Preservation of biological function despite oxidative modification of the apolipoprotein A-I mimetic peptide 4F

• DOI: 10.1194/jlr.m026278
• Publication Date: 2012-05-16T00:28:22Z
• Authors: C. Roger White; Geeta Datta; Amanda K.W. Buck; Manjula Chaddha; Gautam Reddy; Landon Wilson; Mayakonda N. Palg...; Mohammad Abbasi; G.M. Anantharamaiah
• Keywords: Hypochlorous acid; Lipid Oxidation

Myeloperoxidase (MPO)-derived hypochlorous acid induces changes in HDL function via redox modifications at the level of apolipoprotein A-I (apoA-I). As 4F and apoA-I share structural functional properties, we tested hypothesis that acts as a reactive substrate for (HOCl). reduced HOCl-mediated oxidation fluorescent APF concentration-dependent manner (ED50∼ 56 ± 3 μM). This reaction induced physical properties 4F. Addition HOCl to molar ratios ranging from 1:1 3:1 band intensity on SDS-PAGE gels was accompanied by formation higher molecular weight species. Chromatographic studies showed reduction peak area with increasing new products. Mass spectral analyses collected fractions revealed sole tryptophan (Trp) residue equally susceptible lipid-free lipid-bound states. To determine whether Trp influenced its mimetic monitored effects 4F-mediated lipid binding ABCA1-dependent cholesterol efflux. Neither property altered HOCl. These results suggest serves HOCl, an antioxidant response does not influence effluxing capacities peptide. chlorotyrosine dioxindolylalanine myeloperoxidase nitrotyrosine oxindolylalanine phenylalanine scavenger receptor class B1 trifluoroacetic tyrosine (MPO), abundant heme protein released activated leukocytes, catalyzes chloride ions hydrogen peroxide (H2O2) yield MPO has been identified biomarker pathological conditions including sepsis, ischemic injury atherosclerosis (1Zhang R. Brennan M.L. Fu X. Aviles R.J. Pearce G.L. Penn M.S. Topol E.J. Sprecher D.L. Hazen S.L. Association between levels risk coronary artery disease.JAMA. 2001; 286: 2136-2142Crossref PubMed Scopus (765) Google Scholar–4Breckwoldt M.O. Chen J.W. Stangenberg L. Aikawa E. Rodriguez Qiu S. Moskowitz M.A. Weissleder Tracking inflammatory stroke vivo sensing enzyme myeloperoxidase.Proc. Natl. Acad. Sci. USA. 2008; 105: 18584-18589Crossref (251) Scholar). 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Getz G.S. helix number linker anti-atherogenic properties.J. 49: 1268-1283Abstract (62) A recent study demonstrates additional ability avidly bind neutralize lipids (29Van Jung Ruchala Waring Lehrer R.I. Watson A.D. Hama Navab al.Anti-inflammatory apoA-I-mimetic much than apoA-I.J. 2302-2311Abstract (161) amphipathic sensitive motif. goal current study, therefore, test novel how impacts lipid-binding 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) obtained Avanti Polar Lipids. (Alabaster, AL). Sodium Sigma. Superose 6 columns were Amersham Biosciences (NJ). 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]benzoic (APF) Cayman Chemicals. 3H-cholesterol American Radiochemicals (St. Louis, MO). Hypochlorous concentration monitoring absorbance 292 nm (∊= 350 M−1cm−1) 0.1N NaOH using Beckman diode spectrophotometer Model DU 7000. Ac-DWFKAFYDKVAEKFKEAF-NH2, synthesized solid phase synthesis method previously described (30Datta Fogelman A.M. Mishra V.K. Epand R.M. R.F. Lund-Katz al.Effects hydrophobicity physical-chemical biological peptide.J. 42: 1096-1104Abstract Scholar).14C-4F (31Meriwether Imaizumi Grijalva V. Vakili Farias-Eisner al.Enhancement transfer L-4F C57BL/6J mice plasma.J. 2011; 52: 1795-1809Abstract (13) modified [W→F] (Ac-DFFKAFYDKVAEKFKEAF-NH2) substituting (Phe/F) (Trp/W) used control Peptide purity ascertained analysis analytical HPLC. analog ∊280= 7,300 M−1cm−1 ∊264= 1,280 M−1cm−1, respectively. Both endotoxin free Limulus assay QCL-1000 kit (Lonza). studies, assessed. Briefly, or (10–300 μM) added physiological salt solution containing (1.6 10−2 mM). (100 then initiate nonfluorescent fluorescein. Fluorescein fluorescence Bio-Tek Synergy HT microplate reader λex= 480 λem= 515. characteristics [W→F], native analyzed denaturing gel electrophoresis. HOCl:peptide 3:1. Residual quenched addition taurine after 15 min. Native (14 μg) loaded onto 4-20% SDS-Tris glycine stained Coomassie blue. Gels destained MeOH:acetic acid:H2O. See Blue Plus 2 (Invitrogen) identification bands. scanned, performed LabWorks software (Lablogics V4.6). some experiments, 14C-labeled prior electrophoretic separation. Bands cut SDS gels, distribution radioactive counts scintillation counting. expressed percentage absence treatment. further characterize HOCl-induced samples analytical, pressure column chromatography Beckman-Coulter HPLC system. Samples (25 injected C18 (Vydak, 4.6 250 mm, size 5 μm) eluted acetonitrile H2O [0.1% (TFA)] 35–55% gradient (40 min). bond 220 nm. subsequent 600 μg obtain sufficient material spectrometry. Peak (1.0 ml) pooled individual runs lyophilized. relative 280 (1 mg/ml each) ratio time periods 30 s mixed 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC:1:1 mol/mol) complexes addition. At end each point, quench unreacted decay HOCl-treated assessed A280. initially MALDI-TOF Applied Biosystems Voyager DE-Pro reflector positive ion mode (Foster City, CA) 20 Hz. Material peaks resuspended 1:10 dilution α-cyano-4-hydroxycinnamic 0.1% TFA plated analyses. range selected 1,000–4,000 m/z 500 spectra/sample collected. Post processing Explorer (version 3.40). Sequence NanoLC-tandem An aliquot (2 μl) digest cm 75 μm i.d. PepMap100 reverse-phase trap cartridge (Dionex, Sunnyvale, μl/min Eksigent autosampler. After washing 4 min formic ddH20, bound flushed (Dionex) 40 linear (5–50%) acetonitrile-H2O 300 nl/min Nano1D+ LC. (Dublin, CA). washed 90% acetonitrile-0.1% reequilibrated 5% 5600 Triple-TOF spectrometer (AB-Sciex, Toronto, Canada) analyze digest. Eluted subjected time-of-flight survey scan 400–1,250 top twenty most intense MS/MS analysis. Product scans 50 msec carried out tandem spectra parent over 400–2,000. Spectra centroided deisotoped Analyst software, version TF (Applied Biosystems). β-galactosidase trypsin establish confirm accuracy spectrometer. emission spectrum (1:1-3:1) 295 349 turbidity POPC multilamellar vesicles (MLV) measured PBS scattering. For solutions MLVs (1:1 mol/mol ratio), phospholipid clarification every SLM-AMINCO 8100 Spectrofluorometer (SLM Instruments). Representative scattering profiles presented. CD recorded JASCO J-815 spectropolarimeter equipped Peltier-type temperature system (JASCO model PTC-423s/15). nanometer 260 195nm cell 0.1 path length. 25°C average scans. µM (pH 7.4). Mean ellipticity ([θ]MRE) (degcm2/dmol) θ222 calculated following equation: [θ]MRE=(MRW×θ)/(10cl)where MRW mean (peptide divided acids); θ observed helicity degrees; c g/ml; l length centimeters. Percentage equation, Scholar): PercentαHelix=([MREθ222+3,000]/[36,000+3,000])×100 4F- [W→F]mediated procedure Kritharides al. (32Kritharides Christian Stoudt Morel Rothblat G.H. Cholesterol THP-1 macrophages.Arterioscler. 1998; 18: 1589-1599Crossref monocytes seeded 106 cells/well 6-well plates. Acetylated LDL, radiolabeled 3H-cholesterol, cells 24 h 37°C. 8-bromo cAMP (300 expression. Cells incubated 100 μg/ml 37°C lipoprotein-depleted medium BSA. incrementally h. period, media lysates counting amount effluxed cells. Efflux normalized total [W→F]. saturating amounts μg/ml) masking more subtle effect efflux, experiments. (10, 25, 50,