title: Site-specific nitration of apolipoprotein A-I at tyrosine 166 is both abundant within human atherosclerotic plaque and dysfunctional.
abstract: We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼ 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, Ohio 44195. Electronic address: didonaj@ccf.org.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: DiDonato
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Kulwant
authors.initials: K
authors.lastname: Aulak
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Matthew
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Department of Chemistry, Cleveland State University, Cleveland, Ohio 44118.
authors.forename: Gary
authors.initials: G
authors.lastname: Gerstenecker
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Department of Chemistry, Cleveland State University, Cleveland, Ohio 44118.
authors.forename: Celalettin
authors.initials: C
authors.lastname: Topbas
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Department of Chemistry, Cleveland State University, Cleveland, Ohio 44118.
authors.forename: Valentin
authors.initials: V
authors.lastname: Gogonea
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Department of Psychology, John Carroll University, University Heights, Ohio 44118.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: DiDonato
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, Ohio 44195; Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: W H Wilson
authors.initials: WHW
authors.lastname: Tang
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Fox
authors.affiliation: Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Department of Molecular Cardiology, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Edward F
authors.initials: EF
authors.lastname: Plow
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, Ohio 44195; Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Smith
authors.affiliation: Department of Cell Biology and the Leon H. Charney Division of Cardiology, Department of Medicine, New York University School of Medicine, New York, New York 10016.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Fisher
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, Ohio 44195; Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, Ohio 44195; Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195. Electronic address: hazens@ccf.org.
authors.forename: Stanley L
authors.initials: SL
authors.lastname: Hazen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001011:Aorta / Q000378:metabolism; D016632:Apolipoprotein A-I / Q000378:metabolism*; D003331:Coronary Vessels / Q000473:pathology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010544:Peroxidases / Q000378:metabolism; D058226:Plaque, Atherosclerotic / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D026361:Reactive Nitrogen Species; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D026361:Reactive Nitrogen Species; D011994:Recombinant Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine; D010544:Peroxidases
keywords: Aminoacyl-tRNA Synthetase; Apolipoproteins; Atherosclerosis; Dysfunctional HDL; Nitrotyrosine; Orthogonal Amino Acid; Peroxidase; Post-translational Modification; Protein Nitration; Unnatural Amino Acid (uAA)