Apolipoprotein A-1 is a selective target for myeloperoxidase-catalyzed nitration and chlorination

Systemic levels of protein-bound nitrotyrosine (NO2 Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, are independent predictors of atherosclerotic risk, burden, and incidence of cardiac events (JAMA (2001) 286:2136; JAMA (2003) 289:1675). However, the targets of oxidative modifications and the relationship between oxidative modification and any functional alterations are not completely understood. The present studies focus on apolipoprotein A-I (apoA-I), the primary protein constituent of high density lipoprotein (HDL), and show that this protein is a selective target for MPO-catalyzed nitration and chlorination in vivo. Furthermore, the results demonstrate that site-specific oxidation of apoA-I by MPO causes selective inhibition in ABCA1 dependent cholesterol efflux from macrophages. Enrichment in NO2Tyr and ClTyr content was found within apoA-I recovered from serum and human atherosclerotic lesions, and NO2Tyr and ClTyr in apoA-I were found to be higher in individuals with cardiovascular disease (CVD), relative to controls. Analysis of HDL isolated from human plasma revealed that higher NO2Tyr and ClTyr are associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. The oxidative modification of apoA-I by MPO in vivo is apparently facilitated by an ability of MPO to bind to apoA-I, as revealed by (a) reciprocal immuno-precipitation studies in plasma, (b) the recovery of MPO associated with HDL-like particles isolated from human atheroma, and (c) the identification of a specific MPO interaction site in the helix 8 region of apoA-I using hydrogen/deuterium exchange mass spectrometry. Quantitative analyses of site-specific modifications to apoA-I demonstrate that Y192 is the favored apoA-I site for MPO-mediated nitration and chlorination, which lies within the MPO binding site. Further, modification of Y192 is correlated with dose-dependent losses of ABCA1-dependent cholesterol acceptor and lipid-binding activities upon exposure to MPO-generated oxidants. Finally, LC-tandem MS was used to show that apoA-I nitrated at Y192 and Y166 is present in human atheroma tissue. As a whole, these results provide the first direct evidence for apoA-I as a selective target for site-specific oxidative modification by MPO within human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a pro-atherogenic dysfunctional form of HDL in vivo. Finally, these results suggest that apoA-I nitration may be a useful clinical marker for CVD.