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Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis.

  • Zamanian-Daryoush, Maryam1, 2
  • Gogonea, Valentin1, 2, 3
  • DiDonato, Anthony J1, 2
  • Buffa, Jennifer A1, 2
  • Choucair, Ibrahim1, 2, 3
  • Levison, Bruce S1
  • Hughes, Randall A4
  • Ellington, Andrew D5
  • Huang, Ying1, 2
  • Li, Xinmin S1, 2
  • DiDonato, Joseph A1, 2
  • Hazen, Stanley L6, 2, 7
  • 1 Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195.
  • 2 Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, Ohio 44195.
  • 3 Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115.
  • 4 United States Army Research Laboratory South, University of Texas, Austin, Texas 78712. , (United States)
  • 5 Center for Systems and Synthetic Biology, University of Texas, Austin, Texas 78712.
  • 6 Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195 [email protected]
  • 7 Heart and Vascular Institute, Cleveland Clinic, Cleveland, Ohio 44195.
Published Article
Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology
Publication Date
Apr 10, 2020
DOI: 10.1074/jbc.RA119.012092
PMID: 32098873


Apolipoprotein A-I (apoA-I) is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall. In atherosclerotic lesions, apoA-I exhibits marked oxidative modifications at multiple sites, including Trp72 Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp72 of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered Saccharomyces cerevisiae tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair to insert the noncanonical amino acid 5-hydroxytryptophan (5-OHTrp) at position 72 in recombinant human apoA-I and confirmed site-specific incorporation utilizing MS. In functional characterization studies, 5-OHTrp72 apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity in vitro (41.73 ± 6.57% inhibition; p < 0.01). Additionally, 5-OHTrp72 apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (μmol/min/mg) when compared with WT apoA-I and comparable PON1 activation/stabilization compared with reconstituted HDL (WT apoA-I, 1.92 ± 0.04; 5-OHTrp72 apoA-I, 2.35 ± 0.0; and HDL, 2.33 ± 0.1; p < 0.001, p < 0.001, and p < 0.001, respectively). Following injection into apoA-I-deficient mice, 5-OHTrp72 apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; p < 0.01) lipidation and evidence of HDL biogenesis. Collectively, these findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp72 impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo.

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