Characterization of disease-specific chondroitin sulfate nonreducing end accumulation in mucopolysaccharidosis IVA.

Morquio syndrome type A, also known as MPS IVA, is a rare autosomal recessive disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase, a lysosomal hydrolase critical in the degradation of keratan sulfate (KS) and chondroitin sulfate (CS). The CS that accumulates in MPS IVA patients has a disease-specific nonreducing end (NRE) terminating with N-acetyl-D-galactosamine 6-sulfate, which can be specifically quantified after enzymatic depolymerization of CS polysaccharide chains. The abundance of N-acetyl-D-galactosamine 6-sulfate over other possible NRE structures is diagnostic for MPS IVA. Here, we describe an assay for the liberation and measurement of N-acetyl-D-galactosamine 6-sulfate and explore its application to MPS IVA patient samples in pilot studies examining disease detection, effects of age and treatment with enzyme-replacement therapy. This assay complements the existing urinary KS assay by quantifying CS-derived substrates, which represent a distinct biochemical aspect of MPS IVA. A more complete understanding of the disease could help to more definitively detect disease across age ranges and more completely measure the pharmacodynamic efficacy of therapies. Larger studies will be needed to clarify the potential value of this CS-derived substrate to manage disease in MPS IVA patients.

Supplementation with macular carotenoids improves visual performance of transgenic mice.

Carotenoid supplementation can improve human visual performance, but there is still no validated rodent model to test their effects on visual function in laboratory animals. We recently showed that mice deficient in ß-carotene oxygenase 2 (BCO2) and/or ß-carotene oxygenase 1 (BCO1) enzymes can accumulate carotenoids in their retinas, allowing us to investigate the effects of carotenoids on the visual performance of mice. Using OptoMotry, a device to measure visual function in rodents, we examined the effect of zeaxanthin, lutein, and ß-carotene on visual performance of various BCO knockout mice. We then transgenically expressed the human zeaxanthin-binding protein GSTP1 (hGSTP1) in the rods of bco2-/- mice to examine if delivering more zeaxanthin to retina will improve their visual function further. The visual performance of bco2-/- mice fed with zeaxanthin or lutein was significantly improved relative to control mice fed with placebo beadlets. ß-Carotene had no significant effect in bco2-/- mice but modestly improved cone visual function of bco1-/- mice. Expression of hGSTP1 in the rods of bco2-/-mice resulted in a 40% increase of retinal zeaxanthin and further improvement of visual performance. This work demonstrates that these "macular pigment mice" may serve as animal models to study carotenoid function in the retina.

Retinal accumulation of zeaxanthin, lutein, and ß-carotene in mice deficient in carotenoid cleavage enzymes.

Carotenoid supplementation can prevent and reduce the risk of age-related macular degeneration (AMD) and other ocular disease, but until now, there has been no validated and well-characterized mouse model which can be employed to investigate the protective mechanism and relevant metabolism of retinal carotenoids. ß-Carotene oxygenases 1 and 2 (BCO1 and BCO2) are the only two carotenoid cleavage enzymes found in animals. Mutations of the bco2 gene may cause accumulation of xanthophyll carotenoids in animal tissues, and BCO1 is involved in regulation of the intestinal absorption of carotenoids. To determine whether or not mice deficient in BCO1 and/or BCO2 can serve as a macular pigment mouse model, we investigated the retinal accumulation of carotenoids in these mice when fed with zeaxanthin, lutein, or ß-carotene using an optimized carotenoid feeding method. HPLC analysis revealed that all three carotenoids were detected in sera, livers, retinal pigment epithelium (RPE)/choroids, and retinas of all of the mice, except that no carotenoid was detectable in the retinas of wild type (WT) mice. Significantly higher amounts of zeaxanthin and lutein accumulated in the retinas of BCO2 knockout (bco2-/-) mice and BCO1/BCO2 double knockout (bco1-/-/bco2-/-) mice relative to BCO1 knockout (bco1-/-) mice, while bco1-/- mice preferred to take up ß-carotene. The levels of zeaxanthin and lutein were higher than ß-carotene levels in the bco1-/-/bco2-/- retina, consistent with preferential uptake of xanthophyll carotenoids by retina. Oxidative metabolites were detected in mice fed with lutein or zeaxanthin but not in mice fed with ß-carotene. These results indicate that bco2-/- and bco1-/-/bco2-/- mice could serve as reasonable non-primate models for macular pigment function in the vertebrate eye, while bco1-/- mice may be more useful for studies related to ß-carotene.

Inactivity of human ß,ß-carotene-9',10'-dioxygenase (BCO2) underlies retinal accumulation of the human macular carotenoid pigment.

The macula of the primate retina uniquely concentrates high amounts of the xanthophyll carotenoids lutein, zeaxanthin, and meso-zeaxanthin, but the underlying biochemical mechanisms for this spatial- and species-specific localization have not been fully elucidated. For example, despite abundant retinal levels in mice and primates of a binding protein for zeaxanthin and meso-zeaxanthin, the pi isoform of glutathione S-transferase (GSTP1), only human and monkey retinas naturally contain detectable levels of these carotenoids. We therefore investigated whether or not differences in expression, localization, and activity between mouse and primate carotenoid metabolic enzymes could account for this species-specific difference in retinal accumulation. We focused on ß,ß-carotene-9',10'-dioxygenase (BCO2, also known as BCDO2), the only known mammalian xanthophyll cleavage enzyme. RT-PCR, Western blot analysis, and immunohistochemistry (IHC) confirmed that BCO2 is expressed in both mouse and primate retinas. Cotransfection of expression plasmids of human or mouse BCO2 into Escherichia coli strains engineered to produce zeaxanthin demonstrated that only mouse BCO2 is an active zeaxanthin cleavage enzyme. Surface plasmon resonance (SPR) binding studies showed that the binding affinities between human BCO2 and lutein, zeaxanthin, and meso-zeaxanthin are 10- to 40-fold weaker than those for mouse BCO2, implying that ineffective capture of carotenoids by human BCO2 prevents cleavage of xanthophyll carotenoids. Moreover, BCO2 knockout mice, unlike WT mice, accumulate zeaxanthin in their retinas. Our results provide a novel explanation for how primates uniquely concentrate xanthophyll carotenoids at high levels in retinal tissue.

Surface plasmon resonance (SPR)-based biosensor technology for the quantitative characterization of protein-carotenoid interactions.

The surface plasmon resonance (SPR) biosensor method is a highly sensitive, label-free technique to study the non-covalent interactions of biomolecules, especially protein-protein and protein-small molecule interactions. We have explored this robust biosensor platform to study the interactions of carotenoid-binding proteins and their carotenoid ligands to assess the specificity of interaction, kinetics, affinity, and stoichiometry. These characterizations are important to further study uptake and transport of carotenoids to targeted tissues such as the macula of the human eye. In this review, we present an overview of the SPR method and optimization of assay conditions, and we discuss the particular challenges in studying carotenoid-protein interactions using SPR.

Carotenoids as possible interphotoreceptor retinoid-binding protein (IRBP) ligands: a surface plasmon resonance (SPR) based study.

Uptake, transport and stabilization of xanthophylls in the human retina are important components of a complex multistep process that culminates in a non-uniform distribution of these important nutrients in the retina. The process is far from understood; here, we consider the potential role of interphotoreceptor retinoid-binding protein (IRBP) in this process. IRBP is thought to facilitate the exchange of 11-cis-retinal, 11-cis-retinol and all-trans-retinol between the retinal pigment epithelium (RPE), photoreceptors and Müller cells in the visual cycle. Structural and biochemical studies suggest that IRBP has a variety of nonequivalent ligand binding sites that function in this process. IRBP is multifunctional, being able to bind a variety of physiologically significant molecules including fatty acids in the subretinal space. This wide range of binding activities is of particular interest because it is unknown whether the lutein and zeaxanthin found in the macula originate from the choroidal or retinal circulations. If from the choroidal circulation, then IRBP is a likely mediator for their transport across the interphotoreceptor matrix. In this report, we explore the binding interactions of retinoids, fatty acids, and carotenoids with IRBP using surface plasmon resonance (SPR)-based biosensors. IRBP showed similar affinity toward retinoids and carotenoids (1-2 µM), while fatty acids had approximately 10 times less affinity. These results suggest that further studies should be carried out to evaluate whether IRBP has a physiologically relevant role in binding lutein and zeaxanthin in the interphotoreceptor matrix.

Protein-Flavonoid Interaction Studies by a Taylor Dispersion Surface Plasmon Resonance (SPR) Technique: A Novel Method to Assess Biomolecular Interactions.

Flavonoids are common polyphenolic compounds widely distributed in fruits and vegetables. These pigments have important pharmacological relevance because emerging research suggests possible anti-cancer and anti-inflammatory properties as well other beneficial health effects. These compounds are relatively hydrophobic molecules, suggesting the role of blood transport proteins in their delivery to tissues. In this study, we assess the binding interactions of four flavonoids (kaempferol, luteolin, quercetin, and resveratrol) with human serum albumin (HSA), the most abundant protein in the blood, and with glutathione S-transferase pi isoform-1 (GSTP1), an enzyme with well-characterized hydrophobic binding sites that plays an important role in detoxification of xenobiotics with reduced glutathione, using a novel Taylor dispersion surface plasmon resonance (SPR) technique. For the first time, HSA sites revealed a high-affinity binding site for flavonoid interactions. Out of the four flavonoids that we examined, quercetin and kaempferol showed the strongest equilibrium binding affinities (K(D)) of 63 ± 0.03 nM and 37 ± 0.07 nM, respectively. GSTP1 displayed lower affinities in the micromolar range towards all of the flavonoids tested. The interactions of flavonoids with HSA and GSTP1 were studied successfully using this novel SPR assay method. The new method is compatible with both kinetic and equilibrium analyses.

Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease.

The human macula uniquely concentrates three carotenoids: lutein, zeaxanthin, and meso-zeaxanthin. Lutein and zeaxanthin must be obtained from dietary sources such as green leafy vegetables and orange and yellow fruits and vegetables, while meso-zeaxanthin is rarely found in diet and is believed to be formed at the macula by metabolic transformations of ingested carotenoids. Epidemiological studies and large-scale clinical trials such as AREDS2 have brought attention to the potential ocular health and functional benefits of these three xanthophyll carotenoids consumed through the diet or supplements, but the basic science and clinical research underlying recommendations for nutritional interventions against age-related macular degeneration and other eye diseases are underappreciated by clinicians and vision researchers alike. In this review article, we first examine the chemistry, biochemistry, biophysics, and physiology of these yellow pigments that are specifically concentrated in the macula lutea through the means of high-affinity binding proteins and specialized transport and metabolic proteins where they play important roles as short-wavelength (blue) light-absorbers and localized, efficient antioxidants in a region at high risk for light-induced oxidative stress. Next, we turn to clinical evidence supporting functional benefits of these carotenoids in normal eyes and for their potential protective actions against ocular disease from infancy to old age.