A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL
2008; Elsevier BV; Volume: 50; Issue: 3 Linguagem: Inglês
10.1194/jlr.d800042-jlr200
ISSN1539-7262
AutoresXiaosuo Wang, Baohai Shao, Michael N. Oda, Jay W. Heinecke, Stephen M. Mahler, Roland Stocker,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoOxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO86 and MetO112 (termed apoA-I+32) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I+32, apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease. Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO86 and MetO112 (termed apoA-I+32) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I+32, apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease. Atherosclerosis is characterized by heightened oxidative damage to lipids and proteins in the affected arterial wall (1Stocker R. Keaney Jr., J.F. Role of oxidative modifications in atherosclerosis.Physiol. Rev. 2004; 84: 1381-1478Crossref PubMed Scopus (2082) Google Scholar), and the extent of this damage is comparable for different classes of lipoproteins including LDL and HDL (2Niu X. Zammit V. Upston J.M. Dean R.T. Stocker R. Co-existence of oxidized lipids and α-tocopherol in all lipoprotein fractions isolated from advanced human atherosclerotic plaques.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1708-1718Crossref PubMed Scopus (94) Google Scholar). Recent interest has focused on the functional consequences of HDL oxidation. Oxidation could conceivably contribute to the formation of dysfunctional HDL, proposed to be present in humans with cardiovascular disease (3Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1039) Google Scholar). One potentially important pathway for generating dysfunctional HDL via oxidation involves myeloperoxidase, an enzyme that converts hydrogen peroxide (H2O2) and chloride ion to hypochlorous acid (HOCl). Myeloperoxidase is expressed in human atherosclerotic tissue (4Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1115) Google Scholar), and HOCl-modified proteins are present in such lesions (5Hazell L.J. Arnold L. Flowers D. Waeg G. Malle E. Stocker R. Presence of hypochlorite-modified proteins in human atherosclerotic lesions.J. Clin. Invest. 1996; 97: 1535-1544Crossref PubMed Scopus (530) Google Scholar).HOCl converts protein tyrosine residues to 3-chlorotyrosine, and methionine residues to methionine sulfoxide (MetO). In addition, MetO can also be formed from exposure of HDL's major protein, apolipoprotein A-I (apoA-I) to H2O2 (6Anantharamaiah G.M. Hughes T.A. Iqbal M. Gawish A. Neame P.J. Medley M.F. Segrest J.P. Effect of oxidation on the properties of apolipoproteins A-I and A-II.J. Lipid Res. 1988; 29: 309-318Abstract Full Text PDF PubMed Google Scholar) or lipid hydroperoxides (7Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 8Garner B. Waldeck A.R. Witting P.K. Rye K-A. Stocker R. Oxidation of high density lipoproteins. II. Evidence for direct reduction of HDL lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.J. Biol. Chem. 1998; 273: 6088-6095Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), the latter generated during the oxidation of HDL lipids. Oxidation of tyrosine and methionine residues can have dramatic consequences on the functions of apoA-I/HDL, including reverse cholesterol transport, wherein HDL accepts cholesterol from macrophage foam cells in the artery wall and transports it back to the liver for excretion (9Oram J.F. Heinecke J.W. ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease.Physiol. Rev. 2005; 85: 1343-1372Crossref PubMed Scopus (417) Google Scholar). Specifically, apoA-I containing 3-chlorotyrosine and MetO has an impaired ability to promote cholesterol efflux by the ABCA1 pathway (10Bergt C. Pennathur S. Fu X. Byun J. O'Brien K. McDonald T.O. Singh P. Anantharamaiah G.M. Chait A. Brunzell J. et al.The myeloperoxidase product hypochlorous acid oxidizes HDL in the human artery wall and impairs ABCA1-dependent cholesterol transport.Proc. Natl. Acad. Sci. USA. 2004; 101: 13032-13037Crossref PubMed Scopus (379) Google Scholar, 11Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. et al.Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.J. Clin. Invest. 2004; 114: 529-541Crossref PubMed Scopus (641) Google Scholar, 12Peng D.Q. Wu Z. Brubaker G. Zheng L. Settle M. Gross E. Kinter M. Hazen S.L. Smith J.D. Tyrosine modification is not required for myeloperoxidase-induced loss of apolipoprotein A-I functional activities.J. Biol. Chem. 2005; 280: 33775-33784Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar–13Shao B. Oda M.N. Bergt C. Fu X. Green P.S. Brot N. Oram J.F. Heinecke J.W. Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I.J. Biol. Chem. 2006; 14: 9001-9004Abstract Full Text Full Text PDF Scopus (190) Google Scholar). Similarly, oxidation of Met148 impairs apoA-I's ability to activate lecithin-cholesterol acyltransferase (14Shao B. Cavigiolio G. Brot N. Oda M.N. Heinecke J.W. Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I.Proc. Natl. Acad. Sci. USA. 2008; 105: 12224-12229Crossref PubMed Scopus (139) Google Scholar).There is evidence that oxidized apoA-I is present in human blood plasma. Thus, levels of 3-chlorotyrosine are higher in circulating HDL isolated from patients with cardiovascular disease than in HDL isolated from controls (10Bergt C. Pennathur S. Fu X. Byun J. O'Brien K. McDonald T.O. Singh P. Anantharamaiah G.M. Chait A. Brunzell J. et al.The myeloperoxidase product hypochlorous acid oxidizes HDL in the human artery wall and impairs ABCA1-dependent cholesterol transport.Proc. Natl. Acad. Sci. USA. 2004; 101: 13032-13037Crossref PubMed Scopus (379) Google Scholar, 11Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. et al.Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.J. Clin. Invest. 2004; 114: 529-541Crossref PubMed Scopus (641) Google Scholar, 15Pennathur S. Bergt C. Shao B. Byun J. Kassim S.Y. Singh P. Green P.S. McDonald T.O. Brunzell J. Chait A. et al.Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species.J. Biol. Chem. 2004; 279: 42977-42983Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), suggesting that myeloperoxidase may target HDL for oxidation in humans during atherogenesis. Similarly, apoA-I containing methionine residues 86 and 112 as MetO (hereafter referred to as apoA-I+32) has been detected in circulating HDL, and is present at increased concentrations in subjects with a genotype associated with increased coronary artery disease (16Pankhurst G. Wang X.L. Wilcken D.E. Baernthaler G. Panzenböck U. Raftery M. Stocker R. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.J. Lipid Res. 2003; 44: 349-355Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Moreover, it was recently shown that the MetO content of apoA-I is elevated in human type 1 diabetes (17Brock J.W. Jenkins A.J. Lyons T.J. Klein R.L. Yim E. Lopes-Virella M. Carter R.E. Thorpe S.R. Baynes J.W. Increased methionine sulfoxide content of apoA-I in type 1 diabetes.J. Lipid Res. 2008; 49: 847-855Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), a disorder commonly associated with increased oxidative stress (18Baynes J.W. Thorpe S.R. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm.Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2129) Google Scholar).The above findings suggest that oxidized forms of apoA-I containing 3-chlorotyrosine and/or MetO could be clinically relevant. However, testing this possibility is limited by current methods for oxidized apoA-I detection that require time-consuming HDL isolation and HPLC- or mass spectroscopy-based detection steps. We therefore sought to develop a simple method for the specific detection of apoA-I containing MetO. Here we describe a sensitive and specific ELISA that detects MetO-containing apoA-I in HDL and plasma.MATERIALS AND METHODSMaterialsCell culture media and reagents including DMEM, fetal bovine serum, and hybridoma-SFM were purchased from Invitrogen (Carlsbad, CA). EZ-Link Sulfo- NHS-LC-Biotin was obtained from Pierce (Rockford, IL). 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako Pure Chemical Ind. (Osaka, Japan), and 3,3′,5,5′-tetramethylbenzidine, potassium bromide, diethylene triamine pentaacetic acid, ascorbic acid, sydnonimine-1, H2O2, BSA (fraction V), myoglobin, ferritin, and fibrinogen were obtained from Sigma (Saint Louis, MO). Sodium hypochlorite (4% solution) was obtained from Septone (Hemmant, Australia), while PBS pH 7.4 (10 mM) was prepared from tablets (Oxoid, Basingstoke, England). Trifluoroacetic acid (TFA) and HPLC-grade water were obtained from British Drug House (Poole, England). Other HPLC grade solvents were obtained from Merck (Darmstadt, Germany). All other reagents used were of analytical grade. Water was purified by a Milli-Q Ultrapure water system (Millipore, Sydney, Australia).Isolation of HDLFresh blood (200 ml) from healthy and overnight fasted subjects (male, age 30–50 years) was collected into heparinized vacutainers, and plasma obtained by centrifugation at 1,430 g for 20 min at 4°C. HDL was isolated from the plasma by sequential ultracentrifugation (19Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by HPLC postcolumn chemiluminescence.Methods Enzymol. 1994; 233: 469-489Crossref PubMed Scopus (284) Google Scholar). Briefly, the density of pooled plasma was adjusted to 1.24 g/ml with potassium bromide and the plasma then centrifuged for 3 h at 206,360 g and 10°C (Beckman Optima L-90K, VTi50 rotor). The resulting HDL band was aspirated, added to a new quick seal tube, and topped up with 0.9% saline of 1.24 g/ml density (adjusted with potassium bromide, 381.6 mg/ml), and then recentrifuged overnight at 206,360 g and 10°C. HDL (top band) was removed and dialyzed generously against PBS containing 0.1% EDTA and 0.01% chloramphenicol. The obtained HDL was used within 24 h.Oxidation of HDLNative HDL (1–1.5 mg protein/ml) was oxidized in 20 mM PBS containing 100 μM diethylene triamine pentaacetic acid under air and at 37°C by exposure to either the peroxyl radical generator AAPH (2 mM, 3 h) (7Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), H2O2 (100 mM, 24 h), or HOCl (500 μM, 1 h). The reactions were terminated by addition of butylated hydroxytoluene (100 μM), catalase (200 nM), or methionine (2.5 mM), respectively. The reaction mixture (1 ml) was then passed through a gel filtration column (3 ml, NAP-10, GE Healthcare, Uppsala, Sweden) eluted with 1.5 ml of PBS. The oxidized HDL was analyzed within 24 h.HPLC analysis of native and oxidized HDLFreshly isolated HDL (0.06 mg protein) or differently oxidized HDL (1 mg protein) was subjected to a C18 column (250 × 4.6 mm, 5 μm, Vydac) with guard (5 μm, 4.6 ID, Vydac) eluted at 50°C and 0.5 ml/min, with the eluant monitored at 214 nm. The instrument settings were modified slightly from the previously described method (16Pankhurst G. Wang X.L. Wilcken D.E. Baernthaler G. Panzenböck U. Raftery M. Stocker R. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.J. Lipid Res. 2003; 44: 349-355Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, after initial equilibration in 25% solvent A (acetonitrile containing 0.1 vol % TFA) and 75% solvent B (water containing 0.1 vol % TFA) for 10 min, the concentration of solvent A was increased linearly to 45% over 5 min, then to 55% over 32 min, to 95% over 10 min, and finally to 100% in 1 min, after which solvent A was decreased to 25% for column reequilibration. For the purpose of preparation of apolipoprotein standards, a semipreparative RP C18 column (250 × 10 mm, 5 μm, Vydac, flow rate 2 ml/min) was used to allow injection of HDL and differently oxidized HDL (up to 5 mg protein).Apolipoprotein standardsAppropriate protein fractions of HDL and oxidized HDL eluting from the HPLC column were collected on ice, dried under vacuum (AES1010 speedyvac system, Thermo Savant, Waltham, MA) and reconstituted in PBS. The identity of nonoxidized apoA-I and different forms of oxidized apoA-I, containing MetO instead of methionine residues as the only modification(s), was confirmed by mass spectroscopy, as described previously (16Pankhurst G. Wang X.L. Wilcken D.E. Baernthaler G. Panzenböck U. Raftery M. Stocker R. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.J. Lipid Res. 2003; 44: 349-355Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Woodward M. Croft K.D. Mori T.A. Headlam H. Wang X.S. Suarna C. Raftery M.J. Macmahon S.W. Stocker R. The association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population.Clin. Sci. 2009; 116: 53-60Crossref PubMed Scopus (31) Google Scholar). Reconstituted native and oxidized forms of apoA-I and apoA-II were overlaid with argon and stored at −20°C for up to 12 months prior to use. Such storage did not change the nature of the various apoA-I species, as verified by HPLC (data not shown). HPLC-purified apolipoproteins are subsequently referred to as lipid-free forms of apolipoproteins. Concentrations of proteins in each standard were determined using the bicinchoninic acid protein assay with BSA as the standard (Pierce).Anti-human apoA-I+32 monoclonal antibodies (mAb)The generation of anti-human apoA-I+32 monoclonal antibodies was performed by the Centre for Animal Biotechnology (University of Melbourne, Melbourne, Australia) with approval from the local animal ethics committee. Briefly, six Balb/c mice were immunized three times, 4 weeks apart by subcutaneous and intravenous injection of 10 μg HPLC-purified human apoA-I+32 in Freund's adjuvant. The strongest responding mice, as shown by serum Ig levels on an ELISA, were chosen for the fusion. Five days before the fusion and at least 4 weeks after the previous boost immunization, a final boost apoA-I+32 (10 μg in saline) was administered by intravenous injection. On the day of fusion, spleen cells taken from the immunized mice were added to NS-1 cells in serum-free DMEM and the fusion carried out using 50% polyethyleneglycol. Fused cells were plated in 96-well plates with rat thymocyte feeder cells. Seven to 10 days after the fusion, hybridoma supernate was screened for antibodies with solid-phase ELISA using apoA-I+32 as the immunogen. Hybridomas from high producing cell pools were subsequently cloned by limited dilution. Three rounds of clonal isolation were performed to obtain stable cell lines producing monoclonal antibodies. Two hybridoma cell lines (6.4.4.26 and 6.4.4.17.2.2) were selected based on preferential recognition of apoA-I+32 versus apoA-I. Hybridomas initially cultured in DMEM medium containing 10% FBS, were adapted to serum-free medium (Hybridoma-SFM, Invitrogen, Carlsbad, CA). Supernates of the selected hybridoma cell lines were harvested and IgG purified by FPLC using a Hitrap protein G HP affinity column (Amersham Biosciences). To determine the isotype, the mouse immunoglobulin screening/isotyping kit (Zymed Laboratories Inc., San Francisco, CA) was used following the manufacturer's protocol. Purified monoclonal antibodies (mAbs) were biotinylated by mixing with a 20-fold molar excess of Sulfo-NHS-LC-Biotin and incubation at RT for >30 min. The reaction was stopped and biotinylated mAb (B-mAb) dialyzed extensively against PBS at 4°C for 2 days. Finally, the concentration of B-mAb was determined by absorbance at 280 nm.Development of ELISANinety-six-well microplates were coated overnight at 4°C with 100 μl mouse anti-human apoA-I mAb (Chemicon, MAB010-A/11, 1:500 dilution in PBS), plates washed with 0.1% Tween 20 in PBS, followed by incubation for 1 h at 37°C with 2% BSA in PBS as blocking buffer. Plates were washed, samples (lipid-free and lipid-associated apolipoproteins, differently oxidized HDL or other proteins, and plasma) added, and plates incubated for 1 h at 37°C. Following washing, 100 μl B-mAb was added (1:400 dilution with 2% BSA in PBS) before further incubation for 1 h at 37°C. Plates were washed again and streptavidin horseradish peroxidase (Dako, 1:2,500 dilution with 2% BSA in PBS) added and the plate incubated for a further 1 h at 37°C. Following four washes, 100 μl 3,3′,5,5′-tetramethylbenzidine (liquid substrate system solution for ELISA, Sigma) was added and color developed for 5–10 min at RT. The reaction was stopped by addition of 100 μl 20% H2SO4, and the absorbance read at 450 nm in a plate reader.For competition assay, plates were coated with 100 μL goat anti-human apoA-I polyclonal antibody (Rockland, 1:10,000 dilution in PBS), incubated at 4°C overnight, washed and blocked, and 100 μL apoA-I+32 (400 ng/ml) then added. After incubation (37°C) and washing (0.1% Tween 20 in PBS), 100 μL B-mAb17 (see later discussion) and the competing antibody were added, the plate was incubated for 1 h at 37°C, and the color was developed. For detection of oxidized forms of lipid-free apoA-I, respective apolipoproteins were used at serial dilutions (0-500 ng/ml PBS containing 2% BSA), and the sensitivity of the ELISA compared with the HPLC assay. For detection of oxidized apoA-I associated with HDL, 100 μL of AAPH-oxidized HDL (0–15 μg protein/ml) was added either in PBS containing 2% BSA, or in 40% acetonitrile. Signals were compared with readings obtained with lipid-free apoA-I+32, with the amount of apoA-I+32 contained in AAPH-oxidized HDL determined by HPLC. Where indicated, native HDL (0–15 μg/ml) or LDL (0–15 μg/ml) was used.To apply the ELISA to human plasma, plasma samples (10 μl) were filtered (0.22 μm), supplemented with 90 μl oxidized HDL (0–15 μg), and then applied to a hydrated SwellGel Blue column (SwellGel Blue albumin removal kit, Pierce). After 2 min incubation at RT, columns were centrifuged (12,000 g, 1 min). The sample flow-through (100 μl) was reapplied to the column, the column recentrifuged, and the second sample flow-through (100 μl) collected. The column was then washed with 400 μl optimized buffer (25 mM Tris, 300 mM NaCl, pH 7.4), the wash flow-through (400 μl) collected and combined with the above second sample flow-through prior to ELISA.SDS-PAGESDS-PAGE was performed using 10% bis-TRIS Nupage gels (Invitrogen) under reducing conditions. Samples loaded consisted of 1 μl of 10 × diluted plasma without and with an aliquot (15 μg protein) of the oxidized HDL loaded onto the SwellGel Blue, or 5 μl of the combined sample and wash flow-through prepared as previously described. After electrophoresis, proteins were transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences) and immunoblotted using polyclonal goat anti-human apoA-I (Rockland) antibody as the primary and anti-goat horseradish peroxidase (Sigma) as the secondary Ab.Preparation of differently oxidized lipid-free apoA-ISolutions of lipid-free apoA-I (5 μM) in 20 mM PBS containing 100 μM diethylene triamine pentaacetic acid were oxidized under air at 37°C in the presence of different 1e- or 2e-oxidants. Specifically, for the Fenton reaction, 2 h incubation with FeCl3 (10 μM) plus H2O2 (5 mM), or 1 h incubation with FeSO4 (10 μM) plus ascorbic acid (1 mM) was used. Alternatively, apoA-I was oxidized with CuSO4.5H2O (5 μM) or FeCl3 (10 μM) for 2 h. In each case, oxidation was stopped by the addition of EDTA (5 mM final concentration). For 2e-oxidants, H2O2 (5 mM, 24 h incubation stopped with 200 nM catalase), HOCl (50 μM, 1 h incubation stopped with 2.5 mM methionine), or H2O2 (5 mM) plus HOCl (50 μM) (1 h incubation stopped with methionine and catalase) were used. Alternatively, apoA-I was oxidized by 2 h exposure to sydnonimine-1 (1 mM), a generator of nitric oxide plus superoxide anion radical, with the reaction stopped by 100 μM butylated hydroxytoluene. For controls, incubations were carried out in the absence of the respective oxidant. Differently oxidized forms of apoA-I were then subjected to ELISA (250 ng protein/ml) and, after gel filtration, to HPLC (5 μg protein).Wild-type apoA-I and mutants with one, two, or three of the methionine residues replaced with leucine residues were expressed in E. coli as described (14Shao B. Cavigiolio G. Brot N. Oda M.N. Heinecke J.W. Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I.Proc. Natl. Acad. Sci. USA. 2008; 105: 12224-12229Crossref PubMed Scopus (139) Google Scholar, 21Ryan R.O. Forte T.M. Oda M.N. Optimized bacterial expression of human apolipoprotein A-I.Protein Expr. Purif. 2003; 27: 98-103Crossref PubMed Scopus (131) Google Scholar). Individual substitution mutations within human apoA-I cDNA were introduced by primer-directed PCR mutagenesis or mega-primer PCR. All mutations were verified by dideoxy automated fluorescent sequencing of cDNA, and confirmed by MS analysis of the protein (13Shao B. Oda M.N. Bergt C. Fu X. Green P.S. Brot N. Oram J.F. Heinecke J.W. Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I.J. Biol. Chem. 2006; 14: 9001-9004Abstract Full Text Full Text PDF Scopus (190) Google Scholar, 22Shao B. Heinecke J.W. Using tandem mass spectrometry to quantify site-specific chlorination and nitration of proteins: model system studies with high-density lipoprotein oxidized by myeloperoxidase.Methods Enzymol. 2008; 440: 33-63Crossref PubMed Scopus (32) Google Scholar). These mutants (5 μM) were then exposed to 20 mM phosphate buffer pH 7.4 containing 100 μM diethylene triamine pentaacetic acid without (control) and with H2O2 (50 mM) at 37°C for 24 h to convert the remaining methionine residue(s) to the corresponding MetO. The reaction was terminated by addition of 200 nM catalase (to scavenge remaining H2O2) and 10 mM methionine (to inhibit further oxidation). The formation of MetO was confirmed by LC-ESI-MS/MS analysis of tryptic or Glu-C digests of oxidized apoA-I protein and the product yield of individual MetO residues was determined with reconstructed ion chromatograms of product and precursor peptides as described (14Shao B. Cavigiolio G. Brot N. Oda M.N. Heinecke J.W. Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I.Proc. Natl. Acad. Sci. USA. 2008; 105: 12224-12229Crossref PubMed Scopus (139) Google Scholar). Wild-type and mutant native and oxidized apoA-I were then shipped to Sydney for ELISA (500 ng protein/ml) and HPLC analyses (5 μg protein).Preparation of oxidized, methionine residue-containing proteinsNative HDL (1 mg/ml) or 1 mg/ml BSA, myoglobin, ferritin, or fibrinogen were oxidized at 37°C in 20 mM PBS containing 100 μM diethylene triamine pentaacetic acid with either H2O2 (100 mM) or HOCl (500 μM). Samples were incubated for 24 h (H2O2) or 1 h (HOCl) and oxidation terminated by addition of catalase (200 nM) or methionine (2.5 mM), respectively. Oxidized proteins were then subjected to ELISA.Statistical analysisData are shown as mean ± SEM unless otherwise stated. Intra- and interassay reproducibility was determined in four independent analyses each in triplicate.RESULTSNative HDL isolated from freshly obtained human plasma contains apolipoproteins apoA-I and apoA-II as the major proteins, which can be separated by HPLC (Fig. 1A). AAPH-oxidized HDL contained oxidized forms of apoA-I and apoA-II, which exhibit shorter retention times compared with their respective native forms (Fig. 1B). These oxidized forms of apoA-I were designated apoA-I+32 (containing MetO86 plus MetO112 and eluting at ∼23 min) or apoA-I+16, containing MetO112 (∼24 min) or MetO86 (∼30 min). Methionine modifications were the only modifications present in these oxidized forms of apoA-I, as verified by LC-MS/MS (16Pankhurst G. Wang X.L. Wilcken D.E. Baernthaler G. Panzenböck U. Raftery M. Stocker R. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.J. Lipid Res. 2003; 44: 349-355Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Woodward M. Croft K.D. Mori T.A. Headlam H. Wang X.S. Suarna C. Raftery M.J. Macmahon S.W. Stocker R. The association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population.Clin. Sci. 2009; 116: 53-60Crossref PubMed Scopus (31) Google Scholar). Native apoA-II and its oxidized form (apoA-II+16) eluted as three distinct, albeit incompletely resolved species (Fig. 1B), and were characterized recently (20Woodward M. Croft K.D. Mori T.A. Headlam H. Wang X.S. Suarna C. Raftery M.J. Macmahon S.W. Stocker R. The association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population.Clin. Sci. 2009; 116: 53-60Crossref PubMed Scopus (31) Google Scholar).To develop an ELISA for MetO-containing apoA-I, we immunized six mice with HPLC-purified human apoA-I+32 (16Pankhurst G. Wang X.L. Wilcken D.E. Baernthaler G. Panzenböck U. Raftery M. Stocker R. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.J. Lipid Res. 2003; 44: 349-355Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Of the sera from these mice, two showed increased recognition of apoA-I+32 over apoA-I (Fig. 1C). The spleens of these mice were used to generate hybridomas, yielding several different clones, of which two, 6.4.4.26 and 6.4.4.17.2.2 (yielding mAb17) were chosen for isolation of mAbs and subsequent experiments described herein. Of these, 6.4.4.26 (hereafter referred to as MOA-I) demonstrated the highest response to apoA-I+32 (data not shown). Competition experiments revealed that MOA-I and mAb17 raised against apoA-I+32 recognized the same epitope (Fig. 2), as did several commercial polyclonal anti-human apoA-I antibodies (i.e., 600−101−109 from Rockland, A95120H from Biodesign, and AB740 from Chemicon) (data not shown). In contrast, mAb010-A/11 (from Chemicon) and MOA-I bound mutually exclusive epitopes, as absorbance remained unchanged (Fig. 2). Therefore, we used mAb010-A/11 for capture and B-MOA-I for secondary detection in all subse
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