Identification and Characterization of a Pi Isoform of Glutathione S-Transferase (GSTP1) as a Zeaxanthin-binding Protein in the Macula of the Human Eye

Prakash Bhosale,Alexander J Larson,Jeanne M Frederick,Katie Southwick,Craig D Thulin,Paul S Bernstein

doi:10.1074/jbc.m405334200

Prakash Bhosale, Alexander J Larson + Show 4 more

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https://doi.org/10.1074/jbc.m405334200

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Abstract

Uptake, metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs). A membrane-associated XBP was purified from human macula using ion-exchange chromatography followed by gel-exclusion chromatography. Two-dimensional gel electrophoresis showed a prominent spot of 23 kDa and an isoelectric point of 5.7. Using mass spectral sequencing methods and the public NCBI database, it was identified as a Pi isoform of human glutathione S-transferase (GSTP1). Dietary (3R,3'R)-zeaxanthin displayed the highest affinity with an apparent Kd of 0.33 microm, followed by (3R,3'S-meso)-zeaxanthin with an apparent Kd of 0.52 microm. (3R,3'R,6'R)-Lutein did not display any high-affinity binding to GSTP1. Other human recombinant glutathione S-transferase (GST) proteins, GSTA1 and GSTM1, exhibited only low affinity binding of xanthophylls. (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. Like-wise, dietary (3R,3'R)-zeaxanthin displayed alterations in its CD spectrum in association with GSTP1 and XBP. Other mammalian xanthophyll carrier proteins such as tubulin, high-density lipoprotein, low-density lipoprotein, albumin, and beta-lactoglobulin did not bind zeaxanthins with high affinity, and they failed to induce or alter xanthophyll CD spectra to any significant extent. Immunocytochemistry with an antibody to GSTP1 on human macula sections showed highest labeling in the outer and inner plexiform layers. These results indicate that GSTP1 is a specific XBP in human macula that interacts with (3R,3'S-meso)-zeaxanthin and dietary (3R,3'R)-zeaxanthin in contrast to apparently weaker interactions with (3R,3'R,6'R)-lutein.

Highlights

Uptake, metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs)
Other human recombinant glutathione S-transferase (GST) proteins, GSTA1 and GSTM1, exhibited only low affinity binding of xanthophylls. (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
The abbreviations used are: XBP, xanthophyll-binding protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; GSTA, glutathione S-transferase Alpha; GSTM, glutathione S-transferase Mu; GSTP, glutathione Stransferase Pi; HDL, high-density lipoprotein; Isoelectric focusing (IEF), isoelectric focusing; This paper is available on line at http://www.jbc.org

Summary

Introduction

Metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs). (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. We further purify human macular XBP and identify the major protein spot on two-dimensional gels as a Pi isoform of glutathione S-transferase (GSTP1).

Results

Conclusion