(Received for publication, November 6, 1995; and in revised form, January 23, 1996)
From the
The reaction of long lived proteins with reducing sugars has been implicated in the pathophysiology of aging and age-related diseases. A likely intranuclear source of reducing sugar is ADP-ribose, which is generated following DNA damage from the turnover of ADP-ribose polymers. In this study, ADP-ribose has been shown to be a potent histone glycation and glycoxidation agent in vitro. Incubation of ADP-ribose with histones H1, H2A, H2B, and H4 at pH 7.5 resulted in the formation of ketoamine glycation conjugates. Incubation of histone H1 with ADP-ribose also rapidly resulted in the formation of protein carboxymethyllysine residues, protein-protein cross-links, and highly fluorescent products with properties similar to the advanced glycosylation end product pentosidine. The formation of glycoxidation products was related to the degradation of ketoamine glycation conjugates by two different pathways. One pathway resulted in the formation of protein carboxymethyllysine residues and release of an ADP moiety containing a glyceric acid fragment. A second pathway resulted in the release of ADP, and it is postulated that this pathway is involved in the formation of histone-histone cross-links and fluorescent advanced glycosylation end products.
Both intracellular and extracellular proteins are subject to a
variety of non-enzyme-catalyzed chemical modifications (reviewed in (1) ), which can adversely affect function. The accumulation of
chemical modifications in long lived proteins has been implicated in
the pathophysiology of aging (2, 3) and a number of
specific diseases, including diabetes (4, 5) and
Alzheimer's disease(6, 7) . Two interrelated
protein modifications that have received much recent attention are
glycation and oxidation, which lead to the formation of protein
glycoxidation products(2, 3, 8) . Glycation
is initiated by the reaction of a reducing sugar with a protein amino
group to generate Schiff base adducts (9, 10) that can
undergo the Amadori rearrangement to form ketoamine
adducts(11, 12, 13, 14, 15) .
Many of the ketoamine adducts are unstable, and by a complex chemistry
that involves oxidation, glycation often leads to protein glycoxidation
products that have been termed
AGE()(2, 3) . The AGE include protein
carboxymethyllysine (CML) residues (11) and a heterogeneous
group of complex modifications characterized by their high fluorescence
and ability to cause protein-protein
cross-links(3, 16) .
Glucose is assumed to be a major source of glycation and glycoxidation of extracellular proteins in vivo based on its abundance and association with diabetic complications(3, 4, 5) . The sugar sources for the glycoxidation of intracellular proteins are less well understood, but pentoses have been implicated, because they are efficient precursors for the formation of fluorescent AGE(16) . A likely intracellular source of a reducing pentose moiety is ADP-ribose, which is generated from NAD by multiple metabolic pathways (Fig. 1). The cell nucleus is a likely site for ADP-ribose modifications as oxidative stresses and other conditions that cause DNA strand breaks stimulate the synthesis of nuclear polymers of ADP-ribose, which are rapidly turned over generating ADP-ribose in close proximity to the long lived histones rich in lysine residues(17, 18) . Additionally, ADP-ribose can be generated in other cell compartments by the turnover of cyclic ADP-ribose (19, 20) and by the removal of ADP-ribose from proteins modified by the action of protein-mono-ADP-ribosyltransferases(21) .
Figure 1: Pathways for the generation of free ADP-ribose and possible metabolic fates within cells. ADP-ribose is abbreviated ADPR.
The evaluation of a possible role of ADP-ribose in protein glycation is challenging, since the enzyme-catalyzed modification of proteins by ADP-ribose occurs at several different amino acid residues(21, 22) . To distinguish between glycation and enzymatic modification of proteins by ADP-ribose, we have previously prepared model conjugates for ADP-ribose glycation and determined properties that allow the glycation adducts to be distinguished from enzymatic modifications(22) . We report here that ADP-ribose is much more efficient than other possible pentose donors for glycation and glycoxidation of histones. Our results also suggest that previous reports of histone modifications in vivo may represent glycation and glycoxidation reactions initiated by ADP-ribose.
Figure 11: Analysis by reversed-phase HPLC of fluorescent products derived from incubation of ADP-ribose with histone H1. The glycated histone H1 was subjected to acid hydrolysis prior to analysis. The elution position of a pentosidine standard (P) is shown by the arrow. The differences in elution times for pentosidine in Fig. 10and Fig. 11are due to the use of different reversed-phase columns (see ``Experimental Procedures'').
Figure 10:
Analysis by reversed-phase HPLC of
fluorescent products formed by incubation of ADP-ribose with N--Boc arginine (Chromatogram A), N-
-Boc
lysine (Chromatogram B), or both N-
-Boc arginine and N-
-Boc lysine (Chromatogram). The N-
-Boc
protecting groups were removed prior to HPLC analysis. The elution
position of a pentosidine standard (P) is shown by the arrow.
A Varian VXR-400
NMR spectrometer operating at 399.95 MHz for H and 100.58
MHz for
C was used to acquire NMR spectral data. Samples
were lyophilized three times in D
O prior to NMR analysis
and dissolved in D
O at a concentration of approximately 10
mM.
C NMR spectral parameters were as follows:
sweep width, 22,371.36 Hz; acquisition time, 1 s; acquisition delay, 1
s; transients, 70,000 in double precision mode.
C was
referenced using 3-(trimethylsilyl)propionic-2,2,3,3-d
acid, sodium salt. The
C spectrum of G-ADP showed
the following absorptions:
179.98, 155.93, 152.20, 151.80,
143.66, 121.31, 90.04, 86.75, 77.13, 73.03, 71.18, 67.88 ppm.
For analysis of G-ADP by mass spectroscopy, a Kratos CONCEPT 1H two-sector instrument was used. Glycerol was used as a matrix for positive and negative ion determinations, and the sample was spiked with cesium iodide.
For glycation of histones by
[P]ADP-ribose, incubation mixtures (100 µl)
contained 50 µM [
P]ADP-ribose (2.7
10
dpm), 1 mg/ml histone, 50 mM sodium
pyrophosphate, 50 mM potassium phosphate buffer, pH 7.5, with
incubation overnight at 37 °C. After incubation, trichloroacetic
acid was added to 20% (w/v), the mixtures were placed on ice for 15 min
and acid insoluble material was collected by centrifugation. The
pellets were dissolved in 50 mM sodium acetate buffer, pH 5.0,
and applied (Fig. 5) to 15% acid-urea acrylamide gels (24) or used for chemical stability studies (Fig. 6).
Figure 5:
Electrophoretic analysis on acid-urea gels
of histones incubated with [P]ADP-ribose.
Coomassie Blue staining is shown in the left panel, and the
autoradiogram is shown in the right
panel.
Figure 6:
Analysis by reversed-phase HPLC of
radiolabel released at pH 9.0 from histone H1 glycated by
[P]ADP-ribose. The elution positions of G-ADP,
ADP, and AMP are shown.
For glycation of histone H1 by ADP-ribose containing radiolabel in
different positions (see Results), reaction mixtures (100 µl)
contained 500 µM ADP-ribose with either C in
the reducing ribose (1.2
10
dpm) or
P
in the adenosine proximal phosphate (8.2
10
dpm), 1
mg/ml histone H1, 50 mM sodium pyrophosphate, 50 mM potassium phosphate buffer, pH 7.5. The mixture was incubated for
5 h at 37 °C. Following incubation, trichloroacetic acid was added
to 20% (w/v), the mixtures were placed on ice for 15 min and acid
insoluble material was collected by centrifugation. The pellets were
dissolved in 50 mM sodium acetate buffer, pH 5.0 and
radiolabel was determined by liquid scintillation counting.
To examine for the presence of histone H1 cross-linking (Fig. 10), reaction mixtures (1.5 ml) contained 0.67 mg/ml histone H1, 500 µM ADP-ribose, 0.025% SDS, 50 mM sodium acetate, adjusted to pH 9.0 with NaOH, with incubation for 2 h at 37 °C. Following incubation, samples containing approximately 5 µg of protein were applied to 12% SDS-PAGE gels for electrophoresis. Following electrophoresis, gels were stained with Coomassie Blue.
To examine for the fluorescent products formed from histone H1 and ADP-ribose by HPLC (Fig. 11), a reaction mixture (10.0 ml) contained 500 µM ADP-ribose, 0.67 mg/ml histone H1, 0.025% SDS, 50 mM sodium acetate, adjusted to pH 9.0 by addition of NaOH, with incubation overnight at 37 °C. Following incubation, the sample was subjected to lyophilization, 11 M HCl was added, and the sample was purged with nitrogen gas and sealed. After incubation at 110 °C for 24 h, HCl was removed by evaporation, water was added, and the sample was neutralized by the addition of NaOH. An aliquot was subjected to reversed-phase HPLC using a Zorbax column with a gradient elution described elsewhere(25) . Fluorescence was monitored using a Hewlett-Packard programmable detector using an excitation wavelength of 335 nm and an emission wavelength of 385 nm.
Figure 2: Analysis by reversed-phase HPLC of material released at pH 9.0 from a model glycation conjugate derived from ADP-ribose and n-butylamine. Absorbance was monitored at 254 nm. Chromatography of a sample of ketoamine 1 (22) that was stored at pH 5.0, -20 °C and analyzed prior to incubation at pH 9.0 is shown at the top of the figure. The left panels show profiles from samples incubated at pH 9.0 and analyzed at the times indicated. The right panels show profiles from samples incubated at pH 9.0 in the presence of alkaline phosphatase. The elution positions of G-ADP, ADP, and AMP are shown.
The material co-eluting with 5`-ADP was converted to material that co-eluted with 5`-AMP following incubation with venom phosphodiesterase and to material that co-eluted with adenosine following incubation with venom phosphodiesterase and alkaline phosphatase (results not shown), indicating that it was 5`-ADP. The G-ADP was characterized further, since it represented a product potentially unique to protein glycation by ADP-ribose. Snake venom phosphodiesterase treatment of G-ADP resulted in material that co-eluted with 5`-AMP (data not shown), indicating the presence of a phosphodiester linkage.
G-ADP was further characterized by NMR and
mass spectral methods. Fig. 3shows a C NMR
spectrum of G-ADP. Signals corresponding to the five carbons of the
adenine moiety and to the five carbons of ribose were readily
identified by comparison with the spectra of other adenine-containing
nucleotides(22, 26) . Additionally, a signal at a
position expected for the carbonyl carbon of a carboxylate group
(179.98 ppm) and at a position expected for a carbon of a methylene
group linked to a pyrophosphate bridge (71.18 ppm) could be identified.
A weak signal at approximately 75 ppm suggested the possible presence
of a 13th carbon atom present as a hydroxymethyl group, but this could
not be established from the spectrum alone. The
H NMR
spectrum allowed the identification of the adenine protons and the
anomeric proton of the ribose bound to adenine (data not shown). To
determine the molecular weight of G-ADP, the negative ion and positive
ion fast atom bombardment mass spectra of G-ADP were obtained. These
spectra (Fig. 4) showed a negative ion of m/z 514 and a
positive ion of m/z 516, indicating a molecular weight of 515
for G-ADP.
Figure 3:
C NMR spectrum of a product
(G-ADP) released from a model ADP-ribose glycation conjugate (ketoamine
1) by incubation at pH 9.0. The proposed structure and numbering of
G-ADP is shown at the top of the
figure.
Figure 4: Negative ion (top) and positive ion (bottom) mass spectra of a product (G-ADP) released from a model ADP-ribose glycation conjugate (ketoamine 1) by incubation at pH 9.0.
The enzyme sensitivity and NMR data indicated that G-ADP
contained ADP and an additional carbon fragment containing a methylene
carbon and a carboxyl group derived from the second ribose originally
present in the ADP-ribose. If the carbon fragment contained only the
methylene and carboxyl groups, the predicted molecular weight for G-ADP
would be 475. The molecular weight of 515 indicated that G-ADP contains
an additional hydroxymethyl group. Taken together, the data indicate
that the carbon fragment attached to ADP is glyceric acid. The proposed
structure and numbering system is shown at the top of Fig. 3. We postulate that the proton of the 2" carbon of G-ADP
is acidic enough to partially exchange with D0, resulting
in a significantly reduced signal for this carbon in the
C
NMR spectrum, such that it could not be unambiguously detected. In
summary, our results demonstrate that model glycation conjugates of
ADP-ribose degrade at pH 9.0 to form two primary products, 5`-ADP and
G-ADP.
Figure 7: A mechanism proposed for the formation and degradation of a ketoamine adduct by reaction of ADP-ribose with a protein amino group.
Figure 8: Amino acid analysis (11) of histone H1 (A) and histone H1 incubated with ADP-ribose (B). The region of the chromatogram near the elution position of CML is shown.
The study of
Sell and Monnier (16) has shown that pentoses readily generate
a number of different fluorescent AGE. One of these products,
pentosidine, has been structurally characterized(16) . The
formation of these fluorescent AGE begins with the initial glycation of
a lysine amino group but further reactions involve both oxidation and
reaction with protein arginine residues. If the lysine and arginine
residues are present on different polypeptide chains, the formation of
pentosidine results in protein-protein
cross-linking(2, 16) . To determine if ADP-ribose
glycation could lead to glycation products that result in protein
cross-linking, histone H1 was incubated with ADP-ribose and subjected
to analysis by SDS-PAGE. Fig. 9A shows that incubation of
histone H1 with 500 µM ADP-ribose for 2 h at pH 5.0 and
7.0 did not affect its electrophoretic migration, but incubation at pH
8.0 and 9.0 resulted in the appearance of material at the expected
position of a dimer of H1. At pH 9.0, the H1 was almost completely
converted to the dimer position. No differences were observed when
ADP-ribose purified by anion exchange chromatography was used,
indicating that the activity was not due to reactive contaminants in
the commercial preparation of ADP-ribose. With longer times of
incubation, conversion of H1 to the dimer position also was readily
observed at pH 7.0 (data not shown). Since the reaction was most rapid
at pH 9.0, additional experiments were done this pH. Fig. 9B shows that conversion of H1 to a dimer was dependent upon the
concentration of ADP-ribose. When the incubation contained ADP-ribose
with C in the ribose moiety containing the free aldehyde,
radiolabel was incorporated into the dimer of H1 (data not shown).
Aminoguanidine has been shown to serve as an effective inhibitor of
protein glycoxidation(2) . Addition of 500 µM aminoguanidine completely inhibited H1 dimer formation (data not
shown). These results indicated that ADP-ribose was effective in
causing protein cross-links by a mechanism involving glycation.
Figure 9: Analysis by SDS-PAGE of histone H1 incubated with ADP-ribose. A shows Coomassie Blue staining of an unincubated sample (designated C) and samples incubated with 500 µM ADP-ribose where the numbers at the top of the gel indicate pH values. B shows Coomassie Blue staining of a sample incubated at pH 9.0 with the micromolar concentrations of ADP-ribose indicated at the top of the gel and a sample incubated at pH 5.0 with 300 µM ADP-ribose.
To
determine if the protein cross-linking by ADP-ribose involved the
formation of fluorescent AGE, 500 µM ADP-ribose was
incubated with H1, and fluorescence was monitored using the conditions
of excitation and emission typical for pentosidine(16) .
Incubation of histone H1 at pH 9.0 with 500 µM ADP-ribose
resulted in the rapid formation of putative fluorescent AGE (data not
shown). Fluorescent products could be readily detected after a lag of
approximately 8 min and increased in a linear manner as a function of
incubation time. The presence of the lag phase is consistent with a
mechanism by which the formation of ketoamine glycation products
precedes the formation of AGE. Fluorescent products were also observed
when incubations were done at pH 7.4, although the rate of formation
was much slower. Fluorescent products also were observed when histone
H1 was incubated with an ADP-ribose preparation that had been purified
by anion exchange HPLC, but incubation of either histone H1 or
ADP-ribose alone did not result in any detectable fluorescence. When
analyzed after a 20-min incubation, formation of fluorescence was
dependent upon the concentration of ADP-ribose in the incubation
mixtures from 50 to 500 µM. Furthermore, the addition of
100 and 500 µM aminoguanidine inhibited the formation of
fluorescent products by 65 and 98%, respectively. No reaction between
ADP-ribose and aminoguanidine was detected by subjecting reaction
mixtures to anion exchange HPLC, indicating that aminoguanidine was
inhibiting oxidative steps rather than scavenging ADP-ribose and
inhibiting glycation. Reactive oxygen species, whose formation can be
catalyzed by metal ions, have been implicated in the oxidative phase of
glycoxidation(2) . Evidence that the fluorescent products
observed here involved a similar mechanism was provided by the
observation that their formation was inhibited by reduced ascorbate. ()Also, the rate of formation of both fluorescent products
and histone cross-links was increased at either pH 7.0 or 9.0 with
either phosphate or acetate buffer when the buffer concentration was
increased from 10 to 100 mM, consistent with traces of metal
ion-catalyzing aerobic oxidation. (
)
It is noteworthy that ADP-ribose was much more effective than either ribose or ribose 5-phosphate in the formation of fluorescent AGE. While incubation with ADP-ribose with histone H1 resulted in intense fluorescence in minutes, neither ribose or ribose 5-phosphate resulted in detectable fluorescence when incubated under the same conditions for several days.
Many lines of evidence indicate that the non-enzymatic modification of long lived biomolecules plays an important role in the aging process and the pathophysiology of diseases whose incidence increases as a function of age(1, 2, 3, 4, 5, 6, 7) . Protein amino groups are sites of reaction with reducing sugars that result in protein modification by ketoamine adducts(11, 12, 13, 14, 15) . Since ketoamine adducts are not very stable, many, and possibly most, ketoamine adducts are precursors to much more complex protein modifications(11, 16) . Although less reactive, amino groups in DNA bases are also potential sites of modification by reducing sugars(29, 30) .
The results presented in this study have potentially important implications for the non-enzymatic modification of long lived nuclear biomolecules involved in the maintenance of genomic integrity. Age-associated changes in chromatin structure have been described previously(3, 31) , and both individual histones (32) and histones in nucleosomes (33) have been reported to be glycated in vitro. Genomic integrity requires efficient repair of molecular damage to DNA and repair of DNA damage is accompanied by the synthesis of ADP-ribose polymers, which are present only transiently, being rapidly degraded to ADP-ribose(17, 18) . Although the intranuclear levels of ADP-ribose under specific physiological conditions are unknown, intracellular levels of NAD are estimated to be in the range of 100-300 µM and a large fraction of the NAD pool can flow through ADP-ribose polymers to ADP-ribose following DNA damage, indicating that appreciable concentrations of ADP-ribose should occur in the nucleus of DNA-damaged cells(17) . The possibility that ADP-ribose might be a source of a ribose moiety in vivo for protein glycoxidation has been suggested by Sell and Monnier(16) . As described here, ADP-ribose is an efficient source for glycation and glycoxidation of histones in vitro. Many previous studies of protein modification in vitro by reducing sugars have used sugar concentrations in the range of 20-500 mM(12, 32, 33, 34) . In this study, ADP-ribose concentrations of 50-500 µM resulted in readily detectable histone glycation ( Fig. 5and Fig. 6) and glycoxidation ( Fig. 9and Fig. 11). While the reaction rates were much greater at pH 9.0, the lability of the ketoamine glycation conjugates, the cross-linking of histones and the formation of fluorescent AGE also were readily detectable at pH values in the physiological range, indicating that these reactions are likely to occur in intact cells.
Our studies with model ADP-ribose glycation conjugates (Fig. 2Fig. 3Fig. 4) and histone ADP-ribose glycation conjugates ( Fig. 5and Fig. 6) have demonstrated that ketoamines derived from ADP-ribose degrade by two primary pathways, one that generates ADP and a second that generates G-ADP. The efficacy of formation of glycoxidation products, specifically histone cross-links and other fluorescent AGE, likely relates to the degradation pathway that releases ADP ( Fig. 2and Fig. 6). The mechanism for the formation of AGE such as pentosidine postulates the condensation of a ketoamine derived neutral five-carbon fragment with a protein arginine residue(16) . Pathways for the degradation of ADP-ribose derived ketoamines that result in the presence of a residual phosphate would interfere with this condensation, but the release of the ADP moiety results in an uncharged 5 carbon fragment that should readily react with arginine and thus promote histone cross-linking. This pathway may also explain the much greater efficacy of ADP-ribose in the formation of fluorescent products as compared with ribose 5-phosphate as ketoamines formed from ribose 5-phosphate may not degrade as readily to uncharged products. Another factor that may be related to the efficacy of ADP-ribose is the presence of a high affinity binding site for pyrophosphate in histone H1(35) , which may facilitate binding of ADP-ribose to the protein.
Our results suggest that the fluorescent products formed in histone H1 are derived from ADP-ribose, lysine, and arginine, since it was possible to generate a product indistinguishable from pentosidine by incubation of ADP-ribose only with lysine and arginine (Fig. 10). Further evidence that the fluorescent products resulted from glycoxidation was provided by the observation that aminoguanidine, an inhibitor of AGE formation, inhibited the formation of fluorescence. While the results described here have demonstrated histone cross-linking by ADP-ribose, the possibility that ADP-ribose may promote histone-DNA cross-links should also be considered.
The second pathway of degradation of ADP-ribose-derived ketoamines, which leads to the generation of G-ADP, has potential utility for the detection of ADP-ribose-specific glycation of proteins in vivo, since it likely represents a unique glycation product for this nucleotide. Additionally, the release of G-ADP predicted that protein glycation by ADP-ribose can result in the modification of protein lysine residues with carboxymethyl groups (Fig. 7). This prediction was confirmed in the experiments described here (Fig. 8). The occurrence of CML residues in proteins in vivo has been documented(11, 34, 36) . The modification of histone lysine residues by carboxymethyl groups has the net result of converting a positively charged side chain to a negatively charged side chain, which would be expected to alter histone interaction with DNA. A search for the presence of CML in histones in vivo may be useful in assessing the possible role of nuclear generated ADP-ribose in histone glycation in vivo.
While additional studies will be needed to determine if ADP-ribose causes histone glycation in vivo, the results presented here are of interest with regard to a number of previous studies. Hilz and co-workers (37) have described ADP-ribose conjugates of histone H1 in hepatoma cells following DNA damage with chemical stability very similar to the histone glycation conjugates described here, raising the possibility that these conjugates may represent histone glycation. Smulson and co-workers (38) reported a stable complex containing a dimer of histone H1 and ADP-ribose polymers in HeLa cells, although the mechanism by which the histones were covalently cross-linked was not elucidated. Our results raise the possibility that the covalent linkage of the histone molecules is the result of histone glycoxidation initiated by ADP-ribose generated by polymer turnover. Gugliucci and Bendayan (39) have recently detected fluorescent AGE in histones isolated from rats and increased fluorescent AGE in streptozotocin-induced diabetic rats. Streptozotocin is a potent DNA damaging agent known to cause liver DNA damage and to stimulate poly(ADP-ribose) polymerase(17, 18) , raising the possibility that the increases in histone AGE may have resulted from ADP-ribose polymer turnover.
The potentially deleterious effects of protein glycation and glycoxidation suggests that cellular mechanisms to repair or minimize these protein modifications are likely to exist. Several enzyme systems are possible candidates for reversing or limiting protein modifications of this type. Both bacteria (40) and fungi (41) contain glycation removal enzymes. An enzyme that reduces 3-deoxyglucosone, a reactive product released from glucose derived ketoamines, has been described in rat liver(42) . An ADP-ribose pyrophosphatase catalyzes conversion of ADP-ribose to 5`-AMP and ribose 5-phosphate(43) . Our observation that ribose 5-phosphate is much less effective than ADP-ribose in causing protein glycoxidation would indicate that the ADP-ribose pyrophosphatase could function to minimize histone glycoxidation. Protein glycation by glucose leads to the release of 3-deoxyglucosone, and the analogous product released following protein glycation by ADP-ribose would be 3"-ADP-deoxypentosone. It is interesting that 3"-ADP-deoxypentosone has been reported to be the product of the enzyme ADP-ribosylprotein lyase(44) , an enzyme postulated to function in the turnover of ADP-ribose polymers by catalyzing the removal of the protein proximal ADP-ribose residue. In view of the results presented here, the possibility that ADP-ribosyl protein lyase may function in glycation removal should also be considered.