J. Lipid Res. Acyl Labeled PIP's available August 1, 2008
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Journal of Lipid Research, Vol. 43, 1486-1495, September 2002
Copyright © 2002 by Lipid Research, Inc.

{omega}-Carboxyl variants of 7-ketocholesteryl esters are ligands for ß2-glycoprotein I and mediate antibody-dependent uptake of oxidized LDL by macrophages

Qingping Liu1,*, Kazuko Kobayashi*, Jun-ichi Furukawa{dagger}, Junko Inagaki*, Nobuo Sakairi{dagger}, Akimasa Iwado§, Tatsuji Yasuda*, Takao Koike**, Dennis R. Voelker{ddagger} and Eiji Matsuura2,*

* Department of Cell Chemistry, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
{dagger} Division of Bioscience, Graduate School of Environment Earth Science, Hokkaido University, Sapporo 060-0810, Japan
§ Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
** Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
{ddagger} Program in Cell Biology, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206

DOI 10.1194/jlr.M20063-JLR200

1 Q. Liu and K. Kobayashi contributed equally to this work. Back

2 To whom correspondence should be addressed. e-mail: eijimatu{at}md.okayama-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ß2-Glycoprotein I (ß2-GPI) is a major antigen for anticardiolipin antibodies (aCL, Abs) present in patients with antiphospholipid syndrome. We recently reported that ß2-GPI specifically binds to oxidized LDL (oxLDL) and that the ß2-GPI's major ligand, oxLig-1 is 7-ketocholesteryl-9-carboxynonanoate (Kobayashi, K., E. Matsuura, Q. P. Liu, J. Furukawa, K. Kaihara, J. Inagaki, T. Atsumi, N. Sakairi, T. Yasuda, D. R. Voelker, and T. Koike. 2001. A specific ligand for ß2-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages. J. Lipid Res. 42: 697–709). In the present study, we demonstrate that {omega}-carboxylated 7-ketocholesteryl esters are critical for ß2-GPI binding. A positive ion mass spectrum of a novel ligand, designated oxLig-2, showed fragmented ions at m/z 383 and 441 in the presence of acetone, which share features of oxLig-1 and 7-ketocholesterol. In the negative ion mode, ions at m/z 627, 625, and 243 were observed. oxLig-2 was most likely 7-ketocholesteryl-12-carboxy (keto) dodecanoate. These ligands were recognized by ß2-GPI. Liposome binding to macrophages was significantly increased depending on the ligand's concentration, in the presence of ß2-GPI and an anti-ß2-GPI Ab. Synthesized variant, 7-ketocholesteryl-13-carboxytridecanoate (13-COOH-7KC), also showed a significant interaction with ß2-GPI and a similar binding profile with macrophages. Methylation of the carboxyl function diminished all of the specific ligand interactions with ß2-GPI.

Thus, {omega}-carboxyl variants of 7-ketocholesteryl esters can mediate anti-ß2-GPI Ab-dependent uptake of oxLDL by macrophages, and autoimmune atherogenesis linked to ß2-GPI interaction with oxLDL.

Abbreviations: Ab, antibody; APS, antiphospholipid syndrome; ß2-GPI, ß2-glycoprotein I; oxLDL, oxidized LDL; PL, phospholipid; oxLig-1, 7-ketocholesteryl-9-carboxynonanoate; 13-COOH-7KC, 7-ketocholesteryl-13-carboxytridecanoate

Supplementary key words antiphospholipid syndrome • atherosclerosis • autoantibody • ß2-glycoprotein I • oxidized LDL • {omega}-oxidation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The autoimmune disorder, antiphospholipid syndrome (APS), is characterized by the presence of a group of heterogeneous antiphospholipid antibodies (aPL Abs), such as anticardiolipin Abs (aCL) and lupus anticoagulants (LA), in blood, and by occurrence of thromboembolic complications in the arterial and/or venous vasculatures of the patients (1, 2). In 1990, three groups of investigators independently reported that a plasma/serum cofactor complexed with negatively charged phospholipids (PLs), such as cardiolipin, is an antigenic target for aCL (3–5). It is now widely accepted that ß2-glycoprotein I (ß2-GPI) is the major antigen for aCL. However, the mechanisms for interaction between ß2-GPI and anti-ß2-GPI Abs are still uncertain. Two currently proposed mechanisms are: i) Binding of ß2-GPI to PL induces a conformational change in the ß2-GPI molecule, thus exposing a cryptic epitope on the protein for the auto-Ab binding, and/or, ii) ß2-GPI binding to anionic PL increases the local concentration of ß2-GPI, thus promoting an increase in intrinsic affinity and Ab binding to the protein (6–14).

ß2-GPI is a 50 kDa protein present in plasma at approximately 200 µg/ml. It binds to negatively charged molecules, including PLs (15), heparin (16), and plasma membranes of activated platelets, and apoptotic cells on which phosphatidylserine (PS) is exposed (17, 18). ß2-GPI is a member of the short consensus repeats of the complement control protein superfamily, and its fifth domain has a PL binding region. The X-ray crystal analyses (19) showed that the PL binding is provided by a patch consisting of 14 residues of positively charged amino acids and by a flexible loop of S311-K317 in domain V. Recent analysis with domain V's mutant proteins demonstrated interactions of the flexible loop with hydrophobic ligands (20, 21). However, the full details of the structure of in vivo lipid ligands participating in the ß2-GPI binding remains unclear.

ß2-GPI affects not only multiple PL-dependent coagulation pathways but also lipoprotein metabolism (22–24). The oxidation of LDL has been proposed to play a central role in the early phase of atherosclerotic plaque formation, such as the transformation of monocyte-derived macrophages to foam cells (25–30). Accumulating evidence has suggested that the interaction between aPL and malonedialdehyde-modified LDL (MDA-LDL) may be important in relation to the pathogenesis of atherosclerosis and/or atherothrombosis in APS patients (31–33). In 1997, we first reported that ß2-GPI bound directly to oxidized LDL (oxLDL), and that the complex of oxLDL and ß2-GPI was subsequently recognized by anti-ß2-GPI auto-Abs and taken up by macrophages (34). It was further reported that lipid ligands derived from oxLDL were specific for ß2-GPI. The major ligand for ß2-GPI, oxLig-1, was originally reported as 7-ketocholesteryl-9-carboxynonanoate (35). The formal IUPAC name for this compound is 9-oxo-9-(7-ketocholest-5-en-3ß-yloxy) nonanoic acid.

In the present report, we now demonstrate that oxLDL recognition by ß2-GPI and an anti-ß2-GPI Ab is provided by an {omega}-carboxyl function introduced by oxidation of an unsaturated acyl chain of cholesteryl esters.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
L-{alpha}-Dipalmitoylphosphatidylserine (DPPS), 7-ketocholesterol (5-cholesten-3ß-ol-7-one), cholesteryl linoleate (5-cholesten-3ß-ol 3-linoleate), polyinosinic acid [poly(I)], polycytidylic acid [poly(C)], and fucoidan were obtained from Sigma Chemical Co. (St. Louis, MO); dioleoylphosphatidylcholine (DOPC) from Avanti Polar Lipids Inc. (Alabaster, AL); L-3-phosphatidyl-[N-methyl-3H]choline, 1, 2-dipalmitoyl ([3H]-DPPC) (80 Ci/mmol) from Amersham-Pharmacia Biotech (Uppsala, Sweden). All other chemicals were from commercial sources and of reagent-grade quality.

Preparation of human ß2-GPI
ß2-GPI was purified from normal human plasma as described (36) with slight modification. Pooled plasma from healthy subjects was subsequently chromatographed on a heparin-Sepharose column, on a DEAE-cellulose column, and on an anti-ß2-GPI affinity column. To remove any contamination by IgGs, the ß2-GPI-rich fraction was further passed through a protein A-Sepharose column. The final ß2-GPI fraction was delipidated by extensive washing with n-butanol.

Monoclonal antibodies
The monoclonal anti-human ß2-GPI Ab, Cof-22 (IgG1,{kappa}), was established from BALB/c mice immunized with human ß2-GPI (8). A mouse monoclonal anti-ß2-GPI auto-Ab, WB-CAL-1 (IgG2a, {kappa}), was derived from an (NZW x BXSB) F1 mouse (37). A human monoclonal anti-ß2-GPI auto-Ab, EY2C9 (IgM), was established from peripheral blood lymphocytes from an APS patient (38).

Isolation and oxidation of LDL
LDL (d = 1.019–1.063 g/ml) was isolated by preparative ultracentrifugation from fresh normal human plasma, as described (39). The LDL was adjusted to 100 µg/ml and oxidized with 5 µM CuSO4 in PBS for 8 h at 37°C. To terminate the oxidation, 1 mM EDTA was added and dialyzed extensively against PBS containing 1 mM EDTA. The degree of oxidation was estimated as thiobarbituric acid reactive substance (TBARS) value (40) and as migration in agarose electrophoresis.

Lipid extraction and preparative TLC
The lipids from native and oxLDL were isolated, according to the method of Folch, et al. (41). Briefly, lipids were extracted with chloroform-methanol (2:1, v/v) and dried by evaporation. The extracted lipids were spotted on a TLC silica gel-60 plate (2 mm thickness, Merck, Darmstadt, Germany) and developed in chloroform-methanol-30% ammonia-water (120:80:10:5, v/v/v/v, solvent A). Two individual lipid bands, Band-1 and Band-2, containing ligands reactive with ß2-GPI (detected by the ligand blot analysis, as described below), were identified and scraped.

Ligand blot analysis on a TLC plate
For analytical TLC ligand blot, lipids were spotted on a Polygram silica gel G plate (Machery-Nagel, Duren, Germany) and developed in solvent A or in chloroform-methanol (8:1, v/v, solvent B). Ligand blot analysis was performed, as described previously (35). Briefly, after drying and blocking with PBS containing 1% BSA, the plate was simultaneously incubated with ß2-GPI and an anti-ß2-GPI Ab (WB-CAL-1 or EY2C9) for 1 h. In case of Cof-22 Ab, ß2-GPI and the Ab were subsequently incubated for 1 h each. Horseradish peroxidase (HRP)-labeled anti-mouse IgG or HRP-labeled anti-human IgM was then incubated for 1 h. In each step, a plate was extensively washed with PBS. The color was developed with H2O2 and 4-methoxy-1-naphtol (Aldrich, Milwaukee, WI). On control TLC plates, ligands were separated and stained with I2 vapor or with a spray of molybdenum blue.

HPLC
The ß2-GPI-specific ligand, oxLig-2, was purified from the ligand-enriched fraction by a reversed-phase HPLC on a Sephasil Peptide C18 column (4.6 mm x 250 mm; Amersham-Pharmacia Biotech). The scraped band, Band-2, was eluted using a linear gradient of 50–100% solvent C (acetonitrile-isopropanol, 30:70, v/v) against solvent D (water containing 0.2% acetic acid), over 15 min, then 100% solvent C for the following 15 min, at a flow rate of 0.5 ml/min, and absorbance was monitored at 210 nm or 234 nm. The eluate was fractionated every 2 min (1ml/tube). Each fraction was spotted on a TLC plate and subjected to ligand blot analysis with ß2-GPI and EY2C9 Ab.

Synthesis of oxLig-1
To a solution of 7-ketocholesterol (5-cholesten-3ß-ol-7-one, 50.1 mg, 0.13 mmol) and azelaic acid (70.6 mg, 0.38 mmol) in acetone (4 ml) was added 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (WSC; 95.8 mg, 0.50 mmol) and 4-(dimethylamino)pyridine (DMAP; 30.5 mg, 0.25 mmol). The mixture was stirred at room temperature for 2 days, concentrated, and extracted with chloroform. The extract was successively washed with 2 M hydrochloric acid and brine, dried over anhydrous magnesium sulfate, and evaporated. The residue was subjected to column chromatography on silica gel using toluene-ethyl acetate (3:1, v/v) to give synthesized oxLig-1 (36.0 mg, 50.4% yield). 1H-NMR and 13C-NMR spectra were obtained at 300 MHz and 75 MHz, respectively, by an ASX-300 spectrometer (Bruker, Billerica, MA). The field desorption (FD) mass spectrum of synthesized oxLig-1 was recorded on a JMS-SX102A spectrometer (JEOL, Tokyo, Japan). 1H-NMR (300.1 MHz, CDCl3): {delta} = 5.71 (s, 1H, H-6), 4.78–4.69 (m, 1H, H-3); 13C-NMR (75.5 MHz, CDCl3): {delta} = 202.5, 179.7, 173.4, 164.5, 127.1, 72.4, 55.2, 50.4, 50.2, 45.8, 43.5, 39.9, 38.7, 36.6, 36.1, 29.2, 28.9, 28.4, 25.3, 25.0, 24.2, 23.2, 23.0, 19.3, 17.7, 12.4; m/z (FD-MS): 571 [(M+H)+, C36H59O5 requires 571].

Synthesis of 7-ketocholesteryl-13-carboxytridecanoate
To a solution of 7-ketocholesterol (50.1 mg, 0.13 mmol) and tridacanedioic acid (brassylic acid; 61.8 mg, 0.25 mmol) in acetone (4 ml) was added WSC (95.8 mg, 0.50 mmol) and DMAP (30.5 mg, 0.25 mmol). The mixture was stirred at room temperature for 2 days, concentrated, and extracted with chloroform. The extract was successively washed with 2 M hydrochloric acid, aqueous saturated sodium hydrogencarbonate, and brine, dried over anhydrous magnesium sulfate, and evaporated. The residue was subjected to column chromatography on silica gel using toluene-ethyl acetate (3:1, v/v) to give the product (44 mg, 56.0% yield). NMR spectra and FD mass spectra were measured as described above. 1H-NMR (300.1 MHz, CDCl3): {delta} = 5.69 (s, 1H, H-6), 4.80-4.67 (m, 1H, H-3); 13C-NMR (75.5 MHz, CDCl3): {delta} = 202.2, 179.6, 173.1, 164.7, 126.9, 72.3, 55.3, 50.5, 50.1, 45.3, 43.5, 40.6, 39.2, 38.6, 36.5, 36.0, 29.1, 28.8, 28.3, 25.2, 24.9, 24.1, 23.1, 22.9, 19.2, 17.6, 12.3; m/z (FD-MS): 627 [(M+H)+, C40H67O5 requires 627].

Liquid chromatography equipped mass spectrometry
Mass spectra of ß2-GPI-specific ligands, synthesized oxLig-1, oxLig-2 purified from Band-2, and synthesized 7-ketocholesteryl-13-carboxytridecanoate 13-COOH-7KC), were obtained by a liquid chromatography equipped mass spectrometry (LC/MS)-2010 spectrometer (Shimadzu Corp., Kyoto, Japan), equipped with a Shim-pack FC-ODS column (4.6 mm x 30 mm). The column was developed with a linear gradient of 50–100% solvent D (30% acetone in methanol) against water. Positive and negative ionization mass signals were detected in the mass range of 50–750, as ions generated during atomospheric pressure chemical ionization (APCI).

Methylation of lipid ligands
1-Methyl-3-nitro-1-nitrosomethylguanidine (0.20 g) was added to a mixture of 2 M sodium hydroxide (10 ml) and diethyl ether (10 ml) in an ice bath. The mixture was shaken several minutes and the pale yellow ethereal solution separated was used for methylation. The diazomethane solution (2 ml) was added dropwisely to a solution of lipid ligand (1.0 mg) in diethyl ether (1 ml) at 0°C. Each mixture was stored in refrigerator overnight. TLC of the mixture showed complete disappearance of the starting materials. The solvent was removed by blowing air to give the methyl ester as a white amorphous powder.

ELISA for anti-ß2-GPI Ab binding
Anti-ß2-GPI Ab binding was performed as described (35). Briefly, the lipid ligand (50 µg/ ml, 50 µl/well) was adsorbed by evaporation on a plain polystyrene plate (Immulon 1B, Dynex Technologies Inc., Chantilly, VA) and the plate was then blocked with 1% BSA. A monoclonal anti-ß2-GPI Ab (WB-CAL-1, or EY2C9, 1.0 µg/ml, 100 µl/well) was incubated in PBS containing 0.3% BSA with ß2-GPI (15 µg/ml) for 1 h. In case of Cof-22 Ab, ß2-GPI and the Ab were subsequently incubated for each 1 h. Ab binding was probed using HRP-labeled anti-mouse IgG or anti-human IgM. The color was developed with H2O2 and o-phenylenediamine and absorbance was measured at 490 nm. Between each step, extensive washing was performed using PBS containing 0.05% Tween 20.

Preparation of liposomes
Liposomes were prepared as described (42), with the following lipid compositions. Lipid molar ratios of 0, 10, 25, 30, and 50% ligand-containing liposomes were made with DOPC-ligand-[3H]DPPC (80 Ci/mmol). The amount of [3H]DPPC was 0.225%. The ligand component was varied to be either cholesteryl linoleate, DPPS, oxLig-1, oxLig-2, methylated oxLig-2 (Me-oxLig-2), 13-COOH-7KC, or methylated 13-COOH-7KC (Me-13-COOH-7KC).

Cell culture and liposome binding assay
A monolayer culture of mouse macrophage-like cell line, J774A.1 (Riken Cell Bank, Tsukuba, Japan), was used for liposome binding experiments. The cells were maintained in RPMI-1640 medium supplemented with 10% FBS. The cells were plated (1 ml/well) into a 12-well culture plate with RPMI 1640 at 8 x 105 cells/ml and incubated for 24 h at 37°C. The medium was replaced with Celgrosser-P medium (Sumitomo Pharmaceutical Co., Tokyo, Japan). After 2 h preincubation at 37°C, 50 µl of liposomes (50 nmol lipid/well) with/or without ß2-GPI (200 µg/ml) and WB-CAL-1 (100 µg/ml) were added to each well, and the cells were then incubated at 4°C for 2 h. The wells were washed with chilled PBS, and the cells were lysed with 1ml of 0.1 N NaOH. An aliquot was taken to determine cellular proteins and radioactivity associated with the cells. Protein concentration was determined using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of ß2-GPI-specific ligands
We first detected extracted lipids from native and Cu2+-oxdized LDL preparations, by different staining procedures applied to TLC plates, developed in solvent A (Fig. 1A, B) . With the I2 vapor and the molybdenum blue spray, the major change observed due to the Cu2+-oxidation was a small increase in lysophosphatidylcholine (lysoPC). To define the ligands targeted by ß2-GPI and an anti-ß2-GPI auto-Ab (i.e., WB-CAL-1 or EY2C9), ligand blot analysis was performed on the TLC plate. Two major bands and a diffuse lipid band were stained with ß2-GPI and either anti-ß2-GPI auto-Abs. The reactive lipids migrated at similar R f positions to those of cardiolipin and glycolipids, such as galactosylceramide (Gal-Cer) and glucosylceramide (Glu-Cer). The bands detected by ligand blot were not stained with molybdenum blue spray, indicating that they are not phospholipid (Fig. 1A, B). ß2-GPI ligand-enriched lipids (i.e., Band-1 and -2, indicated by arrows in Fig. 1B) were scraped from the TLC plate (in solvent A) and were subjected to another TLC development in solvent B and subsequent ligand blot with ß2-GPI and EY2C9 (Fig. 1C). The ligand corresponding to the upper band (Band-1) has already been reported to contain oxLig-1 (35). The lower band (Band-2) was further purified by reversed-phase HPLC.



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  		Fig. 1. TLC and ligand blot of lipids extracted from LDLs. Lipids extracted from LDLs were spotted on a silica gel plate and developed in solvent A (A and B) and solvent B (C), respectively. The plate was stained with I2 vapor and molybdenum blue (A). Ligand blot of lipids extracted from LDLs and incubated with ß2-GPI and an anti-ß2-GPI Ab (WB-CAL-1 or EY2C9) are shown in B. TLC of the isolated Band-1 and Band-2, in solvent B was followed by ligand blot with ß2-GPI and EY2C9 Ab (C).

 
Purification and characterization of a novel ligand, oxLig-2
The HPLC yielded a novel ligand, we named oxLig-2, from the scraped Band-2 (Fig. 2A, B) . A peak that revealed binding specific for ß2-GPI and EY2C9 was eluted at approximately 26.7 min (equivalent to 13.4 ml of elution volume). To confirm the purity of oxLig-2 (fraction #14), the fraction was re-chromatographed under the same HPLC conditions (Fig. 2C, D) and subjected to the analysis by LC/MS.



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  		Fig. 2. Elution profiles of Band-2 in reversed-phase HPLC. Isolated Band-2 was applied to a Sephasil-Peptide column with a linear gradient of 50–100% solvent C (acetonitrile-isopropanol 30/70, v/v) against solvent D (water containing 0.2% acetic acid). Absorbance was detected at 210 nm (A) and 234 nm (B) using a flow rate of 0.5 ml/min. Fractions (1 ml/tube) were collected and subjected to ligand blot with ß2-GPI and EY2C9 Ab (B, insert). Fraction # 14 from the first HPLC was re-chromatographed on the same HPLC to confirm its purity (C and D).

 
A positive ionization mass spectrum of oxLig-2 showed three signals at m/z 383, 441, and 627 (Fig. 3C) . These two smaller peaks, at m/z 383 (corresponding to 7-ketocholesterol) and 441 [corresponding to 7-ketocholesterol (+acetone)], were identical to those from oxLig-1 and 13-COOH-7KC (Fig. 3A, C, E). The signals at m/z 571, 627, and 627 were detected as a mother ion, [M+H]+, in the positive mass spectra of oxLig-1, oxLig-2, and 13-COOH-7KC, respectively (Fig. 3A, C, E). In contrast, the signals at m/z 569, 625, and 625 were detected as a mother ion, [M-H]-, in the negative mass spectra of oxLig-1, oxLig-2, and 13-COOH-7KC, respectively (Fig. 3B, D, F). In analysis of oxLig-2, another signal at m/z 627 was also detected as a mother ion of dihyro-oxLig-2 (Fig. 3D). In the negative mode, the signals at m/z 187, 243, and 243 were further observed as a daughter ion, [D-H]-, of oxLig-1, oxLig-2, and 13-COOH-7KC, respectively (Fig. 3B, D, F, and Fig. 4) .



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  		Fig. 3. Liquid chromatography equipped mass spectrometry of purified or synthesized ß2-GPI ligands. Left panels indicate positive ionization mass spectra of 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) (A), oxLig-2 (C), and 7-ketocholesteryl-13-carboxytridecanoate (13-COOH-7KC) (E); right panels indicate negative ionization mass spectrum of oxLig-1 (B), oxLig-2 (D), and 13-COOH-7KC (F).

 


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  		Fig. 4. Structures of cholesteryl esters that serve as precursor and ligands for ß2-GPI. Structures of cholesteryl linoleate (A), oxLig-1 (B) (35), oxLig-2 (C), and 13-COOH-7KC are indicated. A proposed fragmentation schemes of these ligands during APCI-MS are also shown and cleavage at the site (indicated by a dotted allow) yields a daughter fragment D.

 
The R f position of oxLig-2 was lower than those of the related {omega}-carboxyl derivatives, i.e., oxLig-1 and 13-COOH-7KC, in TLC-ligand blot in either solvent A or B (Fig. 1 and Fig. 5) , consistent with the deduced difference in polarity. After methylation with diazomethane, the bands corresponding to oxLi-g1, oxLig-2, and 13-COOH-7KC (i.e., Me-oxLig-1, Me-oxLig-2, and Me-13-COOH-7KC, respectively) shifted to higher R f positions than those of the untreated ligands in TLC analysis in solvent B (Fig. 5A). The peak of oxLig-2 appeared earlier (26.7 min) than those of oxLig-1 (27.3 min) and 13-COOH-7KC (28.9 min) when analyzed by the reversed phase HPLC. Further, peaks of Me-oxLig-1 (29.6 min), Me-oxLig-2 (27.1 min), and 13-COOH-7KC (30.0 min) appeared later than those of untreated ligands, respectively. Interestingly, methylation completely diminished both ligand-interactions with ß2-GPI and an anti-ß2-GPI Ab (either Cof-22 and EY2C9) in the TLC-ligand blot (Fig. 5B, C).



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  		Fig. 5. TLC and ligand blot of methylated ligands. Non-treated oxLig-1, oxLig-2, and 13-COOH-7KC, and methylated oxLig-1, oxLig-2, and 13-COOH-7KC (Me-oxLig-1, Me-oxLig-2, and Me-13-COOH-7KC) were spotted on a silica gel plate and developed in solvent B. The plate was stained with I2 vapor (A). Ligand blot was performed with ß2-GPI and an anti-ß2-GPI Ab [Cof-22 (B) or EY2C9 Ab (C)].

 
In ELISA for anti-ß2-GPI Abs using a ligand-coated plate, significant binding of anti-ß2-GPI auto-Abs (WB-CAL-1 and EY2C9) was observed to solid phase oxLig-1, oxLig-2, and 13-COOH-7KC, but not solid phase Me-oxLig-1, Me-oxLig-2, and Me-13-COOH-7KC. Identical results were obtained with Cof-22, a mouse monoclonal anti-ß2-GPI Ab obtained from a human ß2-GPI-immunized mouse (Table 1). All three Abs failed to bind to solid phase cholesterol, 7-ketocholesterol, and cholesteryl linoleate. From all of these results and the previously reported observations (43), the most likely structure of oxLig-2 was concluded to be that of 7-ketocholesteryl-12-carboxy (keto)-dodecanoate, one of the oxidized products derived from cholesteryl linoleate (Fig. 4). However, the exact location of the ketone-group was not assigned.


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  		TABLE 1. Binding of ß2-GPI and anti-ß2-GPI Abs to non-treated or methylated ligands in ELISA

 
Liposome binding to macrophages
Direct binding of liposomes containing oxLig-1, oxLig-2, or 13-COOH-7KC to mouse macrophages, i.e., J774A.1 cells, was compared with that of liposomes containing DPPS. DPPS-containing liposomes showed binding dependent upon the concentration of DPPS. In contrast, the liposomes containing oxLig-1, oxLig-2, or 13-COOH-7KC displayed relatively weak or negligible binding to the cells (Fig. 6A) . Further, we have done inhibition experiments to see whether scavenger receptor(s) is involved in the binding of liposomes containing ß2-GPI ligands. As shown in Fig. 6B and Table 2, binding of oxLig-1-liposomes to macrophages was inhibited by the addition of poly(I) or fucoidan but not by poly(C). Similar results were obtained with DPPS-liposomes. In contrast, the binding of oxLig-2 or 13-COOH-liposomes was not affected even by the addition of poly(I) or poly(C). These results indicate that the scavenger receptor(s) may primarily be involved in binding of liposomes containing DPPS to macrophages but may only be weakly involved in those with ß2-GPI ligand-containing liposomes. Conversely, the uptake of oxLig-1, oxLig-2, and 13-COOH-7KC-containing liposomes by J774A.1 cells was significantly enhanced in the presence of both ß2-GPI and an anti-ß2-GPI Ab (WB-CAL-1), as compared with control binding of cholesteryl linoleate-liposomes (Fig. 7A–D) . In contrast, binding of liposomes was almost completely eradicated by methylation of oxLig-1, oxLig-2, or 13-COOH-7KC (Fig. 7C, D). The ß2-GPI and anti-ß2-GPI Ab-mediated binding of ligand-containing liposomes was not affected either by poly(I) or poly(C).



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  		Fig. 6. Direct binding of ligand-containing liposomes to macrophages. A: A monolayer of J774A.1 cells was incubated for 2 h at 4°C with Celgrosser-P medium containing 3H-labeled liposomes with the indicated concentration of ligand (50 nmol lipid/well). DPPS-containing liposomes (open square); oxLig-1-containing liposomes (closed square); oxLig-2-containing liposomes (closed circle); 13-COOH-7KC-containing liposomes (closed triangle). Data shown are the mean ± SD of triplicate samples. Panel B: In the culture system, indicated concentration of poly(I), poly(C), or fucoidan was added. DPPS (30 mol%)-liposomes with poly(I) (open circle), poly(C) (open square), or fucoidan (open triangle); oxLig-1 (30 mol%)-liposomes with poly(I) (closed circle), poly (C) (closed square), or fucoidan (closed triangle). * Student t-test, P < 0.05.

 

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  		TABLE 2. Effect of scavenger receptor's inhibitors on direct or ß2-GPI/antibody-mediated binding of ligand-containing liposomes to macrophages

 


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  		Fig. 7. ß2-GPI and anti-ß2-GPI Ab-dependent binding of ligand-containing liposomes to macrophage. A monolayer of J774A.1 cells was incubated for 2 h at 4°C with Celgrosser-P medium containing 3H-labeled liposomes containing 30 mol% ligand (50 nmol lipid/well) in the presence (closed square)/or absence (open square) of ß2-GPI (200 µg/ml) and WB-CAL-1 (100 µg/ml). A: Cholesteryl linoleate-containing liposome; B: oxLig-1-containing liposomes; C: oxLig-2-containing liposomes; D: 13-COOH-7KC-containing liposomes. In panels B, C, and D, binding of methylated ligand-liposomes was also compared. Data are indicated as the mean ± SD of triplicate samples.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We previously reported that the major lipid ligand, oxLig-1, specific for ß2-GPI and anti-ß2-GPI auto-Abs derived from the lipid oxidation of LDL (35), is oxLig-1 (Fig. 4). In the present study, we isolated another ligand (oxLig-2), which we characterized to be keto-dodecanoate. Derivatization of such ligands now demonstrate that an {omega}-carboxyl function introduced by Cu2+-oxidation is critical for an interaction with ß2-GPI and its ligands.

Foam cell formation is regarded as the hallmark of early atherogenesis, and LDL is the major source of the lipid deposited in foam cells (26). Native LDL, under normal physiological conditions, cannot induce foam cell formation. The binding of modified LDL to scavenger receptors and possibly other cell surface sites on macrophages leads to unregulated cholesterol accumulation and the formation of foam cells with development of atherosclerotic lesions (44, 45).

Although the nature of the agents responsible for LDL oxidation in vivo is unknown, several candidates have been proposed (46–49). LDL oxidized with Cu2+ ion in vitro exhibits the physicochemical and immunological properties of oxLDL extracted from atherosclerotic lesions (28).

The Cu2+-dependent oxidative products include cholesterol /or oxysterols esterified with 9- or 13-hydroperoxy (or hydroxy)-octadecadienoate, with 9-oxononanoate, or with 9-caboxynonanoate, some of which have also been shown to be present in atherosclerotic plaques (43, 50, 51). In the present study, we provide evidence that oxLig-2 is keto-dodecanoate (Fig. 4). Methylation of oxLig-2 indicated the presence of a carboxyl function on its acyl chain, but the exact location of the ketone-group cannot be assigned by mass spectrometry. Nevertheless, cholesteryl linoleate, which constitutes one of the major cholesteryl esters of LDL, has four carbons with double bonds susceptible to oxygenation at positions C9, C10, C12, and C13.

7-Ketocholesterol is a major oxysterol present in the Cu2+-treated oxLDL (52–55). We have also synthesized a 22-ketocholesterol analog of oxLig-1 (i.e., 22-ketocholesteryl-9-carboxynonanoate; 9-COOH-22KC) in the same manner for synthesizing oxLig-1. However, 9-COOH-22KC did not have the same properties of ß2-GPI ligand (data not shown). The observation suggests that 7-ketocholesterol may also be responsible for binding to ß2-GPI, as well as an {omega}-carboxyl function on shortened fatty acid chains generated by the Cu2+-oxidation of LDL.

It is now well established that anti-ß2-GPI Abs, found in APS patients, bind a complex of ß2-GPI and negatively charged PLs, such as cardiolipin, PS, and phosphatidic acid (36). In our recent (35) and present studies, however, negatively charged PLs are very minor components of oxLDL.

The flexible loop in the C-terminus and a particular cluster consisting of 14 residues of positively charged amino acid residues in domain V of ß2-GPI has a critical role for interaction with amphiphilic compounds such as cardiolipin, PS, phosphatidic acid, and phosphatidylglycerol (19–21, 36). Although ß2-GPI did not bind to cholesterol, 7-ketocholesterol, or cholesteryl linoleate, significant binding was observed to oxLig-1, oxLig-2, and 13-COOH-7KC. Thus, these oxysterol esters having an {omega}-carboxylated acyl chain, constitute a new class of an amphiphilic ligand suitable for ß2-GPI. Further, the observation that methylation of these ligands diminished the ß2-GPI interaction indicates that a free carboxyl residue is required for the recognition.

As previously described, in vivo uptake of oxLDL via scavenger receptor(s) of macrophages may play a central role in atherogenesis. The term, scavenger receptor(s), refers to a number of different cell-surface proteins that can bind chemically or biologically modified lipoproteins. Various scavenger receptors that bind oxLDL have been found on macrophages, including class A scavenger receptors (56), CD36 (57), human homolog CD68 (58), a lectin-like oxLDL receptor-1 (LOX-1) (59), and Fc{gamma} receptor (60).

Although PS-containing liposomes showed significant binding to macrophages dependent upon the PS concentration, those containing oxLig-1, oxLig-2, and 13-COOH-7KC did not (Fig. 6). The simultaneous addition of ß2-GPI and an anti-ß2-GPI Ab significantly increased the binding of liposomes containing oxLig-1, oxLig-2, and 13-COOH-7KC to the macrophages (Fig. 7). Further, poly(I) did not have any inhibitory effect on the binding of ligand-containing liposomes in the presence of ß2-GPI and an anti-ß2-GPI Ab. These results suggest that ß2-GPI and anti-ß2-GPI Ab mediated uptake of oxLDL occurs through Fc{gamma} receptor on macrophages but not via scavenger receptor(s) (61–64).

In the present study, we demonstrate that oxidized cholesteryl esters, especially those with 7-ketocholesterol and an {omega}-carboxyl function in the acyl chain are ligands for ß2-GPI and anti-ß2-GPI auto-Abs. Such auto-Abs are found in APS patients and in an animal model, the WB F1 mouse. Furthermore, one major class of biochemically oxidized compounds derived from plasma LDL consists of {omega}-carboxylated oxysterols such as oxLig-1 and oxLig-2. Although 13-COOH-7KC is an artificially synthesized compound, it also showed significant binding to ß2-GPI as well as oxLig-1 and -2.

Most recently, we observed that high levels of circulating immune complexes containing oxLDL, ß2-GPI, and anti-ß2-GPI auto-Abs in the blood stream, were associated with development of arterial thrombosis in APS patients (unpublished observations). Thus, {omega}-carboxylation of oxysterol esters to form the autoantigenic complex of ß2-GPI bound to oxLDL may have patho-physiologically (etiologically) important roles, especially in development of APS and atherosclerosis.


   ACKNOWLEDGMENTS
 
This work was supported in part by a grant for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Ministry of Health and Welfare of Japan.

Manuscript received February 5, 2002 and in revised form May 21, 2002.


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