Coexpression and Interaction of Wild-type and Missense RS1 Mutants Associated with X-Linked Retinoschisis: Its Relevance to Gene Therapy

Abstract

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. 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. 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. 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.