Biochem. J. (2008) 409, 771–777 (Printed in Great Britain)
771
doi:10.1042/BJ20071006
Effect of methylglyoxal modification on stress-induced aggregation of client
proteins and their chaperoning by human αA-crystallin
Ashis BISWAS*1 , Benlian WANG†‡, Masaru MIYAGI†‡§ and Ram H. NAGARAJ*‡§2
*Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH 44106, U.S.A., †Center for Proteomics, Case Western Reserve University,
Cleveland, OH 44106, U.S.A., ‡Visual Sciences Research Center, Case Western Reserve University, Cleveland, OH 44106, U.S.A., and §Department of Pharmacology,
Case Western Reserve University, Cleveland, OH 44106, U.S.A.
α-Crystallin prevents protein aggregation under various stress
conditions through its chaperone-like properties. Previously, we
demonstrated that MGO (methylglyoxal) modification of αAcrystallin enhances its chaperone function and thus may affect
transparency of the lens. During aging of the lens, not only αAcrystallin, but its client proteins are also likely to be modified
by MGO. We have investigated the role of MGO modification
of four model client proteins (insulin, α-lactalbumin, alcohol
dehydrogenase and γ -crystallin) in their aggregation and structure
and the ability of human αA-crystallin to chaperone them.
We found that MGO modification (10–1000 µM) decreased the
chemical aggregation of insulin and α-lactalbumin and thermal
aggregation of alcohol dehydrogenase and γ -crystallin. Surface
hydrophobicity in MGO-modified proteins decreased slightly
relative to unmodified proteins. HPLC and MS analyses revealed
argpyrimidine and hydroimidazolone in MGO-modified client
proteins. The degree of chaperoning by αA-crystallin towards
MGO-modified and unmodified client proteins was similar.
Co-modification of client proteins and αA-crystallin by MGO
completely inhibited stress-induced aggregation of client proteins.
Our results indicate that minor modifications of client proteins and
αA-crystallin by MGO might prevent protein aggregation and thus
help maintain transparency of the aging lens.
INTRODUCTION
a major post-translational modification; it produces stable adducts
on proteins, which are collectively known as AGEs (advanced
glycation end-products) [17,18]. Previous studies suggest that a
reactive α-dicarbonyl compound, MGO (methylglyoxal), formed
from triose phosphate intermediates of glycolysis, is a major
source of AGEs in the human lens [20,21]. Several years ago, we
established that reaction of MGO with αA-crystallin enhances
its chaperone function [22]. Other groups have studied this
reaction and report similar or opposite findings [23–25]. We
further showed that replacement of selected arginine residues
with alanine mimicked the effects of MGO [26]. Based on those
findings, we proposed that MGO modification of αA-crystallin
might be beneficial to the lens in maintaining transparency during
aging. We believe that in addition to modifying αA-crystallin
during aging, MGO can modify other lens proteins as well.
Other such proteins could be client proteins for αA-crystallin’s
chaperone function. To determine if client protein modification
affected their chaperoning by αA-crystallin, we first established
the effect of MGO modification on chemically or thermally
induced aggregation of four model client proteins, and we then
measured the effect on their binding to MGO-modified and native
αA-crystallins. We also examined the consequences of MGO
modification on the structural characteristics of client proteins.
The main constituents of the mammalian lens are crystallins,
namely α-, β- and γ -crystallins. αA-Crystallin is the major
crystallin, accounting for nearly 50 % of proteins in the lens. It
consists of two subunits, αA- and αB-crystallins, which typically
exist in a molar ratio of approx. 3:1 in the mammalian lens [1].
Although it was believed for many years that α-crystallin was
strictly a lens-specific protein, both αA- and αB-crystallins have
now been found in many non-lenticular tissues [2,3]. α-Crystallin
is a key member of the sHSP (small heat-shock protein) family,
and its structural and functional similarities are conserved from
bacteria to humans [4,5]. Like other sHSPs, α-crystallin acts as a
molecular chaperone in vitro by preventing aggregation of other
proteins under various stress conditions [5–8].
During aging and cataract formation, lens proteins may be
damaged by various factors, including oxidative stress and
UV radiation [9,10]. Damaged proteins thus formed tend to
aggregate, forming large insoluble complexes that compromise
lens transparency. The chaperone activity of α-crystallin is
thought to inhibit aggregation of damaged proteins, thus helping
in maintaining lens transparency.
It is believed that α-crystallin binds to client proteins that
have undergone mild structural perturbation. Previously, several
investigators found that it binds to client proteins that are in their
‘ladder state’ [11–13]. Hydrophobic pockets on the surface of
α-crystallin are thought to interact with client proteins during its
chaperone function [5,7,14].
Many post-translational modifications such as phosphorylation
[15], de-amidation [16], glycation [17,18] and oxidation [19] are
detrimental to the chaperone function of α-crystallin. Glycation is
Key words: αA-crystallin, argpyrimidine, chaperone, client
protein aggregation, human lens, hydroimidazolone.
EXPERIMENTAL
Materials
Bovine insulin, CS (citrate synthase), α-lactalbumin, CA
(carbonic anhydrase) and DTT (dithiothreitol) were obtained
from Sigma Chemical Co. (St. Louis, MO, U.S.A.). TNS
Abbreviations used: AGE, advanced glycation end-product; CA, carbonic anhydrase; DTT, dithiothreitol; HFBA, heptafluorobutyric acid; LC-MS/MS,
liquid chromatography tandem MS; MGO, methylglyoxal; sHSP, small heat-shock protein; TNS, 2-(p -toluidino)naphthalene-6-sulfonic acid, sodium salt.
1
Present address: Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, U.S.A.
2
To whom correspondence should be addressed (email [email protected]).
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772
A. Biswas and others
[2-(p-toluidino)naphthalene-6-sulfonic acid, sodium salt] was
obtained from Molecular Probes (Invitrogen, Carlsbad, CA,
U.S.A.). All other chemicals were of analytical grade.
Cloning, expression and purification of recombinant human
αA-crystallin
Human αA-crystallin with an N-terminal His tag and an enterokinase cleavage site was amplified by PCR and cloned into
pET23d vector [26]. The recombinant protein was expressed
in Escherichia coli BL21(DE3)pLysS cells. The protein was
purified using an Ni–agarose column. The purity was checked
by SDS/PAGE and Western blotting.
Preparation of bovine γ -crystallin
Bovine lenses were homogenized on ice in 10 mM Tris/HCl
(pH 7.2) containing 100 mM NaCl, 1 mM EDTA, 0.02 % sodium
azide and 0.2 mM PMSF and then centrifuged at 18 000 g for
45 min at 4 ◦C. The supernatant fraction was loaded on to
a Sephacryl S300-HR column (95 cm × 1.5 cm). γ -Crystallin
corresponded to the fifth protein peak of the Sephacryl S300
column; the protein was collected, extensively dialysed against
PBS and stored at –20 ◦C. SDS/PAGE separation showed a single
band at ∼ 20 kDa.
Modification of client proteins by MGO
Client proteins (1.0 mg/ml insulin, α-lactalbumin, CA or γ -crystallin) in 50 mM sodium phosphate buffer (pH 7.4) were
incubated with 0–1000 µM MGO for 2 days at 37 ◦C. We then
dialysed all incubation mixtures against 50 mM sodium phosphate
buffer (pH 7.4) to eliminate unreacted MGO.
Co-modification of CA and αA-crystallin by MGO
MGO (100 µM) in 50 mM sodium phosphate buffer (pH 7.4) was
added to a solution containing CA and αA-crystallin (1.0 mg/ml
each). This mixture was then incubated for 2 days at 37 ◦C. After
incubation, the solution was dialysed against 50 mM sodium
phosphate buffer (pH 7.4) to eliminate excess MGO.
Client protein aggregation assay
DTT-induced aggregation of insulin and α-lactalbumin
Freshly prepared DTT (final concentration 20 mM) was added to
80 µg of unmodified and MGO-modified insulin. Light scattering
at 400 nm measured the aggregation for 1 h (at 25 ◦C) [27].
Assays were performed in 96-microwell plates and light scattering
was monitored with a microplate reader (model 190; Molecular
Devices, Sunnyvale, CA, U.S.A.).
Aggregation of α-lactalbumin by DTT was measured in a
similar manner with a microplate reader. The incubation mixture
contained α-lactalbumin (100 µg) in 50 mM sodium phosphate
buffer (pH 7.4) with 100 mM NaCl and 2 mM EDTA. The plates
were incubated at 37 ◦C, and light scattering was monitored at
360 nm [28]. Final reaction volume for both assays was 250 µl.
Thermal aggregation of CA and γ -crystallin
Thermal aggregation assays of unmodified and MGO-modified
CA (150 µg each) were performed in a Beckman DU spectrophotometer (Beckman, Fullerton, CA, U.S.A.). The proteins
were heated to 60 ◦C in 50 mM sodium phosphate buffer
containing 100 mM NaCl (pH 7.4), and light scattering at 400 nm
was monitored for 1 h in the kinetic mode. Thermal aggre
c The Authors Journal compilation c 2008 Biochemical Society
gation of unmodified and MGO-modified γ -crystallin samples
(0.25 mg/ml) was initiated by incubation at 65 ◦C, and light
scattering at 400 nm was monitored for 1 h.
We repeated these assays in the presence of αA-crystallin.
The ratio of α-crystallin to insulin, α-lactalbumin, CA and γ crystallin was 1:20, 1:15, 1:1 and 1:20 (w/w) respectively. For the
experiment involving co-incubation of MGO-modified CA and
MGO-modified αA-crystallin, we used an αA-crystallin/CA ratio
of 1:1 (w/w).
HPLC assay for argpyrimidine
Protein samples (300 µg each) were hydrolysed with 6 M HCl for
20 h at 110 ◦C. The acid was evaporated in a Speed Vac system,
and the pellet was suspended in 200 µl of water and filtered
through a 0.45-µm centrifugal filter. The amino acid content of
each hydrolysate was estimated with ninhydrin as described in
[29]. The samples were then injected into a C18 reversed-phase
column (218TP54; Grace Vydac) and separated with a gradient
system consisting of water and acetonitrile. Solvent A was 0.01 M
HFBA (heptafluorobutyric acid) in water, and solvent B was
70 % acetonitrile in water with 0.01 M HFBA. The solvent programme was as follows: 0–39 min, 16 % B; 40–50 min, 20 % B;
50–60 min, 22 % B; 60–62 min, 28 % B; 62–71 min, 100 %
B; 71–80 min, 16 % B. The column eluate was monitored with
an online fluorescence detector set at λex and λem of 335 and
385 nm respectively. Under these conditions, argpyrimidine had
an Rt (retention time) of ∼ 28 min. Argpyrimidine in the protein
samples was quantified by comparison with peak areas of known
synthetic standards.
HPLC–ESI (electrospray ionization)-MS
Protein reduction, S-alkylation and digestion
MGO-modified insulin (20 µg) was dissolved in 20 µl of 100 mM
Tris/HCl (pH 8.0) containing 8 M urea. The protein was then
reduced with 20 mM DTT for 2 h and treated with 50 mM
iodoacetamide for 30 min at 25 ◦C in the dark. The alkylated
proteins were diluted 10-fold in water and digested with AspN (Roche) for 16 h at 25 ◦C. After heating at 95 ◦C for 5 min
to inactivate Asp-N, the sample was then digested further with
staphylococcal (Staphylococcus aureus) V8 protease (Pierce)
overnight at 25 ◦C. The ratio of protease to protein was 1:50
for both incubations. The resulting peptide solution was vacuumevaporated and reconstituted in 0.1 % formic acid.
Identification of hydroimidazolone
LC-MS/MS (liquid chromatography tandem MS) analyses of the
digests were performed in a Thermo Finnigan linear ion-trap mass
spectrometer (model LTQ-XL) coupled with an Ettan MDLC
system (GE Healthcare). The digests were chromatographed using
a gradient of acetonitrile from 0 to 60 % in aq. 0.1 % formic acid
for 30 min at a flow rate of 300 nl/min. The mass spectrometer was
operated in a data-dependent MS to MS/MS switching mode, with
the four most intense precursor ions in each MS scan subjected
to MS/MS analysis. Data thus obtained were searched with
Mascot Daemon software for modification of arginine residues.
Further interpretation of the MS/MS spectrum of the modified
peptide R22 *GFFYTPKA30 (the asterisk represents modification
of the residue) was performed with MS-Product software
(http://www.prospector. ucsf.edu/ucsfhtml4.0/msprod.htm).
TNS fluorescence measurements
Unmodified and MGO-modified client proteins (100 µg/ml each)
were incubated separately with 20-fold molar excess of TNS
Role of methylglyoxal modification in the aggregation of client proteins
Figure 1
773
Aggregation of client proteins
(A) Percentage aggregation of insulin at 25 ◦C in the presence of 20 mM DTT; (B) aggregation of
α-lactalbumin (0.4 mg/ml) at 37 ◦C. (C, D) Percentage thermal aggregation of CA (0.25 mg/ml)
at 60 ◦C and γ -crystallin at 65 ◦C. The numbers at the bottom of each column indicate the
concentration (µM) of MGO added. Each bar represents the average from three different assays.
*The value for percentage aggregation was 0 %.
Figure 2
◦
[in DMF (dimethylformamide)] for 2 h at 25 C. Fluorescence
emission spectra of TNS-bound samples were recorded between
350 and 520 nm with an LS-55 PerkinElmer spectrofluorimeter at
25 ◦C with excitation and emission band passes of 10 and 20 nm.
TNS emission was measured after excitation at 320 nm.
Client proteins cross-linking by MGO
RESULTS
α-Lactalbumin, CA and γ -crystallin (1.0 mg/ml) were incubated separately with 0–1000 µM
MGO for 48 h, then dialysed and analysed by SDS/PAGE under denaturing conditions. (A)
Lane 1, molecular-mass markers; lane 2, α-lactalbumin alone; lane 3, α-lactalbumin + 10 µM
MGO; lane 4, α-lactalbumin + 25 µM MGO; lane 5, α-lactalbumin + 100 µM MGO; lane 6,
α-lactalbumin + 1000 µM MGO. (B) Lane 1, CA alone; lane 2, CA + 10 µM MGO; lane 3,
CA + 25 µM MGO; lane 4, CA + 100 µM MGO; lane 5, CA + 1000 µM MGO. (C) Lane 1,
molecular-mass markers; lane 2, γ -crystallin alone; lane 3, γ -crystallin + 10 µM MGO;
lane 4, γ -crystallin + 25 µM MGO; lane 5, γ -crystallin + 100 µM MGO; lane 6, γ -crystallin
+ 1000 µM MGO.
We previously showed that modification by MGO improves αAcrystallin’s chaperone function [22], and we thought that this
phenomenon might be important for maintaining lens transparency. Presumably, along with αA-crystallin in the aging lens,
client proteins are also modified by MGO. To determine how
MGO modification affects aggregation of client proteins, we first
examined the aggregation profiles in the presence or absence of
MGO (10–1000 µM) for four proteins: insulin, α-lactalbumin,
CA and γ -crystallin. We chose these particular client proteins to
compare the impact of MGO modification on both chemically and
thermally induced aggregation.
Surprisingly, we found that MGO modification inhibited both
chemically and thermally induced aggregation of the proteins,
and the extent of inhibition depended on MGO concentration
(Figure 1). When the proteins were modified with 10–25 µM
MGO, aggregation was inhibited by only 10–20 %. However, the
inhibition increased substantially with 100 µM MGO. While
the DTT-induced aggregation was completely suppressed when
insulin was modified with 1000 µM MGO (Figure 1A), aggregation of α-lactalbumin was completely suppressed at only
100 µM MGO (Figure 1B). Thermal aggregation of γ -crystallin was completely suppressed when modified with 1000 µM
MGO. These observations led us to conclude that modification
by MGO renders client proteins resistant to chemical and thermal
aggregation.
We considered that MGO may induce cross-linking of client
proteins, which would thus attain a rigid structure that would resist
unfolding. To test this possibility, we incubated client proteins
with various concentrations (10–1000 µM) of MGO for 48 h.
In samples incubated with MGO up to 100 µM, we found no
apparent cross-linking of client proteins, but at 1000 µM MGO
it became apparent (Figure 2). Because protein aggregation was
inhibited at 100 µM MGO (Figure 1) in the absence of protein
cross-linking, we assume that other changes are more likely to be
the cause.
Our previous study had established that MGO-modified αAcrystallin binds more client proteins than unmodified αA-crystallin when compared on a molar basis [22]. Because of this
finding, we wanted to investigate how MGO modification affected
the chaperone function of αA-crystallin. Because modification
by 100 µM MGO either partially or almost completely inhibited
client protein aggregation, we used proteins modified with less
than 100 µM MGO for these experiments. Figure 3 shows that
inhibition of aggregation (P) increased in unmodified and MGOmodified client proteins after addition of human αA-crystallin.
The plots indicate that αA-crystallin’s chaperoning efficiency
remained unaltered, even though MGO modification of client
proteins inhibited their aggregation. For example, αA-crystallin,
whether MGO-modified or not, inhibited insulin aggregation
by ∼ 45 %. Likewise, γ -crystallin aggregation was inhibited by
∼ 90 % for both unmodified and MGO-modified protein. From
these results, we concluded that modification of client proteins
with low concentrations of MGO (< 100 µM) did not affect
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A. Biswas and others
Figure 5 Estimation of argpyrimidine by HPLC: argpyrimidine was
identified in all four client proteins that had been incubated with MGO
Figure 3
Chaperone function of human αA-crystallin
Suppression of the aggregation (P) of unmodified and MGO-modified client proteins
when αA-crystallin was included. The ratios of αA-crystallin to the client proteins (insulin,
α-lactalbumin, CA and γ -crystallin) were 1:20, 1:15, 1:1 and 1:20 (w/w) respectively. Each bar
represents the average of three different assays. *Not determined.
Figure 4 Co-modification of αA-crystallin and CA by MGO: effect on thermal
aggregation of CA
Protein aggregation of unmodified and MGO-modified CA (0.25 mg/ml) was measured as
described in the text. The proteins were incubated in 50 mM phosphate buffer (pH 7.2) containing
100 mM NaCl for 1 h at 60 ◦C in the presence or absence of 100 µM MGO-modified 0.25 mg/ml
αA-crystallin, and light scattering due to protein aggregation was monitored at 400 nm. This
assay was also performed with co-modified CA and αA-crystallin (100 µM MGO each) at a
chaperone/client protein ratio of 1:1 (w/w).
chaperoning by αA-crystallin during either thermal or chemical
aggregation.
MGO presumably modifies both αA-crystallin and its client
proteins simultaneously within the aging lens. Accordingly, we
measured the effect of modification of both αA-crystallin and
CA (chosen as a representative client protein) by 100 µM MGO.
We found that MGO-modified αA-crystallin inhibits CA thermal
aggregation by ∼ 50 % (Figure 4). Similarly, modification of
CA with MGO inhibited its aggregation by ∼ 66 %. When αA
c The Authors Journal compilation c 2008 Biochemical Society
All samples were incubated with 10 or 100 µM MGO or with buffer alone for 48 h at 37 ◦C, and
adduct formation was measured by reversed-phase HPLC with an online fluorescence detector.
Each bar represents the average of three different assays. *Argpyrimidine not detected.
crystallin and CA were both modified by MGO, inhibition was
nearly complete (92 %). Taken together, these findings indicate
that MGO modification of αA-crystallin and its client proteins
helps to keep client proteins soluble. Because protein turnover in
the lens is extremely low, this phenomenon could be important
for maintaining lens transparency.
In two earlier studies, we found that MGO modification of
Arg21 and Arg103 to argpyrimidine improved the chaperone function of human αA-crystallin [22,26]. We now wanted to determine if these modifications correlated with resistance to thermal
and chemical aggregation of client proteins. We noted a
higher concentration of argpyrimidine in proteins incubated with
100 µM MGO than in those incubated with 10 µM MGO. We
found that the extent of resistance to aggregation with MGO
treatment related directly to the amount of argpyrimidine in client
proteins: the higher the argpyrimidine concentration (Figure 5),
the greater the resistance to aggregation (Figure 1).
Hydroimidazolone is another MGO-derived arginine modification on proteins. We could not measure this modification directly
because of limitations in our HPLC instrumentation. However,
we were able to detect it in MGO-modified insulin by MS. We
sequentially digested 100 µM MGO-modified insulin with Asp-N
and V8 and analysed the product by LC-MS/MS. The precursor
ion mass increment of 54 Da between the modified and unmodified
peptides suggests the modification adduct of hydroimidazolone.
Figure 6 shows the tandem mass spectra of the arginineresidue-containing peptide, R22 GFFYTPKA30 , from unmodified
and MGO-modified insulin B-chains. The results indicate that
MGO modification of hydroimidazolone occurs at Arg22 . We did
not detect argpyrimidine, although it was identified by HPLC in
another experiment (Figure 5). The low abundance (∼ 6 pmol/
100 µmol of amino acid) of this modification probably precluded
its detection. We also noted a loss of 44 Da, which is consistent
with decarboxylation during the collision-induced dissociation.
Because insulin has only one arginine residue, modification of this
single residue to hydroimidazolone (and possibly argpyrimidine
as well) appears to prevent aggregation by DTT. Formation
Role of methylglyoxal modification in the aggregation of client proteins
Figure 6
775
MS/MS spectra of peptides derived from MGO-treated and untreated insulin
MS/MS spectra of (A) the peptide R22 GFFYTPKA30 (with an m /z of 543.5) from untreated insulin and (B) R22 *GFFYTPKA30 (with an m /z of 570.9) from MGO-modified insulin. Both precursor ions
are doubly charged.
of hydroimidazolone as well as argpyrimidine likely prevents
aggregation in other proteins as well.
In addition to formation of argpyrimidine and hydroimidazolone, MGO modification could influence aggregation of proteins
through structural perturbations. Two reports assert that MGO
modification perturbs the structure of α-crystallin so as to significantly increase surface hydrophobicity, although these studies
failed to correlate surface hydrophobicity with chaperone function
[22,24]. We used far- and near-UV CD spectroscopy to examine
secondary and tertiary structural perturbation by MGO (10 and
100 µM) in all four client proteins. We found no apparent change
in either the secondary or tertiary structure in any of these proteins
(results not shown), suggesting that the resistance to thermal and
chemical aggregation is not due to structural perturbation.
We used the hydrophobic probe, TNS, to define how MGO
modification influences the surface hydrophobicity of the four
client proteins. TNS-bound insulin, α-lactalbumin, CA and γ crystallin displayed fluorescence with maximum emission (λmax )
at 441, 459, 425 and 461 nm respectively. The λmax did not
change when these four proteins were modified by 10 and
100 µM MGO. However, we noted a slight reduction in fluorescence (from ∼ 6 % to 23 %), indicating a decrease in surface
hydrophobicity. Argpyrimidine fluoresces at 380 nm when excited
at 335 nm. We believe that the amount of argpyrimidine formed
was insufficient to produce a shoulder second peak relative to
the TNS fluorescence at 380 nm. From these findings, we assume
that even a slight decrease in surface hydrophobicity together
with the formation of the MGO adducts, argpyrimidine and
hydroimidazolone, conveys resistance to protein aggregation.
However, at this point, we are unable to determine which of these
factors is the most important.
The presence of argpyrimidine (Figure 5) and mild reduction of
surface hydrophobicity suggest that the TNS- and MGO-binding
sites are located in different regions in the four client proteins.
Therefore we determined whether the TNS- and MGO-binding
sites are mutually exclusive. We found argpyrimidine in CA and
γ -crystallin (not treated with TNS, but incubated with MGO) to
be ∼ 450 and ∼ 1150 nmol/µmol of amino acid. Surprisingly, we
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776
A. Biswas and others
Figure 7 Effect of post-treatment of MGO on the fluorescence spectra of
TNS-bound client protein
CA (1.0 mg/ml) was incubated with 20-fold molar excess of TNS at 25 ◦C for 2 h. After dialysis,
TNS-bound CA was incubated either with a buffer or with 100 µM MGO at 37 ◦C for 48 h.
Fluorescence spectra of the MGO-modified and unmodified samples (0.5 mg/ml) were recorded
at 25 ◦C from 350 to 520 nm. The λex was 320 nm. The excitation and emission band passes
were 10 and 20 nm.
found similar concentrations in proteins treated first with TNS and
then modified with MGO. This would suggest mutually exclusive
binding sites for TNS and MGO on these two proteins.
We considered the possibility that MGO could have displaced
TNS and modified arginine to the same extent as in the MGOmodified protein not treated with TNS. Accordingly, we determined the fluorescence of TNS-bound MGO-modified CA (with
or without prior TNS treatment) (Figure 7). If MGO displaced
TNS, it would result in lower fluorescence. However, the
fluorescence intensity of TNS-treated and MGO-modified protein
(λmax = 404 nm) was higher than that of TNS-treated CA (λmax =
420 nm); therefore we do not believe that MGO displaced TNS
during incubation. These results also confirm that the MGO- and
TNS-binding sites are mutually exclusive.
DISCUSSION
Our objective was to determine the impact of MGO modification
of client proteins on chaperoning by αA-crystallin. Because
MGO is a rapidly reacting molecule and it induces structural
changes in proteins through formation of chemical adducts, we
expected MGO modification to enhance thermally and chemically
induced aggregation of those proteins and to decrease the chaperone function of αA-crystallin. Contrary to these expectations,
we found that MGO modification inhibited in a concentrationdependent manner the chemically and thermally induced
aggregation of four different proteins while having no effect
on chaperoning by αA-crystallin. Our findings are similar to
those of Wang and Spector [30], who found that oxidation of
client proteins failed to influence chaperoning by α-crystallin.
Even more intriguing is the fact that co-modification of both αAcrystallin and client protein with MGO affords greater protection
against aggregation of a client protein than modification of either
protein alone.
α-Crystallin binds to proteins that have undergone mild
structural perturbation (molten globule state) and prevents their
further denaturation and aggregation [31–34]. By this mechanism,
α-crystallin helps to maintain lens transparency during aging.
c The Authors Journal compilation c 2008 Biochemical Society
This protein protective mechanism is particularly important in
the lens where protein turnover is negligible. However, lens
proteins can be altered by post-translational modifications such
as oxidation, glycation, truncation and de-amidation, all of which
contribute to protein cross-linking and aggregation [35]. The
loss of α-crystallin chaperone function as a consequence of such
modifications would diminish the protection that it affords against
protein aggregation. In this context, modification of αA-crystallin
by MGO is unique, because it enhances the chaperone function.
Our research suggests that this enhancement is likely due to
formation of hydroimidazolone and argpyrimidine adducts on
selected arginine residues of αA-crystallin.
Both of these important MGO-induced modifications [36,37]
are found in the human lens, where they constitute two of the major
modifications to lens proteins. Although exactly which specific
human lens proteins are modified is not yet clear, the major protein
of the lens, i.e. α-crystallin, is likely included.
It is puzzling how MGO modification can afford increased protection against thermal and chemical denaturation of client
proteins in the absence of any observable structural perturbation.
A similar observation was made with β L -crystallin; it had
increased resistance to thermal denaturation after H2 O2 -induced
oxidation [30]. We considered that MGO modification might
decrease surface hydrophobicity. In fact, we noted a slight, but
not significant, decrease in TNS fluorescence in MGO-modified
client proteins. Whether this slight decrease can account for the
resistance to aggregation remains to be verified.
The fact that MGO modification occurred even after TNS
binding and that MGO failed to displace bound TNS on CA
suggests that these two molecules bind to different regions of
this protein. Although MGO- and TNS-binding sites are mutually
exclusive, the TNS fluorescence intensity decreased slightly in
MGO-modified client proteins compared with unmodified client
proteins. Because MGO-binding sites influence the overall surface
hydrophobicity of a protein, such a subtle change may make these
client proteins resistant to thermal and chemical stress.
Importantly, modification of the single arginine in insulin was
sufficient to increase its resistance to DTT-induced denaturation.
One possibility is that arginine modification might prevent
reduction of the disulfide bond by DTT, thus improving protection.
A more likely scenario is that modification of positively
charged arginine residues by MGO and conversion of these into
neutral adducts decreases hydrophobic interaction during protein
aggregation. This phenomenon may also occur in γ -crystallin and
α-lactalbumin, both of which had increased thermal resistance
after MGO modification. Taken together, it is evident that
resistance to thermal and chemical stress by MGO modification
is not restricted to a single protein or one type of stress.
We considered that MGO modification might mildly perturb
client proteins sufficiently to enhance their binding to αA-crystallin, thus improving resistance to aggregation, as we found in
the co-incubation experiments. However, our UV-CD spectroscopy data showed no change in either the secondary or the
tertiary structure in client proteins that had been modified by MGO
(< 100 µM). MGO modification of αA-crystallin increases its
surface hydrophobicity, which we believe underlies its enhanced
chaperone function. Our observation that co-modification of
αA-crystallin and client proteins further enhances resistance to
thermal and chemical denaturation beyond that of either modified
protein alone suggests a complex protective system. Such a
protective system may be beneficial in the aging eye.
Finally, whether MGO in the lens is beneficial or harmful is
difficult to predict. On the one hand, MGO-mediated lysine–lysine
and lysine–arginine cross-linking (at high MGO concentrations)
is likely to contribute to protein aggregation during aging. On
Role of methylglyoxal modification in the aggregation of client proteins
the other hand, MGO modification of arginine residues on αAcrystallin and its chaperoning client proteins (at low MGO concentrations) might prevent aggregation of proteins during lens
aging. An additional observation that MGO modification inhibits
glycation-mediated loss in chaperone function and synthesis of
pentosidine in α-crystallin [38] further suggests a beneficial role.
Therefore, based on these observations, it is tempting to speculate
that MGO modifications in the aging lens might benefit the lens
by enhancing the chaperone function of αA-crystallin along with
improving the resistance of client proteins against stress-induced
aggregation.
This study was supported by NIH (National Institutes of Health) grants R01EY-016219
and R01EY-09912 (R. H. N.) and P30EY-11373 (Visual Sciences Research Center of Case
Western Reserve University), Carl F. Asseff, M. D. Professorship to R. H. N., RPB (Research
to Prevent Blindness; New York, NY, U.S.A.) and Ohio Lions Eye Research Foundation.
We thank Michael Zagorski and Krzysztof Palczewski of Case Western Reserve University
for use of the CD spectropolarimeter and the fluorescence spectrofluorimeter.
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Received 26 July 2007/9 October 2007; accepted 17 October 2007
Published as BJ Immediate Publication 17 October 2007, doi:10.1042/BJ20071006
c The Authors Journal compilation c 2008 Biochemical Society