Abstract
Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.
Highlights
Oxidation of LDL by the myeloperoxidase (MPO)H2O2-chloride system is a key event in the development of atherosclerosis
In order to investigate whether an increased consumption of H2O2 could result from scavenging of hypochlorous acid (HOCl) by LDL, Met was added
In order to investigate whether modifications of apoB-100 differed when LDL was oxidized under several conditions, we investigated the formation of Cl-Tyr, O-Met,oxidized tryptophan (OxTrp), and aminoadipic acid, a degradation product of N-chloramine Lys [35, 36]
Summary
Materials and reagents Ammonium bicarbonate, formic acid (FA), NaCl, K2HPO4, methanol, and acetonitrile were from Merck (Darmstadt, Germany). LDL (1 mg) was dispersed in 1 ml PBS (150 mM ClϪ and 10 mM phosphate, pH 7.4) and incubated for 2 h at 37°C with NaOCl (added as a single addition with gentle vortexing) at oxidant/LDL molar ratios in between 25:1 and 333:1. MPO-LDL was generated by mixing 8 μl of HCl 1 M (final concentration: 4 mM), 45 μl of MPO (final concentration: 250 nM), a volume containing 1.6 mg LDL (final concentration: 0.8 mg/ml in PBS, pH 7.4), and 40 μl of H2O2 50 mM (final concentration: 1 mM). LDL was precipitated with 500 μl trichloroacetic acid (10%, v/v) and centrifuged for 10 min at 4,500 g. Four hundred microliters of LDL solution from each patient/volunteer was treated as described previously.
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