Increased methionine sulfoxide content of apoA-I in type 1 diabetes (original) (raw)
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Translational Proteomics, 2014
The oxidative modification of apolipoprotein A-I's methionine148 (M148) is associated with defective HDL function in vitro. Multiple reaction monitoring (MRM) is a mass spectrometric technique that can be used to quantitate post-translational modifications. In this study, we developed an MRM assay to monitor the abundance ratio of the peptide containing oxidized M148 to the native peptide in ApoA-I. Measurement of the oxidized-to-unoxidized-M148 ratio was reproducible (CV < 5%). The extent of methionine M148 oxidation in the HDL of healthy controls, and type 2 diabetic participants with and without prior cardiovascular events (CVD) were then examined. The results suggest a significant increase in the relative ratio of the peptide containing oxidized M148 to the unmodified peptide in the HDL of participants with diabetes and CVD (p < 0.001), compared to participants without CVD. Monitoring the abundance ratio of the peptides containing oxidized and unoxidized M148 by MRM provides a means of examining the relationship between M148 oxidation and vascular complications in CVD.
A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL
Journal of Lipid Research, 2009
Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO 86 and MetO 112 (termed apoA-I 132) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I 132 , apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e-or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease.
Journal of Lipid Research, 2002
Atherosclerosis is a state of heightened oxidative stress. Oxidized LDL is present in atherosclerotic lesions and used as marker for coronary artery disease, although in human lesions lipids associated with HDL are as oxidized as those of LDL. Here we investigated specific changes occurring to apolipoprotein A-I (apoA-I) and apoA-II, as isolated HDL and human plasma undergo mild, chemically induced oxidation, or autoxidation. During such oxidation, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. Placing plasma at ؊ 20 ؇ C prevents autoxidation, whereas metal chelators and butylated hydroxytoluene offer partial protection. Independent of the oxidation conditions, apoA-I and apoA-II (dimer) with two MetO residues accumulate as relatively stable oxidation products. Compared to controls, serum samples from subjects with the endothelial cell nitric oxide synthase a/b genotype that is associated with increased coronary artery disease contain increased concentrations of apoA-I with two MetO residues. Our results show that during the early stages, oxidation of HDL gives rise to specifically oxidized forms of apoA-I and apoA-II, some of which may be useful markers of in vivo HDL oxidation, and hence potentially atherosclerosis.
Methionine Oxidation Impairs Reverse Cholesterol Transport by Apolipoprotein AI
Proceedings of the …, 2008
HDL protects against vascular disease by accepting free cholesterol from macrophage foam cells in the artery wall. This pathway is critically dependent on lecithin:cholesterol acyltransferase (LCAT), which rapidly converts cholesterol to cholesteryl ester. The physiological activator of LCAT is apolipoprotein A-I (apoA-I), the major HDL protein. However, cholesterol removal is compromised if apoA-I is exposed to reactive intermediates. In humans with established cardiovascular disease, myeloperoxidase (MPO) oxidizes HDL, and oxidation by MPO impairs apoA-I’s ability to activate LCAT in vitro. Because a single methionine residue in apoA-I, Met-148, resides near the center of the protein’s LCAT activation domain, we determined whether its oxidation by MPO could account for the loss of LCAT activity. Mass spectrometric analysis demonstrated that oxidation of Met-148 to methionine sulfoxide associated quantitatively with loss of LCAT activity in both discoidal HDL and HDL3, the enzyme’s physiological substrates. Reversing oxidation with methionine sulfoxide reductase restored HDL’s ability to activate LCAT. Discoidal HDL prepared with apoA-I containing a Met-1483Leu mutation was significantly resistant to inactivation by MPO. Based on structural data in the literature, we propose that oxidation of Met-148 disrupts apoA-I’s central loop, which overlaps the LCAT activation domain. These observations implicate oxidation of a single Met in apoA-I in impaired LCAT activation, a critical early step in reverse cholesterol transport.
Circulation Journal, 2011
Although high-density lipoprotein-cholesterol (HDL-C) levels in large epidemiological studies are inversely related to the risk of coronary heart disease (CHD), increasing the level of circulating HDL-C does not necessarily decrease the risk of CHD events, CHD deaths, or mortality, HDL can act as an anti-or a proinflammatory molecule, depending on the context and environment. Based on a number of recent studies, it appears that the anti-or proinflammatory nature of HDL may be a more sensitive indicator of the presence or absence of atherosclerosis than HDL-C levels. The HDL proteome has been suggested to be a marker, and perhaps a mediator, of CHD. Apolipoprotein A-1 (apoA-I), the major protein in HDL is a selective target for oxidation by myeloperoxidase, which results in impaired HDL function. Improving HDL function through modification of its lipid and/or protein content maybe a therapeutic target for the treatment of CHD and many inflammatory disorders. HDL/apoA-I mimetic peptides may have the ability to modify the lipid and protein content of HDL and convert dysfunctional HDL to functional HDL. This review focuses on recent studies of dysfunctional HDL in animal models and human disease, and the potential of apoA-I mimetic peptides to normalize the composition and (function of lipoproteins.
Increased Lipid Peroxidation in LDL from Type-2 Diabetic Patients
Lipids, 2010
Increased oxidative stress is associated with type-2 diabetes and related cardiovascular diseases but oxidative modification of LDL has been partially characterized. Our aim was to compare the lipid and fatty acid composition as well as the redox status of LDL from diabetic patients and healthy subjects. First, to ensure that isolation of LDL by sequential ultracentrifugation did not result in lipid modifications, lipid composition and peroxide content were determined in LDL isolated either by ultracentrifugation or fast-protein liquid chromatography. Both methods resulted in similar concentrations of lipids, fatty acids, hydroxy-octadecadienoic acid (HODE) and malondialdehyde (MDA). Then, LDL were isolated by ultracentrifugation from 8 type-2 diabetic patients and 8 control subjects. Compared to control LDL, diabetic LDL contained decreased cholesteryl esters and increased triglyceride concentrations. Ethanolamine plasmalogens decreased by 49%. Proportions of linoleic acid decreased in all lipid classes while proportions of arachidonic acid increased in cholesteryl esters. Total HODE concentrations increased by 56%, 12-and 15-hydroxyeicosatetraenoic acid by 161 and 86%, respectively, and MDA levels increased by 2-fold. αtocopherol concentrations, expressed relative to triglycerides, were lower in LDL from patients compared to controls while γ-tocopherol did not differ. Overall, LDL from type-2 diabetic patients displayed increased oxidative stress. Determination of hydroxylated fatty acids and ethanolamine plasmalogen depletion could be especially relevant in diabetes.
The Journal of Lipid Research, 2002
The aim of the study was to assess the isolation of HDL by fast protein liquid chromatography (FPLC) to perform kinetics studies of apolipoprotein (apo)A-I-HDL labelled with a stable isotope. Comparison between FPLC and ultracentrifugation has been made. ApoA-I-HDL kinetics were studied by infusion of [5.5.5-2 H 3 ]leucine for 14 h in five subjects. Using FPLC, pre  1 HDL and ␣ HDL (HDL 2 and HDL 3 ) were separated from 200 l of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in pre  1 HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to ␣ HDL suggested the existence of a mixture of apoA-I-HDL sub-classes. From this difference in enrichments, the turnover rate of total HDL-UC, usually assumed to be ␣ HDL, was probably overestimated in previous studies. To our knowledge, this study is the first report which provides a convenient tool to distinguish enrichments of apoA-I in pre  1 HDL and ␣ HDL from total HDL previously used for kinetic measurements. This original and new method should help to understand the kinetics of HDL in humans and the reverse cholesterol transport dynamics. -Chétiveaux, M., H. Nazih, V. Ferchaud-Roucher, G. Lambert, Y. Zaïr, M. Masson, K. Ouguerram, D. Bouhours, and M. Krempf. The differential apoA-I enrichment of pre  1 and ␣ HDL is detectable by gel filtration separation.