Michael N. Oda

Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I.

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, protein-bound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo.

Optimized bacterial expression of human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product proteins primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.

Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I

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-Is ability to activate LCAT in vitro. Because a single methionine residue in apoA-I, Met-148, resides near the center of the proteins 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 enzymes physiological substrates. Reversing oxidation with methionine sulfoxide reductase restored HDLs ability to activate LCAT. Discoidal HDL prepared with apoA-I containing a Met-148→Leu mutation was significantly resistant to inactivation by MPO. Based on structural data in the literature, we propose that oxidation of Met-148 disrupts apoA-Is 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.

Myeloperoxidase: An Oxidative Pathway for Generating Dysfunctional High-Density Lipoprotein

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by artery wall macrophages triggers atherosclerosis, the leading cause of cardiovascular disease. Conversely, high-density lipoprotein (HDL) retards atherosclerosis by promoting cholesterol efflux from macrophages by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway. HDL has been proposed to lose its cardioprotective effects in subjects with atherosclerosis, but the underlying mechanisms are poorly understood. One potential pathway involves oxidative damage by myeloperoxidase (MPO), a heme enzyme secreted by human artery wall macrophages. We used mass spectrometry to demonstrate that HDL isolated from patients with established cardiovascular disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of MPO. When apolipoprotein A-I (apoA-I), the major HDL protein, was oxidized by MPO, its ability to promote cellular cholesterol efflux by ABCA1 was impaired. Moreover, oxidized apoA-I was unable to activate lecithin:cholesterol acyltransferase (LCAT), which rapidly converts free cholesterol to cholesteryl ester, a critical step in HDL maturation. Biochemical studies implicated tyrosine chlorination and methionine oxygenation in the loss of ABCA1 and LCAT activity by oxidized apoA-I. Oxidation of specific residues in apoA-I inhibited two key steps in cholesterol efflux from macrophages, raising the possibility that MPO initiates a pathway for generating dysfunctional HDL in humans.

The C-terminal domain of apolipoprotein A-I contains a lipid-sensitive conformational trigger

Exchangeable apolipoproteins can convert between lipid-free and lipid-associated states. The C-terminal domain of human apolipoprotein A-I (apoA-I) plays a role in both lipid binding and self-association. Site-directed spin-label electron paramagnetic resonance spectroscopy was used to examine the structure of the apoA-I C terminus in lipid-free and lipid-associated states. Nitroxide spin-labels positioned at defined locations throughout the C terminus were used to define discrete secondary structural elements. Magnetic interactions between probes localized at positions 163, 217 and 226 in singly and doubly labeled apoA-I gave inter- and intramolecular distance information, providing a basis for mapping apoA-I tertiary and quaternary structure. Spectra of apoA-I in reconstituted HDL revealed a lipid-induced transition of defined random coils and β-strands into α-helices. This conformational switch is analogous to triggered events in viral fusion proteins and may serve as a means to overcome the energy barriers of lipid sequestration, a critical step in cholesterol efflux and HDL assembly.

Apolipoprotein A-I Assumes a “Looped Belt” Conformation on Reconstituted High Density Lipoprotein

Apolipoprotein A-I (apoA-I) plays a central role in the reverse cholesterol transport pathway; however, the structural basis for its antiatherogenic effects remains poorly understood. Here we employ EPR spectroscopy and fluorescence resonance energy transfer to elucidate the conformation and relative alignment of apoA-I monomers on discoidal (9.4 nm) reconstituted high density lipoprotein (rHDL). EPR spectroscopy provided evidence for an extended helical secondary structure. Position 139 since it was the only residue examined to display a dynamic motional character consistent with a flexible loop structure. The EPR spectra of nitroxide probes at positions 133 and 146 exhibit spin coupling, indicating that these positions are proximal to an apoA-I paired counterpart on the perimeter of rHDL. fluorescence resonance energy transfer studies employing engineered apoA-I variants possessing a single tryptophan (energy donor) and/or a single cysteine (whose thiol moiety was covalently labeled with an extrinsic energy acceptor) provided evidence that paired apoA-I molecules around the perimeter of rHDL align in an extended antiparallel conformation. Taken together with the observation that the EPR spectra of nitroxide probes positioned at intervening sequence positions (134-145) do not exhibit spin coupling, this has led us to propose a “looped belt” model, wherein residues 133-146 comprise a flexible loop segment that confers to apoA-I an intrinsic ability to adapt its structure to accommodate changing particle lipid content. Specifically, in the looped belt model, with the exception of amino acids 134-145, apoA-I aligns with its counterpart in a helix 5-helix 5 registry, centered at position 139.

Multiple-Reaction Monitoring–Mass Spectrometric Assays Can Accurately Measure the Relative Protein Abundance in Complex Mixtures

BACKGROUND Mass spectrometric assays could potentially replace protein immunoassays in many applications. Previous studies have demonstrated the utility of liquid chromatography-multiple-reaction monitoring-mass spectrometry (LC-MRM/MS) for the quantification of proteins in biological samples, and many examples of the accuracy of these approaches to quantify supplemented analytes have been reported. However, a direct comparison of multiplexed assays that use LC-MRM/MS with established immunoassays to measure endogenous proteins has not been reported. METHODS We purified HDL from the plasma of 30 human donors and used label-free shotgun proteomics approaches to analyze each sample. We then developed 2 different isotope-dilution LC-MRM/MS 6-plex assays (for apoliporoteins A-I, C-II, C-III, E, B, and J): 1 assay used stable isotope-labeled peptides and the other used stable isotope-labeled apolipoprotein A-I (an abundant HDL protein) as an internal standard to control for matrix effects and mass spectrometer performance. The shotgun and LC-MRM/MS assays were then compared with commercially available immunoassays for each of the 6 analytes. RESULTS Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. In contrast, the isotope dilution LC-MRM/MS approaches showed correlations with immunoassays of r = 0.61-0.96. The LC-MRM/MS approaches had acceptable reproducibility (<13% CV) and linearity (r ≥0.99). Strikingly, a single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards. CONCLUSIONS Because peak area ratios measured in multiplexed LC-MRM/MS assays correlate well with immunochemical measurements and have acceptable operating characteristics, we propose that LC-MRM/MS could be used to replace immunoassays in a variety of settings.

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B

The polyene antibiotic amphotericin B (AMB) is an effective antifungal agent whose therapeutic potential is limited by poor aqueous solubility and toxicity toward host tissues. Addition of apolipoprotein A-I to a multilamellar phospholipid vesicle dispersion containing 20% (w/w) AMB induces the formation of reconstituted high density lipoprotein (rHDL), with solubilization of the antibiotic. Density gradient ultracentrifugation resulted in flotation of the complexes to a density of 1.16 g/ml, and negative stain electron microscopy revealed a population of disk-shaped particles. Native gradient polyacrylamide gel electrophoresis indicated a particle diameter of ∼8.5 nm. Absorbance spectroscopy provided evidence for AMB integration into the lipid milieu. AMB-rHDLs were potent inhibitors of Saccharomyces cerevisiae growth, yielding 90% growth inhibition at <1 μg/ml yeast culture. In studies with pathogenic fungal species, similar growth inhibition characteristics were observed. Compared with AMB-deoxycholate micelles, AMB-rHDL displayed greatly attenuated red blood cell hemolytic activity and decreased toxicity toward cultured hepatoma cells. In in vivo studies in immunocompetent mice, AMB-rHDLs were nontoxic at 10 mg/kg, and they showed efficacy in a mouse model of candidiasis at concentrations as low as 0.25 mg/kg. These results indicate that AMB-rHDLs constitute a novel formulation that effectively solubilizes the antibiotic and elicits strong in vitro and in vivo antifungal activity with no observed toxicity at therapeutic doses.

Modifying Apolipoprotein A-I by Malondialdehyde, but Not by an Array of Other Reactive Carbonyls, Blocks Cholesterol Efflux by the ABCA1 Pathway

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.