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Xingjun Fan; A Putative glutathione transporter identified in mouse lens with glutathione de novo synthesis deficiency (LEGSKO) is also elevated in aged human lens. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5737.
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Glutathione (GSH) is the most abundant anti-oxidative compound found in the lens. It fulfils numerous cellular functions, including protection against oxidative stress, control of cellular redox potential and detoxification of xenobiotics. In aging, human lens GSH levels are impaired and accompanied with increased oxidation, protein disulfide formation and protein cross-link. Lens GSH concentrations are believed tightly regulated by two main mechanisms: by adjusting the rates of synthesis and transporter systems. This is confirmed by our lens CKO mouse study (Fan et al, PLOS ONE 2012(7):e50832). However, the molecular identity of GSH transporters has remained elusive; LEGSKO mouse provided a unique model for GSH transporter identification and characterization.
The RNA from wild type and homozygous LEGSKO mouse lenses (n=3) was extracted, and 84 mouse drug transporters were screened using RT2-PCR array (Qiagen). Only genes with more than 2 fold elevations were further validated (n=10). The candidate protein expression was determined by western-blot in LEGSKO and WT lenses. The candidate protein expression was also determined in collected human lenses in various ages by immuneprecipitation and western-blot.
: Initial screening from 84 drug transporters genes, seven putative GSH transporters were identified with at least 2 fold increase of mRNA level. Among seven candidates, solute transporter gene Slco4a1 mRNA was more than seven fold elevated in LEGSKO vs. wt lenses. Similarly, over five folds increase in protein expression in lens cortex was founded in LEGSKO lens compared to wt. Preliminary data based on IP from 20-70yrs old human lenses (n=5), SLCO4A1 protein was significantly elevated in lenses older than 60yr compared to 20-30yr lenses.
Homozygous LEGSKO mouse, after complete shutdown of GSH de novo synthesis has unexpectedly become a unique platform for studies of GSH transporter and GSH homeostasis. This is significant since the former field has been in need of breakthroughs. Large transporter genes screening and validation is currently on the way. This study is expected to bring more insight into our understanding of lens GSH homeostasis and regulation in relationship to age-related nuclear cataract formation.
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