Abstract
Purpose:
Glutathione (GSH) is the most abundant anti-oxidative compound found in the lens. In age-related nuclear cataract (ARNC) and the LEGSKO mouse lens GSH levels are impaired and accompanied with increased crystallin oxidation, protein disulfide formation and protein cross-linking. To understand the role of disulfides for protein stability in ARNC, we have determined the human cataractous lens disulfidome and compared the results with that of the LEGSKO mouse and in vitro oxidative stressed lens protein homogenate.
Methods:
Protein homogenates from young (n =3), aged normal (n =3) and level II to V (based on LOCS III) human cataract lens nucleus (n= 3/each group), as well as from LEGSKO mouse lens (n=3) and WT mouse treated with H2O2 were processed for identification of cysteine residues involved in disulfide formation using isotope-coded affinity-tag (ICAT) labelling and peptide mapping with LTQ Orbitrap mass spectrometry, and results were compared.
Results:
Three types of cysteine residues were discovered. 1) Cysteine residues that are not prone to oxidation with age and cataract, such as cys67 in βB2, cys19 in γD, cys109 in γC and cys99 in βA4; 2) cysteine residues that are relative susceptible to oxidation with age and more significantly increased in cataract, such as cys33 in βA4, cys42 in ΥD, cys52 and cys170 in βA1/3, cys80 in βB1, cys38 in βB2 and cys83 in γS; 3) cysteine residues that are barely oxidized with age but significantly oxidized in cataract, such as Cys154 in γC, cys117 in βA1/3, cys115, cys130 in γS, cys42 and cys78/cys79 in γC. Conserved cysteine residues that were also highly oxidized in vitro and in LEGSKO mouse were cys52, cys117, cys185 in βA1/3, cys33 in βA4, cys78 in βB1, cys19 in γD and cys83, cys130 in γS. These results indicate a high homology between human, mouse and in vitro oxidation sites, validating thereby the LEGSKO mouse as a model for ARNC.
Conclusions:
These studies form the ground work for the engineering and testing of oxidation and hopefully cataract resistant recombinant crystallins using the LEGSKO mouse for knock-in strategies.