Abstract
Retinoschisin or RS1 is a discoidin domain-containing protein encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration characterized by a splitting of the retina. Retinoschisin, expressed and secreted from photoreceptors and bipolar cells as a homo-octameric complex, associates with the surface of these cells where it serves to maintain the cellular organization of the retina and the photoreceptor-bipolar synaptic structure. To gain insight into the role of retinoschisin in retinal cell adhesion and the pathogenesis of XLRS, we have investigated membrane components in retinal extracts that interact with retinoschisin. Unlike the discoidin domain-containing blood coagulation proteins Factor V and Factor VIII, retinoschisin did not bind to phospholipids or retinal lipids reconstituted into unilamellar vesicles or immobilized on microtiter plates. Instead, co-immunoprecipitation studies together with mass spectrometric-based proteomics and Western blotting showed that retinoschisin is associated with a complex consisting of Na/K ATPase (alpha3, beta2 isoforms) and the sterile alpha and TIR motif-containing protein SARM1. Double labeling studies for immunofluorescence microscopy confirmed the co-localization of retinoschisin with Na/K ATPase and SARM1 in photoreceptors and bipolar cells of retina tissue. We conclude that retinoschisin binds to Na/K ATPase on photoreceptor and bipolar cells. This interaction may be part of a novel SARM1-mediated cell signaling pathway required for the maintenance of retinal cell organization and photoreceptor-bipolar synaptic structure.
Highlights
(XLRS), a leading cause of early onset macular degeneration in males [1]
Homology modeling and molecular dynamics simulations of the retinoschisin discoidin domain has led to the suggestion that retinoschisin like Factor V may bind to the phospholipid bilayer through the interaction of conserved hydrophobic residues within the spike region of the discoidin domain [22]
To gain insight into the molecular and cellular mechanisms underlying the role of retinoschisin in the normal retina structure and function and the pathogenesis of X-linked retinoschisis (XLRS), we examined the interaction of retinoschisin with retinal membrane lipids and proteins
Summary
Materials—1,2-dioleoyl-sn-glycero-3-phospho-choline (DOPC), 1,2,-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) were purchased from Avanti Polar Lipids (Alabaster, AL). Preparation of Retinal Membranes—Two bovine or human retinas were incubated in 2.0 ml of TBS (20 mM Tris, pH 7.4, 150 mM NaCl) containing 1 mM MgCl2 and Complete protease inhibitor (Roche Applied Sciences, Basel, Switzerland) for 25 min on ice. Retina cells were homogenized using a Dounce homogenizer and subsequently passed through a 22-gauge needle. For the preparation of mouse retinal membranes, 16 retinas from wild-type C57BL/6 or retinoschisin-deficient mice [15] were suspended in 20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2 containing Complete protease inhibitor. The sections were blocked and permeabilized in 10% goat serum and 0.2% Triton X-100 in PB for 20 min and subsequently labeled overnight at room temperature with the primary polyclonal retinoschisin antibody and monoclonal antibody to Na/K ATPase diluted in PB containing 2.5% goat serum and 0.1% Triton X-100 (1:5000 anti-retinoschisin; 1:200 anti-Na/K ATPase (␣3-subunit)). Samples were visualized under a Zeiss Axioplan fluorescence microscope equipped with an Eclipse image analyzer
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