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Vincent M Monnier, Benlian Wang, Grant Hom, Xingjun Fan; Identification of Crystallin Cysteine Oxidation Sites in Vitro, in LEGSKO Mouse and Old Human Lens. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5726.
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© ARVO (1962-2015); The Authors (2016-present)
Low glutathione levels in the lens nucleus have been associated with increased crystallin oxidation and the formation of age-related nuclear cataract (ARNC). To better understand this paradigm, we have created a conditional GSH synthesis knockout (LEGSKO) mouse that displays several features of ARNC, including protein oxidation, aggregation and opacification. We now investigated the extent to which these changes can be mimicked through in vitro oxidation and how these apply to the aging mouse and human lens.
Dialyzed mouse lens protein homogenate (1-14mg/ml) in K+/PO4 buffer was incubated with 1 and 5 mM H2O2 for several hours until opacification was observed. These solutions and those from freshly prepared LEGSKO mouse and human lenses (19 and 70 yr old) were processed for labeling of free and disulfide -linked sulfhydryls with the ICAT labeling method, followed by proteomic analysis by LC/MS/MS of labeled tryptic peptides.
Analysis of LEGSKO crystallins by 2D-PAGE revealed an age-related shift from mostly intramolecular to intermolecular disulfide crosslinks which mimicked the pattern observed at 20 months in wild type mouse lenses. 29 disulfide formation sites were identified in H2O2 treated lens of which 25 were found in LEGSKO lens. These included CRYAB: C154, Cryba1/3 : C52,117,142,185, Cryba2 C33,119, Cryba4 C99; Crybb1 C78,150,178,240; Crybb2 38,67; Crybb3 C207; Cryga 42,Crygb C42,110, Crygc C42,131, Crygf C19,42,79,119, Crygs C115,130. Some sites were oxidized only in LEGSKO lens, such as Crygs C130. In old normal human lens, only 14 oxidation sites were found, of which 9 were correctly predicted in Cryba1/3. Cryba4, Crybb2. Most of the gamma crystallin oxidation sites were not predicted, except for Crygd C19 and 42, and Crygs 115.
In vitro oxidation closely predicts oxidation sites in LEGSKO mouse lens. More data are needed in human lens to clearly separate effects of aging vs. cataractogenesis and potential artefactual oxidation.
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