Oxidation of methionine residues to methionine sulfoxides does not decrease potential antiatherogenic properties of apolipoprotein A-I

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.     

Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein

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 degrees 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.     

Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1, a key cardioprotective function of HDL

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine - two characteristic products of MPO - are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1, a key cardioprotective function of HDL

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine - two characteristic products of MPO - are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Methionine oxidation and aging

It is well established that many amino acid residues of proteins are susceptible to oxidation by various forms of reactive oxygen species (ROS), and that oxidatively modified proteins accumulate during aging, oxidative stress, and in a number of age-related diseases. Methionine residues and cysteine residues of proteins are particularly sensitive to oxidation by ROS. However, unlike oxidation of other amino acid residues, the oxidation of these sulfur amino acids is reversible. Oxidation of methionine residues leads to the formation of both R- and S-stereoisomers of methionine sulfoxide (MetO) and most cells contain stereospecific methionine sulfoxide reductases (Msr's) that catalyze the thioredoxin-dependent reduction of MetO residues back to methionine residues. We summarize here results of studies, by many workers, showing that the MetO content of proteins increases with age in a number of different aging models, including replicative senescence and erythrocyte aging, but not in mouse tissues during aging. The change in levels of MetO may reflect alterations in any one or more of many different mechanisms, including (i) an increase in the rate of ROS generation; (ii) a decrease in the antioxidant capacity; (iii) a decrease in proteolytic activities that preferentially degrade oxidized proteins; or (iv) a decrease in the ability to convert MetO residues back to Met residues, due either to a direct loss of Msr enzyme levels or indirectly to a loss in the availability of the reducing equivalents (thioredoxin, thioredoxin reductase, NADPH generation) involved. The importance of Msr activity is highlighted by the fact that aging is associated with a loss of Msr activities in a number of animal tissues, and mutations in mice leading to a decrease in the Msr levels lead to a decrease in the maximum life span, whereas overexpression of Msr leads to a dramatic increase in the maximum life span.     

Formation of methionine sulfoxide-containing specific forms of oxidized high-density lipoproteins

Atherosclerosis is characterized by the accumulation of both lipoprotein-derived lipids and inflammatory cells in the affected vascular wall that results in a state of heightened oxidative stress and that is reflected by the accumulation of oxidized lipoproteins. Circulating oxidized low-density lipoprotein (oxLDL) is used as a surrogate marker for coronary artery disease, although the 'escape' of oxLDL from the vessel wall is hindered by the large size of this lipoprotein and its specific retention by the extracellular matrix. Also, the oxidation of lipoproteins in human atherosclerotic lesions is not limited to LDL. In fact, the lipids of all classes of lipoproteins are oxidized to a comparable extent. Examining the fate of lipid hydroperoxides, the primary lipid peroxidation products, in high-density lipoproteins (HDL) undergoing oxidation, revealed that they become reduced to the corresponding alcohols by specific Met residues of apolipoprotein A-I (apoA-I) and apoA-II. As a consequence, 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. This review describes the characterization of specifically oxidized HDL with an emphasis on MetO formation, the structural and functional consequences of such oxidation, and the potential utility of specifically oxidized HDL as a surrogate marker of atherosclerosis.     

Acrolein impairs ATP binding cassette transporter A1-dependent cholesterol export from cells through site-specific modification of apolipoprotein A-I

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde, but the factors that control its reactions with nucleophilic groups on proteins remain poorly understood. Lipid peroxidation and threonine oxidation by myeloperoxidase are potential sources of acrolein during inflammation. Because both pathways are implicated in atherogenesis and high density lipoprotein (HDL) is anti-atherogenic, we investigated the possibility that acrolein might target the major protein of HDL, apolipoprotein A-I (apoA-I), for modification. Tandem mass spectrometric analysis demonstrated that lysine 226, located near the center of helix 10 in apoA-I, was the major site modified by acrolein. Importantly, this region plays a critical role in the cellular interactions and ability of apoA-I to transport lipid. Indeed, we found that conversion of Lys-226 to N(epsilon)-(3-methylpyridinium)lysine by acrolein associated quantitatively with decreased cholesterol efflux from cells via the ATP-binding cassette transporter A1 pathway. In the crystal structure of truncated apoA-I, Glu-234 lies adjacent to Lys-226, suggesting that negatively charged residues might direct the modification of specific lysine residues in proteins. Finally, immunohistochemical studies with a monoclonal antibody revealed co-localization of apoA-I with acrolein adducts in human atherosclerotic lesions. Our observations suggest that acrolein might interfere with normal reverse cholesterol transport by HDL by modifying specific sites in apoA-I. Thus, acrolein might contribute to atherogenesis by impairing cholesterol removal from the artery wall.     

Increased methionine sulfoxide content of apoA-I in type 1 diabetes

Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.     

Oxidatively modified high density lipoprotein promotes inflammatory response in human monocytes–macrophages by enhanced production of ROS,TNF-α, MMP-9, and MMP-2

It has been proposed that high-density lipoprotein (HDL) loses its cardioprotective ability through oxidative modifications by reactive oxygen species (ROS) and promote atherogenesis. However, the pro-atherogenic pathways undergone by oxidized HDL remain poorly understood. Since monocytes play a crucial role in atherogenesis, this study was aimed to investigate the influence of both native and oxidized HDL (oxHDL) on monocytes-macrophages functions relevant to atherogenesis. HDL particles were isolated from human blood samples by ultracentrifugation and subjected to in vitro oxidation with CuSO(4). The extent of oxidation was quantitated by measurement of lipid peroxides. Human peripheral blood mononuclear cells were isolated and cultured under standard conditions. Cells were treated with native and oxHDL at varying concentrations for different time intervals and used for several analyses. Intracellular ROS production was assessed based on ROS-mediated DCFH fluorescence of the cells. The release of TNF-α and matrix metalloproteinases (MMPs) was quantitated using ELISA kit and gelatine zymography, respectively. Treatment of cells with oxidized HDL enhanced the production of ROS in a concentration-dependent way, while native HDL had no such effect. Further, the release of TNF-α, MMP-9, and MMP-2 was found to be remarkably higher in cells incubated with oxHDL than that of native HDL. Results demonstrate that oxidative modification of HDL induces pro-inflammatory response and oxidative stress in human monocytes-macrophages.     

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.     

Enhanced macrophage uptake of elastase-modified high-density lipoproteins

Incubation of human HDL (d = 1.063-1.21 g/ml) with monocyte-derived elastase causes selective proteolysis of apoA-II and apoA-I apolipoproteins. We have found that elastase-digested HDL (ED-HDL) bind to J774-A1 murine macrophages with enhanced affinity and are internalized and degraded at a rate threefold higher than that of native HDL. Unlike oxidized LDL and HDL and proteolytically modified LDL, the uptake of ED-HDL lipoproteins does not affect the cellular lipid biosynthesis nor modify the cell lipid content. The cell surface binding of (125)I-ED-HDL can be competed by native HDL but not by acetylated LDL, consistent with the idea that ED-HDL are recognized by the class B type I scavenger receptor. The liberation of elastase by lipid-engorging macrophages is regarded as an important event during atherogenesis. By enhancing the cellular uptake of HDL this process can lead to a local decrease of antiatherogenic HDL particles.     

The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/anti-inflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential.     

Properties of chemically oxidized kininogens

Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.     

Recharging oxidative protein repair: Catalysis by methionine sulfoxide reductases towards their amino acid, protein, and model substrates

The sulfur-containing amino acid methionine (Met) in its free and amino acid residue forms can be readily oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Methionine sulfoxide reductases A (MSRA) and B (MSRB) reduce MetO back to Met in a stereospecific manner, acting on the S and R forms, respectively. A third MSR type, fRMSR, reduces the R form of free MetO. MSRA and MSRB are spread across the three domains of life, whereas fRMSR is restricted to bacteria and unicellular eukaryotes. These enzymes protect against abiotic and biotic stresses and regulate lifespan. MSRs are thiol oxidoreductases containing catalytic redox-active cysteine or selenocysteine residues, which become oxidized by the substrate, requiring regeneration for the next catalytic cycle. These enzymes can be classified according to the number of redox-active cysteines (selenocysteines) and the strategies to regenerate their active forms by thioredoxin and glutaredoxin systems. For each MSR type, we review catalytic parameters for the reduction of free MetO, low molecular weight MetO-containing compounds, and oxidized proteins. Analysis of these data reinforces the concept that MSRAs reduce various types of MetO-containing substrates with similar efficiency, whereas MSRBs are specialized for the reduction of MetO in proteins.     

Role of individual methionines in the fibrillation of methionine-oxidized alpha-synuclein

The aggregation of normally soluble alpha-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. We have previously established that oxidation of all four methionine residues in alpha-synuclein (to the sulfoxide, MetO) inhibits fibrillation of this protein in vitro and that the MetO protein also inhibits fibrillation of unmodified alpha-synuclein. Here we show that the degree of inhibition of fibrillation by MetO alpha-synuclein is proportional to the number of oxidized methionines. This was accomplished be selectively converting Met residues into Leu, prior to Met oxidation. The results showed that with one oxidized Met the kinetics of fibrillation were comparable to those for the control (nonoxidized), and with increasing numbers of methionine sulfoxides the kinetics of fibrillation became progressively slower. Electron microscope images showed that the fibril morphology was similar for all species examined, although fewer fibrils were observed with the oxidized forms. The presence of zinc was shown to overcome the Met oxidation-induced inhibition. Interestingly, substitution of Met by Leu led to increased propensity for aggregation (soluble oligomers) but slower formation of fibrils.     

Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding

Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K(m) lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

Transthyretin in high density lipoproteins: association with apolipoprotein A-I

Previous studies have revealed the presence of transthyretin (TTR) on lipoproteins. To further address this issue, we fractionated plasma lipoproteins from 9 normal individuals, 10 familial amyloidotic polyneuropathy (FAP) patients, and 19 hyperlipidemic subjects using gel filtration. In the majority of the subjects, as well as in 9 of the 10 FAP patients and 14 of the 19 patients with hyperlipidemia, TTR was detected by ELISA in the high density lipoprotein (HDL) fraction. The presence of TTR in HDL was confirmed by direct sequencing and by immunoblotting; using non-reducing conditions, TTR was found by immunoblotting in a high molecular weight complex, which reacted also for apolipoprotein A-I (apoA-I). The amount of TTR present in HDL (HDL-TTR), as quantified by ELISA corresponded to 1;-2% of total plasma TTR. However, no detectable TTR levels were found in HDL fraction from 6 of the hyperlipidemic subjects. No correlation was found between the lack of TTR in HDL and plasma levels of total, LDL-, or HDL-associated cholesterol as well as levels of apoA-I and total plasma TTR. Ligand binding experiments showed that radiolabeled TTR binds to the HDL fraction of individuals with HDL-TTR but not to the corresponding fractions of individuals devoid of HDL-TTR, suggesting that HDL composition may interfere with TTR binding. The component(s) to which TTR binds in the HDL fraction were investigated. Polyclonal antibody against apoA-I was able to block the interaction of TTR with HDL, suggesting that the interaction of TTR with the HDL particle occurs via apoA-I. This hypothesis was further demonstrated by showing the formation of a complex of TTR with HDL and apoA-I by crosslinking experiments. Furthermore, anti-apoA-I immunoblot under native conditions suggested the existence of differences in HDL particle properties and/or stability between individuals with and without HDL-TTR.     

Comparison of the cytolytic effects in vitro on Trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein A-I from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection.

The African trypanosome, Trypanosoma brucei brucei causes a fatal wasting disease in livestock but does not ordinarily infect humans, apparently because this unicellular parasite is lysed by high density lipoproteins (HDL) in human serum. To assess whether there is a specific active constituent in trypanolytic HDL, we have systematically compared the cytotoxic action on T.b.brucei in vitro of native and delipidated HDL, and of individual apolipoproteins, from nonpermissive hosts (human and baboon) with their counterparts from susceptible hosts (cattle and sheep). When suspensions of trypanosomes were incubated for 2 h at 37 degrees C with human or baboon plasma most cells were lysed, but not with bovine or sheep plasma. Similarly, HDL isolated from human and baboon plasma were trypanolytic (typically about 95% and 60% lysis, respectively, at 1 mg protein/ml), whereas bovine and sheep HDL were benign (less than 8% lysis). Subfractionation of human HDL by serial isopycnic ultracentrifugation and by heparin-Sepharose affinity chromatography established that the denser and smaller particles had greater trypanolytic activity both in vitro and in vivo. When human HDL was delipidated, the trypanocidal activity was associated with the water-soluble protein (apolipoprotein) fraction and not with the lipid constituents. Bovine apolipoproteins were also weakly trypanolytic in free solution (20-40% lysis), but not when complexed with cholesterol-phospholipid liposomes (less than 10% lysis). The major apolipoprotein of human HDL, apolipoprotein (apo) A-I had full trypanolytic activity (89-95% lysis at 1 mg protein/ml) when purified, whether in solution or incorporated into liposomes, but other apolipoproteins isolated from human HDL, including apoA-II, apoC, and apoE, were nontrypanolytic. Purified baboon apoA-I was also trypanolytic, though less potent than human apoA-I, but apoA-I from permissive hosts (cattle and sheep) was inactive when presented in liposomes. Incubation of bovine or sheep HDL with purified human apoA-I, and subsequent separation of the HDL by ultracentrifugation, produced chimeric HDL containing significant amounts of the human apolipoprotein; these particles showed appreciable trypanolytic activity. By contrast, human HDL particles in which about 70% of the apoA-I had been displaced with apoA-II had markedly reduced lytic properties compared to the native HDL (30% versus 80% lysis at 0.6 mg total protein/ml). We tentatively conclude that the trypanolytic activity of native human or baboon plasma resides in the apoA-I content of the HDL particles and that, conversely, bovine and sheep plasma are inactive because the apoA-I polypeptide present in their HDL lacks trypanocidal activity.     

In vivo studies of HDL assembly and metabolism using adenovirus-mediated transfer of ApoA-I mutants in ApoA-I-deficient mice.

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.