Purification and Partial Characterization of a Lutein-Binding Protein from Human Retina

Abstract

Dietary intake of lutein and zeaxanthin appears to be advantageous for protecting human retinal and macular tissues from degenerative disorders such as age-related macular degeneration. Selective concentration of just two of the many dietary carotenoids suggests that uptake and transport of these xanthophyll carotenoids into the human foveal region are mediated by specific xanthophyll-binding proteins such as GSTP1 which has previously been identified as the zeaxanthin-binding protein of the primate macula. Here, a membrane-associated human retinal lutein-binding protein (HR-LBP) was purified from human peripheral retina using ion-exchange chromatography followed by size-exclusion chromatography. After attaining 83-fold enrichment of HR-LBP, this protein exhibited a significant bathochromic shift of approximately 90 nm in association with lutein, and equilibrium binding studies demonstrated saturable, specific binding toward lutein with a K(D) of 0.45 muM. Examination for cross-reactivity with antibodies raised against known lutein-binding proteins from other organisms revealed consistent labeling of a major protein band of purified HR-LBP at approximately 29 kDa with an antibody raised against silkworm (Bombyx mori) carotenoid-binding protein (CBP), a member of steroidogenic acute regulatory (StAR) protein family with significant homology to many human StAR proteins. Immunolocalization with antibodies directed against either CBP or GSTP1 showed specific labeling of rod and cone inner segments, especially in the mitochondria-rich ellipsoid region. There was also strong labeling of the outer plexiform (Henle fiber) layer with anti-GSTP1. Such localizations compare favorably with the distribution of macular carotenoids as revealed by resonance Raman microscopy. Our results suggest that HR-LBP may facilitate lutein's localization to a region of the cell subject to considerable oxidative stress.

Heritability of the spatial distribution and peak density of macular pigment: a classical twin study

“…Taken with the previous reports of heritability in excess of 70%, these findings suggest that genetic factors exert an overwhelming influence on the amount of MP that is deposited in the eye. Given that several specific xanthrophyll-binding proteins have been identified, 38,39 including the Pi isoform of gluthathione S-transferase, 38 it is possible that specific polymorphisms may exist that influence the uptake, metabolism or deposition of L and Z. Indeed, genetic factors are likely to account for part of the variance unaccounted for in studies investigating the relationships between dietary, serum, and macular carotenoid concentrations.…”

“…A recent population-based study that looked specifically at the spatial profile of MP using dual-wavelength analysis of autofluorescence images found that a ring-like structure of MP was present in almost 20% of the participants, tended to be bilateral and was significantly more common in females and never smokers. 40 Although substantial progress has been made in understanding mechanisms involved in the uptake, metabolism, and deposition of L and Z, 39,41 it is clearly a complex process. The ratio of L/Z/meso-zeaxanthin varies with retinal profile with Z predominant at the central peak and L predominant in the peripheral retina.…”

Can the silkworm (<i>Bombyx mori</i>) be used as a human disease model?

“…The antibody to B. mori CBP has cross-reactivity to macular carotenoid transporter identified from human retina and layers of the primate macula where the macular carotenoid pigment is at its highest concentration (15). The human macular carotenoid transporter StARD3 (also known as MLN64) was discovered by Bernstein et al in 2009 (16).…”

Intracellular Uptake Mechanism of Lutein in Retinal Pigment Epithelial Cells

“…Bhosale et al characterized a zeaxanthin-binding protein Pi isoform of human glutathione S-transferase (GSTP1) (26). Steroidogenic acute regulatory domain 3 (stARD3) was identified as a lutein-binding protein (27,28). stARD was shown to be expressed in mitochondria (29).…”