Oxidation of Low Density Lipoprotein Particles Decreases Their Ability to Bind to Human Aortic Proteoglycans
1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês
10.1074/jbc.272.34.21303
ISSN1083-351X
AutoresKatariina Öörni, Markku O. Pentikäinen, Arto Annila, Petri T. Kovanen,
Tópico(s)Atherosclerosis and Cardiovascular Diseases
ResumoOxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis. Oxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis. Human atherosclerosis is characterized by accumulation of low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; apo, apolipoprotein; MDA, malondialdehyde; BHT, butylated hydroxytoluene; AAPH, 2,2′-azobis(2-amidinopropane)hydrochloride; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; TBARS, thiobarbituric acid-reactive substances. in the arterial intima. In atherosclerostic lesions, LDL is modified and accumulates both intra- and extracellularly. The LDL modification that has been most studied is oxidation. The role of LDL oxidation in intracellular accumulation is well established. Thus, oxidized LDL is taken up by macrophage scavenger receptors, which transform the macrophages into foam cells (1Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). In contrast, the role of oxidation of LDL in extracellular accumulation of LDL is not known. An important component of extracellular accumulation of LDL is binding of LDL to the extracellular matrix consisting mainly of proteoglycans (2Nievelstein P.F.E.M. Fogelman A.M. Mottino G. Frank J.S. Arterioscler. Thromb. 1991; 11: 1795-1805Crossref PubMed Google Scholar, 3Frank J.S. Fogelman A.M. J. Lipid Res. 1989; 30: 967-978Abstract Full Text PDF PubMed Google Scholar). Native LDL binds proteoglycans via ionic interactions between the positively charged lysine and arginine residues of the apolipoprotein component of LDL, the apoB-100, and the negatively charged sulfate and carboxyl groups of glycosaminoglycan chains of the proteoglycans (4Iverius P.-H. J. Biol. Chem. 1972; 247: 2607-2613Abstract Full Text PDF PubMed Google Scholar, 5Weisgraber K.H. Rall Jr., S.C. J. Biol. Chem. 1987; 262: 11097-11103Abstract Full Text PDF PubMed Google Scholar, 6Camejo G. Olofsson S.-O. López F. Carlsson P. Bondjers G. Arteriosclerosis. 1988; 8: 368-377Crossref PubMed Google Scholar, 7Cardin A.D. Weintraub H.J.R. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Upon oxidation of LDL, the unsaturated fatty acids of LDL lipids are decomposed, for instance, to malondialdehyde (MDA) and 4-hydroxynonenal. These compounds can then react with the lysine residues of apoB-100, thereby neutralizing them and so interfering with their ability to interact with other molecules. Indeed, Haberland et al. (8Haberland M.E. Fogelman A.M. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1712-1716Crossref PubMed Scopus (240) Google Scholar) found that MDA-modified LDL failed to bind to heparin. In this study we oxidized LDL in vitro by several methods and analyzed the effect of oxidation on its degree of binding to proteoglycans isolated from human aortas. We also studied the effects of lysine modification on the degree of binding, by specifically modifying the lysine residues of apoB-100, and by preventing lysine oxidation by prior methylation of the lysine residues. Since the lysine residues of apoB-100 are of two types having different pKa values, 8.9 and 10.5 (9Lund-Katz S. Ibdah J.A. Letizia J.Y. Thomas M.T. Phillips M.C. J. Biol. Chem. 1988; 263: 13831-13838Abstract Full Text PDF PubMed Google Scholar), of which the former "active lysines" are thought to be located in the areas of apoB-100 that bind to proteoglycans, we also investigated the effect of oxidation on this population of lysine residues in LDL. Bovine serum albumin, butylated hydroxytoluene (BHT), cholesteryl linoleate, ε-aminocaproic acid, and soybean lipoxygenase type V were from Sigma; copper(II) sulfate pentahydrate, EDTA, and CHOD-iodide kit (catalog no 14359) for peroxide measurement were from Merck; malondialdehyde bis(dimethyl acetal) was from Aldrich; and 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH) was from Polysciences. Chondroitinase ABC and AC, chondroitin-6-sulfate, and the unsaturated chondro-disaccharide kit for high performance liquid chromatography (HPLC) were from Seikagaku Kogyo. [1,2-3H]Cholesteryl linoleate was from Amersham. Phenylmethylsulfonyl fluoride was from Boehringer Mannheim, Celite 545 (acid-washed) was from Fluka, and the Schrynel nylon filter from Zürcher Beuteltuchfabrik AG. [13C]Formaldehyde (99% isotope enrichment) as a 20% solution in water was from Isotec Inc., and [14C]formaldehyde from DuPont. NaCNBH3 from Sigma was purified by recrystallization from dichloromethane before use (10Jentoft N. Dearborn D.G. Methods Enzymol. 1983; 91: 570-579Crossref PubMed Scopus (180) Google Scholar). Dulbecco's phosphate-buffered saline (PBS), RPMI 1640 culture medium with 25 mm HEPES, fetal calf serum, penicillin, and streptomycin were from Life Technologies, Inc. Female NMRI mice (20–30 g) were obtained from a licensed animal center (Poikkijoki). Superose 6 HR 10/30 columns, HiTrapN-hydroxysuccinimide activated columns, HiTrap heparin columns, HiTrap Q columns, and PD-10 columns were from Pharmacia LKB Biotechnology; Bio-Gel A-0.5m gel filtration medium was from Bio-Rad; the 5-μm NH2 (0.3 × 25 cm) and S5 ODS (0.3 × 25 cm) columns were from Spherisorb. Cholesteryl ester transfer protein was a kind gift from Drs. C. Ehnholm and M. Jauhiainen, National Public Health Institute, Helsinki, Finland. Human LDL (d = 1.019–1.050) was isolated from plasma of healthy volunteers by sequential ultracentrifugation in the presence of 3 mm EDTA (11Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 77: 757-761Google Scholar). [3H]Cholesteryl linoleate-LDL ([3H]CL-LDL) was prepared by incubating LDL and cholesteryl ester transfer protein with solid dispersions of [3H]cholesteryl linoleate on Celite for 18–24 h as described previously (12Paananen K. Saarinen J. Annila A. Kovanen P.T. J. Biol. Chem. 1995; 270: 12257-12262Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The amount of LDL is expressed in terms of its protein concentration. LDL was oxidized with copper by incubating LDL or [3H]CL-LDL (1 mg/ml) in PBS supplemented with 5 μm copper sulfate at 37 °C for the time periods indicated (13Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3883-3887Crossref PubMed Scopus (1465) Google Scholar). LDL was oxidized with AAPH by incubating [3H]CL-LDL (1 mg/ml) in buffer A (150 mmNaCl, 1 mm EDTA, pH 7.4) supplemented with 25 mm AAPH at 37 °C for the time periods indicated (14Stocker R. Bowry V.W. Frei B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1646-1650Crossref PubMed Scopus (788) Google Scholar). Oxidation was terminated by addition of BHT and EDTA (see below) and cooling the sample on ice. AAPH was removed from the sample over a PD-10 column equilibrated and eluted with ice-cold buffer A. LDL was oxidized by mouse peritoneal macrophages essentially as described elsewhere (15Pentikäinen M.O. Lindstedt K.A. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 740-747Crossref PubMed Scopus (26) Google Scholar). Briefly, [3H]CL-LDL (100 μg/ml) was incubated in 300 μl of RPMI 1640 cell culture medium supplemented with 300 nm copper sulfate in a 24-well plastic culture plate seeded with 1 × 106 macrophages in a humidified CO2 incubator (5% CO2 in air) at 37 °C for the time periods indicated. LDL was oxidized with soybean lipoxygenase by incubating [3H]CL-LDL (2.5 mg/ml) in buffer A containing 1.25 mm linoleate-albumin and 60 units/μl soybean lipoxygenase at 37 °C for 18 h (16Sparrow C.P. Parthasarathy S. Steinberg D. J. Lipid Res. 1988; 29: 745-753Abstract Full Text PDF PubMed Google Scholar). LDL was reisolated by gel filtration chromatography on an A-0.5m column (1 × 50 cm). In all systems, oxidation was terminated by addition of BHT and EDTA to give final concentrations of 20 μm and 1 mm, respectively. LDL was modified with MDA by incubating [3H]CL-LDL (1 mg/ml in PBS) with increasing amounts of MDA on an ice bath for 3 h (17Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Witztum J.L. Steinberg D. J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar). The final concentrations of MDA are given in the figure legends. After incubation, LDL was dialyzed extensively against buffer A. To 2 mg of [3H]CL-LDL in 2 ml of buffer A (150 mm NaCl, 1 mm EDTA, pH 7.4), 0.2 m NaCNBH3 was added to give a final concentration of 20 mm. After addition of 15 × 10−6 mol of formaldehyde to the sample, the mixture was incubated at 4 °C for 18 h (10Jentoft N. Dearborn D.G. Methods Enzymol. 1983; 91: 570-579Crossref PubMed Scopus (180) Google Scholar). β-Carotene was analyzed spectrophotometrically by measuring the absorbance of the β-carotene in LDL (1 mg/ml) at 482 nm (18Chang G.J. Woo P. Honda H.M. Ignarro L.J. Young L. Berliner J.A. Demer L.L. Arterioscler. Thromb. 1994; 14: 1808-1814Crossref PubMed Google Scholar). Reactive lysine residues (ε-amino groups of lysine residues) were determined with trinitrobenzenesulfonic acid (19Habeeb A.F.S.A. Anal. Biochem. 1966; 14: 328-336Crossref PubMed Scopus (1956) Google Scholar), using valine as standard, and expressed as percentages of the value for native LDL. The quantity of conjugated dienes in LDL was assessed by measuring the absorbance of LDL (0.1 mg/ml) at 234 nm with a spectrophotometer fitted with a cuvette with a 1-cm light path (20Esterbauer H. Striegl G. Puhl H. Rotheneder M. Free Radical Res. Commun. 1989; 6: 67-75Crossref PubMed Scopus (1705) Google Scholar). Lipid peroxides were measured as described by El-Saadani et al. (21El-Saadani M. Esterbauer H. El-Sayed M. Goher M. Nassar A.Y. Jurgens G. J. Lipid Res. 1989; 30: 627-630Abstract Full Text PDF PubMed Google Scholar) with a commercial CHOD-iodide kit. Thiobarbituric acid-reactive substances (TBARS) were measured essentially as described by Hessler et al. (22Hessler J.R. Morel D.W. Lewis L.J. Chisolm G.M. Arteriosclerosis. 1983; 3: 215-222Crossref PubMed Google Scholar), and expressed as MDA equivalents, with 1,1,3,3-tetramethoxypropane as standard. The electrophoretic mobility of LDL was determined on cellulose acetate plates (Helena Laboratories). The extent of LDL aggregation was determined by analyzing oxidized [3H]CL-LDL with gel filtration column chromatography on two Superose 6 HR 10/30 columns connected in series. The degree of aggregation was measured as the proportion of3H radioactivity eluting in the void volume of the column. Proteoglycans from intima-media of human aortas obtained at autopsy within 24 h of accidental death were prepared essentially as described by Hurt-Camejo et al. (23Hurt-Camejo E. Camejo G. Rosengren B. López F. Wiklund O. Bondjers G. J. Lipid Res. 1990; 31: 1397-1398Abstract Full Text PDF Google Scholar). Briefly, proteoglycans were extracted from the intima-media at 4 °C for 24 h with 15 volumes of 6 m urea, 1 mNaCl in the presence of 10 mm EDTA, 10 mmε-aminocaproic acid, 0.2 mm phenylmethyl sulfonyl fluoride, and 0.02% (w/v) NaN3. After extraction, the mixture was centrifuged at 100,000 × g for 60 min. The supernatant was diluted with 6 m urea to give a final concentration of 0.25 m NaCl and loaded on a HiTrap Q column (5 ml) equilibrated with 6 m urea, 0.25m NaCl, 10 mm CaCl2, and 50 mm acetate, pH 6.2, and the protease inhibitors. The column was washed with the above buffer, and the proteoglycans were eluted with a linear gradient of 0.25 to 1.0 m NaCl in the buffer (120 ml) at a flow rate of 2 ml/min. The peaks at 280 nm were collected, dialyzed against water, and lyophilized. The disaccharide composition of the proteoglycans was analyzed by HPLC, using a 5-μm NH2 column, after treatment of the proteoglycans with chondroitinase ABC and AC (24Macek J. Krajickova J. Adam M. J. Chromatogr. 1987; 414: 156-160Crossref PubMed Scopus (20) Google Scholar). The proteoglycan preparation used here contained 56% of chondroitin-6-sulfate, 25% of chondroitin-4-sulfate, and 19% of dermatan sulfate. The amounts of the proteoglycans are expressed in terms of their glycosaminoglycan content. Human arterial proteoglycans or chondroitin-6-sulfate were coupled to anN-hydroxysuccinimide-activated HiTrap column (1 ml or 5 ml, respectively) according to the manufacturer's instructions. For this purpose, 1.0 mg of proteoglycans or 10 mg of chondroitin-6-sulfate in 0.2 m NaHCO3 and 0.5 m NaCl, pH 8.3, were coupled to the column at 25 °C for 2 h. The column was blocked with 0.5 m ethanolamine, pH 8.3, containing 0.5m NaCl. Under these conditions, 0.7 mg of the proteoglycans and 7.4 mg of the chondroitin-6-sulfate were found to be coupled to the column. The columns were equilibrated with buffer B (10 mmHEPES, 2 mm CaCl2, 2 mmMgCl2, pH 7.4) before use. LDL or [3H]CL-LDL was oxidized as described above, and 20–30-μl samples of the incubation mixtures corresponding to 20–30 μg of LDL were analyzed on the proteoglycan or heparin affinity columns by elution with a linear gradient of NaCl (0 to 250 mm or 0 to 500 mm, respectively, in 10 min) in buffer B. Chromatography was performed at a flow rate of 1 ml/min. LDL was eluted from the chondroitin-6-sulfate column at a flow rate of 2 ml/min with buffer B containing 250 mm NaCl. Proteins were detected by UV absorbance at 280 nm or, in some experiments in which radiolabeled LDL was used, by collecting fractions and determining their radioactivity. The conductivity of the eluent was also monitored. The chromatographic apparatus was Smart system (Pharmacia). LDL (30 mg) was incubated for 6 h with 5 μm CuSO4 in 30 ml of PBS at 37 °C. Oxidation was terminated by addition of BHT and EDTA to give final concentrations of 20 μm and 1 mm, respectively. For NMR analysis, the amino groups of the free lysine residues of apoB-100 of native or oxidized LDL were13C-labeled by reductive methylation with [13C]formaldehyde (9Lund-Katz S. Ibdah J.A. Letizia J.Y. Thomas M.T. Phillips M.C. J. Biol. Chem. 1988; 263: 13831-13838Abstract Full Text PDF PubMed Google Scholar, 10Jentoft N. Dearborn D.G. Methods Enzymol. 1983; 91: 570-579Crossref PubMed Scopus (180) Google Scholar). First, 0.2 mNaCNBH3 was added to 30 mg of native or oxidized LDL in 30 ml of buffer A to give a final concentration of 20 mm. After addition of 2 × 10−4 mol of [13C]formaldehyde to the samples, the mixtures were incubated for 18 h at 4 °C. The reactions were stopped by extensive dialysis against buffer A, and the labeled LDL solutions were concentrated in Amicon 100 concentrators with a 100-kDa cut-off membrane. The H13CHO was doped with a trace of [14C]formaldehyde to give a known specific radioactivity. The degree of reductive methylation was calculated by counting the amount of [14C]formaldehyde incorporated into the lysine residues of apoB-100. Broad-band proton-decoupled13C NMR spectra were measured from LDL samples comprising 11.2–11.4 mg of protein/ml in a solution containing 150 mmNaCl, 1 mm EDTA, and 0.02% NaN3, pH 7.4, and 10% of D2O for the spectrometer field-lock.13C NMR spectra were obtained at 150.8 MHz with a Varian Unity 600 NMR spectrometer. All experiments were recorded at 37 ± 0.5 °C, and the spectral width was 250 ppm, corresponding to 16,000 points in 0.219 s. Pulse length was 8 μs. Proton decoupling was performed with a GARP sequence (25Shaka A.J. Barker P. Freeman R. J. Magn. Reson. 1985; 64: 547Google Scholar). Total running times varied from 4 to 6 h, depending on the sample. Relaxation delay was 1.0 s to gain a good signal-to-noise ratio. The repetition time of these measurements was on the order of the longitudinal relaxation time of the active lysines (26Lund-Katz S. Innerarity T.L. Arnold K.S. Curtiss L.K. Phillips M.C. J. Biol. Chem. 1991; 266: 2701-2704Abstract Full Text PDF PubMed Google Scholar). Consequently, the number of active lysines in these measurements was underestimated compared with the normal lysines which relax faster. We therefore measured 13C-labeled LDL using relaxation delays of 1.0 and 5.0 s and obtained a correction factor of 1.47 to subsequently multiply the integrated dimethyl-lysine resonances of the active lysines. Prior to Fourier transformations, free induction decays were filled to 32,000 and weighted by 2-Hz line broadening. Macrophages from unstimulated NMRI mice were harvested into Dulbecco's PBS containing 1 mg/ml bovine serum albumin (27Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (2023) Google Scholar). The peritoneal cells were resuspended in RPMI 1640 medium containing 20% of heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells (1–2 × 106) were seeded into plastic 24-well plates (Falcon) and incubated overnight in a humidified CO2 (5% CO2 in air) incubator. Before the experiments, the dishes were washed to remove nonadherent cells. Each monolayer received 300 μl of RPMI medium containing 10 mg/ml bovine serum albumin, 100 IU/ml penicillin, 100 μg/ml streptomycin, 200 μmoleate-albumin, and 30 μg of native or oxidized LDL. After incubation for 18 h at 37 °C, the amount of cholesteryl oleate in the cells was determined with reverse phase HPLC, using an S5 ODS (0.3 × 25 cm) column as described previously (28Piha M. Lindstedt L. Kovanen P.T. Biochemistry. 1995; 34: 10120-10129Crossref PubMed Scopus (67) Google Scholar). Protein was determined by the method of Lowryet al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), with bovine serum albumin as standard. Glycosaminoglycans were determined by the method of Bartold and Page (30Bartold P.M. Page R.C. Anal. Biochem. 1985; 150: 320-324Crossref PubMed Scopus (59) Google Scholar). To study the effect of LDL oxidation on the amount of LDL bound to human aortic proteoglycans, LDL was oxidized by incubation with 5 μm CuSO4 at 37 °C for various periods of time up to 18 h. At the indicated time points, the degree of LDL oxidation was analyzed by measuring changes in several parameters known to be associated with LDL oxidation (Fig. 1, left panels): loss of β-carotene and of ε-amino groups (lysine); formation of conjugated dienes, TBARS, and peroxides; and increase in the electrophoretic mobility of LDL. The amount of oxidized LDL bound to aortic proteoglycans was determined by applying aliquots of the incubation mixtures to a proteoglycan affinity column. The column was then washed for 1 min with buffer B, and the bound LDL was eluted with a 10-min linear NaCl gradient (0–250 mm NaCl) in buffer B. Elution was monitored with UV absorbance at 280 nm, and the gradient was checked by measuring the changes in conductivity (Fig. 1, right panels, dotted lines). Of the 20 μg of native LDL applied, 95% bound to the column and eluted as a single peak at 65 mm NaCl (Fig. 1, right panels, top). As shown by the elution profiles of the samples treated with CuSO4 for various time periods, the higher the degree of oxidation (Fig. 1, left panels), the smaller was the amount of LDL bound to the proteoglycans, until, after oxidation for 18 h, the modified LDL particles failed to bind to the proteoglycans (Fig. 1, right panels, bottom). Progressive copper-induced oxidation of LDL generates aggregation of the lipoprotein (31Lougheed M. Steinbrecher U.P. J. Biol. Chem. 1996; 271: 11798-11805Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To assess whether the observed loss in the ability of LDL to bind to proteoglycans is due to aggregation of LDL, we determined the degree of particle aggregation in our oxidized LDL samples by gel filtration chromatography. When LDL was oxidized for 18 h, which fully abolishes binding of LDL to proteoglycans (Fig. 1), the amount of LDL aggregated (eluting in the void volume of the column) was 24%. Since only a fraction of the oxidized LDL particles had aggregated, aggregation cannot be responsible for the loss in the ability of LDL to bind to proteoglycans. As oxidation progressed, the electrophoretic mobility of LDL increased (Fig. 1, left panels). Since only one band of LDL was formed, the particles must have been modified to roughly the same extent at any given time point. Yet, when analyzed with the proteoglycan affinity column, two populations of LDL emerged, one eluting without binding to the proteoglycans, and the other binding to the column and eluting only at the higher NaCl concentrations (Fig. 1,right panels). To examine whether these two populations of LDL had been differently oxidized, we performed an experiment in which LDL was oxidized by incubation with CuSO4 for 6 h. The sample was then applied to a chondroitin-6-sulfate affinity column, the unbound LDL was collected in buffer B, and the bound LDL was eluted with buffer B containing 250 mm NaCl. Both fractions were concentrated in Amicon 10 concentrators with a 10-kDa cut-off membrane. Aliquots of the fractions were then rechromatographed on the same affinity column: most (90%) of the bound LDL did bind again, and most (85%) of the unbound LDL remained unbound. The same result was obtained when a proteoglycan affinity column was used. The bound and unbound oxidized LDL fractions were examined for their contents of peroxides and number of free ε-amino groups in lysines. No difference in peroxide content was found between the two fractions. In striking contrast, the amount of free lysine residues was 90% in the bound LDL and only 65% in the unbound LDL. When LDL was oxidized for 18 h, a similar difference in the number of lysine residues was observed between the bound and unbound LDL fractions, although the proportion of LDL that was bound was smaller than after oxidation for 6 h (above). In an additional experiment, LDL was oxidized for 18 h and chromatographed on a heparin HiTrap (1 ml) affinity column. Again, although there was no difference in peroxide content between the two oxidized LDL fractions, the bound LDL contained a higher proportion (75%) of free lysine residues than the unbound LDL (58%). As expected, the relative electrophoretic mobility of the unbound LDL was higher than that of the bound LDL. Taken together, the above results are compatible with the idea that the factor which had rendered LDL particles unable to bind to glycosaminoglycans or to proteoglycans was the reduction in the number of free lysine residues of apoB-100. LDL was next oxidized with AAPH for various time periods. At the indicated time points, one sample of the incubation mixture was taken for analysis of the amounts of ε-amino groups, TBARS, peroxides, and relative electrophoretic mobility (Fig. 2, left panels), while another was applied to the proteoglycan affinity column (Fig. 2, right panels). As with copper-modified LDL, it was found that as oxidation advanced, the binding of LDL to the column decreased. After oxidation for 12 h, when 60% of the lysine residues were modified, the LDL failed to bind to the proteoglycans. Finally, AAPH-oxidized LDL, which contained 70% of free lysines, was applied to a chondroitin-6-sulfate affinity column, and the percentages of the free lysine residues in the bound and the unbound LDL fractions were found to be 72 and 64%, respectively. Thus, in the AAPH-oxidized, as in the copper-oxidized LDL preparations, the number of free lysine residues was higher in the bound fraction than in the fraction that failed to bind to the glycosaminoglycans. To obtain LDL oxidized in a more physiological way, LDL was incubated with mouse peritoneal macrophages for up to 12 h. At the indicated times, formation of TBARS and the relative electrophoretic mobility of the LDL particles were determined (Fig. 3, left panels), and the degree of binding to proteoglycans was determined on the proteoglycan column (Fig. 3, right panels). As the incubation time increased, LDL was progressively oxidized, the degree of oxidation being comparable to that obtained with CuSO4 or AAPH. As in the previous experiments, the increasing extent of oxidation progressively decreased the amount of binding, so that after incubation with the macrophages for 12 h, when the production of TBARS had reached a value of about 10 nmol of MDA/mg of LDL protein, no LDL was bound to the proteoglycan column. Oxidation of LDL by lipoxygenases has recently received attention as a possible mode of oxidation in the arterial wall (17Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Witztum J.L. Steinberg D. J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar). To investigate whether lipoxygenase-mediated oxidation of LDL might play a role in the interaction of LDL with arterial proteoglycans, LDL was next oxidized with soybean lipoxygenase in buffer A containing 1.25 mmlinoleate-albumin. After oxidation for 18 h, the samples were chromatographed on an A-0.5m column (1 × 50 cm) to separate the linoleate-albumin from the LDL. Fractions containing LDL were pooled and concentrated, and their peroxide content, number of ε-amino groups, electrophoretic mobility, and degree of binding to proteoglycans were determined. As shown in Table I, soybean lipoxygenase oxidized LDL in the presence of linoleate-albumin. The peroxide content of the oxidized samples was similar to that observed with the other methods of oxidation. However, the changes in electrophoretic mobility were smaller and the differences in the degree of lysine modification less pronounced than with the other methods of oxidation, and, as expected, the amount of binding to proteoglycans was only slightly decreased.Table IOxidation of LDL with soybean lipoxygenaseLDLɛ-Amino groupsPeroxidesElectrophoretic mobilityLDL bound to proteoglycan column% of native LDLA365/50 μg of LDLrelative to LDL%Control LDL1000.051.192Lipoxygenase-treated LDL870.241.275After incubation for 18 h of [3H]CL-LDL (2.5 mg/ml) in buffer A containing 1.25 mm linoleate-albumin and 60 units/μl soybean lipoxygenase at 37°C, the amounts of ɛ-amino groups and the relative electrophoretic mobility were measured as described under "Experimental Procedures." The ratio of 3H radioactivity eluting from the proteoglycan column in peak II (see Fig. 1) to the total eluted 3H radioactivity is used as a measure of the LDL bound to the prote
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