Thiolation of the gamma B-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide*

Stacy R. A. HansonDagger , Andrew A. Chen§, Jean B. SmithDagger , and Marjorie F. Lou§

From the Dagger  Department of Chemistry and § Center for Biotechnology, Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0905

    ABSTRACT
Top
Abstract
Introduction
References

Oxidative damage of the lens causes disulfide bonds between cysteinyl residues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H2O2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H2O2. The H2O2 treatment decreased the glutathione and increased the protein-glutathione and protein-cysteine disulfides in the lens. The molecular mass of the gamma B-crystallin isolated from lenses, not treated with H2O2, agreed with the published sequence (Mr 20,966). Some lenses also had a less abundant gamma B-crystallin component 305 Da higher (Mr 21,270), suggesting the presence of a glutathione adduct. The gamma B-crystallins from H2O2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified gamma B-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of gamma B-crystallins from different lenses indicated that the +305 Da modifications were not at a specific cysteine. For the lenses incubated without H2O2, there was evidence of adducts at Cys-41 and in peptide 10-31, which includes 3 cysteines. Analysis of modified peptide 10-31 by tandem mass spectrometry showed GSH adducts at Cys-15, Cys-18, and Cys-22. In addition, gamma B-crystallins from H2O2-treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.

    INTRODUCTION
Top
Abstract
Introduction
References

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, gamma B-crystallins were isolated from untreated bovine lenses and from lenses incubated with and without H2O2 in organ culture. gamma 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 gamma B-crystallin are known. In this investigation, the modifications of gamma 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.

    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 alpha -, beta H-, beta L-, and gamma -crystallins fractions. In addition, three fresh lenses were homogenized, and the gamma -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 alpha -, beta H-, beta L-, and gamma -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 alpha -, beta H-, beta L-, and gamma -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 gamma -Crystallins-- The gamma -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 gamma -crystallin subfamilies were pooled and stored at -20 °C until analysis by mass spectrometry.

Analysis of gamma -Crystallins by Electrospray Ionization Mass Spectrometry (ESIMS)-- After isolation of the gamma -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 gamma B-Crystallins by ESIMS-- The gamma 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 gamma 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 gamma B-crystallin, which had been calculated from the known sequence of bovine gamma B-crystallin (35).

Analysis of Peptic Peptides of gamma B-Crystallin (10-31) by ESIMS-- Approximately 1-2 nmol of gamma 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 gamma B-Crystallin Peptide 10-31 by Tandem Mass Spectrometry-- Tandem mass spectrometric experiments were performed on gamma B peptide 10-31 previously isolated from a tryptic digest of intact gamma 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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Table I
Glutathione and protein-thiol mixed disulfides in bovine lensesa

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 gamma -Crystallins-- Size exclusion fractionation of the water-soluble proteins of both the fresh and control lenses showed five peaks corresponding to alpha -, beta H-, beta L-, and gamma -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 alpha - and beta H-crystallin peaks appeared to merge into one larger peak, and the absorbances for beta L- and gamma -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 alpha  and beta H fractions have merged in the chromatogram for the H2O2-treated lens, and the absorbance of the gamma -crystallin peak is 30% lower.

The bovine gamma -crystallins collected from the size exclusion fractionation were further fractionated by reversed phase HPLC. There were five peaks for the gamma -crystallins from the fresh and control lenses; the peaks for the gamma -crystallins from the H2O2-incubated lenses were not as well resolved (Fig. 3, A and B). Although the gamma 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 gamma -crystallins. A, control lens. The proteins in the peaks were identified by their molecular weights, determined by ESIMS. In order of elution, they are gamma S, gamma B, gamma D, gamma C, and gamma E. B, lens treated with 30 mM H2O2. The proteins were identified as gamma B, gamma D, gamma C, and gamma E. gamma S-Crystallin was not found in these fractions.

Analysis of gamma -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 gamma S-crystallin (Mr 20,835), the second fraction was gamma B-crystallin (Mr 20,966), the third fraction was gamma D-crystallin (Mr 20,749), the fourth fraction contained gamma D- and gamma C-crystallins (Mr 21,008), and the fifth fraction was gamma 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 gamma -crystallins or because a modified crystallin was also present. For example, the mass spectrum for fraction 2 shows that, in addition to gamma B-crystallin, there are minor components with molecular masses corresponding to gamma S-crystallin and gamma B-crystallin plus an additional 305 Da, suggesting the presence of a GSH adduct of gamma 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 gamma -crystallins from the control lenses, the only other molecular mass indicating modification was a very minor component in a mass spectrum of gamma S-crystallin, also 305 Da higher, suggesting the presence of a GSH adduct to gamma S-crystallin (data not shown).


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Fig. 4.   Reconstructed electrospray ionization mass spectra of bovine gamma B-crystallins. A, gamma B-crystallins isolated from a control lens (Fig. 3A, second peak). B, gamma B-crystallins isolated from a lens treated with 30 mM H2O2 (Fig. 3B, second peak). Note the increase in gamma B-crystallin with a GSH adduct, the presence of gamma B-crystallin with two GSH adducts and a species suggesting addition of seven oxygens (+7 × 16 Da) to gamma B-crystallin.

Mass spectra of the gamma -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 gamma S-crystallin. The major component of the second fraction collected from HPLC was gamma 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 gamma B-crystallin (Mr 20,964), gamma B-crystallin plus two GSH adducts (Mr 21,576), and gamma 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 gamma D-, gamma C-, and gamma E-crystallins, not modified.

Analysis of Peptides from Tryptic Digestion of gamma B-Crystallin-- To confirm the identities of the modifications and to locate the specific sites that had been modified, gamma 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 gamma 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.

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 gamma 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 gamma 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.

                              
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Table II
Peptides found in tryptic digest of gamma B-crystallins from bovine lenses incubated in 30 mM H2O2

A mass spectrum showing peptides in an HPLC fraction from the tryptic digest of the gamma 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 gamma 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.

In addition to the modifications at cysteinyl residues, there was evidence of partial oxidation of all 7 methionines of gamma 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 gamma 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.


    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), gamma 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 gamma 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 gamma 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, beta H merged with alpha -crystallin, and the peak sizes of both beta L- and gamma -crystallins were diminished (Fig. 2B).

The most important contribution of this investigation is the specific, detailed information about the modifications of gamma B-crystallins caused by in situ oxidative stress. The principal sites of modification were the cysteinyl and methionine residues. gamma B-Crystallin was chosen for observation of these modifications because it is a major protein of the gamma -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 gamma B-crystallin, only two GSH adducts per molecule were formed. Slingsby and Miller (44), incubating isolated gamma 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 gamma 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 gamma 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 gamma 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 gamma B-crystallin agrees with an in vitro incubation study using isolated bovine alpha -crystallins (30). In that study, isolated alpha -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 alpha -crystallins.

The x-ray crystal structure of gamma 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 gamma 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.

    ACKNOWLEDGEMENTS

Mass spectral analyses were performed by the Nebraska Center for Mass Spectrometry. We express our appreciation to Fine Wu for assistance in preparing the figures.

    FOOTNOTES

* This work was supported by National Institutes of Health Research Grants EY RO1 10595 (to M. F. L.) and EY RO1 07609 (to J. B. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Veterinary and Biomedical Sciences, 134 VBS, University of Nebraska, Lincoln, NE 68583-0905. Tel.: 402-472-0307; Fax: 402-472-9690.

    ABBREVIATIONS

The abbreviations used are: PSSG, protein disulfide-bonded glutathione; PSSC, protein disulfide bonded cysteine; ESIMS, electrospray ionization mass spectrometry; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography.

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Top
Abstract
Introduction
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