Coexpression and interaction of wild-type and missense RS1 mutants associated with X-linked retinoschisis: Its relevance to gene therapy

PURPOSE. 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 wildtype 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.