Title:
Development of conformational myocilin antibodies, and biophysical characterisation of the mouse myocilin olfactomedin domain
Development of conformational myocilin antibodies, and biophysical characterisation of the mouse myocilin olfactomedin domain
Author(s)
Patterson-Orazem, Athena Capucine
Advisor(s)
Lieberman, Raquel L.
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Abstract
Mutations in myocilin are causative for the heritable form of open angle glaucoma in humans yet, almost 20 years after its discovery, the function of myocilin within the trabecular meshwork (TM) eye remains elusive. In this thesis, structural insights into the myocilin unique Y-shaped tetrameric architecture composed of N-terminal coiled coils and C-terminal olfactomedin (OLF) domain were employed to identify the target epitope and conformational specificity of popular myocilin-targeted antibodies. While available commercial antibodies target a range of structural domains, none were specific to natively folded myocilin. This prompted the development of new antibodies targeting folded epitopes across myocilin that are further cross-reactive for human and mouse proteins, to streamline reagent use in the laboratory. As part of the development of antibodies targeting the OLF domain, the crystal structure of mouse OLF was solved to 1.5 Å resolution. Further characterization of wild-type mouse OLF and mutants associated with glaucoma phenotypes in humans, revealed similar structure and stability between mouse and human OLF. However, aggregation kinetics for mouse OLF differ from humans, with implications for the relevance of mouse models for myocilin-associated glaucoma. For the N-terminal coiled coil regions, three new antibodies were developed. All three exhibit conformational selectivity and immunoprecipitate endogenous myocilin secreted from TM primary cells, and two are cross-reactive to both human and mouse myocilin, offering the opportunity to track folded myocilin in a variety of mouse and human tissues with a single reagent. These new antibodies offer the broader glaucoma research community the opportunity to track functional N-terminal myocilin in mouse and human samples, towards a deeper molecular understanding of both healthy and glaucomatous eye physiology.
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Date Issued
2019-04-02
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Text
Resource Subtype
Dissertation