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      Structural and Mutational Characterisation of Human Retinoschisin

      Ramsay, Ewan

      [Thesis]. Manchester, UK: The University of Manchester; 2017.

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      Abstract

      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.

      Bibliographic metadata

      Type of resource:
      Content type:
      Form of thesis:
      Type of submission:
      Degree type:
      Doctor of Philosophy
      Degree programme:
      PhD Wellcome Trust - Molecular and Cell Biology 4yr (CMB)
      Publication date:
      Location:
      Manchester, UK
      Total pages:
      188
      Abstract:
      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.
      Thesis main supervisor(s):
      Thesis co-supervisor(s):
      Funder(s):
      Language:
      en

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        Record metadata

        Manchester eScholar ID:
        uk-ac-man-scw:306992
        Created by:
        Ramsay, Ewan
        Created:
        18th January, 2017, 14:48:44
        Last modified by:
        Ramsay, Ewan
        Last modified:
        3rd February, 2017, 10:54:35

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