Abstract
Purpose: :
Previous studies of guinea pigs treated with hyperbaric O2 (HBO) showed elevated levels of protein S-thiolation in the lens nucleus. Here we identified sites of S-glutathiolation and/or S-cysteinylation in lens nuclear crystallins of guinea pigs treated with HBO.
Methods: :
Guinea pigs, initially 18 months old were treated 3x per week for 10 weeks with HBO. Lens nuclei were homogenized in a N2 atmosphere in buffer containing EDTA and IAA to prevent artifactual disulfide formation. Trypsin digests of lens nuclear water soluble (WS) proteins were analyzed by Multidimensional Protein Identification Technology (MudPIT). Sequences of lens crystallins were derived through NEIBank by creation and sequence analysis of a guinea pig lens cDNA library. Analysis of MS/MS spectra was performed using Sequest software, with further processing conducted with in house software and the program Scaffold.
Results: :
MS/MS spectra indicated that all detected guinea pig WS crystallins participated in S-thiolation except B, which contains no -SH. O2-treatment produced significant S-thiolation of γA,B,C and binding of glutathione by βA3 and βB1 (P<0.05), but no significant O2-induced increase in S-thiolation was observed for A, B, βA2, βA4, βB2, βB3, γS and ζ-crystallins, compared to controls. O2-induced S-thiolation of γB at C16 and C23 was significantly more than controls, with C16>C23. For βA3, C142 was more susceptible to glutathiolation than C165. To explain the reactivity of certain -SH groups, 3D models of γB and βA3 were generated by homology modeling using 3D-JIGSAW software based on mammalian lens crystallin X-ray structures. Guinea pig γB and βA3 had sequence identities >90% compared to other lens crystallins. 3D representations of γB and βA3 were space filled using DS ViewerPro software. γB showed C16 was more exposed and susceptible to S-thiolation compared to C23. Similarly, βA3 revealed C142 was exposed and close to positively charged lysine residues, which presumably attracted negatively charged glutathione disulfide.
Keywords: crystallins • oxidation/oxidative or free radical damage • cataract