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

摘要

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 Trp(72). Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp(72) 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-OHTrp(72) 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-OHTrp(72) apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (?mol/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-OHTrp(72) 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-OHTrp(72) 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 Trp(72) impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo.