Abstract
Purpose::
Lutein and zeaxanthin are found in millimolar levels in the human macula. While increasing evidence suggests that high levels of these xanthophyll carotenoids may be protective against age-related maculopathy, the biochemical processes by which they are concentrated in the retina have been poorly understood. We have demonstrated previously that the pi isoform of glutathione S-transferase (GSTP1) is the specific binding protein in the primate macula for zeaxanthin. Here we present the biochemistry and tissue distribution of the analogous human retinal lutein binding protein (HR-LBP).
Methods::
Postmortem human peripheral retinas were homogenized in phosphate buffer, and the yellow membrane fraction was solubilized in CHAPS detergent. Proteins associated with endogenous carotenoids were purified by ion-exchange and gel-filtration chromatography. Major protein spots were separated and immunoblotted with an antibody to silkworm gut lutein binding protein (SG-LBP, also known as CBP and BmStART), a recently purified insect lutein binding protein of the steroidogenic acute regulatory (StAR) family, a group of proteins involved in intracellular transport and binding of small hydrophobic molecules.
Results::
The most highly purified carotenoid-associated fraction of human peripheral retinal extracts yielded one major protein at ~56 kDa (HR-LBP). Lutein accounted for 95% of the endogenously associated carotenoids. The lutein binding protein exhibited a typical three peak carotenoid spectrum but with an 80 nm shift of its absorbance maximum to 537 nm. HR-LBP is strongly immunoreactive with anti-SG-LBP and other anti-StAR antibodies, indicating that HR-LBP is a StAR family protein. Localization of antibody directed against SG-LBP in adult Macaca retina revealed strong immunoreactivity in the inner segments (ellipsoids) and synaptic terminals of both rod and cone photoreceptors. This distribution overlaps, in part, with that shown previously for GSTP1.
Conclusions::
Identification of specific binding proteins for retinal lutein and zeaxanthin establishes the biochemical basis for the selective uptake and retention of the macular pigment carotenoids. It is remarkable that StAR family proteins have been utilized to bind lutein in organisms as widely divergent as silkworms and humans.
Keywords: carotenoids/carotenoid binding proteins • macular pigment • protein purification and characterization