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A1/A3-crystallins include S-methylation, glutathiolation, and truncation
Department of Chemistry, University of Nebraska–Lincoln, Lincoln, Nebraska 68588-0304, USA
Reprint requests to: Jean B. Smith, Department of Chemistry, Hamilton Hall, University of Nebraska–Lincoln, Lincoln, NE 68588-0304, USA; e-mail: jbsmith{at}unlserve.unl.edu; fax: (402) 472-9402.
(RECEIVED March 14, 2004; FINAL REVISION September 2, 2004; ACCEPTED September 3, 2004)
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Abstract |
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-crystallins undergo S-methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that 
A1/A3-crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of A1-crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post-translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N-terminal truncations were also found. Keywords: human lens crystallins; cataract; in vivo protein modification; S-methylation; glutathiolation
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04738505.
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Introduction |
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-, - and -crystallins. Because lens crystallins undergo little or no turnover, there is opportunity for numerous post-translational modifications as the lens ages (David et al. 1996; Lampi et al. 1998; Ma et al. 1998; Takemoto and Boyle 1998; Slingsby and Clout 1999; Hanson et al. 2000). Some modifications, identified in young clear lenses, may reflect normal development and maturation of the lens, while others, associated with aged lenses, could negatively impact crystallin conformation, aggregation state, or solubility, resulting in increased light scattering and eventual loss of lens transparency.
The free sulfhydryls of cysteine residues are among the most reactive functional groups in proteins. Cysteine residues in lens crystallins are susceptible to oxidation forming mixed disulfides with low molecular weight thiols such as glutathione, cysteine, and -glutamylcysteine (S-thiolation) (Dickerson and Lou 1993), and protein–protein disulfide bonds (Spector and Roy 1978; Lapko et al. 2002a). Although the physiological role of mixed disulfides is not well understood (Lou and Dickerson 1992; Kamei 1993; Feng et al. 2000), there is a correlation between these modifications and the color and opalescence of the lens nuclei (Lou et al. 1999). Also, the association of protein–protein disulfide bonds with crystallin aggregation and insolubilization suggests that intermolecular disulfide bonding has a role in cataractogenesis (Kodama and Takemoto 1988; Kodama et al. 1988; Stephan et al. 1999; Pande et al. 2000; Lapko et al. 2002a).
In a normal lens, the reduced form of glutathione (GSH) is the major factor in maintaining protein sulfhydryl groups in the reduced form (Lou and Dickerson 1992; Cotgreave and Gerdes 1998; Klatt and Lamas 2000). This role is achieved by direct scavenging of reactive oxygen species (Coan et al. 1992) as well as by producing reducing equivalents for enzymes involved in maintenance of redox equilibria (Hayes and McLellan 1999; Klatt and Lamas 2000). Although lens GSH concentration is normally ~5 mM (Dickerson and Lou 1997), GSH synthesis is slower in aged lenses (Rathbun and Murray 1991) and lower GSH levels have been reported in the lens nuclei of most forms of cataract (Lou et al. 1999; Bova et al. 2001). Recent data showing methylated cysteines in human lens crystallins (Lapko et al. 2002b, 2003a; Searle et al. 2004) suggest that other reactions may also protect reactive sulfhydryls. The high levels of cysteine methylation in S- and D-crystallins isolated from young clear lenses indicate that S-methylation does not negatively affect the functions of these proteins and, perhaps, may be beneficial in preventing disulfide bonding.
In the present paper, we describe previously unreported modifications of A1/A3-crystallins including methylation and glutathiolation of cysteines. These modifications were detected using the same methodology that permitted identification of methylation as a major modification of human lens S-crystallins (Lapko et al. 2002b). Key to identification of cysteine modifications is comparison of protein mass spectra of nuclear and cortical crystallins from human lenses of different ages, both prior to and after derivatization with cysteine-specific reagents such as 4-vinylpyridine and iodoacetamide. A1- and A3-crystallins are products of the same HuA1/A3 gene containing two in-frame initiation codons (Hogg et al. 1986). Protein synthesis initiated from these two codons generates the two proteins, which are identical except that A3-crystallin has 18 more amino acids at the N terminus than A1-crystallin has. The N-terminal methionine of A3-crystallin is acetylated. The N-terminal residue of A1-crystallin is an acetylated alanine corresponding to residue 19 of A3 (Lampi et al. 1997). Both A1- and A3-crystallins are truncated even in newborn lenses, with a loss of four and 22 residues from their N termini, respectively, yielding identical truncated forms (Lampi et al. 1998; Ma et al. 1998). These forms will be designated here as 
4 with the residues numbered according to their positions in A1-crystallin. Further truncations, which occur during lens maturation, will also be designated as followed by the number of residues missing from the N terminus of A1-crystallin. Among the -crystallins, substantial levels of methylated cysteine were found only in A1/A3-crystallins. Similar to -crystallins, A1/A3-crystallins are methylated at specific cysteine residues. A1/A3-Crystallins are also glutathiolated even in young lenses. Both methylation and glutathiolation may be important in maintaining crystallin structure.
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A1/A3-crystallins were determined by mass spectral analysis of A1/A3-crystallins isolated by size exclusion chromatography (Fig. 1A
) followed by reversed-phase HPLC of the -crystallins (Fig. 1B). Only the water-soluble proteins were examined in this study, but for lenses up to 19 yr old, the water-soluble portion includes at least 93% of the crystallins. Therefore, these spectra include nearly all the A1/A3-crystallins.
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A1- and A3-crystallins are the major forms; yet nearly a fourth of the proteins have lost four (from A1) or 22 (from A3) residues from the N terminus (Table 1). Additional truncations at the N termini along with other post-translational modifications make interpretation of mass spectra of A1/A3-crystallins from older lenses rather difficult. However, comparative analysis of proteins isolated from lenses of different ages facilitates identification of the mass spectral peaks. As described in the Materials and Methods section, the nuclear crystallins were obtained by boring through the center of the lens and then trimming the ends of the cylinder. These ends combined with the remainder of the lens are referred to as the cortex.
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A1/A3-crystallins were easily detected among the crystallins from the cortex of an 11-yr-old lens (Fig. 2A, peaks at 23,102 Da and 25,192 Da) and in 19-yr-old lenses (spectra not shown). The major peak in this spectrum (Fig. 2A) at 22,646 Da corresponds to the previously described 4 truncated protein. Peaks at 22,351 Da and 22,294 Da are due to further truncation of the proteins with losses of seven and eight residues from the N terminus of A1-crystallin (or 25 and 26 residues of A3). Small peaks with mass increases of 14 Da at 22,660 Da and 22,308 Da (Fig. 2A) indicate possible methylation.
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A1/A3-crystallins isolated from the nucleus of the same lens (Fig. 2B
) shows an increase in truncated forms 7 and 8 (peaks at 22,352 Da and 22,294 Da) as well as small amounts of proteins missing nine and 11 residues. The truncation sites are illustrated in Figure 3. The peaks with an additional 14 Da are more prominent among the nuclear proteins (Fig. 2B) than the cortical proteins (Fig. 2A), and further methylation is indicated by additional peaks at +28 Da and +42 Da.
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). Well resolved peaks with molecular masses 91 Da less than the expected masses of the derivatized forms of A1/A3-crystallins indicate methylation of cysteine residue(s). Dimethylated species were also easily identified among 4-vinylpyridine-derivatized proteins (Fig. 2C). Identification of trimethylated proteins was more problematic because the mass of underivatized proteins with three methylations differs by only one mass unit from the carbamylated species. Derivatization separated the masses of the trimethylated proteins and carbamylated proteins, but the mass spectral peaks for the trimethylated forms were then barely detectable.
The presence of a small peak at 22,951 Da (Fig. 2A,B) with a molecular mass 305 Da higher than the major 4 truncated form, and its absence after reduction of the proteins suggests the presence of a glutathione adduct. These glutathiolated species, which have similar abundances in the nuclei and cortex (Fig. 2A,B), are the major component of samples collected from the front shoulder of the A1/A3-crystallin HPLC fraction (Fig. 1B).
Sites of major post-translational modifications of A1/A3-crystallins
Truncations of A1/A3-crystallins, evident in the mass spectra of undigested proteins (Fig. 2A,B), were confirmed by MS/MS analysis of isolated truncated N-terminal tryptic peptides (residues 5–14, 8–14, and 9–14 of A1-crystallin) and Asp-N peptides 10–18 and 12–18. Three of five truncations were adjacent to proline residues (Fig. 3). Methylation of cysteine residues was evaluated from MALDI MS spectra of tryptic digests of the proteins after derivatization with 4-vinylpyridine. Three of the five cysteine-containing peptides of A1/A3-crystallins from the nucleus of an 11-yr-old lens had the signature of methylation: additional peaks with a mass 91 Da lower than the expected masses of the derivatized peptides. This 91-Da decrease due to derivatization with 4-vinylpyridine (Friedman 2001) helped identify methylated proteins and the methylated sites in tryptic peptides. Peptides that MALDI indicated might be methylated were further analyzed by LC/MS/MS, confirming methylation at Cys 64 (Fig. 4), Cys 99, and Cys 167 (Table 2).
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A1-crystallin peptide 92–104 with methylation at Cys 99 and peptide 7–18 of S-crystallin. Even when contamination by S-crystallin was small, the strong MS response of S peptide 7–18 interfered with direct estimation of methylation at A1 Cys 99. Therefore, separation of A1 92–104 and S 7–18 by online LC/MS was important for estimation of the extent of methylation at Cys 99. Iodoacetamide-derivatized peptides were chosen for estimation of abundance by LC/ESI MS/MS analysis because methylated and iodoacetamide-derivatized peptides have similar ionization efficiencies and charged state distributions. Peptides derivatized with 4-vinylpyridine are less satisfactory for quantitation because they are not stable during MS/MS analysis, yielding complicated MS/MS spectra, and because their charged state distributions differ from the methylated peptides.
MALDI mass spectra of tryptic peptides from A1/A3-crystallins isolated from an 11-d-old lens indicated the presence of GSH adducts in peptides 47–72 and 92–107, each containing one cysteine residue (Cys 64 and Cys 99, respectively). Analysis of these peptides by LC/MS/MS confirmed the assignments (spectra not shown).
The peptides in the tryptic, Asp-N, and chymotryptic digests were searched for evidence of several previously reported modifications in A1/A3-crystallins including phosphorylation at Ser 142 and Thr 109, methylation at Arg 119, and acetylation of Lys 104, Lys 107, and Lys 113 (MacCoss et al. 2002). Even with specific ion monitoring, peptides with these modifications were not detected, suggesting that if they are present, the abundances are very low. Carbamylation of the free N termini of truncated A1/A3-crystallins was found in 11- and 19-yr-old lenses.
Changes in abundances of the major post-translational modifications
Both truncation and methylation of A1/A3-crystallins increase with age and are higher in the nucleus than in the cortex. Intact A1/A3-crystallins are found in the cortex of lenses as old as 70 yr (data not included in this study), but only truncated proteins are detected in the nuclei of 11-yr-old (Fig. 2B) and older lenses. Comparison of spectra for nuclear and cortical A1/A3-crystallins from 11-yr-old and 19-yr-old lenses shows more truncation among nuclear than cortical proteins from both ages.
Both nuclear and cortical proteins from 19-yr-old lenses are more methylated than those from 11-yr-old lenses (Table 2). In the nuclei of 11-yr-old lenses, the abundance of methylated 4 and 8 A1-crystallins is similar to the nonmethylated forms (Fig. 2B), while in the nuclei of the 19-yr-old lenses, methylated 8 A1-crystallin is the predominant form. Nonmethylated 4 is the most abundant form in the cortex of lenses from both ages (Table 1). Total methylation of nuclear A1/A3-crystallins is almost twice the methylation of cortical A1/A3-crystallins from 11- and 19-yr-old lenses (Table 2).
These trends of increased methylation with age and differences between the cortex and nucleus are illustrated by the 4 form. Methylation of this form is nearly 30% in the cortex of 11-yr-old lenses (Fig. 2A), ~35% in the cortex of 19-yr-old lenses (data not shown), 47% in the nuclei of 11-yr-old lenses (Table 2), and >75% in the nuclei of 19-yr-old lenses (data not shown).
Our data showed no changes in the specificity of methylation with aging. For example, Cys 99 was the major methylation site in A1/A3-crystallins isolated from both 11- and 19-yr-old lenses (Table 2). Approximately 10% of the truncated forms in the nucleus of an 11-yr-old lens are carbamylated at the N terminus (Fig. 2C).
Contrary to S-methylation and truncation, glutathiolation is similar among A1/A3-crystallins isolated from lenses of different ages. Even in an 11-d-old lens, ~15% of A1/A3-crystallins are glutathiolated. Derivatization with iodoacetamide without reduction of disulfide bonds was used to analyze the GSH-containing fractions from 11-d-old and 19-yr-old lenses. With this approach, much better recovery of disulfide-bonded peptides was obtained than when non-derivatized proteins were digested. Both in 11-d-old and 19-yr-old lenses, Cys 64 and Cys 99 were the major sites of glutathiolation. The possibility of glutathiolation at Cys 167 was suggested by the presence of a very minor peak at 2682.0 Da corresponding to tryptic peptide 160–178 plus glutathione (spectra not shown). This peptide without glutathione produced a strong response in MALDI mass spectra.
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Discussion |
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A1/A3-crystallins as it is in S- and D-crystallins (Lapko et al. 2002b, 2003a) and that methylation occurs even in young lenses. Exposed cysteines are among the most reactive residues in proteins. They are extremely sensitive to oxidation. Accessible cysteines can also form mixed disulfides (with low molecular weight thiols) and both intra- and intermolecular disulfide bonds. Such cross-linking of crystallin subunits is a physiologically abnormal process leading to aggregation and precipitation of the proteins and development of cataract (Kodama et al. 1988; Stephan et al. 1999; Pande et al. 2000). In vitro studies have demonstrated formation of protein disulfide cross-links and lens opacification under conditions of oxidative stress such as hyperbaric oxygen (Giblin et al. 1988, 1995) and exposure to hydrogen peroxide (Brigelius et al. 1983; Hanson et al. 1999). Modification of cysteines by methylation or glutathiolation could be beneficial by inhibiting formation of protein–protein disulfide bonds.
Identification of a mechanism explaining in vivo methylation of crystallins is a challenging task. The major sites of S-methylation in human D- and S-crystallins share certain structural similarities such as nearby cysteine residues (Fig. 5). The lack of homology both among A1/A3-crystallin sequences containing methylated cysteines and among regions of A1/A3- and -crystallins with methylated cysteines (Fig. 5) supports an argument against a highly specialized methylase as the methylating agent. However, no methylated residue other than cysteine has been detected. Comparison of the sequences of - and A1/A3-crystallins with the sequence of B2, whose structure is known (Chirgadze et al. 1991), suggests that the methylated cysteines in - and A1/A3-crystallins all have high solvent-accessibility. Cysteine 34 of A1-crystallin, which is conserved in all human -crystallins, is not methylated. The three-dimensional structure of B2-crystallin shows that this cysteine is buried.
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A1/A3, B2, and A, but not in -crystallins (Smith et al. 1995; Feng et al. 2000; Lapko et al. 2003a). Formation of protein disulfides with glutathione (and cysteine) has been proposed as a precursor of protein–protein disulfide cross-links (Lou 2000). This hypothesis is supported by evidence that bovine lenses exposed to 30 mM hydrogen peroxide form glutathiolated B-crystallins and become opaque (Hanson et al. 1999). Also increased oxidized glutathione and protein-glutathione disulfide bonds have been reported in nuclei of aged lenses (Lou 2000; Bova et al. 2001), perhaps because reduced glutathione is diminished in the nucleus (Lou et al. 1999; Bova et al. 2001). This decrease in reduced GSH may be due to an internal barrier to the movement of small molecules that develops by middle age (Truscott 2003). In contrast, it has also been suggested that glutathiolation is a protective mechanism, preventing irreversible oxidation, disulfide bond formation, and insolubility (Kamei 1993; Cappiello et al. 2000). This hypothesis is supported by evidence that glutathiolated B2-crystallins are less prone to precipitation (Feng et al. 2000). Our evidence of a relatively high level of glutathiolation of A1/A3-crystallins from a newborn lens suggests that glutathiolation is not detrimental. Although the role of glutathiolation of A1/A3-crystallins in young lenses is not clear, this modification results in a species more stable to oxidation than nonglutathiolated proteins, and capable of regenerating the reduced protein. Regeneration of the thiol groups from glutathiolated proteins may occur either chemically or enzymatically (Thomas et al. 1995; Jung and Thomas 1996). In human lenses, thioltransferase and glutathione S-transferase are the major enzymes involved in the dethiolation process (Raghavachari et al. 1999; Lou 2000). A possibility for direct chemical reduction of protein-glutathione disulfide also was shown using intact rabbit lenses (Willis and Schleich 1996). In aged lenses, which have increased levels of oxidized glutathione and a decreased ability to regenerate sulfhydryl groups, accumulation of proteinthiol disulfides may lead to protein–protein disulfide cross-links.
Among the post-translational modifications of A1/A3-crystallins that occur during maturation are multiple N-terminal truncations. Loss of four N-terminal residues of A1-and 18-residues of A3-crystallin at an early stage of life has been previously established (Lampi et al. 1998; Ma et al. 1998), but this is the first report of further truncations corresponding to loss of seven, eight, nine, and 11 residues of A1-crystallin. Three of the truncations are adjacent to proline. Truncations C-terminal to proline residues and their functional consequences are well known, being widespread in CD26/DPPIV and homolog proteases (Boonacker and Van Noorden 2003). Also, enzymes that cleave proteins to release proteins with an N-terminal proline have been described (Yoshimoto et al. 1983), but the mechanism(s) for these truncations is not known.
The truncated proteins were abundant among the water-soluble crystallins, suggesting that removal of residues at the N termini of A1/A3-crystallins has no adverse affect on solubility. Our data showing these truncations in A1/A3-crystallins from young lenses support the idea that such truncations are more likely functional than harmful (Werten et al. 1999b). It has been suggested that loss of the N-terminal portion of the proteins allows modulation of protein repulsion during lens maturation and maintains the protein concentration gradient required for transparency of the lens (Werten et al. 1999b). In vitro studies of truncated forms of B1-crystallins also indicate that loss of the N terminus is not as detrimental to its stability as some other post-translational modifications, such as deamidation (Kim et al. 2002).
A1- and A3-crystallins are among very few examples of expression of multiple eukaryotic proteins from a single mRNA by a process called leaky ribosomal scanning (Werten et al. 1999a). Occasional skipping of the first start codon results in initiating translation at a second codon and expression of two proteins differing only in the N-terminal region. Analysis of cortical A1A3-crystallins isolated from lenses of different ages showed very similar relative levels of A1- and A3-crystallins throughout life. The N-terminal truncations of these two proteins most likely proceed through the 4 form (Fig. 3) resulting in the 7, 8, 9, or 11 proteins. Despite the presence of the additional extension of 18 amino acid residues in A3-crystallins, no truncated intermediates due to cleavage among the first 18 residues of A3 were detected.
With the exception of C-, D, and B-crystallins, all the human lens crystallins are cotranslationally acetylated at their N termini. We recently showed that some C-, D-, and B-crystallins in mature human lenses are post-translationally carbamylated. We report here that truncation of portions of the N-terminal regions of A1/A3-crystallins results in 4, 7, and 8 forms with nonprotected N termini, which may then be N-carbamylated. Due to the relatively short lifetimes of these truncated forms, the level of the carbamylation is lower than carbamylation of C-, D and B-crystallins. Carbamylation of accessible N-termini appears to be a common post-translational modification in lens proteins.
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Materials and methods |
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A1/A3-crystallinsA1/A3-crystallins have the same elution time as A2-crystallin on reversed-phase HPLC, a 25 mM sodium citrate buffer containing 100 mM NaCl, 1 mM EDTA (pH 6.2), was used in the size exclusion chromatography isolating glutathiolated A1/A3-crystallins. With these conditions, the majority of A1/A3-crystallins eluted in the H-fraction, while A2-crystallins, which form only dimers, eluted in the L-fraction (Lapko et al. 2003b). The total -crystallins or individual H and L fractions (Ma et al. 1998) were further separated by reversed-phase HPLC using a 20% to 55% gradient of acetonitrile in water with 0.1% trifluoroacetic acid (TFA). The A1/A3-crystallin fractions were concentrated to dryness, redissolved in 50% acetonitrile-containing 0.3% formic acid, and analyzed by mass spectrometry.
Derivatization of sulfhydryl groups of A1/A3-crystallins
The A1/A3-crystallins (20–200 pmol) were dissolved in 300 µL of buffer (250 mM Tris-HCl, 6 M guanidine hydrochloride [GdHCl], 1 mM EDTA, 5 mM dithiothreitol [DTT] at pH 8.5). After incubation for 1 h, the sulfhydryl groups were derivatized by reaction with 15 mM iodoacetamide for 30 min or with 25 mM 4-vinylpyridine (Friedman 2001) for 90 min. The reactions were quenched by adding 30 µL of 2-mercaptoethanol.
When the GSH adducts to A1/A3-crystallins were to be determined, the crystallins were derivatized with iodoacetamide in a buffer without DTT. A solution of 30 mM iodoacetamide in a 6 M GndHCl buffer (pH 8.5) was added to dry protein samples and allowed to react for 15 min at room temperature. After derivatization, the proteins were desalted by RP HPLC.
Enzymatic digestions of A1/A3-crystallins
The A1/A3-crystallins, derivatized with iodoacetamide or 4-vinypyridine, were dissolved in 100 mM ammonium bicarbonate (pH 7.8) and digested at 37°C with modified trypsin (Promega) or chymotrypsin (Sigma) for 8 h or with Asp-N protease (Sigma) for 24 h at an enzyme:protein ratio of 1:50. Digests were freeze-dried and dissolved in 0.1% TFA for online mass spectrometric analysis or in 50% acetonitrile, 0.1% TFA for matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Proteins with GSH-adducts were digested at trypsin:protein ratio of 1:30 for 3 h at 37°C.
Electrospray ionization mass spectrometric analysis of proteins
The A1/A3-crystallins were dissolved in 50% acetonitrile, 0.3% formic acid, and injected directly into a Q-Tof mass spectrometer (Micromass) using a solvent flow of 5 µL/min. The typical uncertainty in protein mass determinations was 0.005%.
Mass spectrometric analysis of peptides
Peptides produced by enzymatic digestions were analyzed by online capillary reversed-phase HPLC (0.3 x 250 mm, C18-PM; LC-Packings) connected directly to an electrospray ionization ion trap mass spectrometer (Finnigan MAT LCQ). The HPLC gradient was 0%–50% acetonitrile in water, both with 0.01% TFA, over 50 min. The flow rate was 5 µL/min. Peptide elution was monitored at 214 nm. For peptide mapping, the mass spectrometer was routinely operated in the full-scan MS mode with the three most abundant ions of each scan analyzed by MS/MS. A collision energy of 35%–45% was used for the MS/MS analyses. The uncertainty in the peptide mass determinations was ±0.2 Da over the mass range of 100–2000 Da. The zoom mode of operation for a specific mass/charge with a window of ±5 m/z was used for detection of peptides containing methylation, glutathiolation, truncation, and carbamylation. Nano-flow reversed-phase HPLC-ESIMS using a Q-Tof Ultima mass spectrometer (Micromass) was also used for peptide mapping when the amount of sample was limited.
The peptides from enzymatic digestions were also analyzed by MALDI MS (Voyager-DE Pro mass spectrometer, Applied Bio-systems). Typically, peptides were detected in the reflectron mode of operation. Large peptides were analyzed in the linear mode. -Cyano-4-hydroxycinnamic acid was the matrix.
Estimation of the abundance of truncated A1/A3-crystallins
The sites of truncation of A1/A3-crystallins were confirmed by detecting the corresponding peptides from the N terminus of A1-crystallins (Ion Trap mass spectrometer; Finnigan MAT LCQ). Carbamylated forms of the N-terminal peptides were identified by detecting peptides with masses increased by 43 Da. The sequences of the N-terminal peptides were confirmed by MS/MS.
Estimates of the abundances of truncated A1/A3-crystallins were calculated from the relative intensities of the mass spectral peaks for the proteins derivatized with 4-vinylpyridine as described previously (Lapko et al. 2003a). Calculations of the abundances of the truncated forms as well as other post-translational modifications should be considered only estimates because the mass spectral responses of the modified and unmodified proteins (or peptides) may not be equivalent. In addition, some selective losses could occur during the chromatographic isolations. The proteins included in estimations of truncated A1/A3-crystallins were intact A1/A3-crystallins, the forms missing four, seven, and eight residues from the N terminus, and the corresponding monomethylated species of each.
Estimation of abundance of S-methylation in A1/A3-crystallins
Total methylation in nuclear A1/A3-crystallin was calculated as the sum of estimated abundances of methylations at Cys 64, Cys 99, and Cys 167. The intensities of the peaks in MALDI mass spectra of tryptic digests of the A1/A3-crystallins A1/A3-crystallin were used to estimate the extent of methylation at Cys 64 and Cys 167. S-Methylation was recognized as a 91-Da decrease in the mass of a Cys-containing peptide derivatized with 4-vinylpyridine. Methylation at Cys 64 of A1-crystallin was estimated from relative intensities of peaks for methylated and 4-vinylpyridinylated tryptic peptides of residues 50–72 (peaks at 2672.2 and 2763.2 Da, respectively) as well as the peptides of residues 47–72 (3000.4 and 3091.4 Da). Methylation at Cys 167 was estimated from the tryptic peptides of residues 160–175 or 160–178 of A1-crystallin. Methylation at Cys 99 was estimated from the relative abundances of peptides from residues 92–107 derived from iodoacetamide-derivatized proteins by monitoring ions at 932.1 and 953.6 m/z (doubly charged species) using LC/ESI MS.
Because the abundance of methylation was lower in cortical A1/A3-crystallins than in the nuclear proteins, the total methylation in cortical A1/A3-crystallins was estimated from the relative intensities of the mass spectral peaks for the proteins derivatized with 4-vinylpyridine as described previously (Lapko et al. 2003a).
Analysis of glutathiolation of A1/A3-crystallins
A1/A3-Crystallins with GSH-adducts (A1/A3+GSH) were isolated by reversed phase HPLC of the H fraction obtained after gel chromatography of -crystallins from an 11-d-old lens in a 25 mM sodium citrate buffer. Di- and monoglutathiolated A1/A3-crystallins eluted as separate peaks in front of the A1/A3-crystallin peak. For older lenses, separation of glutathiolated A1/A3-crystallins was relatively poor, although the front shoulder of A1/A3-crystallins peak was enriched in the glutathiolated species.
The sites of glutathiolation in 11-d-old and 19-yr-old lenses were determined by MALDI MS of tryptic digests of the proteins derivatized with iodoacetamide without reduction of disulfide bonds. Tentative assignments of GSH-peptides 47–72 (3289.4 Da), 92–107 (2152.0 Da), and 92–104 (1808 Da) were confirmed by selective ion monitoring with MS/MS analyses of the expected ions. Changes in the abundance of glutathiolation with aging were estimated from mass spectra of nonreduced A1/A3-crystallins isolated from nuclear and cortical -crystallins of 11-d-old, 11-yr-old, and 19-yr-old lenses.
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References |
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