation with 10 mmol/L sodium chlorate for 5 days reduced 125I-LDL cell association and degradation (Fig 3). At 10 mmol/L sodium chlorate, no change in the amount of protein or DNA per dish was observed. Quantitative estimation of 35S radioactivity released from enzyme-digested cells indicated that the digestion of GAGs was extensive. Most of the radioactivity appeared as a single band with a molecular mass <3000 D after sodium dodecyl sulfate–polyacrylamide electrophoresis. The control medium from undigested cells contained only radioactive material that did not enter the gel (>200 000 D).
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Because preliminary experiments indicated that the binding of 125I-labeled apoB heterodimer peptide to plastic in six-well plates was substantial, direct binding of this peptide was not possible in adhering fibroblasts. This was measured instead with THP-1 cells in suspension in low-protein-binding tubes by incubation at 4°C with increasing amounts of 125I-labeled peptide. Nonspecific binding was determined in the presence of a large excess concentration (50 mmol/L) of unlabeled peptide. A Scatchard plot of this data (not shown) indicated a Kd of 6.2x10-8 mol/L (r=.76) and 1.3x106 apoB heterodimers bound/cell. The binding appeared to be specific, because when fibroblast cells were incubated with 100 000 cpm 125I-labeled apoB heterodimer tracer and increasing concentrations of unlabeled peptide, the cold apoB heterodimer displaced the binding. On the other hand, adding an apoB-derived peptide of the same size, segment 455 through 481 (EDYLILRVIGNMGQTMEQLTPELKS), did not inhibit the binding. Association of the 125 I-labeled apoB heterodimer with fibroblasts was reduced from 0.042±0.01 to 0.017±0.01 nmol/mg cell protein by preincubating the cells with mAb C-7, which binds LDL receptors of both bovine and human origin. A control mAb directed against an irrelevant antigen (Aspergillus niger glucose oxidase) showed no effect. This suggests that >50% of the binding of the heterodimer to the cells is with structures at or near the LDL receptor. Control experiments with labeled LDL indicated a similar level of inhibition. Further evidence that the apoB heterodimer binds to the apoB/E receptor was obtained by using detergent-solubilized membrane fractions from fibroblasts. Electrophoresis of the receptor-enriched fraction, followed by ligand blotting and autoradiography, showed a weak but detectable binding of both 125I-labeled apoB heterodimer peptide and LDL to a band with a molecular weight in the same range as the LDL receptor (160 kD). The band was visible in the presence but not the absence of 5 mmol/L Ca2+ (50 mmol/L Tris-HCl, pH 8, 90 mmol/L NaCl, 50 mg/mL BSA, and 5 mmol/L Ca2+ for 60 minutes at 20°C). To gain information on the relative amount of low-affinity, cell-associated LDL and receptor-bound, high-affinity bound LDL, we used the procedure developed by Moscatelli.27 The results indicated that at different levels of LDL in the medium 30% of the cell-associated LDL resisted washing with 2 mol/L NaCl at a neutral pH (Fig 4), which suggests a receptor-mediated, high-affinity association. 70% of the LDL was released by such treatment, a condition that should dissociate LDL bound by low-affinity bonds to PGs. The fact that treatment with chondroitinase ABC and heparitinase provoked an appreciable reduction of the released low-affinity bound LDL supports this conclusion.
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Experiments With Purified PGs and GAGs
Binding of the apoB heterodimer to human arterial CSPGs by using frontal elution chromatography showed an affinity of 2±1.5 µmol/L (average of four measurements). This affinity suggested that the heterodimer could be used in displacement experiments to study LDL binding to purified GAGs. We examined how LDL of different sizes was displaced from C6S–Affigel beads in the microtiter plate procedure described in "Methods." The most evident difference was that the smaller LDL (25 nm) had a binding that was 1.4 times that of the larger LDL (27 nm) subfraction (Fig 5). The amount of peptide required to displace the binding was somewhat less for achieving 50% displacement of the large LDL subfraction. However, 20 times less apoB heterodimer was enough to displace 50% of the binding compared with monomeric segment 3359 through 3367 (Fig 5). It appears, then, that the apoB heterodimer has 10 to 20 times greater affinity for CSPGs or C6S than for the monomeric apoB segments, and it retains its affinity for GAGs at physiological salt concentrations.
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Discussion |
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In apoB-100 several positive segments appear to conform to the apoB/E receptor binding domain.4 5 12 Segments 3145 through 3157 and 3359 through 3367 are the most positive sequences flanking the putative U-turn induced by the disulfide bond between 3167 and 3297.4 5 12 This suggests that formation of positive patches formed by linearly distant segments brought together by turns of the apoB-100 may contribute to form the extended receptor-binding and GAG-binding region of LDL. A synthetic dimeric tandem peptide (a 144 through 155 repeat) from apoE binds the apoB/E receptor, whereas the monomeric peptide is inactive.2 In analogy with apoE, we used the apoB heterodimer SVKAQYKKNKHRHGGGRLTRKRGLK (segments 3145 through 3157 and 3359 through 3367) as a model of a configuration in which these two segments are in proximity. This may be a minimal model because other regions of apoB-100 that also bind GAGs, such as 4230 through 4253 and 2106 through 2121, may contribute to the lipoprotein association. However, in vitro these last segments show lower affinity for GAGs.10 11 The ability of the apoB heterodimer to inhibit degradation of 125I-LDL by cells (Fig 1
and Table 2) was expected given the high homology between this model and the apoE dimer. Our results indicated that the apoB heterodimer that competed for the LDL receptor also contained the ligands for binding to GAGs of biological relevance, mostly chondroitin sulfates, dermatan sulfate, and probably heparan sulfate. An additional finding was that cell-surface PGs contributed to cell association and internalization of LDL, probably by creating a pericellular compartment that concentrates the particles in the immediacy of the apoB/E receptor. A similar situation has been postulated to exist in the uptake of VLDL remnants by hepatocytes. In this last process apoE plays the role of sequential ligand for pericellular heparan and chondroitin sulfates and the specific membrane receptor.18 19 These conclusions, like ours, are supported by the decreased binding and degradation of lipoproteins by cells after hydrolysis of cell-surface GAGs.
The reversible association of LDL with arterial CSPGs alters the organization of the lipid and protein moiety of the particle and appears to increase the surface exposure of basic residues of apoB-100.32 These effects are more marked in small dense LDL particles than in large ones.14 The GAG-induced alterations are associated with an increase in LDL binding and degradation by human monocyte–derived macrophages and smooth muscle cells.14 33 34 The reduced LDL degradation by fibroblasts observed after digestion of the pericellular PGs may be related to elimination of possible alterations in LDL surface structure that may be present when the pericellular PGs are intact. A second but less likely possibility is that the enzyme treatment activates the cells and in some way downregulates the expression of receptors. The complex shape in the binding isotherm for 125I-LDL to fibroblasts is well established and has been interpreted as steric hindrance for binding to the receptor.35 The curve in Fig 2 better fitted a model with a single class of binding sites after preincubation with heparitinase and chondroitinase. At low LDL concentrations in the treated cells the equilibrium appears shifted to higher binding. One explanation may be that removal of GAGs uncovered high-affinity sites but reduced those contributing to binding at high LDL concentrations. However, this hypothesis would need documentation by experiments aided to evaluate number and classes of binding sites with different extents of digestion. We interpret the results as an indication that GAGs are a second class of binding sites with higher Bmax and lower affinity for binding of LDL to the cells than the apoB/E receptor. Reduction of LDL binding and degradation after diminishing PG sulfation with chlorate (Fig 3) was expected because negative-charge density is one of the main factors contributing to the association of GAGs with LDL and other basic proteins.7 8 9 However, here we did not observe the suggested "uncovering" of high-affinity sites detected after treatment with chondroitinase ABC, but the reduction of binding and degradation at saturating levels of LDL was clearer. It is important to indicate that chlorate treatment does not eliminate GAG chains but rather reduces their sulfation.28 The results of the experiments with the sequential washing procedure, as proposed by Moscatelli,27 evaluate low- and high-affinity cell association (Fig 4). These results support the importance of a pericellular PG layer as a region of accretion of LDL modulating cell uptake via the receptors. Chondroitinase ABC decreased 125I-LDL degradation more than heparitinase. Perhaps there are more and larger chondroitin/dermatan sulfate GAGs that interact with the LDL. The lack of effect of heparitinase on the degradation of 125I-LDL in fibroblasts reported by Williams et al21 and Ji et al18 19 does not contradict the results presented here, since they used only heparitinase or heparinase to treat the cells and tissue. Williams et al21 found that the lipoprotein lipase–mediated increase of LDL degradation by fibroblasts was independent of the apoB/E receptor. Our cell system contained no added lipase. It is feasible that binary complexes of LDL-GAG(s) modulate receptor-mediated uptake but that those ternary complexes involving LDL–lipoprotein lipase–GAG(s) enter the cell by alternate pathways. The complexity of these phenomena is increased by the possible competition of the basic segments of apoE and apoB-100 for the binding of lipoprotein lipase immobilized in the extracellular matrix20
The affinity constants obtained for direct binding of 125I-labeled apoB heterodimer to THP-1 cells indicated 10 times lower affinity with the synthetic peptide than LDL. The binding appeared saturable, calcium dependent, and was blocked by mAbs to the LDL receptor. The LDL receptors contain repeated sequences of negatively charged amino acids that are clustered in coated pits.36 A small molecule with some of the charge and sequence required for interaction, like the apoB heterodimer, could perhaps bind in large numbers to each receptor. This could be one way to explain why the total number of binding sites for the apoB heterodimer is several orders of magnitude larger than that for LDL. The experiments with extracellular GAGs indicated that the apoB heterodimer has an affinity for CSPGs and C6S in a range similar to that of LDL. This was 10 to 20 times higher than that of its linear monomeric components. These results (Fig 5) also indicated that smaller LDL subfractions have a higher total binding to C6S than larger subfractions. This is consistent with previous data showing that smaller, dense LDL has a higher affinity for CSPGs, which suggests an increased exposure of positive segments.14 34
The dimeric sequence motif may be a plausible mechanism by which association with lipids could increase the binding of apoE to the LDL receptor without changing the secondary structure of apoE.2 The affinity of lipid-free apoE is >2500 times lower than that of the lipid-complexed protein (Karl H. Weisgraber, letter, June 1994). Our results show differences in the affinities of the apoE and apoB heterodimer for the receptor. This was not unexpected, since the receptor binding site in apoE (142 through 152) contains six positive charges (in the dimer, 12), whereas the proposed LDL receptor binding sites in apoB (3359 through 3367 and 3147 through 3157) contain five positive charges each (10 in the heterodimer). Natural variants of apoE for positions 142, 145, and 146, in which the basic amino acid is substituted by a neutral or acidic amino acid, display 20% to 40% of normal binding.36 37 Thus, eliminating only one of the positive charges in the LDL receptor–binding segment of apoE may reduce the affinity of apoE (dimer) in VLDL or a liposome to the level of affinity of apoB-100 in LDL for the LDL receptor.
The above results and ours indicate that tertiary structure and charge densities, not secondary structure, contribute to the receptor- and GAG-binding properties of lipoproteins. It is possible that linear segments that are components of an LDL receptor–binding region on the LDL particle are too far apart in large particles. Differences in the distribution of VLDL and LDL subfractions with dissimilarities in size, composition, and surface charge could alter their binding to pericellular PGs and receptors.14 38 A general regulatory mechanism is associated with the above ideas. ApoB would become a ligand for the LDL receptor when the lipoprotein reached a size at which the receptor-binding regions of apoB adopted the proper surface location; this could possibly be a structure in which positive segments coalesce on the LDL surface.11 14 The data presented here and from other studies indicate that the LDL receptor–binding regions of apoB-100 are also the segments that bind GAGs most strongly.10 11 In addition, heparin and other sulfated GAGs can compete for LDL with their membrane receptors.39 These findings raise interesting questions concerning the distribution and availability of LDL and apoE-containing particles interacting with pericellular PGs and cell membrane–specific receptors.20 These associations may modulate the extracellular deposition and cellular uptake of LDL in normal tissues and the arterial wall.40
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Acknowledgments |
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This work was supported by grants from the Swedish Medical Research Council (project No. 4531), the Swedish Heart- Lung Foundation, and Astra Hassle AB. We thank Ulla Ruetschi, Department of Clinical Chemistry, Sahlgren's Hospital, Goteborg, for sequencing the synthetic peptides and Ulla-Britt Rignell for her excellent technical assistance.
Received ;
revision received ;
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References |
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