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J Biol Chem, Vol. 274, Issue 26, 18492-18502, June 25, 1999
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From the Laboratory of Food and Biodynamics, Methylglyoxal (MG), an endogenous metabolite that
increases in diabetes and is a common intermediate in the Maillard
reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation of
N On the other hand, as an immunochemical approach to the detection of
these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B)
directed to the MG-modified protein and found that they specifically
recognized the major fluorescent product, argpyrimidine, as the
dominant epitope. The immunohistochemical analysis of the kidneys from
diabetic patients revealed the localization of argpyrimidine in intima
and media of small artery walls. Furthermore, the accumulation of
argpyrimidine was also observed in some arterial walls of the rat brain
after middle cerebral artery occlusion followed by reperfusion. These
results suggest that argpyrimidine may contribute to the progression
of not only long term diabetic complications, such as nephropathy
and atherosclerosis, but also the tissue injury caused by
ischemia/reperfusion.
Nonenzymatic glycation (Maillard reaction) is a complex series of
reactions between reducing sugars and amino groups of proteins, which
leads to browning, fluorescence, and cross-linking of the proteins. The
reaction is initiated with the reversible formation of a Schiff's
base, which undergoes a rearrangement to form a relatively stable
Amadori product. The Amadori product further undergoes a series of
reactions through dicarbonyl intermediates to form advanced glycation
end products (AGEs)1 (1). It
has been shown that the formation of AGEs in vivo contributes to the pathophysiologies associated with aging and the long
term complications of diabetes (2).
A number of aldehydes and ketones, in addition to sugars, are known to
form AGEs. Methylglyoxal (MG), among them, has recently received
considerable attention as a mediator to form AGEs. MG is known to be
formed nonenzymically by amine-catalyzed sugar fragmentation reactions
(3-5) and by spontaneous decomposition of triose phosphate
intermediates in glycolysis (5). It is also a product of the metabolism
of acetol, an intermediate in the catabolism of both threonine (6) and
the ketone body acetone (7). A recent study on the formation of AGEs in
endothelial cells cultured under hyperglycemic conditions indicated
that MG was the major precursor of AGEs (8). Chaplen et al.
(9) have shown that high levels of MG are indeed present in cultured Chinese hamster ovary cells. In addition, increased levels of MG are
also found in blood from diabetic patients and in the lens of
streptozotosin-induced diabetic rats (10, 11). Che et al. (12) have reported that MG induces gene expression of heparin-binding epidermal growth factor-like growth factor by provoking oxidative stress. It is also known that MG is the physiological substrate of the
glyoxalase system, which catalyzes the conversion of MG to
D-lactate via the intermediate
S-D-lactoylgluthathione (13).
MG irreversibly modifies proteins under physiological conditions (14).
The reactions proceed even at physiological concentrations of MG (14)
and form fluorescent products, characteristics of which resemble those
occurring in proteins in aging and diabetes (15). It has been reported
that MG binds and modifies a number of proteins, including bovine serum
albumin (15-18), ribonuclease A (19, 20), lysozyme (21), and collagen
(22). MG modification of protein may be closely associated with
cellular toxicity. It has been shown that MG selectively inhibits
mitochondrial respiration and glycolysis in the cells (23). In relation
to this, MG has been shown to inactivate membrane ATPases and
glyceraldehyde-3-phosphate dehydrogenase, a key enzyme of the
glycolytic pathway (24). The high reactivity of MG with proteins and
its relatively high concentration in the plasma (25) suggest that MG
represents a common intermediate in the formation of AGEs in
vivo. This assumption may be supported by the fact that
macrophages have a specific receptor for proteins modified by reaction
with MG, as well as for glucose-derived AGEs (18). It has been reported
that MG primarily reacts with arginine residues to form
N Here, we report the chemical and immunochemical characterization
of the MG modification of arginine. On the basis of chemical and
spectroscopic evidence, we identify a novel MG-arginine adduct having
the tetrahydropyrimidine structure and also demonstrate that the major
fluorescent product, originally identified as 5methylimidazolone, is argpyrimidine. In addition, as an immunochemical approach to the
detection of MG-arginine adducts in vivo, we raised the
monoclonal antibodies and characterize their specificities. An attempt
to detect antigenic materials in vivo was also made in the
kidneys from diabetic patients and in the rat brain after
ischemia/reperfusion.
Materials
MG (40% aqueous solution), bovine serum albumin (BSA)
(essentially fatty acid-free) and
N General Procedure
Separation of MG-treated
N Reaction of N N HPLC Analysis
Separation was carried out in a Develosil ODS-HG-5 column
(4.6 × 250 mm, Nomura Chemicals Co., Seto, Japan) by applying a linear gradient of 10-50% methanol in 50 mM acetic acid
from 15 to 30 min after the isocratic condition (0.1% methanol in 50 mM acetic acid) for 15 min at a flow rate of 0.8 ml/min.
The eluent was monitored at 230 nm. For preparative HPLC, the reaction
mixture was applied to a Develosil ODS-HG-5 column (8.0 × 250 mm), equilibrated in a solution of 15% methanol in 50 mM
acetic acid, and eluted at a flow rate of 2.0 ml/min. The elution
profiles were monitored by absorbance at 215 nm.
Amino Acid Analysis
BSA (1 mg/ml) in 100 mM sodium phosphate buffer (pH
7.4) was treated with 100 mM MG at 37 °C for 24 h.
After incubation, the reaction mixtures were treated with 10%
trichloroacetic acid. Precipitated proteins were separated by
centrifugation at 10,000 × g for 10 min. The pellets
were collected and washed with 200 µl of diethyl ether. The resultant
pellets were dried and subjected to acid hydrolysis with 6 N HCl at 105 °C for 24 h. The hydrolysate was
concentrated and dissolved with 50 mM sodium citrate buffer (pH 2.2). The amino acid analysis was performed with a JEOL JLC-500 amino acid analyzer equipped with a JEOL LC30-DK20 data analyzing system.
Monoclonal Antibodies
Female BALB/c mice were immunized three times with the
MG-treated keyhole limpet hemocyanin. Spleen cells from the immunized mice were fused with P3/U1 murine myeloma cells and cultured in hypoxantine/aminopterin/thymidine selection medium. Culture
supernatants of the hybridoma were screened using an enzyme-linked
immunosorbent assay (ELISA), employing pairs of wells of microtiter
plates on which were absorbed MG-treated BSA and native BSA as antigen
(1 µg of protein/well). After incubation of 50 µl of hybridoma
supernatants, and with intervening washes with phosphate-buffered
saline, pH 7.8, containing 0.05% Tween 20 (PBS-Tween), the wells were
incubated with alkaline phosphatase-conjugated goat antimouse IgG,
followed by a substrate solution containing 1 mg/ml
p-nitrophenyl phosphate. Hybridoma cells corresponding to
supernatants that were positive on MG-modified BSA and negative on
native BSA were then cloned by limiting dilution. After repeated
screening, three clones were obtained. Among them, clones 3C and 6B
showed the most distinctive recognition of MG-modified BSA.
ELISA
Direct ELISA--
A coating antigen was prepared by incubating 1 mg of BSA with 10 mM aldehydic compounds in 1 ml of 50 mM sodium phosphate buffer, pH 7.2, for 24 h at
37 °C. A 100-µl aliquot of the antigen solution containing 0.4 mg
of protein was added to each well of a 96-well microtiter plate and
incubated overnight at 4 °C. The antigen solution was then removed,
and the plate was washed with PBS-Tween. Each well was filled with 200 µl of 0.5% gelatin solution for 1 h at 37 °C. The primary
antibody was then added to the wells at 100 µl/well of 1 µg/ml
solution for 3 h at 37 °C. The plate was then washed once with
PBS-Tween. After discarding the supernatants and washing three times
with PBS-Tween, 100 µl of a 5 × 103 dilution of
goat anti-mouse IgG conjugated to horseradish peroxidase in PBS-Tween
was added. After incubation for 1 h at 37 °C, the supernatant
was discarded, and the plates were washed three times with PBS-Tween.
Enzyme-linked antibody bound to the well was revealed by adding 100 µl/well 1,2-phenylenediamine (0.5 mg/ml) in 0.1 M
citrate/phosphate buffer (pH 5.0) containing 0.003%
H2O2. The reaction was terminated by the
addition of 50 µl of 2 M sulfuric acid, and the
absorbance at 492 nm was read on a micro-ELISA plate reader.
Competitive ELISA--
For characterization of the antibody, a
competitor was incubated with the antibody for 20 h at 4 °C to
yield competitor/antibody mixtures containing antibody at 1 µg/ml and
variable concentrations of the competitor. A 100-µl aliquot of the
competitor/antibody mixture was added to each well and incubated for
1 h at 37 °C. After discarding the supernatants and washing
three times with PBS-Tween, the second antibody was added, and the
enzyme-linked antibody bound to the well was revealed as described
previously. Results were expressed as the ratio
B/Bo, where B = (absorbance in
the presence of the competitor Animal Experiments
The details of the operative method have been previously
reported (32). Animal experiments were carried out according to a
protocol approved by the Animal Care Committee of Juntendo University. Adults male Wistar rats (SPF) weighing 250-300 g were used (Japan SLC,
Shizuoka, Japan). In anesthetized rats, the right middle cerebral
artery was occluded at its origin by the insertion of a thin nylon
surgical thread via the external and internal carotid arteries
according to the method of Longa et al. (33) with slight modifications. After 3 h of right middle cerebral artery
occlusion, the thread was removed to allow reperfusion. The body
(rectal) temperature was maintained at 37 °C using a heating pad and
lamp-heating. All rats exhibited neurogenic deficits characterized by
severe left hemiparesis, more in the upper extremity, and right
Horner's syndrome, and most of the rats died after 48 h due to
severe brain edema. Five rats were perfused transcardially with 4%
paraformaldehyde in PBS each at 6 and 24 h after reperfusion.
Immunohistochemistry
Human Kidneys--
Formalin-fixed and paraffin-embedded renal
tissues derived from autopsy samples of diabetic and nondiabetic
patients were prepared. The immunohistochemical localization of
argpyrimidine in the tissues was examined by a labeled streptavidin
biotin method using Maxitags immunoenzyme universal kit (Shandon
Lipshaw, Pittsburgh, PA). In brief, the tissue sections were
deparaffinized with xylene and hydrated in a series of increasing water
concentrations in ethanol. The slides were then placed in a chamber
containing 0.3% H2O2 solution in methanol for
20 min at room temperature to inhibit the endogenous peroxidase
activity, followed by the blocking step for 15 min. Subsequently, the
slides were incubated with a 1:100 dilution of anti-argpyrimidine mouse
monoclonal antibody (mAb6B) for 2 h at room temperature. To
perform immunoabsorption experiments, the antibody was preincubated
with and without free N-acetylargpyrimidine (final
concentration, 25 µM) overnight at 4 °C before
application to the tissue section as described above. After the
reaction with primary antibody, the slides were washed with water and
PBS and incubated with biotinylated anti-mouse IgG for 30 min at room temperature, followed by another wash. In the next step, the slides were incubated with peroxidase-conjugated streptavidin for 30 min at
room temperature and then washed with water. The color reaction was
carried out by incubating the slides with freshly prepared
3,3'-diaminobenzidine reagent. After stopping the reaction by washing
the slides with water, the nuclei were counterstained with hematoxylin
for 20 s.
Rat Brain after Middle Cerebral Artery Occlusion Followed by
Reperfusion--
The brains were removed and then postfixed with 4%
paraformaldehyde in PBS. The paraffin-embedded brains were cut coronary at a thickness of 5 mm, deparaffinized with xylene, and dehydrated with
100% ethanol. Immunostaining was performed by the
avidin-biotin-peroxidase complex (ABC) method as previously reported
(32, 34). After blocking endogenous peroxidase activities by treatment
with 3% H2O2 in methanol, the sections were
incubated with 10% normal goat serum in 0.01 M PBS
containing 2% BSA for 10 min at room temperature to block nonspecific
binding. After washing with PBS, the sections were incubated with the
monoclonal antibody (mAb6B) overnight at 4 °C. After being rinsed in
0.01 M PBS, the sections were incubated with biotinylated
anti-mouse IgG (1:100 dilution; Vectastain ABC kit, Vector
Laboratories, Burlingame, CA) for 60 min at room temperature. They were
then incubated with ABC (1:100 dilution; Vectastain ABC kit, Vector
Laboratories) for 1 h. After rinsing, sections were finally
incubated with 0.02% 3,3-diaminobenzidine and 0.03%
H2O2 in distilled water for 7-10 min. To
confirm the specificity of immunostaining, competition experiments were
performed with the mAb6B that was preincubated for 2 h at 37 °C
with an excess of argpyrimidine. Control sections were treated in the same manner with omission of the primary antibodies or with nonimmune rabbit or mouse IgG as a negative control.
Characterization of Major Nonfluorescent and Fluorescent
MG-Arginine Adducts
The selectivity of the binding of MG to arginine residues was
assessed by changes in the amino acid composition of MG-treated proteins. When BSA (1 mg/ml) was incubated with 100 mM MG
for 24 h at 37 °C, about 78% of the arginine and 27% of
lysine residues were lost, and no significant change in other amino
acids was observed. The data suggested that the arginine residues of
proteins represented primary targets for reaction with MG. Hence, to
determine the structure of the MG-arginine adduct generated in the
protein, the reaction of MG with
N Nonfluorescent Products--
The LC-MS analysis of the peaks
(product b) eluted at 22-24 min exhibited the same
molecular weight of 361 (M+H) (data not shown). After purification, the
product was characterized by FAB-MS and 1H and
13C NMR. The FAB-MS on the glycerol matrix showed a
molecular ion at m/z of 361 (M+H) (Fig.
2). High resolution FAB-MS in the
positive ion mode showed a molecular weight of 361.1729 (S.E., +1.6
ppm), that corresponded to the molecular formula of
C14H25O7N4. The 1H NMR spectrum exhibited the following signals:
All of the peaks (product a) eluted at 13-15 min in Fig.
1A showed an absorption maximum at 220 nm and a molecular
weight of 271 upon LC-MS analysis, suggesting that they were a mixture of isomers. The 1H NMR spectrum of the product showed the
following signals; Fluorescent Product--
This compound (product c in
Fig. 1) had absorption maxima at 240 and 340 nm (Fig. 1B).
It was fluorescent, with an excitation at 320 nm and an emission at 380 nm. The 1H NMR spectrum of the product isolated as a major
fluorescent product showed a singlet at 2.60 ppm (f) corresponding to
three protons in D2O (Fig.
4A). The other signals
corresponded to protons of arginine; Department of Neurology,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-acetyl-L-arginine with MG at
37 °C, two nonfluorescent products and one fluorescent product were
detected as the major products. The nonfluorescent products were
identified as the
N
-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine
derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine
adduct having a tetrahydropyrimidine moiety
(N-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine).On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified as
N-(5-methylimidazolon-2-yl)-L-ornithine
(5-methylimidazolone), was found to be identical to
N-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-L-ornithine
(argpyrimidine): (i) the low and high resolution fast atom
bombardment-mass spectrometry gave a molecular ion peak at
m/z of 297 (M+H) and a molecular formula of
C10H25O6N4,
respectively, which coincided with argpyrimidine; (ii) the
1H NMR spectrum of this product in
d6-Me2SO showed a singlet at 2.10 ppm
corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid
chromatography-mass spectrometry with the mode of selected ion
monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 °C for 14 days scarcely generated 5-methylimidazolone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-(5-methyl-4-imidazolon-2-yl)-L-ornithine
(5-methylimidazolone) and
N-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine
(5-hydro-5-methylimidazolone) (26). Shipanova et al.
(27) have recently identified a novel MG-arginine adduct,
N-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-L-ornithine
(argpyrimidine), as a major fluorescent product, and the presence of
this adduct in the human serum and cornea has also been demonstrated
(28). MG also reacts with lysine residues to generate an MG-derived lysine-lysine cross-link (imidazolysine) (29) and
N
-carboxyethyllysine (CEL) (30), which
accumulate with aging and its related diseases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-acetyl-L-arginine were
purchased from Sigma. MG was purified by distillation under reduced
pressure, and the concentration of MG in stock solutions was determined
by HPLC as 2-methylquinoxaline (31). An authentic sample of CEL was
kindly provided by Dr. J. W. Baynes (University of South
Carolina). 3-Deoxyglucosone was kindly provided by Dr. F. Hayase (Meiji
University). Keyhole limpet hemocyanin was obtained from Pierce.
Horseradish peroxidase-conjugated goat IgG fraction to mouse IgG was
obtained from Organon Teknika Co. (Durham, NC).
-acetylarginine was carried out on a Jasco
Gulliver HPLC with a Jasco MD-910 UV-visible photodiode array detector.
Low and high resolution fast atom bombardment-mass spectrometry
(FAB-MS) was measured with a JEOL JMS-700 (MStation) instrument. NMR
spectra were recorded with a Bruker AMX600 (600 MHz) instrument.
Ultraviolet absorption spectra were measured with a Hitachi U-Best-50
spectrophotometer, and fluorescence spectra were recorded with a
Hitachi F-2000 spectrometer. Liquid chromatography-mass spectrometry
(LC-MS) was measured with a Jasco PlatformII-LC instrument.
-Acetyl-L-arginine with
MG
-Acetyl-L-arginine (100 mM) was incubated with MG (100 mM) in 100 mM sodium phosphate buffer, pH 7.4, at 37 °C for 14 days, with adjustment of the pH to 7.4 with sodium hydroxide solution (5 M) as required. The reaction mixture was then
lyophilized to dryness and extracted with ethanol. After evaporation of
the ethanol under vacuum, the residual solid product was purified by
reversed-phase HPLC.
background absorbance (no antibody)) and Bo = (absorbance in the absence of the
competitor background absorbance).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-acetylarginine was carried out. Upon
incubation of N-acetylarginine with MG for 14 days at 37 °C, two nonfluorescent products (a and
b) and one fluorescent product (c) were detected (Fig. 1). After purification, they were
characterized by 1H and 13C NMR, FAB-MS, and
LC-MS along with N-acetylarginine.
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Fig. 1.
HPLC profile (A) and
two-dimensional spectrum (B) of the reaction mixture
of
N -acetyl-L-arginine
with MG. N-Acetyl-L-arginine
(100 mM) was incubated with MG (100 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C for 14 days.
H
(D2O): 1.48 (g, 3H, s), 1.51 (g',
3H, s), 1.53 (f, 3H, s), 1.57 (f',
3H, s), 1.62 (d, 1H, m), 1.68 (d, 1H, m), 1.83 (c, 2H, m),
1.98 (a, 3H, s), 3.24 (e, 2H, t, Jd, e = 6.8 Hz), 3.75 (h, 1H, s), 3.94 (h', 1H, s), 4.22 (b, 1H, m). The
13C NMR spectrum showed the following signals; C
(D2O): 22.5 (C-a), 22.6 (C-f), 25.2 (C-d), 25.4 (C-g), 29.0 (C-c), 41.1 (C-e), 54.4 (C-b), 58.4 (C-l), 63.0 (C-l'), 72.5 (C-h'),
75.4 (C-h), 80.2 (C-m), 81.8 (C-m'), 151.6 (C-k), 174.8 (C-i), 177.1 (C-n', COOH), 178.0 (C-j, COOH), 178.7 (C-n, COOH). Compared with the
1H and 13C NMR spectra between
N-acetylarginine and the product, the
following signals were assigned for the presence of native
N-acetylarginine structure in the product;
H 1.98 (a), 4.22 (b), 1.83 (c), 1.62 (d), 1.68 (d), 3.24 (e); C
22.5 (C-a), 54.4 (C-b), 29.0 (C-c), 25.2 (C-d), 41.1 (C-e), 174.8 (C-i), 178.0 (C-j, COOH), 151.6 (C-k). The 1H and
13C NMR spectra of the product showed the presence of one
methine (h or h'). The 13C NMR spectrum showed the presence
of two quaternary carbons linked with the guanidino group (C-l and C-m)
and one carboxylic acid carbon (C-n or C-n'). It was suggested that the
product had isomers, because C-m was an asymmetric carbon. Each signal
of two methyl (f and g) and methine (h) showed two peaks by the effects
of a chiral center. The methyl protons (f or f') was correlated with C-h (or C-h'), C-l (or C-l') and C-n (or C-n') in the
1H-detected multiple-bond heteronuclear multiple quantum
coherence spectrum (Fig. 3). The
two-dimensional spectra also showed the correlation of another methyl
protons (g or g') and C-m (or C-m'), and of methine (h or h') and C-f
(or C-f'), C-g (or C-g'), C-l (or C-l'), and C-m (or C-m'). Six signals
of carbon (C-f, C-g, C-h, C-l, C-m, and C-n) seemed to have originated
from two molecules of MG at the reaction with the guanidino group in
N-acetylarginine. Based on these
characteristics, it was determined that the purified product was a
novel tetrahydropyrimidine-type MG-arginine adduct,
N-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine (Fig. 3). The product was generated in the yield of 16.0% when N-acetylarginine was incubated with MG
for 14 days at 37 °C. It is also notable that a similar
tetrahydropyrimidine derivative was also isolated from the reaction
mixture of N-benzoylarginine with MG (data
not shown).
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Fig. 2.
FAB-MS spectrum of product
b.
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Fig. 3.
Assignment of spectroscopic data from
1H-detected multiple-bond heteronuclear multiple quantum
coherence spectrum experiments to structure elements of product
b.
H (D2O): 1.40 (f, 3H, d,
Jf, g = 7.2 Hz), 1.64-1.84 (c and d, 4H, m), 2.01 (a,
3H, s), 3.32, 3.65 (e, 2H, m), 4.15 (b, 1H, m), 4.41 (g,
1H, q, Jf, g = 7.2 Hz). The 13C NMR
spectrum showed the following signals; C (D2O): 16.2 (C-f), 22.7 (C-a), 24.3 (C-d), 29.1 (C-c), 40.2 (C-e), 54.9 (C-g), 55.3 (C-b), 157.6 (C-j), 158.6 (C-h), 174.5 (C-i, COOH), 178.6 (C-k). The
FAB-MS on the glycerol matrix in the positive ion mode showed a
molecular ion at m/z of 271 (M+H). The 1H and
13C NMR and FAB-MS indicated that the products were
identical to 5-hydro-5-methylimidazolone derivatives (35). This
compound was formed in a 12.3% yield from the reaction of
N-acetylarginine with MG.
H (D2O): 1.59-1.98
(c and d, 4H, m), 2.18 (a, 3H, s), 3.52 (e, 2H, m), 4.25 (b, 1H, m). 13C NMR spectrum showed the following signals;
C (D2O): 19.0 (C-f), 22.6 (C-a), 26.0 (C-d), 29.4 (C-c),
41.0 (C-e), 52.7 (C-b), 138.8 (C-j), 155.2 (C-i), 156.6 (C-k), 161.0 (C-g), 174.1 (C-h, COOH). These data seemed to be consistent with those
of the 5-methylimidazolone derivative previously reported (14).
However, several lines of evidence suggested that the structure had
been erroneously reported. Low and high resolution FAB-MS gave a
molecular ion peak at m/z 297 (M+H) and a molecular formula
of C10H25O6N4,
respectively, which did not coincide with 5-methylimidazolone but with
N-acetyl-N-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-L-ornithine
(argpyrimidine), which has been recently reported by Shipanova et
al. (27). This was supported by the data that the 1H
NMR spectrum of this product in d6-Me2SO showed
a singlet at 2.10 ppm (f) corresponding to six protons (Fig.
4B). In addition, we noted that the chemical shift of 156.6 ppm (C-k) in 13C NMR spectrum is too far upfield for the
carbonyl carbon of the 5-methylimidazolone ring. The carbon signals
of 138.8 and 156.6 ppm were reasonably assigned to the carbon adjacent
to hydroxy and methyl groups in argpyrimidine, respectively. We
attempted to detect the
N-acetyl-5-methylimidazolone derivative
(molecular weight, 269) by LC-MS with the mode of selected ion
monitoring, but the peak corresponding to the product was not detected
at all (Fig. 5). Furthermore, because the
formation of 5-methylimidazolone has been suggested to involve a
spontaneous autoxidation of the intermediate 5-hydro-5-methylimidazolone (26), we attempted to detect
5-methylimidazolone upon incubation of
5-hydro-5-methylimidazolone in sodium phosphate buffer (pH 7.4) at
37 °C for 14 days; however, 5-methylimidazolone was scarcely
detected by LC-MS (data not shown). Finally, we prepared "5-methylimidazolone" following the method previously reported (14), but the reaction provided one major fluorescent product that was
chromatographically and spectrophotometrically identical to the product
we isolated (data not shown). When these data were taken together, it
was seen that the fluorescent product, putatively identified as
5-methylimidazolone, was identical to argpyrimidine (27). Argpyrimidine
was generated in a 3.95% yield from the reaction of
N-acetylarginine with MG for 2 weeks at
37 °C.
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Fig. 4.
1H NMR spectra of product
c in D2O (A) and in
Me2SO (B).
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Fig. 5.
The LC-MS analysis of the reaction mixture of
N -acetyl-L-arginine
(100 mM) incubated with MG (100 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C for 14 days. The ion mode was positive electrospray. A,
spectrum monitored by the selecting molecular ion at m/z of
297 for detecting argpyrimidine; B, spectrum monitored by
the selecting molecular ion at m/z of 269 for detecting
5-methylimidazolone; C, spectrum monitored by UV absorbance
at 215 nm.
The Monoclonal Antibodies Raised against MG-modified Protein
We demonstrated that MG reacted with the arginine derivatives and
generated a number of adducts, including the
5-hydro-5-methylimidazolone-type and tetrahydropyrimidine-type
nonfluorescent adducts and the argpyrimidine-type fluorescent adduct.
To specifically detect these MG-arginine adducts generated in
vivo, we raised monoclonal antibodies directed to the MG-modified
protein. During preparation of the monoclonal antibody, hybridomas were
selected by comparing the reactivities of the culture supernatant to
MG-modified BSA. Among three clones obtained, clones 3C and 6B showed
the most distinctive recognition of MG-modified BSA. The
immunoreactivity of mAb3C and mAb6B to the protein treated with
aldehydes or ketones was examined by ELISA. As shown in Fig.
6, among the compounds tested, both
monoclonal antibodies recognized not only the MG-modified proteins but
also the proteins treated with the trioses, including hydroxyacetone, dihydroxyacetone, glycolaldehyde, and glyceraldehyde. The result also
revealed that the peroxidation products of polyunsaturated fatty acids
are not the source of antigens. In addition, binding of the MG-modified
protein to both antibodies was scarcely inhibited by the MG-lysine
adducts (imidazolysine and CEL) and the MG-arginine adducts
(5-hydro-5-methylimidazolone and tetrahydropyrimidine-type adduct) but
significantly inhibited by argpyrimidine (Fig.
7), indicating that a dominant epitope of
both monoclonal antibodies is argpyrimidine. When BSA (1 mg/ml) was
incubated with MG for 24 h at 37 °C, there was a progressive
increase in the number of argpyrimidine (from 0 to 0.118 mol/mol of
protein) formed as the concentration of MG in the reaction mixture was
varied from 0 to 100 mM. This result was accompanied by a
progressive increase in the immunoreactivity of the protein with mAb6B.
Antibody reactivity appeared proportional to the yield of argpyrimidine
(Fig. 8).
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Immunohistochemical Detection of Argpyrimidine in Renal Tissues from Diabetic Patients
It has been demonstrated that the rate of MG production increases
during hyperglycemia (36). Hence, the formation of argpyrimidine in vivo was evaluated in the kidneys from diabetic patients
by immunohistochemistry using anti-argpyrimidine monoclonal antibody (mAb6B). We found significant argpyrimidine immunoreactivity in intima
and media of small artery walls of a diabetic kidney (57-year-old male)
(Fig. 9B), whereas vascular
walls of nondiabetic kidneys were scarcely immunostained with the
monoclonal antibody (75-year-old male) (Fig. 9A).
Preadsorption of mAb6B by free
N-acetylargpyrimidine abolished the
immunostaining (data not shown), indicating the specific reactivity of
the antibody with epitopes. It is of interest to note that the staining
patterns of argpyrimidine were almost identical with those of the AGEs
(data not shown). Therefore, long term exposure of tissue proteins with
MG in diabetic patients leads to increased tissue levels of
argpyrimidine, accounting for argpyrimidine deposition in their aortic
tissues.
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Postischemic Accumulation of Argpyrimidine in the Rat Brain
Vascular endothelial injury, which occurs in association with ischemia/reperfusion, inflammation, xenobiotic metabolism, and other circumstances, is believed to play an important role in the initiation and progression of various vascular diseases. We tested whether antigenic materials stained with the anti-argpyrimidine monoclonal antibody (mAb6B) are formed in the rat brain after middle cerebral artery occlusion followed by reperfusion. Histological changes in the rat brain 3 h after middle cerebral artery occlusion followed by reperfusion have been previously reported (37). In brief, after 1 h of reperfusion, some striatal neurons become shrunken when compared with intact neurons, whereas the frontoparietal cortical neurons supplied by the middle cerebral artery do not show any pathological changes. The number of shrunken or triangular neurons increases in the lateral striatam after 3 h of reperfusion, and these atrophic neurons were further increased after 6 h. The infarcted zone spread more widely and is well demarcated. The neurons in the infarcted zone become blurred at 24 h after reperfusion.
Immunoreactivity with mAb6B was not detected in normal rat brain (data
not shown), and it was also not detected at 6 h after reperfusion
(Fig. 10A). The accumulation
of antigenic materials was observed in some arterial walls within the
infarcted zone at 24 h after reperfusion (Figs. 10B and
11). No immunoreactivity was identified
in neuritic plaques, in glial cytoplasm, or nuclei from any cell type.
It was observed that co-incubation with authentic argpyrimidine blocked
the immunoreactivity of mAb6B with arteriolar walls and that nonimmune
mouse IgG gave no immunostaining pattern (data not shown). The
noninfarcted zone in the same section did not show any significant
immunostaining (Fig. 10C). It has been suggested that
reperfusion injury after ischemia is linked to the generation of oxygen
free radicals that have the capacity to initiate the destruction of
biological membranes and cellular structures by activating lipid
peroxidation and altering membrane phospholipids. However, the staining
patterns of argpyrimidine in the brains were inconsistent with those of
lipid peroxidation-specific epitopes that primarily localized in
neurons, glia cells, and neuropils (36), suggesting that the source of
MG in this experimental model of ischemia/reperfusion is independent of
lipid peroxidation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The Nonfluorescent MG-Arginine Adducts--
In the present study,
we identified a novel MG-arginine adduct having a tetrahydropyrimidine
moiety, which is expected to be formed from the reaction of the
guanidino group of arginine with two MG molecules. Formation of this
adduct appears to be reasonably explained by the following mechanism
(Scheme 1). The carbonyl carbon in the
monohydrate form of MG is attacked by the guanidino group of arginine
to form the Schiff's base adduct after dehydration. Another carbonyl
carbon in the monohydrate form of MG is then attacked by the primary
amino group of the Schiff's base intermediate, and the reaction is
completed after dehydration followed by aldose condensation. Although
the detailed mechanism remains unclear, it is postulated that the
secondary amine derivative is an unstable intermediate that
preferentially reacts with MG, leading to the formation of the
tetrahydropyrimidine derivative. Alternatively, it may not be unlikely
that an MG dimer having free aldehyde groups reacts with the guanidino
group of arginine, generating the formation of a condensed-ring
product. To assess the formation of this adduct in proteins, we
attempted to analyze it in the hydrolyzed samples of protein that had
been treated with MG; however, it turned out to be unfeasible because
acid hydrolysis, even after sodium borohydride (NaBH4) treatment of the
adduct, led to quantitative release of the arginine moiety as free
arginine.
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It has been proposed that the 5-hydro-5-methylimidazolone derivative
consists of three tautomeric pairs of isomers that are all
interconvertible by the three processes of tautomerism (35). Such a
tautomerism was also suggested from the reversed-phase HPLC analysis of
the reaction mixture of MG and
N-acetylarginine (Fig. 1) and from the and J values in 1H NMR analysis of the isolated
products. It has been reported that 5-hydro-5-methylimidazolone
competes with MG-treated human serum albumin for binding to receptors
on human monocytes, which provide a convenient monocytic model for
studying receptor-mediated endocytosis of oxidized and glycated
proteins (35). A similar 5-hydro-5-methylimidazolone derivative is also
formed from other 
-oxoaldehydes, such as 3-deoxyglucosone, in
biological systems (38).
The Fluorescent MG-Arginine Adducts--
Lo et al. (14)
have reported that the reaction of MG with arginine residues proceeds
via the reversible formation of a glycosylamine and a
dihydroxyimidazolidine, with slow conversion to form a fluorescent 5-methylimidazolone. Since then, 5-methylimidazolone has been suggested
to represent a common structure in MG-mediated advanced glycation
reactions (26). In vitro glycation of proteins in fact
generates a similar imidazolone derivative,
N-(5-(2, 3, 4-trihydroxybutyl)-4-imidazolon-2-yl)-L-ornithine (39). We
first believed that the major fluorescent MG-arginine adduct should be
5-methylimidazolone (40), because incubation of
N-acetylarginine with MG almost exclusively
generated a single fluorescent product (Fig. 1) and the 1H
NMR data in D2O (Fig. 4A) agreed completely with
those reported previously (14); in particular, the singlet at 2.60 ppm,
corresponding to three protons of 5-methyl group, was decisive.
However, it was doubtful that the aminoimidazolonyl structure of
5-methylimidazolone was fluorescent. This led us to reexamine the
structure of the adduct. Various lines of evidence consequently proved
that the fluorescent product was not 5-methylimidazolone but was
identical to argpyrimidine. A number of points that contradict the
5-methylimidazolone structure are summarized as follows: the low and
high resolution FAB-MS gave a molecular ion peak at m/z of
297 (M+H) and a molecular formula of
C10H25O6N4,
respectively, which coincided with argpyrimidine. The 1H
NMR spectrum of this product in d6-Me2SO showed
a singlet at 2.10 ppm corresponding to six protons (Fig.
4B). The chemical shift of 156.6 ppm in the 13C
NMR spectrum is too upfield for the carbonyl carbon of the
5-methylimidazolone ring, whereas the carbon signals of 138.8 and 156.6 ppm could be reasonably assigned to the carbon adjacent to hydroxy and
methyl groups in argpyrimidine, respectively. The peak corresponding to
the 5-methylimidazolone derivative (molecular weight, 269) was not
detected by LC-MS with the mode of selected ion monitoring (Fig. 5). In
addition to this definitive evidence, we have also observed that the
incubation of 5-hydro-5-methylimidazolone, a putative precursor of
5-methylimidazolone, does not generate 5-methylimidazolone.
Immunochemical Detection of Argpyrimidine-- In this study, we obtained new murine monoclonal antibodies, mAb3C and mAb6B, that clearly distinguished the MG-modified protein from the native protein. It appears that both monoclonal antibodies are specific to argpyrimidine. The observation (Fig. 6) that the anti-argpyrimidine monoclonal IgG cross-reacted with proteins that had been treated with trioses, such as hydroxyacetone, dihydroxyacetone, and glyceraldehyde, suggested that these compounds produced similar derivatives in the protein to those generated in the MG-modified protein. In fact, proteins modified with dihydroxyacetone produce a derivative with identical fluorescence characteristics to MG-modified proteins (15). Alternatively, they may produce MG during incubations, leading to the formation of MG-modified proteins. This may be supported by the previous observations that triose phosphates, such as glyceraldehyde-3-phosphate and dihydroxyacetonephosphate, degrade to MG (5, 41).
The immunohistochemical analysis of the kidneys from diabetic patients
revealed the formation of argpyrimidine in the arterial walls of a
diabetic kidney (Fig. 9), leading to the assumption that the formation
of argpyrimidine is associated with the development of atherosclerosis
in diabetic complications. Westwood et al. (18) have
reported that both MG-modified proteins and glucose-derived AGEs
bind to a common receptor in macrophages. Recently, one of the major epitopes of the anti-AGE antibody has been identified as
N-(carboxymethyl)lysine, which is formed
upon the reaction of lysine residues with glyoxal generated during the
glycation reaction. In addition,
N-(carboxymethyl)lysine has also been
reported as a product of lipid peroxidation (42): a mechanism has been
proposed in which the metal-catalyzed oxidation of polyunsaturated
fatty acids in the presence of protein leads to the formation of
glyoxal, an intermediate formed during lipid peroxidations,
reacting with lysine residues to generate
N-(carboxymethyl)lysine. The observation that
the staining patterns of argpyrimidine in these deposits were almost
identical with those of the
AGEs2 also suggests that
dicarbonyl intermediates, such as MG and glyoxal, may contribute to the
development of atherosclerosis.
Postischemic reperfusion injury is caused by several interacting factors. The immunohistochemical analysis of the rat brain after middle cerebral artery occlusion followed by reperfusion has shown that formation of argpyrimidine occurs in ischemic regions of the brain and that it is localized to the arterial walls (Figs. 10 and 11). These data led to the assumption that MG and/or other trioses may play a significant role in acute arterial injury due to ischemia/reperfusion. These short chain sugars generated during reperfusion may react with matrix tissue or cell surface proteins and result in the alteration of the structure and function of matrix proteins or in the stimulation of cellular responses due to the cross-linking of cell surface proteins. Our preliminary studies have also revealed the formation of argpyrimidine in some arterial walls of human brains.3 It is therefore likely that trioses, such as MG, play an important role in vascular endothelial injury and contribute to the progression of vascular diseases, whereas neurons that are generally considered to be vulnerable to ischemic injury, such as striatal neurons, were negative for argpyrimidine during the reperfusion period. Even as the infarcted zone enlarged with prolongation of the reperfusion period, very few cortical neurons in the ischemic areas were positively stained.
In addition to arginine, lysine residues of proteins are potential targets of dicarbonyl compounds. It has been shown that MG forms a numbers of adducts with lysine residues. Brinkman et al. (43) have described an imidazolium cross-link structure, imidazolysine. This product was also detected in proteins reacted with MG in vitro and in plasma proteins (29). In addition, Ahmed et al. (44) have recently reported a new MG-lysine adduct, CEL, that has also been shown to accumulate with aging and its related diseases. Although we were unable to raise the monoclonal antibody against these MG-lysine adducts, we have recently raised the rabbit polyclonal antiserum consisting of at least one subpopulation that reacts with imidazolysine.4 This specific antiserum may enable us to detect the MG-lysine adduct in vivo.
In summary, we identified a novel MG-arginine adduct
(tetrahydropyrimidine) and also proved that the major fluorescent
product, which had been putatively identified as 5-methylimidazolone,
was argpyrimidine. In addition, we raised monoclonal antibodies
specific for the fluorescent adduct (argpyrimidine). Using the
anti-argpyrimidine monoclonal antibody (mAb6B), the accumulation of
antigenic materials in human atherosclerotic lesions and in the rat
brain after middle cerebral artery occlusion followed by reperfusion
was demonstrated. Because MG is known to be involved in widespread
biological processes, the MG modification of arginine may contribute to
the pathophysiologies associated with aging and its related diseases.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. John W. Baynes (University of South Carolina) for supplying CEL and to Dr. Fumitaka Hayase (Meiji University) for supplying 3-deoxyglucosone. We also thank Izumi Iwamoto (Second Department of Pathology, Kobe University School of Medicine) for technical support on immunohistochemistry.
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FOOTNOTES |
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* This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences.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.
To whom correspondence should be addressed. Tel.:
81-52-789-4127; Fax: 81-52-789-5741; E-mail:
uchidak{at}agr.nagoya-u.ac.jp.
2 S. Miyata, S. Maeda, and K. Uchida, K., unpublished data.
3 N. Hattori, K. Uchida, and Y. Mizuno, unpublished data.
4 T. Oya, T. Osawa, and K. Uchida, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
AGE, advanced
glycation end product;
MG, methylglyoxal;
BSA, bovine serum albumin;
HPLC, high performance liquid chromatography;
FAB-MS, fast atom
bombardment-mass spectrometry;
LC-MS, liquid chromatography-mass
spectrometry;
CEL, N-carboxyethyllysine;
PBS, phosphate-buffered saline;
ELISA, enzyme-linked immunosorbent assay;
mAb, monoclonal antibody.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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N
,
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tetrahydropyrimidine; 
, 5-hydro-5-methylimidazolone; 
,
argpyrimidine; 
, imidazolysine; ×, carboxyethyllysine.
