Characterization of covalent modifications of HDL apoproteins by endogenous oxidized phospholipids

摘要

The atheroprotective functions of high density lipoprotein (HDL) are compromised under oxidative stress. We have carried out a systematic analysis of modifications of HDL apoproteins by endogenous oxidized phospholipids (oxPL) known to be generated under oxidative stress. Human HDL and plasma were oxidized using several methods, or were exposed to synthetic oxPL. Protein adduction by oxPL was assessed using LC-MS/MS and MALDI-TOF MS. The pattern of HDL apoprotein modification by oxPL was found to be independent of the oxidation systems used. The major modification targets, apoA-I (helix 5-8) and apoA-II (central domain), were mainly modified by oxPL with γ-hydroxy (or oxo)-α,β-unsaturated aldehyde at sn-2 position via formation of Michael adduct (the most abundant), cyclic hemiacetal and Schiff base adduct. Modifications by oxPL having alkyl aldehyde group via Schiff base formation were also identified. Experiments on plasma exposed to oxidation or synthetic oxPL demonstrated that oxPL modification is highly selective, and the four histidines (H155, H162, H193 and H199) in helices 6-8 of apoA-I are the main modification target. H710 and H3613 in apoB-100 of LDL and K190 of human serum albumin were also modified by oxPL but to a lesser extent. Comparison of oxPL with short chain aldehyde HNE using MALDI-TOF MS demonstrated high selectivity and efficiency of oxPL in the modification of HDL apoproteins. These findings provide a novel insight into a potential mechanism for the loss of the atheroprotective function of HDL in conditions of oxidative stress.