methodology-for-studying-interactions-vitamin-membrane-receptors

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...

University of Minnesota

We investigated the association between  (rs3742330) and  (rs10719) polymorphisms and pseudoexfoliation glaucoma (PXG) and related clinical phenotypes in a Saudi cohort. In a retrospective case-control study, TaqMan real-time, PCR-based genotyping was performed in 340 participants with 246 controls and 94 PXG cases. The minor (G) allele frequency of rs3742330 in PXG (0.03) was significantly different from that in the controls (0.08) and protective against PXG (odds ratio (OR) = 0.38, 95% confidence interval (CI) = 0.16-0.92),  = 0.017). Similarly, the rs3742330 genotypes showed a significant protective association with PXG in dominant ( = 0.019, OR = 0.38, 95% CI = 0.15-0.92), over-dominant ( = 0.024, OR = 0.39, 95% CI = 0.16-0.95), and log-additive models ( = 0.017, OR = 0.38, 95% CI = 0.16-0.92). However, none remained significant after an adjustment for age, sex, and multiple testing. Rs10719 in DROSHA did not show any significant allelic or genotype association with PXG. However, a protective effect of the GA haplotype in  and  and PXG ( = 0.034) was observed. Both polymorphisms showed no significant effect on intraocular pressure and the cup-disk ratio. In conclusion, we report a significant genetic association between variant rs3742330 in , a gene involved in miRNA biogenesis, and PXG. Further investigation in a larger group of patients of different ethnicities and functional studies are warranted to replicate and validate its potential role in PXG.

Polymorphism rs3742330 in microRNA Biogenesis Gene  Is Associated with Pseudoexfoliation Glaucoma in Saudi Cohort.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, wherein the perception of vision is initiated when these neurons respond to photons in the light stimuli. The photoreceptor cell is structurally studied as outer segments (OS) and inner segments (IS) where proper protein sorting, localization, and compartmentalization are critical for phototransduction, visual function, and survival. In human retinal diseases, improper protein transport to the OS or mislocalization of proteins to the IS and other cellular compartments could lead to impaired visual responses and photoreceptor cell degeneration that ultimately cause loss of visual function.

The role of motor proteins in photoreceptor protein transport and visual function.

The systemic transport of dietary vitamin A/all- retinol bound to RBP4 into peripheral tissues for storage is an essential physiological process that continuously provides visual chromophore precursors to the retina under fasting conditions. This mechanism is critical for phototransduction, photoreceptor cell maintenance and survival, and in the support of visual function. While the membrane receptor STRA6 facilitates the blood transport of lipophilic vitamin A into the eye, it is not expressed in most peripheral organs, which are proposed to express a second membrane receptor for the uptake of vitamin A from circulating RBP4. The discovery of a novel vitamin A receptor, RBPR2, which is expressed in the liver and intestine, but not in the eye, alluded to this long-sort non-ocular membrane receptor for systemic RBP4-ROL uptake and transport. We have previously shown in zebrafish that the retinol-binding protein receptor 2 (Rbpr2) plays an important role in the transport of yolk vitamin A to the eye. Mutant  zebrafish lines manifested in decreased ocular retinoid concentrations and retinal phenotypes. To investigate a physiological role for the second vitamin A receptor, RBPR2, in mammals and to analyze the metabolic basis of systemic vitamin A transport for retinoid homeostasis, we established a whole-body  knockout mouse () model. These mice were viable on both vitamin A-sufficient and -deficient diets.  mice that were fed a vitamin A-sufficient diet displayed lower ocular retinoid levels, decreased opsins, and manifested in decrease visual function, as measured by electroretinography. Interestingly, when  mice were fed a vitamin A-deficient diet, they additionally showed shorter photoreceptor outer segment phenotypes, altogether manifesting in a significant loss of visual function. Thus, under conditions replicating vitamin A sufficiency and deficiency, our analyses revealed that RBPR2-mediated systemic vitamin A transport is a regulated process that is important for vitamin A delivery to the eye when RBP4-bound ROL is the only transport pathway in the fasting condition or under vitamin A deficiency conditions.

Mice Lacking the Systemic Vitamin A Receptor RBPR2 Show Decreased Ocular Retinoids and Loss of Visual Function.

The distribution of dietary vitamin A/all- retinol/ROL throughout the body is critical for maintaining retinoid function in peripheral tissues and for retinoid delivery to the eye in the support of visual function. In the circulation, all--retinol bound to the RBP4 protein is transported and sequestered into target tissues for long-term storage. Two membrane receptors that facilitate all- retinol uptake from RBP4 have been proposed. While it is well established that the membrane receptor, STRA6, binds to circulatory RBP4 for ROL transport into the eye, the second vitamin A receptor, RBPR2, which is expressed in non-ocular tissues, is less characterized. Based on the structural homology between these two RBP4 receptors, published literature, and from our recent work in   deficient mice, we hypothesized that RBPR2 might also have high-binding affinity for RBP4 and this mechanism facilitates ROL transport. Herein, we aimed to elucidate the membrane topology and putative RBP4 binding residues on RBPR2 to understand its physiological function for retinoid homeostasis. Using  analysis and site-directed mutagenesis, we identified a potential RBP4 binding domain on RBPR2. We employed an  cell-based system and confirmed that mutations of these residues on RBPR2 affected its binding to exogenous RBP4 and subsequently vitamin A uptake. Using Surface Plasmon Resonance assays, we analyzed both the binding affinities and kinetic parameters of wild-type RBPR2 and individual mutants affecting the RBPR2-RBP4 binding domain with its physiological ligand RBP4. These studies not only revealed a putative RBP4 binding domain on RBPR2 but also provided new structural, biochemical, and critical information on its proposed role in RBP4 binding for ROL transport and retinoid homeostasis.

Mapping of the extracellular RBP4 ligand binding domain on the RBPR2 receptor for Vitamin A transport.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.

In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function.

In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.

The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters.

This study investigated the association between apolipoprotein E () gene polymorphisms (rs429358 and rs7412) and primary angle-closure glaucoma (PACG) and pseudoexfoliation glaucoma (PXG) in a Saudi cohort. Genotyping of 437 DNA samples (251 controls, 92 PACG, 94 PXG) was conducted using PCR-based Sanger sequencing. The results showed no significant differences in the allele and genotype frequencies of rs429358 and rs7412 between the PACG/PXG cases and controls. Haplotype analysis revealed ε3 as predominant, followed by ε4 and ε2 alleles, with no significant variance in PACG/PXG. However,  genotype analysis indicated a significant association between ε2-carriers and PACG (odds ratio = 4.82, 95% CI 1.52-15.26,  = 0.007), whereas no notable association was observed with PXG. Logistic regression confirmed ε2-carriers as a significant predictor for PACG ( = 0.008), while age emerged as significant for PXG ( < 0.001). These findings suggest a potential role of ε2-carriers in PACG risk within the Saudi cohort. Further validation and larger-scale investigations are essential to elucidate the precise role of  in PACG pathogenesis and progression.

Rakesh Radhakrishnan

Department of Ophthalmology and Visual Neurosciences

University of Minnesota

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Rakesh Radhakrishnan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology and Visual Neurosciences

University of Minnesota

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Department of Ophthalmology and Visual Neurosciences