Effects of Reagent and Enzymatically Generated Hypochlorite on Physicochemical and Metabolic Properties of High Density Lipoproteins

Abstract

Myeloperoxidase (MPO),In contrast to low density lipoproteins (LDL), 1 high plasma concentrations of high density lipoproteins (HDL) are associated with a decreased risk for the development of coronary artery disease, an effect commonly attributed to their central role in reverse cholesterol transport (1). During this process, HDL is able to promote efflux of cholesterol from peripheral tissues, and the accepted cholesterol is (at least in part) esterified by the action of lecithin-cholesterol acyltransferase (2). Cholesteryl esters (CEs) formed by the lecithin-cholesterol acyltransferase reaction are then transferred from HDL to other lipoproteins mediated by the cholesterol ester transfer protein or are delivered to the liver for bilary secretion or reutilization during lipoprotein assembling (3).Within the oxidative theory, HDL appears to be Janus-faced (4 -6). The majority of lipid hydroperoxides (the first detectable products of lipoprotein oxidation) in plasma are transported in the HDL fraction (7), and HDL was suggested to act as a sink for preformed lipid hydro(pero)xides. HDL-associated cholesteryl ester hydroperoxides, which are probably transferred to HDL by the action of the cholesterol ester transfer protein (8), are preferentially catabolized over nonoxidized CEs; this was shown in HepG2 cells (9), in situ perfused rat liver (10), and intact rats (11). HDL was also shown to protect LDL from lipid peroxidation, inhibiting the formation of lipid hydroperoxides but not the formation of conjugated dienes (12). On the other hand, HDL is more easily oxidized than LDL (7), HDLassociated lecithin-cholesterol acyltransferase is modified by reactive short and long chain aldehydes (13), and the ability of oxidatively modified HDL to promote cellular cholesterol efflux is diminished (14 -16), most probably due to alterations in its apolipoprotein moiety. During the acute phase response, up to 80% of apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, is displaced by serum amyloid A (17), and due to this exchange in the HDL apolipoprotein domain, native, anti-inflammatory HDL became proinflammatory during the acute phase response (18). The above mentioned results imply that HDL can protect LDL against oxidative modifications and could serve as a vehicle for detoxification of potentially (cyto)-toxic lipid hydroperoxides. However, under certain circumstances many of the important physiological properties of HDL can be lost during oxidation/modification, transforming HDL into a proatherogenic lipoprotein particle.

Effects of Protein Oxidation on the Structure and Stability of Model Discoidal High-Density Lipoproteins

“…Increasing oxidant:protein ratios to 25:1 and beyond resulted in near-complete conversion of monomeric to cross-linked protein on discoidal particles (lanes 3, 4); lipid-free apoA-I (data not shown) undergoes extensive intramolecular cross-linking under these conditions (44). At lower oxidant:protein ratios (≤10:1) that encompass the range of physiologically relevant conditions (26), lipid-bound and lipid-free apoA-I show comparable population distribution of monomeric, dimeric and trimeric species (lanes 1, 2 and 1′, 2′ compared); therefore, we used these relatively low ratios in our analysis of (oxA-I):DMPC complexes and their comparison with ox(A-I:DMPC) disks.…”

“…This destabilization was attributed to reduced protein affinity for lipid upon MetO formation in the apolar lipid-binding faces of the amphipathic apolipoprotein α-helices (29). Such MetO formation occurs at early stages of oxidation and is followed by modifications of other protein groups, such as aromatic residues and Lys, and their cross-linking (24)(25)(26)(27)(28)(29)(30)(31). Correlation of rHDL destabilization (Fig.…”

“…Physiological concentrations of HOCl have not been accurately determined, although some in-vivo-based reports suggest that, at sites of acute inflammation, the molar ratio of oxidant to HDL may be as high as 30:1 (26 and references therein). At these and lower HOCl levels and near-neutral pH, oxidative modifications in HDL are mainly limited to apolipoproteins (26,27). We use this property of HOCl to test the effects of apolipoprotein oxidation on the structure and stability of model discoidal HDL.…”

“…We use this property of HOCl to test the effects of apolipoprotein oxidation on the structure and stability of model discoidal HDL. Dose-dependent HOCl-induced modifications in the major HDL proteins, apoA-I and apoA-II, have been well-documented by using limited proteolysis, mass spectrometry and liquid chromatography; the general consensus is that oxidation of Met and Cys occurs first, followed by modifications of Lys and aromatic residues and, eventually, protein cross-linking into homoor heterodimers and trimers (24,(26)(27)(28)(29)(30). Specifically, limited proteolysis and mass spectrometry studies of apoA-I in solution or on HDL suggest that, at a 5:1 HOCl:protein molar ratio, Met 86, Met 112 and Met 148 are converted to methionine sulfoxides (MetO); at a 10:1 ratio, Phe 37 and Phe 71 are the primary targets of oxidation; and at a 25:1 ratio, Tyr 192 is chlorinated (25,28).…”

Mild Oxidation Promotes and Advanced Oxidation Impairs Remodeling of Human High-Density Lipoprotein In Vitro

“…6-8. 44 Thus, oxidation to stages 1, 2 in OCl-treated HDL involves almost exclusively protein modifications, while similar stages in Cu-treated HDL also involve significant modifications to the lipids. This suggests that maximal destabilization of HDL observed at oxidation stage 2 by either reagent is mainly due to protein modifications.…”