Methylation and carbamylation of human gamma-crystallins

Abstract

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 gammaS-crystallins, S-methylation of cysteine residues, can prevent disulfide formation. The aim of this study was to determine whether cysteines in gammaC-, gammaD-, and gammaB-crystallins are also S-methylated. Our data show that all the gamma-crystallins are S-methylated, but only at specific cysteines. In gammaD-crystallin, methylation is exclusively at Cys 110, whereas in gammaC- and gammaB-crystallins, the principal methylation site is Cys 22 with minor methylation at Cys 79. gammaD-crystallin is the most heavily methylated gamma-crystallin. gammaD-Crystallins from adult lenses are 37%-70% methylated, whereas gammaC and gammaB are approximately 12% methylated. The specificity of gamma-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 gammaB-, gammaC-, gammaD-crystallins. N-terminal carbamylation is likely a developmentally related modification that does not negatively impact crystallin function.

Figure 1.

Reconstructed ESI mass spectra of γC-crystallin isolated from the nucleus of a 19-year-old lens. (A) Underivatized. The mass at 20,747 Da corresponds to unmodified γC-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.

Figure 2.

MS/MS spectra of the N-terminal chymotryptic peptide 1–6 of γC-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.

Figure 3.

Reconstructed ESI mass spectra of γD-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.

Figure 4.

MS/MS spectra of peptides corresponding to 107–112 from an Asp-N digest of iodoacetamide-derivatized γD-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.

Figure 5.

(A) Reconstructed ESI mass spectrum of γB-crystallin isolated from an 11-day-old lens. The mass at 20,776 Da corresponds to unmodified γB-crystallin. Small peaks due to γS, γC, and γD are also evident. (B) Mass spectrum of γB-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 γB-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 γD-crystallins.

Figure 6.

Amino acid sequences of homologous regions of human γ-crystallins containing sites of S-methylation indicated by an asterisk. The major methylation sites, Cys 26 of γS- and Cys 110 of γD-crystallin, are shown in bold.