title: Coexpression and interaction of wild-type and missense RS1 mutants associated with X-linked retinoschisis: its relevance to gene therapy.
abstract: OBJECTIVE
  X-linked retinoschisis (XLRS) is an early-onset retinal disease caused by mutations in retinoschisin (RS1), a multisubunit, extracellular protein implicated in retinal cell adhesion. Delivery of the normal RS1 gene to photoreceptors of retinoschisin-deficient mice results in prolonged protein expression and rescue of retinal structure and function. However, most persons with XLRS harbor a missense mutation in the RS1 gene leading to expression of a nonfunctional protein. The purpose of this study was to examine the effect that coexpression of wild-type RS1 with disease-causing mutants has on RS1 expression, oligomerization, and secretion to further evaluate gene therapy as a possible treatment for XLRS.
  
  
  METHODS
  RS1 mutants (C59S, D158N, C142W, C142S, T185K, R141H, R141G) were individually expressed or coexpressed with myc-tagged wild-type RS1 (myc-RS1) in EBNA293 cells. Protein expression, secretion, and subunit assembly of wild-type and mutant RS1 were analyzed by Western blotting and coimmunoprecipitation. Immunofluorescence was used to examine the cellular distribution of RS1.
  
  
  RESULTS
  Myc-RS1 was identical to untagged, wild-type RS1 with respect to cellular localization, disulfide-linked octamer formation, and secretion. In coexpression studies, myc-RS1 assembled into a disulfide-linked octameric complex and was secreted from cells independent of all disease-linked RS1 mutants studied except the R141H mutant.
  
  
  CONCLUSIONS
  When wild-type RS1 is expressed in the same cells as disease-causing mutants, the wild-type protein undergoes protein folding, subunit assembly, and secretion independent of all disease-causing RS1 mutants studied except R141H. These studies suggest that gene therapy may be an effective treatment for most persons with XLRS.
authors.affiliation: Department of Biochemistry and Molecular Biology, Centre for Macular Research, University of British Columbia, Vancouver, Canada.
authors.forename: Frank M
authors.initials: FM
authors.lastname: Dyka
authors.forename: Robert S
authors.initials: RS
authors.lastname: Molday
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D015316:Genetic Therapy; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism; D017510:Protein Folding; D041441:Retinoschisis / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D019309:Epstein-Barr Virus Nuclear Antigens; D005136:Eye Proteins; C108726:RS1 protein, human