From the Center for Vision Research, Department of
Ophthalmology and the ** Institute of Pathology, Case Western Reserve
University and University Hospitals of Cleveland, Cleveland, Ohio 44106 and the
Institute of Food Chemistry, Technical University of
Berlin, Berlin 13355, Germany
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ABSTRACT |
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The Maillard reaction, a
non-enzymatic reaction of ketones and aldehydes with amino groups of
proteins, contributes to the aging of proteins and to complications
associated with diabetes. Methylglyoxal (MG) is a 2-oxoaldehyde derived
from glycolytic intermediates and produced during the Maillard
reaction. We reported previously the formation of a lysine-lysine
protein cross-linking structure (imidazolysine) and a fluorescent
arginine modification (argpyrimidine) from the Maillard reaction of MG.
Here we show that rabbit antibodies to MG-modified ribonuclease A
identify proteins modified by the Maillard reaction of glucose,
fructose, ribose, glyceraldehyde, glyoxal, ascorbate, and ascorbate
oxidation products (dehydroascorbate, 2,3-diketogulonate,
L-xylosone, and L-threose) in addition to those
modified by MG. The antibody recognized imidazolysine and argpyrimidine
and a glyoxal-derived lysine-lysine cross-link. It did not react with
N-carboxymethyllysine. Incubations with amino acids
revealed strongest reactivity with
N
-t-butoxycarbonylarginine and MG, and we
identified argpyrimidine as one of the epitopes from this incubation
mixture. Serum proteins from human diabetics reacted more strongly with
the antibody than those from normal individuals, and the levels
correlated with glycemic control. Collagen from human corneas contained
MG-derived modifications, with those from older subjects containing
higher levels of modified proteins than those from younger ones. An
immunoaffinity-purified antibody showed higher reactivity with old
corneas than with younger ones and localized the antigens primarily
within the stromal region of the cornea. These results confirm reported
MG-derived modifications in tissue proteins and show that
dicarbonyl-mediated protein modification occurs during Maillard
reactions in vivo.
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INTRODUCTION |
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The non-enzymatic reaction of aldehydes and ketones with amino groups of proteins via the Maillard reaction forms irreversible adducts on proteins known as advanced glycation end products (AGEs).1 Long lived proteins such as eye lens crystallins and extracellular collagen accumulate significant amounts of AGEs over time (1-3). However, even short lived proteins such as serum and intracellular proteins accumulate smaller amounts of AGEs (4-6).
Immunochemical and chromatographic methods established a direct correlation between tissue age and accumulation of AGEs (7-9). Sell et al. (7) reported an inverse correlation between age and accumulation of skin collagen pentosidine, an AGE found in relatively large amounts in collagen. AGE-mediated cross-linking could contribute to decreased solubility of collagen in the extracellular matrix in humans and animals (8-10). AGE levels are generally enhanced 2-3-fold in individuals with diabetes (11-13), and these compounds may play a role in other diseases as well. Anti-AGE antibodies identified AGEs in diabetic kidneys, lesions associated with atherosclerosis, cataractous lenses, and amyloid deposits (14-16).
Numerous studies indicate a direct relationship between tissue AGEs and the severity of diabetic complications (17, 18). AGE synthesis appears to depend on the degree of glycemia and duration of diabetes (12, 19, 20); in experimental diabetes some synthetic pathways may be triggered by a glycemic threshold (21). In addition, it is claimed that AGEs ingested in food can enhance serum AGE levels and contribute to the development of diabetic nephropathy (22).
Specific receptors for AGEs occur in many cell types and have been implicated in vascular complications of diabetes and Alzheimer's disease (23, 24). For example, AGEs and their receptors may contribute to loss of pericytes during diabetic microangiopathy (25). A recent study showed that AGEs are angiogenic and induce vascular endothelial growth factor in cultured endothelial cells (26). Additional support for a role of AGEs in diabetes comes from experiments in which the blocking of AGE synthesis inhibits diabetic complications (27).
Some investigators believe that it is not the sugar per se but dicarbonyl compounds derived from Maillard reactions, autoxidation of sugars, and other metabolic pathways which cause modification of proteins in diabetes. In this regard, Niwa et al. (28) showed 3-deoxyglucosone-modified arginine residues in tissue proteins. The other dicarbonyls, methylglyoxal and glyoxal, were also shown to modify proteins through the Maillard reaction. Although formation of cross-linking structures by glyoxal occurs in vitro (29, 30), there is still no evidence for such cross-linking in vivo.
Methylglyoxal (pyruvaldehyde) (MG) is an -ketoaldehyde that
originates from dephosphorylation of the glycolytic intermediates, polyol pathway metabolites and acetone (31). Several tissues and plasma
of diabetic individuals exhibit increased levels of MG (31-33), and
even the normal human lens has relatively high levels (34). MG is a
potential player in various pathological processes. It can modify amino
acids, nucleic acids, and proteins (31, 35, 36), including numerous
intra- and extracellular membrane proteins (31). A few MG-derived
modifications have been defined chemically. Binding of MG-modified
proteins to receptors on macrophages (37) and monocytic (THP-1) cells
can induce cytokines such as interleukin-1
(38) and
monocyte-macrophage colony-stimulating factor (39). We reported
formation of an MG-derived lysine-lysine cross-link in proteins and
showed that it could be formed in vivo (40). We recently
identified a major fluorescent arginine adduct derived from MG, but we
have not yet shown it in tissue proteins (41). Ahmed et al.
(42) identified carboxyethyllysine, a lysine modification of MG in
human lens proteins, and showed that it accumulated with aging.
In addition to its protein-altering effects, MG is a potential mutagen. Carboxyethylguanine was identified as a modification caused by MG (43), and other studies indicate that MG can damage cells (31). Taken together, these observations suggest that increased MG concentrations in diabetes alter various macromolecules, which, in turn, contribute to long term complications of diabetes. Additionally, chronic exposure to MG may deteriorate protein structure and cause progressive protein aging and age-associated impairments.
To understand better the role of MG in protein modifications during aging and diabetes, we developed antibodies that recognize MG-derived modifications in human tissues and serum. These antibodies enabled us to identify MG-mediated Maillard reactions in vivo and to define further specific epitopes involved in the modified proteins.
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EXPERIMENTAL PROCEDURE |
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Chemicals
Bovine serum albumin (BSA), ascorbate, L-threose, MG (40% aqueous solution), Pronase E (from Streptomyces griseus), leucine aminopeptidase M (type IV-S from porcine kidney microsomes), carboxypeptidase A (type II from bovine pancreas), collagenase (type VII from Clostridium histolyticum), rabbit IgG, and pepsin (from porcine stomach mucosa) were obtained from Sigma. Ribonuclease A was from Boehringer Mannheim. Glyoxal (40% w/v) and dehydroascorbate were from Aldrich. 3-Deoxyglucosone was synthesized according to Madson and Feather (44). 2,3-Diketogulonate was synthesized by the method of Otsuka et al. (45). Xylosone was a gift from Dr. Milton Feather, University of Missouri, Columbia, MO. MG was purified by vacuum distillation (46). All other chemicals were commercially available reagents. Human corneas were kindly supplied by the Cleveland Eye Bank. Human plasma samples were obtained from the University Hospitals of Cleveland and volunteers.
N-Carboxymethyllysine was a generous gift from Dr. John
Baynes, University of South Carolina. Imidazolysine (also known as MOLD
(methylglyoxal-lysine dimer) and GOLD (glyoxal-lysine dimer) were
purified by methods described elsewhere (40, 47).
Modification of RNase A and BSA
BSA and RNase A in PBS (both 100 mg/ml) were incubated with 50 mM MG (purified as described above) for 3 weeks at 37 °C under sterile conditions. Unbound MG was removed by dialysis against PBS, and the proteins, BSA-MG and RNase-MG, were lyophilized. Incubations were done over different time periods with BSA (50 mg/ml of 0.2 M sodium phosphate buffer, pH 7.4) and different concentrations of MG (0, 5, 10, and 25 mM). Aliquots were withdrawn on days 0, 5, 10, 15, and 30 and dialyzed against 2 liters of PBS for 24 h.
Immunization of Rabbits
New Zealand White rabbits were immunized by subcutaneous injection with a total of 1.0 mg each of RNase-MG emulsified with Freund's complete adjuvant and boosted with injections in Freund's incomplete adjuvant every 2 weeks. After the fourth booster injection, the rabbits were bled, and the serum was separated. The IgG fraction was purified on a protein-G Sepharose column (Pharmacia Biotech Inc.) according to the manufacturer's instructions.
Purification of Antibodies for MG-derived Modifications
BSA-MG was coupled to EAH-Sepharose (Pharmacia) as recommended by the manufacturer. Briefly, 25 mg of BSA-MG in 4.0 ml of water was adjusted to pH 7.0 with 1 N HCl. This solution was added to a 3.25-ml suspension of EAH-Sepharose containing 40 mg of EDC. The mixture was shaken at room temperature for 2 h followed by incubation at 4 °C overnight. The gel was then packed in a column and washed twice, first with 30 ml of 0.1 M sodium acetate buffer (pH 4.0; buffer A) and then with 30.0 ml of 0.1 M Tris/HCl (pH 8.0; buffer B), each containing 0.5 M NaCl. The gel was finally washed with 80.0 ml of PBS. The IgG fraction (obtained as described above) (0.5 ml) was mixed with the gel and reacted at 4 °C overnight while shaking. The gel was loaded onto a column and washed with 40.0 ml of PBS. 1-ml fractions were collected, and the absorption at 280 nm was recorded. When the absorbance reached base line, the column was eluted with 0.2 M glycine-HCl buffer (pH 2.28), and 1-ml fractions were collected in glass tubes containing 150 µl of 1 M Tris-HCl (pH 9.0) to bring the pH to neutral. The protein-containing fractions were pooled and dialyzed overnight against 2 liters of PBS and concentrated by ultrafiltration using a 10,000 molecular weight cutoff filter (Amicon, Inc., Beverly, CA). The column was reused after sequential washing with buffers A and B.
Enzyme-linked Immunosorbent Assay (ELISA)
Microtiter plates were coated with 1 µg/well BSA-MG in PBS for 2 h at room temperature in a humid chamber. The wells were blocked with 300 µl of 3% BSA in PBS for 1.5 h and washed twice with PBS. Direct ELISA used 50 µl of the diluted antibody, and competitive ELISA used diluted antibody that was preincubated with the test sample for 1 h at 37 °C before addition to the plate. After incubation for 2 h and washing four times with PBS, the plates were developed for 2 h with 1:60,000 diluted goat anti-rabbit antibody coupled to alkaline phosphatase, washed four times with PBS, and incubated with 100 µl of substrate (p-nitrophenylphosphate) in 1.0 M diethylamine with 0.5 M MgCl2 and 0.2% sodium azide (pH 9.8). Samples were incubated for 1.0-1.5 h at 37 °C until A410 nm reached 0.6-0.8. The plates were read on a Dynatech microplate reader (model MR 5000, Dynatech Laboratories, Inc., Chantilly, VA).
Incubation of Amino Acids with Carbohydrates
50 mM N-acetyllysine or
N
-acetylarginine alone or in combination was incubated
with a 50 mM concentration of the designated carbohydrate
in 0.2 M sodium phosphate buffer (pH 7.4) at 37 °C for
0, 3, 7, and 21 days. Carbohydrates were glucose, fructose, 3-deoxyglucosone, D-ribose, DL-glyceraldehyde,
MG, glyoxal, ascorbate, dehydroascorbate, 2,3-diketogulonate,
L-xylosone, and L-threose.
Incubation of BSA with Carbohydrates
BSA (50 mg/ml) in 0.2 M sodium phosphate buffer (pH 7.4) was incubated with 100 mM glucose, fructose, ribose, MG, glyoxal, glyceraldehyde, ascorbate, dehydroascorbate, 2,3-diketogulonate, or L-threose at 37 °C. The samples were incubated for 2 weeks, except those containing glucose and fructose, which were incubated for 1 month. The protein from each incubation was dialyzed against PBS for 48 h and lyophilized.
Immunoreactivity of Human Proteins
Corneal Collagen-- Collagen was isolated from human corneas by scraping off the endothelial and epithelial layers and mincing the remaining tissue. The mince was suspended in 3.0 ml of PBS and homogenized in a Polytron homogenizer (Brinkmann Instruments) three times for 10 s each at a speed setting of 8. This homogenate was lyophilized, and the dried sample was extracted twice with 1.5 ml of 0.05 M Tris-HCl (pH 7.5) and 1 M NaCl by stirring overnight at room temperature and centrifugation at 15,000 rpm in an Eppendorf microcentrifuge (Brinkmann Instruments). The supernatant fraction was discarded from each extraction. The pellet was then extracted twice with 3.0 ml of 0.5 M acetic acid (stirring at 4 °C) followed by centrifugation at 20,000 × g. The supernatant fraction was discarded, and the pellet was lyophilized, reconstituted in 10% pepsin (w/w) in 0.4 M acetic acid, and stirred overnight at 4 °C. Centrifugation at 20,000 × g for 90 min separated the pepsin-soluble fraction (supernatant) and the pepsin-insoluble fraction (pellet). The pepsin-insoluble fraction was then digested with 1% collagenase (w/w) in 0.02 M HEPES containing 0.1 M CaCl2 at 37 °C for 16 h. The digested material was filtered through a 10,000 molecular weight cutoff centrifugal filter (Gelman Sciences). Amino acid estimation, with L-leucine as the standard, was done by the ninhydrin reaction method of Moore and Stein (48). Fractions corresponding to 0.2 µmol of leucine equivalent were used for competitive ELISA in which the antibody was reacted with corneal collagen extract for 1 h at 37 °C before addition to the microplate.
Immunofluorescent Staining of Human Cornea-- Human corneas were fixed with 4% paraformaldehyde, embedded in paraffin, and 5-µm sections were cut in a microtome. The sections were deparafinized with xylene and hydrated in a series of increasing water concentrations in ethyl alcohol. They were allowed to stand in water for 3 min and washed twice with PBS before blocking with 10% goat serum for 1 h in a humid chamber. The sections were then reacted overnight at 4 °C with IgG specific for MG modifications (1:500 dilution in 10% goat serum), washed three times (5 min each time) with PBS, and incubated with 20 µg/ml rhodamine isothiocyanate-conjugated goat anti-rabbit IgG (Sigma) for 1 h in a humid chamber. The sections were washed three times (5 min each time) with PBS and then further developed for 60 min with fluorescein isothiocyanate conjugated to wheat germ agglutinin (Sigma) (2.5 µg/ml). After washing three times (5 min each time) with PBS, the specimens were equilibrated for 10 min in Slow Fade buffer (Molecular Probes, Eugene, OR), stabilized with one drop of Slow Fade antifading reagent, and mounted in Gel/Mount (Biomeda Corp., Foster City, CA). They were examined at a magnification of × 20 with a Zeiss LSM 410 scanning microscope (Carl Zeiss, Germany) equipped with an Axiovert 100 inverted microscope, an argon-krypton external laser, and rhodamine and fluorescein isothiocyanate filters. Images were processed with Adobe Photoshop 4.0 software for the Power Macintosh computer.
Human Plasma Proteins--
Serum samples were obtained
from 12 normal subjects (age range 22-50 years) and 12 diabetic
patients (age range 45-75 years). Diabetic sera had glycated
hemoglobin levels of more than 10% (range 10-18%), and controls had
levels between 3.5 and 5%. Serum proteins were precipitated with an
equal volume of cold 10% trichloroacetic acid, and the precipitate was
treated with 5.0 ml of diethyl ether (5 min). The ether was evaporated,
and the residue was desiccated and stored at 80 °C. The proteins
(5 mg) were digested with Pronase E (2% w/w in 0.5 ml of PBS) for
16 h followed by another addition of Pronase E (2% w/w) and
further incubation for 8 h. Further digestion was accomplished by
51 units of carboxypeptidase A and 50 units of leucine aminopeptidase
M. The final digested material was filtered through a 10,000 molecular
weight cutoff filter (Gelman Sciences). Amino acid estimation was by
the ninhydrin reaction as described above. 100-µl samples were taken
from each specimen for competitive ELISA.
Western Blotting-- Human serum protein samples were electrophoresed on a 12% SDS-gel (60 min, 40 mA, 13 µg of protein/lane). The proteins were transferred electrophoretically to a nitrocellulose membrane (Bio-Rad) for 2 h at 90 V. The membrane was blocked with 5% non-fat dry milk (blotto) in TBST (Tris-buffered saline and Tween 20) buffer (pH 7.0) overnight at 4 °C, reacted for 2 h with the immunoaffinity-purified antibody (1:2,500 diluted in blotto), washed three times (15 min each time) with TBST buffer, incubated for 1 h in blotto with 1:30,000 diluted anti-rabbit IgG coupled to horseradish peroxidase (Sigma), washed 8-10 times (10-15 min each time) with TBST buffer (10 min each time), then incubated with a 1:1 mixture of solution A and B (Supersignal chemiluminescence substrate kit; Pierce) for 3-5 min and exposed to x-ray film (reflection/autoradiography film from NEN Life Science Products).
Statistical Analysis-- Data were analyzed by Student's t test, and the results were expressed as mean ± S.D.
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RESULTS |
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Antibody Characterization
BSA-MG Incubation-- To establish specificity of the antibodies for MG-derived protein modifications, microplates were coated with 1 µg of BSA modified with 0, 5, 10, and 25 mM MG. The immunoreactivity of BSA-MG was directly related to the fluorescence (excitation/emission 370/440 nm) of the protein (Fig. 1, upper panel), suggesting that antibody binding is directly proportional to AGE formation and that the fluorescent AGEs are antigenic epitopes. As can been seen in Fig. 1, lower panel, the immunoreactivity increased with time of incubation and MG concentration. The reaction was rapid initially at all three concentrations of MG and was maximal after 15 days. Thereafter, the slight decrease in reactivity might indicate extensive modification of the protein with decreased availability of epitopes. Nonspecific binding to carrier protein is unlikely because the plate was coated with BSA-MG, and the antibodies were directed against RNase-MG.
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Amino Acid Incubations--
Mixtures of
N-acetyllysine or N
-acetylarginine with
various sugars, MG, glyoxal, and ascorbate oxidation products helped us
to characterize the antibody with competitive ELISAs. In the incubations with N
-acetyllysine, the antibody was
inhibited slightly (10-20%) in incubation mixtures containing
glucose, fructose, ascorbate, dehydroascorbate, and 2,3-diketogulonate
and more in samples containing ribose, L-threose, MG, and
glyoxal (10-30%) (Fig. 2A).
Samples combining N
-acetylarginine with carbohydrates showed varying degrees of inhibition. The greatest inhibition was
achieved in samples with MG (Fig. 2B). A rapid formation of reactive antigens occurred in samples with MG, as revealed by 74%
inhibition with the sample taken out immediately after starting the
reaction. This inhibition declined to 62% on day 3 and stayed almost
at that level throughout the incubation. Inhibition was also observed
in samples containing glyceraldehyde (63% after 21 days of
incubation); reactive antigens formed within 3 days and remained almost
at that level. Other carbohydrates tested showed varying degrees of
inhibition from 0 to 52%.
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BSA Incubation with Sugars and Ascorbate Oxidation Products-- To establish whether MG-derived modifications on proteins serve as epitopes, we incubated BSA with various carbohydrates. The inhibition pattern is shown in Fig. 4. BSA alone caused little inhibition throughout the incubation, but incubation with MG and glyceraldehyde produced antigens that caused almost 100% inhibition with 1-mg test samples. All ascorbate oxidation products caused equivalent inhibition, but incubation with glucose produced more antigens than in incubations with ribose.
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Structurally Characterized AGEs--
The antibodies were tested
for immunoreactivity with our structurally characterized MG-derived
AGEs. N-Carboxymethyllysine did not react, even at a
concentration as high as 50 nmol (Table I). The antibody recognized both
imidazolysine and argpyrimidine, but it was 94 times more effective
with argpyrimidine than imidazolysine at a concentration of 25 nmoles.
GOLD also served as a weak antigen; its inhibition was about 42 times
lower than argpyrimidine. Taken together, these results suggest that
arginine modification(s) are the major antigenic epitopes for the
antibody.
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MG-derived Modification in Human Proteins
Corneal Collagen-- Human corneal collagen digests became turbid upon incubation in PBS during ELISA. However, when HEPES buffer (pH 7.4) was substituted for PBS, they remained clear. As can been seen in Fig. 5, old corneas showed higher immunoreactivity than young ones, indicating a trend toward an increase in AGEs with age.
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Human Plasma Proteins-- Human plasma protein digests contained reactive antigens as assessed by the competitive ELISA. Because the digests were filtered through a centrifugal filter with a molecular weight cut-off of 10,000, it is unlikely that these preparations contain enzymes that could cleave the antibody during incubation. Fig. 7A shows that plasma proteins from diabetic donors were significantly more inhibitory (38-88% inhibition) than their normal counterparts (15-48%) (p < 0.0001). The diabetic samples were from poorly controlled diabetic donors whose glycated hemoglobin levels ranged from 10 to 18%. Glycated hemoglobin levels were related linearly to immunoreactivity (p = 0.0002) as shown in Fig. 7B, suggesting that MG-mediated modification of plasma proteins is influenced by the extent of glycemia.
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DISCUSSION |
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The purpose of this study was to identify overall protein
modification by MG in aging and diabetes with specific antibodies and
to determine the chemical nature of antigenic epitopes. Previous studies determined the chemical structure of only a few MG products of
the Maillard reaction. Two lysine modifications, imidazolysine (also
known as MOLD) and carboxyethyllysine have been described and
identified in vivo (40, 42). The immunoreactivity of the antibodies with N-acetyllysine and MG incubation
may in part be due to carboxyethyllysine. However, when MG reacts
with proteins, arginine is predominately modified. Structure of three
arginine modifications, argpyrimidine (41), 5-hydro-5-methylimidazolone and 5-methylimidazolone (49) have been reported, and recently Uchida
et al. (50) identified 5-methylimidazolone in
vivo. In addition to arginine and lysine, MG can modify cysteine
residues (35), but the chemical nature of the products remain
unknown.
One difficulty in detecting arginine modifications is that they are invariably acid-labile; enzyme digestion is most often used to detect them. However, highly modified proteins that contain large amounts of AGEs are often digested poorly by enzymes. We used antibodies to circumvent these problems and to study Maillard reactions in vivo. Immunological methods enabled us to detect MG-derived modifications in modified proteins without digestion.
It is not surprising that arginine residues are major epitopes for
the antibody because dicarbonyls like MG have a high propensity to
react with arginine residues. In fact, reaction of dicarbonyl compounds
with proteins has been employed for many years as a means of modifying
arginine residues. To study such modifications further, we tried to
purify products from incubation mixtures of
N-t-butoxycarbonylarginine and MG. We detected
only three ninhydrin-reactive products from the reaction, where
argpyrimidine was the major fluorescent product. Henle et
al. (51) and Westwood et al. (49) reported that
5-methylimidazolone is one of the major products of this reaction, and
we assumed that it could be one of the epitopes for the antibody.
However, we were unable to detect or isolate this product by our
purification procedure.
The formation of antibody-reactive products by several sugars and ascorbate oxidation products suggests that MG is a common intermediate in Maillard reactions involving these carbohydrates. Antibody recognition of products structurally related to MG modifications is unlikely in our experiments, but we cannot rule it out entirely. We propose that MG forms during the Maillard reaction and sugar oxidation as shown in Fig. 8. In support of this scheme, several other studies indicate formation of MG during sugar autoxidation and Maillard reactions (52, 53). Shinohara et al. (54) reported that inhibition of glyoxalase in cultured cells under hyperglycemic conditions decreases formation of AGEs. Further, the receptor for the MG-modified protein can also bind a glucose-modified protein (55). Finally, Uchida et al. (50) showed that the antibodies against 5-methylimidazolone also react with sugar-modified proteins. In addition, we found that argpyrimidine could be synthesized by other sugars and ascorbate oxidation products in addition to MG.
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The rapid formation of MG-derived modifications in mixtures of BSA or
N-acetylarginine with DL-glyceraldehyde is
not unexpected. Dehydration of glyceraldehyde could lead to the
formation of MG. The next best precursors of MG-derived modifications
were ribose and L-threose, suggesting that these sugars
could be a source of MG in vivo. This coincides with our
previous observation that for argpyrimidine synthesis
DL-glyceraldehyde is the next best precursor after MG, and
both D-ribose and L-threose are also effective. However, the mechanism of MG formation from D-ribose and
L-threose remains unknown.
The formation of reactive antigens in a mixture of BSA + glyoxal was
unexpected, although glyoxal appears to be formed during Maillard
reactions of MG. The antibody is unlikely to recognize glyoxal-derived
N-carboxymethyllysine since our experiments indicate that
it is not a reactive epitope (Table I). Glyoxal-derived arginine
modification(s) are more likely candidates.
Our demonstration of immunoreactive antigens in plasma proteins and corneal collagen further confirms that MG-mediated Maillard reactions occur in vivo. Corneal collagen accumulates AGEs with age and diabetes, as revealed by an increase in collagen-bound fluorescence and pentosidine levels (56-58). Our study takes this observation a step further by showing that MG-derived products contribute to accumulation of AGEs in this tissue.
Identification of antibody-reactive substances in plasma proteins and their increase in diabetes along with a positive correlation with glycated hemoglobin further underscore the importance of MG-derived modifications in vivo. These results support previous findings that MG levels in red blood cells correlate with the degree of glycemia (59). Although imidazolysine might contribute to the immunoreactivity with plasma proteins, it is not a major epitope for the antibody and could not account entirely for the observed results.
Dicarbonyl compounds are major intermediates in Maillard reactions, and aminoguanidine, an inhibitor of AGE formation, is thought to work through its reaction with dicarbonyl compounds. We assume, therefore, that inhibition of AGE formation from aminoguanidine is in part because of its reaction with MG.
One obvious question is what is the significance of Maillard reactions by MG when MG is present only in nanomolar amounts? First, the actual tissue concentration of MG may be much higher than reported because the rapid reaction of MG with proteins might deplete free MG levels. Second, MG may be more effective than glucose in modification of proteins because reported MG levels (256 ± 92 nM in healthy controls and 479 ± 49 nM in diabetics) (33) exceed the open chain reactive form of glucose (about 100 nM in healthy controls and 200-500 nM in diabetics). Finally, MG can be produced by various mechanisms, many of which depend on the degree of glycemia, such as glycation, sugar autoxidation, and the polyol pathway. All of these mechanisms are likely to be enhanced in diabetes. Thus, our findings in this study support the notion that MG plays a significant role in protein modifications in vivo and suggest that it contributes to complications of diabetes.
If MG is such a rapid modifier of protein, how do cells manage to survive MG formation? Several enzymes may modulate MG-mediated reactions; glyoxalase, aldose reductase, and aldehyde reductase metabolize MG to less toxic or inactive products (60-62). However, any decline in the function of these enzymes, alterations in their synthesis or post-translational modifications, as well as loss of required cofactors could shift the balance toward MG-mediated Maillard reactions. Contrary to this view, Ratliff et al. (63) reported increased levels of MG-metabolizing enzymes in mononuclear and polymorphonuclear cells from insulin-dependent diabetic patients who had developed complications. It is not clear whether this results from an attempt to cope with elevated MG levels or whether it signals a concomitant elevation in MG levels and enzymes as part of the pathological process. However, Beisswenger et al. (64) noted decreased MG metabolism (through glyoxalase) with progression of retinopathy in type I diabetic patients.
In summary, this study provides immunochemical evidence for MG-mediated protein modifications in vivo. This is a primary step toward determination of how such modifications affect age and diabetes-associated complications.
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ACKNOWLEDGEMENTS |
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We thank the Cleveland Eye Bank for supplying human corneas and Adrian Valeriu for performing NMR analyses.
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FOOTNOTES |
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* This study was supported in part by United States Public Health Service Grant EY 09912 (to R. H. N.), P-30 EY11373 Core Grant to the Case Western Reserve University Visual Science Center from the National Eye Institute, and in part by Research to Prevent Blindness, New York.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.
§ Recipient of a postdoctoral fellowship from Juvenile Diabetes Foundation International, New York.
¶ Recipient of a fellowship from the Hartford Foundation/American Federation of Aging Research Medical Student Geriatrics Scholars Program, New York.
To whom correspondence should be addressed: Dept. of
Ophthalmology, Wearn Bldg., Rm. 637, Case Western Reserve University, Cleveland, OH 44106. Tel.: 216-844-1132; Fax: 216-844-5812; E-mail: nhr{at}po.cwru.edu.
1 The abbreviations used are: AGEs, advanced glycation end products; MG, methylglyoxal; BSA, bovine serum albumin; MOLD, methylglyoxal-lysine dimer; GOLD, glyoxal-lysine dimer; PBS, phosphate-buffered saline; EDC, N-ethyl-N-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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