modification of proteins in vitro by physiological …paul a. voziyan# , raja g. khalifah‡,...

Modification of Proteins In Vitro by Physiological Levels of Glucose: Pyridoxamine Inhibits Conversion of Amadori Intermediate to Advanced Glycation End-products

Through Binding of Redox Metal Ions

Paul A. Voziyan#§, Raja G. Khalifah‡, Christophe Thibaudeau£, Alaattin Yildiz#, Jaison Jacob¶, Anthony S. Serianni£, and Billy G. Hudson#§

From the Departments of #Medicine and ¶Biochemistry, Vanderbilt University Medical

Center, Nashville, TN 37232, £Department of Chemistry and Biochemistry, University of

Notre Dame, Notre Dame, IN 46556, and ‡BioStratum, Inc., Durham, NC 27703

Running title: Pyridoxamine inhibition of AGE pathways

§ To whom correspondence should be addressed: Division of Nephrology, Vanderbilt

University Medical Center, S-3223 MCN, 1161 21st Avenue South, Nashville,

Tennessee 37232-2372. Tel.: 615-322-2089 Fax: 615-343-7156; E-mail:

[email protected] or [email protected]

* This work was supported by NIDDKD Research Grants DK065138 and P60DK20593,

and by a Research Grant from BioStratum, Inc.

1Abbreviations: : AGE, advanced glycation end-product; AG, aminoguanidine; ALE,

advanced lipoxidation end-product; BSA, bovine serum albumin; CML, Nε-

(carboxymethyl)lysine; DTPA, diethylenetriaminepentaacetic acid; FL, fructosyllysine;

GO, glyoxal; GLA, glycolaldehyde; MGO, methylglyoxal; MOLD, methylglyoxal-lysine

dimer; PM, pyridoxamine; RNase, bovine pancreatic ribonuclease A.

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Hyperglycemic conditions of diabetes accelerate protein modifications by glucose

leading to the accumulation of advanced glycation end-products (AGEs). The role of

AGEs in the development of serious complications of diabetes and other diseases

emphasizes the necessity for understanding the mechanisms of their formation and

inhibition. We have investigated the conversion of protein-Amadori intermediate to

protein-AGE and the mechanism of its inhibition by pyridoxamine (PM), a potent AGE

inhibitor that has been shown to prevent diabetic complications in animal models.

During incubation of proteins with physiological diabetic concentration of glucose, PM

prevented the degradation of the protein glycation intermediate identified as

fructosyllysine (Amadori) by 13C NMR using [2-13C]-enriched glucose. Subsequent

removal of glucose and PM led to conversion of protein-Amadori to AGE Nε-

carboxymethyllysine (CML). We utilized this inhibition of post-Amadori reactions by PM

to isolate protein-Amadori intermediate and to study the inhibitory effect of PM on its

degradation to protein-CML. We first tested the hypothesis that PM blocks Amadori-to-

CML conversion by interfering with the catalytic role of redox metal ions that are

required for this glycoxidative reaction. Support for this hypothesis was obtained by

examining structural analogs of PM in which its known bidentate metal ion binding sites

were modified and by determining the effect of endogenous metal ions on PM inhibition.

We also tested the alternative hypothesis that the inhibitory mechanism involves

formation of covalent adducts between PM and protein-Amadori. However, our 13C NMR

studies demonstrated that PM does not react with the Amadori. Because the

mechanism of interference with redox metal catalysis in post-Amadori AGE formation is

operative under the conditions closely mimicking diabetic state, it may contribute

significantly to PM efficacy in preventing diabetic complications in vivo. Inhibition of

protein-Amadori degradation by PM also provides a simple procedure for the isolation of

protein-Amadori intermediate, prepared at physiological levels of glucose for relevancy,

to study both the biological effects and the chemistry of post-Amadori pathways of AGE

formation.


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Chemical modifications of circulating, cellular and matrix proteins by glucose are

thought to be a major factor in pathogenesis of diabetes, atherosclerosis, and

neurodegenerative diseases, as well as in the process of ageing (1-4). These

modifications derive, in part, from glycation reactions, i.e. the reversible condensation of

the aldehyde group of glucose with a protein amino group, forming a Schiff base

followed by an essentially irreversible rearrangement to an Amadori intermediate

(Fig.1). This intermediate undergoes cycles of condensations with additional amines,

dehydrations, and oxidative fragmentations to yield heterogeneous chemical

compounds collectively referred to as advanced glycation end-products (AGEs)1.

Formation of chemically stable AGEs in this Maillard reaction can permanently alter

protein structure and function (for review, see ref. 5-7). They range from CML (the

pathway is shown in Fig.1) to more complex structures such as pentosidine (a lysine-

arginine crosslink) and pyralline (8-10). More AGEs have been identified in the recent

years, among them inter-lysine cross-links crosslines (11) and vesperlysines (12).

Some Maillard products are antigenic, and it has been shown that anti-AGE sera

predominantly recognize CML attached to protein (13). A number of pathways of AGE

formation include oxidative steps catalyzed by redox-active transition metal ions, as

exemplified by the conversion of protein-Amadori intermediate to CML (Fig.1).

CML is a major AGE modification associated with different human pathologic

states. It can be derived either directly from the glycated protein (Fig. 1), or from protein

modification by small reactive carbonyl and dicarbonyl compounds that are either

metabolites or autoxidation products of glucose, ascorbate, Schiff bases, Amadori

intermediates or polyunsaturated lipids (14-16). CML-modified proteins have been found

in plasma, renal tissues, retinas, and collagen of diabetic patients (17-22). CML is the

predominant AGE in intracellular neurofibrillary deposits in patients with Alzheimer

disease (23) and in macrophage-derived foam cells in human atherosclerotic plaques

(24). Its concentration in human tissues increases significantly with age (25). CML-

modified protein has also been reported to be a ligand for RAGE, an AGE receptor that

mediates cellular pro-inflammatory responses (26).


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We earlier reported a novel way to isolate ribose-Amadori intermediate using

high concentration of ribose (27, 28). This ribose-derived intermediate, although not

physiologically relevant, provided an experimental strategy to elucidate mechanisms of

AGE formation and to search for inhibitors that block it. This led to discovery that

pyridoxamine, a natural intermediate of vitamin B6 metabolism, is an efficient in vitro

inhibitor of conversion of the Amadori intermediate to AGEs, particularly CML (28-30).

Under in vivo conditions, pyridoxamine inhibited the accumulation of CML modifications

in skin collagen and in retina, and prevented the development of early renal disease and

retinopathy in the streptozotocin rat and db/db mouse models of diabetes (31-33). In

non-diabetic Zucker obese (fa/fa) rats, the protection of PM against renal and vascular

pathology was also accompanied by the inhibition of CML formation (34).

Pyridoxamine can form moderately stable complexes with a number of transition

metal ions (35, 36). Recently, Baynes et al. reported that PM and other AGE inhibitors

can inhibit copper-dependent oxidation of ascorbic acid and proposed that metal

chelation may play a major role in the inhibition of glycoxidation reactions (37).

However, it remains to be demonstrated whether or not the redox metal ion requirement

and its inhibition by ligand binding are identical in these two reactions. It also has not

been established whether other mechanisms of inhibition, such as covalent adduct

formation of PM with the Amadori, could significantly contribute to AGE inhibition. In the

present work, we demonstrate for the first time that PM can inhibit degradation of a

glucose-derived protein-Amadori intermediate and have utilized this inhibition to isolate

the glucose-derived Amadori at physiological glucose concentrations. The isolation of

this intermediate has enabled the study of the mechanism of PM inhibition of post-

Amadori pathway leading to CML formation, including demonstration by 13C NMR that

no adducts are formed between PM and Amadori. Our overall results support the

hypothesis that PM inhibits post-Amadori glycoxidation reactions by binding to required

redox metal ions and interfering with their catalytic role in these reactions. Since this

PM inhibition occurred in vitro under physiological diabetic concentrations of glucose,

we propose that this PM mechanism contributes to inhibition of AGE formation by PM

observed in diabetic animal models (31-33).


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Experimental procedures Materials. D-ribose, pyridoxamine dihydrochloride, 1-deoxy-1-morpholino-D-

fructose, 3-hydroxypyridine, 4-aminomethylpyridine, and bovine serum albumin were

purchased from Sigma-Aldrich. 5-Nitro-2-hydroxybenzylamine was synthesized by

ChemSyn Laboratories (Lenexa, KS), and its structure and purity were confirmed by

proton NMR and mass spectrometry. D-Glucose was purchased from Gibco BRL.

RNase A was obtained from Worthington Biochemical Co. D-[2-13C]Glucose (99 atom-

% 13C) was purchased from Omicron Biochemicals.

Preparation of glucose-derived and ribose-derived protein-Amadori. For the

preparation of protein-Amadori intermediate under physiologically relevant conditions,

BSA or RNase (20 mg/ml) was incubated with either 5 mM or 30 mM glucose and 20

mM pyridoxamine in 200 mM Na-phosphate pH 7.5 buffer, containing 0.02% sodium

azide, at 37 °C for 80 days. In some cases, protein-Amadori intermediate was prepared

using 100 mM glucose and the conditions described above. Accumulation of BSA-

Amadori was followed using the nitroblue tetrazolium method described below. When

the concentration of Amadori intermediate reached a maximum constant value, glucose

and pyridoxamine were removed by dialysis against 100 volumes of the same Na-

phosphate buffer with 6 buffer changes over a 24 h period. The buffer was equilibrated

at 4°C and dialysis was carried out at this temperature. The complete removal of PM

was confirmed by the absence of PM fluorescence (Ex=324 nm, Em=393 nm).

Ribose-Amadori modified BSA was prepared as described earlier (29). Briefly,

protein was incubated in the presence of 500 mM ribose in 200 mM Na-phosphate pH

7.5 buffer at 37°C for 24 h. Sodium azide (0.02%) was added to the incubations to

prevent bacterial growth. At the end of the incubation, free and Schiff-base ribose was

removed by dialysis at 4°C.

Detection of protein-CML using ELISA. Modifications of protein lysine residues

to CML were measured by ELISA using polyclonal anti-AGE antibody R618 as

described earlier (28-30). Protein-CML was the antigenic epitope in our ELISA

measurements (38).

Quantitative measurements of protein-Amadori. The formation of protein-

Amadori was analyzed using a Sigma Fructosamine kit. The assay is based on the


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ability of Amadori intermediates to reduce nitroblue tetrazolium leading to the formation

of formazan that can be monitored spectrophotometrically at 550 nm. We modified the

Sigma protocol by filtering the working solution prior to use in the assay. This

modification significantly reduced the background signal and improved the

reproducibility of the measurements. Aliquots (40 µl) of protein-Amadori (5 mg/ml) were

mixed with 0.7 ml of Sigma Fructosamine assay working solution and incubated at 37°C

for 10 min. The absorbance of each sample was measured and incubation at 37°C was

resumed. After 10 min, a second absorbance measurement was taken. Amadori

intermediate was quantified by comparing the differences in absorbance between two

measurements for each sample with those of a 1-deoxy-1-morpholino-D-fructose

standard. The protein concentration was determined using a second derivative analysis

of the protein absorbance spectrum to minimize contributions from glycation-related

spectral components (39).

Determination of pKa. The apparent pKa values for 5-nitro-2-hydroxybenzylamine

were determined by spectrophotometric titration at 22°C using an Agilent 8453 UV-

visible spectrophotometer. The value of pKa1 (phenolic group), obtained by least

squares curve fitting of absorbance titration data, was 6.12±0.01. These data indicate

that at physiological pH, the phenolic group of 5-nitro-2-hydroxybenzylamine is present

mostly in the deprotonated state, a condition favoring complex formation between PM

and metal ions (35).

Solution 13C NMR. Amadori-RNase was prepared using D-[2-13C]glucose under

experimental conditions described earlier. After the removal of free and Schiff-base [2-13C]glucose and PM by dialysis at 4 ºC, the buffer was replaced by 50 mM Na-

phosphate buffer prepared with D2O, pD 7.5, using a Centricon-3 centrifugation

concentrator. NMR experiments were performed at 21 ºC on a Varian UnityPlus 600

MHz spectrometer. FID transients (20,000) were collected over spectral widths of 225

ppm (Fig. 6A, B) or 300 ppm (Fig. 6C-E). Larger spectral widths were used in Fig. 6C-E

to determine whether the adducts between PM and acyclic [2-13C]-labeled Amadori

intermediate may have formed upon incubation with 20 mM PM for 8 days at 37ºC. No

signals were detected at δ > 180 ppm in Fig. 6D, E compared to Fig. 6C. In all NMR

experiments, the recycle time was 4.7 s, which was judged sufficient to ensure a return


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to magnetization equilibrium between pulses. All NMR experiments were performed

under waltz-16 decoupling conditions. The FIDs were zero-filled (final digital resolution

0.2 Hz/ data point), apodized with an exponential window function (line broadening = 15

Hz) and Fourier transformed to give the spectra shown in Fig. 6. Reprocessing the

FIDs with a Gaussian-to-Lorentzian window function showed that the apparent singlets

at ~ 97.7 ppm and ~ 95.5 ppm contained significant fine structure. At least 3 resonances

were observed between 97.6 and 97.8 ppm, and at least 6 signals were observed

between 95.3 and 95.6 ppm. Presumably this multiplicity is due to the small effect of

protein site environments on Amadori chemical shifts.

Absorbance measurements. Single-wavelength absorbance measurements,

absorbance spectra, and second derivative analyses were performed on a Hewlett

Packard 8452A diode array spectrophotometer equipped with a Peltier temperature

control unit.

Results

Reversible inhibition of CML formation by pyridoxamine. We initially described a

method to prepare Amadori intermediate in the AGE pathways using high

concentrations of ribose (27). This ribose-derived intermediate provided an

experimental strategy to search for inhibitors that block AGE formation and led to the

discovery of PM inhibition of post-Amadori reactions (28-30). In the present work, we

have used PM to trap and identify glucose-derived protein-Amadori intermediate. As

shown in Fig. 2A, PM completely inhibited formation of immunoreactive BSA-CML

during the initial 35 days of incubation of BSA with 200 mM glucose. When PM and

glucose were removed, the formation of BSA-CML from the putative intermediate was

observed (Fig. 2A, triangles). After 35 days of incubation with glucose, significant

accumulation of fructosyllysine (Amadori) intermediate was detected only in the sample

containing PM (Fig. 2B, bar graph). The structural identity of protein Amadori

intermediate was confirmed using 13C NMR as described vide infra in Fig. 6. Upon the

removal of PM and glucose, BSA-Amadori degraded (Fig. 2B, inset) and converted to

BSA-CML (Fig. 2A, triangles). It is noteworthy that CML is the major but not the only

AGE product of degradation of Amadori intermediate. This complexity of post-Amadori


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reactions may account for the apparent differences in kinetics of Amadori degradation

and formation of Amadori-derived CML (Fig. 2).

The data in Fig. 2 present two important findings. 1) PM inhibits the formation of

glucose-derived protein-CML by inhibiting degradation of protein-bound Amadori

intermediate; 2) this PM property provides a strategy to specifically follow the formation

and degradation of protein-Amadori intermediate.

We utilized the isolation of the glucose Amadori by PM to determine the kinetics

of formation and degradation of BSA-Amadori under physiologically relevant

concentrations of glucose found in the plasma of diabetic patients (>5 mM and up to 30

mM). As shown in Fig. 3A, incubation of BSA with 30 mM glucose at 37°C in the

presence of PM resulted in significant increase in the rate of accumulation of BSA-

Amadori intermediate compared to that at 5 mM glucose. The kinetics was

characterized by t1/2 ~ 9 days, consistent with the notion that relatively short-lived

proteins (t1/2 ~ days) can be modified by glucose in vivo (40, 41). The degree of

modification of protein lysine residues in the albumin-Amadori prepared with diabetic

concentration of glucose was about 9% (Fig. 3A), or ~5 lysine residues per molecule out

of total 58 Lys. This result is in good agreement with preferential modification of 4 lysine

residues found in albumin purified from diabetic patients (42).

The removal of unreacted glucose and PM by extensive dialysis at 4°C followed

by incubation again at 37°C led to the disappearance of ~90% of protein-Amadori

intermediate (FL) after 80 days (Fig. 3A). This process was accompanied by formation

of BSA-CML. Different concentrations of freshly added PM inhibited this CML formation

(Fig. 3B). This inhibition of post-Amadori protein-CML formation is consistent with the

results of our earlier experiments using ribose-derived protein-Amadori intermediate

(28, 30). At 1 mM PM (a 2.5-fold molar excess of PM over Amadori moiety), BSA-CML

formation was completely inhibited (Fig. 3B). Even at the substoichiometric

concentration of PM (0.1 mM), a significant inhibition was still evident (Fig. 3B).

Importantly, this PM concentration is similar to a steady-state pyridoxamine level in

plasma of PM-treated animals (31). The data in Fig. 3 establish that PM can inhibit

CML formation under physiologically relevant solution conditions. Since PM prevented

the degradation of protein-Amadori intermediate, the target of PM action must be post-


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Amadori steps in the CML pathway that lead to irreversible oxidation (Fig. 1). We set

out to establish which of these steps is targeted by PM.

Role of metal ions in inhibition of the post-Amadori CML pathway by PM.

Pyridoxamine is known to form complexes with transition metal ions such as Fe3+ and

Cu2+ and to also inhibit copper-dependent oxidation of ascorbic acid (35-37). We

checked if PM interferes with the oxidative steps in the CML pathway through binding

catalytic metal ions (Fig. 1). The redox metal ion catalysts required for CML formation

occur naturally in the Na-phosphate buffer used in our experiments (43). Electron pairs

on oxygen and nitrogen of the aminomethyl and phenol moieties of PM participate in

bidentate coordination with the metal ion (Fig. 4). If pyridoxamine-metal complex

formation is critical for the inhibition of the conversion of protein-Amadori intermediate to

protein-CML, then modification of these PM functional groups would abolish inhibition.

We utilized ribose-derived BSA-Amadori, a model that exhibits a more rapid rate of

Amadori-to-CML conversion due to the higher abundance of reactive acyclic form of

ribose-Amadori compared to glucose-Amadori (28, 30). We tested three PM structural

analogs to determine their inhibitory potential. 3-Hydroxypyridine and 4-

aminomethylpyridine lack one of the two moieties critical for bidentate complex

formation with the metal ion (Fig. 4A and B). These PM analogs showed no inhibition,

even at very high concentrations (Fig. 4A and B). On the other hand, 5-nitro-2-

hydroxybenzylamine, a compound that has both 3-hydroxy and 4-aminomethyl groups,

showed strong inhibition similar to that reported previously for PM (28-30), even though

its other aromatic ring differs from that of PM (Fig. 4C). These results suggest that PM

inhibits the post-Amadori CML pathway through complex formation with catalytic metal

ions. This conclusion is further supported by the effects of addition of CuCl2 to

incubations of protein-Amadori and PM (Fig. 5). In these experiments, metal ion

contaminants naturally present in phosphate buffer were not removed prior to

incubation. As shown in Fig. 5, the increase in metal concentration significantly

diminished the inhibitory effect of PM on protein-CML formation in a concentration-

dependent fashion, consistent with the importance of metal ion complexation in the PM

inhibition of post-Amadori reactions.


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Lack of reactivity of PM with protein-Amadori intermediate. We demonstrated

recently that PM forms relatively stable five-ring adduct structures with carbonyl groups

of low molecular weight compounds such as glycolaldehyde (38). Since the protein-

bound Amadori moiety, a ketoamine, contains a carbonyl group (Fig. 1), we checked

whether the amino group of PM may form a stable adduct with an acyclic Amadori

carbonyl. In such AGE inhibition mechanism, originally hypothesized for

aminoguanidine (44) but later refuted (45), the adduct formation would inhibit the

conversion of the reactive form of the protein-Amadori intermediate to protein-CML. The

formation of a stable adduct with PM, whether covalent or reversible, would necessarily

shift the tautomeric equilibrium towards the acyclic form of the Amadori intermediate,

thus depleting the cyclic forms (Fig. 1).

This possibility was investigated using 13C NMR and RNase as the model protein,

since resonance assignments for the Amadori moieties have been established

previously (46). Like with BSA, RNase-Amadori intermediate was forming in the

presence of 30 mM glucose and PM, t1/2~22 d (data not shown). For the 13C NMR

experiments the RNase-Amadori intermediate was prepared with 30 mM D-[2-13C]glucose and also with 100 mM D-[2-13C]glucose to achieve a higher degree of lysine

modification and improve the signal-to-noise ratio. The 13C NMR spectra of RNase-[2-13C]-Amadori (Fig. 6A, C) were in good agreement with previously reported resonance

assignments for RNase-[2-13C]-Amadori in the anomeric carbon region (46).

Characteristic resonances (Fig. 6C) arising from the prevailing cyclic β-pyranose forms

(at 95.5, 96.1 and 97.7 ppm) of Amadori intermediates on the ε-amino group of lysine-

41, on ε-amino groups of remaining lysines, and on the N-terminal α-amino group were

observed. Corresponding signals arising from the minor α- and β-furanose forms of

Amadori intermediates on similar amino groups were also detected (101.9, 102.8

(broad) and 104.1 ppm for α; 98.9 (ε-amino group) and 101.4 ppm (ε-amino group of

Lys-41) for β). The signal at 95.8 ppm previously assigned (46) to the α-pyranose

Amadori on ε-amino groups may overlap with that at 95.5 ppm assigned to β-pyranose

Amadori on similar groups. Neither the unmodified RNase nor PM contributed to the

anomeric region of 13C NMR spectrum (data not shown). In RNase-Amadori prepared

under physiological diabetic conditions (30 mM glucose), the observed ratio of modified


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ε-amino to α-amino groups of ~1 appears significantly smaller than reported in RNase-

Amadori prepared with 250 mM glucose (46).

In the presence of either 3 mM or 20 mM PM, no significant change in the

intensities of resonances corresponding to cyclic Amadori forms was observed even

after prolonged incubation (8 days). Importantly, in the experiments shown in Fig. 6B,

6D and 6E, the same PM-to-Amadori molar ratio was used at which PM inhibited

conversion of protein-Amadori to protein-CML (Fig. 3B, data for 0.1 mM and 1mM PM).

If the PM inhibitory effect was due to Amadori-PM adduct formation, it would cause a

shift of the C2 Amadori signals out of the anomeric region of the 13C NMR spectrum,

thus decreasing their intensities. The observed lack of intensity changes and the

absence of additional 13C NMR signals suggests that Amadori-PM adducts, if formed,

are produced in minor amounts. The content of these adducts would be substantially

less than 5% of total protein-Amadori as determined by the sensitivity of 13C NMR

experiments (Fig. 6). Therefore, the formation of such adducts cannot significantly

contribute to PM inhibition of Amadori-to-CML conversion.

Discussion Glucose is a ubiquitous natural metabolite present in all human tissues. In its

aldehyde form it reacts non-enzymatically with nucleophilic groups on proteins,

preferentially with ε-amino groups of lysines and N-terminal α-amino groups. An early

product of this reaction, protein-Amadori, is converted to a variety of stable advanced

glycation end-products or AGEs. Although this reaction is very slow due to the low

abundance of the reactive aldehyde form of glucose at equilibrium (0.003%), the

gradual accumulation of stable AGEs alters protein conformation and function. This

protein modification by glucose is significantly accelerated under hyperglycemic

conditions of diabetes and is considered one of the major factors in the development of

diabetic complications.

Our previous work showed that pyridoxamine, unlike other AGE inhibitors such

as aminoguanidine, blocks the conversion of ribose-derived Amadori intermediate to

protein-CML (28-30). In the present work, we identified the nature of the protein-bound

intermediate formed from glucose in the presence of PM and investigated the


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mechanism of inhibition of post-Amadori protein-CML formation by PM. In the presence

of PM and diabetic levels of glucose (30 mM), the CML precursor was identified as

protein-Amadori intermediate using 13C NMR (Fig. 6). When glucose and PM were

removed, the protein-bound Amadori moiety underwent oxidative degradation and

formed CML, indicating that PM acted on one of the post-Amadori steps of AGE

formation (Fig. 3). The absence of reversible or irreversible adducts between protein-

Amadori intermediate and PM was deduced also using 13C NMR (Fig. 6). The slow

physiologically relevant kinetics of degradation, however, presented an experimental

challenge. Therefore, once the inhibitory effects of PM were established under the

physiological solution conditions, we utilized the experimental system based on ribose-

derived Amadori that we introduced earlier, which allows for significantly higher rates of

post-Amadori reactions (28-30).

We utilized the ribose-derived Amadori to demonstrate that pyridoxamine inhibits

the conversion of protein-Amadori intermediate to protein-CML primarily by binding with

catalytic metal ions and blocking the oxidative steps in the pathway (Fig. 1). This

conclusion is based on several experimental observations: pyridoxamine did not react

with the carbonyl group of protein-Amadori (Fig. 4); the removal of either one of the PM

pyridine ring substituents that participate in bidentate metal ion coordination caused a

complete loss of PM inhibition of post-Amadori CML formation (Fig. 5); the inhibitory

effect of PM can be diminished by increasing concentration of metal ions (Fig. 6).

Importantly, this inhibition occurred at PM concentrations similar to those reported in

preclinical studies of PM efficacy (31) and with the level of protein modification similar to

that found in diabetic patients (Fig. 3).

The inhibition of degradation of important protein glycation intermediate by PM,

which can be reversed by removal of PM, provides an experimental tool to study the

formation and the degradation of protein-Amadori intermediate. Blocking of the post-

Amadori reactions enables the rates and levels of formation of protein-Amadori

intermediate to be easily measurable, even at low physiologically relevant levels of

glucose. Upon the removal of PM and glucose, post-Amadori AGE pathways could be

investigated without the interference from glucose or Schiff-base oxidation products that

may contribute to AGE formation (47, 48). Often, high concentrations of glucose (up to


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1 M) are used in protein glycation reactions in vitro. These reaction conditions lead to

high levels of protein modifications by glucose and enhance additional modifications of

protein by reactive products of glucose degradation. As an illustration, the amount of

albumin-Amadori formed at 200 mM glucose was about 7-fold greater than that formed

at more physiological 30 mM glucose (Fig. 2 and 3). The level of Amadori and AGE

moieties formed at high non-physiological concentration of glucose is likely to have

different impact on structure, function and cellular effects of the proteins compared to

more subtle modifications occurred at physiologically relevant glucose concentrations.

In our experiments, the formation of albumin-Amadori at diabetic glucose concentration

(30 mM) was consistent with elevated levels of glucose-modified proteins in diabetes

(40, 41), while the degree of modification of lysine residues was in good agreement with

that found in albumin purified from diabetic patients (42). Thus the inhibition by PM

provides a simple method for isolation of protein-Amadori intermediates, prepared at

physiological levels of glucose, for studies of its biological effects in cell culture or in

vitro experiments. Under in vivo conditions, Cu2+ and Fe3+ are the most important metal ion

catalysts of oxidative reactions. In blood plasma and in cellular cytoplasm virtually all of

these ions are sequestered in specific metal transporters and other metalloproteins (49,

50). However, copper metalloproteins such as ceruloplasmin and Zn,Cu-superoxide

dismutase and the iron metalloproteins such as hemoglobin and myoglobin have been

shown to undergo significant conformational change and even fragmentation during

glycation reactions, causing the release of bound metal (51-53). Pyridoxamine can form

moderately stable complexes with a number of transition metal ions but has a

preference for Cu2+ and Fe3+ (36, 54). By forming such complexes, PM can reduce

catalytic activity of metal ions released from damaged proteins.

Our results demonstrate that metal binding appears to be critical for PM inhibition

of post-Amadori AGE formation. However, the observation that aminoguanidine also

binds catalytic metal ions (37) but does not inhibit post-Amadori AGE formation (28)

indicates that there are other factors required for efficient inhibition of post-Amadori

reactions by PM. For example, it has been suggested that metal ions could also be

bound by CML clusters on glycated proteins or by native proteins, such as serum


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albumin. Such bound metal ions may retain a significant redox activity for catalysis of

oxidative reactions (55-58). Uniquely, PM may form ternary complexes with such

protein-bound metal ions, making them significantly less active as oxidation catalysts.

Previously, we and others have demonstrated that PM can prevent protein

modifications by chemically trapping free low molecular weight reactive carbonyl

products of glucose and lipid degradation (38, 59-61, Fig. 7). In the present study, we

determined that PM inhibition of post-Amadori CML formation (28-30) is due primarily to

its interference with metal ion catalysis (Fig. 7). It is noteworthy that metal ion

complexation may also contribute to PM inhibition of AGEs derived from free low

molecular weight carbonyl compounds (Fig. 7). Some of these reactions, such as the

formation of CML from glycolaldehyde, require oxidative steps and can be catalyzed by

redox metal ions (14). In this case, complexation of metal ions could be complementary

to the PM carbonyl trapping mechanism as suggested by our previous work (38).

It is clear that the effective inhibition of protein AGE modifications has an

important therapeutic potential. In recent reports, PM alleviated renal pathology in the

streptozotocin rat and spontaneous db/db mouse models of diabetes (31-33), and it

showed successful progress through safety and efficacy clinical trials for diabetic

nephropathy (62). It is possible that different inhibitory mechanisms such as

scavenging of toxic carbonyl metabolites from carbohydrates (38, 59) and lipids (60,

61), and interfering with metal ion catalysis (37 and this study) may operate in vivo. Supporting this notion, PM treatment of diabetic animals has resulted in lower levels of

protein-CML, as well as MOLD, a dicarbonyl-derived protein crosslink (31, 32, 59). The

relative importance of these mechanisms is difficult to estimate because pyridoxamine

action in vivo will be influenced by factors such as local tissue and intracellular

concentrations of redox metal ions, reactive carbonyls, and PM itself. It may also be

affected by competition from endogenous carbonyl and metal scavengers. However,

the greater efficacy of PM, compared to carbonyl scavengers, in diabetic animal studies

(31) suggests that the inhibition of degradation of protein-Amadori via interference with

redox metal ion catalysis may play a more important role in vivo.

The pathogenicity of protein-Amadori intermediate itself remains an open

question. Damaging effects of early protein glycation intermediates in diabetic animals


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and cell culture systems have been reported (63). However, the transient nature of

such intermediates makes it difficult to attribute these effects to specific chemical

structures. The observed effects may reflect the conversion of these early

intermediates to the chemically stable AGEs or may be mediated by the byproducts of

this conversion, such as superoxide. Interestingly, the level of the circulating protein-

Amadori intermediates remained unchanged in diabetic animals treated with PM (31),

even though their conversion to AGEs was blocked. These data suggest the existence

of mechanisms that confer a rapid catabolism of protein-Amadori intermediate in vivo.

Regardless of whether this intermediate is pathogenic itself or simply a precursor to

pathogenic AGEs, the rate of its accumulation at diabetic glucose concentration

suggests that proteins with relatively short lifetimes (days) may undergo modifications in

diabetes (Fig. 3).

The combination of different inhibitory mechanisms may place PM at an

advantage over the compounds functioning primarily as either scavengers of metal ions

or carbonyl trapping agents. For instance, PM reactivity towards dicarbonyl compounds

is significantly lower than that of aminoguanidine (P. A. Voziyan and B. G. Hudson,

unpublished data), but PM exhibits greater efficacy in animal models (31). PM also

exhibits relatively weak metal binding, with a stability constant for Cu2+ several orders of

magnitude lower than that of strong chelators such as EDTA or DTPA (50). Hence, PM

is a potent inhibitor of formation of protein-AGEs that is less likely to interfere with

metabolic pathways that are not the intended targets.

Asknowledgements – We thank Ms. Parvin Todd for excellent technical assistance.


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Figure legends

Figure 1. Pathway of formation of glucose-derived protein-Amadori and protein-

CML (from ref. 5, 47, and 64). Reversible condensation of the aldehyde group of

glucose with a protein amino group results in formation of a Schiff base followed by

essentially irreversible rearrangement to an Amadori intermediate. The Amadori

intermediate enolizes to the 2,3-enediol in a reaction catalyzed by phosphate anions

(65). The 2,3-enediol undergoes spontaneous autoxidation involving the formation of

superoxide, catalyzed by transition metal ions; hydrogen peroxide, formed by

superoxide dismutation, regenerates the catalytic metal oxidation state. The putative

dicarbonyl product of 2,3-enediol autoxidation undergoes further oxidative degradation,

producing Nε-carboxymethyllysine and D-erythronic acid.

Figure 2. Reversible inhibition of the formation of glucose-derived BSA-CML by

pyridoxamine. (A) BSA (5 mg/ml) was incubated with 200 mM glucose and 20 mM

pyridoxamine in 200 mM Na-phosphate buffer containing 0.02% sodium azide, pH 7.5,

at 37°C for 35 days. Glucose and pyridoxamine were removed by dialysis at 4°C; the

complete removal of PM was confirmed by the absence of a fluorescence signal at

Ex=324 nm, Em=393 nm. After dialysis, the protein solution was again incubated at

37°C in the same buffer for up to 120 days (triangles). An aliquot from this sample was

incubated continuously without the removal of PM or glucose (squares). Control sample

did not contain PM (circles). Modifications of protein lysine residues to CML were

measured by ELISA as described under Experimental procedures. Each data point

represents an average of two measurements. (B) After 35 days of incubation, the

content of fructosyllysine in the samples was determined using the nitroblue tetrazolium

method described under Experimental procedures. Inset: after the complete removal of

glucose and PM, the protein solution was again incubated at 37°C in the same buffer for

up to 120 days followed by the determination of fructosyllysine. Each data point

represents an average of two measurements.


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Figure 3. Kinetics of formation and degradation of BSA-Amadori at physiological

levels of glucose. (A) BSA (20 mg/ml) was incubated with either 5 mM (open circles) or

30 mM glucose (filled circles) and 20 mM pyridoxamine in 200 mM Na-phosphate buffer

containing 0.02% sodium azide, pH 7.5, at 37°C. Accumulation of BSA-Amadori

(fructosyllysine, FL) was followed using the nitroblue tetrazolium method described

under Experimental procedures with the baseline readings (BSA alone incubated under

the same conditions) subtracted. For the incubations with 30 mM glucose: when the

concentration of Amadori reached a maximum constant value, glucose and

pyridoxamine were removed by dialysis at 4°C; the complete removal of PM was

confirmed by the absence of a fluorescence signal at Ex=324 nm, Em=393 nm. After

dialysis, the protein solution was again incubated at 37°C in the same buffer and

degradation of Amadori-BSA was followed for up to 160 days. (B) BSA-Amadori was

prepared with 30 mM glucose as described above. After dialysis, BSA-Amadori (5

mg/ml) was again incubated at 37°C in the same buffer either without PM (circles) or

with different concentrations of freshly added PM: 0.1 mM (diamonds), 1 mM (squares)

or 15 mM (triangles). Inverted triangles represent the data from incubations of

unmodified BSA. Modifications of protein lysine residues to CML were measured by

ELISA as described under Experimental procedures. Each data point represents an

average of two measurements.

Figure 4. Inhibition of post-Amadori CML formation by structural analogs of PM.

Ribose-derived BSA-Amadori intermediate was prepared as described under

Experimental procedures. After the removal of sugar by extensive dialysis at 4°C,

incubation at 37°C was resumed in the presence of the indicated concentrations of 3-

hydroxypyridine (A), 4-aminomethylpyridine (B), and 5-nitro-2-hydroxybenzylamine (C).

CML was measured using ELISA; each experimental point represents an average of

two measurements. Note the differences in the concentration range used in (A) and (B)

compared to that in (C).


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Figure 5. Inhibition of post-Amadori CML formation by pyridoxamine at different

metal concentrations. For the preparation of protein-Amadori intermediate, BSA (10

mg/ml) was incubated with 0.5 M ribose in 200 mM Na-phosphate buffer containing

0.02% sodium azide, pH 7.5, at 37°C for 24 h. After the removal of sugar by an

extensive dialysis at 4°C, incubation at 37°C was resumed in the presence of the

indicated concentrations of PM in buffer alone (circles), buffer containing 10 µM CuCl2

(triangles) or buffer containing 100 µM CuCl2 (diamonds). After 48 h, BSA-CML was

measured by ELISA; each symbol represents an average of two measurements.

Figure 6. 13C NMR spectra of RNase-[2-13C]-Amadori with and without PM.

Amadori-RNase was prepared using either 30 mM D-[2-13C]glucose (A and B) or 100

mM D-[2-13C]glucose (C, D and E) and experimental conditions described earlier. After

removal of the free [2-13C]glucose and PM by dialysis, the buffer was replaced by 50

mM Na-phosphate buffer (pD 7.5), prepared with D2O. NMR experiments were

performed on a Varian UnityPlus 600 MHz NMR spectrometer at 21ºC, collecting

20,000 transients per spectrum (see Experimental Procedures). (A) and (C) Spectra of

RNase-[2-13C]-Amadori without PM using two samples at 4.1 mM (A; Sample 1) and

3.8 mM (C; Sample 2) protein concentration. (B) Spectrum of Sample 1 after the

addition of 3 mM PM and incubation for 24 h (final protein concentration = 4.0 mM). (D)

and (E) Spectra of Sample 2 after incubation with 20 mM PM for 1 day (D) or 8 days

(E). RNase concentration was 3.1 mM in D and E. Abbreviations pyr and fur,

respectively, designate the pyranose and the furanose forms of the protein-Amadori

intermediate.

Figure 7. A model for inhibition of AGE/ALE formation by PM. The numbers

represent steps in AGE pathways inhibited by PM. Step 1. PM prevents conversion of

protein-Amadori to protein-CML via complexation of catalytic metal ions (present work).

Step 2. PM inhibits protein modifications by scavenging free carbonyl products of

glucose and lipid degradation (38, 59-61). Step 3. PM may also inhibit metal-catalyzed

oxidative steps in protein modifications by low molecular weight carbonyl compounds

(ref. 38 and present work).


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C=OH-C-OH

--H

-HO-C-H

-

glucose aldehyde

Schiff base Amadori intermediate

Mn+ M(n-1)+

O2

2,3-enediol

C=OH2C-NH-protein-

OH

-

Nε -carboxymethyllysine (CML)

erythronic acid

H2N-protein

C3H7O3

OH

-C=O

-

C3H7O3

C=H-C-OH

--H

-HO-C-H

-

C3H7O3

N-protein H2C-NH-proteinC=O

--HO-C-H

-

C3H7O3

C-OHH2C-NH-protein-

-C-OH

=

C3H7O3

C=OH2C-NH-protein-

-C=O

-

C3H7O2

Mn+

H2O2

M(n-1)+

1-amino-1-deoxy-fructofuranose form of protein-Amadori

1-amino-1-deoxy-fructopyranose form of protein-Amadori

O

CH2NH

OH

protein

OHOCH2 CH2NH

OH

protein

Voziyan et al., Fig.1

OHOH OH

HOOH

H2O2

OH.

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Time (d)0 20 40 60 80 100 120

BSA

-CM

L(E

LISA

read

ings

at 4

10 n

m)

0.5

1.0

1.5

2.0

FL (A

bs a

t 550

nm

)

0.0

0.1

0.2

0.3

0.4

A

B

Time (d)

40 60 80 100 120

FL (m

ol/m

ol L

ys)

0.2

0.4

0.6

BSA aloneafter 35 d

BSA/glucose after 35 d

BSA/glucose+PM after 35 d

BSA/glucose

BSA/glucose+PM

Glucose and PM removed


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Incubation time (d)0 20 40 60 80 100 120 140 160

BSA

-CM

L(E

LISA

read

ings

at 4

10 n

m)

0.1

0.2

0.3

Incubation time (d)0 20 40 60 80 100 120 140 160

FL (m

ol/m

ol L

ys)

0.00

0.02

0.04

0.06

0.08

0.10 30 mM Glucose and 20 mM PM removed

30 mM Glucose and 20 mM PM removed

A

B


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Time (days)0 1 2 3 4 5 6

ELIS

A R

eadi

ng (4

10 n

m)

0.0

0.1

0.2

0.3

0.4

0.5

0.6 3 mM NHBA 1 mM NHBA 0.5 mM NHBA 0.1 mM NHBA none

O

CH2

NH2

O

H

N2

O

CH2

NH2

O

H

N2

0 2 4 6 8 10 12

0.0

0.5

1.0

1.5

2.0 none3 mM 3 - HP15 mM 3 - HP50 mM 3 - HP

ELIS

A R

eadi

ng (4

10 n

m)

Time (days)

N

OH

N

OH

0 2 4 6 8 10

0.0

0.5

1.0

1.5

2.0none3 mM 4- AMP15 mM 4 - AMP50 mM 4 -AMP

Time (days)

N

CH2

NH2

N

CH2

NH2

ELIS

A R

eadi

ng (4

10 n

m)

A

B

3-hydroxypyridine

4-aminomethylpyridine

5-nitro-2-hydroxybenzylamine

C

H2NH2C

O

O

NH2

H

N

C 2

H3C

OH

M2+

CH3

N

OH

Pyridoxamine metal complex (from ref. 35 and 36)

Voziyan et al., Fig. 4


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Concentration of PM (mM)0 2 4 6 8 10

Nor

mal

ized

ELI

SA re

adin

gs

0.2

0.4

0.6

0.8

1.0


100 µM CuCl2

10 µM CuCl2

No CuCl2 added


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Reducing sugars

Protein-Amadori adducts

Protein

Protein

Reactive carbonyl compounds

AGE/ALE-modified proteins

PM-carbonyl adducts

Mn+ / [

O 2]

M n+/ [O2 ]

PM

(Mn+)PM2

PM

(Mn+)PM2


13

2 PM

Lipids


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Anthony S. Serianni and Billy G. HudsonPaul A. Voziyan, Raja G. Khalifah, Christophe Thibaudeau, Alaattin Yildiz, Jaison Jacob,

through binding of redox metal ionsinhibits conversion of amadori intermediate to advanced glycation end-products Modification of proteins In vitro by physiological levels of glucose: Pyridoxamine

published online September 15, 2003J. Biol. Chem.

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http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

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