Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

• Published in: The Journal of biological chemistry 2014-04, Vol.289 (15), p.10276-10292
• Main Authors: DiDonato, Joseph A., Aulak, Kulwant, Huang, Ying, Wagner, Matthew, Gerstenecker, Gary, Topbas, Celalettin, Gogonea, Valentin, DiDonato, Anthony J., Tang, W.H.Wilson, Mehl, Ryan A., Fox, Paul L., Plow, Edward F., Smith, Jonathan D., Fisher, Edward A., Hazen, Stanley L.
• Format: Article
• Language: English
• Subjects: Aminoacyl-tRNA Synthetase; Animals; Antibodies, Monoclonal - chemistry; Aorta - metabolism; Apolipoprotein A-I - metabolism; Apolipoproteins; Atherosclerosis; Coronary Vessels - pathology; Dysfunctional HDL; Escherichia coli - metabolism; Humans; Male; Mice; Mice, Inbred BALB C; Molecular Bases of Disease; Nitrotyrosine; Orthogonal Amino Acid; Peroxidase; Peroxidases - metabolism; Plaque, Atherosclerotic - metabolism; Post-translational Modification; Protein Nitration; Protein Processing, Post-Translational; Reactive Nitrogen Species; Recombinant Proteins - metabolism; Tyrosine - analogs & derivatives; Tyrosine - metabolism; Ultracentrifugation; Unnatural Amino Acid (uAA)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.556506

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr166), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr166 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-Tyr166-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-Tyr166-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-Tyr166-apoA-I existed as a lipid-poor lipoprotein with