Abstract
Abstract: :
Purpose:Uptake, metabolism, and stabilization of xanthophyll carotenoids in the retina is thought to be mediated by specific xanthophyll binding proteins (XBP). A membrane–associated XBP was purified from human macula using ion–exchange chromatography followed by size–exclusion chromatography. Two–dimensional gel electrophoresis showed a prominent spot of 23 Kda (IEP 5.2). Using MALDI–TOF and electrospray LC/MS/MS sequencing methods with in–gel tryptic digestion and the public NCBI database, it was identified as the pi isoform of human glutathione S–transferase (GSTP1). GST proteins are well–known for their interactions with hydrophobic molecules such as steroids and retinoids, but carotenoid–GST interactions have not been reported previously. Methods: Xanthophyll interactions with recombinant human GSTP1 were studied using binding assays and circular dichroism (CD) spectroscopy. Exogenous xanthophyll carotenoid ligands were added to the proteins in 1% tetrahydrofuran (THF), incubated overnight at 4°C, and unbound carotenoids were extracted into hexane. Results: (3R,3'R)–Zeaxanthin displayed saturable binding with an apparent dissociation constant (Kd) of 0.33 µM, while (3R,3'S–meso)–zeaxanthin had a Kd of 0.58 µM. Lutein did not display any appreciable binding affinity to GSTP1. (3R,3'S–meso)–zeaxanthin, an optically inactive nondietary xanthophyll carotenoid present in the human macula, exhibited a strong induced CD spectrum in association with human macular XBP that was nearly identical to the CD spectrum induced by GSTP1. Likewise, dietary (3R,3'R)–zeaxanthin displayed substantial alterations in its CD spectrum in association with GSTP1 and XBP, while dietary (3R,3'R,6'R)–lutein did not. Xanthophyll–GSTP1 associations were further compared with other mammalian xanthophyll carrier proteins such as tubulin, high–density lipoprotein (HDL), low–density lipoprotein (LDL), albumin, and ß–lactoglobulin. The other mammalian proteins studied failed to induce or alter xanthophyll CD spectra to any significant extent. Conclusions: These results indicate that GSTP1 is a specific XBP in human macula that interacts with dietary (3R,3'R)–zeaxanthin and (3R,3'S–meso)–zeaxanthin. The physiological role of GSTP1 in human macular carotenoid metabolism is under investigation.
Keywords: carotenoids/carotenoid binding proteins • protein purification and characterization • macular pigment