Abstract
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.
Highlights
Apolipoprotein A-I is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall
We report a significant impairment of in vitro cholesterol acceptor activity and in vivo HDL biogenesis function observed with 5-OHTrp72 Apolipoprotein A-I (apoA-I) relative to WT apoA-I
We first examined the specificity of this orthogonal pair for oxidized tryptophan by co-transforming the plasmid encoding the engineered machinery with either one of two reporters encoding superfolder GFP (sfGFP)-150TAG (Fig. 1, A and B), or apoA-I-72TAG (Fig. 1C), as described under “Experimental procedures.”
Summary
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. Following injection into apoA-I– deficient mice, 5-OHTrp 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 These findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo. 5-OHTrp apoA-I has impaired atheroprotective activities has been the subject of intense investigation with efforts focused on recapitulating the physiological modifications in vitro using MPO-catalyzed modifications with chlorinating and nitrating oxidation systems These studies are limited by oxidation on all susceptible residues, thereby confounding interpretation of functional studies and assignment of impaired activities to specific sites. The functional consequence of site-specific (single-site) oxidation of apoA-I remains poorly understood
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