© 1997 American Heart Association, Inc.
Articles |
Copper Ions Promote Peroxidation of Low Density Lipoprotein Lipid by Binding to Histidine Residues of Apolipoprotein B100, But They Are Reduced at Other Sites on LDL
From the Departments of Internal Medicine (P.W., J.W.H.) and Molecular Biology and Pharmacology (J.W.H.), Washington University School of Medicine, St Louis, Mo.
Correspondence to Dr Jay W. Heinecke, Division of Atherosclerosis, Nutrition and Lipid Research, Box 8046, 660 South Euclid Ave, St. Louis, MO 63110. E-mail heinecke{at}im.wustl.edu
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Abstract |
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Abstract Oxidized LDL is implicated in the pathogenesis of atherosclerosis. A widely studied model for oxidation of the lipid in LDL involves Cu2+. Recent studies suggest that Cu2+ may be reduced to Cu1+ by
-tocopherol to initiate LDL lipid peroxidation. LDL
demonstrates binding sites for Cu2+, but the nature of
these binding sites, as well their role in promoting Cu2+
reduction and lipid peroxidation, has not been established. In the
current studies, we used diethylpyrocarbonate (DEPC) to modify the
histidine residues of apolipoprotein B100, the major protein in LDL.
First, we demonstrated that histidine residues were preferentially
modified by DEPC under our experimental conditions. Then we monitored
the kinetics of Cu2+-promoted oxidation of LDL and
DEPC-modified LDL. In both cases, the progress curve of lipid
peroxidation exhibited a lag phase and a propagation phase. However,
when LDL was modified with DEPC, the length of the lag phase was
prolonged whereas the rate of lipid peroxidation during the propagation
phase was lower. Studies with LDL oxidized by 2,2'-azobis
(2-amidinopropane) hydrochloride and phosphatidylcholine liposomes
oxidized with hydroxyl radical established that DEPC was not acting
simply as a nonspecific inhibitor of lipid peroxidation.
DEPC treatment of LDL almost completely inhibited its ability to bind
Cu2+. These observations suggest that peroxidation of the
lipids in LDL can proceed with normal kinetics only when
Cu2+ binds preferentially to sites on apolipoprotein B100
that contain histidine residues. We also compared the kinetics of
Cu2+ reduction in the absence and presence of DEPC. There
was no effect of DEPC modification on either the rate or extent of
Cu2+ reduction by LDL. Therefore LDL is likely to contain a
second class of binding sites for Cu2+ that does not
involve histidine residues. Thus, LDL appears to contain at least two
classes of Cu2+-binding sites: histidine containing sites,
which are responsible in part for promoting lipid peroxidation during
the propagation phase, and sites at which Cu2+ is reduced
without binding to histidine.
Key Words: LDL oxidation • atherosclerosis • lipid peroxidation • metal binding • vitamin E
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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An elevated blood level of LDL is a major risk factor for atherosclerosis.1 However, many lines of evidence suggest that LDL must be oxidized before it can exert its atherogenic effects (reviewed in 2 3 4 5 ). Much evidence indicates that lipoprotein oxidation occurs in vivo2 3 4 5 6 7 8 and is of central importance in the pathogenesis of the disease.2 3 4 5 9 10 Effects of oxidized LDL in vitro include stimulation of monocyte adhesion to endothelial cells,11 promotion of vascular thrombosis,12 and cytotoxicity to cells of the artery wall.13 Moreover, macrophage scavenger receptors recognize LDL modified by decomposition products of peroxidized lipid,14 15 16 suggesting a possible mechanism for the uptake of oxidized LDL. Because LDL is the major carrier of blood cholesterol, unregulated entry of oxidized LDL into macrophages could transform the phagocytes into cholesterol-laden foam cells whose accumulation in the artery wall marks the initial stage of atherosclerosis. This potential link between LDL oxidation and foam cell formation suggests that knowledge of the relevant mechanisms may be critical to understanding the development of atherosclerotic lesions.2 3 4 5
Early studies demonstrated that increasing concentrations of iron or copper promoted the modification of LDL that was incubated in vitro with smooth muscle cells.17 Moreover, LDL that had been modified in the presence of either metal ion stimulated human macrophages to accumulate cholesteryl ester.17 Metal chelators inhibited LDL oxidation by cultured cells of the arterial wall,17 18 19 suggesting that iron or copper represents one pathway for LDL oxidation. Iron or copper also modified LDL in the absence of cells if present at sufficiently high concentrations.17 19 Subsequent studies have shown that Cu2+ binds to LDL20 21 22 and that LDL can reduce Cu2+ to Cu1+.23 24
Although the physiological relevance of metal ions
has not yet been established, in vitro modification of LDL by metal
ions is now a major model for LDL oxidation. The component reactions
are poorly understood, but one possibility is that reduced metal ions
decompose preexisting lipid hydroperoxides (LOOH) into
reactive alkoxyl radicals (RO·). The
hydroperoxide-derived radicals then are scavenged by antioxidants or
attack polyunsaturated fatty acids (LH) to form
carbon-centered radicals (L·) that initiate
the radical chain reaction of lipid peroxidation (scheme
1).4 25 26 The reaction cycle continues until antioxidants
or radical-radical cross-linking reactions terminate lipid
peroxidation:
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The observation that metal ions stimulate lipid peroxidation in
the absence of detectable hydroperoxides27 28 suggests
that this is not an important pathway early in LDL oxidation.
Alternatively, metal ions may be reduced by exogenous29 30
or endogenous reductants,31 32 33 , which become
converted to free radicals that can peroxidize LDL lipid. In this
scheme, one potential reductant is -tocopherol, which is
converted to -tocopherol radical. This radical then
attacks a polyunsaturated fatty acid to initiate lipid
peroxidation:
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Studies with a widely used model, in vitro oxidation of LDL by
free copper ions, provide support for this
idea.28 32 34 35 Reduction of bound Cu2+ to
Cu1+ by endogenous vitamin E, for example,
appears to be a key step in the initiation of LDL lipid
peroxidation.23 24 Cu2+ converts
-tocopherol to tocopherol radical in
detergent suspensions.34 Moreover, the initial
concentration of the vitamin determines the rate at which phospholipids
added to the detergent undergo lipid peroxidation.34
Tocopherol-mediated lipid peroxidation of LDL also takes
place in a wide variety of oxidation systems.28 31 32 33 34 35
These observations have led to the proposal that
-tocopherol is converted to tocopherol
radical during a reaction that reduces Cu2+. The
tocopherol radical then initiates lipid peroxidation
(scheme 2).
Copper forms complexes with LDL,20 21 22 23 24 and most of this binding apparently involves apolipoprotein B100,20 the major protein in LDL. The binding is poorly understood, but histidine residues may be involved because of their high affinity for divalent metals.36 37 To investigate the possible role of histidine residues in the binding of Cu2+ to LDL and the resulting peroxidation of LDL lipid, we used DEPC to modify this amino acid. We show that modifying the histidine residues of LDL with DEPC inhibits the binding of Cu2+ and retards the oxidation of LDL by copper. However, histidine modification does not prevent the reduction of Cu2+ to Cu1+. These results suggest that preferential binding of Cu2+ to histidine residues of apolipoprotein B100 plays a role in lipid peroxidation but that these sites are distinct from sites on LDL that promote Cu2+ reduction.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Unless otherwise indicated, all chemicals were purchased from Sigma Chemical.
Lipoproteins
LDL (d=1.019 to 1.063 g/mL) was prepared by
discontinuous density gradient ultracentrifugation from
human plasma (4 mmol/L EDTA) treated with 10 µmol/L
phenylmethylsulfonylfluoride.30 All solutions
contained 1 mmol/L EDTA and 0.1 mmol/L diethylenetriamine pentaacetic
acid. The isolated LDL was dialyzed against 150 mmol/L NaCl, 1 mmol/L
EDTA (pH 7.4) at 4°C under N2, stored at 4°C under
N2 in the dark, and used for experiments within 2 weeks
of preparation.
Size exclusion chromatography was used to reisolate LDL and to remove low-molecular-weight components of reaction mixtures. LDL and DEPC-LDL were passed over a 1.5 cm2x10 cm column of Bio-Gel P-6 DG (Econo-Pac 10 DG column; Bio-Rad Laboratories) equilibrated with the indicated buffer at room temperature and immediately used for experiments. Protein concentrations of LDL solutions subjected to size exclusion chromatography were determined by absorbance at 280 nm.
Modification of LDL With DEPC
DEPC was stored at 4°C under argon. Stock solutions of DEPC
were prepared freshly in ice-cold anhydrous ethanol. The concentration
of DEPC was determined by adding one aliquot (2 to 5 µL) of stock
solution to 3 mL of 10 mmol/L imidazole in 100 mmol/L phosphate buffer
(pH 6.8) and incubating the solution for 2 minutes at 25°C. DEPC
concentration was calculated from the increase in absorbance at 230 nm
(
=3000 mol/L-1cm-1).38
Hydroxylamine solutions were prepared by dissolving solid reagent in 50
mmol/L sodium phosphate buffer and titrating the solution to pH 6.5
with 1 mol/L KOH.
LDL was incubated at room temperature in 50 mmol/L phosphate buffer (pH
6.8) containing the indicated concentration of DEPC. Previous studies
have demonstrated that under these conditions, the modification
reaction is specific for histidine residues.39 The
progress curve of the reaction of DEPC with histidine was monitored as
the change in absorbance at 245 nm (=3200
M-1cm-1).40 The reaction was
terminated by subjecting the DEPC-modified LDL (DEPC-LDL) to size
exclusion chromatography on a column equilibrated with
buffer A (140 mmol/L NaCl, 10 mmol/L sodium phosphate, pH 7.4) as
described above. The reisolated DEPC-LDL was immediately transferred to
ice and rendered oxygen free by nitrogen sparging. Elapsed time between
lipoprotein reisolation and oxidation experiments never exceeded 30
minutes.
LDL Oxidation
LDL and DEPC-LDL (70 µg of protein/mL) were incubated with
either 5 µmol/L CuSO4 or 1 mM AAPH in buffer A (140
mmol/L NaCl, 10 mmol/L sodium phosphate, pH 7.4) at 25°C. AAPH
thermally decomposes to generate aqueous phase peroxyl radicals that
promote LDL lipid peroxidation by a nonmetal ion-dependent mechanism.
At the indicated times, 100 µmol/L DTPA and 20 µmol/L BHT were
added to inhibit further lipid peroxidation, and the reaction mixtures
were immediately placed on ice until analysis at the end of the
experiment.
Measurement of LDL Oxidation
Conjugated diene formation was measured as the change in
absorbance at 234 nm (=29 500
M-1cm-1).41 Results are
expressed as nmol of lipid oxidation products per mg of LDL
proteins. Reactive aldehydes derived from lipid peroxidation were
monitored as TBARS30 . Samples (0.5 mL; 35 µg of LDL
protein) were mixed with 1 mL of TBARS stock solution, vortexed, and
heated in boiling water for 15 minutes. After cooling to room
temperature, the samples were vortexed and centrifuged for 5
minutes x10 000g, and the absorbance of the supernatant
was determined at 532 nm. TBARS are expressed as malondialdehyde
equivalent content (nmol per mg of LDL protein) using an extinction
coefficient determined from malondialdehyde prepared by acid hydrolysis
of malondialdehyde tetramethyl acetal (Eastman Kodak). Lipid peroxides
were measured as the oxidation of iodide to triiodide using CHOD color
reagent (Merck, Darmstadt, Germany) as described by El-Saadani et
al.41 LDL solution (0.5 mL; 35 µg of protein) was mixed
with 1 mL of color reagent, the sample was incubated for 30 minutes in
the dark at room temperature, and the absorbance of the solution at 365
nm (=17 300 M-1cm-1)41 was
then measured. Results are expressed as nmol of lipid hydroperoxides
per mg of LDL protein. The lag phase and rate of lipid peroxidation
during the propagation phase were determined graphically from the
progress curve of lipid peroxidation as described.4 41
Determination of Copper Binding by Membrane Filtration
LDL and DEPC-LDL (250 µg of protein/mL) were incubated for 10
minutes at 25°C with the indicated concentration of Cu2+
in buffer A. Free Cu2+ was removed from the LDL using
membrane filtration by concentrating the sample to <10% of its
original volume in a centrifugal concentrator (10 000
Mr cutoff; Centricon- 10; Amicon, Inc), diluting
the retentate solution to its original volume with buffer A and
repeating the procedure. The concentrations of protein and
Cu2+ in aliquots of the retentate were then measured.
Control experiments demonstrated that this procedure removed >99% of
free Cu2+ in solution. To minimize LDL oxidation, buffer A
was supplemented with 20 µmol/L BHT, and all procedures after the
initial incubation of LDL with Cu2+ were carried out at
4°C. Cu2+ bound to LDL was quantified using bathocuproine
disulfonate,23 which specifically binds to the reduced
(Cu1+) but not to the oxidized (Cu2+) form of
copper; this complex has an absorbance maximum at 480
nm.23 42 43 Ascorbic acid was used to reduce
Cu2+.23 Ascorbic acid (1 mmol/L) and
bathocuproine disulfonate (400 µmol/L) were added to the LDL
solution. After a 30-minute incubation at 37°C, the increase in
absorbance at 480 nm was measured and the concentration of copper was
determined by comparison with a standard curve of CuSO4
prepared in buffer A and subjected to the same procedure.
Determination of Copper Binding by Equilibrium Dialysis
LDL (2 mg of protein/mL) was incubated for 15 minutes at 25°C
in buffer A alone or in the same buffer supplemented with 2 mmol/L DEPC
and reisolated by size exclusion chromatography as
described above. The reisolated LDLs (1 mg of protein/mL) were dialyzed
at 4°C for 4 hours against 20 mmol/L HEPES, 150 mmol/L NaCl buffer
(pH 7.4) supplemented with the indicated final concentration of
CuSO4 using a Spectra/Por membrane (apparent MW cutoff
12 000 to 14 000 daltons; Spectrum Medical Industries). BHT (100
µmol/L) was included to inhibit LDL lipid peroxidation. The copper
concentrations of the dialysis solution, LDL solution, and DEPC-LDL
solution were then determined using ascorbic acid and bathocuproine
disulfonate as described above.
Reduction of Cu2+ by LDL
LDL and DEPC-LDL (20 µg of protein/mL) were incubated with 10
µmol/L Cu2+ in the presence of 400 µmol/L bathocuproine
disulfonate in buffer A at 25°C. The change in absorbance at 480 nm
of the Cu1+-bathocuproine disulfonate complex was
monitored.23
Liposomes
A solution of soybean phosphatidylcholine (1 mg/mL in
anhydrous ethanol; 72% unsaturated fatty acyl side chains) was
evaporated to dryness under N2. Liposomes were prepared by
adding 0.8 mL of buffer A and incubating the solution for 1 hour at
37°C, followed by 10 seconds of sonication in a bath
sonicator.
Other Procedures
Protein was determined using the method of Lowry et
al44 with bovine serum albumin as the standard.
All solutions were prepared with double-distilled, deionized water, and
all buffers were treated with Chelex-100 resin (BioRad) to remove
contaminating metal ions. All results are
representative of those observed in at least three
independent experiments.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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DEPC Modification of Histidine Residues
DEPC is known to react preferentially with histidine residues in proteins over the pH range of 5.5-7.5,38 39 although it also can bind to lysine and tyrosine under basic conditions.45 46 We therefore first demonstrated that under our experimental conditions, DEPC reacts predominantly with histidine residues of apolipoprotein B100 and not with its tyrosine or lysine residues.
The product of the reaction between histidine and DEPC is
N-carbethoxyhistidine, which absorbs maximally at 245
nm.40 The difference spectrum between LDL and LDL exposed
to DEPC at pH 6.8 showed a large peak at this wavelength (Fig 1A
), strongly suggesting that DEPC
modifies histidine residues of apolipoprotein B100. Using the
extinction coefficient of
N-carbethoxyhistidine40 and the magnitude of the
245-nm peak in the difference spectrum (Fig 1A), we estimated that
70% of the 115 histidine residues in apolipoprotein B100 were
available to react with DEPC.
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Because the absorption peak of N-carbethoxylysine also is 245 nm, we
distinguished between binding to histidine and to lysine by adding
hydroxylamine to the reaction mixture 12 minutes after the addition of
DEPC. Hydroxylamine reverses the binding of histidine to DEPC, and it
lowered the increase in absorbance at 245 nm of DEPC modified LDL by
>80% (Fig 2A). This reversal would not
have occurred if DEPC had been attached to lysine
residues.40
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final concentration 1 mol/L) or an equal volume of buffer alone
(
) was added to the reaction mixture. Changes in absorbance at
245 nm are corrected for changes in volume of the reaction mixture. (B)
In a parallel incubation, LDL, LDL treated with DEPC (DEPC-LDL), and
LDL treated with DEPC and then hydroxylamine (DEPC/HA-LDL) were
reisolated by size exclusion chromatography. The LDLs
(100 µg of protein/mL) were incubated at 25°C in buffer A
supplemented with 5 µmol/L CuSO4, and the rate of LDL
oxidation was monitored as the increase in absorbance at 234 nm.
The product of the reaction between tyrosine and DEPC is
O-carbethoxytyrosine, which has a much lower absorbance at 278 nm than
tyrosine itself.45 There was no evidence for a decrease in
absorbance at this wavelength when LDL was treated with DEPC (Fig 1A
),
suggesting that DEPC was not modifying a significant fraction of
tyrosine residues. Collectively, these observations suggest that DEPC
is reacting predominantly with histidine residues, but that lysine
residues of apolipoprotein B100 may also be targets for
modification.
Treatment of LDL with DEPC for varying times affected the progress
curve of Cu2+-promoted LDL oxidation as measured by the
appearance of conjugated dienes, a marker of peroxidized lipid. The
reaction typically produces an S-shaped curve that, for operational
purposes, can be divided into a lag phase, a faster propagation phase,
and a termination phase.4 26 41 This description of the
kinetics of lipid oxidation is not meant to imply a specific mechanism
of lipid peroxidation.25 26 31 32 33 When LDL was exposed to
DEPC for 4 minutes, the propagation phase was delayed; a 12-minute
exposure delayed it even more (Fig 1B). However, the total yield of
oxidized LDL after prolonged exposure to Cu2+ remained
constant despite the extent of histidine modification. Therefore the
chemical modification of apolipoprotein B100 histidine residues affects
the rate of LDL oxidation but not the final yield of LDL lipid
peroxidation products as monitored by diene conjugation.
Adding hydroxylamine to LDL that had been exposed to DEPC largely
reversed the effect of DEPC on the lag phase (Fig 2B). Hydroxylamine
similarly reversed the increase in absorbance at 245 nm of DEPC-LDL
(Fig 2A), suggesting that modification of histidine residues was
responsible in part for the effects of DEPC on the kinetics of
Cu2+-catalyzed LDL oxidation. However, the progress curve
of lipid peroxidation was not completely normalized by hydroxylamine,
suggesting that lysine residues might also be involved in promoting LDL
oxidation. Collectively, these data suggest that both the length of the
lag phase and the rate of lipid peroxidation during the propagation
phase are related in part to the number of histidine residues of
apolipoprotein B100 available to react with DEPC.
Lag Phase Length and Lipid Peroxidation Rate
To further explore this idea, we determined the relationship
between the length of the lag phase of Cu2+-promoted LDL
oxidation and the number of histidine residues modified by DEPC (as
estimated by the change in absorbance at 245 nm of DEPC-modified LDL).
A positive relationship between the two was observed (Fig 3), suggesting that the number of
unmodified histidine residues available to Cu2+ determines
in part the lag phase of LDL oxidation. There was an inverse
relationship between the propagation rate and the extent of histidine
modification (Fig 3), suggesting that histidine residues also play a
role in catalyzing LDL oxidation during the propagation phase. The
relationship between the length of the lag phase and the rate of the
propagation phase was curvilinear (Fig 4). This latter observation suggests that
different Cu2+-binding/histidine-containing sites
may participate in the lag phase and propagation phase of LDL
oxidation.
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To further characterize the effect of DEPC on lipid oxidation, we
determined the length of the lag phase and the rate of the propagation
phase of LDL and DEPC-LDL exposed to different Cu2+
concentrations (Fig 5). DEPC treatment of
LDL prolonged the length of the lag phase (Fig 5A) over the range of
Cu2+ concentrations tested. Moreover, it almost completely
blocked the increase in the rate of oxidation during the propagation
phase induced by increasing concentration of Cu2+ (Fig 5B).
These results strongly support the suggestion that histidine-containing
sites make an important contribution to the propagation phase of LDL
oxidation.
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Antioxidant Action of DEPC
To ensure that DEPC affects the time course of LDL oxidation by
blocking the binding of Cu2+ to histidine residues rather
than by preventing oxidation in a nonspecific manner, we compared the
effects of DEPC on the oxidation of LDL by Cu2+ and by
AAPH. The latter generates peroxyl radicals in the aqueous phase that
initiate the peroxidation of LDL lipid without the involvement of metal
ions. Using three different assays for oxidized LDL—the appearance of
conjugated dienes at 234 nm, the TBARS assay, and hydroperoxide
measurement—we found that DEPC attenuates Cu2+-promoted
LDL oxidation (Fig 6, left panel) without
affecting the oxidation of LDL by AAPH (Fig 6, right panel).
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The oxidizing intermediates formed during Cu2+ promoted LDL
lipid peroxidation might be more reactive than peroxyl radical. To
establish that DEPC was not inhibiting LDL oxidation by scavenging such
species, we determined whether DEPC affected the rate of lipid
peroxidation of phosphatidylcholine liposomes exposed to hydroxyl
radical, generated using a
Cu2+-H2O2 system. DEPC had no
effect on the kinetics of lipid peroxidation in this system as
monitored by TBARS and the formation of lipid hydroperoxides (Fig 7). These findings indicate that DEPC is
not acting as a nonspecific lipid-soluble antioxidant. Instead, they
suggest that distinct Cu2+-binding sites provided by
histidine residues that are accessible to modification by DEPC play a
critical role in LDL lipid peroxidation.
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Copper Binding by DEPC-LDL
To determine whether DEPC blocks the interaction of
Cu2+ with LDL, we measured the number of
Cu2+-binding sites on LDL and on DEPC-LDL exposed at 25°C
to different concentrations of Cu2+. To minimize the
possibility that oxidation of LDL by Cu2+ was altering the
results, the lipid-soluble antioxidant BHT was included in the reaction
mixture, and free low-molecular-weight components were rapidly
separated at 4°C from the LDL at the end of the incubation by
membrane filtration. Under these conditions, LDL exposed to DEPC (Fig 8) exhibited a dramatic decrease in
Cu2+ binding.
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We used equilibrium dialysis to test further the role of DEPC-sensitive
histidine residues in Cu2+ binding by LDL. LDL and DEPC-LDL
were dialyzed versus either 5, 10, or 20 µmol/L CuCl2 at
4°C for 4 hours, and the total concentration of Cu2+ in
the dialysate fluid and the LDL solutions was determined (Fig 9). The presence of LDL in the buffer
increased the concentration of Cu2+ by 45-80%, indicating
that the metal ion was binding to the lipoprotein. The increase in
concentration of Cu2+ was almost completely inhibited by
DEPC treatment of LDL. This finding, together with the results of
membrane filtration, suggests that the histidine residues on
apolipoprotein B100 are the major sites where Cu2+ binds to
LDL.
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Several lines of evidence indicate that LDL oxidation is promoted by the reduction of metal ions.23 24 29 30 To determine whether the histidine sites we identified above participate in Cu2+ reduction as well as in Cu2+ binding, we monitored the rate of conversion of Cu2+ to Cu1+ by LDL, using bathocuproine disulfonate as an indicator. This molecule selectively chelates Cu1+, forming a complex that absorbs light strongly at 480 nm.
The progress curve for the reduction of Cu2+ by LDL was
virtually identical to that for the reduction of Cu2+ by
LDL-DEPC (Fig 10A). Both curves rapidly
reached a plateau, presumably as endogenous reductants in
LDL became depleted. The absorption spectrum of each reaction mixture
after a 900-minute incubation was virtually identical to that for
Cu2+ reduced by ascorbic acid (Fig 10B), confirming that
the chromophore was a complex of bathocuproine disulfonate with
Cu1+. These results indicate that Cu2+ is
reduced by LDL but that the histidine-binding sites on apolipoprotein
B100 sensitive to modification by DEPC are not involved in this
reduction.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We used DEPC to investigate the identity of sites on LDL that bind Cu2+ and to explore the role of these sites in Cu2+-promoted lipid peroxidation of LDL. We showed that modification of apolipoprotein B100 with DEPC inhibited Cu2+ binding and slowed lipid peroxidation during the propagation phase of LDL oxidation. These results suggest that these metal binding sites contain an amino acid residue that reacts with DEPC.
Several lines of evidence suggest that histidine is the amino acid residue that defines in part the Cu2+-binding sites on apolipoprotein B100. First, many previous studies have shown that histidine residues react selectively with DEPC under the conditions used in our experiments.38 39 40 42 45 Second, the difference spectrum between LDL and LDL modified with DEPC exhibited a maximum at 245 nm, the absorption peak of N-carbethoxylated histidine or N-carbethoxylated lysine.40 N-carboxethylation of histidine is reversible, whereas the covalent modification of lysine is not.40 46 The reaction of DEPC with apolipoprotein B100 was reversed by >80% by hydroxylamine, suggesting that histidine was the major target for modification. Third, the interaction between LDL and DEPC did not trigger a decrease in absorbance at 270-280 nm, which would indicate binding to tyrosine.45 Finally, we ascertained that DEPC was not acting as a nonspecific inhibitor of lipid peroxidation by demonstrating that LDL oxidation by Cu2+, but not AAPH, was inhibited by the chemical-modifying reagent.
Modification by DEPC resulted in almost complete inhibition of
Cu2+ binding by LDL as assessed by membrane filtration and
equilibrium dialysis. Under these conditions, 70% of the histidines
of apolipoprotein B100 were N-carbethoxylated as monitored by the
change in absorbance at 245 nm. Collectively, these results suggest
that histidine residues that are susceptible to chemical modification
by DEPC account for most of the Cu2+-binding sites on LDL.
However, because hydroxylamine did not completely reverse the binding
of DEPC to LDL, it is possible that lysine residues are also involved
in binding Cu2+. Modification of amino acid residues by
derivatization may alter the secondary and tertiary structure of
proteins. Thus, changes in the conformation of apolipoprotein B100
might also play a role in altering Cu2+ binding by LDL.
The structural nature of the DEPC-sensitive Cu2+ binding
sites on LDL remains to be defined. High-affinity binding of metal ions
requires a minimum of two ligands. Bidentate sites can be formed by
His-X3-His and His-X3-Cys on an -helix and
by His-X-His on a ß-pleated sheet.36 37 47
Binding constants for Cu2+ binding as high as
106 M-1 have been measured for
His-X3-His sites in proteins.47 A wide range
of other geometries and coordination numbers mediate metal binding in
proteins36 37 47 ; such sites may contain a variety of
ligands, including imidazole groups, amino groups, negatively charged
side chains of other amino acids, and carbonyl oxygens of the peptide
bond. Whether amino acid side chains such as the amino group of
lysine and the sulfur group of cysteine23 48 play a role
in binding Cu2+ by LDL has not yet been clearly
determined.
Modification of the histidine residues on LDL with DEPC also altered the progress curve of LDL lipid peroxidation by Cu2+. There was a small but consistent increase in the initial rate, and the lag phase lengthened. Moreover, in DEPC-LDL the propagation rate decreased, and increasing the concentration of Cu2+ in the reaction mixture had little effect on the rate of lipid peroxidation during the propagation phase. The increase in the initial rate of oxidation may occur because DEPC modification of histidine residues results in displacement of Cu2+ from relatively high-affinity sites on apolipoprotein B100 to sites of lower affinity that promote lipid peroxidation during the lag phase. The ability of DEPC to prolong the lag phase and decrease the propagation rate suggests that the binding of Cu2+ to histidine sites on apolipoprotein B100 plays an important role in promoting reactions involved in the propagation phase of LDL oxidation. Lysine residues may also be involved because hydroxylamine only partially reversed the inhibitory effects of DEPC on the propagation phase of Cu2+-catalyzed LDL oxidation.
The existence of a second class of Cu2+-binding sites was revealed by the finding that Cu2+ reduction was linked to LDL oxidation23 24 but did not appear to involve histidine residues of apolipoprotein B100 sensitive to DEPC modification. Because the latter sites bind Cu2+ with relatively high affinity under our experimental conditions, the Cu2+-reducing sites most likely are low-affinity sites. Cu2+-promoted LDL oxidation therefore appears to involve at least two types of Cu2+-binding sites: a histidine-containing site, which binds copper with relatively high affinity but does not reduce the metal ion, and a histidine-free site, which binds Cu2+ with relatively low affinity and reduces it to Cu1+. The latter may lie on apolipoprotein B100 or on the lipid moiety of LDL. The cellular oxidation of LDL similarly may involve reductants because the production of superoxide and thiols has been implicated in LDL oxidation by smooth muscle cells, phagocytes, and endothelial cells.5 29 30
It is not known how the binding of Cu2+ stimulates LDL oxidation, but our data are compatible with the following possibilities. One class of binding sites on LDL interact with Cu2+ to promote the formation of tocopherol radical, which in turn may act to initiate lipid peroxidation (scheme 2).23 24 26 31 32 33 34 35 As lipid hydroperoxides accumulate in LDL, a second class of Cu2+-binding sites may become involved in LDL oxidation as indicated by the observation that DEPC decreases the propagation rate of lipid peroxidation. These DEPC-sensitive sites preferentially involve histidine residues and may stimulate LDL oxidation by favoring the conversion of lipid hydroperoxides to alkoxyl radicals in a reaction involving Cu1+ (scheme 1). Alternatively, histidine residues may bind Cu2+, and the divalent metal ion may then interact with Cu1+, promoting lipid peroxidation during the propagation phase. Previous studies with iron have shown that both reduced and oxidized forms are required for the metal ion-dependent oxidation of microsomes.49 50
In summary, our results indicate that at least two different classes of Cu2+-binding sites may participate in the oxidation of LDL lipids by Cu2+. One class involves histidine residues of apolipoprotein B100. These sites appear to play an important role in the propagation phase of LDL oxidation. A second class promotes the reduction of Cu2+ and may be located either on apolipoprotein B100 or on LDL lipid. These sites may participate in the initial stages of LDL lipid peroxidation. In future studies, it will be important to identify the nature of these binding sites, which may reside on the phospholipid of LDL. These observations indicate that distinct copper binding sites may play a role in the promotion of lipid peroxidation during different phases of LDL oxidation as well as in the reduction of Cu2+.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Drs L. Sage and S. Hazen for critical reading of the manuscript. This work was supported by grant R01- AG12293 from the National Institutes of Health. J.W.H. is an Established Investigator of the American Heart Association.
Received December 13, 1996; accepted September 12, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986;232:34-47.
2. Steinbrecher UP, Zhang HF, Lougheed M. Role of oxidatively modified LDL in atherosclerosis. Free Rad Biol Med.. 1990;9:155-168.[Medline] [Order article via Infotrieve]
3. Witztum JL, Seinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest.. 1991;88:1785-1792.
4. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med.. 1991;13:341-390.
5. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Rad Biol Med.. 1996;20:707-727.[Medline] [Order article via Infotrieve]
6.
Haberland ME, Fong D, Cheng C.
Malondialdehyde-altered protein occurs in atheroma
of Watanabe heritable hyperlipidemic rabbits.
Science. 1988;241:215-218.
7.
Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S,
Witztum JL. Distribution of oxidation specific lipid-protein
adducts and apolipoprotein-B in atherosclerotic lesions of varying
severity from WHHL rabbits.
Arteriosclerosis. 1990;10:336-349.
8. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parathasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest.. 1989;84:1086-1095.
9.
Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima
A, Yoshida H, Kawai C. Probucol prevents the progression of
atherosclerosis in Watanabe heritable
hyperlipidemic rabbit, an animal model for familial
hypercholesterolemia. Proc Natl
Acad Sci U S A. 1987;84:5928-5931.
10.
Carew TE, Schwenke DC, Steinberg D.
Antiatherogenic effects of probucol unrelated to its
hypercholesterolemic effect: evidence that antioxidants
in vivo can effectively inhibit low density lipoprotein degradation in
macrophage-rich fatty streaks and slow the progression of
atherosclerosis in the Watanabe heritable
hyperlipidemic rabbit. Proc Natl Acad Sci
U S A. 1987;84:7725-7729.
11. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest.. 1990;85:1260-1266.
12.
Fei H, Berliner JA, Parhan F, Drake TA.
Regulation of endothelial cell tissue factor
expression by minimally oxidized LDL and
lipopolysaccharide. Arterioscler Thromb.. 1993;13:1711-1717.
13.
Chisolm GM, Ma GP, Irwin KC, Martn LL, Gunderson KG,
Linberg LF, Morel DW, Dicorleto PE. 7
Beta-hydroperoxycholest-5-en-3 beta-ol, a component of human
atherosclerotic lesions, is the primary cytotoxin of oxidized human low
density lipoprotein. Proc Natl Acad Sci U S A. 1994;91:11452-11456.
14.
Henriken T, Mahoney EM, Steinberg D. Enhanced
macrophage degradation of biologically modified low density
lipoprotein. Arteriosclerosis. 1983;3:149-159.
15.
Zhang H, Yang Y, Steinbrecher VP. Structural
requirements for binding of modified proteins to the scavenger receptor
of macrophages. J Biol Chem.. 1993;268:5535-5542.
16.
Steinbrecher UP, Lougheed M, Kwan WC, Dirks M.
Recognition of oxidized low density lipoprotein by the scavenger
receptor of macrophages results from derivatization of
apolipoprotein B by products of fatty acid peroxidation.
J Biol Chem.. 1989;264:15216-15223.
17. Heinecke JW, Rosen H, Chait A. Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J Clin Invest.. 1984;74:1890-1894.
18.
Morel DW, DiCorleto PE, Chisolm GM.
Endothelial and smooth muscle cells alter low
density lipoprotein in vitro by free radical oxidation.
Arteriosclerosis. 1984;4:357-364.
19.
Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL,
Steinberg D. Modification of low density lipoprotein by
endothelial cells involves lipid peroxidation and
degradation of low density lipoprotein phospholipids.
Proc Natl Acad Sci U S A. 1984;81:3883-3887.
20. Kuzuya M, Yamada K, Hayashi T, Funaki C, Naito M, Asai K, Kuzuya F. Role of lipoprotein-copper complex in copper catalyzed-peroxidation of low-density lipoprotein. Biochem Biophys Acta.. 1992;1123:334-341.[Medline] [Order article via Infotrieve]
21. Gieseg SP, Esterbauer H. Low density lipoprotein is saturable by pro-oxidant copper. FEBS Lett.. 1994;343:188-194.[Medline] [Order article via Infotrieve]
22. Ziouzenkova O, Gieseg SP, Ramos P, Esterbauer H. Factors affecting resistance of low density lipoproteins to oxidation. Lipids. 1996;31:S71–S76.
23.
Lynch SM, Frei B. Reduction of copper, but not
iron, by human low density lipoprotein (LDL)—implications for metal
ion-dependent oxidative modification of LDL. J Biol
Chem.. 1995;270:5158-5163.
24.
Kontush A, Meyer S, finckh B, Kohlschutter A, Beisiegel
U. Alpha-tocopherol as a reductant for Cu(II) in
human lipoproteins—triggering role in the initiation of lipoprotein
oxidation. J Biol Chem.. 1996;271:11106-11112.
25. Porter NA. Chemistry of lipid peroxidation. Methods Enzymol.. 1984;105:273-277.[Medline] [Order article via Infotrieve]
26. Abuja PM, Esterbauer H. Simulation of lipid peroxidation in low density lipoprotein by a basic skeleton of reactions. Chem Res Toxicol.. 1995;8:753-763.[Medline] [Order article via Infotrieve]
27. Frei B, Gaziano JM. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation. J Lipid Res.. 1993;34:2135-2145.[Abstract]
28. Lynch SM, Frei B. Mechanisms of copper-dependent and iron-dependent oxidative modification of human low density lipoprotein. J Lipid Res.. 1993;34:1745-1753.[Abstract]
29. Heinecke JW, Baker L, Rosen H, Chait A. Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells. J Clin Invest.. 1986;77:757-761.
30.
Heinecke JW, Rosen H, Suzuki LA, Chait A. The
role of sulfur-containing amino acids in superoxide production
and modification of low density lipoprotein by arterial
smooth muscle cells. J Biol Chem.. 1987;262:10098-10103.
31. Bowry VW, Stocker R. Tocopherol-mediated peroxidation: the prooxidant effect of vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J Am Chem Soc.. 1993;115:6029-6044.
32. Bowry VW, Mohr D, Cleary J, Stocker R. Prevention of tocopherol-mediated peroxidation in ubiquinol-10-free human low density lipoprotein. J Biol Chem.. 1995;270:56-63.
33.
Ingold KU, Bowry VW, Stocker R, Walling C.
Autoxidation of lipids and antioxidation by
alpha-tocopherol and ubiquinol in homogeneous
solution and in aqueous dispersions of lipids—unrecognized
consequences of lipid particle size as exemplified by oxidation of
human low density lipoprotein. Proc Natl Acad Sci
U S A. 1993;90:45-49.
34. Maiorino M, Zamburlini A, Roveri A, Ursini F. Prooxidant role of vitamin E in copper induced lipid peroxidation. FEBS Lett.. 1993;330:174-176.[Medline] [Order article via Infotrieve]
35. Yoshida Y, Tsuchiya J, Niki E. Interaction of alpha-tocopherol with copper and its effect on lipid peroxidation. Biochim Biophys Acta.. 1994;1200:85-92.[Medline] [Order article via Infotrieve]
36.
Arnold HF, Haymore LB. Engineered metal-binding
proteins: purification to protein folding. Science. 1991;252:1796-1797.
37. Reagan L. Protein design: novel metal binding sites. Trends Biochem Sci.. 1995;20:280-284.[Medline] [Order article via Infotrieve]
38. Melchior WB, Fahrney D. Ethoxyformylation of proteins: reaction of ethoxyformic anhydride with alpha-chymotrypsin, pepsin, and pancreatic ribonuclease at pH 4. Biochemistry. 1970;9:251-258.[Medline] [Order article via Infotrieve]
39. Lundblad RL, Noyes CM. Chemical Reagents for Protein Modification. Vol 1. Boca Raton, FL: CRC Press, Inc, 1900.
40. Miles EW. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol.. 1977;47:431-442.[Medline] [Order article via Infotrieve]
41. El-Saadani M, Estyerbauer M, El-Sayad H, Hoger M, Jurgens AY, Jurgens GA. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res.. 1989;30:627-630.[Abstract]
42.
Joselow M, Dawson CR. The copper of ascorbic
acid oxidase: exchange studies with radioactive copper.
J Biol Chem.. 1951;191:11-20.
43.
Rahman A, Shahabuddin, Hadi SM, Parish JH, Ainley K.
Strand scission in DNA induced by quercetin and Cu(II): role of
Cu(I) and oxygen free radicals. Carcinogenesis. 1989;10:1833-1839.
44.
Lowry OH, Rosenbough NJ, Farr AL, Randall RJ.
Protein Measurement with the Folin phenol reagent.
J Biol Chem.. 1951;193:265-275.
45. Burstein Y, Walsh KA, Neurath H. Evidence of an essential histidine residue in thermolysin. Biochemistry. 1974;13:205-210.[Medline] [Order article via Infotrieve]
46. Muhlrad A, Hegyi G, Toth G. The adenosine triphosphatase activity of the meromyosins. Biochim Biophys Acta. 1967;2:19-26.
47. Arnold FH. Metal-affinity separations: a new dimension in protein processing. BioTech.. 1991;9:151-156.
48. Ferguson E, Singh RJ, Hogg N, Kalyanaraman B. The mechanism of apolipoprotein B100 thiol depletion during oxidative modification of low-density lipoprotein. Arch Biochem Biophys.. 1997;341:287-294.[Medline] [Order article via Infotrieve]
49. Miller DM, Grover TA, Nayini N, Aust SD. Xanthine oxidase- and iron-dependent lipid peroxidation. Arch Biochem Biophys. 1993;301;1-7.
50.
Miotti G, Aust SD. The requirement for iron(III)
in the initiation of lipid peroxidation by iron(II) and hydrogen
peroxide. J Biol Chem.. 1987;262:1098-1104.
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