Retinoschisin is a peripheral membrane protein with affinity for anionic phospholipids and affected by divalent cations.
OBJECTIVE
Retinoschisin (RS) is a retina-specific, secreted protein implicated in X-linked juvenile retinoschisis and essential for the structural and functional integrity of the retina. This biochemical characterization and ultrastructural localization of RS in intact murine retina was performed to further understanding of the molecular basis of its function.


METHODS
Subcellular fractions and fractions enriched in photoreceptor inner and outer segments were prepared from mouse retina by differential or density gradient ultracentrifugation. Immunoblot analysis was used to assess the expression of RS in various subcellular compartments and its fractionation into soluble phase on treatment of retinal cell membranes with several solubilizing reagents. RS-lipid interactions were evaluated by a protein-lipid overlay assay that used wild-type and mutant forms of RS discoidin domain glutathione S-transferase (GST) fusion proteins. The subcellular localization of RS in mouse retina was visualized by pre-embedding immunogold electron microscopy. Ultrastructure was evaluated by transmission electron microscopy.


RESULTS
RS was intimately associated with cell membranes of the retina. It was found to cluster on the outer leaflet of the plasma membrane of the photoreceptor inner segments, which synthesize and secrete it. It was released from the membrane at high pH, which is characteristic of a peripheral membrane protein. It was extracted from the membrane by the nonionic detergent NP-40, together with glycerophospholipids. Protein-lipid overlay assays indicated a preferential interaction between RS and anioic phospholipids. Extraction of RS from the membrane was inhibited by divalent cations. Photoreceptor inner segment morphology was markedly affected in RS(-)(/y) mice, which failed to express RS protein.


CONCLUSIONS
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's localization in photoreceptors and its biochemical properties suggest a functional role locally, at the site of secretion and membrane adhesion, in maintaining the photoreceptor inner segment stability and architecture.
Vijayasarathy
Camasamudram
C
Section for Translational Research in Retinal and Macular Degeneration (STRRMD), National Institute on Deafness and Other Communication Disorders (NIDCD), National Institutes of Health, Bethesda, Maryland, USA. camasamv@nidcd.nih.gov
Takada
Yuichiro
Y
Zeng
Yong
Y
Bush
Ronald A
RA
Sieving
Paul A
PA
D000818:Animals; D000838:Anions; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism* / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000378:metabolism* / Q000648:ultrastructure; D005982:Glutathione Transferase / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron; D010743:Phospholipids / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D041441:Retinoschisis / Q000378:metabolism; D013347:Subcellular Fractions
D000838:Anions; D015815:Cell Adhesion Molecules; D005136:Eye Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; C491843:RS1 protein, mouse; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase