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INTRODUCTION |
Age-related cataract is one of the major causes of blindness in
humans. The pathology of such a condition involves opacification and
decreased transparency of the lens, which can lead to loss of vision.
Although the mechanisms for age-related cataractogenesis are not
understood, oxidation of the lens proteins, known as crystallins, is
associated with cataract formation in humans (1, 2). Protein
thiolation, which involves the formation of disulfide bonds between the
cysteinyl residues of lens proteins and other low molecular weight
thiols in the lens, is one of the modifications caused by oxidative
stress of the lens (3). Thiols in the lens that participate in this
reaction are GSH and cysteine, which form
protein-S-S-glutathione
(PSSG)1 and
protein-S-S-cysteine (PSSC), respectively (4).
These protein-thiol products are referred to as mixed disulfides.
The conformational changes caused by protein thiolation (5, 6) may
allow some of the buried functional groups to be exposed and modified.
This may cause proteins to aggregate and disrupt the close packing of
the crystallins, decreasing their solubility (7) and leading to
cataract formation. In the H2O2-induced
cataract model, the progression of cataract is associated with
sequential events involving first the formation of PSSG, followed by
protein-protein disulfide cross-links, decreased protein solubility,
and finally an increase in the formation of high molecular weight
aggregates (8). Protein-thiol mixed disulfides accumulate in older
human lenses (9) and in all types of human cataractous lenses (10, 11).
Elevated PSSG or PSSC or both have been found in lenses from animals
treated with oxidants such as UV radiation (12), photodynamic systems (13), naphthalene (14), and hyperbaric oxygen (15). The extent of
thiolation in lens proteins either as PSSG or PSSC can be quantified as
the released product of glutathione sulfonic acid (GSO3H)
or cysteic acid (CSO3H), respectively, after oxidation with
performic acid (16), but previously available techniques have not
permitted determination of the exact site of thiolation. Recently, mass
spectrometry has been used to locate and identify sites of a variety of
post-translational modifications of bovine lens crystallins (17-22)
and has, thus far, been the only technique that has permitted specific
location of GSH adducts (23). Mass spectrometry has also been used to
detect protein-protein disulfide bonds (24-28) and methionine
oxidation (29, 30), as well as other modifications of human lens
crystallins not associated with oxidation. Because picomole quantities
are sufficient for these analyses, a single lens is adequate to obtain
information about a variety of post-translational modifications.
To understand the role of protein thiolation in
H2O2-induced cataract,
B-crystallins were
isolated from untreated bovine lenses and from lenses incubated with
and without H2O2 in organ culture.
B-Crystallin was chosen because it contains 7 cysteine residues with
various degrees of exposure and reactivity, making it naturally
susceptible to thiolation under oxidative stress. In addition, the
amino acid sequence and the conformation of
B-crystallin are known.
In this investigation, the modifications of
B-crystallin caused by
H2O2 treatment of the whole lens were
determined to be GSH adducts at some cysteinyl residues, partial
oxidation of all the methionines, and the formation of two
intramolecular disulfide bonds.
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MATERIALS AND METHODS |
Chemicals--
The following chemicals were purchased from
Sigma: 30% H2O2, Tris-HCl, GSH,
trichloroacetic acid (TCA), trifluoroacetic acid (TFA), sodium
bicarbonate, L-glutamine, glucose oxidase, penicillin, streptomycin, trypsin
(L-1-tosylamide-2-phenylethylchloromethyl ketone-treated,
from bovine pancreas), TC-199 medium with Earle's salts,
dithio-nitrobenzoic acid, and EDTA. Bicinchoninic acid was obtained
from Pierce. HPLC grade acetonitrile was purchased from Mallinkrodt
Baker (Paris, KY). Water used in the HPLC system was purchased from
Baxter Diagnostics, Inc. (Deerfield, IL) and/or Sigma or was deionized
on-site using a Millipore SuperQ water treatment system (Millipore
Corp., Bedford, MA). The dialysis tubing was CelluSepT2, with a
8000-10,000 molecular weight cutoff (Membrane Filter Products, Inc.,
San Antonio, TX).
Lens Organ Culture--
Eyes from 18-24-month-old cows were
obtained from BeefAmerica, Inc. (Norfolk, NE). Each lens was surgically
removed within 4 h postmortem. Three lenses were analyzed without
any previous incubation or H2O2 exposure
(referred to as fresh lenses). An additional two pairs of lenses were
analyzed after organ culture in a CO2 incubator at
37 °C, following the method of Zigler and Hess (31). One lens of
each pair was placed in 9 ml of TC-199 medium with no
H2O2 and used as a control. The contralateral
lens of each pair was placed in medium containing 30 mM
H2O2 and sufficient glucose oxidase to maintain
the 30 mM H2O2 concentration. After 24 h of incubation, morphological characteristics and changes were
recorded, and the lenses were photographed. The incubated lenses were
rinsed with saline solution, blotted on filter paper, weighed, and then
processed immediately for biochemical analysis.
Preparation of Lens Homogenate and Water-soluble and
Water-insoluble Protein Fractions--
Each lens was homogenized in 10 ml of an ice-cold solution of 0.1 mM EDTA. Approximately 1 ml of the homogenate from each of the incubated lenses was removed for
determination of protein-thiol mixed disulfides and GSH. The remainder
was centrifuged for 30 min at 12,000 × g to separate
the water-soluble and water insoluble proteins. The water-insoluble
pellet was redissolved in 10 M urea. The protein
concentrations of both the water-soluble and water-insoluble fractions
were determined by the bicinchoninic acid method (32). The remaining
supernatant was separated by size exclusion chromatography into
-,
H-,
L-, and
-crystallins fractions. In
addition, three fresh lenses were homogenized, and the
-crystallins
were immediately isolated from the water-soluble portion.
Glutathione and Protein-Thiol Mixed Disulfide Analyses--
The
proteins in the 1-ml aliquot of supernatant were precipitated in
ice-cold 10% TCA and removed after centrifugation at 1600 × g for 15 min. The supernatant from the TCA precipitation was
immediately assayed for GSH with dithio-nitrobenzoic acid, following
the method of Ellman (33). The pellet from the TCA precipitation was
immediately washed three times with 10% TCA solution followed by one
wash with a 1:1 solution of methanol and ether. The precipitate was
dried overnight in a dry bath at 60 °C and then stored at
20 °C
pending analysis for protein-thiol mixed disulfides.
The concentrations of PSSG and PSSC in the pellet were determined
according to a previously published procedure (16). Glutathione and
cysteine were released from the protein adducts by performic acid
oxidation, forming glutathione sulfonic acid and cysteic acid,
respectively. These acids were separated and quantified using a Dionex
(Sunnyvale, CA) LC system equipped with an anion exchange column
(AminoPac PA1, 4 × 250 mm) and postcolumn monitoring of the
products at 570 nm after reaction with ninhydrin. Data were processed
with PeakNet 4.3 software (Dionex Corp.).
Size Exclusion Chromatography of Lens
Crystallins--
Approximately 1 ml (50 mg/ml) of the water-soluble
portion of the homogenate from each lens, fresh as well as incubated
with and without H2O2, was fractionated into
-,
H-,
L-, and
-crystallins by size
exclusion chromatography (Superdex 200 column, 1.6 × 60 cm;
Amersham Pharmacia Biotech) using an eluting buffer (0.02 M
Tris, 0.1 M NaCl, 0.001 M NaN3, pH
7.6) at a flow of 0.5 ml/min (8). Fractions were collected
automatically at intervals of 2 min/tube, and the absorbance of each
tube was measured at 280 nm. Fractions corresponding to
-,
H-,
L-, and
-crystallins were pooled,
lyophilized, reconstituted in water, dialyzed in 8000-10,000 molecular
weight cutoff tubing, and lyophilized again. The samples were stored at
80 °C for future use.
Reversed Phase HPLC of
-Crystallins--
The
-crystallins
were further separated using a reversed phase HPLC system (Waters,
Milford, MA) consisting of a C18 column (4.6 × 150 mm, 5 µm)
and a linear gradient of 30-45% solvent B in 30 min. Solvent A was
water with 0.1% TFA, and solvent B was acetonitrile with 0.1% TFA.
The eluate was monitored with a UV detector at 280 nm (Amersham
Pharmacia Biotech). Fractions for the
-crystallin subfamilies were
pooled and stored at
20 °C until analysis by mass spectrometry.
Analysis of
-Crystallins by Electrospray Ionization Mass
Spectrometry (ESIMS)--
After isolation of the
-crystallins by
reversed phase HPLC, their molecular weights were determined by direct
injection of the protein solution (0.5-1.0 nmol) into a quadrupole
electrospray mass spectrometer (Platform II; Micromass, Manchester,
UK). The sample was delivered to the mass spectrometer in a solution of 50:50 acetonitrile/water at a flow rate of 5 µl/min. The ESIMS raw
data for a protein consist of peaks due to several multiply protonated
forms. The instrument was calibrated for protein analysis with the
multiply charged peaks of myoglobin over a range of 700-1800 Da; data
were processed with Mass Lynx 2.0 software to obtain reconstructed mass
spectra showing the protein masses consistent with these multiply
charged species. The mass uncertainty was ±2 Da.
Analysis of Tryptic Peptides of
B-Crystallins by
ESIMS--
The
B-crystallins from the lenses that had been
incubated with or without H2O2 were further
analyzed after tryptic digestion to locate the sites of modification.
Approximately 10 nmol of
B-crystallin, isolated by reversed phase
HPLC, was dissolved in 300 µl of 0.1 M Tris buffer, pH
7.8, and digested with trypsin using a 25:1 ratio of protein to enzyme
with a 4-h incubation at 37 °C. The digest was stored at
20 °C
until analysis. An on-line HPLC ESIMS system was used to fractionate
the peptides and to determine their molecular weights. The on-line HPLC
consisted of a C18 microbore column (Zorbax, 1 × 50 mm, 5 µm;
Micro-Tech Scientific, Sunnyvale, CA) with a binary gradient system
(Gilson, Middleton, WI). Solvent A was water with 0.1% TFA, and
solvent B was acetonitrile with 0.1% TFA. A gradient of 2-50%
solvent B in 45 min eluted the peptides.
The effluent from the HPLC was split with 10% diverted to the mass
spectrometer, described previously, and 90% diverted through a UV
detector (Applied Biosystems, Foster City, CA), to a fraction collector, and saved for further analyses. The sample was delivered to
the ESIMS in the HPLC effluent. The mass spectrometer was calibrated with NaI according to standard procedures suggested by the
manufacturer. NaI forms a series of cluster ions
(NanIn
1)+, which can be observed
to ~4000 Da (34). The uncertainty of the mass calibration was ±0.3 Da.
Depending on the size and number of basic residues in a peptide,
singly, doubly, and triply protonated peptides may be evident in an
electrospray ionization mass spectrum. Peptides were identified by
matching their molecular masses with the masses of peptides expected
from tryptic digestion of
B-crystallin, which had been calculated
from the known sequence of bovine
B-crystallin (35).
Analysis of Peptic Peptides of
B-Crystallin (10-31) by
ESIMS--
Approximately 1-2 nmol of
B peptide 10-31, which had
been previously isolated from the reversed phase HPLC separation of a
tryptic digest of the intact protein, was digested with pepsin (Sigma).
The pepsin digest was performed at an enzyme/substrate ratio of ~1:25
in water with 0.1% TFA, pH 2, at room temperature for 5 min.
After enzymatic digestion, the subpeptides of peptide 10-31 were
fractionated on a C18 capillary column (Hypersil C18, 300 µm inner
diameter × 15 cm, 3 µm; LC Packings, San Francisco, CA) using an
HPLC binary gradient system (Shimadzu Scientific Instrument Inc.,
Columbia, MD). Solvent A was water with 0.05% TFA, and solvent B was
acetonitrile with 0.05% TFA. A gradient of 2-50% solvent B in 48 min
was used for peptide elution. The sample was delivered to the ESIMS in
the HPLC effluent.
Analysis of
B-Crystallin Peptide 10-31 by Tandem Mass
Spectrometry--
Tandem mass spectrometric experiments were performed
on
B peptide 10-31 previously isolated from a tryptic digest of
intact
B-crystallin (see above) using a Finnigan MAT (San Jose, CA) LCQ mass spectrometer equipped with an electrospray source. The sample
was delivered to the mass spectrometer in a 50:50 mixture of
acetonitrile and water at a flow rate of 5 µl/min. A collisional energy of 22% was used for peptide fragmentation with helium. The
isolation width used was 1.5 Da. This instrument was calibrated with
Ultramark over a mass range of 50-2000 Da with a mass uncertainty of
±0.3 Da.
 |
RESULTS |
Lens Morphological Changes--
After the 24-h incubation, the
lenses incubated without H2O2 (referred to as
control lenses) retained their clarity, but lenses exposed to 30 mM H2O2 had become uniformly cloudy
in the entire outer cortical region and appeared swollen (Fig.
1). The wet weight was increased from
1.82 ± 0.05 g for the control lenses to 2.08 ± 0.12 g for the H2O2-treated lenses (Table
I).

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Fig. 1.
Photographs of 18-month-old calf lenses
cultured in TC 199 medium for 24 h. A, control lens, no
H2O2. B, lens incubated in medium
with 30 mM H2O2
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Glutathione and Protein-Thiol Mixed Disulfide
Quantification--
Treatment of lenses with 30 mM
H2O2 decreased the free GSH and increased both
protein-thiol mixed disulfides (Table I). GSH decreased from 7.54 ± 0.59 µmol/g wet weight to 0.05 ± 0.01 µmol/g wet weight, a
99% loss. PSSG showed a 30-fold increase, from 0.18 ± 0.07 to
5.12 ± 0.09 µmol/g wet weight; PSSC had a 4-fold increase, from
0.11 ± 0.02 to 0.50 ± 0.04 µmol/g wet weight.
Concentration of Protein in the Water-soluble and Water-insoluble
Fractions--
The water-insoluble protein constituted only 5% of the
total protein in a control lens but increased to 44% of the proteins of a lens that had been treated with H2O2
(Table I).
Isolation of
-Crystallins--
Size exclusion fractionation of
the water-soluble proteins of both the fresh and control lenses showed
five peaks corresponding to
-,
H-,
L-,
and
-crystallins and a low molecular weight fraction (Fig.
2A). The chromatograms for the
proteins from the H2O2-treated lenses were
substantially different from those for the fresh and control lenses in
that the
- and
H-crystallin peaks appeared to merge
into one larger peak, and the absorbances for
L- and
-crystallins were less than half of those for the fresh and control
lenses (Fig. 2B).

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Fig. 2.
Size exclusion chromatograms of water soluble
lens proteins. A, control lens. B, lens treated
with 30 mM H2O2. Note that the and H fractions have merged in the chromatogram for the
H2O2-treated lens, and the absorbance of the
-crystallin peak is 30% lower.
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The bovine
-crystallins collected from the size exclusion
fractionation were further fractionated by reversed phase HPLC. There
were five peaks for the
-crystallins from the fresh and control
lenses; the peaks for the
-crystallins from the
H2O2-incubated lenses were not as well resolved
(Fig. 3, A and B).
Although the
B-crystallins from the
H2O2-treated lenses eluted at the same percent
acetonitrile as those from the control lenses, the absorbance was
considerably smaller.

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Fig. 3.
Chromatograms of the reversed phase HPLC
fractionation of the water-soluble -crystallins. A,
control lens. The proteins in the peaks were identified by their
molecular weights, determined by ESIMS. In order of elution, they are
S, B, D, C, and E. B, lens treated with 30 mM H2O2. The proteins were
identified as B, D, C, and E. S-Crystallin was not found
in these fractions.
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Analysis of
-Crystallins by ESIMS--
The molecular masses of
the proteins separated by reversed phase chromatography from the fresh
and control lenses indicated that the primary component of the first
fraction was
S-crystallin (Mr 20,835), the
second fraction was
B-crystallin (Mr 20,966), the third fraction was
D-crystallin (Mr
20,749), the fourth fraction contained
D- and
C-crystallins
(Mr 21,008), and the fifth fraction was
E-crystallin (Mr 20,953). These experimental
molecular masses are within 2 Da of those reported by Kilby et
al. (36). Some fractions contained more than one protein either
because there was incomplete separation of the
-crystallins or
because a modified crystallin was also present. For example, the
mass spectrum for fraction 2 shows that, in addition to
B-crystallin, there are minor components with molecular masses
corresponding to
S-crystallin and
B-crystallin plus an additional
305 Da, suggesting the presence of a GSH adduct of
B-crystallin
(Fig. 4A). When GSH, with a
molecular mass of 307 Da, forms a disulfide bond with a cysteinyl
residue, 2 hydrogens are eliminated, resulting in an increase to the
protein of 305 Da. The GSH adduct was evident in only one of the three fresh lenses as 4% of the signal and in the control lenses as 36 and
44% of the signal. Among other
-crystallins from the control lenses, the only other molecular mass indicating modification was a
very minor component in a mass spectrum of
S-crystallin, also 305 Da
higher, suggesting the presence of a GSH adduct to
S-crystallin
(data not shown).

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Fig. 4.
Reconstructed electrospray ionization mass
spectra of bovine B-crystallins. A, B-crystallins
isolated from a control lens (Fig. 3A, second peak).
B, B-crystallins isolated from a lens treated with 30 mM H2O2 (Fig. 3B, second
peak). Note the increase in B-crystallin with a GSH adduct, the
presence of B-crystallin with two GSH adducts and a species
suggesting addition of seven oxygens (+7 × 16 Da) to
B-crystallin.
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Mass spectra of the
-crystallins isolated from the lenses incubated
in 30 mM H2O2 indicated the
presence of several modifications (Fig. 4). The mass spectrum of the
first peak eluting from reversed phase HPLC yielded a molecular weight
consistent with unmodified
S-crystallin. The major component of the
second fraction collected from HPLC was
B-crystallin with one GSH
adduct (Mr 21,270). For one lens this peak
constituted 62% of the signal; for the other lens, it was 58%. Other
proteins in this fraction had molecular weights corresponding to
B-crystallin (Mr 20,964),
B-crystallin plus two GSH adducts (Mr 21,576), and
B-crystallin plus 7 oxygens (Mr 21,078) (Fig.
4B). The peak representing addition of two GSH adducts was
8% for one lens and 15% for the other. The later eluting fractions
contained proteins with molecular masses corresponding to
D-,
C-,
and
E-crystallins, not modified.
Analysis of Peptides from Tryptic Digestion of
B-Crystallin--
To confirm the identities of the modifications
and to locate the specific sites that had been modified,
B-crystallins from the control and
H2O2-treated lenses were digested with trypsin, and the resulting peptides were separated and analyzed by on-line reversed phase HPLC and ESIMS. The masses of these peptides were compared with masses of expected peptides calculated from the known
sequence (35) (Fig. 5).

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Fig. 5.
Sequence of B-crystallin showing the
masses of expected peptides in a tryptic digest of the unmodified
protein. The masses of peptides formed by cleavage C-terminal to
Lys and Arg are given above the sequence.
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Control Lenses--
For both control lenses, masses corresponding
to all of the expected peptides, except peptide 32-36, were found. The
tryptic digest of one control lens also included a peptide
corresponding to residues 10-31 plus 305 Da, indicating that some of
peptide 10-31 had a GSH adduct. Peptide 10-31 contains three Cys
residues, Cys-15, Cys-18, and Cys-22, all possible sites for the
adduct. Analysis of the tryptic digest of the other control lens
indicated that the GSH adduct was at Cys-41. No other modifications
were noted for the control lenses.
H2O2-treated Lenses--
Several
additional modifications were found in the
B-crystallins isolated
from the H2O2-treated lens. There were masses
corresponding to modification of peptide 100-115 and peptide 37-58 by
GSH, partial oxidation of all Met-containing peptides, and the
formation of two intramolecular disulfide bonds. The analysis of one of
the H2O2-treated lenses with two GSH adducts
showed one adduct present on peptide 10-31 and the other at Cys-109.
Analysis of the other H2O2-treated lens
indicated the two GSH adducts were located on peptide 10-31 and at
Cys-41. Because peptide 10-31 contains 3 Cys residues, Cys-15, Cys-18,
and Cys-22, this peptide was further analyzed both by tandem mass
spectrometry and by on-line reversed phase HPLC and ESIMS after further
digestion with pepsin. Both analyses indicated that all three Cys
residues were partially modified, but no peptide included more than one modification.
In the
B-crystallins from the H2O2-treated
lenses, intramolecular disulfide bonds were found between peptide
10-31 and peptide 77-79 and between peptide 37-58 and peptide
77-79. Because Cys-41 and Cys-78 are the only cysteinyl residues in
peptides 37-58 and 77-79, the participation of Cys-41 and Cys-78 is
certain. Note that Cys-78 of peptide 77-79 was found both in a
disulfide bond with peptide 10-31 and bonded to Cys-41. A summary of
these modifications is given in Table II.
A mass spectrum showing peptides in an HPLC fraction from the tryptic
digest of the
B-crystallins isolated from an
H2O2-treated lens is shown in Fig.
6. Present in this fraction is unmodified peptide 10-31 (calculated Mr 2537.0), which can
be seen as the 2+ and 3+ charge states at m/z
1269.4 and 846.8, respectively. Peptide 10-31 is also evident with a
GSH adduct, again as the 2+ and 3+ charge states at
m/z 1422.3 and 948.2, respectively. The 2+ and 3+
charge states indicate a peptide of molecular weight 2842.6, in
agreement with the calculated molecular weight of 2842.3 (2537.0 for
peptide 10-31 plus 305.3 for the GSH adduct). The peaks at
m/z 1450.2 and 967.4 in Fig. 6 are the 2+ and 3+
charge states of the dipeptide (Mr 2899.1)
formed by a disulfide bond between peptide 10-31 and peptide
77-79.

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Fig. 6.
Electrospray ionization mass spectrum of a
fraction from a tryptic digest of B-crystallin from an
H2O2-treated lens illustrating the
modifications of peptide 10-31. The unmodified peptide 10-31
(calculated Mr 2537.0) is present as the 2+ and
3+ charge states at m/z 1269.4 and 846.8, respectively. Peptide 10-31 with a GSH adduct (+305 Da) is present as
the 2+ and 3+ charge states at m/z 1422.3 and
948.2, respectively. Peptide 10-31 disulfide bonded to peptide 77-79
(calculated Mr 364.1) is also evident as the 2+
and 3+ charge states at m/z 1450.2 and 967.4. These masses are consistent with a peptide with
Mr 2899.1.
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In addition to the modifications at cysteinyl residues, there was
evidence of partial oxidation of all 7 methionines of
B-crystallins isolated from the H2O2-treated lenses. A
representative mass spectrum showing evidence of oxidation at Met 171 is shown in Fig. 7. The mass of peptide
169-174 is 829.5, yielding a protonated molecular ion of 830.5. The
peak at m/z 846.4 corresponds to addition of 16 Da. Because mass spectrometric studies have shown that methionine is
preferentially oxidized by H2O2 (30), Met-171
was assumed to be the modified site. Similar masses with an increase of
16 Da were found for all seven of the methionine-containing peptides. Even though peptide 100-115 contained both Met and Cys, oxidation of
Met and the GSH adduct were not found on the same molecule; peptide
100-115 was seen unmodified, oxidized at Met-102, or with the GSH
adduct.

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Fig. 7.
Electrospray ionization mass spectrum of
peptides from the tryptic digest of B-crystallin from an
H2O2-treated lens illustrating evidence of
oxidation at Met 170. The expected protonated molecular ion of
peptide 169-174 is 830.5. The peak at m/z 846.4 corresponds to the presence of a peptide 16 Da larger, indicating the
addition of an oxygen.
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|
 |
DISCUSSION |
Bovine lenses (37), as well as lenses from pigs (38) and monkeys
(39), have a higher resistance to oxidative stress than rodent lenses
(16). The degree of morphological change in bovine lenses exposed to 30 mM H2O2 (Fig. 1) was similar to that of rat lenses exposed to 0.5 mM
H2O2 (8), but the biochemical damage to the
thiols was more severe. Although thiolation of proteins in rat lenses
has been extensively studied, the sites of thiolation and other protein
modifications attributable to oxidation have never been determined.
Bovine lenses were more suitable than rat lenses for this study,
because one bovine lens is 200-fold larger than a rat lens and can
therefore provide enough purified crystallins for a detailed
investigation of the modifications by mass spectrometry. With the organ
culture conditions used in this study, 30 mM
H2O2 was the threshold concentration for
inducing a visible morphological change in the lens. A higher
concentration, 150 mM H2O2, induced total lens opacification (data not shown). Because thiolation is an
early event in the oxidation of lens proteins (8),
B-crystallin, which contains seven cysteines, was chosen as a model to study the
relation of thiolation to cataract formation. A high concentration of
H2O2 (30 mM) was used to ensure
that enough H2O2 could enter the lens for
sufficient protein modification to be detected by mass spectrometry.
In contrast to the fresh lenses, which had only 0-4% with a GSH
adduct, the extent of GSH adduct formation in the control lenses was 36 and 44% even though they had not been exposed to H2O2. This suggests that the conditions for
organ culture, although closely mimicking the physiological environment
of a lens, allow oxidation of some proteins because of the differences
in the O2 concentrations of organ culture and in
vivo conditions. Although organ culture is useful for some
studies, it cannot exactly replicate the in vivo system.
Determination of PSSG and PSSC in the total lens after oxidation using
a Dionex LC system does not show increases after 24 h of organ
culture (data not shown). Perhaps only
B-crystallin is oxidized, and
the amount of this protein in the total lens is too small to be
detected in analysis of total protein-thiol mixed disulfides. The
location of the GSH adduct was not at the same cysteine residue in the
B-crystallins from different lenses, indicating that its formation
is not attributable to a specific reaction.
In this investigation, the H2O2 treatment
caused a large decrease in lens GSH. The majority (60%) of the
decrease could be accounted for by the increase in PSSG (see Table I),
indicating extensive thiolation of the lens proteins. It has been
established previously in rat lenses that free GSH is the source of GSH
for the adduct PSSG. It is believed that GSH is first oxidized to GSSG
and then binds nonenzymatically to the thiol group of a lens protein
(16). The increase in PSSC, in contrast, was relatively minor. The
source for the cysteine adduct in PSSC is not clear, although the free
cysteine pool in the lens is a likely source (40). The level of
cysteine in a normal lens is very low (3-5% of GSH), with most of it
concentrated in the nuclear region (40, 41). Because of this, PSSC can
be appreciably elevated only if sufficient oxidant penetrates into the
inner region of the lens, as is the case with hyperbaric oxygen-treated
guinea pig lenses (15) and with rat lenses exposed to
H2O2 for
48 h (8).
Exposure of bovine lenses to 30 mM
H2O2 induced a number of modifications in the
lens crystallins, resulting in decreased solubility of the proteins
(the portion of water-insoluble proteins increased from 5 to 44%) and
changed the elution profiles of the lens crystallins. For instance,
H merged with
-crystallin, and the peak sizes of both
L- and
-crystallins were diminished (Fig. 2B).
The most important contribution of this investigation is the specific,
detailed information about the modifications of
B-crystallins caused
by in situ oxidative stress. The principal sites of
modification were the cysteinyl and methionine residues.
B-Crystallin was chosen for observation of these modifications
because it is a major protein of the
-crystallin family containing 7 cysteine and 7 methionine residues. Among the 7 thiols, Cys-32 and
Cys-78 are buried and inaccessible to bulky reagents, Cys-18 and
Cys-109 are partially exposed, and Cys-15, Cys-22, and Cys-41 are
exposed (42, 43). Of the possible oxidative modifications, thiolation by GSH at the three Cys residues in peptide 10-31 (Cys-15, Cys-18, or
Cys-22) and/or Cys-41 appears to occur most readily, because these are
the sites of modification found in the control lenses. After oxidation
by H2O2, GSH adducts were also found at Cys-109 (Fig. 4B). This suggests that the thiolation sites in
peptide 10-31 and Cys-41 are exposed and thus easily modified, in
agreement with the x-ray crystallographic data (42). It is interesting to note that even though there are 7 thiols in
B-crystallin, only
two GSH adducts per molecule were formed. Slingsby and Miller (44),
incubating isolated
B-crystallin with a large excess of GSH at a
physiological pH, induced a maximum of three GSH adducts per molecule,
although the major product was
B-crystallin with only two GSH
adducts. This discrepancy may be attributable to the fact that the
thiolation in the current study was done with an intact lens, whereas
the study by Slingsby and Miller (44) used isolated proteins.
The second protein-thiol species, PSSC, was not observed in the
B-crystallins from the H2O2-treated lenses.
As shown in Table I, the levels of PSSC in both the control and
H2O2-treated lenses were much lower than that
of PSSG. PSSC may not have formed, or the extent of modification in
B-crystallin may have been too low to be detected by the current procedure.
The evidence for oxidation of all of the methionine residues of
B-crystallin agrees with an in vitro incubation study
using isolated bovine
-crystallins (30). In that study, isolated
-crystallins, incubated with 1 mM
H2O2 and 0.1 mM FeCl3,
showed oxidation at all the methionines but at no other locations. The difference between those results and the present data, which show GSH
adducts as well as oxidized methionines, can be attributed to the fact
that no GSH was present in the incubation medium used with the isolated
-crystallins.
The x-ray crystal structure of
B-crystallin (42) suggests that
Cys-18 and Cys-22 are favorably positioned for easy formation of a
disulfide bond, and a disulfide bond between these residues has
previously been observed (20). In this investigation, however, there
was no evidence for a disulfide bond between Cys-18 and Cys-22 in any
of the lenses studied. Intramolecular disulfide bonds between Cys-78
and Cys-41 and between Cys-78 and peptide 10-31 were found in
B-crystallin after the lens was exposed to H2O2. Perhaps the initial thiolation in peptide
10-31 or at Cys-41 caused a conformational change that exposed the
previously buried Cys-78, making it more accessible for disulfide bond
formation. This suggestion of a thiolation-induced conformational
change, along with previous evidence that GSH adducts induce
conformational changes (5, 6, 45), further supports the hypothesis (16) that oxidative stress-induced thiolation plays a pivotal role in the
mechanism of cataract formation.