From the Laboratory of Food and Biodynamics, Graduate
School of Bioagricultural Sciences, Nagoya University, Nagoya
464-8601, the § Faculty of Regional Studies, Gifu
University, Gifu 501-1193, and the ¶ Department of Pathology and
Biology of diseases, Graduate School of Medicine, Kyoto University,
Sakyo-ku, Kyoto 606, Japan
Received for publication, October 3, 2002, and in revised form, December 5, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
4-Hydroxy-2-nonenal (HNE), a major
racemic product of lipid peroxidation, reacts with histidine to form a
stable HNE-histidine Michael addition-type adduct possessing three
chiral centers in the cyclic hemiacetal structure. In the present
study, we characterized configurational isomers of a
HNE-N Several lines of evidence indicate that the oxidative modification
of proteins and the subsequent accumulation of the modified proteins
have been found in cells during aging, oxidative stress, and in various
pathological states, including premature diseases, muscular dystrophy,
rheumatoid arthritis, and atherosclerosis (1, 2). The important agents
that give rise to the modification of a protein may be represented by
lipid metabolites, such as 4-hydroxy-2-alkenals (3, 4). These
metabolites are considered important mediators of cell damage due to
their ability to covalently modify biomolecules, which can disrupt
important cellular functions and can cause mutations (3). Furthermore,
the adduction of aldehydes to apolipoprotein B in low density
lipoproteins has been strongly implicated in the mechanism by which low
density lipoprotein is converted to an atherogenic form that is taken up by macrophages, leading to the formation of foam cells (5, 6).
4-Hydroxy-2-nonenal (HNE),1
among reactive aldehydes, is a major product of lipid peroxidation (3,
7-9) and is believed to be largely responsible for cytopathological
effects observed during oxidative stress (3). HNE exerts these effects
because of its facile reactivity with biological materials,
particularly the sulfhydryl groups of proteins (10). The reaction of
HNE with sulfhydryl groups leads to the formation of thioether adducts that further undergo cyclization to form cyclic hemiacetals (3, 11).
Formation of thiol-derived Michael adducts, stabilized as cyclic
hemiacetals, was initially considered to constitute the main reactivity
of HNE (3). However, other studies led to the realization that HNE
could form Michael adducts also with the imidazole moiety of histidine
residues (12, 13) and the Because HNE generated in lipid peroxidation is a racemic mixture of
4R- and 4S-enantiomers (15), the HNE-histidine
Michael adduct, possessing three chiral centers at C-2, C-4, and C-5 in the tetrahydrofuran moiety, is presumed to be composed of at least eight isomers (see Scheme 1). However, the structures of HNE-histidine adducts in solution have not been fully characterized as yet with complicating 1H NMR diastereoscopic splittings by three
chiral centers. In the present study, we characterized the
configurational isomers of the
HNE-N Materials--
N Racemic and Enantioisomeric HNE--
The stock solutions of HNE
were prepared by the acid-treatment (1 mM HCl) of
4-hydroxy-2-nonenal dimethylacetal, which was synthesized according to
the procedure of De Montarby et al. (17). (R)-
and (S)-HNEs were prepared by the enzymatic resolution of racemic HNE (18) and purified by a chiral-phase HPLC on a ChiralPak AD-RH column (0.46 × 15 cm) (Daicel Chemical Industries, Ltd., Osaka, Japan) eluted with a linear gradient of
acetonitrile/water/acetic acid (90/10/0.01, v/v) (solvent
A)-acetonitrile (solvent B) (time = 0-5 min, 100% A; 60 min, 0%
A), at a flow rate of 0.8 ml/min. The elution profiles were monitored
by UV absorbance at 224 nm. The concentrations of racemic and
enantioisomeric HNE stock solutions were determined by the measurement
of UV absorbance at 224 nm (19).
Reaction of N In Vitro Modification of BSA--
Modification of protein by HNE
enantiomers was performed by incubating BSA (1.0 mg/ml) with 1-10
mM (R)-HNE or (S)-HNE in 1 ml of 0.1 M sodium phosphate buffer (pH 7.2) at 37 °C for 24 h.
General Procedures--
NMR spectra were recorded using a Bruker
AMX600 (600 MHz) instrument. Liquid chromatography-mass spectrometry
was carried out with a Jasco PlatformII-LC instrument.
Molecular Orbital Calculations--
The restricted Hartree-Fock
(RHF) and density functional theory (DFT) with B3LYP
exchange-correlation functional calculations for eight isomers of the
model compound
(5'-methyl-N Antibody Preparation--
Female BALB/c mice were immunized
three times with (R)-HNE-treated or
(S)-HNE-treated KLH. Spleen cells from the immunized mice
were fused with P3/U1 murine myeloma cells and cultured in hypoxantine/amethopterin/thymidine selection medium. Culture
supernatants of the hybridoma were screened using ELISA, employing
pairs of wells of microtiter plates on which were absorbed
(R)-HNE-treated or (S)-HNE-treated
BSA as the antigen (1 µg of protein/well). After incubation with 100 µl of the hybridoma supernatants, and with intervening washes with
Tris-buffered saline, pH 7.8, containing 0.05% Tween 20 (TBS-Tween),
the wells were incubated with alkaline phosphatase-conjugated goat
anti-mouse IgG, followed by a substrate solution containing 1 mg/ml
p-nitrophenyl phosphate. Hybridoma cells, corresponding to
the supernatants that were positive on (R)-HNE-modified or
(S)-HNE-modified BSA, were then cloned by limiting dilution.
After repeated screening, four clones were obtained. Among them, clones
R310 and S412 showed the most distinctive recognition of the
(R)-HNE-modified and (S)-HNE-modified BSA, respectively.
Enzyme-linked Immunosorbent Assay--
Cross-reactivity of
antibodies for aldehyde-treated proteins was determined by a
non-competitive ELISA. A coating antigen was prepared by incubating 1 mg of BSA with 2 mM aldehyde in 1 ml of 50 mM
sodium phosphate buffer, pH 7.2, for 2 h at 37 °C. A 100-µl
aliquot of the antigen solution was added to each well of a 96-well
microtiter plate and incubated for 20 h at 4 °C. The antigen
solution was then removed, and the plate was washed with Tris-buffered
saline (TBS) containing 10% Tween 20 (TBS/Tween). Each well was
incubated with 200 µl of 1% BSA in TBS/Tween for 30 min at 37 °C
in a moist chamber to block the unsaturated plastic surface. The plate
was then washed once with TBS/Tween. A 100-µl aliquot of antibody was
added to each well and incubated for 1 h at 37 °C. After
discarding the supernatants and washing three times with TBS/Tween, 100 µl of a 5 × 104 dilution of goat anti-rabbit IgG
conjugated to horseradish peroxidase in TBS/Tween was added. After
incubation for 1 h at 37 °C, the supernatant was discarded, and
the plates were washed three times with TBS/Tween. Enzyme-linked
antibody bound to the well was revealed by adding 100 µl/well of
1,2-phenylenediamine in 0.1 M citrate/phosphate buffer (pH
5.0) containing 0.003% hydrogen peroxide. The reaction was terminated
by the addition of 50 µl of 2 M sulfuric acid, and
absorbance at 492 nm was read on a micro-ELISA plate reader.
In a competitive ELISA study, a competitor was incubated with antibody
for 20 h at 4 °C to yield competitor/antibody mixtures containing antibody at 25 ng/ml and variable concentrations of the
competitor. A 100-µl aliquot of competitor/antibody mixtures was
added to each well and incubated for 1 h at 37 °C. After
discarding the supernatants and washing three times with TBS/Tween, the
second antibody was added and the enzyme-linked antibody bound to the well was revealed as described above. Results were expressed as the
ratio, B/Bo, where
B = absorbance in the presence of competitor SDS-PAGE--
SDS-PAGE was performed according to Laemmli (20).
The protein was stained with Coomassie Blue
Immunoblot Analysis--
The gel was transblotted onto a
nitrocellulose membrane, incubated with Block Ace (40 mg/ml) for
blocking, washed, and treated with the primary antibody. This procedure
was followed by the addition of horseradish peroxidase conjugated to a
goat anti-mouse IgG F(ab')2 fragment and ECL reagents
(Amersham Biosciences, Buckinghamshire, UK). The bands were visualized
by exposure of the membranes to autoradiography film.
Animal Experiments--
The ferric nitrilotriacetate
(Fe3+-NTA) solution was prepared immediately before use by
the method described by Toyokuni et al. (21) with a slight
modification. Briefly, ferric nitrate enneahydrate and the
nitrilotriacetic acid disodium salt were each dissolved in deionized
water to form 80 and 160 mM solutions, respectively. They
were mixed at the volume ratio of 1:2 (molar ratio, 1:4), and the pH
was adjusted with sodium hydrogen carbonate to 7.4. Male SPF slc:
Wistar rats (Shizuoka Laboratory Animal Center, Shizuoka), weighing
130-150 g (6 weeks of age), were used. They were kept in a stainless
steel cage and given commercial rat chow (Funabashi F-2, Chiba) as well
as deionized water (Millipore Japan, Osaka) ad libitum.
Animals received a single intraperitoneal injection of
Fe3+-NTA (15 mg of Fe3+/kg body weight). They
were sacrificed at 0, 24, and 48 h after the administration. The
animals were sacrificed by decapitation. Both kidneys of each animal
were immediately removed. One of them was fixed in Bouin's solution,
embedded in paraffin, cut at 3-µm thickness, and used for
immunohistochemical analyses by an avidin-biotin complex method with
alkaline phosphatase (22). Briefly, after deparaffinization with xylene
and ethanol, normal rabbit serum (Dako Japan Co., Ltd., Kyoto; diluted
to 1:75) for the inhibition of the nonspecific binding of the secondary
antibody, a monoclonal antibody (mAbR310 or mAbS412) (0.5 µg/ml),
biotin-labeled rabbit anti-mouse IgG serum (Vector Laboratories;
diluted 1:300), and avidin-biotin complex (Vector; diluted 1:100) were
sequentially used. Procedures, using phosphate-buffered saline or the
IgG fraction (0.5 µg/ml) of normal mouse serum instead of mAbR310 and
mAbS412 antibodies, showed no or negligible positive responses.
Reactions of N NMR Characterization of HNE-Histidine Michael Adducts--
The
four peaks (Ra, Rb, Sa, and
Sb) obtained from (R)-HNE-histidine and
(S)-HNE-histidine adducts were then subjected to
characterization by 600-MHz NMR. The assignment of the proton signals
and stereochemistry could be made by analyzing 1H-1H-correlation spectroscopy (COSY),
1H-detected multiple-bond heteronuclear multiple quantum
coherence (HMBC), 1H-detected multiple-bond heteronuclear
multiple quantum coherence (HMQC), and nuclear Overhauser and exchange
spectroscopy (NOESY) spectrum recorded in D2O. In all
samples, we observed the correlation between the methine proton
(C-4) of the tetrahydrofuran moiety and two imidazole vinyls (C-2' and
C-4') in the HMBC spectrum (data not shown), indicating that the
reaction exclusively occurred at the N Molecular Orbital Calculations--
To elucidate the fundamental
structural principles that govern the tetrahydrofuran ring in the
HNE-histidine adducts and to relate the results to experimental
observations in the NMR analysis, molecular orbital calculations on the
eight isomers of the model compound
(5'-methyl-N Monoclonal Antibodies against (R)-HNE-histidine and
(S)-HNE-histidine Michael Adducts--
We have previously raised a
monoclonal antibody (mAbHNEJ2) against HNE-modified protein and found
that it recognizes the HNE-histidine adduct as the epitope (21). This
antibody has been widely used for assessing oxidative stress in
vitro (24) and in vivo (25-28). We examined the
binding of the antibody to the (R)-HNE-histidine and
(S)-HNE-histidine adducts and found that it equally
recognized both adducts.2
Hence, to evaluate the distribution of the (R)-HNE-histidine and (S)-HNE-histidine adducts separately, we attempted to
raise a monoclonal antibody directed toward each adduct. To this end, mice were immunized individually with (R)-HNE-modified and
(S)-HNE-modified keyhole limpet hemocyanins. During the
preparation of the monoclonal antibodies, hybridomas were selected by
the reactivities of the culture supernatant to the
(R)-HNE-modified and (S)-HNE-modified BSA,
respectively. We finally obtained the monoclonal antibodies, mAbR310
and mAbS412, which specifically recognized (R)-HNE-modified and (S)-HNE-modified proteins, respectively (Fig.
4, A and B). The
ELISA study attested that, among reactive aldehydes tested, only
(R)-HNE and (S)-HNE generated the epitopes that
could be recognized by mAbR310 and mAbS412, respectively (Fig.
4C).
Then, we characterized the antibodies' ability to recognize specific
molecular targets in their native three-dimensional structure. As shown
in Fig. 5A, binding of the
HNE-modified protein to mAbR310 and mAbS412 was scarcely inhibited by
the HNE-cysteine and HNE-lysine adducts but significantly inhibited by
the HNE-histidine adduct. Approximately 5 nmol of the HNE-histidine
adduct per well (100 µl) caused 50% inhibition of antibody binding
to the HNE-modified protein, whereas at least 10-fold higher
concentrations of HNE-lysine or HNE-cysteine adduct were necessary for
the same inhibition. These data indicate that both antibodies represent
the anti-HNE-histidine monoclonal antibodies. To further examine
whether configuration of the tetrahydrofuran moiety of HNE-histidine is
involved in the antibody binding, immunoreactivity of mAbR310 and
mAbS412 to the mixtures (Ra, Rb, Sa,
and Sb) of two diastereomers was tested. As shown in Fig.
5B, mAbR310 showed the highest immunoreactivity to
Rb (the mixture of 2R,4S,5R
and 2S,4S,5R isomers), whereas mAbS412
preferentially recognized Sa (the mixture of
2R,4S,5S and
2S,4S,5S isomers). Based on the common
configurations in each sample, the 4S,5R and 4S,5S configurations of the tetrahydrofuran
moiety were suggested to be critical in the biding of mAbR310 and
mAbS412, respectively.
In Vivo Distribution of (R)-HNE and (S)-HNE-derived
Epitopes--
Formation of (R)- and
(S)-HNE-derived epitopes in vivo was
immunohistochemically assessed in a rat renal carcinogenesis model with
Fe3+-NTA. The monoclonal antibody mAbHNEJ2 (16), which
recognizes the (R)-HNE-histidine and
(S)-HNE-histidine adducts equally, was also used for
comparison. It has been shown that iron overload using
Fe3+-NTA induces acute renal proximal tubular necrosis, a
consequence of oxidative tissue damage, that eventually
leads to a high incidence of renal adenocarcinoma in rodents (29, 30).
The kidneys were excised at the time of sacrifice and then fixed with
Bouin's fixative. The hematoxylin and eosin-stained sections of the
paraffin-embedded tissues were analyzed for histological damage. The
morphological changes in the kidneys of rats treated with
Fe3+-NTA versus time are very similar to
previous reports on ddY mice (21, 31). In the control rat kidney, an
almost negligible level of immunoreactivity was observed (data not
shown). The immunoreactivities appeared in some of the renal proximal
tubular cells 3 h after the administration of 15 mg of
Fe3+/kg of body weight of Fe3+-NTA,
whereas the patterns of distribution of (R)- and
(S)-HNE epitopes appeared to be significantly different. As
shown in Fig. 6 (Top),
consistently with the previous observation (21), the (R,S)-HNE epitopes immunoreactive with mAbHNEJ2
were mainly detected in the cytoplasm and in some of the nuclei. This
pattern of distribution in the rat kidney was consistent with that of
the distribution of other lipid peroxidation products and their
conjugates with cytosolic proteins (32, 33), suggesting a correlation
between the production of racemic HNE and oxidative stress. Similarly to the (R,S)-HNE epitopes, the distribution of
(S)-HNE epitopes in the proximal tubular cells is consistent
with that of the (R,S)-HNE epitopes (Fig. 6,
bottom). However, the (R)-HNE epitopes were mainly located in the nuclei (Fig. 6, middle).
Pre-absorption of the antibodies with HNE-histidine adducts completely
abolished the immunostainings (data not shown), indicating the specific reactivity of these antibodies with their epitopes. These data suggest
that (R)- and (S)-HNE may exert distinct effects
on cellular functions under oxidative stress. The nuclear staining of
(R)-HNE epitopes, in particular, may reflect the mutagenic
and cytotoxic potential of HNE.
Structural information on the nature of the HNE modification of
histidine side chain was first reported by Uchida and Stadtman (12).
The observation, that reduction of the aldehyde group of the primary
Michael addition product with sodium borohydride converts them to the
hydroxy derivatives that are stable to strong acid hydrolysis, formed
the basis of methods for the identification and quantification of the
HNE-histidine Michael adduct of proteins by conventional amino acid
analytical techniques (12). It was shown by means of these techniques
that at least 80% of the histidine residues that were lost when human
plasma low density lipoprotein was treated with HNE were accounted for
as the Michael addition product (34). Based on these findings, it had
been proposed that the Michael addition of imidazole groups to the
double bond of HNE represents the dominant reaction pathway for the
histidine residue, existing as a cyclic hemiacetal. Later, definitive
evidence for the structure of the HNE-histidine Michael adduct was
provided (23). However, until this study, the stereochemistry of
the HNE-histidine adduct in solution had remained to be investigated.
Due to the presence of three chiral centers at C-2, C-4, and C-5 in the
tetrahydrofuran moiety, the HNE-histidine Michael adduct was suggested
to be composed of at least eight configurational isomers (Scheme
1) (12, 23). In addition, the previous
observations that (i) the
HNE-N-acetylhistidine adduct by NMR
spectroscopy and by molecular orbital calculations. In addition, we
raised monoclonal antibodies against (R)-HNE-histidine and
(S)-HNE-histidine adducts, characterized their
specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural
characterization of the configurational isomers of an HNE-histidine
adduct, we prepared the (R)-HNE-histidine and
(S)-HNE-histidine adducts by incubating
N
-acetylhistidine with each HNE enantiomer,
both of which provided two peaks (Ra and Rb from
(R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase
high-performance liquid chromatography. The NMR analysis showed that
each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole
limpet hemocyanins as the antigens, we raised the monoclonal
antibodies, mAbR310 and mAbS412, which enantioselectively recognized
the (R)-HNE-histidine and (S)-HNE-histidine
adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of
diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the
mixture of 2R,4S,5R and
2S,4S,5R isomers), whereas mAbS412
preferentially recognized Sa (the mixture of
2R,4S,5S and
2S,4S,5S isomers). The presence of
(R)-HNE and (S)-HNE epitopes in
vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate, by which
nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively,
were detected.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of lysine residues (14). The
HNE-histidine Michael adduct is readily isolable and is stabilized
toward retro-Michael reaction, because of the poorer leaving group
ability of imidazole over amine at neutral conditions.
-acetylhistidine adduct by NMR
spectroscopy and by molecular orbital calculations. Moreover, we raised
monoclonal antibodies against (R)-HNE-histidine and
(S)-HNE-histidine adducts, characterized their
specificities, and investigated the in vivo distributions of
each adduct under oxidative stress.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Acetyl-L-histidine
and bovine serum albumin (BSA) were obtained from Sigma. Keyhole limpet
hemocyanin (KLH) was obtained from Pierce. Ferric nitrate
nonahydrate and sodium carbonate were from Wako (Osaka, Japan);
nitrilotriacetic acid disodium salt was from Nacalai Tesque, Inc.
(Kyoto, Japan). Anti-HNE-histidine monoclonal antibody (mAbHNEJ2) was
kindly provided by Nihon Oil Factory Co. (Tokyo, Japan) (16).
Horseradish peroxidase-linked anti-rabbit IgG immunoglobulin and ECL
(enhanced chemiluminescence) Western blotting detection reagents were
obtained from Amersham Biosciences (Buckinghamshire, UK).
-Acetylhistidine with Racemic or
Enantioisomeric HNE--
The reaction mixture (10 ml) containing 40 mM N
-acetylhistidine was
incubated with 20 mM (R,S)-HNE,
(R)-HNE, or (S)-HNE in 50 mM sodium
phosphate buffer (pH 7.2). After incubation for 24 h at 37 °C,
the reaction mixtures were analyzed with a reverse-phase HPLC on a
Develosil ODS-HG-5 column (4.6 × 250 mm, Nomura Chemicals, Aichi,
Japan) eluted with a linear gradient of
acetonitrile/water/trifluoroacetic acid (90/10/0.01, v/v) (solvent
A)
acetonitrile/trifluoroacetic acid (100/0.01, v/v) (solvent
B) (time = 0 - 5 min, 100% A; 60 min, 0% A), at a flow rate of
1.0 ml/min. The elution profiles were monitored by absorbance at
200-400 nm.
-(2-hydroxy-5-methyltetrahydrofuran-4-yl)imidazole)
were performed by using the Gaussian 98 program package. All molecular
geometries were fully optimized with RHF/6-31G* and DFT/6-31G* levels
at C1 symmetry, respectively. After molecular geometries of
each species were optimized, vibrational analysis calculations were also carried out to check whether they were correctly energy-minimized structures. All calculations were made on the HIT alpha 667d computer at Gifu University.
background absorbance (no antibody) and Bo = absorbance in the absence of competitor
background absorbance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
Acetylhistidine with Racemic and
Enantioisomeric HNE
To facilitate structural characterization of
configurational isomers of the HNE-histidine adduct, we prepared
(R)-HNE-histidine and (S)-HNE-histidine adducts
by incubating N
-acetylhistidine with
(R)-HNE and (S)-HNE, respectively. As shown in
Fig. 1, reversed-phase HPLC demonstrated
that the reaction of N
-acetylhistidine with
(S)-HNE in sodium phosphate buffer (pH 7.2) for 24 h at
37 °C gave two peaks (Sa and Sb). The reaction
with (R)-HNE also provided two peaks (Ra and
Rb). These HPLC profiles were similar to those observed in
the reaction of N
-acetylhistidine with
racemic HNE, providing RSa and RSb. The liquid
chromatography-mass spectrometry analysis of these peaks gave a
pseudomolecular ion peak at m/z 354 (M+H)+ (data
not shown), which would be expected from the Michael addition-type HNE-histidine adducts, suggesting that they all represent the products
derived from the Michael addition of the imidazole nitrogen atom to the
C-3 of HNE.
View larger version (15K):
[in a new window]
Fig. 1.
Reaction of
N-acetylhistidine with racemic and enantioisomeric
HNE. The reactions were performed as described under
"Experimental Procedures."
position of the imidazole ring. This agrees with the previous assignment on the basis of the coupling constant between two imidazole vinyls (23). The COSY spectrum of Sa suggested that the peak
was a mixture of two diastereomers (Sa1 and Sa2) (Supplementary Data Fig. 1). Similarly, other peaks (Ra, Rb, and Sb) also appeared to contain a pair of
diastereomers (Supplementary Data Tables I and II). Identification of
the proton signals of the tetrahydrofuran moiety in Ra,
Rb, Sa, and Sb follows from the
analysis of NOESY spectra collected in D2O. In
Sa, proton signals at 4.24 and 4.00 ppm, which were assigned
to the methine protons at C-5 of Sa1 and Sa2,
respectively, displayed NOE cross-peaks to the methine proton at C-4,
whereas there were no such NOE cross-peaks in Sb
(Sb1 + Sb2) (Supplementary Data Fig. 2),
suggesting that the configurations at C-4 of Sa and Sb are S and R, respectively. Absolute
configurations at C-2 and C-3 were determined by the NOE connectivity
between the C-2 methine proton and C-3 methylene protons (H-3
and
H-3
). We observed NOE cross-peaks between C-4 proton and C-3 proton (H-3
) and between C-3 proton (H-3
) and C-2 proton in
Sa1, whereas the presence of NOE cross-peaks between the C-4
proton and C-3 (H-3
) proton and between the C-3 proton (H-3
) and
C-2 proton were observed in Sa2. These data suggest that
Sa1 and Sa2 correspond to
2R,4S,5S and
2S,4S,5S isomers, respectively. Similarly, the absolute configuration of the tetrahydrofuran moiety of
Sb1 and Sb2 was determined to be
2S,4R,5S and 2R,4R,5S, respectively. Based on the
assignment of these signals, relative amounts (%) of Sa1,
Sa2, Sb1, and Sb2 in D2O
were found to be 25, 8, 25, and 42, respectively. In a similar manner
to the assignment of the (S)-HNE-histidine isomers, the
absolute configuration of the four isomers (Ra1, Ra2, Rb1, and Rb2) of the
(R)-HNE-histidine adduct were determined to be
2S,4R,5R,
2R,4R,5R,
2R,4S,5R, and 2S,4S,5R, respectively. In addition,
their relative amounts (%) of (R)-HNE-histidine isomers
(Ra1:Ra2:Rb1:Rb2 = 25:8:25:42) were exactly comparable to those of the
(S)-HNE-histidine isomers. Fig.
2 summarizes the configurations of the
eight isomers of the HNE-N
-acetylhistidine
adduct. For each of the isomers, the (R)-HNE-histidine adduct structure is essentially a mirror image of the
(S)-HNE-histidine adduct. Furthermore, the NMR data on the
eight isomers allowed assignment of the signals in the one-dimensional
1H-NMR analysis of the
(R,S)-HNE-N
-acetylhistidine
adduct (Supplementary Data Fig. 3).
View larger version (18K):
[in a new window]
Fig. 2.
Chemical structures of the configurational
isomers of the
(R,S)-HNE-N -acetylhistidine
adduct.
-(2-hydroxy-5-methyltetrahydrofuran-4-yl)imidazole)
were performed. Table I shows the total
energies in atomic units and relative energies in
kilocalories/mol. There is a similar trend in the relative energies
between RHF/6-31G* and DFT/6-31G* levels of calculation. Notably, the
relative energies calculated by molecular orbital methods correlated
well with the relative amounts of the isomers of the
(R)-HNE-histidine and (S)-HNE-histidine adducts found in the NMR analysis. Fig. 3 shows
optimized structures of eight isomers calculated by the DFT/6-31G*
method. The values in parentheses are electronic charges on
atoms. The instability of each molecule increases in the orders of
Rb2, Rb1, Ra1, Ra2 and of
Sb2, Sb1, Sa1, Sa2. There
is the correlation between the trend of the relative stability and the
O-C-2 bond length indicated by arrows: 1.411 A
(Rb2 and Sb2), 1.416 A (Rb1 and
Sb1), 1.418 A (Ra1 and Sa1), and 1.419 A (Ra2 and Sa2).
Total and relative energies for the model compounds of eight
configurational isomers of the HNE-histidine adduct
-(2-hydroxy-5-methyltetrahydrofuran-4-yl)imidazole)
were performed by using the Gaussian 98 program package. All molecular
geometries were fully optimized with RHF/6-31G* and DFT/6-31G* levels
at C1 symmetry, respectively. After molecular geometries of
each species were optimized, vibrational analysis calculations were
also carried out to check whether they are correctly energy minimized
structures.
View larger version (28K):
[in a new window]
Fig. 3.
Optimized structures of the model compound
(5'-methyl-N -(2-hydroxy-5-methyltetrahydrofuran-4-yl)imidazole)
of the HNE-histidine configurational isomers. The values in
parentheses are electronic charges on atoms.
View larger version (49K):
[in a new window]
Fig. 4.
Specificity of mAbR310 and mAbS412 to the
(R)-HNE-modified and (S)-HNE-modified
protein. A, immunoreactivity of mAbR310 toward
(R)-HNE-modified and (S)-HNE-modified BSA.
Upper, SDS-PAGE; lower, immunoblot. B,
immunoreactivity of mAbS412 toward (R)-HNE-modified and
(S)-HNE-modified BSA. Upper, SDS-PAGE;
lower, immunoblot. In A and B, BSA (1 mg/ml) was incubated with (R)- or (S)-HNE (0-5
mM) in 1 ml of 50 mM sodium phosphate buffer,
pH 7.4, for 24 h at 37 °C. C, immunoreactivity of
mAbR310 and mAbS412 to the aldehyde-treated protein. Affinity was
determined by a direct ELISA. A coating antigen was prepared as
described under "Experimental Procedures."
View larger version (31K):
[in a new window]
Fig. 5.
Binding of mAbR310 and mAbS412 to the
configurational isomers of HNE-histidine adducts. A,
competitive ELISA with the Michael addition-type HNE adducts.
Left, mAbR310; right, mAbS412. The HNE-modified
BSA was used as the absorbed antigens. Competitors were as follows:
, HNE-N
-acetylhistidine;
,
HNE-N
-acetyllysine;
,
HNE-N
-acetylcysteine;
,
N
-acetylhistidine;
,
N
-acetyllysine;
,
N
-acetylcysteine. B, competitive
ELISA with the four peaks (Ra, Rb, Sa,
and Sb) obtained from the (R)-HNE-histidine and
(S)-HNE-N
-acetylhistidine adducts.
Left, mAbR310; right, mAbS412. The HNE-modified
BSA was used as the absorbed antigens. Competitors were as follows:
, Sa;
, Sb;
, Ra; and
,
Rb.
View larger version (100K):
[in a new window]
Fig. 6.
Immunohistochemistry of renal cortex with
mAbHNEJ2 (top), mAbR310 (middle), and
mAbS412 (bottom) (serial sections, ×400
magnification). The immunoreactivities appeared in some of the
renal proximal tubular cells 3 h after the administration of 15 mg
Fe3+/kg body weight of Fe3+-NTA. The
immunoreactivities with mAbHNEJ2 and mAbS412 were mainly detected in
the cytoplasm and in some of the nuclei, whereas the immunoreactivities
with mAbR310 were mainly detected in the nuclei.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-acetylhistidine Michael adduct was
detected as two peaks upon reverse-phase HPLC analysis and (ii) four
peaks were detected in the acid-hydrolysis followed by the amino acid
analysis of their o-phthaldehyde derivatives (12)
also suggested the multiplicity of primary products in the
HNE/N
-acetylhistidine reaction. However, with
complicating diastereoscopic splittings by three chiral centers, the
proton NMR spectrum of (R,S)-HNE-histidine adduct
was too complex to analyze directly (23). In the present study, to
facilitate the structural characterization of HNE-histidine isomers, we
prepared (R)-HNE- and
(S)-HNE-N
-acetylhistidine adducts
separately, both of which provided two peaks (Ra and
Rb from (R)-HNE-histidine and Sa and
Sb from (S)-HNE-histidine) with relative amounts
of 1:2 in the reverse-phase HPLC analysis (Fig. 1). With regard to the
reactivity of HNE enantiomers toward the histidine derivative, we could
not see any differences in the rate of formation of the Michael adducts
between two HNE enantiomers,2 suggesting that the
chirality of the C-4 hydroxy group may not affect the reactivity
at the C-3 double bond with the imidazole group of the histidine
derivative. The 600-MHz NMR analysis of the four peaks (Sa,
Sb, Ra, and Rb) revealed that each
peak contained a pair of diastereomers (Sa1 and
Sa2, Sb1 and Sb2, Ra1 and
Ra2, and Rb1 and Rb2). In addition, we
determined the absolute configurations of eight isomers by NOE analysis
(Supplementary Data Tables I and II) and finally assigned the signals
of the isomers in the one-dimensional proton NMR spectrum of the
(R,S)-HNE-N
-acetylhistidine
Michael adduct (Supplementary Data Fig. 3). Our assignment of the eight
isomers of the HNE-N
-acetylhistidine Michael
adducts confirms the recent proposal of Nadkarni and Sayre (23) made on
the basis of an indirect comparative experiment. The NMR analysis also
revealed the presence of ring-opened structures in the four samples
dissolved in 100% CD3OD.2 It was observed
that, in methanol solution, relative amounts of the ring-opened
structure were similar to those of the ring-closed adducts in the
fractions Ra and Sa, whereas the ring-opened
structure was much more predominant than the closed structure in the
fractions Rb and Sb. These observations suggest
that the configurations in the ring-opened structure may be critical in
the equilibrium between the ring-opened and ring-closed structures.
View larger version (11K):
[in a new window]
Scheme 1.
Reaction of histidine residue with
HNE. A, chemical structures of R- and
S-enantiomers of HNE; B, formation of the
HNE-histidine Michael adduct, possessing three chiral centers
(asterisks).
In molecular orbital calculation of the model compound of the
HNE-histidine adduct
(5'-methyl-N-(2-hydroxy-5-methyltetrahydrofuran-4-yl)imidazole),
there was a correlation between the trend of the relative stability and the O-C-2 bond length in the tetrahydrofuran moiety (Table I and Fig.
3). In addition, we found that the relative energies calculated by
molecular orbital methods correlated well with the relative amounts of
the eight isomers in the NMR analysis. These observations suggest that
the electron delocalization features on the oxygen atom of
tetrahydrofuran moiety may reflect the relative amounts of isomers. The
delocalization of the electron pair on the oxygen atom of the
tetrahydrofuran ring increases the strength of the covalent bonding
nature and in turn decreases the polarization of the bond and
stabilizes the whole molecule. Thus, the configuration of the
tetrahydrofuran ring may affect the electron delocalization features,
which contribute to the stability of the HNE-histidine adduct.
Immunological detection is a powerful tool that can be used to evaluate the presence of a desired target and its subcellular localization. Major advantages of this technique over biochemical approaches are the evaluation of small numbers of cells or archival tissues that may otherwise not be subject to analysis. Because of the increasing interest in HNE and HNE modification of proteins under oxidative stress, it seemed useful to prepare an antibody interacting specifically with the HNE moiety or with the HNE-amino acid conjugates in proteins; such antibodies have been prepared by immunizing rabbits with HNE-treated KLH (35), in which HNE adducts of histidine, lysine, and cysteine serve as the antigenic sites. Later, Toyokuni et al. (21) raised the monoclonal antibody (mAbHNEJ2) against HNE-modified KLH and found that the antibody cross-reacted specifically with HNE-modified proteins and had a higher affinity for HNE-histidine adduct than for HNE-lysine and HNE-cysteine adducts (21). However, most of the antibodies, including mAbHNEJ2, directed to the HNE-modified protein have been raised against a protein treated with racemic HNE. Therefore, nothing is known about which of the two enantiomers bound to proteins is generated in vitro and in vivo. Hence, in the present study, we raised novel monoclonal antibodies, mAbR310 and mAbS412, against (R)-HNE-treated and (S)-HNE-modified KLH, respectively. It was observed that mAbR310 and mAbS412 showed the highest affinity for the (R)-HNE-treated and (S)-HNE-treated proteins, respectively (Fig. 4, A and B), and scarcely reacted with the proteins treated with other aldehydes (Fig. 4C). The lack of cross-reactivity of the antibodies for the 2-nonenal-treated protein can be ascribed to the absence of the 4-hydroxy group, which leads the primary Michael adduct to the tetrahydrofuran derivative through an intramolecular cyclization. This and the observation (Fig. 4C) that both antibodies did not cross-react with the proteins that had been treated with the HNE analogs, such as 4-hydroxy-2-pentenal, 4-hydroxy-2-hexenal, 4-hydroxy-2-heptenal, 4-hydroxy-2-octenal, and 4-hydroxy-2-decenal, suggest that both tetrahydrofuran and butyl moieties of the HNE-histidine adduct may be critical for the antibody binding. In addition, the binding of these antibodies to the HNE-treated proteins was selectively inhibited by HNE-histidine adducts, suggesting that the imidazole ring is also involved in the antibody binding. Thus, we propose that mAbR310 and mAbS412 recognize the Nt-(2-hydroxy-5-butyltetrahydrofuran-4-yl)imidazole as the common epitopes. Furthermore, we characterized the antibodies' ability to recognize the configurations of the tetrahydrofuran moiety of the HNE-histidine adduct and found that mAbR310 and mAbS412 preferentially reacted with the mixture (Rb) of 2R,4S,5R and 2S,4S,5R isomers and the mixture (Sa) of 2R,4S,5S and 2S,4S,5S isomers, respectively (Fig. 5). Based on the common configurations in each mixture, we suggested that both antibodies might recognize the configurations at C-4 and C-5 of the tetrahydrofuran ring in the adduct, i.e. mAbR310 and mAbS412 recognize the 4S,5R and 4S,5S configurations, respectively.
The presence of immunoreactive materials with mAbR310 and mAbS412
in vivo was demonstrated in the kidney of rats exposed to Fe3+-NTA. The iron chelate was originally used for an
experimental model of iron overload (36). Repeated intraperitoneal
injections of Fe3+-NTA were reported to induce acute and
subacute renal proximal tubular necrosis and a subsequent high
incidence (60-92%) of renal adenocarcinoma in male rats and mice (28,
29). A single injection of Fe3+-NTA causes a number of
time-dependent morphological alterations in the structure
and the function of the renal proximal tubular cells and their
mitochondria. During the early stage of injury, typical cellular
changes are the loss of brush border, cytoplasmic vesicles,
mitochondrial disorganization, and dense cytoplasmic deposits in the
proximal tubular cells. Most of the damaged epithelia show the typical
appearance of necrotic cells, and more than half of the proximal
tubular cells are removed. It has been suggested that oxidative stress
is one of the basic mechanisms of Fe3+-NTA-induced acute
renal injury and is closely associated with renal carcinogenesis (37).
The present study using mAbR310 and mAbS412 demonstrated that, in
agreement with the previous observation on racemic HNE (21), both
(R)-HNE and (S)-HNE epitopes were mainly detected
in the proximal tubules, the target organs of Fe3+-NTA
(Fig. 6). However, the intracellular localizations of these epitopes
were markedly different. The (S)-HNE epitopes were detected in cytosols and in some of the nuclei, whereas the (R)-HNE
epitopes were mainly detected in the nuclei. Although the mechanism of the distinct localization of (R)- and (S)-HNE
epitopes is currently unknown, these results invite speculation that
there may be a differential mechanism for production of (R)-
and (S)-HNE or there may be specific targets of each
enantiomer in the cells. Clearly, it is important to establish the
mechanism for the differential cellular distributions of
(R)- and (S)-HNE epitopes in the cells. Furthermore, the characterization of the biological consequences of
their distributions also merit immediate attention.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Ojika for his helpful advice and K. Onodera for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology and by the Program for Promotion of Basic Research Activities for Innovative Biosciences in Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains two tables and three figures.
To whom correspondence should be addressed: Laboratory of Food
and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya
University, Nagoya 464-8601, Japan. Tel.: 81-52-789-4127; Fax:
81-52-789-5741; E-mail: uchidak@agr.nagoya-u.ac.jp.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M210129200
2 M. Hashimoto and K. Uchida, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HNE, 4-hydroxy-2-nonenal; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; DFT, density functional theory; RHF, restricted Hartree-Fock; Fe3+-NTA, ferric nitrilotriacetate; HMBC, 1H-detected multiple-bond heteronuclear multiple quantum coherence spectrum; HMQC, 1H-detected multiple-bond heteronuclear multiple quantum coherence; NOESY, nuclear Overhauser and exchange; COSY, correlation spectroscopy; KLH, keyhole limpet hemocyanin; TBS, Tris-buffered saline; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Shacter, E. (2000) Drug. Metab. Rev. 32, 307-326[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Stadtman, E. R.,
and Levine, R. L.
(2000)
Ann. N. Y. Acad. Sci.
899,
191-208 |
3. | Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Free Radic. Biol. Med. 11, 81-128[CrossRef][Medline] [Order article via Infotrieve] |
4. | Uchida, K. (2000) Free Radic. Biol. Med. 28, 1685-1696[CrossRef][Medline] [Order article via Infotrieve] |
5. | Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C., and Witztum, J. L. (1989) N. Engl. J. Med. 320, 915-924[Medline] [Order article via Infotrieve] |
6. | Steinberg, D. (1995) Adv. Exp. Med. Biol. 369, 39-48[Medline] [Order article via Infotrieve] |
7. | Esterbauer, H., Cheeseman, K. H., Dianzani, M. U., Poli, G., and Slater, T. F. (1982) Biochem. J. 208, 129-140[Medline] [Order article via Infotrieve] |
8. | Benedetti, A., Comporti, M., and Esterbauer, H. (1980) Biochim. Biophys. Acta 620, 281-296[Medline] [Order article via Infotrieve] |
9. | Benedetti, A., Pompella, A., Fulceri, R., Ramani, A., and Comporti, M. (1986) Biochim. Biophys. Acta 876, 658-666[Medline] [Order article via Infotrieve] |
10. | Esterbauer, H., Zollner, H., and Scholz, N. (1975) Z. Naturforsch. C 30, 466-473[Medline] [Order article via Infotrieve] |
11. | Schauenstein, E., and Esterbauer, H. (1979) Ciba Found. Symp. 67, 225-244 |
12. | Uchida, K., and Stadtman, E. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4544-4548[Abstract] |
13. |
Uchida, K.,
and Stadtman, E. R.
(1993)
J. Biol. Chem.
268,
6388-6393 |
14. |
Szweda, L. I.,
Uchida, K.,
Tsai, L.,
and Stadtman, E. R.
(1993)
J. Biol. Chem.
268,
3342-3347 |
15. |
Schneider, C.,
Tallman, K. A.,
Porter, N. A.,
and Brash, A. R.
(2001)
J. Biol. Chem.
276,
20831-20838 |
16. | Toyokuni, S., Miyake, N., Hiai, H., Hagiwara, M., Kawakishi, S., Osawa, T., and Uchida, K. (1995) FEBS Lett. 359, 189-191[CrossRef][Medline] [Order article via Infotrieve] |
17. | De Montarby, L., Mosset, P., and Gree, R. (1988) Tetrahedron Lett. 29, 3895[CrossRef] |
18. | Allevi, P., Anastasia, M., Cajone, F., Ciuffreda, P., and Sanvito, A. M. (1993) J. Org. Chem. 58, 5000-5002 |
19. | Schauenstein, E., Taufer, M., Esterbauer, H., Kylianek, A., and Seelich, T. (1971) Monatsh. Chem. 102, 517-529 |
20. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
21. | Toyokuni, S., Uchida, K., Okamoto, K., Hattori-Nakakuki, Y., Hiai, H., and Stadtman, E. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2616-2620[Abstract] |
22. | Toyokuni, S., Tanaka, T., Hattori, Y., Nishiyama, Y., Yoshida, A., Uchida, K., Hiai, H., Ochi, H., and Osawa, T. (1997) Lab. Invest. 76, 365-374[Medline] [Order article via Infotrieve] |
23. | Nadkarni, D. V., and Sayre, L. M. (1995) Chem. Res. Toxicol. 8, 284-291[Medline] [Order article via Infotrieve] |
24. |
Kondo, M.,
Oya-Ito, T.,
Kumagai, T.,
Osawa, T.,
and Uchida, K.
(2001)
J. Biol. Chem.
276,
12076-12083 |
25. |
Okada, K.,
Wangpoengtrakul, C.,
Osawa, T.,
Toyokuni, S.,
Tanaka, K.,
and Uchida, K.
(1999)
J. Biol. Chem.
274,
23787-23793 |
26. | Oberley, T. D., Toyokuni, S., and Szweda, L. I. (1999) Free Radic. Biol. Med. 27, 695-703[CrossRef][Medline] [Order article via Infotrieve] |
27. | Toyokuni, S., Yamada, S., Kashima, M., Ihara, Y., Yamada, Y., Tanaka, T., Hiai, H., Seino, Y., and Uchida, K. (2000) Antioxid. Redox Signal. 2, 681-685[Medline] [Order article via Infotrieve] |
28. |
Nakamura, K.,
Kusano, K.,
Nakamura, Y.,
Kakishita, M.,
Ohta, K.,
Nagase, S.,
Yamamoto, M.,
Miyaji, K.,
Saito, H.,
Morita, H.,
Emori, T.,
Matsubara, H.,
Toyokuni, S.,
and Ohe, T.
(2002)
Circulation
105,
2867-2871 |
29. | Ebina, Y., Okada, S., Hamazaki, S., Ogino, F., Li, J.-L., and Midorikawa, O. (1986) J. Natl. Cancer Inst. 76, 107-113[Medline] [Order article via Infotrieve] |
30. | Li, J.-L., Okada, S., Hamazaki, S., Ebina, Y., and Midorikawa, O. (1987) Cancer Res. 47, 1867-1869[Abstract] |
31. | Toyokuni, S., Okada, S., Hamazaki, S., Minamiyama, Y., Yamada, Y., Liang, P., Fukunaga, Y., and Midorikawa, O. (1990) Cancer Res. 50, 5574-5580[Abstract] |
32. | Uchida, K., Fukuda, A., Kawakishi, S., Hiai, H., and Toyokuni, S. (1995) Arch. Biochem. Biophys. 317, 405-411[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Ichihashi, K.,
Osawa, T.,
Toyokuni, S.,
and Uchida, K.
(2001)
J. Biol. Chem.
276,
23903-23913 |
34. |
Uchida, K.,
Szweda, L. I.,
Chae, H. Z.,
and Stadtman, E. R.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8742-8746 |
35. | Uchida, K., Toyokuni, S., Nishikawa, K., Kawakishi, S., Oda, H., Hiai, H., and Stadtman, E. R. (1994) Biochemistry 33, 12487-12494[Medline] [Order article via Infotrieve] |
36. | Awai, M., Narasaki, M., Yamanoi, Y., and Seno, S. (1979) Am. J. Pathol. 95, 663-674[Abstract] |
37. | Toyokuni, S. (1996) Free Radic. Biol. Med. 20, 553-566[CrossRef][Medline] [Order article via Infotrieve] |