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INTRODUCTION |
The Maillard reaction involves reaction of amino groups on
proteins with aldehydes and ketones to produce advanced glycation end-products, or AGEs.1 The
reaction products include amino acid cross-links, fluorophores, and
chromophores on proteins (1, 2). AGEs can bind to specific receptors on
cells to cause intracellular oxidative stress as well as the synthesis
of growth factors and cytokines (3-6), and the end result is usually
damage to the affected tissues (7, 8).
Many tissues have increased AGE content with aging, but increased AGEs
are also associated with specific pathologies such as formation of
cataracts (9), Alzheimer's disease (10), amyloidosis (11),
atherosclerosis (12), and diabetic retinopathy (13, 14). A direct role
for AGEs in diabetes was proposed when it was found that AGE infusion
into normal rats produced diabetic changes (15). In addition,
pharmacological intervention to block the effects of AGEs appears to
prevent certain complications of diabetes (16, 17).
Cataract formation is a multifactorial process. One mechanism by which
glycation damages proteins is through production of reactive oxygen
species (ROS), and various studies link the production of glycation
products and the formation of ROS (18-22). Ample evidence indicates
that ROS contribute to lens protein damage (23-25), but it is not
clear how ROS could be produced within the lens. Recent studies suggest
that lens pigments, in the presence (26) or absence of ultraviolet
light (27), could generate ROS.
The Amadori product, the initial reaction product in the Maillard
reaction, remains almost constant within normal aging lenses, but it
occurs at much higher levels in diabetic and senile cataractous lenses
(28, 29). Numerous studies document the formation of AGEs in lens
proteins during aging and cataractogenesis (9, 30-32). In fact,
Ortwerth et al. (33) found that ascorbate-derived AGEs can
produce ROS, and human lenses contain relatively large amounts of
ascorbate. Oxidation of ascorbate produces highly reactive compounds,
including dicarbonyls that can form AGEs (34, 35).
We reasoned that, if glycation products are a source of ROS in the
lens, continued formation of ROS could account for cumulative damage to
lens crystallins during aging and cataractogenesis. However, we made
the surprising discovery that glycated proteins can produce
pentosidine, a protein cross-linking AGE on native unmodified lens
proteins. Accordingly, we investigated the formation of pentosidine
from early and advanced glycation products on
-crystallin, and we
propose a pathway for pentosidine synthesis from short-chain carbohydrates derived from degradation of early glycation products.
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine lenses were obtained from Pel-Freez
Biologicals, Roger, AR. EAH-Sepharose was purchased from Amersham
Pharmacia Biotech, Piscataway, NJ. Lysozyme, cytochrome c,
Chelex-100, deferoxamine,
-ketoglutarate, luminol, thiourea,
N-
-acetyllysine, and N-
-acetylarginine were
obtained from Sigma Chemical Co. EDC and salicylate were purchased from
Aldrich. Furosine standard was from Neosystem, France. Normal and
diabetic human lenses were obtained from the National Disease Research
Interchange and the Cleveland Eye Bank. Human brunescent lenses
were obtained from India (J. N. Medical College Hospital, Aligarh,
India). All buffers (unless otherwise mentioned) were prepared
with Chelex-100-treated water and, after preparation, were passed
through a Chelex-100 column to remove any remaining metal ions.
Purification of
-Crystallin--
After decapsulation, bovine
lenses were homogenized with a hand-held glass homogenizer in 50 mM Tris/HCl buffer (pH 7.4) containing 0.2 M
KCl, 1.0 mM EDTA, 10 mM mercaptoethanol, and
0.05% sodium azide. The homogenate was centrifuged for 20 min at
20,000 × g. The supernatant containing the bovine lens
proteins (BLP) was dialyzed against water for 24 h.
-Crystallin
(mixture of
A and
B) was isolated from BLP by gel filtration
chromatography on Sephacryl S-200 (36). The pooled
-crystallin
fraction from the column was dialyzed against 4 liters of
phosphate-buffered saline for 48 h and lyophilized.
Immobilization of Proteins on EAH-Sepharose--
-Crystallin
or lysozyme was immobilized on EAH-Sepharose gel (illustrated in Scheme
I). Five milliliters of EAH-Sepharose was
washed three times with water and adjusted to pH 4.5 with dilute HCl.
The gel was then collected in a conical tube and suspended in 10.0 ml
of water with 100 mg of
-crystallin and 191 mg of EDC. The contents
were gently mixed at 4 °C for 12 h. The pH of the mixture was
maintained by adding dilute NaOH. At the end of the reaction, the gel
was filtered and thoroughly washed five times, alternating 0.1 M sodium acetate buffer (pH 4.0) with 0.5 M
NaCl and 0.1 M Tris-HCl buffer (pH 8) with 0.5 M NaCl. The washed and immobilized protein preparation was
stored at 4 °C until use.

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Scheme 1.
Preparation of STGP and LTGP gels.
-Crystallin was immobilized on EAH-Sepharose in the presence of EDC.
It was then glycated with 500 mM D-ribose for 2 days in the case of STGP gel and with 100 mM for 15 days in
the case of LTGP gel. The ST and LT glycated gels were incubated with
BLP or lysozyme or amino acids. The supernatant was used for
analysis.
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Preparation of STGP and LTG Gels--
STGP gel (
-crystallin
or lysozyme) was prepared by reacting EAH-Sepharose-immobilized
proteins with 500 mM D-ribose for 2 days at
37 °C in 50 mM phosphate buffer (pH 7.4), and LTGP gel was prepared by reacting the proteins with 100 mM
D-ribose for 15 days at 37 °C. After glycation, the gels
were washed extensively with water to remove unreacted ribose and
stored at 4 °C until use.
Formation of Superoxide Anion from Glycated
-Crystallin--
Superoxide anion production by STGP and LTGP gels
was measured by cytochrome c reduction, as described by
Bhuyan and Bhuyan (37). Superoxide dismutase-inhibitable cytochrome
c reduction was taken to be a measure of superoxide anion.
STGP and LTGP gels (100 mg each) were incubated in 50 mM
sodium phosphate buffer (pH 7.4) at room temperature with slow
stirring. Aliquots were withdrawn at fixed time intervals for
cytochrome c reduction assay.
Formation of Hydroxyl Radical from Glycated
-Crystallin--
Hydroxyl radical production by glycated gels (from
-crystallin) was measured by the salicylate hydroxylation method.
Typically, 100 mg of STGP- or LTGP gel was incubated with salicylate.
The amount of 2,3-dihydroxy benzoate formed was assayed by a
spectrometric method (38) described by Bhuyan and Bhuyan (37).
Estimation of Pentosidine in Glycated Protein--
STGP and LTGP
gels (100 mg each) (from
-crystallin) were acid hydrolyzed in 6 N HCl for 16 h at 110 °C. The contents were dried
in a Speed-Vac concentrator (Savant Instruments, Inc. Farmingdale, NY),
rehydrated in 200 µl of water, and filtered through a 0.2-µm filter. Pentosidine was measured by the HPLC method as described previously (9) and quantified by comparison with a standard curve for
synthetic pentosidine.
Incubation of BLP or Lyzozyme with STGP and LTGP Gels--
The
STGP and LTGP gels (from
-crystallin or lyzozyme) were washed 10 times with 50 mM sodium phosphate buffer (pH 7.4) before incubation. BLP or lyzozyme (2 mg/ml) was incubated with the STGP- or
LTGP gels (100 mg) in 50 mM phosphate buffer (pH 7.4) at
37 °C with gentle mixing for up to 7 days. The final volume of the reaction mixture was 1.0 ml. BLP incubated with unglycated
-crystallin (EAH-Sepharose bound) served as control. Aliquots (0.2 ml) were drawn at regular time intervals. In some experiments with
-crystallin immobilized gels, we added ROS quenchers (1.0 mM luminol, 5 mM
-ketoglutarate, 5 mM mannitol), metal ion chelator (1 mM
deferoxamine), or FeCl3 (100 µM) at the start
of the reaction. Pentosidine was measured by HPLC.
Incubation of N-
-Acetyllysine + N-
-Acetylarginine with STGP
and LTGP gels--
N-
-Acetyllysine (5 mM)
and N-
-acetylarginine (5 mM) were incubated
at 37 °C in 50 mM phosphate buffer (pH 7.4) as described above for BLP. Aliquots of the supernatant were taken after 3 and 7 days for pentosidine measurement.
Pentosidine Formation under Anaerobic Conditions--
In some
experiments, air-tight screw cap tubes with an inlet for argon were
used to incubate 100-mg STGP- or LTGP gel with BLP under anaerobic
conditions. Otherwise, incubation conditions were as indicated earlier.
Argon was bubbled through the mixture for 20 min and the argon-air
mixture was removed with a vacuum pump. The tubes were then tightly
sealed and incubated at 37 °C for 3 days. Controls included
nonglycated protein gels and glycated gels incubated without proteins.
In addition, samples were also incubated simultaneously under aerobic
conditions for comparison.
Formation of Pentosidine from Water-insoluble Human Lens
Proteins--
Water-insoluble proteins (WI) from nondiabetic aged
(age: 67 years) or brunescent lenses (age: 76 years), were prepared as described by Shamsi et al. (39). 5 mg of WI was washed
thoroughly five times with 1 ml of 50 mM sodium phosphate
buffer (pH 7.4). After the final wash, 2.0 mg of BLP was added, and the
sample was incubated at 37 °C for 7 days. Aliquots (0.2 ml) were
taken at 3 and 7 days to measure pentosidine. Furosine content in the WI before and after incubation was measured by HPLC (Alltech, Dearfield, IL) using a furosine-dedicated column (40). For some incubations, the WI protein extract was reduced with sodium
borohydride. Incubations of WI with sodium phosphate buffer alone
served as a control.
Binding of Early Glycation Products from Human Lenses to Affinity
Matrix--
Water-soluble lens protein (WS) was isolated (34) from
age- and wet-weight-matched normal and diabetic (nuclear cataractous lenses from patients with Type II diabetes, diabetes duration: >10
years) human lenses. Two lenses (200-240 mg) from donors of 60-70
years of age were used for each sample. The samples were dialyzed
against 50 mM sodium phosphate buffer (pH 7.8), and loaded onto a previously equilibrated (50 mM phosphate buffer)
Affi-Gel-601 boronate column (Bio-Rad Laboratories, CA). The amount of
protein bound to the column was calculated by subtracting the amount of unbound proteins from the amount loaded on the column. After binding of
WS samples, the gel was thoroughly washed with buffer and then used in
incubations with 2.0 mg of BLP in sodium phosphate buffer at 37 °C
for up to 7 days. Some experiments used N-
-acetylarginine (5 mM) + N-
-acetyllysine (5 mM)
in the place of BLP. In other experiments, the gel was reduced with
sodium borohydride before incubation. Aliquots from the supernatant
were assayed for pentosidine.
Formation of Pentosidine from Short-chain
Carbohydrates--
N-
-Acetylarginine and
N-
-acetyllysine were incubated with two and three carbon
carbohydrates. N-
-Acetylarginine and
N-
-acetyllysine (5 mM each) were incubated in
50 mM phosphate buffer (pH 7.4) with 5 mM
carbohydrate at 37 °C. Aliquots were
drawn at 24 and 48 h, and pentosidine was measured after
acid hydrolysis (6 N HCl, 110 °C, 16 h).
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Table I
Effect of ± oxygen in the formation of pentosidine from STGP and
LTGP gels
BLP (2 mg/ml in 50 mM phosphate buffer, pH 7.4) was
incubated with 100 mg of STGP and LTGP gel at 37 °C in the presence
and absence of oxygen for 3 days. Pentosidine in BLP was estimated by
HPLC.
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Other Procedures--
SDS-PAGE used 15% gels under reducing
conditions according to the method of Laemmli (41). Amino acid content
of acid-hydrolyzed proteins was measured by the ninhydrin reaction
(34), and protein was measured by the Bradford method using a Bio-Rad
protein assay kit with bovine serum albumin as a standard.
Sodium borohydride (SBH) reductions involved incubating 5 mg of protein
with 5 mM SBH for 2 h at room temperature at pH 9.0 with slow stirring. Unreacted SBH was removed by washing the protein (WI).
Statistics--
Mean differences were evaluated by one-way
analysis of variance using StatView Software (SAS Institute, Inc.,
Cary, NC). Fisher's protected least significant difference test was
employed in these calculations. We considered a p value of
<0.05 statistically significant. In all figures the means that do not
share a common superscript are statistically different at
p < 0.05.
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RESULTS |
We found no pentosidine in control incubations where EAH-Sepharose
immobilized proteins with or without glycation were incubated with
buffer alone. These controls rule out the possibility of pentosidine
leaching from the gel or the formation of pentosidine on the gel itself.
Pentosidine Content of Immobilized Glycated
-Crystallin--
Pentosidine was measured by HPLC and expressed as
nanomoles per gram of gel. LTGP had nearly twice the amount of
pentosidine (4.8 ± 0.3 nmol/g of gel) as compared with STGP
(2.5 ± 0.1 nmol/g of gel). We were unable to quantify the Amadori
product of the ribose reaction with protein amino groups, because there
are no specific assays for it. Measurements of pentosidine indicate
that LTGP accumulated more AGEs than did STGP. However, as suggested by
the studies of Booth et al. (42), we expected STGP to
accumulate more Amadori product under the incubation conditions of our experiments.
Superoxide Anion and Hydroxyl Radicals from Immobilized Glycated
-Crystallin--
We used superoxide dismutase-inhibitable reduction
of cytochrome c as a measure of superoxide anion. Fig.
1A shows that superoxide anion
was minimal with unmodified
-crystallin (coupled to gel) but was
significant in glycated
-crystallin (coupled to gel). SOD inhibited
superoxide anion production from both STGP and LTGP gels. Based on the
data, we calculated that the amount produced by STGP gel and LTGP gel
was 9.5 ± 2.1 nmol/h/g of gel and 7.8 ± 2.0 nmol/h/g of
gel, respectively. Hydroxyl radical production was twice that in STGP
gel (6.2 ± 2.3 µmol/h/g of gel) compared with the LTGP gel
(3.0 ± 0.9 µmol/h/g of gel) (Fig. 1B).

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Fig. 1.
A, formation of superoxide anion from
STGP and LTGP gels. Superoxide dismutase-inhibitable reduction of
cytochrome c was measured at 550 nm. A typical experiment
used 100 mg of glycated gel. Incubation conditions are described in the
text. Means that do not share a common superscript letter
are statistically significant at p < 0.05. B, formation of hydroxyl radicals from STGP and LTGP gels.
STGP and LTGP gels were incubated with salicylate, and the amount of
2,3-dihydroxybenzoate formed was measured spectrophotometrically.
Control experiments contained the gel coupled to unglycated
-crystallin. Means that do not share a common superscript
letter are statistically significant at p < 0.05.
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Pentosidine Formation in BLP Incubated with Glycated
-Crystallin--
The STGP and LTGP gels prepared from incubation of
EAH-Sepharose-
-crystallin with ribose were washed thoroughly before
every incubation to remove loosely bound glycated proteins and any
breakdown products formed during storage. The gels were then incubated
with BLP. Pentosidine was measured after acid hydrolysis of an aliquot of the incubation mixture. HPLC chromatograms showed pentosidine as a
well-resolved peak from samples in which BLP incubated with STGP and
LTGP gels (Fig. 2A). Fig.
2B shows that the pentosidine content in both incubations
nearly doubled at 7 days compared with the amount at 3 days. The STGP
gel produced two to three times as much pentosidine as did the LTGP gel
(p < 0.05). These results indicate that pentosidine is
produced from carbohydrates derived from glycated proteins, and the
early glycation products (Amadori and Schiff's base) are the
precursors.

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Fig. 2.
Formation of pentosidine in BLP incubated
with STGP and LTGP gels. A, pentosidine was
estimated by HPLC. BLP (2.0 mg/ml) was incubated with 100 mg of STGP or
LTGP gels for 3 or 7 days as described in the text. Control experiments
contained unglycated EAH- -crystallin + BLP. Means that do not share
a common superscript letter are statistically significant at
p < 0.05. A, HPLC tracing of pentosidine
standard and pentosidine produced on BLP. B, Pentosidine in
BLP incubated with glycated gels. BLP incubated with unglycated
-crystallin (EAH-Sepharose-bound) served as control. Means that do
not share a common superscript letter are statistically
significant at p < 0.05.
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Effect of ROS Quenchers and a Metal Ion Chelator on Pentosidine
Formation--
STGP and LTGP gels were incubated with BLP for 7 days
in the presence or absence of a ROS quencher or deferoxamine or
FeCl3. As can be seen in Fig.
3, addition of
-ketoglutarate (a
scavenger of H2O2) most effectively inhibited
pentosidine formation in BLP incubated with SGTP gels; pentosidine was
approximately half that in samples incubated without the ROS quencher.
Luminol, an O
2 scavenger, only marginally inhibited
pentosidine formation, and thiourea, an OH· scavenger, inhibited
it by ~30%. The effects were not as great in incubations with LTGP
gels, but
-ketoglutarate and thiourea caused significant
(p < 0.05) inhibition. The metal ion chelator, deferoxamine, was ineffective. Addition of 100 µM
FeCl3 enhanced pentosidine formation in both STGP and LTGP
gels. The increase was 17% with STGP and 73% with LTGP gel. These
results suggest that H2O2 is an important
constituent in pentosidine synthesis from glycated proteins.

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Fig. 3.
Effect of ROS quenchers on the formation of
pentosidine. BLP (2.0 mg/ml) was incubated with 100 mg each of
STGP and LTGP gel in the presence or absence of indicated ROS quenchers
for 7 days. Pentosidine was measured in BLP after the incubation. Means
that do not share a common superscript letter are
statistically significant at p < 0.05.
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Effect of Lack of Oxygen--
To assess the importance of oxygen
in the formation of pentosidine, we did some incubations in the absence
of oxygen. Table I shows that pentosidine
was decreased ~64 and 98% in STGP and LTGP gels in the absence of
oxygen, which indicates that oxidative reactions mediated by ROS are
necessary for pentosidine formation.
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Table II
Formation of pentosidine from short-chain carbohydrates
N- -Acetyllysine and N- -acetylarginine (5 mM each) were incubated with 5 mM each of the
indicated carbohydrate in 50 mM sodium phosphate buffer, pH
7.4, at 37 °C for 1 and 2 days. Pentosidine was measured after acid
hydrolysis of the incubation mixture.
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Covalent Cross-linking of BLP Incubated with STGP and LTGP
Gels--
SDS-PAGE revealed that BLP underwent covalent cross-linking
upon incubation with glycated gels. We found cross-linked proteins of
molecular mass 40-45 kDa in both STGP and LTGP gel incubated proteins (Fig. 4A). STGP gels
incubated with BLP produced higher levels of these proteins than
comparable incubations with LTGP gels, suggesting that covalent
cross-linking possibly occurred as a result of pentosidine and other
AGEs in BLP. Results of densitometric analysis showed that, after 7 days of incubation, STGP gel produced nearly three times as much
cross-linked proteins as did the LTGP gel (Fig. 4B).

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Fig. 4.
A, covalent cross-linking of BLP on incubation with
STGP and LTGP gels. BLP (2 mg/ml) was incubated with STGP or LTGP gel
in phosphate buffer, and aliquots were drawn at 3 and 7 days and
analyzed by SDS-PAGE (15% gel). Lanes 1-4, 3 days
incubation; lanes 5-8, 7 days incubation; lane
9, molecular weight markers. Lanes 1 and 5,
BLP incubated with unglycated EAH-Sepharose- -crystallin for 3 and 7 days; lanes 2 and 6, BLP incubated
with STGP gel. Lanes 3 and 7, BLP incubated with
LTGP gel; lanes 4 and 8, BLP alone. STGP and LTGP
gels with buffer alone was run simultaneously but showed no protein
bands. B, densitometric analysis of cross-linked proteins of
40-45 kDa.
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Lyzozyme and N-
-Acetylarginine + N-
-Acetyllysine Incubated
with Gels--
The amount of pentosidine formed from lysozyme after 3 and 7 days of incubation with STGP gel was 9.7 ± 1.3 and
13.2 ± 0.9 pmol/µmol of amino acids, whereas in LTGP gel
measured at the same time, it was 2.5 ± 0.8 and 5.3 ± 0.6 pmol/µmol of amino acids (Fig.
5A). Again, the amounts
measured in STGP gel were higher than those in LTGP gel. The
pentosidine levels in the STGP gel samples after 7 days incubation with
lyzozyme were about half those measured in BLP incubated under similar
conditions.

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Fig. 5.
Pentosidine formation in Lyzozyme
(A) and in
N- -acetylarginine + N- -acetyllysine incubation
mixture (B). STGP and LTCP gels (100 mg) were
incubated with lysozyme (2 mg/ml) or with
N- -acetylarginine + N- -acetyllysine (5 mM each) for 3 and 7 days. Lyzozyme (or amino acid mixture)
incubated with unglycated -crystallin (EAH-Sepharose-bound) served
as control. Means that do not share a common superscript
letter are statistically significant at p < 0.05.
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In another set of experiments, gels were incubated with
N-
-acetylarginine + N-
-acetyllysine instead
of BLP. Arginine and lysine are precursor amino acids for pentosidine,
and we found that significant amounts of pentosidine were produced in
these incubations. The levels were 30-44% higher following incubation with STGP gel than with the LTGP gel (Fig. 5B). Taken
together, these results show that pentosidine formation is not specific to BLP alone and that it forms in other proteins as well.
Pentosidine in BLP Incubated with Glycated Lysozyme--
Lysozyme
was immobilized on EAH-Sepharose and glycated using
D-ribose. STGP and LTGP gels were prepared by incubating
with D-ribose for 2 and 15 days. As shown in Fig.
6, incubations with the STGP gel
consistently yielded more pentosidine than did the LTGP gel, but
pentosidine formation in both increased from 3 to 7 days.

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Fig. 6.
Pentosidine formation in BLP and
(N- -acetylarginine + N- -acetyllysine) incubated
with lyzozyme STGP and LTGP gels. STGP and LTGP gels were
prepared by coupling lyzozyme with EAH-Sepharose followed by short-term
and long-term incubation with D-ribose (see "Experimental
Procedures"). Other details are same as in Fig. 2. A,
pentosidine in BLP incubated with lysozyme-glycated gel, B,
pentosidine in amino acids incubated with lysozyme-glycated gel. BLP
incubated with EAH-Sepharose-bound unglycated lyzozyme served as
control. Means that do not share a common superscript letter
are statistically significant at p < 0.05.
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STGP or LTGP gels were also incubated with a mixture of
N-
-acetylarginine + N-
-acetyllysine, and
the results were similar to those obtained with BLP (Fig.
6B). Together these incubation experiments confirm that
pentosidine formation on unmodified native proteins occurs not only
with glycated
-crystallin but also with other proteins.
Formation of Pentosidine in BLP Incubated with Water-insoluble
Human Lens Proteins--
To determine whether these lens proteins
could mimic the effects of in vitro glycated proteins, we
incubated WI proteins from aged or brunescent lenses with BLP. This
experiment also helped us to establish that glucose-derived early
glycation products produce pentosidine. We washed the WI thoroughly
with water before incubation to remove all soluble protein and
incubated them with BLP (2 mg/ml) for 3 and 7 days. Analysis of BLP
after incubation revealed that proteins from aged lenses produced
nearly 3-fold higher levels of pentosidine (Fig.
7, A and B) than
proteins from brunescent lenses. Sodium borohydride treatment of WI
before incubation with BLP almost completely suppressed pentosidine
formation (Fig. 7B). Furosine content of WI protein of aged
and brunescent lenses was estimated before and after incubation. The
amount of furosine in WI of aged and brunescent lenses were similar
(~500 pmol/µmol of amino acids) (Fig. 7C) but was
completely degraded after 7 days of incubation (Fig. 7D). As
expected, sodium borohydride treatment resulted in the reduction of the
Amadori, and therefore, furosine was not detected in these samples
(Fig. 7D). These results show that proteins glycated
in vivo can produce pentosidine on nonglycated proteins and
that early glycation products are pentosidine precursors.

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Fig. 7.
A and B, formation of
pentosidine in BLP incubated with WI proteins from aged and brunescent
cataractous human lenses. Washed WI proteins (5 mg) were incubated with
BLP (2 mg/ml) as described in the text. C and D,
furosine content of WI before and after incubation with BLP. Furosine
was estimated by HPLC. For some experiments, WI was reduced by SBH
before incubation with BLP. Unreacted SBH was removed by washing
proteins several times with buffer. Means that do not share a common
superscript letter are statistically significant at
p < 0.05.
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Incubation with Affinity Matrix-bound Early Glycation
Products--
We calculated that 18.5 and 28.8 mg of protein from
early glycation products were bound to a boronate affinity gel from
incubations with aged and diabetic lenses. The gel was then incubated
with BLP or a mixture of N-
-acetylarginine + N-
-acetyllysine. As Fig. 8
shows, the bound proteins produced pentosidine in BLP. The amounts
formed from diabetic lens proteins were higher than those formed by
aged lenses after either 3 or 7 days of incubation. Similarly, when a
mixture of N-
-acetylarginine + N-
-acetyllysine was added to the matrix-bound diabetic
lens proteins, pentosidine formation was nearly twice that from
comparable samples from aged lenses. Neither control incubations of BLP
with gel, but without bound lens proteins, nor incubations with amino
acids or BLP produced pentosidine. We concluded that diabetic lenses
produce more pentosidine than nondiabetic lenses of comparable age, and
that early glycation products promote pentosidine formation.

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Fig. 8.
Formation of pentosidine in BLP from affinity
matrix-bound early glycation products of human lenses. Early
glycation products from WS extracts of diabetic and aged lenses were
bound to a boronate affinity gel by passing through the column. The
matrix was extensively washed with sodium phosphate buffer, and 100 mg
of gel was incubated with 2 mg of BLP or with a mixture of
N- -acetylarginine + N- -acetyllysine (5 mM each) for 3 and 7 days as described in the text. We used
100 µg of BLP and 833 nmol of amino acids for pentosidine
measurements. The results are expressed as picomoles of pentosidine per
gram of gel.
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Pentosidine Formation from Short-chain Carbohydrates--
To
determine which of the fragments derived from early glycation products
might serve as precursors of pentosidine, we incubated two and three
carbon carbohydrates with a mixture of N-
-acetylarginine + N-
-acetyllysine and measured the amount of pentosidine
formed. Among the carbohydrates tested, glyceraldehyde produced the
highest levels of pentosidine followed by glycolaldehyde and glyoxal
(Table II). 1H NMR (D2O) and UV absorption
spectra of the product from glyceraldehyde were fully compatible with
those reported for ribose-derived pentosidine (43) (data not shown). In
the case of glycolaldehyde and glyoxal, spectroscopic
characterization is necessary to confirm the formation of pentosidine.
Because glyceraldehyde was the major precursor, we have proposed a
pathway for pentosidine formation from this compound (Fig. 9). First, condensation of the aldehydic
group of glyceraldehyde with the guanidino group of arginine and
dehydration yields 1. Elimination of a molecule of
formaldehyde yields 2. Oxidation of 2 followed by
condensation with a molecule of lysine gives 4, which by
cyclization and reaction with a second molecule of glyceraldehyde forms
6. Intramolecular cyclization of 6 followed by
elimination of two molecules of water yields pentosidine. In fact, a
structure similar to the proposed intermediate 6 has been
reported from the reaction of short-chain carbohydrates with arginine
and lysine (44).
 |
DISCUSSION |
We reasoned that Amadori products could accumulate in the lens,
where proteins are long-lived. These products could continually generate the reactive oxygen species and damage lens proteins. Other
investigators had shown that, when high amounts of D-ribose are used for glycation, proteins accumulate the Amadori product, and
its degradation is inhibited (42, 45). Therefore, we used a novel
preparation in which
-crystallin was coupled to EAH-Sepharose and
glycated to enrich the lens proteins with Amadori products or AGEs.
Our studies confirm the formation of O
2 and OH·
radicals from the glycated proteins. Previous studies from other
laboratories demonstrated O
2 production from early glycation
products that include 1,2- and 2,3-enolization of the Schiff's base
and oxidation of the enolate anion (46-49). We expected higher levels
of O
2 from the short-term glycated
-crystallin (500 mM ribose, 2 days) than the long-term glycated
-crystallin (100 mM, 15 days), but we observed only
modest increases. This may be due to the presence of early glycation
products in the long-term glycated
-crystallin.
A puzzle was the production of OH· from glycated proteins. This
radical could be produced via the Fenton reaction from
H2O2. Phosphate buffer has trace amounts of
cupric and ferric ions. To remove metal ions we passed our buffers
through a Chelex-100 column, but this may not have removed all trace
metal ions. In fact, a recent study showed that buffers passed through
Chelex-100 resin retain some cupric ions (50). This may have initiated the Fenton reaction and produced OH·. Additionally, lens
proteins themselves may have initiated the Fenton reaction. It is known
that lens proteins can bind metal ions in vivo (51), which
raises the possibility that the
-crystallins from bovine eyes may
have redox-active metal ions bound to it.
We found cross-linked proteins of 40-45 kDa in bovine lens proteins
that were incubated with glycated proteins, and lens proteins incubated
with short-term glycated proteins had higher levels than those
incubated with long-term glycated proteins. These observations suggest
that the cross-linked proteins are produced by reactions mediated
primarily by early glycation products, not likely by AGEs. The finding
that pentosidine is formed in BLP, that was incubated with glycated
-crystallin, supports this view. In addition, Araki et
al. (30) demonstrated immunoreactivity with 40- to 45-kDa proteins
in aged and cataractous lenses with antibodies against glucose-derived
AGEs. SDS-PAGE studies of lens proteins showed that aged and
cataractous lenses have proteins of 40-45 kDa. Spector and colleagues
(52, 53) isolated a fluorescent 43-kDa protein from human lenses. They
also reported that proteins of 40-45 kDa could be formed from the
transitional metal-catalyzed reaction in lens crystallins incubated
with ascorbate (54).
We were surprised by the finding of pentosidine in proteins that were
incubated with glycated
-crystallin. According to the conventional
dogma, a pentose sugar, such as ribose, reacts first with a lysine
residue on proteins to form an Amadori product, which then reacts with
an arginine residue to form pentosidine (43). Based upon results from
our own experiments, we believe that pentosidine can form from
short-chain carbohydrates. Our experiments show that glyceraldehyde,
glycolaldehyde, and glyoxal can produce pentosidine when incubated with
N-
-acetyllysine + N-
-acetylarginine.
Whether these carbohydrates were ultimately responsible for pentosidine
formation in BLP or lysozyme still needs to be established, probably by
analysis of carbohydrates after chemical trapping during the reaction.
We believe that the Amadori product or Schiff's base underwent
fragmentation through reactive oxygen species-mediated reactions to
produce the short-chain carbohydrate intermediates, which then reacted
sequentially with lysine and arginine residues to produce pentosidine.
The total absence of pentosidine in glycated proteins that were reduced with SBH supports this view. In confirmation of our proposed pathway, Farboud et al. (55) observed pentosidine formation from
glycolaldehyde, as we did. Formation of glycolaldehyde and glyoxal as
intermediates in glycation was also documented (56, 57).
Our observations with human lens proteins are noteworthy, because they
have important implications for human aging and disease. First, we
incubated bovine lens proteins with water-insoluble lens proteins
isolated from brunescent cataractous lenses. These damaged lenses are
completely opaque and highly pigmented, and they contain relatively
large amounts of Amadori products and AGEs (9, 51, 58). We found that
the furosine content decreased after incubation, indicating degradation
of the Amadori product, but loss of this intermediate did not
correspond to the formation of pentosidine. Only 26 and 9% of the
Amadori product in aged and cataractous lenses could account for the
formed pentosidine. We assume that other products were produced along
with pentosidine.
In our experiments with boronate affinity column, as expected, diabetic
lenses had more Amadori product than aged lenses, as indicated by the
amount of affinity gel-bound protein. Bovine lens proteins incubated
with the gel formed pentosidine. We did not expect boronate-bound
Amadori product to cleave and produce pentosidine. It is possible that
early glycation products other than those bound to boronate may have
served as pentosidine precursors in these incubations. Nevertheless,
these results suggest that the higher levels of pentosidine in diabetic
lenses compared with normal lenses (59) is due, in part, to degradation
of the relatively higher levels of Amadori product.
We found that oxygen is necessary for pentosidine formation from the
Amadori product in the lens. Because there is low oxygen tension in the
lens (60), such reactions probably occur only at a very slow rate. In
addition, the lens may have other defenses against pentosidine
formation, such as aldose reductase, which metabolizes glyceraldehyde,
a potential precursor for pentosidine, as shown by our studies.
The formation of pentosidine was not an isolated phenomenon that
occurred only from glycated
-crystallin. We could reproduce those
results by using glycated lysozyme in place of glycated
-crystallin
and lysozyme in place of BLP. Our results suggest that pentosidine
formation from glycated proteins occurs not only in the lens but may
also occur in other tissue proteins in the body as well.
In vitro incubation experiments have shown progression of
cross-linking of glycated proteins in the absence of sugar (61, 62). In
diabetes, certain complications progress during periods of good
glycemic control (63). These observations suggest that biochemical
processes that are set in motion in the presence of sugar or under poor
glycemic control can continue even after a return to normoglycemia. Our
finding that pentosidine and possibly other AGEs can be formed from
degradation of early glycation products in the absence of sugar offers
one potential scenario.
In summary, our studies focused upon the formation of pentosidine from
glycation. We propose a novel pathway of pentosidine formation that
could illuminate mechanisms of AGE formation in diabetes and aging as
well as suggest potential therapeutic interventions.