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Kevin L Schey, Zhen Wang, Frank Joseph Giblin; Quantification of irreversible thioether GSH modification in human and guinea pig lens proteins. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5597.
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© ARVO (1962-2015); The Authors (2016-present)
Glutathione can react with dehydroamino acids, such as dehydroalanine (DHA) and dehydrobutyrine (DHB), resulting in irreversible glutathionylation through a thioether bond. The purpose of this study was to measure the abundance of GSH modifications in different regions and different fractions of human lenses to better understand the location and solubility effects of this irreversible modification. Further, the level of irreversible glutathionylation was also measured in a hyperbaric oxygen guinea pig model of nuclear cataract to quantitate its levels in a well-characterized cataract model.
Cortex, outer nucleus and inner nucleus regions from human lenses were isolated. Lens proteins in water-soluble fraction (WSF), urea-soluble fraction (USF) and urea-insoluble fraction (UIF) were digested by trypsin and analyzed by mass spectrometry. The level of GSH modification at six modification sites on six different proteins was quantified relative to the respective nonmodified peptides. The levels of glutathionylation were compared between different lens regions, between different lens ages, as well as between normal and cataract lenses. The abundance of irreversible glutathionylation was also measured in hyperbaric oxygen treated guinea pigs.
The results show variability in abundances of GSH modifications for different lens proteins. Cys5 of βA4-crystallin undergoes extensive modification (>50% of total βA4 protein) whereas βB1-crystallin is modified to less than 1%. Generally the level of modification increases from cortex to outer nucleus regions. For proteins that undergo degradation with age, GSH abundance tends to decrease in the inner nucleus suggesting further modification. The results also suggest modification on some proteins induces protein solubility changes such as modification on S59 of αA- and αB-crystallin. Comparing the level of GSH modification on Cys5 of βA4-crystallin in HBO treated and control guinea pig lenses suggests that oxidative stress increases irreversible GSH thioether formation on cysteine residues.
Irreversible glutathionylation occurs on many lens proteins and modification increases with increasing age. GSH alters protein solubility in a protein dependent manner. The level of modification in cataract lenses is not significantly increased.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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