Shotgun proteomic analysis of S-thiolation sites of guinea pig lens nuclear crystallins following oxidative stress in vivo

Abstract

Purpose: To compare levels of S-glutathiolation and S-cysteinylation occurring at more than 60 cysteine residues of 12 different guinea pig lens water-soluble nuclear crystallins following treatment of the animals with hyperbaric oxygen (HBO).

Methods: Guinea pigs (initially 18 months old) were treated 30X (3X per week for 10 weeks) with HBO (2.5 atm 100% O(2) for 2.5 h) as a model to study the formation of nuclear cataract. This treatment produces a moderate increase in lens nuclear light scatter (compared to denser scatter occurring after 80 HBO treatments), with five- to sixfold increases in levels of protein-bound glutathione (PSSG) and protein-bound cysteine (PSSC). Trypsin digests of lens nuclear water-soluble proteins were analyzed with two-dimensional liquid chromatography and mass spectrometry to identify specific cysteine residues binding either glutathione or cysteine. Lens nuclei of age-matched untreated animals were used as controls.

Results: All major crystallins, except αB, were modified to some extent by either S-glutathiolation or S-cysteinylation. Overall, 72% of the cysteine residues of guinea pig lens nuclear crystallins were shown to be capable of binding glutathione, cysteine, or both molecules. The crystallin with the highest level of modification was βA1/A3 (six of eight -SH groups), and that with the lowest (two of five -SH groups) was βA2. O(2)-induced increases in PSSG levels were 2.8, 2.4, and 4.1 times control for γA-, γB-, and γC-crystallins, respectively. Comparable increases in PSSC levels for the three γ-crystallins were 2.3, 2.7, and 2.4 times control, respectively. βB2-crystallin showed the highest amount of O(2)-induced PSSG formation of any of the crystallins, as well as a substantial level of control PSSG, and nearly all of this was due to a single residue, C67, a site also present in human βB2-crystallin. Overall, 32 of the 44 modified cysteine residues were homologous with the human.

Conclusions: This large-scale study successfully identified lens crystallin cysteine residues that bound glutathione and/or cysteine under normal or oxidative stress conditions. The high percentage of protein -SH groups that are modified by S-thiolation in the guinea pig lens nucleus demonstrates the substantial protein sulfhydryl redox buffer capability present in the center of the lens. The results suggest that PSSG and PSSC formation may act to delay O(2)-induced insolubilization of γA-, γB-, and γC-crystallins, and β-crystallins, but with a greater effect on the γ-crystallins at an early stage of oxidative stress. The study has shown that technological approaches are now available to investigate in considerable detail the role of specific lens -SH groups in nuclear cataractogenesis.

Figure 1

The numbers of tandem mass (MS/MS) spectra are shown for peptides of each guinea pig lens water-soluble nuclear crystallin. A shows total counts for peptides containing an -SH group; B shows peptides containing protein-bound glutathione (PSSG) expressed as per -SH peptide; and C shows peptides containing protein-bound cysteine (PSSC) expressed as per -SH peptide. Open bars are counts from age-matched controls and solid black bars are counts after 30 treatments of the animals with hyperbaric oxygen. There is a different vertical scale for A, compared to those for B and C, which are identical. Results for γN-crystallin are not shown because of a low number of detected total peptides (<50 counts). The counts in A correspond to a soluble protein sample of 0.4 mg. Seven crystallins (βA1/A3, βA4, βB1, βB2, βB3, γC, and γS) showed an O₂-induced decrease in counts for peptides containing a cysteine residue, while 5 crystallins (αA, βA2, γA, γB and ζ) showed an increase (A). All the crystallins except αA and βA4 showed an O₂-induced increase in PSSG level (B). βB2-crystallin exhibited the highest levels of control as well as O₂-induced PSSG. In C, all the crystallins except αA and βB3 showed an O₂-induced increase in PSSC level. γC-crystallin exhibited the highest levels of control as well as O₂-induced PSSC.

Figure 2

The numbers of tandem mass (MS/MS) spectra are shown for the 8 -SH group-containing peptides of water-soluble nuclear βA1/A3-crystallin. A shows total counts for each of the 8 peptides containing an -SH group; B shows peptides containing protein-bound glutathione (PSSG) expressed as per -SH peptide; and C shows peptides containing protein-bound cysteine (PSSC) expressed as per -SH peptide. Open bars are counts from age-matched controls and solid black bars are counts after 30 treatments of the animals with hyperbaric oxygen. There is a different vertical scale for A, compared to those for B and C, which are identical. The counts in A correspond to a soluble protein sample of 0.4 mg. Peptides for each of the 8 cysteine residues in panel A showed an O₂-induced decrease in number of counts. A relatively low number of counts were detected for the C117 control and O₂-treated peptides. The majority of O₂-induced PSSG (B), and to a lesser extent PSSC (C), was shown by residues C142 and C165.

Figure 3

The numbers of tandem mass (MS/MS) spectra are shown for the 7 -SH group-containing peptides of water-soluble nuclear γB -crystallin. A shows total counts for each of the 7 peptides containing an −SH group; B shows peptides containing protein-bound glutathione (PSSG) expressed as per −SH peptide; and C shows peptides containing protein-bound cysteine (PSSC) expressed as per −SH peptide. Open bars are counts from age-matched controls and solid black bars are counts after 30 treatments of the animals with hyperbaric oxygen. There is a different vertical scale for A, compared to those for B and C, which are identical. The counts in panel A correspond to a soluble protein sample of 0.4 mg. Residue C42 showed a relatively high number of peptide counts for control and O₂-treated samples due to the peptides containing C42 being identical in sequence for γA-, γB- and γC-crystallins (A). No detectable counts were obtained for residues C33 and C79 for either the control or O₂-treated samples. Residues C16 and C23 accounted for the majority of O₂-induced PSSG (B) and PSSC (C).

Figure 4

The numbers of tandem mass (MS/MS) spectra are shown for the 5 -SH group-containing peptides of guinea pig lens water-soluble nuclear βB1-crystallin. A shows total counts for each of the 5 peptides containing an -SH group; B shows peptides containing protein-bound glutathione (PSSG) expressed as per -SH peptide; and C shows peptides containing protein-bound cysteine (PSSC) expressed as per -SH peptide. Open bars are counts from age-matched controls and solid black bars are counts after 30 treatments of the animals with hyperbaric oxygen. There is a different vertical scale for A, compared to those for B and C, which are identical. The counts in A correspond to a soluble protein sample of 0.4 mg. Peptides for each of the 5 cysteine residues showed an O₂-induced decrease in number of counts (A). The majority of O₂-induced PSSG (B) and PSSC (C) were shown by residues C148 and C176.

Figure 5

The numbers of tandem mass (MS/MS) spectra are shown for the 2 -SH group-containing peptides of water-soluble nuclear βB2-crystallin. A shows total counts for each of the 2 peptides containing an −SH group; B shows peptides containing protein-bound glutathione (PSSG) expressed as per −SH peptide; and C shows peptides containing protein-bound cysteine (PSSC) expressed as per −SH peptide. Open bars are counts from age-matched controls and solid black bars are counts after 30 treatments of the animals with hyperbaric oxygen. There is a different vertical scale for A, compared to those for B and C, which are identical. The counts in panel A correspond to a soluble protein sample of 0.4 mg. A shows that peptides for each of the 2 cysteine residues showed a slight O₂-induced decrease in number of counts. The majority of control and O₂-induced PSSG was shown by residue C67 (B). Note that this single residue of βB2 produced the highest levels of control and O₂-induced PSSG, compared to the other 11 crystallins (see Figure 1B). Residue C67 produced relatively little control or O₂-induced PSSC (C), compared to the amounts observed for PSSG (B).