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Xingjun Fan, Benlian Wang, Vincent M. Monnier; Age-related Conversion Of Intra- To Intermolecular Disulfide Crystallin Crosslinking In The Human Lens Is Mimicked By Glutathione Depletion In The Legsko Mouse Lens. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1053.
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© ARVO (1962-2015); The Authors (2016-present)
Glutathione (GSH) is the most abundant anti-oxidative compound of the lens. In age-related nuclear cataract (ARNC) GSH levels are impaired and accompanied by increased protein oxidation, protein disulfide formation and cross-linking. Here we have determined the pattern of disulfide crosslinking in young and old human lens and compared the results with those from the LEGSKO mouse in which lenticular GSH levels were conditionally knocked down by targeting of gamma glutamate cystine ligase (Gclc).
: Lens protein extracts were analyzed by 2D SDS-gel electrophoresis for intra- and inter-molecular disulfide formation, and proteomic analysis was carried out by tandem mass sequencing to identify individual proteins. The crystallin type and its disulfide crosslink were also confirmed by Western blot.
14 of 19 of cysteine-baring lens proteins were present as intramolecular crosslinks in 4 yr old human lens. At 67 yrs, however, 12 of 14 proteins were present as intermolecular crosslinks indicating a shift toward oxidation with age. In the C57BL/6 control (WT) mouse lens, 12 of 17 proteins were present as intramolecular crosslinks. , and 19 of 21 were present as inter-molecular crosslinks at 20 months, confirming a similar age-related trend in the mouse and human lens. However, GSH depletion at 5 months resulted in quasi total conversion of intra- into intermolecular crosslinks in the LEGSKO lens. The major types of crystallins detected via proteomics were gamma and beta crystallins. In the aging human lens, predominant crosslinks involved mostly beta-beta crosslinks and mixes of alpha A γA, γC,γD and beta S crosslinks. In the LEGSKO cataractous mouse, most crosslinks involved γ-γ, β-β and some α-γC or γD/E crosslinks. Western-blot using gamma D crystallin antibody identified multiple spots of gamma D crystallins involved in intra-disulfide in young LEGSKO (1mos old) or wild type mouse lens, and in inter-disulfide in older LEGSKO mice ( over 5mos ). The gamma D crystallins formed inter-disulfide crosslink in the size of 30 to 70KD molecular weight, indicating that at least two or three proteins are involved in the crosslink. These results indicate that intermolecular crystallin disulfides may play a critical role in ARNC via oxidation, and that glutathione is one of major defenses in this process.
Homozygous LEGSKO mouse, like old human lenses, have a 50% drop in lenticular GSH levels and develop age-related nuclear cataract that is characterized by protein-protein disulfides. This novel animal model is expected to be very fruitful for the development of pharmacological agents against ANC.
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