Abstract
Oxidative damage by myeloperoxidase (MPO) has been proposed to deprive HDL of its cardioprotective effects. In vitro studies reveal that MPO chlorinates and nitrates specific tyrosine residues of apoA-I, the major HDL protein. After Tyr-192 is chlorinated, apoA-I is less able to promote cholesterol efflux by the ABCA1 pathway. To investigate the potential role of this pathway in vivo, we used tandem mass spectrometry with selected reaction monitoring to quantify the regiospecific oxidation of apoA-I. This approach demonstrated that Tyr-192 is the major chlorination site in apoA-I in both plasma and lesion HDL of humans. We also found that Tyr-192 is the major nitration site in apoA-I of circulating HDL but that Tyr-18 is the major site in lesion HDL. Levels of 3-nitrotyrosine strongly correlated with levels of 3-chlorotyrosine in lesion HDL, and Tyr-18 of apoA-I was the major nitration site in HDL exposed to MPO in vitro, suggesting that MPO is the major pathway for chlorination and nitration of HDL in human atherosclerotic tissue. These observations may have implications for treating cardiovascular disease, because recombinant apoA-I is under investigation as a therapeutic agent and mutant forms of apoA-I that resist oxidation might be more cardioprotective than the native form.
Highlights
Oxidation of apolipoprotein A-I by myeloperoxidase has been proposed to deprive high density lipoprotein (HDL) of its cardioprotective effects
We found that tyrosine residue 192 (Tyr-192) is the major nitration site in apolipoprotein A-I (apoA-I) of circulating HDL but that Tyr-18 is the major site in lesion HDL
To maximize analytical sensitivity and quantify site-specific modifications of apoA-I, we used a triple quadrupole mass spectrometer coupled to a nanoACQUITY UltraPerformance liquid chromatography (nano-LC) system to detect peptides and included hypochlorous acid (HOCl)- and peroxynitrite-oxidized [15N]apoA-I in the digestion reaction as an internal standard
Summary
Oxidation of apolipoprotein A-I by myeloperoxidase has been proposed to deprive HDL of its cardioprotective effects. To investigate the potential role of this pathway in vivo, we used tandem mass spectrometry with selected reaction monitoring to quantify the regiospecific oxidation of apoA-I This approach demonstrated that Tyr-192 is the major chlorination site in apoA-I in both plasma and lesion HDL of humans. To further increase its power and obtain a quantitative measure of site-specific oxidation, we generated isotope-labeled [15N]apoA-I protein for use as an the internal standard [22, 53] This quantitative analytical approach demonstrated that Tyr-192 is the major chlorination site in apoA-I of HDL isolated from human plasma and atherosclerotic tissue. Our observations support the proposal that chlorination of apoA-I by MPO may generate a dysfunctional form of HDL in vivo
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