Abstract
Purpose: :
Retinoschiscin (RS1) is an extracellular protein encoded by the gene responsible for X-linked retinoschisis, an early onset retinal degeneration disease characterized by a splitting of the retina. In recent studies we have shown that RS1 does not bind to phospholipids, but instead, interacts with a Na/K ATPase-SARM1 complex on photoreceptor and bipolar cells. The goal of this study is to further investigate the interaction of RS1 with the Na/K ATPase-SARM1 complex and identify the component of this complex which directly binds RS1 as an important step in understanding the molecular and cellular basis for the functioning of RS1 as a retinal cell adhesion protein and its role in X-linked retinoschisis.
Methods: :
Anti-retinoschisin RS1 3R10 monoclonal antibody coupled to Sepharose 2B was used to immunoprecipitate RS1 from detergent-solubilized retinal extracts of Rs1h knockout and wild-type (WT) mice with or without the prior addition of purified RS1. Co-immunoprecipitation studies were also carried out on detergent-solubilized extracts from HEK293 cells singly or co-expressing RS1 with the alpha3 or beta2 subunit of Na/K ATPase. Proteins that co-immunoprecipitate with RS1 were analyzed by western blotting. Co-localization of these proteins was visualized by immunofluorescence microscopy.
Results: :
Immunoprecipitation and immunofluorescence labeling studies indicated that RS1 specifically interacted and co-localized with the Na/K ATPase-SARM1 complex on photoreceptor and bipolar cells of WT mice. Preliminary studies indicate that the Na/K ATPase is co-immunoprecipitated with RS1 when purified RS1 is added to retina membrane extracts from Rs1h knockout mice. In HEK 293 cell expression studies, RS1 co-localized and co-immunoprecipitated with the beta2 subunit of Na/K ATPase.
Conclusions: :
These studies provide additional evidence for the specific interaction of RS1 with the Na/K ATPase-SARM1 complex on photoreceptor and bipolar cells. This interaction appears to be mediated by the beta2 subunit of the Na/K ATPase.
Keywords: protein purification and characterization • retina: distal (photoreceptors, horizontal cells, bipolar cells) • retinal degenerations: hereditary