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Methylation and carbamylation of human -crystallins

Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588, USA

Reprint requests to: Jean B. Smith, Department of Chemistry, Hamilton Hall, University of Nebraska, Lincoln, NE 68588, USA; e-mail: jbsmith{at}unlserve.unl.edu; fax: (402) 472-9402.

(RECEIVED January 31, 2003; FINAL REVISION May 1, 2003; ACCEPTED May 1, 2003)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0305403.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Accessible sulfhydryls of cysteine residues are likely sites of reaction in long-lived proteins such as human lens crystallins. Disulfide bonding between cysteines is a major contributor to intermolecular cross-linking and aggregation of crystallins. A recently reported modification of S-crystallins, S-methylation of cysteine residues, can prevent disulfide formation. The aim of this study was to determine whether cysteines in C-, D-, and B-crystallins are also S-methylated. Our data show that all the -crystallins are S-methylated, but only at specific cysteines. In D-crystallin, methylation is exclusively at Cys 110, whereas in C- and B-crystallins, the principal methylation site is Cys 22 with minor methylation at Cys 79. D-crystallin is the most heavily methylated -crystallin. D-Crystallins from adult lenses are 37%–70% methylated, whereas C and B are ~12% methylated. The specificity of -crystallin methylation and its occurrence in young clear lenses supports the idea that inhibition of disulfide bonding by S-methylation may play a protective role against cataract. Another modification, not reported previously, is carbamylation of the N termini of B-, C-, D-crystallins. N-terminal carbamylation is likely a developmentally related modification that does not negatively impact crystallin function.

Keywords: Human lens crystallins; cataract; cysteine S-methylation; N-terminal carbamylation; N-terminal acetylation

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
The primary function of the eye lens is to focus images on the retina. This function is possible because of the unique composition of the lens, where closely packed proteins account for ~38% of the wet mass. More than 90% of the lens proteins are structural proteins, called crystallins. The crystallins are organized into three main classes, -, ß-, and -crystallins, on the basis of the size of assemblies they form and their sequence homologies. The -crystallins, monomers with molecular masses of ~21 kD, constitute ~30% of the proteins in the nuclei of adult human lenses. Four -crystallin genes are expressed in humans: S, B, C, and D (Meakin et al. 1985; Heon et al. 1999). Several types of hereditary cataracts have been linked to mutations in human -crystallin genes (Heon et al. 1999; Stephan et al. 1999; Kmoch et al. 2000; Ren et al. 2000; Santhiya et al. 2002). A single base alteration in the D-crystallin gene resulting in cysteine rather than arginine at residue 14 is associated with a type of hereditary, juvenile-onset punctate cataract (Stephan et al. 1999). This mutation in D-crystallin does not cause conformational changes or destabilize the protein, but the presence of the additional exposed cysteine creates an opportunity for disulfide cross-linked oligomerization of the mutated protein that leads to cataracts (Pande et al. 2000). -Crystallins, which are rich in cysteine residues, are the most abundant disulfide-bonded crystallins present in the high molecular weight aggregates typical of senile nuclear cataracts (Takemoto and Azari 1977; Kodama and Takemoto 1988; Hejtmancik 1998; Lapko et al. 2002a). In addition, cysteine residues of lens crystallins are susceptible to other post-translational modifications that can lead to nondisulfide cross-linking (Garner et al. 2000).

Recent evidence of methylation at two specific cysteines of S-crystallins suggests a possible protective mechanism against cataracts (Lapko et al. 2002b). Methylation of these exposed cysteine residues prevents their participation in the intermolecular disulfide bonding that leads to protein cross-linking, aggregation, and eventually cataract. To determine whether other members of the -crystallin family also have this post-translational modification, C-, B-, and D-crystallins were isolated and analyzed by mass spectrometry. S-methylation was found among all -crystallins. In addition, carbamylation of the N termini of C-, D-, and B-crystallins was established as a major post-translational modification during lens aging.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
This study identified several major post-translational modifications of human -crystallins and the age-related changes in these modifications. Quantitation of the post-translational modifications is approximate because the mass spectral responses of the modified and unmodified proteins and/or peptides may not be equivalent. The responses may also be affected by signal suppression due to coeluting components. In addition, there are undoubtedly some selective losses during the chromatographic isolations. A 19-year-old lens was chosen to illustrate these data (Figs. 1–4, below) because it has the major modifications of an adult lens (Datiles et al. 1992; Lampi et al. 1998; Ma et al. 1998; Lapko et al. 2002b), but fewer of the minor modifications that make isolation of proteins from older lenses more difficult and complicate the mass spectra. In addition, as much as 95% of the nuclear proteins from a 19-year-old lens are soluble, allowing characterization of the crystallins using soluble extracts. In this report, the term modification refers to in vivo post-translational reactions, whereas the term derivatization refers to in vitro reactions used in the analysis.

View larger version (18K): [in this window] [in a new window]   Figure 1. Reconstructed ESI mass spectra of C-crystallin isolated from the nucleus of a 19-year-old lens. (A) Underivatized. The mass at 20,747 Da corresponds to unmodified C-crystallin, whereas the mass at 20,790 Da indicates carbamylation or acetylation. (B) Derivatized with 4-vinylpyridine. The masses at 21,496 and 21,539 Da are 91 Da lower than the major peaks, indicating the presence of a methylated cysteine.

View larger version (21K): [in this window] [in a new window]   Figure 4. MS/MS spectra of peptides corresponding to 107–112 from an Asp-N digest of iodoacetamide-derivatized D-crystallin isolated from the nucleus of a 19-year-old lens containing (A) no methylated cysteines (m/z 782.3) and (B) one methylated Cys residue (m/z 739.3). Because Cys 110 was methylated (+14 Da) and could not be derivatized by iodoacetamide (+57 Da), all fragments (b4, b5, y3, y4, y5) that included Cys 110 showed a net loss of 43 Da. The expected y- and b-fragments for the peptide with both cysteines derivatized are given at the top.

Post-translational modifications of C-crystallin

To identify the major site(s) of modification and distinguish between carbamylation and acetylation, peptides from C-crystallins digested with trypsin, chymotrypsin, or AspN proteases were analyzed. The accuracy in determining peptide masses (±0.2 Da) allows a 43 Da increase due to carbamylation to be distinguished from a 42 Da increase due to acetylation. Both reactions can occur at amino groups in the side chains of lysines or at free N termini of proteins. Peptide 1–6 of C-crystallin was found both with and without addition of 43 Da. Tandem mass spectrometric (MS/MS) analysis of the unmodified (Fig. 2A) and the modified (Fig. 2B) peptides confirmed that the modification was carbamylation of the N terminus. A search for other sites of carbamylation or acetylation gave evidence that the N terminus was also acetylated. The acetylated peptide eluted after the carbamylated one during on-line HPLC/MS analysis. The total ion current for the carbamylated N-terminal peptide was 8–10 times higher than for the same peptide with acetylation. These data indicated that the mass spectral peak ~43 Da higher than C was primarily due to N-terminal carbamylation.

View larger version (25K): [in this window] [in a new window]   Figure 2. MS/MS spectra of the N-terminal chymotryptic peptide 1–6 of C-crystallin with (A) a free N terminus (m/z 728.4) and (B) with a carbamylated N terminus (m/z 771.4). The b- and y-fragments at m/z 229.3 and m/z 671.4 are critical for identification of the carbamylation site. The presence of b2 +43 ion and y5 without modification, as well as absence of a peak at m/z 714.4, which would have indicated carbamylation at Lys 2, show that the N-terminal Gly is carbamylated. The presence of some b-series ions without the modification is consistent with previous observations that the carbamyl group is unstable in MS/MS analysis (Lapko et al. 2001; Van Driessche et al. 2002). The amino acid sequence and expected b- and y-fragments for peptide 1–6 are given at the top.

Post-translational modifications of D-crystallin
In contrast to the simple mass spectra of C- and S-crystallins, the mass spectrum of D-crystallins from the nucleus of a 19-year-old lens had eight major components ranging from 20,521 to 20,665 Da (Fig. 3A, Table 1). The complicated spectrum is due to combinations of three major post-translational modifications, that is, truncation of the C-terminal serine, carbamylation of the N terminus, and S-methylation of cysteines. Peaks 1–4 are due to full-length D-crystallin (M_r 20,607) and its methylated and/or carbamylated forms, whereas peaks 5–8 are truncated D-crystallin (M_r 20,520) and its methylated and/or carbamylated species. Molecular masses and identities of these forms and their derivatives are given in Table 1.

View larger version (21K): [in this window] [in a new window]   Figure 3. Reconstructed ESI mass spectra of D-crystallins isolated from the nucleus of a 19 year-old lens. (A) Underivatized. (B) Derivatized with 4-vinylpyridine. The masses and identities of each numbered peak are given in Table 1.

View this table: [in this window] [in a new window]   Table 1. Experimentally determined masses (Da) of the major human D-crystallins

View this table: [in this window] [in a new window]   Table 2. Estimated abundance (%) of soluble D-crystallins from human lenses of different ages

Because all of the tryptic peptides of 4-vinylpyridine derivatized D-crystallins produced strong peaks in MALDI MS, the sites of methylation and the extent of methylation were determined by use of tryptic peptides. In addition, peptide 107–112 from Asp-N digestion of iodoacetamide-derivatized D was used to determine the methylation status of Cys 108 and Cys 110. A selected ion plot for methylated peptide 107–112 (m/z 787.2, data not shown) showed only one peak and the MS/MS spectrum of this peak demonstrated that Cys 110 is the only methylated residue in this peptide (Fig. 4).

In addition to S-methylation, N-terminal carbamylation, N-terminal acetylation, and truncation of the C-terminal serine were found in the peptides from D-crystallin. Both carbamylation and acetylation of the N-terminal Gly residue of D-crystallin were present, with the peaks due to carbamylation approximately eight times larger than those due to acetylation. Truncation of the C-terminal Ser was confirmed by MS/MS analyses of tryptic peptides 168–172 and 169–172 (data not shown). The various masses of D-crystallins (Fig. 3; Table 1) could thus be attributed to combinations of methylation at Cys 110, carbamylation (or acetylation) of the N terminus, and truncation of the C-terminal serine.

B-crystallin and its major modifications
Although molecular biology data indicate that there are at least six closely related human -crystallins (Wistow and Piatigorsky 1988; Graw 1997), only three -crystallins—S, C, and D—are abundant. In a recent study analyzing peptides from digests of the total proteins of a 4-year-old cataractous lens, peptides derived from B-crystallin were detected (MacCoss et al. 2002). In the present study, the presence of a protein with a molecular mass of 20,776 Da (Fig. 5A) corresponding to the sequence of B-crystallin, is supporting evidence that B is expressed. This protein, which accounted for ~4% of the total -crystallins, eluted in reversed-phase HPLC as a small peak between D- and C-crystallins with some overlap with both. Identification of the peptides from digestion of this protein with trypsin and Asp-N proteases confirmed the published sequence (den Dunnen et al. 1985).

View larger version (18K): [in this window] [in a new window]   Figure 5. (A) Reconstructed ESI mass spectrum of B-crystallin isolated from an 11-day-old lens. The mass at 20,776 Da corresponds to unmodified B-crystallin. Small peaks due to S, C, and D are also evident. (B) Mass spectrum of B-crystallin from the nucleus of a 19-year-old lens after derivatization with 4-vinylpyridine and isolation by reversed-phase HPLC. The mass at 21,511 Da corresponds to B-crystallin with all seven Cys residues derivatized. The mass at 21,554 Da is the carbamylated protein. The peaks 91 Da lower than the major peaks at 21,420 and 21,463 Da indicate the presence of minor S-methylation. The small peaks at 21,150 and 21,194 Da are D-crystallins.

In vitro methylation of human -crystallins
The possibility that the enzyme betaine-homocysteine methyltransferase (BHMT) could methylate -crystallins was tested using BHMT isolated from monkey lenses and recombinant human BHMT. For these experiments, total -crystallins from an 11-day-old human lens were the substrate, and both betaine and dimethylacetothetin were tested as donors of methyl groups (Garrow 1996). The initial level of methylation of S- and D-crystallins in the 11-day-old lens sample was ~5%. No increase in methylation was detected, even after 8 h of incubation.

Age-related changes in abundances of major species -crystallins
The inner nuclei and the remaining portions of lenses were analyzed separately to determine age-related changes in the modifications of C- and D-crystallins. For C-crystallin, carbamylation increased with age to 50%–64% in adult lenses (Table 3). In lenses of 11 years and older, N-terminal carbamylation was 8–10 times more abundant than acetylation, but in the 11-day-old lens, carbamylation and acetylation were both about 2%–3%. Methylation of C-crystallins increased to 12% in adult lenses. In all lenses, modifications were similar for C-crystallins from the inner nucleus and the remainder of the lens.

View this table: [in this window] [in a new window]   Table 3. Estimated abundance (%) of modified human C-crystallins from human lenses of different ages

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

 
Although -crystallins account for ~30% of the soluble protein of adult lenses, the post-translational modifications of these crystallins are not yet completely characterized. Previous publications have reported truncations, disulfide bonding, methionine oxidation, and deamidation of -crystallins (Abbasi et al. 1998; Hanson et al. 1998, 2000; Lapko et al. 2002a). In addition, phosphorylation and oxidation of tyrosines and methylation of lysines were reported in a 4-year-old cataractous lens (MacCoss et al. 2002). Recently, methylation of specific cysteine residues was identified as one of the most abundant post-translational modifications in clear human lenses (Lapko et al. 2002b). We now report that S-methylation is a major post-translational modification of all of the -crystallins. In addition, we found N-carbamylation of C-, D-, and B-crystallins. Identification of these post-translational modifications in -crystallins was facilitated by instrumental developments that have improved the resolution of protein mass spectra.

S-methylation of cysteine residues in crystallins is of particular interest because methylated cysteines are not capable of forming the disulfide bonds that lead to high molecular weight aggregates typical of cataract. Protein methylation, which has been implicated previously in numerous functions such as signal transduction, regulation of gene activity, and protein sorting (Armitage 1999; Davie and Dent 2002; Kouzarides 2002), typically occurs at side chains of lysine, arginine, glutamic, and aspartic acids, and at some N- and C-terminal residues (Park and Paik 1990). Beside our report of S-methylation of S-crystallin (Lapko et al. 2002b), the only other reports of in vivo methylation of cysteine are S-methylation of hemoglobin following exposure to methylating agents (Bailey et al. 1981; Ferranti et al. 1996), self-methylation of methyltransferases involved in methylation of cytosine in DNA (Szilak et al. 1994), and repair of DNA damage caused by alkylating agents (Olsson and Lindahl 1980). Only about 0.02% of human hemoglobin is S-methylated (Tornqvist et al. 1988), whereas about 50% of D-crystallin in older lenses is methylated.

Methylation in -crystallins is highly specific. D-, S-, C-, and B-Crystallins have six, seven, eight, and seven cysteine residues, respectively. Among these cysteines, only Cys 26 of S-crystallin and Cys 110 of D-crystallin are heavily methylated. Comparison of the sites of S-methylation and the amino acid sequences (Fig. 6) suggests certain patterns in the methylation of -crystallins. Two major sites of methylation, Cys 26 of S-crystallin and Cys 110 of D-crystallin have the sequence: Asp-Cys-X-Cys, in which X = Asp in S or Ser in D. Both sites are within acidic clusters. The cysteine residue corresponding to Cys 26 of S-crystallin is conserved in both C- and B-crystallins (Fig. 6), but the level of methylation at this Cys residue in C- and B-crystallins is much lower than in S-crystallin.

View larger version (37K): [in this window] [in a new window]   Figure 6. Amino acid sequences of homologous regions of human -crystallins containing sites of S-methylation indicated by an asterisk. The major methylation sites, Cys 26 of S- and Cys 110 of D-crystallin, are shown in bold.

Although direct nonenzymatic action of a methylating agent in the lens is possible, the high specificity of methylation and the presence of methylated -crystallins even in very young lenses suggest that the reaction is enzymatic. The presence of several methyltransferases and methionine adenosyltransferase, a S-adenosylmethionine synthesizing enzyme, in human lenses is consistent with enzymatic methylation in the lens (Geller et al. 1986; Rao et al. 1998; Cornish et al. 2002). One of the most abundant proteins in the nucleus of monkey lenses is betaine-homocysteine methyltransferase (BHMT; Rao et al. 1998). This enzyme, which is involved in the biosynthesis of methionine, is also expressed in human lenses (Rao et al. 1998). A novel isozyme, betaine-homocysteine methyl transferase 2 (BHMT2) was identified recently in several human tissues and its expression was found in fetal eyes (Chadwick et al. 2000). Both protein extracts from monkey lenses and recombinant human BHMT were tested for their ability to methylate human -crystallins in vitro. No detectable methylation was found, leaving unanswered the question of what enzyme(s) might be involved in the methylation of -crystallins. In vivo S-methylation is a very slow reaction developed over decades. Finding conditions that accelerate the reaction in vitro could facilitate detection of S-methylation.

Another abundant post-translational modification identified in this study is carbamylation of the N termini of B-, C-, and D-crystallins. Carbamylation is often associated with the reaction of proteins with isocyanate. Because urea decomposes to isocyanate, carbamylation can occur in urea solutions. The product of the reaction is a protein with -CONH₂ (+43 Da) attached to an amino group. The development of cataracts in patients with renal failure was speculated to be the result of increased concentrations of cyanate in the body leading to carbamylation of the lens crystallins (Harding and Crabbe 1984; van Heyningen and Harding 1988), but examination of -crystallins from uremic patients gave no evidence of carbamylation (Smith et al. 1995). Carbamylation is not necessarily due to the presence of urea or cyanate/isocyanate. Enzymatic carbamylation involving carbamylphosphate as a carbamylating agent is also known. N-carbamylation of aspartate is the first step in the biosynthesis of pyrimidine, and carbamylation of ornithine is involved in the biosynthesis of arginine. The term carbamylation has also been used to describe addition of CO₂ (Lorimer and Miziorko 1980; Golemi et al. 2001). The product of this reaction has a -COOH group (+44 Da) attached. An example of this type of carbamylation is the activation of ribulose-1,5-bisphosphate carboxilase/oxigenase (Rubisco) by covalent binding of carbon dioxide to a specific Lys residue in the active site of the enzyme (Lorimer and Miziorko 1980). Another example is the reversible binding of carbon dioxide to the N-terminal amino group of hemoglobin.

Carbamylation due to addition of -CONH₂, as observed in -crystallins, occurs most readily at the free N termini of proteins (Martin and Harding 1989) and then at -amino groups of lysines. Like the majority of proteins, (Brown 1970; Driessen et al. 1985; Kendall et al. 1990), -, ß-, and S-crystallins are N-terminally acetylated during translation, but C-, D-, and B-crystallins have free N termini, which are susceptible to carbamylation. The reasons for differences in the status of the N termini of -crystallins are not clear. The human -crystallins are highly homologous. Even though they have similar molecular masses, S-crystallin elutes slightly earlier than the other -crystallins on size exclusion chromatography, suggesting that the conformation of S-crystallin is less compact. S-crystallin has a four-residue insert, TGTK (residues 2–6) not present in the other -crystallins. It is possible that the presence of this insert results in conformational differences and affects the status of the N-terminal residue.

Carbamylation of C- and D-crystallins, at 45%–60% in adult human lenses is among the more abundant modifications. To our knowledge, the only previously reported in vivo N-terminal carbamylation is in a bacterial high-potential iron-sulfur protein (Van Driessche et al. 2002). The level of carbamylation of this bacterial protein, ~50%, is comparable with the carbamylation of C- and D-crystallins. The importance of carbamylation of lens crystallins, its affect on protein conformation and aggregation, and potential to induce cataract have been discussed extensively (Beswick and Harding 1984, 1987; Harding and Crabbe 1984; Crompton et al. 1985; Qin et al. 1992; Smith et al. 1995; Derham and Harding 1999). However, in these studies, the carbamylation was at the -amino groups of lysines, which likely affects protein structure very differently from N-terminal carbamylation. The presence of N-carbamylation in -crystallins from young lenses suggests that this modification is not detrimental to protein solubility and function.

N-acetylation of B-, C-, and D-crystallins was also detected. In adult lenses, N-acetylation is ~10%–12% of the level of N-terminal carbamylation, but in the 11-day-old lens, acetylation and carbamylation of both C and D were comparable (2%–3%). Whether this N-acetylation is due to co- or post-translational acetylation is not known. Usually, N-terminal acetylation of proteins is a cotranslational event, but there are examples of N-terminal acetylation occurring after translation (Dores et al. 1990). N-terminal acetylation is performed by N-terminal acetyltransferases distinct from transferases carrying out internal post-translational acetylation of proteins (Polevoda et al. 1999). Internal post-translational acetylation may have a role in signaling and other cellular functions, similar to that of phosphorylation (Magnaghi-Jaulin et al. 2000; Sterner and Berger 2000; Kouzarides 2002). Despite its wide occurrence, the biological role of N-terminal acetylation remains obscure (Smyth and Zakarian 1980; Tsunasawa and Sakiyama 1984; Tercero et al. 1993).

In addition to S-methylation, N-carbamylation, and N-acetylation of D-crystallin, we observed the previously reported truncation of the C-terminal Ser residue (Abbasi et al. 1998). This truncation is analogous to truncation of the C-terminal Ser of A-crystallin (Miesbauer et al. 1994; Takemoto and Gopalarishnan 1994). It has been suggested that truncation of the C terminus of A-crystallin may contribute to cataractogenesis by affecting the aggregation state and chaperone activity of the protein (Kelley et al. 1993; Takemoto 1999; Thampi et al. 2002). The high level of truncated D-crystallins in clear lenses, especially in the nucleus, argues against a similar role for this modification in D.

In contrast to methylation of S-crystallin, which is higher in the nucleus than cortex (Lapko et al. 2002b), methylation and carbamylation of C-crystallins are similar throughout the lens. Although the spatial distribution of modifications may be due to differential localization of enzymes (Rao et al. 1998; Chadwick et al. 2000) or other reactive species, the main factor determining the extent of modifications is probably the age of the protein. Our data on the abundance of -crystallins in human lenses (Table 4) are in agreement with previous data that showed no C- nor B-crystallin transcripts in 10-year-old lenses (Brakenhoff et al. 1990), but increasing expression of S-crystallin as the lens ages (Ma et al. 1998). Because the lens grows by adding layers of cells at the periphery, accumulating the oldest proteins in the nucleus and the more recently synthesized in the cortex, cortical S-crystallins will not have had as much opportunity to become methylated as those in the inner nucleus. In contrast, as there is little biosynthesis of C-crystallins after birth, all C-crystallins are of approximately the same age and have similar methylation, regardless of their location in the lens. The abundances of -crystallins (Table 4) indicate some redistribution of C- and other -crystallins between the inner nucleus and the remainder of the lens. These data suggest a dynamic organization of the lens allowing diffusion or exchange of proteins from the nucleus.

View this table: [in this window] [in a new window]   Table 4. Abundance (mg) of major soluble -crystallins in human lenses of different ages

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

Soluble proteins obtained from the inner nuclei and the remaining portions were each fractionated by size exclusion chromatography (Superose 12 HR 10/30 column, Pharmacia Biotech) equilibrated with the homogenization buffer at flow rate of 0.4 mL/min. The total -crystallins were collected, and individual -crystallins isolated by reversed-phase HPLC (Vydac 4.6 x 150 mm C4 column) equipped with a protein Macrotrap (Michrom BioResources) using a gradient of 20%–55% acetonitrile with 0.1% trifluoroacetic acid (TFA) over 35 min (Hanson et al. 1998). The proteins were concentrated to dryness, redissolved in 50% acetonitrile containing 0.3% formic acid, and analyzed by mass spectrometry.

Determination of abundances of -crystallins in human lenses
Protein concentrations in lens extracts and in the -crystallin fractions obtained after gel-filtration chromatography were determined using Bradford Reagent (Sigma Chemical Co.). The quantity of individual -crystallins (S-, C-, D-, and B) was estimated from their UV-absorbances using extinction coefficients calculated from their amino acid sequences (Pace et al. 1995). When mass spectra indicated that -crystallin fractions were not pure, the crystallins were derivatized with 4-vinylpyridine and separated by reversed-phase HPLC, in which the derivatized crystallins eluted as separate peaks. Each crystallin was quantified by its UV-absorbance.

In vitro experiments on methylation of -crystallins
The ability of recombinant BHMT and monkey lens crystallins to methylate lens proteins was examined using the total -crystallin fraction from a 11-day-old human lens isolated by size-exclusion chromatography. Recombinant human BHMT, with a specific activity ~2000 units/mg (Garrow 1996), was a gift from Dr. T. Garrow. Monkey BHMT was prepared from Rhesus monkey lenses as described by Rao et al. (1998). The reaction mixture included human -crystallins (0.8 mg/mL), 5 mM betaine or dimethylacetothetin as the donor of methyl groups, 0.5 or 5 mM DTT, 20 µL BHMT (1 mg/mL), or 20 µL of the ß_H fraction of monkey lenses (1 mg/mL), 25 mM NaCl and 50 mM Tris-HCl (pH 7.8). The final reaction volume was 110 µL. After incubation for 8 h at 30°C, the -crystallins were isolated by reversed-phase HPLC, and the level of methylation was determined by mass spectrometry.

Derivatization of sulfhydryl groups of -crystallins
-Crystallins (40–200 pmole) were dissolved in 300 µL of a buffer (250 mM Tris-HCl, 6 M guanidine hydrochloride, 1 mM EDTA, 5 mM dithiothreitol at pH 8.5). After incubation for 1 h, the sulfhydryl groups were derivatized by reaction with 15 mM iodoacetamide or iodoacetic acid for 30 min or with 25 mM 4-vinylpyridine for 90 min (Friedman 2001). The reactions were quenched by adding excess dithiothreitol. The derivatized proteins were desalted by reversed-phase HPLC, and analyzed by ESIMS.

Enzymatic digestions of -crystallins
-Crystallins derivatized with iodoacetamide or 4-vinypyridine were digested with trypsin or chymotrypsin in 100 mM ammonium bicarbonate at 37°C for 8 h or with Asp-N protease for 24 h at an enzyme:protein ratio of 1:50. Asp-N digestion at an enzyme:protein ratio 1:100 for 18 h was used to generate large C-terminal fragments of D-crystallins. Digests were freeze-dried and dissolved in 0.1% TFA for on-line mass spectrometric analysis or in 50% acetonitrile, 0.1% TFA for MALDI MS.

Analysis of -crystallins by ESIMS
Intact -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%. Peptides produced by enzymatic digestions were analyzed by on-line capillary reversed-phase HPLC-ESIMS using an ion trap mass spectrometer (Finnigan MAT LCQ). 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 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 specified mass/charge with a window of ±5 m/z was used for detection of carbamylation and acetylation in -crystallins and for analysis of methylation in D-crystallins. Resolution of mass spectra of proteins was enhanced using maximum entropy software (Micromass).

Analysis of peptides by MALDI
Enzymatic digests were also analyzed by MALDI MS (Voyager-DE Pro mass spectrometer, Applied Biosystems). Typically, peptides were detected in the reflectron mode of operation. Large C-terminal fragments of D-crystallins were analyzed in the linear mode. In all experiments, -cyano-4-hydroxycinnamic acid was the matrix.

Quantification of major modifications of -crystallins
Mass spectral peak intensities of the C-crystallins derivatized with 4-vinylpyridine were used to calculate the abundance of each modification. In this calculation, the intensity due to each particular modification was divided by the sum of the intensities of the peaks due to all C-crystallin forms. The calculated values for carbamylation include an ~10% contribution from acetylation due to overlap of the peaks for acetylated and carbamylated C.

Analyses of protein digests, as well as undigested proteins, were used to estimate the level of D-crystallin modification. For D-crystallins from the 50 and 72-year-old lenses, methylation and truncation were estimated from MALDI analysis of the appropriate peptides. Peptide 99–114 in a tryptic digest of 4-vinylpyridine derivatized D was used to estimate methylation, and Asp-N peptides 113–172 and 113–173 were used to estimate truncation. N-terminal carbamylation was estimated from peaks in the zoom mode of ESIMS for peptides 1–7, unmodified at m/z 694.5 and carbamylated at m/z 737.5.

The relative abundance of the modified forms of D-crystallin from lenses, 11 days old to 19 years old, were estimated from the size of the mass spectral peaks for each protein. These major forms accounted for ~85% of the total D-crystallins. The masses of each protein are given in Table 1. The values for the methylated species include both the methylated form (+14 Da) and the oxidized (+16 Da) forms. Corrections calculated from mass spectra of the 4-vinylpyridine derivatives, in which the methylated and oxidized forms were separated, indicated that these forms contributed <3%. Once the abundance of each D-crystallin form had been estimated, the total methylation was calculated as the sum of the four species that included this modification (methylated D, methylated and carbamylated D, methylated and truncated D, and methylated, truncated, and carbamylated D). The total level of carbamylation and truncation were calculated similarly.

Estimation of the ratio of N-terminal acetylation to N-terminal carbamylation
The relative abundance of N-terminal carbamylation and acetylation in C- crystallin was estimated from the ratio of the peaks for peptide 1–6 with carbamylation and acetylation at m/z 771.3 and m/z 770.3, respectively, using the zoom mode of the ion trap mass spectrometer at m/z 770.8 ± 5. Similarly, the ratio of N-carbamylated and N-acetylated D-crystallins was estimated from the peaks for carbamylated and acetylated peptide 1–6 at m/z 736.8 ± 5.

Accessibility calculations
The surface accessibilities of the cysteine residues in -crystallins were calculated with the program GETAREA 1.1 (http://www.scsb.utmb.edu/cgi-gin/get_a_form.tcl) developed at The Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX as described previously (Lapko et al. 2002a). The sequence of each -crystallin was superimposed on the X-ray structure of bovine D-crystallin for these calculations (Chirgadze et al. 1996).

	Acknowledgments

 
We thank Dr. T. Garrow (University of Illinois) for providing dimethylacetothetin and recombinant BHMT and Dr. S. Zigler (NIH) for samples of monkey lenses. We thank Dr. D. Berkowitz (University of Nebraska) for helpful discussions. This research was supported by a grant (EY RO1 07609) from the NIH and the Nebraska Center for Mass Spectrometry.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

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