Abstract
Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most XLRS-associated mutations cause intracellular retention, however a subset are secreted as octamers and the cause of their pathology is ill-defined. Therefore, here we investigated the solution structure of the retinoschisin monomer and the impact of two XLRS-causing mutants using a combinatorial approach of biophysics and cryo-EM. The retinoschisin monomer has an elongated structure which persists in the octameric assembly. Retinoschisin forms a dimer of octamers with each octameric ring adopting a planar propeller structure. Comparison of the octamer with the hexadecamer structure indicated little conformational change in the retinoschisin octamer upon dimerization, suggesting that the octamer provides a stable interface for the construction of the hexadecamer. The H207Q XLRS-associated mutation was found in the interface between octamers and destabilized both monomeric and octameric retinoschisin. Octamer dimerization is consistent with the adhesive function of retinoschisin supporting interactions between retinal cell layers, so disassembly would prevent structural coupling between opposing membranes. In contrast, cryo-EM structural analysis of the R141H mutation at ∼4.2Å resolution was found to only cause a subtle conformational change in the propeller tips, potentially perturbing an interaction site. Together, these findings support distinct mechanisms of pathology for two classes of XLRS-associated mutations in the retinoschisin assembly.
Highlights
The retina is a unique neural tissue, possessing pronounced laminar architecture with maintenance of retinal structure critical to neural processing [1]
Multiangle Light Scattering (MALS) analysis of retinoschisin monomer revealed a molecular weight of approximately 27 kDa consistent with sequence predictions (Fig. 1A)
Retinoschisin was further probed through Small Angle X-Ray Scattering (SAXS) analysis of the discoidin domain (Fig. S3), which formed a smaller globular structure with an radius of gyration (Rg) of 15.6 Å and a Dmax of 55 Å (Figs. 1C and D and Fig. S3)
Summary
The retina is a unique neural tissue, possessing pronounced laminar architecture with maintenance of retinal structure critical to neural processing [1]. XLRS is caused by over 230 mutations in the RS1 gene, as reported by the HMGD Professional database [3], which encodes the protein retinoschisin [4, 5]. Retinoschisin, a 24kDa protein secreted by photoreceptors, consists of a retinoschisin (Rs1) domain and a discoidin domain with a small C-terminal extension [6,7,8]. The resulting octameric complex is secreted and diffuses throughout the retina, attaching to the outer plasma membrane leaflet [11, 12]. This is crucial for maintenance of normal retinal cytoarchitecture. Despite observations that retinoschisin binds Na+/K+-ATPase [24] and L-type Voltage Gated Calcium ion Channels (L-VGCCs) [25], the molecular mechanism of retinoschisin function remains elusive
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