Abstract
The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.
Highlights
High density lipoproteins (HDL)1 are generally regarded as antiatherogenic, an activity commonly attributed to the removal of extrahepatic cholesterol by HDL particles [1, 2] and apolipoproteins, mainly apolipoprotein A-I, that dissociate from HDL [3, 4]
Cholesteryl ester transfer protein (CETP) transfers oxidized lipids from low density lipoproteins (LDL) to HDL [7], and HDL carries the majority of cholesteryl ester hydroperoxides (CE-OOH, the first and major products formed during lipoprotein oxidation) in human plasma [5]
Oxidized LDL is generally thought to contribute to atherogenesis, because it can cause lipid accumulation in macrophages and has a number of other potentially proatherogenic properties [43]
Summary
Isolation and Selective Oxidation of HDL—Isolated HDL was prepared from fresh plasma obtained from normolipidemic donors [18], and its protein content was determined by the bicinchoninic acid method (Sigma) using bovine serum albumin (BSA) (Sigma) as standard. Ethanol (20 l) and drHDL containing cholesterol were added, and the mixture was incubated at 37 °C for 30 min before dilution with buffer and supplementation with -mercaptoethanol (4 mM, final concentration) and fatty acid-free BSA (4 mg/ml). HMDM were incubated with acLDL as described above and labeled during the 18-h equilibration period with 5 Ci/ml methyl-[3H]choline chloride (Amersham Pharmacia Biotech) in the presence of 0.1% BSA [29]. Cells were washed twice with PBS containing 0.1% BSA, washed twice with PBS, and lysed in 0.2 N NaOH for 10 min on ice, and aliquots of the lysates used to count radioactivity and to determine protein using the bicinchoninic acid assay. The cellular phospholipid mass at time 0 was determined using an enzymatic kit (Roche Molecular Biochemicals)
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