RS1 structure-function relationships: Roles in retinal adhesion and X-linked retinoschisis

X-linked retinoschisis is a form of macular degeneration that can result in visual loss in young males or in females with both copies of the gene defective. The RS1 gene associated with X-linked retinoschisis was positionally cloned in 1997. The gene was found to encode RS1, a 224-amino acid protein containing a discoidin domain that spans most of the protein. Discoidin domains are found in a wide variety of proteins that are involved mainly in cell adhesion. This thesis investigation examined the structure-function relationships in RS1 and their roles in maintaining retinal cell adhesion and in causing X-linked retinoschisis. Results showed that RS1 exists as a single disulfide-linked homo-octamer formed by intermolecular disulfide bonds between C59 and C223. C40-C40 intermolecular disulfide bonds further result in disulfide-linked dimer formation within this octameric complex. Within the discoidin domain, C63-C219 and C110-C142 form intramolecular disulfide bonds to allow for proper protein folding and stability of the discoidin domain, and C83 exists as a free cysteine. To allow for RS1 secretion into the extracellular matrix, each RS1 subunit is cleaved after S23 by signal peptidase. The main molecular mechanisms that cause X-linked retinoschisis can be grouped into four categories: mutations in the leader sequence prevent proper RS1 targeting to the endoplasmic reticulum; most mutations in the discoidin domain prevent proper protein folding and secretion; mutations in C59 or C223 prevent octameric assembly; and R141 H within the discoidin domain causes abnormal oligomer formation. A suspected polymorphism D1 58N was shown to behave similarly to wildtype RS1. To identify the component that RS1 interacts with, known ligands of discoidin family proteins were tested for their ability to bind RS1. Unlike Factor V, RS1 did not bind phospholipids; however, similar to discoidin I, RS1 bound to D-galactose. This interaction depended on the octameric form of RS1, as dimers or monomers interacted weakly with galactose. Isopropyl beta-D-1-thiogalactopyranoside eluted RS1 from a galactose-agarose column. This property was used to purify RS1 from retinal membranes. In summary, RS1 is a lectin whose function critically depends on the proper folding of its discoidin domain and disulfide-linked octamerization.