Abstract
Purpose :
Oxidation of susceptible eye lens crystallin proteins is a key process in lens opacification and the development of cataract, which increases with age as glutathione levels in lens fiber cells are depleted, especially in the nuclear region. Here, the cysteine-rich and abundant γ-crystallins may provide the last line of defense against oxidative protein misfolding and aggregation by serving as redox sinks. Previous work from our laboratory demonstrated in vitro the transfer of a disulfide bond from one γ-crystallin protein to another, i.e., from wildtype γD-crystallin (γD) to its destabilized tryptophan-oxidation mimicking variant, W42Q, a redox reaction that initiates an oxidative aggregation cascade. This study aims to characterize the disulfide transfer pathways between γ-crystallins that may contribute, either favorably or unfavorably, to the progression of age-related nuclear cataract.
Methods :
Wildtype human γS-crystallin (γS WT) and six cysteine knockouts thereof, along with γD W42Q, were produced and purified. Disulfide-linked dimer-forming propensity was assessed via size-exclusion chromatography and SDS-PAGE. Disulfide transfer was assessed via a turbidity assay monitoring protein aggregation of the redox-sensitive variant, γD W42Q.
Results :
Disulfide-linked dimers were formed by γS WT and all cysteine knockouts except C24S, verifying previous findings that C24 mediates the formation of an intermolecular disulfide. Oxidized γS WT triggered the aggregation of γD W42Q to a degree comparable to oxidized glutathione (Fig. 1). Dimerization of γS via C24 reduced the magnitude of this effect but did not abolish it. Moreover, both fully reduced γS WT and the C24S mutant were completely inert. The prerequisite of C24 for disulfide transfer suggests that it occurs between one or more conformationally strained, intramolecular disulfides in the CXCXC motif of γS.
Conclusions :
The major eye lens protein, γS, participates in disulfide exchange in the aging, glutathione-depleted lens to modulate oxidative protein aggregation and the progression of age-related nuclear cataract.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.