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P. Bhosale, J.M. Frederick, K. Southwick, C.D. Thulin, P.S. Bernstein; A Proteomic Approach to the Biochemistry of Xanthophyll Carotenoids in Human and Avian Ocular and Nonocular Tissues . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1756.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Uptake, metabolism, stabilization, and antioxidant functions of xanthophyll carotenoids in the retina are mediated by specific xanthophyll–binding proteins (XBP). We have recently identified and characterized glutathione S–transferase P1 (GSTP1) as a zeaxanthin–binding protein in the human macula (Bhosale, et al., J. Biol. Chem. 2004, 279:49447). In this study the physiological distribution of the GSTP1 in the macula is explored, and in a parallel proteomic approach, an apparently novel XBP was purified from the liver of Japanese quail. METHODS: Xanthophyll binding proteins from human macula and quail liver were purified using stepwise ion exchange and silica gel size exclusion chromatography coupled with inline photodiode array detection. Prominent protein spots on 1–D and 2–D gels were identified by LC–MS/MS analysis. Identified proteins were studied for xanthophyll–binding properties using classical binding assays and several spectroscopic techniques. Confocal immunolocalization of GSTP1 was also performed. Results:The major protein in our most highly purified macular xanthophyll–binding protein preparations was identified as a pi isoform of glutathione S–transferase (GSTP1). Binding and spectroscopic studies using recombinant human GSTP1 prove that this protein binds zeaxanthin, but not lutein, with high specificity and affinity. Immunocytochemistry studies with antibodies to GSTP1 demonstrate that its tissue distribution is consistent with the known distribution of zeaxanthin in the retina. A 50 kDa XBP was purified from quail liver that upon binding with endogenous lutein and zeaxanthin displayed a strong bathochromoic shift of 80 nm. We are currently characterizing this protein further and elucidating its role as an XBP, with particular attention to its potential role as a specific lutein–binding protein. Conclusions:We have identified GSTP1 as the first specific xanthophyll–binding protein in any vertebrate tissue, and we have evidence that a novel XBP is present in quail liver. GSTP1, the newly discovered quail liver XBP, and other related xanthophyll–binding proteins are likely to mediate the uptake and protective functions of lutein and zeaxanthin in the human macula and other tissues. Defects in these XBPs may predispose individuals to visual loss from age–related macular degeneration and other systemic disorders related to oxidative damage.
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