Tyrosine 192 in Apolipoprotein A-I Is the Major Site of Nitration and Chlorination by Myeloperoxidase, but Only Chlorination Markedly Impairs ABCA1-dependent Cholesterol Transport*
- Baohai Shao‡,
- Constanze Bergt‡,
- Xiaoyun Fu‡,
- Pattie Green‡,
- John C. Voss§,
- Michael N. Oda¶,
- John F. Oram‡ and
- Jay W. Heinecke‡∥
- ‡Department of Medicine, University of Washington, Seattle, Washington 98195, the §Department of Biological Chemistry, University of California, Davis, California 95616, and ¶Children's Hospital Research Institute, Oakland, California 94609
- ↵∥ To whom correspondence should be addressed: Division of Metabolism, Endocrinology, and Nutrition, Box 356426, University of Washington, Seattle, WA 98195. E-mail: heinecke{at}u.washington.edu.
Abstract
High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO-). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO-. Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr192 of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO-, suggesting that solvent accessibility accounted in part for the reactivity of Tyr192. The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr192. Both ONOO- and MPO nitrated Tyr192 in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr192 is the predominant site of nitration and chlorination when MPO or ONOO- oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease.
- Received October 8, 2004.
- Revision received November 29, 2004.
- The American Society for Biochemistry and Molecular Biology, Inc.
