Site-specific nitration of apolipoprotein A-I at tyrosine 166 is both abundant within human atherosclerotic plaque and dysfunctional [Metabolism]

February 20th, 2014 by DiDonato, J. A., Aulak, K., Huang, Y., Wagner, M., Gerstenecker, G., Topbas, C., Gogonea, V., DiDonato, A. J., Tang, W. H. W., Mehl, R. A., Fox, P. L., Plow, E. F., Smith, J. D., Fisher, E. A., Hazen, S. L.

We previously reported that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Y166), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Y166 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 site-specifically nitrated at residue 166 (166YNO2-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 nitroY-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 show 166YNO2-apoA-I is easily detected in atherosclerotic human coronary arteries, accounts for ~8% of total apoA-I within the artery wall, but is nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses shows 166YNO2-apoA-I exists as a lipid-poor lipoprotein, with <3% recovered within the HDL-like fraction (d=1.063-1.21). 166YNO2-apoA-I in plasma shows similar distribution. Recovery of 166YNO2-apoA-I using immobilized mAb 4G11.2 shows an apoA-I form with 88.1+/-8.5% reduction in LCAT activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Site-specific nitration of apoA-I at Y166 is thus 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.
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