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C. Vijayasarathy, Y. Takada, Y. Zeng, R. A. Bush, P. A. Sieving; Retinoschisin Is a Peripheral Membrane Protein With Affinity for Anionic Phospholipids and Affected by Divalent Cations. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4482.
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Loss of retinoschisin (RS) function is the cause of X-linked retinoschisis (XLRS), a form of macular degeneration characterized by splitting within the retinal layers, and progressive loss of vision in young males. RS is predicted to serve as adhesion protein in maintaining the architecture of the retina, but a precise molecular description of RS function is not yet known. Hence, we investigated (a) the molecular interactions of RS in intact retina (b) the ultrastructural localization of RS in photoreceptors and (C) structural organization of the photoreceptor cells in adult mice with a retinal phenotype resembling that of human XLRS (RS1-/y).
Subcellular fractions were prepared from mice retinas by differential or density gradient ultracentrifugation. The expression of RS was evaluated using immunoblotting. The nature of RS-membrane association and orientation were studied by alkaline, high salt and detergent extractions of membrane fractions and protease protection assays. RS-lipid interactions were evaluated by protein-lipid overlay assay that employed wild type and mutant forms of RS discoidin domain GST fusion proteins. The subcellular localization of RS in mouse retina was visualized by preembedding immunogold electron microscopy. Ultrastructure was evaluated by transmission electron microscopy.
RS is intimately associated with cell membranes of the retina and, clustered primarily on the outer leaflet of the plasma membrane of the photoreceptor inner segments, which synthesize and secrete RS. Characteristic of peripheral membrane proteins, RS was released from the membrane at high pH. RS was extracted from the membrane by non-ionic detergent NP-40 together with glycerophospholipids. Protein-lipid overlay assays indicated a preferential interaction between RS and anioic phospholipids. RS extraction from the membrane was inhibited by divalent cations. Photoreceptor inner segment morphology is markedly affected in RS1- / y mice, which fail to express RS protein.
RS in intact retina is a peripheral membrane protein. Although distributed over the two membrane faces, RS is associated primarily with the outer leaflet of the inner segment plasma membrane through anionic phospholipids and divalent cations. RS localization in photoreceptors and its biochemical properties, together with the photoreceptor anomalies seen in RS1- / y mice, suggest that RS has a functional role locally, at the site of secretion and membrane adhesion, in maintaining the photoreceptor inner segment stability and architecture.
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