[Thesis]. Manchester, UK: The University of Manchester; 2017.
X-Linked Retinoschisis (XLRS) is a currently incurable, progressive retinal degeneration
that affects approximately 1:20,000 males. Sufferers have a loss of retinal structure
and visual acuity, leading to blindness. The condition is caused by mutation of the
RS1 gene encoding the retinal-specific protein retinoschisin. Retinoschisin is critical
in maintaining the normal, ordered retinal architecture, with deletion in mice models
leading to loss of both structure and visual processing, analogous to XLRS sufferers.
However, re-introduction of retinoschisin using adeno-associated viral vectors leads
to complete rescue in these models. Despite the importance of retinoschisin in maintaining
retinal architecture, the mechanism by which it maintains this structure remains unknown.
As a result, this study aimed to structurally characterise retinoschisin and XLRS-associated
point mutants R141H and H207Q to gain insight into the mechanism of retinoschisin
action. To this end, retinoschisin was expressed and purified from HEK 293-EBNA cells
and the structure of both monomeric and octameric retinoschisin was investigated using
Small-Angle X-Ray Scattering (SAXS) and Cryo-electron microscopy (Cryo-EM). Monomeric
retinoschisin was found to adopt an elongated structure that allowed for the tight
association of the subunits into a planer propeller structure. However, in solution
conditions the octamer also stably self-assembled into a dimer of octamers, for which
the structure was solved using cryo-EM. This allowed for construction of a quasi-atomic
model, enabling mapping of XLRS-associated point mutations on the complex. Two major
classes of mutation were identified, in the intra-octamer and inter-octamer interfaces,
suggesting a mechanism of pathology for these mutants. Observation of clustered conservative
mutations at the inter-octamer interface suggested the dimer of octamers may be physiologically
relevant. Furthermore, comparison of the R141H mutant to the wild-type revealed an
additional mutated site in the propeller tips. Here, R141H was suggested to induce
a small conformational change and alter an interaction site. Another mutant, H207Q,
however, induced a destabilization of the assembled retinoschisin molecule.In conclusion,
we purified and structurally characterised human retinoschisin, identifying a new
hexadecameric oligomer. The structure of this allowed for identification of distinct
classes of mutations on the assembled molecule and a hypothesis of the mechanism of
retinoschisin action in the retina.