Modifying apolipoprotein A-I by malondialdehyde, but not by an array of other reactive carbonyls, blocks cholesterol efflux by the ABCA1 pathway

Article abstract of DOI:10.1074/jbc.M110.118182

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

Full text of DOI:10.1074/jbc.M110.118182

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 24, pp. 18473–18484, June 11, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Modifying Apolipoprotein A-I by Malondialdehyde, but Not
by an Array of Other Reactive Carbonyls, Blocks Cholesterol
Efflux by the ABCA1 Pathway*
Received for publication, February 25, 2010 Published, JBC Papers in Press, April 8, 2010, DOI 10.1074/jbc.M110.118182
Baohai Shao‡1, Subramaniam Pennathur§2, Ioanna Pagani¶, Michael N. Oda¶3, Joseph L. Witztumʈ, John F. Oram†,
and Jay W. Heinecke‡
From the ‡Department of Medicine, University of Washington, Seattle, Washington 98195, the §Department of Internal Medicine,
University of Michigan, Ann Arbor, Michigan 48109, the ¶Children’s Hospital Oakland Research Institute, Oakland, California
94609, and the ʈDepartment of Medicine, University of California at San Diego, La Jolla, California 92093
Dysfunctional high density lipoprotein (HDL) is implicated
in the pathogenesis of cardiovascular disease, but the under-
lying pathways remain poorly understood. One potential
mechanism involves covalent modification by reactive car-
bonyls of apolipoprotein A-I (apoA-I), the major HDL pro-
tein. We therefore determined whether carbonyls resulting
from lipid peroxidation (malondialdehyde (MDA) and
hydroxynonenal) or carbohydrate oxidation (glycolaldehyde,
glyoxal, and methylglyoxal) covalently modify lipid-free
apoA-I and inhibit its ability to promote cellular cholesterol
efflux by the ABCA1 pathway. MDA markedly impaired the
ABCA1 activity of apoA-I. In striking contrast, none of the
other four carbonyls were effective. Liquid chromatography-
electrospray ionization-tandem mass spectrometry of MDA-
modified apoA-I revealed that Lys residues at specific sites
had been modified. The chief adducts were MDA-Lys and a
Lys-MDA-Lys cross-link. Lys residues in the C terminus of
apoA-I were targeted for cross-linking in high yield, and this
process may hinder the interaction of apoA-I with lipids and
ABCA1, two key steps in reverse cholesterol transport. More-
over, levels of MDA-protein adducts were elevated in HDL
isolated from human atherosclerotic lesions, suggesting that
lipid peroxidation might render HDL dysfunctional in vivo.
Taken together, our observations indicate that MDA dam-
ages apoA-I by a pathway that generates lysine adducts at
specific sites on the protein. Such damage may facilitate the
formation of macrophage foam cells by impairing cholesterol
efflux by the ABCA1 pathway.
The cardioprotective effects of high density lipoprotein
(HDL)4 are generally attributed to reverse cholesterol transport
(1, 2). In this scenario, HDL removes excess cholesterol from
artery wall macrophages and transports it back to the liver for
excretion in bile. Apolipoprotein A-I (apoA-I), the major pro-
tein of HDL, plays a critical role in the first step of reverse
cholesterol transport by enhancing the sterol efflux from
macrophages that is accomplished by the ATP-binding cassette
transporter A1 (ABCA1) (3, 4). The ligand for ABCA1 is lipid-
free apoA-I produced by remodeling HDL (5, 6). Activation of
ABCA1 depends critically on repeats 1 and 10 of apoA-I, which
are located at the N- and C-terminal regions of the protein,
respectively (7–9). ApoA-I also activates lecithin:cholesterol
acyltransferase, which converts free cholesterol to cholesteryl
ester, a subsequent step in the maturation of HDL particles
(10–12).
It has been proposed that HDL loses its cardioprotective
effects in humans with established coronary artery disease,
although the underlying mechanisms are unclear (13). One
potential pathway involves modification of apoA-I by reactive
intermediates, perhaps in the artery wall. Indeed, oxidation of
apoA-I by myeloperoxidase, a heme protein that generates an
array of reactive oxygen and nitrogen species, severely impairs
cholesterol efflux by the ABCA1 pathway and inhibits the abil-
ity of the protein to activate lecithin:cholesterol acyltransferase
(14–20). However, little is known about the ability of other
reactive species to impair the ABCA1 activity of apoA-I.
One mechanism for modifying proteins involves reactive
carbonyls (21, 22), which have been implicated in the pathogen-
esis of atherosclerosis and diabetic vascular disease (23–25).
They result from oxidation of carbohydrates or amino acids and
peroxidation of lipids (21, 22, 26). Major carbonyl products
of carbohydrate oxidation in vivo are thought to be glyoxal,
methylglyoxal, and glycolaldehyde, which are precursors of
advanced glycation end products. Lipid peroxidation yields a
different spectrum of reactive carbonyl compounds, including
malondialdehyde (MDA), 4-hydroxynonenal (HNE), and acro-
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grants HL086798, HL77268, P30ES07083, HL086559, P01HL088093,
HL092237, and P01HL030086. This work was also supported by Grant
P30DK017047 from the Diabetes Education and Research Center, Univer-
sity of Washington.
† This article is dedicated to the memory of Jack Oram (deceased March 31,
2010) and the pioneering contributions he made to our understanding of
HDL biology.
1 Supported by National Institutes of Health K99/R00 Award K99HL091055
from the NHLBI. To whom correspondence should be addressed: Box
358055, Division of Metabolism, Endocrinology, and Nutrition, Dept. of
Medicine, 815 Mercer St., University of Washington, South Lake Union, 4 The abbreviations used are: HDL, high density lipoprotein; apoA-I, apoli-
Seattle, WA 98109. E-mail: bhshao@u.washington.edu.
2 Supported by a clinical scientist development award from The Doris Duke
Foundation.
poprotein A-I; DTPA, diethylenetriaminepentaacetic acid; ESI, electrospray
ionization; HNE, 4-hydroxynonenal; LC, liquid chromatography; MALDI-
TOF-MS, matrix-assisted laser desorption ionization-time-of-flight mass
spectrometry; MDA, malondialdehyde; MS, mass spectrometry; RLU, rela-
tive light units; BHK, baby hamster kidney.
3 Supported by Grant 16FT-0054 from the Tobacco-related Disease Research
Program of California.
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Modification of ApoA-I by Carbonyls
lein (24, 26), which have been termed advanced lipoxidation
end products (21). Importantly, advanced lipoxidation and
advanced glycation end product levels are elevated in diabetes,
which greatly increases the risk for coronary artery disease (21,
27). Reactive carbonyls can also be produced by oxidation of
hydroxyamino acids (28). For example, myeloperoxidase uses
hydrogen peroxide produced by the NADPH oxidase of phago-
cytes to convert L-serine to glycolaldehyde, which in turn can
react with proteins to form N-(carboxymethyl)lysine (28). This
pathway may be physiologically relevant, because N-(car-
boxymethyl)lysine formation during acute inflammation is
impaired in NADPH oxidase-deficient mice (29).
HDL may be constantly exposed to reactive carbonyls,
because it is the major carrier of lipid hydroperoxides in plasma
(30). Moreover, when plasma is oxidized ex vivo, apoA-I is a
major target for covalent modification by lipid oxidation prod-
ucts (31). Reactive carbonyls are also known to impair HDL
function in vitro. Thus, high concentrations of carbonyls mod-
ified apoA-I and inhibited lecithin:cholesterol acyltransferase
activation by HDL, but the underlying mechanisms were not
identified (32). When apoA-I incorporated into discoidal,
reconstituted HDL was exposed to methylglyoxal, it lost its abil-
ity to activate lecithin:cholesterol acyltransferase (33). This loss
was associated with modification of arginine, lysine, and tryp-
tophan residues. We previously showed that acrolein (an ␣,␤-
unsaturated carbonyl derived from oxidized lipids and
myeloperoxidase-oxidized threonine) modifies a single lysine
residue in repeat 10 of apoA-I. That specific modification asso-
ciated quantitatively with impaired cholesterol transport by
ABCA1 (34).
Neither the structures of the protein adducts nor the specific
amino acid residues that MDA and other reactive carbonyls
modify in apoA-I have been identified. In this study, we inves-
tigated the reactivity of carbonyls with apoA-I, and we deter-
mined whether these compounds altered the ability of the pro-
tein to promote cholesterol efflux by the ABCA1 pathway. We
focused on five carbonyls implicated in the pathogenesis of car-
diovascular disease (21, 23, 24, 35) as follows: MDA, HNE,
glyoxal, methylglyoxal, and glycolaldehyde (Fig. 1A). We found
that MDA selectively modifies Lys residues and dramatically
impairs the ability of apoA-I to transport cellular cholesterol by
the ABCA1 pathway. Moreover, we detected elevated levels of
MDA adducts in HDL isolated from human atherosclerotic
lesions, raising the possibility that MDA interferes with normal
HDL cholesterol transport by modifying Lys residues in apoA-I
in the artery wall.
FIGURE 1. Structures of carbonyls and carbonyl adducts.
by ion-exchange chromatography (36). Protein concentration
was determined using the Lowry assay (Bio-Rad), with albumin
as the standard. The human studies committees at the Univer-
sity of Washington School of Medicine and University of Mich-
igan approved all protocols involving human material.
Isolation of Lesion HDL—Atherosclerotic tissue was har-
vested at endarterectomy, snap-frozen, and stored frozen at
Ϫ80 °C until analysis. Lesions from a single individual (ϳ0.5 g
wet weight) were mixed with dry ice and pulverized in a stain-
less steel mortar and pestle. Lesion HDL was isolated by ultra-
centrifugation from extracts of tissue powder (14, 16), using
buffers supplemented with 100 ␮M diethylenetriaminepenta-
acetic acid (DTPA), 100 ␮M butylated hydroxytoluene, and pro-
tease inhibitor mixture (Sigma). ApoA-I was detected by
immunoblotting, using a rabbit IgG polyclonal antibody to
human apoA-I (Calbiochem) followed by a horseradish perox-
idase-conjugated goat anti-rabbit IgG. Detection was enhanced
EXPERIMENTAL PROCEDURES
Materials—All organic solvents were high performance liquid
chromatography grade. 4-Hydroxynonenal (HNE) was purchased
from Cayman Chemical (Ann Arbor, MI). Unless otherwise indi-
cated, all other materials were purchased from Sigma.
Isolation of HDL and ApoA-I—Plasma was prepared from
EDTA-anticoagulated blood collected from healthy adult sub-
jects who had fasted overnight. HDL (density 1.125–1.210
g/ml) was isolated from plasma by sequential ultracentrifuga-
tion and depleted of apolipoproteins E and B100 by heparin-
agarose chromatography (36). ApoA-I was purified from HDL by chemiluminescence.
18474 JOURNAL OF BIOLOGICAL CHEMISTRY
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Modification of ApoA-I by Carbonyls
Preparation of Malondialdehyde (MDA)—MDA was pre- MS/MS analyses were performed in the positive ion mode
pared immediately before use by rapid acid hydrolysis of mal- (mass range 200–2,000 Da) with a Thermo-Finnigan LCQ
oncarbonyl bis-(dimethylacetal) (37, 38). Briefly, 20 ␮l of 1 M Deca XP Plus instrument (40).
HCl was mixed with 200 ␮l of maloncarbonyl bis-(dimethylac-
Quantifying ApoA-I Modifications with 15N-Labeled ApoA-I—
etal), and the mixture was incubated at room temperature for Loss of precursor peptides and product yields of modified
45 min. The reaction mixture was diluted with 980 ␮l of 10 mM peptides were quantified by isotope dilution (41) with recon-
phosphate buffer (pH 7.4). After further dilution, the MDA structed ion chromatograms of precursor and product pep-
concentration of the stock solution was determined by absorb- tides, using 15N-labeled apoA-I as internal standard (18). An
ance at 245 nm, using ⑀ ϭ 13,700 MϪ1 cmϪ1 (39).
equal amount of 15N-labeled apoA-I was added to control or
Modification of ApoA-I by MDA and Other Carbonyls—Re- modified apoA-I prior to digestion. Loss of precursor peptide
actions of lipid-free apoA-I (5 ␮M, 0.14 mg of protein/ml) with was calculated from the ratio of the peak area of precursor pep-
MDA or other carbonyls (HNE, glyoxal, methylglyoxal, and gly- tide of apoA-I from control or modified apoA-I to that of the
colaldehyde) were carried out at 37 °C for 24 h in 50 mM sodium corresponding 15N-labeled peptide from 15N-labeled apoA-I.
phosphate buffer (pH 7.4) containing 100 ␮M DTPA. Reactions Thus, precursor peptide loss (%) ϭ (1 Ϫ (peak area of precursor
were initiated by adding MDA or other carbonyls and termi- ion from modified apoA-I/peak area of precursor ion from 15N-
nated by adding a 20-fold molar excess (relative to carbonyls) of apoA-I) ϫ (peak area of precursor ion from 15N-apoA-I/peak
aminoguanidine. When indicated, reaction mixtures were area of precursor ion from control apoA-I)) ϫ 100.
reduced with 10 mM NaBH4.
Product yields of modified peptides were calculated from the
Modification of HDL by MDA—Reactions with HDL (0.2 ratio of the peak area of product peptide of apoA-I relative to
mg of protein/ml) were carried out at 37 °C for 24 h in 50 mM that of the corresponding 15N-labeled peptide from 15N-labeled
sodium phosphate buffer (pH 7.4) containing 100 ␮M DTPA. apoA-I. Thus, product yield (%) ϭ 100 ϫ (peak area of product
Reactions were initiated by adding MDA and terminated peptide in modified apoA-I/peak area of 15N-labeled precursor
by adding a 20-fold molar excess (relative to MDA) of peptide) ϫ (peak area of 15N-labeled precursor peptide/peak
aminoguanidine.
area of precursor peptide from control apoA-I). When the prod-
Efflux of Cellular Cholesterol—Baby hamster kidney (BHK) uct peptide was cross-linked, the product ion peak area was com-
cells expressing mifepristone-inducible human ABCA1 were pared with the sum of the peak areas of all precursor peptides that
radiolabeled with [3H]cholesterol. Expression of ABCA1 was formed the cross-linked product peptide. Thus, product yield
induced by incubating the cells for 20 h with Dulbecco’s mod- (%) ϭ 100 ϫ (peak area of cross-linked product peptide in modi-
ified Eagle’s medium containing 1 mg/ml bovine serum albu- fied apoA-I/sum of peak area of 15N-labeled precursor pep-
min (Dulbecco’s modified Eagle’s medium/bovine serum albu- tides) ϫ (sum of peak areas of 15N-labeled precursor peptides/
min) and 10 nM mifepristone. Efflux of [3H]cholesterol was sum of peak areas of precursor peptides from control apoA-I).
measured after a 2-h incubation with Dulbecco’s modified This method assumes that the precursor ions and product ions
Eagle’s medium/bovine serum albumin without or with apoA-I have similar MS response characteristics (40).
(17). Cholesterol efflux mediated by apoA-I was calculated as
Immunochemical Detection of MDA Adducts—A chemilumi-
the percentage of total [3H]cholesterol (medium plus cell) nescence immunoassay, using monoclonal MDA2 that detects
released into the medium after the value obtained with Dulbec- MDA-lysine adducts, was performed with modifications as
co’s modified Eagle’s medium/bovine serum albumin alone was described previously (42). In brief, 96-well Microfluor-2 White
subtracted. BHK cells incubated with native or carbonyl-mod- plates (Thermo Electron Corp.) were coated overnight at 4 °C
ified apoA-I for up to 4 h showed no changes in morphology or with HDL (3 ␮g/ml protein; isolated from plasma or atheroscle-
in cell protein or cholesterol content per well.
rotic carotid lesions) in phosphate-buffered saline buffer sup-
Matrix-assisted Laser Desorption Ionization-Time-of-Flight- plemented with 0.27 mM EDTA and 20 ␮M butylated hydroxy-
Mass Spectrometry (MALDI-TOF-MS)—MALDI-TOF-MS was toluene. The wells were then washed with TBS buffer (150 mM
performed using a Voyager-DE STR system equipped with NaCl, 50 mM Tris base, 0.27 mM EDTA (pH 7.4)) containing 1%
delayed extraction (Applied Biosystems, Foster City, CA) (34). bovine serum albumin before biotinylated MDA2 (2 ␮g/ml)
The matrices were ␣-cyano-4-hydroxycinnamic acid for pep- was added for 1 h at room temperature. After additional wash-
tide digests and sinapinic acid for intact proteins. Samples were ings, bound MDA2 was detected by adding NeutrAvidin alka-
desalted with a ZipTip pipette tip (ZipTipC18 or ZipTipC4 for line phosphatase (Pierce) and Lumi-Phos 530 (Lumingen, Inc.)
peptides or proteins, respectively; Millipore Corp., Billerica, and monitoring chemiluminescence (Dynex Luminometer,
MA) before they were applied to the MALDI plate. Spectra of Thermo Electron Corp.). Each sample was assayed in triplicate.
peptide digests were obtained in the positive reflector mode, Results are expressed in relative light units/100 ms (RLU/ms).
using an accelerating voltage of 20 kV. The spectra of intact All samples were measured in a single assay.
proteins were obtained in the positive linear mode, using an
accelerating voltage of 25 kV.
Modeling MDA-modified ApoA-I—Energy minimization cal-
culations (Accelrys Discovery Studio Client 2.5) used a hybrid
Proteolytic Digestion of ApoA-I and LC-ESI-MS—Native model combining the conformational information for amino
or carbonyl-modified lipid-free apoA-I was digested with acids 1–180 (43) and amino acids 181–243 (44). To examine the
sequencing grade modified trypsin (Promega) or sequencing effect of cross-linking on the apoA-I conformation, we imposed
grade endoproteinase Glu-C (Roche Applied Science) and frac- 2.5–5.6 Å distance constraints (5.6 Å is the estimated maxi-
tionated by LC as described previously (17, 34, 40). MS and mum length of the MDA cross-link) among the N⑀ atoms of Lys
JUNE 11, 2010•VOLUME 285•NUMBER 24
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Modification of ApoA-I by Carbonyls
residues 182, 195, 206, 208, 226, and 239, and CHARMM force-
field was applied to the apoA-I model with a CFF partial charge
forcefield (45) based on the distance constraints. To evaluate
the effect of restrictions in mobility induced by cross-links, we
performed two minimization protocols on our initial model as
follows: the steepest descent method (maximum number of
steps 500, root mean square gradient of 0.1, and energy change
0.0 kT/e) followed by the conjugate gradient method (maxi-
mum number of steps 500, root mean square gradient of 0.0001,
and energy change 0.0 kT/e). After energy minimization mod-
eling, we executed equilibration molecular dynamics with 1,000
steps of 0.001 time steps and a target temperature of 310 K. The
final model was evaluated with the MODELER protein verifica-
tion tool of Discovery Studio (45) to ensure that it was energet-
ically more favorable than the initial conformation.
Statistical Analysis—Unless indicated otherwise, results repre-
sent means and S.D. of triplicate determinations and are repre-
sentative of at least two independent experiments.
RESULTS
Modification of ApoA-I by MDA, but Not by Four Other Reac-
tive Carbonyls, Impairs Cholesterol Efflux by the ABCA1
Pathway—To determine whether any of the five reactive car-
bonyls affect cellular lipid metabolism, we measured the ability
of apoA-I that had been modified by MDA, HNE, glyoxal,
FIGURE 2. Impact of carbonyl modification on ability of apoA-I to promote
cholesterol efflux by ABCA1. ApoA-I (5 ␮M) was incubated with 250 ␮M glyoxal,
methylglyoxal, glycolaldehyde, MDA, or HNE (A), or with the indicated concen-
trations of MDA for 24 h (Band D), or with 250 ␮M MDA for the indicated times (C)
methylglyoxal, or glycolaldehyde to promote cholesterol efflux at 37 °C in 50 mM sodium phosphate buffer (pH 7.4) containing 100 ␮M DTPA.
Reactions were initiated by adding carbonyl and terminated by adding a 20-fold
from cultured cells expressing high levels of ABCA1. Reactions
with carbonyls were carried out at neutral pH in phosphate
buffer supplemented with DTPA to chelate redox-active metal
ions (46). When ABCA1-transfected BHK cells were incubated
with 3 ␮g/ml apoA-I that had been exposed for 24 h at 37 °C to
a 50:1 ratio of carbonyl (mol/mol), the apolipoprotein that had
been incubated with MDA was markedly less able to promote
cholesterol efflux (Fig. 2A). In striking contrast, apoA-I incu-
bated with the same molar ratio of each of the other carbonyls
did not lose cholesterol efflux activity (Fig. 2A).
molar excess (relative to carbonyl) of aminoguanidine. A–C, [3H]cholesterol-la-
beled ABCA1-transfected BHK cells were incubated for 2 h with 3 ␮g/ml of con-
trol (0 ␮M carbonyls) or carbonyl-modified apoA-I. D, [3H]cholesterol-labeled
ABCA1-transfected BHK cells were incubated with the indicated concentrations
of untreated or MDA-treated apoA-I (20:1 or 50:1, mol/mol, MDA/protein) for 2 h.
At the end of the incubation, [3H]cholesterol efflux to the acceptor apolipopro-
tein was measured. HNE was added to apoA-I in ethanol. Control experiments
showedthatthesamefinalconcentrationofethanol(ϳ1␮lper200␮l)increased
the efflux activity of native apoA-I by ϳ10%. Results represent two independent
experiments.
detect an increase in ions with the anticipated m/z of multimers
of native apoA-I, even though the carbonyls we investigated
had two electrophilic centers. On the other hand, MALDI-
TOF-MS showed that the molecular weight of apoA-I
increased steadily as the molar ratio of MDA rose from 0 to 100
(Fig. 3, solid squares). At a 50:1 molar ratio, the increase was
ϳ350 atomic mass units. These results indicate that MDA
covalently modifies apoA-I and progressively increases the
molecular weight of the protein. HNE and methylglyoxal also
significantly increased the molecular weight of apoA-I (Fig. 3,
solid circles and empty triangles, respectively), indicating that
they covalently modify apoA-I. At a 50:1 molar ratio, the
increase was ϳ700 atomic mass units with HNE and ϳ350
atomic mass units with methylglyoxal. In contrast, glyoxal and
glycolaldehyde had little effect on the apparent molecular
weight of apoA-I, indicating that they fail to yield adducts of
apoA-I that are detectable under our experimental conditions.
However, it is important to note that certain glycolaldehyde
adduction reactions are reversible (47).
Exposure to increasing concentrations of MDA progressively
and dramatically deprived apoA-I of its ability to promote cho-
lesterol efflux (Fig. 2B). A 20:1 molar ratio of MDA reduced
activity by ϳ50%, and the effect was time-dependent. At a 50:1
molar ratio, the loss was ϳ25% at 2 h and ϳ50% at 5 h (Fig. 2C),
suggesting that the carbonyl reacts rapidly with apoA-I. Both
the apparent Km and Vmax values for cholesterol efflux changed
as apoA-I was exposed to increasing concentrations of MDA
(Fig. 2D). These results demonstrate that MDA significantly
reduces the ability of apoA-I to promote cholesterol efflux by
the ABCA1 pathway. In contrast, modification by HNE,
glyoxal, methylglyoxal, or glycolaldehyde had little effect.
ApoA-I Exhibits Increased Molecular Weight When Modified
by Carbonyls—All of the carbonyls we investigated have two
electrophilic centers (Fig. 1A). Therefore, they have the poten-
tial to form mono-adducts as well as intra- and intermolecular
cross-links. To better understand the potential reactions of
apoA-I, we exposed the protein to up to a 100:1 molar ratio of
carbonyl and analyzed the intact protein with MALDI-TOF-
MS. Using this approach, we observed a progressive decrease in
the ion intensity of material with the mass-to-charge ratio (m/z)
Based on these observations and the molecular weights of the
carbonyl adducts we detected in apoA-I (Fig. 1B; see below), we
of native apoA-I (data not shown). However, we were unable to estimate that a 50:1 molar ratio of carbonyl yielded ϳ7–8
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Modification of ApoA-I by Carbonyls
Lys106, and Lys107; and (iii) and Lys12 and the free N-terminal
amino group. In contrast, there was little loss of other Lys-
containing peptides (e.g. Lys77 or Lys88), suggesting that MDA
does not modify certain Lys residues in apoA-I (Fig. 4A).
MDA Converts Free Amino Groups in ApoA-I to the Propenal
Adduct (Kϩ54) but Not to the Dihydropyridine Adduct—In
vitro studies have identified a number of adducts between MDA
and Lys residues (Fig. 1B), including N-propenal-lysine (Kϩ54)
and dihydropyridine (DHP)-lysine (Kϩ134 (48)). To determine
which adducts form in apoA-I, we exposed the protein to MDA,
digested it, and analyzed the resulting peptides by LC-ESI-MS.
MS/MS detected Kϩ54 in multiple peptides, strongly suggest-
ing that N-propenal-Lys is a major adduct when MDA modifies
Lys. In contrast, we failed to detect DHP-Lys when we exposed
apoA-I to a wide range of MDA concentrations.
To confirm that Kϩ54 was generated when apoA-I was
exposed to MDA, we applied LC-ESI-MS/MS to Glu-C or tryp-
sin digests. Fig. 5A shows a spectrum from a representative
Glu-C peptide of apoA-I: LYRQKVEPLRAE, where K was
FIGURE 3. MALDI-TOF-MS of carbonyl-modified apoA-I. ApoA-I was
exposed to the indicated concentrations of carbonyl for 24 h, as described in
the legend to Fig. 2. After the reaction was terminated with aminoguanidine
and reaction products reduced by NaBH4, intact proteins were desalted and
analyzed by MALDI-TOF-MS. The masses of the intact native or carbonyl-
modified proteins were determined, using myoglobin and bovine serum
albumin as the internal standards.
ϩ
Lys118 in the native protein (peptide ϩ H ; m/z 1501.8). When
apoA-I was incubated with MDA and digested, we detected a
major product peptide at m/z 1555.8. This was consistent with
the formation of LYRQ(Kϩ54)VEPLRAE. This ion was not
detectable in control apoA-I. In the MDA-treated protein, the
b5–b7 ions had gained 54 atomic mass units, but the y5 and y6
MDA, 4–5 HNE, and 5–6 methylglyoxal adducts, respectively, ions were unmodified (Fig. 5B), strongly suggesting that Lys118
per mol of apoA-I.
had been converted to N-propenal-Lys. Because trypsin fails to
Peptides Containing Lys Residues Are Lost in High Yield cleave peptide bonds C-terminal to a modified Lys residue, we
When Lipid-free ApoA-I Is Exposed to MDA—ApoA-I contains used the missing cleavages in tryptic digests to confirm our
21 Lys, 16 Arg, and 5 His residues that could potentially react observations. Both tryptic and Glu-C digestion also identified
with carbonyls. To determine which residues are actually mod- the N-propenal adduct of the N-terminal amino group of
ified, we exposed the lipid-free protein to MDA, digested the apoA-I. Thus, MS analysis of Glu-C and tryptic digests demon-
modified protein with trypsin or Glu-C, and used LC-ESI- strated that Lys residues that contained an ⑀-amino group as
MS/MS to analyze the resulting peptide mixture. Because well as the N-terminal ␣-amino group of apoA-I could form the
apoA-I is rich in Lys and Arg residues, a tryptic digest yields N-propenal adduct when lipid-free apoA-I was exposed MDA.
several small peptides that are poorly retained on a reverse-
MDA Converts a Subset of Residues to N-Propenal-Lys in
phase column. To ensure complete coverage and to unambig- High Yield—To determine whether MDA targets specific Lys
uously identify the site at which each peptide is modified, we residues in apoA-I, we exposed the protein to a 20:1 molar ratio
also used the proteolytic enzyme Glu-C, which cleaves peptide of the carbonyl for 24 h and used isotope dilution LC-ESI-MS to
bonds C-terminal to glutamic acid in ammonium bicarbonate quantify the product yields of modified Lys residues. The esti-
buffer (40). Used in concert, peptides from tryptic and Glu-C mated product yield for Kϩ54 is shown in Fig. 4B. Lys118
,
digests span the entire sequence of apoA-I.
Lys133, and Lys195, as well as the N-terminal amino group, were
To determine which apoA-I residues MDA modifies, we selectively converted to the N-propenal adduct in relatively
used isotope dilution to monitor loss of precursor peptides. high yields. It is noteworthy that of the two precursor peptides
This approach provides a global, unbiased view of protein dam- lost in highest yield (Fig. 4A), one contained Lys226 and the
age (18). Peptide loss was quantified using 15N-labeled apoA-I other contained Lys94, Lys96, Lys106, and Lys107. However, those
as internal standard. Loss of precursor peptide was calculated two peptides were converted to N-propenal-Lys in relatively
using the ratio of the peak area of precursor peptide of apoA-I low yield (compare Fig. 4, A with B). These observations suggest
from control or modified apoA-I to that of the corresponding that Kϩ54 accounts for only a subset of the peptides that are
15N-labeled peptide from 15N-labeled apoA-I.
lost in high yield when apoA-I is exposed to MDA.
We focused on Lys residues first, because previous studies
MDA Forms Lys-1-Amino-3-iminopropene-Lys Cross-links
identified them as major targets of MDA (24, 48). When apoA-I (Lys-MDA-Lys, Kϩ36ϩK) in ApoA-I—The N-propenal-Lys
was exposed to a 20-fold molar ratio of MDA for 24 h, isotope adduct contains a carbonyl group that could cross-link with a
dilution revealed that multiple peptides containing one or more second Lys residue to form the Lys-MDA-Lys adduct (Fig. 1B).
Lys residues were lost in high yield. This observation suggests Indeed, when apoA-I was exposed to low molar ratios of MDA,
that the amino side chain of the residue was a major target for LC-ESI-MS/MS identified Kϩ36ϩK, the Lys-1-amino-3-imi-
MDA (Fig. 4A). We detected Ͻ50% of three native peptides nopropene-Lys (Lys-MDA-Lys) cross-link in which two Lys
containing Lys residues as follows: (i) Lys226; (ii) Lys94, Lys96, residues have gained 36 atomic mass units (39). For example,
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Modification of ApoA-I by Carbonyls
fied all four potential cross-links in
adjacent Lys residues: Lys94-MDA-
Lys96, Lys106-MDA-Lys107, Lys206
-
MDA-Lys208
,
and Lys238-MDA-
Lys239 (Fig. 6B, bottom panel).
We also identified 27 cross-links
between more distant Lys residues
in lipid-free apoA-I and between
the free N-terminal amino group
and Lys12 (Fig. 6A). It is noteworthy
that the C terminus of apoA-I
readily formed Kϩ36ϩK adducts
between residues that were far apart
in the primary sequence. For exam-
ple, most of the peptide containing
Lys226 was lost from apoA-I exposed
to MDA (Fig. 4A), and we identified
cross-links between that residue
and Lys118, Lys133, Lys182, Lys195
,
and Lys206 (Fig. 6A). Those peaks of
material exhibited high ion cur-
rents, suggesting that Lys-MDA-
Lys might be an important product
when MDA modifies apoA-I.
Lys-MDA-Lys Cross-link Is
a
Major Product When ApoA-I Is
Exposed to MDA—To determine
the relative yields of Kϩ36ϩK, we
exposed apoA-I to a 50:1 molar ratio
of MDA, digested the modified pro-
tein with Glu-C or trypsin, and used
reconstructed ion chromatograms
with isotope dilution to quantify the
ion current of each product peptide.
All four pairs of closely situated Lys
FIGURE 4. Precursor loss and product yields of N-propenal-Lys (K؉54), N-propenal-Trp (W؉54), and
N-propenal-His (H؉54) in MDA-modified apoA-I. ApoA-I was exposed to a 20-fold molar ratio of MDA, as
described in the legend to Fig. 2. A mixture of 5 ␮g of modified or control apoA-I and 2.5 ␮g of 15N-labeled
apoA-I was digested with trypsin or Glu-C. A, following LC-ESI-MS/MS analysis, the ratio of precursor peptides
derived from modified or control apoA-I relative to 15N-labeled peptides derived from 15N-labeled apoA-I was
determined from extracted ion chromatograms. Precursor peptide loss was calculated as described under
“Experimental Procedures.” B, product yields of Kϩ54, Wϩ54, and Hϩ54 were calculated from the ratio of peak
area of product peptide from modified apoA-I to that of the corresponding 15N-labeled peptide from 15N-
labeled apoA-I, as described under “Experimental Procedures.” The N-propenal adducts were identified by
LC-ESI-MS/MS analysis. N-t, N-terminal amino group. Results represent three independent experiments.
peptide KAKPALE (m/z 756.5), containing Lys206 and Lys208
,
residues (Lys94 and Lys96, Lys106 and Lys107, Lys206 and Lys208
,
was readily detected in Glu-C digests of lipid-free apoA-I. After Lys238 and Lys239) formed Kϩ36ϩK in high yield (Fig. 6B, bot-
apoA-I was exposed to MDA, the intensity of this precursor tom panel). However, certain Lys residues that were widely sep-
peptide fell significantly, although two new peaks of material arated in the primary sequence of apoA-I also produced a high
appeared of m/z 810.5 and 792.5. This observation is consistent yield of Lys-MDA-Lys. Thus, Lys133 and Lys140, Lys118 and
with the formation of Kϩ54 and Kϩ36ϩK. In the product Lys140, and Lys195 and Lys206 were cross-linked to about the
peptide of m/z 810.5, MS/MS indicated that Lys206 or Lys208 same extent as juxtaposed Lys residues (Fig. 6B, top panel).
was converted to Kϩ54, although in the product peptide of
We next compared the product yields of N-propenal adducts
m/z 792.5, Lys206 and Lys208 formed the Kϩ36ϩK cross-link of Lys (Kϩ54) and amino-3-iminopropene cross-links of Lys
(Fig. 5D). In the peptide containing Kϩ36ϩK, the b3 to b5 (Kϩ36ϩK) in apoA-I incubated with different concentrations
ions gained 36 atomic mass units, whereas the y1 to y4 ions of MDA for different times. We found that when the concen-
were unchanged (compare Fig. 5, C and D). These findings are tration of MDA or the incubation time was increased, the prod-
consistent with the conversion of Lys206 and Lys208 to uct yield of Kϩ36ϩK for Lys206 and Lys208 was much greater
Lys-MDA-Lys.
than that of the individual Kϩ54 adducts (data not show).
MDA Cross-links Both Juxtaposed and Distant Lys Residues These observations strongly suggest that Lys-MDA-Lys is the
in ApoA-I Primary Sequence—The estimated distance between major product when MDA modifies apoA-I.
the two N⑀ atoms of Lys-MDA-Lys is ϳ5.6 Å. If the lysine side
Lys Residues in the C Terminus of ApoA-I Form Multiple
chain (ϳ7.6 Å) is included, the total distance of Lys-MDA-Lys Kϩ36ϩK Adducts—Several individual Lys residues formed
cross-linking is ϳ20.8 Å, suggesting that Lys residues lying 0–4 cross-links with multiple Lys residues. Thus, Lys133 linked to
amino acids apart in the primary sequence of apoA-I might be five Lys residues, Lys182 to five Lys residues, Lys195 to seven Lys
the primary targets of MDA. To test this proposal, we used residues, and Lys226 to five Lys residues (Fig. 6A). Importantly,
LC-ESI-MS/MS to identify the sites of Kϩ36ϩK in Glu-C and most of those Lys residues lie in the C-terminal domain of
tryptic digests of MDA-treated apoA-I. This approach identi- apoA-I.
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Modification of ApoA-I by Carbonyls
MDA, we noted the 8 residues flanking each of the most and
least reactive Lys residues (Table 1). Of the most reactive Lys
residues, 60% (6 of 10 residues) had a positively charged amino
acid located 2 residues toward the N terminus (Ϫ2 position),
whereas 0% (0 of 8 residues) of the least reactive Lys residues
exhibited this feature. We also observed that 60% (5 of 8) of
poorly reactive Lys residues had a negatively charged residue 3
amino acids toward the C terminus (ϩ3 position), whereas only
20% (2 of 10) of the highly reactive ones exhibited that feature.
Furthermore, the 4 most reactive Lys residues all had a posi-
tively charged residue at the Ϫ2 position, whereas the 3 least
reactive Lys residues all had a negatively charged residue at the
ϩ3 position. Our observations suggest that, in apoA-I, nearby
positively charged amino acids help MDA modify Lys, whereas
negatively charged amino acids might inhibit that reaction.
Energy Minimization Model of C Terminus of ApoA-I That
Has Been Cross-linked by MDA—We used molecular dynamics
energy minimization together with previous structural models
(43, 44) to determine how Kϩ36ϩK cross-links in repeats 9 and
10 affect the structure of lipid-free apoA-I. When the cross-
links that we observed between residues Lys182, Lys195, Lys206
,
Lys208, Lys226, and Lys239 (Fig. 7A) were incorporated into
apoA-I with 2.5–5.6 Å distance constraints between the Lys N⑀
atoms, the C terminus of apoA-I became highly condensed (Fig.
7B). The overall structure of the cross-linked C terminus can be
represented schematically as a hexagonal shape (Fig. 7, C and
D). These observations, which represent an array of potential
conformers of the various cross-linked forms of apoA-I, suggest
that intramolecular cross-linking of the C terminus limits con-
formational adaptability as well as the ability of the domain to
interact with ABCA1.
HDL Isolated from Human Atherosclerotic Lesions Is
Enriched in Proteins That Have Been Modified by MDA—To
quantitatively assess whether MDA modifies HDL in vivo, we
isolated HDL by sequential density gradient ultracentrifugation
from human plasma and carotid atherosclerotic tissue recov-
FIGURE 5. MS/MS analysis of N-propenal-Lys (K؉54) and Lys-MDA-Lys
(K؉36؉K) in MDA-modified apoA-I. ApoA-I was incubated in buffer
alone (A and C) or exposed to a 20-fold molar excess of MDA (B and D), as
described in the legend to Fig. 2. A Glu-C peptide digest was analyzed by
LC-ESI-MS/MS, as described under “Experimental Procedures.” A, MS/MS ered at surgery. To prevent artifactual oxidation of lipoproteins,
spectrum of [LYRQKVEPLRAE ϩ H]ϩ (m/z 1501.8) where K is Lys118. B,
we used buffers containing high concentrations of DTPA (a
metal chelator) and butylated hydroxytoluene (a lipid-soluble
antioxidant). Immunoblotting with a monospecific rabbit anti-
body demonstrated that apoA-I accounted for Ͼ50% of the
protein in lesion HDL.
MS/MS spectrum of [LYRQ(Kϩ54)VEPLRAE ϩ H]ϩ (m/z 1555.8), where K is
Lys118. C, MS/MS spectrum of [KAKPALE ϩ H]ϩ, where the first K is Lys206
and the second
K
is Lys208 (m/z 756.5). D, MS/MS spectrum of
[KAK(Kϩ36ϩK)PALE ϩ H]ϩ, where the first K is Lys206 and the second K is
Lys208 (m/z 792.5, Lys206ϩLys208ϩ36).
Lys Residues in Repeats 9 and 10 of ApoA-I Are Converted to
We used the monoclonal antibody MDA2, which detects
Kϩ54 and Kϩ36ϩK in High Yield—By using apoA-I exposed MDA-lysine adducts on a wide variety of modified proteins
to three different concentrations of MDA, we identified the 10 (42), to quantify MDA-HDL. We first showed that HDL
most reactive and 8 least reactive Lys residues in apoA-I (Table exposed to MDA under the conditions used for our in vitro
1). Six of the 10 most reactive Lys residues were in repeats 8–10 studies yielded material that was immunoreactive with MDA2
at the C terminus (Lys195, Lys206, Lys208, Lys226, Lys238, and (Fig. 8A). Using MDA2 in an immunoassay, we found that the
Lys239), whereas all 8 of the least reactive were located at the N level of MDA-modified proteins in HDL isolated from the ath-
terminus (Lys23, Lys40, Lys45, Lys59, Lys77, Lys88, Lys106, and erosclerotic lesions (407 Ϯ 167 RLU/100 ms; n ϭ 10) was 3.6-
Lys107). Importantly, repeats 9 and 10 of the C-terminal domain fold higher than in circulating HDL (113 Ϯ 63 RLU/100 ms; n ϭ
are needed to promote sterol efflux by ABCA1 (7–9). Our 5; p Ͻ 0.0005) (Fig. 8B). These observations provide strong
observations suggest that MDA adduction and cross-linking of evidence that HDL can be modified by MDA in the human
Lys residues in the C terminus might be particularly detrimen- artery wall.
tal to the ability of apoA-I to transport cholesterol by ABCA1.
HNE Modifies ApoA-I His Residues in High Yield—His and
Suitably Juxtaposed, Positively Charged Amino Acids May Lys residues are the major targets for 4-hydroxynonenal in vitro
Help MDA Modify Lys—To determine whether the local amino (24, 49). To determine whether they are targeted in apoA-I, we
acid sequence of apoA-I might direct Lys modification by exposed the protein to a 50:1 molar ratio of HNE and analyzed
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Modification of ApoA-I by Carbonyls
Methylglyoxal Modifies Arg Resi-
dues in ApoA-I—Arg residues are
the major target for methylglyoxal
in vitro (50). LC-ESI-MS analysis of
a Glu-C digest of native apoA-I
demonstrated that the 18 Arg resi-
dues of the protein were distributed
among 12 peptides. When we
exposed apoA-I to a 50:1 molar ratio
of methylglyoxal, we detected all 12
peptides containing Rϩ54 and/or
Rϩ72. MS/MS analysis confirmed
that the Arg residues in those pep-
tides had gained 54 or 72 atomic
mass units, suggesting the forma-
tion of imidazolone adducts (Fig. 1B)
(50). We determined the product
yields of methylglyoxal-Arg adducts
with reconstructed ion chromato-
grams of product and precursor pep-
tides. Arg61 was the major target of
methylglyoxal in apoA-I. When the
protein was exposed to a 50:1 molar
ratio of the carbonyl, the product
yield of imidazolone adducts involv-
ing Arg61 was ϳ80% (Fig. 9, yellow
bars). However, modifying apoA-I
with a 50:1 molar ratio of methyl-
glyoxal failed to affect the ability of
the protein to promote cellular cho-
lesterol efflux by ABCA1.
Glycolaldehyde Fails to Yield De-
tectable Adducts in ApoA-I—Gly-
colaldehyde converts Lys residues
⑀
FIGURE 6. Product yield of Lys-MDA-Lys (K؉36؉K) and cross-linking map between Lys residues in MDA-
modifiedapoA-I.ApoA-Iwasexposedtoa50-foldmolarratioofMDA,asdescribedinthelegendtoFig.2.Amixture
of control or modified apoA-I (5 ␮g) and 15N-labeled apoA-I (2.5 ␮g) was digested with trypsin or Glu-C. Following
LC-ESI-MS/MS analysis, the product yields of Lys-MDA-Lys (Kϩ36ϩK) between Lys residues were calculated from
the ratio of peak area of product peptide from modified apoA-I to that of the corresponding 15N-labeled peptide
from15N-labeledapoA-I,asdescribedunder“ExperimentalProcedures.”Allcross-linkedadductswereconfirmedby
LC-ESI-MS/MS analysis. A, distant cross-linking map between Lys residues in MDA-modified apoA-I; B, product yield
of adjacent cross-linking (bottom panel) and product yield of distant cross-linking (top panel).
to N -(carboxymethyl)lysine (21,
51) and glycolaldehyde-pyridine in
vitro (52). To identify the major tar-
gets in apoA-I, we exposed the pro-
tein to a 50:1 molar ratio of glycolal-
dehyde, reduced the protein with
NaBH4, and analyzed a proteolytic
a proteolytic digest by LC-ESI-MS/MS. All five peptides that digest with LC-ESI-MS/MS. However, we were unable to detect
contained His were lost in near-quantitative yield. MS/MS any major products. These results are consistent with our
analysis revealed that the His residue in each peptide gained 158 MALDI-TOF-MS analysis, which failed to detect an increase in
or 156 atomic mass units without NaBH4 reduction, suggesting molecular weight after apoA-I was exposed to glycolaldehyde.
that His was converted to the hemiacetal adduct (Fig. 1B) (49). Our observations suggest that glycolaldehyde does not generate
In contrast, we were unable to detect HNE-Lys or HNE-Arg a high yield of stable adducts in apoA-I, even though we readily
after exposing apoA-I to HNE.
detected glycolaldehyde-Arg adducts when we studied model
We determined the product yield of HNE-His with recon- peptides.
structed ion chromatograms of product and precursor peptides
DISCUSSION
Conversion of HDL to a dysfunctional form has been pro-
from apoA-I exposed to a 50:1 molar ratio of HNE (Fig. 9, pur-
ple bars). This concentration of HNE does not affect the ability
of the protein to promote cholesterol efflux by the ABCA1 posed to impair the cardioprotective properties of the lipopro-
pathway. All five His residues were modified in near-quantita- tein, but the underlying mechanisms remain poorly understood
tive yield (ϳ90%). This observation strongly suggests that the (13, 53). However, many lines of evidence implicate reactive
His residues of apoA-I are the major target of HNE and that carbonyls in the pathogenesis of atherosclerotic and diabetic
HNE adducts of His have little impact on the ability of the vascular disease (21, 27, 54, 55). Therefore, we determined
protein to promote cholesterol efflux by the ABCA1 pathway.
whether pathophysiologically relevant carbonyls, MDA, HNE,
18480 JOURNAL OF BIOLOGICAL CHEMISTRY
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Modification of ApoA-I by Carbonyls
TABLE 1
Analysis of amino acid sequences flanking the Lys residues most
reactive and least reactive with MDA in apoA-I
ApoA-I (5 ␮M) was exposed to 10, 100, or 250 ␮M MDA, as described in the legend
to Fig. 2. Product yields were calculated by isotope dilutions using 15N-labeled
apoA-I. Results represent two independent experiments.
FIGURE 8. Quantification of MDA-modified proteins in HDL isolated from
plasma and atherosclerotic tissue of humans. A, HDL (from healthy
plasma, 0.2 mg of protein/ml) was exposed to indicated concentrations of
MDAasdescribedinthelegendtoFig. 2. B, plasmawasobtainedfromhealthy
humans. Human atherosclerotic tissue was obtained at surgery from subjects
undergoing carotid endarterectomy. HDL was isolated from plasma and tis-
sue by ultracentrifugation, and equal amounts of HDL protein were added to
each well, and then MDA-modified proteins were quantified by enzyme-
linked immunosorbent assay using the MDA2 monoclonal antibody (RLU/
ms ϭ relative light units/ms). Each value shown is the average of triplicate
measurements as described under “Experimental Procedures.”
glyoxal, methylglyoxal, and glycolaldehyde, might impair a key
aspect of cardioprotective effects of apoA-I, i.e. cholesterol
removal by the ABCA1 pathway. Remarkably, we found that
MDA, unlike the other carbonyls we tested, potently damages
this critical step in reverse cholesterol transport. These obser-
vations may be biologically important because we showed that
HDL isolated from human atherosclerotic lesions had been
modified by MDA.
The nature of selective vulnerability of apoA-I to MDA is
a key question. Three lines of evidence strongly support the
proposal that MDA modifies the ⑀-amino group of Lys resi-
dues in the C terminus of apoA-I and that the resulting Lys-
MDA adducts impair cholesterol efflux by the ABCA1 path-
way. First, quantitative analysis of proteolytic digests of
apoA-I by isotope dilution MS revealed that certain Lys res-
idues were converted in high yield to the MDA-Lys or Lys-
MDA-Lys adduct. Six of the 10 most reactive Lys residues
resided in repeats 7–10 of the C terminus of apoA-I (Fig. 9),
suggesting that modification of this region might be an impor-
tant mechanism for impairing ABCA1 activity. Second, HNE
failed to affect the ability of apoA-I to promote cholesterol
efflux even though it modified all five His residues of the central
domain of apoA-I in high yield (Fig. 9). Glyoxal and methylg-
lyoxal, which likewise failed to affect the ABCA1 activity of
apoA-I, modified residues in the N terminus of the protein and
the central region of the protein but not its C terminus (Fig. 9).
Third, the C terminus of apoA-I was the major site for cross-
linking by MDA (Fig. 9). A model based on molecular dynamics
energy minimization suggested that cross-linking caused the C
terminus of apoA-I to become highly condensed, which should
markedly affect its conformation and conformational adapta-
bility. Importantly, studies of deletion mutants of apoA-I sug-
gest that repeat 10 of the C terminus of the protein helps acti-
vate ABCA1 (7, 9). Taken together, these observations support
the proposal that modification of the C terminus of apoA-I by
MDA deprives the protein of its ability to promote cholesterol
efflux by the ABCA1 pathway.
FIGURE 7. Hexagon model of MDA-modified C terminus of apoA-I. A, loca-
tion of Lys residues modified in high yield by MDA in the C-terminal region of
lipid-free apoA-I. B, energy minimization model of repeats 7–10 of apoA-I
cross-linked by MDA. C, hexagon model of cross-links between C-terminal
lysine residues in MDA-modified apoA-I. D, close-up view of C-terminal in the
energy minimization model of apoA-I cross-linked by MDA.
JUNE 11, 2010•VOLUME 285•NUMBER 24
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Modification of ApoA-I by Carbonyls
minus, reducing the amount of free
energy available for the lipid-free to
lipid-bound transition of apoA-I.
Modification of apoA-I could be
physiologically relevant because
MDA is one of the most abundant
carbonyls generated through lipid
peroxidation (24). Indeed, immuno-
chemical analyses of HDL with
MDA2, a monoclonal antibody that
binds with high affinity to a variety
of MDA-modified proteins (42),
demonstrated that HDL isolated
from atherosclerotic tissue con-
tained a 3.6-fold higher concentra-
tion of MDA than HDL from the
plasma of apparently healthy
humans. Moreover, MDA concen-
tration appears to increase in dis-
eases, such as diabetes mellitus, that
increase the risk of cardiovascular
disease (54). Importantly, MDA
adducts have also been detected in
low density lipoproteins isolated
from atherosclerotic lesions (62),
and modification by MDA of Lys
residues in apoB-100 of low density
lipoprotein is implicated in macro-
phage foam cell formation (35, 63), a
critical step in atherogenesis.
These observations suggest that
modification of Lys residues by
MDA generated during lipid per-
oxidation could promote the forma-
tion of macrophage foam cells by
two distinct pathways as follows: (i)
FIGURE 9. Summary of amino acid residues modified by MDA, HNE, or methylglyoxal in lipid-free apoA-I.
The product yields of MDA-Lys (Kϩ54) and Lys-MDA-Lys (Kϩ36ϩK) (red), HNE-His (Hϩ158, purple), or methyl-
glyoxal-Arg (Rϩ54 and Rϩ72, yellow) were determined by isotope dilution MS. High yield cross-linking
between lysine residues by MDA are indicated (red arc).
The C-terminal region of lipid-free apoA-I contains a stretch conversion of low density lipoprotein into a ligand for scav-
of random coil (residues 188–208) and a stretch of ␤-strand enger receptors that admit cholesterol into macrophages; and
(residues 209–220) (44, 56–58). The random coil is thought to (ii) modification of lipid-free apoA-I derived from HDL in a
contribute to the energy requirements of lipid-free to lipid- manner that impairs cholesterol efflux by the ABCA1 pathway.
bound conformational transition, and the ␤-strand stabilizes
Most studies of protein modification have focused on the
the soluble lipid-free conformation of apoA-I (44, 59). When vulnerability of individual amino acid side chains. Remarkably
the protein binds lipid, however, residues 188–220 undergo little is known about the influence of nearby residues, although
a transition to an ␣-helical structure (44). This conformational it is clear that reactive intermediates can modify specific amino
transition has been proposed to account for most of the free acid residues in proteins (19, 34, 64, 65). When we exposed
energy required to transform apoA-I from its lipid-free to lipid- apoA-I to low concentrations of MDA, isotope dilution MS
bound states (44, 59). Molecular dynamics energy minimiza- revealed that certain Lys residues were modified in high yield,
tion modeling, based on the Lys-MDA-Lys adducts we whereas others were almost completely resistant. Thus, MDA
observed in repeats 7–10, suggested that cross-linking limited modifies Lys residues only at specific sites in lipid-free apoA-I.
the conformational adaptability of the C terminus of lipid-free Analysis of nearby amino acids in the apoA-I sequence demon-
apoA-I. Cross-linking would therefore minimize the enthalpic strated that a positively charged amino acid located two resi-
contribution of this region to lipid binding.
dues away (toward the N terminus of the protein) from a Lys
Collectively, our observations suggest that MDA alters the residue strongly associated with increased reactivity with
ability of apoA-I to promote sterol efflux by impairing two key MDA. In contrast, a negatively charged amino acid residue
steps in the ABCA1 pathway (60). First, MDA-Lys adducts alter located three amino acids toward the C terminus associated
the structure and conformation of repeat 10, preventing pro- with decreased modification by MDA. It is important to note
ductive interactions with ABCA1 (61). Second, Lys-MDA-Lys that most of apoA-I has an ␣-helical structure and that amino
cross-links limit the conformational adaptability of the C ter- acids two and three residues apart in a primary ␣-helical
18482 JOURNAL OF BIOLOGICAL CHEMISTRY
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Modification of ApoA-I by Carbonyls
4. Tall, A. R., Yvan-Charvet, L., Terasaka, N., Pagler, T., and Wang, N. (2008)
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sequence are closely located in three-dimensional space. Our
observations suggest that positively charged amino acids that
are appropriately juxtaposed to Lys residues in apoA-I may
favor the formation of MDA-Lys adducts. Interestingly, posi-
tively charged surfaces in albumin were recently proposed to
direct site-specific modification of Lys residues by MDA (66).
HNE is perhaps the best studied product of lipid peroxida-
tion (24). It can react with imidazoles (His), thiols (Cys), or free
amino (Lys) groups of proteins to form stable Michael adducts
with hemiacetal structures (39, 67). We readily detected His
adducts after we exposed apoA-I to HNE. Quantitative analysis
by isotope dilution MS revealed that all five His residues in
apoA-I were modified in high yield (ϳ90%) when the protein
was exposed to a 50:1 molar ratio of HNE. Remarkably, this
concentration of HNE had no effect on the ability of apoA-I to
promote cholesterol efflux by the ABCA1 pathway, likely
because all five His residues are located in the central region of
the protein, which does not appear to play a major role in acti-
vating the transporter.
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