RS1, a discoidin domain-containing retinal cell adhesion protein associated with X-linked retinoschisis, exists as a novel disulfide-linked octamer.
RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. Our results indicate that RS1 exists as a novel octamer in which the eight subunits are joined together by Cys(59)-Cys(223) intermolecular disulfide bonds. Subunits within the octamer are further organized into dimers mediated by Cys(40)-Cys(40) bonds. These cysteines lie just outside the discoidin domain indicating that these flanking segments primarily function in the octamerization of RS1. Within the discoidin domain, two cysteine pairs (Cys(63)-Cys(219) and Cys(110)-Cys(142)) form intramolecular disulfide bonds that are important in protein folding, and one cysteine (Cys(83)) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein.
Wu
Winco W H
WW
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
Wong
Julie P
JP
Kast
Juergen
J
Molday
Robert S
RS
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002448:Cell Adhesion; D002460:Cell Line; D041321:Chromosomes, Human, X / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D000070818:Discoidin Domain Receptors; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry; D011975:Receptors, Mitogen / Q000737:chemistry; D012160:Retina / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D041441:Retinoschisis / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
D018076:DNA, Complementary; D003902:Detergents; D004220:Disulfides; D005136:Eye Proteins; D010455:Peptides; C108726:RS1 protein, human; D011975:Receptors, Mitogen; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D014357:Trypsin; D003545:Cysteine; D000645:Ammonium Sulfate