From the Biochemistry Group, The Heart Research Institute, Sydney
New South Wales 2050 and
Cardiovascular Investigation
Unit, Royal Adelaide Hospital,
Adelaide South Australia 5000, Australia
Human high density lipoproteins (HDL) can reduce
cholesteryl ester hydroperoxides to the corresponding hydroxides
(Sattler W., Christison J. K., and Stocker, R. (1995) Free
Radical Biol. & Med. 18, 421-429). Here we demonstrate that this
reducing activity extended to hydroperoxides of phosphatidylcholine,
was similar in HDL2 and HDL3, was independent
of arylesterase and lecithin:cholesteryl acyltransferase activity, was
unaffected by sulfhydryl reagents, and was expressed by reconstituted
particles containing apoAI or apoAII only, as well as isolated human
apoAI. Concomitant with the reduction of lipid hydroperoxides specific
oxidized forms of apoAI and apoAII formed in blood-derived and
reconstituted HDL. Similarly, specific oxidized forms of apoAI
accumulated upon treatment of isolated apoAI with authentic cholesteryl
linoleate hydroperoxide. These specific oxidized forms of apoAI and
apoAII have been shown previously to contain Met sulfoxide (Met(O)) at Met residues and are also formed when HDL is exposed to
Cu2+ or soybean lipoxygenase. Lipid hydroperoxide reduction
and the associated formation of specific oxidized forms of apoAI and
apoAII were inhibited by solubilizing HDL with SDS or by pretreatment of HDL with chloramine T. The inhibitory effect of chloramine T was
dose-dependent and accompanied by the conversion of
specific Met residues of apoAI and apoAII into Met(O). Canine HDL,
which contains apoAI as the predominant apolipoprotein and which lacks the oxidation-sensitive Met residues Met112 and
Met148, showed much weaker lipid hydroperoxide reducing
activity and lower extents of formation of oxidized forms of apoAI than
human HDL. We conclude that the oxidation of specific Met residues of apoAI and apoAII to Met(O) plays a significant role in the 2-electron reduction of hydroperoxides of cholesteryl esters and
phosphatidylcholine associated with human HDL.
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INTRODUCTION |
Plasma levels of high density lipoprotein (HDL)1
cholesterol and apolipoprotein
(apo)1 AI inversely correlate
with the risk of developing coronary heart disease (1, 2). This is
generally thought to be due to the participation of HDL in removal of
cholesterol from peripheral tissues and its ensuing transport to the
liver for excretion (3). HDL has other potential anti-atherogenic
properties, such as its ability to inhibit the oxidation of low density
lipoproteins (LDL) (4) and the pro-atherogenic activities of oxidized
LDL (5, 6). LDL oxidation appears to be a key event in atherogenesis (see e.g. Ref. 7), and oxidized lipids, particularly those of cholesteryl esters, are present in human atherosclerotic lesions (8).
HDL, but less so LDL, can reduce cholesteryl ester hydroperoxides
(CE-OOH) to the corresponding cholesteryl ester hydroxides (CE-OH) (9).
We have proposed (9, 10) that this reducing activity of HDL is
potentially anti-atherogenic based on the following: (i) by reducing
CE-OOH to CE-OH, HDL converts potentially reactive species to
relatively inert species, less likely to give rise to further radicals
and hence secondary radical reactions (9); (ii) HDL also reduces CE-OOH
transferred from oxidized LDL via cholesteryl ester transfer protein
(11) and thus can potentially detoxify pro-atherogenic oxidized LDL;
(iii) the reduction of HDL CE-OOH occurs in and is selectively
accelerated by rat liver perfusate (10); (iv) HDL CE-O(O)H are rapidly
and selectively removed by Hep G2 cells in culture (12) and in
situ perfused rat liver (10); and (v) in rats, HDL CE-OH are
rapidly cleared from the circulation by the liver and secreted into
bile (13), indicating a physiologically plausible (3) detoxification
and exit route for oxidized lipids associated with HDL.
The underlying mechanism(s) for HDL's CE-OOH reducing activity has not
been elucidated. Here we report evidence for lipid hydroperoxides
(LOOH) being reduced by specific Met residues of apoAI and apoAII in
HDL.
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EXPERIMENTAL PROCEDURES |
Materials--
Chloramine T, phenylacetate,
5,5'-dithiobis-2-nitrobenzoic acid (DTNB), and
p-chloromercuriphenylsulfonic acid (PCMPS) were purchased
from Sigma. All other chemicals, solvents, and materials were obtained
from the sources indicated in the accompanying paper (14).
Isolation of HDL and ApoAI--
Human and canine HDL and human
HDL2 and HDL3 were isolated rapidly from
freshly obtained EDTA plasma by the 4-h two-step density gradient
ultracentrifugation using a TL100.4 rotor (Beckman Instruments, Palo
Alto, CA) described previously (15). This method produces two major HDL
bands that have been shown previously to correspond to the
HDL2 and HDL3 subfractions (15). In experiments
where total human HDL was used both subfractions were collected and pooled. This rapid isolation method results in minimal oxidative damage
to HDL during isolation (14, 15). After isolation, HDL was
gel-filtered, supplemented with 1 mM EDTA, and kept on ice
under argon overnight before use (14). Where HDL
paraoxonase/arylesterase (EC 3.1.8.1) activity was studied, HDL was
isolated from heparinized human plasma into 50 mM Tris/HCl,
pH 7.4, and not exposed to either phosphate buffer or EDTA unless
specifically indicated. HDL protein concentrations were estimated and
apoAI isolated as described (14).
Preparation of Spheroidal Reconstituted HDL--
Spheroidal
reconstituted HDL containing apoAI only (3 molecules of apoAI/particle,
rHDLAI) or apoAII only (6 molecules of apoAII
dimer/particle, rHDLAII) were generated by incubating
discoidal rHDL prepared by cholate dialysis, with purified
lecithin:cholesterol acyltransferase (LCAT) and LDL (16). As judged by
nondenaturing polyacrylamide gradient gel electrophoresis, the
rHDLAI and rHDLAII each comprised a single,
homogeneous population of particles. The molar ratio of
phospholipids/cholesteryl esters/unesterified cholesterol/protein of
the particles were 32.4:18.9:5.0:1 for rHDLAI and
32.6:19.1:5.1:2 for rHDLAII. All compositional analyses were performed on a Cobas Fara autoanalyzer (Roche Diagnostics, Switzerland). The stoichiometry varied by <5% for all components in
two independent preparations of each rHDL. The majority of the
cholesteryl esters was cholesteryl palmitate and cholesteryl oleate;
however, cholesteryl linoleate was also present at
0.65 mol/mol
unesterified cholesterol, presumably derived from LDL during the
conversion of the discoidal rHDL to spheroidal rHDLAI (16).
HDL Reducing Activity--
Isolated HDL (1.5-2.0 mg of
protein/ml) was oxidized in PBS containing 1 mM EDTA by
aerobic incubation for 2 h at 37 °C in the presence of AAPH
(4-7.5 mM), a generator of aqueous peroxyl radicals. After
oxidation, AAPH was removed by gel filtration and HDL supplemented with
1 mM EDTA, unless specified otherwise.
To selectively oxidize Met residues (17), HDL (~1.5 mg of protein/ml)
was incubated for 1 h at 22 °C in PBS, 1 mM EDTA
and in the presence of chloramine T (50-500 µM).
Unreacted chloramine T was removed subsequently by gel filtration and
HDL was then either analyzed immediately or oxidized with AAPH as
above, the radical generator removed and CE-OOH reducing activity
assessed by incubation at 37 °C in the presence of 1 mM
EDTA under argon and in the dark. In other experiments HDL was
pretreated with H2O2 (1- 200 mM) or
HOCl (50 µM) instead of chloramine T.
HDL (~1.5 mg of protein/ml) was also incubated for 4 h at
22 °C in PBS (1 mM EDTA) with 2 mM DTNB or 2 mM PCMPS to derivatize thiols associated with the
lipoprotein. Since apoAI does not contain Cys and the sole Cys residue
of apoAII forms a disulfide giving rise to the apoAII homodimer, the
major targets for these reagents were most likely to be the
quantitatively minor HDL apolipoproteins and HDL-associated proteins,
notably LCAT.
Loss of endogenous fluorescence of Trp residues was determined in
SDS-solubilized HDL or different apoAI forms after HPLC separation
(14). Analysis of HDL's unoxidized lipids, CE-OOH, CE-OH, and
phosphatidylcholine hydroperoxides (PC-OOH), was by HPLC as described
(14). For determination of PC-OOH plus phosphatidylcholine hydroxides
(PC-O(O)H), HPLC with UV234 nm instead of
chemiluminescence detection was used. Unoxidized apoAI and apoAII and
their oxidized forms were determined by HPLC as described (14).
Measurement of HDL Arylesterase Activity--
HDL (20-40 µg
of protein), isolated in the absence of EDTA, was added to 950 µl of
25 mM Tris/HCl, 1 mM CaCl2, 1 mM phenylacetate, and the increase in absorbance at 270 nm
was recorded for at least 60 s (6, 18). Arylesterase activity was
calculated using the molar extinction coefficient of 1,310 M
1 cm
1 and defined as 1 µmol
of phenylacetate hydrolyzed per min, with the specific activity given
as units/mg HDL protein. Arylesterase activity was inhibited by
pretreating HDL with 3 mM EDTA for 90 min at 37 °C under
argon (6). EDTA was then removed by gel filtration, the HDL oxidized,
and CE-OOH and PC-OOH reduction assessed as described above but in 25 mM Tris/HCl, pH 8.0, containing 1 mM
CaCl2. Reducing activity and arylesterase activity were
assessed in parallel with HDL or "chelated HDL" in the presence of
1 mM CaCl2 or 1 mM EDTA,
respectively. Esterase activity was determined before and after
AAPH-induced oxidation of HDL and at the end of the time course
experiments where CE-OOH and PC-OOH reduction was also assessed.
Fast Protein Liquid Chromatography--
HDL (1.2 mg of protein
in 200 µl), in the absence and presence of 1% SDS, was subjected to
size exclusion chromatography using a fast protein liquid
chromatography system (Pharmacia Biotech Inc.) fitted with a
Superose-12 (Pharmacia) column (30 × 1.5 cm inner diameter)
eluted with 20 mM sodium phosphate buffer (pH 7.8; 4 °C)
at 0.25 ml/min and 279 nm.
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RESULTS |
Reduction of HDL CE-OOH and PC-OOH--
By using radiolabeled
CE-OOH, we have demonstrated previously that HDL (much more effectively
than LDL) reduces this class of LOOH in a stoichiometric fashion to
CE-OH (9). We sought here to confirm this and in addition to determine
whether PC-OOH was also reduced by HDL incubated at 37 °C. For this,
we first mildly oxidized HDL using AAPH and assessed reduction of
CE-OOH and PC-OOH subsequent to removal of AAPH (see "Experimental
Procedures"). In agreement with previous results (9), the proportion
of total oxidized cholesteryl esters present as CE-OH increased as time progressed (Fig. 1A), and
there was a near stoichiometric conversion of CE-OOH to CE-OH (Fig.
1A, inset). Fig. 1B shows, for the first time,
that the proportion of total oxidized phosphatidylcholine present as
phosphatidylcholine hydroxides (PC-OH) also increased as time
progressed. By contrast, the levels of PC-OOH decreased rapidly and
those of PC-OOH plus PC-OH (i.e. PC-O(O)H) remained unaltered (Fig. 1B, inset), indicating that PC-OOH was
reduced to PC-OH.

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Fig. 1.
Reduction of LOOH by HDL in the presence and
absence of SDS. HDL was pre-oxidized with 7.5 mM AAPH
for 2 h at 37 °C, AAPH removed, and the subsequent reduction of
CE-OOH and PC-OOH determined as described under "Experimental
Procedures." A, increase in the proportion of CE-OH of
CE-OOH plus CE-OH (CE-O(O)H) in the absence ( ) and presence ( ) of
1% (w/v) SDS. The inset shows the loss of CE-OOH
(circles) and gain of CE-OH (squares) in the absence (closed symbols) and presence (open
symbols) of SDS. B, increase in PC-OH as a proportion
of the PC-OOH plus PC-OH (PC-O(O)H) in the absence (closed
circles) and presence (open circles) of 1% SDS. The
inset shows the loss of PC-O(O)H in the absence of SDS ( )
and that of PC-OOH in the absence ( ) and presence of SDS ( ). Loss
of PC-O(O)H was not affected by SDS. Data are means ± S.E. or
range where appropriate of n = 6, 2, 3, and 1 experiments for CE-OH/CE-O(O)H SDS, CE-OH/CE-O(O)H
+SDS, PC-OH/PC-O(O)H SDS, and PC-OH/PC-O(O)H
+SDS, respectively.
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The addition of 1% (w/v) SDS to HDL inhibited both CE-OOH and PC-OOH
reduction (Fig. 1). The presence of SDS disrupted the structural
integrity of HDL. Thus, in the absence of SDS, a single major peak,
corresponding to native HDL particles, eluted from the gel filtration
column, whereas in the presence of 1% SDS, a series of broad and
ill-defined peaks were observed (Fig. 2). This suggests that an intact HDL particle was required in order for
both CE-OOH and PC-OOH reduction to occur efficiently. Studies performed using HDL2 and HDL3 subfractions
showed that the CE-OOH reducing activity of both subfractions was
similar to that of HDL (Fig. 3).

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Fig. 2.
Disruption of HDL structure by 1% SDS.
HDL was prepared in PBS (A) or PBS containing 1% (w/v) SDS
(B). After gentle mixing, the samples were subjected to fast
protein liquid chromatography as described under "Experimental
Procedures."
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Fig. 3.
HDL CE-OOH reducing activity is similar in
HDL2 and HDL3. HDL2 and
HDL3 were pre-oxidized with 4 mM AAPH for
2 h at 37 °C, the AAPH removed, and the subsequent reduction of
CE-OOH determined as described under "Experimental Procedures." The
increase in the proportion of CE-OH of CE-OOH plus CE-OH (CE-O(O)H) in HDL2 ( ) and HDL3 ( ) is shown. Data are
means ± S.E. of three experiments each using HDL from different
donors.
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To understand the mechanism(s) by which HDL reduces CE-OOH and PC-OOH,
we first investigated a possible contribution of HDL-associated paraoxonase/arylesterase (EC 3.1.8.1). This enzyme hydrolyzes aryl
esters of carboxylic acids and organophosphates (18) and oxidized (but
not native) fatty acyl chains of phospholipids (6), requires
Ca2+ for its activity, and is inhibited effectively by EDTA
(19). We therefore treated isolated HDL with EDTA (referred to as
chelated HDL) and determined its impact on CE-OOH and PC-OOH reducing
and arylesterase activities. Chelated HDL was almost totally devoid of
arylesterase activity (Fig.
4A), yet maintained its CE-OOH or PC-OOH reducing activity (Fig. 4, B and C).
Arylesterase activity in freshly isolated HDL was 5.2 ± 1.7 units/mg (mean ± S.D., n = 3) and decreased to
45 ± 12% of that (mean ± SD, n = 3) after AAPH-induced oxidation and a further 19 h incubation at 37 °C, demonstrating that although the absolute activity was lower,
arylesterase remained active throughout the entire time course
studied.

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Fig. 4.
Dissociation of HDL LOOH reducing activity
and arylesterase activity. HDL was isolated in the absence of EDTA
or PBS and assessed for arylesterase activity (A), CE-OOH
(B), and PC-OOH reducing activity (C).
Arylesterase activity was determined using phenyl acetate as a
substrate without ( EDTA) and with prior treatment of a
portion of the HDL with 3 mM EDTA (+EDTA) as
described under "Experimental Procedures." For B and
C, HDL samples were prepared as for A and then
pre-oxidized with 7.5 mM AAPH for 2 h at 37 °C and
the CE-OOH (B) and PC-OOH (C) reducing activity
determined as described in the legend to Fig. 1. Data are means of a
single experiment representative of three (A) and are
means ± S.E. of three separate experiments ((B) and
(C)).
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In separate experiments, HDL was incubated with 2 mM DTNB
to inhibit the activity of HDL-associated LCAT. DTNB oxidizes Cys residues which are thought to be critical for enzyme activity (20), and
others have shown that 0.2-1.5 mM DTNB inhibits LCAT activity (see e.g. Ref. 21). However, treatment of HDL with 2 mM DTNB had no discernible effect on subsequent CE-OOH
reducing activity (Fig. 5). Similarly,
PCMPS, another LCAT inhibitor, also failed to affect CE-OOH reducing
activity (Fig. 5). Thus, neither arylesterase nor LCAT activities play
a role in the CE-OOH or PC-OOH reducing activity of HDL.

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Fig. 5.
Treatment of HDL with LCAT activity
inhibitors does not inhibit CE-OOH reducing activity. HDL ( 1.5
mg of protein/ml) was incubated for 4 h at 22 °C in PBS
containing 1 mM EDTA and, where indicated, 2 mM
PCMPS or 2 mM DTNB. The thiol-reactive reagents were then
removed and HDL pre-oxidized with 7.5 mM AAPH for 2 h
at 37 °C, AAPH removed, and CE-OOH reducing activity assessed as
described under "Experimental Procedures." Data represent control HDL not treated with LCAT inhibitor ( ) or HDL treated with DTNB ( ) or PCMPS ( ).
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To prove unambiguously that HDL's apolipoproteins rather than
associated enzymes/proteins support the observed reducing activity, we
tested CE-OOH reduction by rHDLAI and rHDLAII.
The purity of the protein composition of these rHDL was confirmed by
SDS-polyacrylamide gel electrophoresis with silver staining (data not
shown) and by reversed phase HPLC. rHDLAI contained only
apoAI, whereas rHDLAII contained apoAII with traces (<5%)
of apoAI, and both particles clearly reduced CE-OOH to CE-OH, with
rHDLAII being somewhat more efficient than
rHDLAI (Fig. 6). These
results imply that apoAI and apoAII are responsible for the LOOH
reducing activity associated with native human HDL. Since apoAI does
not contain Cys and apoAII does not contain Trp, these results also
demonstrate that Cys and Trp are not essential for CE-OOH reduction.
Cys is thought to be present almost exclusively as a disulfide in
apoAII, giving rise to the homodimeric structure of human apoAII
(22).

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Fig. 6.
Reduction of CE-OOH by rHDL containing either
apoAI or apoAII. rHDL containing only apoAI (rHDLAI)
or apoAII (rHDLAII) were prepared, pre-oxidized with
7.5-10 mM AAPH for 2 h at 37 °C, and subsequently
assessed for CE-OOH reducing activity, as described under
"Experimental Procedures." The initial CE-OOH concentrations for
both rHDL were ~5 µM. Data are means ± range of
two experiments using separate preparations of rHDL.
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Oxidation of Met Residues of ApoAI and ApoAII Is Associated with
the Reduction of LOOH by HDL--
Previous reports have documented the
sensitivity of certain Met residues of apoAI and apoAII to oxidation
(14, 17, 23). Based on this evidence, we speculated that Met oxidation
in apoAI and apoAII might be responsible for the reduction of LOOH. To investigate this possibility, pre-oxidized HDL was incubated at 37 °C under argon (i.e. conditions that support CE-OOH
and PC-OOH reducing activity) and the formation of oxidized forms of
both apoAI and apoAII followed by HPLC (see "Experimental
Procedures"). A representative chromatogram of apolipoproteins in
native HDL is given in Fig.
7A. As shown previously (14,
17, 23), apoAI and apoAII were the major apolipoproteins in HDL, with
apoCs as minor components.

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Fig. 7.
HPLC chromatograms of HDL apolipoproteins
during LOOH reduction. HDL was pre-oxidized with 7.5 mM AAPH for 2 h at 37 °C, AAPH removed, and HDL
incubated subsequently at 37 °C before analysis of apolipoproteins
as described under "Experimental Procedures." ApoAI+16
and apoAI+32 refer to oxidized forms 16 and 32 mass units
greater, respectively, than apoAI. ApoAIIa contains 1 molecule of
Met(O) per apoAII dimer (25). Typical chromatograms are shown for
native HDL before (A) and directly after pre-oxidation and
AAPH removal (B, t = 0 h) and after
subsequent incubation in the absence of AAPH for 3 (C) and
7 h, respectively (D). Note, only half the amount of
HDL was injected in A versus B-D to allow easier
assessment of consumption of apoAI/AII and formation of oxidized
apoAI/AII.
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The accompanying paper (14) describes the time-dependent
decrease in the content of apoAI and apoAII and concomitant formation apoAI+16, apoAI+32, and apoAIIa during
AAPH-induced oxidation of HDL. ApoAIIa contains a single Met(O) in
place of one of the two Met26 residues of apoAII dimer
(23), whereas apoAI+16 and apoAI+32 contain one
and two Met(O) residues per apoAI monomer, respectively (14, 17). The
chromatogram shown in Fig. 7B is derived from pre-oxidized
HDL and is representative of changes occurring to apoAI and apoAII when
the intact lipoprotein is exposed AAPH for restricted periods (14). The
observed specific formation of apoAI+16,
apoAI+32, and apoAIIa during exposure of HDL to AAPH is not
due to direct oxidation of the apolipoproteins by AAPH-derived radicals
(14). Incubation of such pre-oxidized HDL subsequent to the removal of
all AAPH resulted in a further time-dependent decrease in
unoxidized apoAI and apoAII and a concomitant increase in the formation
of apoAI+16, apoAI+32, and apoAIIa (Fig. 7,
B-D). Inclusion of 1% (w/v) SDS significantly inhibited the formation of oxidized forms of apoAI and apoAII (Fig.
8), to an extent comparable with the
inhibition of LOOH reduction (cf. Fig. 1).

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Fig. 8.
Dissociation of HDL with SDS inhibits the
formation of oxidized forms of apoAI and apoAII. HDL was
pre-oxidized with 7.5 mM AAPH for 2 h at 37 °C,
AAPH removed, and the subsequent formation of oxidized forms of apoAI
and apoAII (see Fig. 7) determined as described under "Experimental
Procedures." The increase in the proportion of either oxidized apoAI
(A) or oxidized apoAII (B) of native plus
oxidized apolipoprotein content is shown in the absence ( ) and
presence ( ) of 1% (w/v) SDS. Data are mean ± range of
duplicates from two experiments.
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We next confirmed that formation of the oxidized forms of apoAI and
apoAII occurred concomitantly with HDL LOOH reduction in both
rHDLAI and rHDLAII. As shown in Fig.
9, the rate of apoAII oxidation was
greater than that of apoAI in the corresponding rHDL. This result is in
agreement with the faster rate of CE-OOH reduction observed with
rHDLAII versus rHDLAI (Fig. 6).
Similar to the situation with intact HDL, reduction of LOOH by
rHDLAI and rHDLAII resulted in a
time-dependent decrease in unoxidized apoAI and apoAII and
a concomitant increase in (apoAI+16 and
apoAI+32) and apoAIIa, respectively (not shown). Together, these results demonstrate that the reduction of LOOH in HDL is related
to the formation of oxidized forms of apoAI and apoAII, characterized
by the conversion of Met residues to Met(O).

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Fig. 9.
Oxidation of apoAI and apoAII in
rHDLAI and rHDLAII accompanying LOOH
reduction. rHDLAI and rHDLAII were
pre-oxidized with 7.5-10 mM AAPH for 2 h at 37 °C
and subsequently assessed for formation of oxidized forms of apoAI and
apoAII under conditions associated with LOOH reducing activity as
described in the legend to Fig. 3. Results, expressed as described in
the legend to Fig. 8, are representative of two separate experiments
using different preparations of rHDL.
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Inhibition of CE-OOH Reduction by Pre-oxidation of Met Residues of
ApoAI and ApoAII--
In the absence of sulfhydryl groups, chloramine
T selectively oxidizes Met residues (24), including Met112
and Met148 in apoAI of intact HDL (17). To investigate the
role of apoAI and apoAII Met residues in CE-OOH reduction, we
pretreated HDL with chloramine T before pre-oxidation with AAPH and
subsequent assessment of CE-OOH reducing activity. As expected,
treatment of HDL with increasing concentrations of chloramine T caused
an increased proportion of the Met(O)-containing oxidized forms of apoAI and apoAII (Fig. 10A),
reminiscent of the situation with AAPH-oxidized HDL incubated at
37 °C after removal of the radical generator. There was no
discernible decrease in the Trp fluorescence in the
chloramine-T-oxidized samples, as indicated by unaltered ratios of
UV214 nm absorbance to Trp fluorescence in the unoxidized
and oxidized forms of apoAI after HPLC separation (see "Experimental
Procedures"). In addition, Trp fluorescence in intact HDL directly
after treatment with 50, 100, and 500 µM chloramine T was
92 ± 9, 94 ± 4, and 90 ± 5% (mean ± S.D.,
n = 4) of the nontreated, native HDL, respectively.
This indicated that Trp residues in apoAI were not appreciably oxidized
upon treatment of HDL with chloramine T. Furthermore, such treatment
did not result in severe lipid damage. Thus, even at the highest
chloramine-T concentration used, the levels of cholesteryl linoleate
and
-tocopherol were
95 and
75% of control values.
Importantly, there was a dose-dependent decrease in both
the levels of CE-OH formed during the 2-h pre-oxidation period and the
CE-OOH reducing activity when HDL was treated with increasing
concentrations of chloramine T (Fig. 10B). This effect was
already evident at the zero time point as illustrated by the lower
degree of CE-OH detected at the higher chloramine-T concentrations used
(Fig. 10B). Pretreatment of HDL with the Met-oxidizing
species H2O2 (1-200 mM) or HOCl (50 µM) similarly inhibited HDL CE-OOH reducing activity
(data not shown). Together, these data show that prior selective
oxidation of Met residues of HDL's apoAI and apoAII results in a
dose-dependent inhibition of subsequent LOOH reducing
activity.

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Fig. 10.
Dose-dependent inhibition of HDL
CE-OOH reducing activity and oxidation of apoAI and apoAII by
pretreatment with chloramine T. HDL was incubated with increasing
concentrations of chloramine T for 60 min at 22 °C and the formation
of oxidized forms of apoAI and apoAII (A) and CE-OOH
reducing activity (B) assessed after removal of unreacted
chloramine T and a subsequent 2-h oxidation with 7.5 mM
AAPH as described under "Experimental Procedures." Oxidation of
apoAI (solid bars) and apoAII (open bars) is
expressed as described in the legend to Fig. 8. Data in A
are means ± range from two experiments (100 and 500 µM chloramine T) and a single experiment (50 µM chloramine T). The data in B are from a
single experiment representative of two experiments obtained with
different HDL preparations.
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CE-OOH Reducing Activity of Canine HDL--
If Met residues of
apoAI and apoAII were important for the CE-OOH reducing activity
associated with HDL, the prediction would be that isoforms of apoAI and
apoAII lacking Met112 and Met148 would exhibit
decreased CE-OOH reducing activity. ApoAI is the predominant
apolipoprotein in canine HDL (Ref. 25, verified by SDS-polyacrylamide
gel electrophoresis, data not shown) and shares
85% sequence
homology with human apoAI (29), and Met112 and
Met148 are substituted by Val and Leu, respectively (29).
Oxidation of canine HDL (2.0 mg of protein/ml) with AAPH (7.5 mM for 2 h at 37 °C) resulted in similar initial
levels of CE-OOH in the reaction mixtures compared with human HDL
(10.2 ± 1.3 µM, mean ± range,
n = 2 versus 13.1 ± 2.1, mean ± S.D., n = 6, for canine versus human HDL,
respectively). In contrast, CE-OOH reducing activity was substantially
decreased, although not absent (Fig. 11A). The low degree of
CE-OOH reduction observed in canine HDL was comparable with that seen
in human HDL in the presence of 1% (w/v) SDS (cf. Fig. 1);
it was accompanied by a depletion of native canine apoAI and the
formation of a less hydrophobic form of apoAI (data not shown).
Although we have not characterized this new (oxidized) form, canine
apoAI was clearly less reactive than apoAI of human HDL (Fig.
11B).

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Fig. 11.
Reduction of CE-OOH by canine HDL.
Canine and human HDL were pre-oxidized with 7.5 mM AAPH for
2 h at 37 °C, AAPH removed, and CE-OOH reduction (A)
and apolipoprotein oxidation (B) assessed as described under
"Experimental Procedures." B, increase in less hydrophobic forms of apoAI (canine HDL) or ratio of oxidized to total
apoAI (human HDL). Data are means of six and two separate experiments
for human and canine HDL, respectively.
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CE-OOH Reducing Activity of Isolated ApoAI--
To test whether
formation of Met(O) in HDL is due to reaction of Met112 and
Met148 with CE-OOH directly, thus yielding CE-OH, we
incubated isolated apoAI with CE-OOH. This resulted in a
time-dependent decrease in the concentration of CE-OOH
while CE-OH accumulated (Fig.
12A). A linear approximation
showed that the rate of CE-OH formation was ~64% of the rate of
CE-OOH depletion. The absence of a strict 1:1 stoichiometry may be
explained by the formation of products other than hydroxides. Exposure
of isolated apoAI to CE-OOH also resulted in an increase in
apoAI+32 (Fig. 12B) and apoAI+16, the latter as a shoulder prior to the unoxidized apoAI peak (not shown). Under these conditions, the rate of apoAI+32
formation was not correlated quantitatively to the rates of CE-OOH
depletion and CE-OH accumulation. Irrespective of the issue of
quantitation, however, these results demonstrate unequivocally that
Met112 and Met148 in apoAI can reduce CE-OOH to
CE-OH. It was found necessary to have CE-OOH present in excess to apoAI
to obtain an appreciable rate of product formation. This was in
contrast to the experiments with native HDL where the molar ratio of
apoAI/CE-OOH was ~3:1 (e.g. for Figs. 3 and 7). It is
conceivable that the native structure of apoAI in HDL facilitates a
direct interaction with the peroxide moiety of CE-OOH due to immediate
apposition of the two reactive groups, consistent with the much lower
rate of CE-OOH reduction in SDS-treated versus intact HDL
(see Fig. 1). In any case, CE-OOH were not appreciably reduced to CE-OH
during the time course studied when apoAI was replaced with the same
mass of bovine serum albumin or when the peroxide was incubated in the
absence of apoAI (not shown).

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Fig. 12.
Time course of the reaction of isolated
human apoAI with CE-OOH at 37 °C. The initial concentrations of
apoAI and CE-OOH were 25 and 63 µM, respectively.
A, loss of CE-OOH ( ) and concomitant accumulation of
CE-OH ( ). The reaction rates (linear approximations; solid
lines) of CE-OOH depletion and CE-OH formation, corrected with
respect to control samples (no apoAI), were 20 and 12 nM/min, respectively. The time 0 concentrations were
"normalized" to 0 µM to correct for slight
differences in the concentrations of CE-O(O)H between the experimental
and control samples. The experiment was performed in duplicate, and the
mean coefficients of variation of the CE-OOH and CE-OH data were 18 and
34%, respectively. B, formation of apoAI+32;
peak area normalized to zero absorbance units at t = 0 min. The error bars (S.D.) associated with the apoAI+32 fell within the size of the symbols. Qualitatively similar results were obtained in three separate experiments.
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DISCUSSION |
The present study strongly supports a role for Met residues of
apoAI and apoAII as the principal mechanism by which HDL reduces LOOH
to lipid hydroxides. Under all circumstances examined, the reduction of
LOOH by HDL was accompanied by the formation of specifically oxidized
apoAI and apoAII that contained Met(O) as the selective oxidation
product. Also, selective oxidation of HDL's Met residues with
nucleophilic oxidants such as chloramine T inhibited subsequent LOOH
reducing activity in a dose-dependent manner. Furthermore, canine HDL, the apoAI of which lacks the oxidation-sensitive
Met112 and Met148 (26), showed much less
reducing activity. Finally, apoAI in solution was capable of reducing
CE-OOH to CE-OH with concomitant fomation of Met(O). Together, these
results provide evidence for Met112 and Met148
of apoAI and Met26 of apoAII oxidizing to Met(O) as LOOH
are reduced to the corresponding lipid hydroxides in HDL
(Scheme I).

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Scheme I.
Proposed mechanism for the reduction of
HDL-associated LOOH by human apoAI and apoAII. HDL's LOOH are
reduced to the corresponding lipid hydroxides (LOH) via
direct 2-electron transfer from the sulfide of thioethers of the
oxidation-prone Met112 and Met148 residues of
human apoAI and Met26 of apoAII, resulting in formation of
their respective Met(O).
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The possibility that HDL-associated proteins other than apolipoproteins
are responsible for the observed reduction of LOOH was addressed using
several different approaches. Arylesterase activity was inhibited by
EDTA treatment, yet this had no inhibitory effect on LOOH reduction
(Fig. 4). Also, the lack of effect of treatment of HDL with DTNB or
PCMPS (Fig. 5) indicated that neither LCAT (27) nor accessible thiols
are required for the LOOH reducing activity. A potentially relevant
activity associated with HDL not investigated here is the trypanosome
lytic factor, which has been reported to exhibit a peroxidase-like
activity in the presence of H2O2 (28). However,
this activity is located almost exclusively on the very dense
subfractions of human HDL and is largely absent from HDL2
(29), whereas HDL2 and HDL3 showed equal CE-OOH
reducing activity (Fig. 3). Therefore, a role for trypanosome lytic
factor in LOOH reduction by HDL is also not likely. Finally, our
observation that rHDL containing either apoAI or apoAII alone fully
supports LOOH reduction demonstrates unambiguously that both of the
major apolipoproteins of human HDL are able to reduce LOOH, independent of any other protein activity.
The chemical reactivity of sulfides toward peroxides is well
established (30). It is also known that H2O2,
chloramine T, and t-butyl hydroperoxide can selectively
oxidize Met residues in proteins to yield Met(O) (17, 24, 31, 32).
However, the reactivity of more complex LOOH with protein Met residues, as shown here with CE-OOH, is less well studied. In studies on rabbit
reticulocyte lipoxygenase, Rapoport et al. (33) reported the
selective oxidation of a single Met residue to Met(O) by
13(S)-hydroperoxylinoleic acid. Gan et al. (34)
recently confirmed this for reticulocyte and human 15-lipoxygenase and
demonstrated that the single (out of six) Met residue to become
oxidized is located in the substrate binding pocket, where it interacts
with the enzymic product. These studies suggest that for more complex
LOOH to cause oxidation of hydrophobic Met residues within proteins,
specific interaction is required. This is consistent with our findings
that dissociation of HDL by SDS strongly inhibited LOOH-induced Met
oxidation, that the CE-OOH reducing activity of HDL is much greater
than that of LDL (9), that canine HDL has low reducing activity, and that only Met112 and Met148 but not
Met86 residue(s) in apoAI become oxidized during LOOH
reduction (14, 17, 23).
The finding that canine HDL, which lacks the oxidation-sensitive
Met112 and Met148 of human apoAI, exhibited
only slight CE-OOH reducing activity (Fig. 11) is consistent with our
hypothesis that these Met residues are of significant importance. The
remaining low reducing activity of canine HDL may be due to an
interaction of LOOH with other redox-active amino acids, possibly
including Met86, although previous studies have shown that
it is resistant to autoxidation (17) and AAPH-induced oxidation of
human HDL.2
The fact that Met(O) formed in proteins can be reduced by peptide
Met(O) reductase (EC 1.8.4.6) (24, 35, 39) raises the possibility that
apoAI and apoAII could act as redox-cycling "active sites" in a
pseudo-enzymatic way, providing a peroxidase-like activity. The implied
reduction of HDL's LOOH to lipid hydroxides by the proposed 2-electron
reaction (Scheme I) would represent an antioxidant defense, as it
prevents the participation of LOOH in secondary radical or other
potentially damaging reactions. Peptide Met(O) reductase, present in
virtually all mammalian tissues (37), is assumed to be an intracellular
enzyme, consistent with its requirement for thioredoxin and thioredoxin
reductase (38), and the fact that it reduces Met(O) in extracellular
-1-proteinase inhibitor in vitro (38) but not in blood
(39). Future studies will show whether Met(O) reductase is capable of
reducing Met(O) formed in apoAI and apoAII and thereby regenerate
HDL's LOOH reducing activity.
On the other hand, if extracellular oxidation of Met to Met(O) in HDL
is not a reversible process in biological systems, its formation can be
considered as oxidative damage, and apoAI/AII containing Met(O) would
be expected to accumulate at sites of oxidative stress. Consistent with
this, lipoprotein fractions of HDL, density-isolated from advanced
human atherosclerotic plaques by ultracentrifugation, contain
detectable amounts of oxidized isoforms of both apoAI and
apoAII.2
The question whether the LOOH reducing activity associated with Met
residues of HDL represents an antioxidant defense or oxidative damage
has potential ramifications for a number of processes relevant to
atherosclerosis. As discussed above, limitation of the progression of
oxidative modification of LDL by reductive removal of LOOH is one
important example. In addition, in vitro oxidation of HDL by
copper can result in a loss of the lipoprotein's ability to stimulate
the efflux of cholesterol from foam cells (40-42), the first step in
reverse cholesterol transport. It is possible that the specific protein
oxidation resulting from the reduction of LOOH is also sufficient to
cause changes in HDL's ability to promote cholesterol efflux from
cells. Of possible significance, Met112 and
Met148 (of apoAI) and Met26 (apoAII) are
located within the known lipid binding domains of the respective
apolipoproteins (43). In addition, the presence of Met(O) in oxidized
peptide analogs of apoAI and apoAII alters their secondary structure
and affects their ability to interact with lipids (23) which, at least
for apoAI, is crucial for both cholesterol removal and activation of
LCAT. In light of these considerations and our present findings, it
seems possible that selective oxidation of Met residues, as can be
expected whenever lipid peroxidation occurs, may affect the biological
activities of HDL.
In summary, the present studies provide evidence that the ability of
HDL to reduce and thereby detoxify potentially pro-atherogenic LOOH is
due, to a significant degree, to a reaction with specific Met residues
on apoAI and apoAII.
We thank Dr. Mike Davies for helpful
discussions and comments, Drs. Roger Dean and Wendy Jessup for critical
reading of the manuscript, and Julie Brauman for technical assistance.