Abstract
Purpose :
We have recently reported that deamidations associated with cataracts increase aggregation. The purpose of this study was to quantify the disulfides in wild type and deamidated gammaS-crystallin upon oxidation and correlate levels to aggregation.
Methods :
Wild type gammaS and deamidated gammaS (N14D, N76D, N143D) crystallins were reduced and then rapidly exchanged into phosphate buffer containing amounts of GSH:GSSH ranging from 5 mM:0 mM to 0 mM:2.5 mM. This approximated the loss of both reduced and total glutathione in normal vs cataractous lenses and mimicked the oxidative environment found during cataract formation. Proteins were then incubated for 2-24 hours at 37 or 60 degrees C before whole mass measurement using an Orbitrap Fusion mass spectrometer (Thermo Scientific). The overall numbers of disulfide bonds and glutathione adducts were determined and correlated to changes in aggregation. Aggregation was measured by dynamic light scattering using a DynaPro NanoStar Reader (Wyatt Tech. Co.).
Results :
We identified an increase in non-native disulfide bonds in oxidized, deamidated gammaS-crystallin. The intermolecular C24-C24 crosslink was increased in deamidated gammaS during oxidation in vitro, as was the C144-C114 crosslink. After 2 h incubation at 1:1 ratio of GSH:GSSH, the deamidated gammaS had increased radii of the main monomer peak and increased aggregates at radii > 10 nm compared to wild type. The monomer peak was 3.2 nm compared to 2.5 nm of the wild type and the higher radii species contributed 3% of the mass compared to none detected in the wild type. The overall shape of the light scattering autocorrelation curves suggested multimodal distribution for both proteins, but the curves were distinctly different with the curve for the deamidated gammaS not reaching baseline as fast suggesting larger species.
Conclusions :
Our findings support that deamidation predisposes crystallins to further modifications that then lead to higher-ordered oligomers and eventually large light scattering aggregates associated with cataracts.
This is a 2021 ARVO Annual Meeting abstract.