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

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.

The Integration of Metabolomic and Proteomic Analyses Revealed Alterations in Inflammatory-Related Protein Metabolites in Endothelial Progenitor Cells Subjected to Oscillatory Shear Stress

“…After OSS intervention in EPCs, PSPH expression was upregulated, regulating cysteine and methionine metabolism, eventually leading to a significant decrease in Betaine. A study showed that after cells were subjected to OSS, the betaine level decreased significantly and that 5-hydroxytryptophan, a potential biomarker of DEM with reduced concentrations after experiencing OSS intervention, was incorporated into apolipoprotein A-I, impairing cholesterol efflux activity and high-density lipoprotein biogenesis ( Zamanian-Daryoush et al, 2020 ), promoting the occurrence and development of AS. β-Alanine and L-histidine are known to be involved in the synthesis of carnosine, which can delay inflammation and fight AS ( Caruso et al, 2019 ).…”

Unraveling the Complexity of HDL Remodeling: On the Hunt to Restore HDL Quality

“…Especially, chlorination of Tyr192 has been shown to impair ABCA1-dependent RCT [56][57][58] and markedly increase atherosclerotic plaque instability [59]. ABCA1-dependent RCT was also shown to be impaired by MPO-mediated oxidation of the Trp72 residue (Trp72(O)-ApoA-I) [60], which, interestingly, accounted for 20% of the ApoA-I in atherosclerotic arteries, while the overall abundance in circulation was low [61]. Elevated Trp72(O)-ApoA-I levels demonstrated a potent pro-inflammatory activity on endothelial cells and were associated with increased CV risk [61].…”

Roles of Myeloperoxidase in the Oxidation of Apolipoproteins: Interest of Monitoring Myeloperoxidase Oxidation of Apolipoproteins A-1 and B-100 to Improve the Estimation of Lipoprotein Quality in Cardiovascular Diseases

“…For example, differences between control subjects and patients with stable CAD or acute coronary syndrome have been established in relation to the presence of Cl-Tyr (at Tyr192) and the oxidation of Met (at Met 148) of apoA-1 [68]. The residue of Trp 72 also seems to be promising as a biomarker, since it is the subject of a multitude of studies that highlighted its importance in cholesterol efflux [44,[68][69][70][71]. It must be noted that cholesterol efflux capacity is not completely dependent on levels of functional apoA-1 [72].…”

“…More recently, incorporation of site-specific oxTrp (including Trp 72) into apoA-1 has been performed [71]. By using genetic code expansion technology, noncanonical amino acid 5-hydroxytryptophan (5-OH-Trp) has been inserted at position 72 in recombinant human apoA-1.…”