title: The role of the medaka protein O-mannosyltransferase 2 across tissues, development and dystroglycanopathies
creator: Cornean, Alex
subject: 570
subject: 570 Life sciences
description: Protein O-mannosylation is a conserved modification of proteins with the sugar mannose. Defective O-mannosylation of the peripheral membrane protein α-dystroglycan (α-DG) results in a spectrum of congenital diseases called dystroglycanopathies (DGpathies). DGpathies manifest a broad range of symptoms from serious, prenatal changes in the morphology of the brain to adult-onset muscular dystrophy. Consequently, mutations in the genes encoding the protein O-mannosyltransferases 1 and 2 (POMT1/2), which catalyse the first steps of O-mannosylation, cause the most severe forms of DGpathies. Why different organs are distinctly affected in patients with varying grades of DGpathies is not clear. Moreover, the precise contribution of the known substrates of the POMT1-POMT2 complex, α-DG, SUCO and KIAA1549, to the pathology in DGpathies remains ill-defined. Therefore, the aim of this thesis was to resolve the organismal role of the POMT1-POMT2 complex in DGpathies and to disentangle the contributions of its substrates.    To address these questions, I created a framework for highly efficient base editing to mutate virtually any cytosine or adenine in the teleost fish genome. Using CRISPR/Cas9 and base editors, I established pomt2 DGpathy models in medaka (Oryzias latipes) and characterised these using behavioural, biochemical, histological and transcriptomic analyses. I could show that organs such as the eye and the brain are only affected when POMT-complex function was substantially disrupted. By contrast, the muscles and spine are much more prone to minute changes in the enzymatic properties of the POMTs. Next, I disrupted the POMT-substrates individually using a CRISPR/Cas9 approach. I identified a previously unknown cardiovascular function of α-DG and SUCO, and revealed that SUCO plays an important role in notochord development. Finally, I employed base editing to mutate O-mannosylation glycosites on α-DG (T330) and SUCO (S806 and T811), which partially recapitulated respective loss-of-function phenotypes. With this first-ever evidence for the study of glycosites using base editing in vivo, I demonstrate the feasibility of this new approach to provide functional insights in a developing organism.    Future studies linking structure-function relationships on the substrate level to tissue-wide developmental consequences in model systems will be instrumental to enable tailored, preclinical drug screens and to provide reliable predictions of disease progressions in DGpathies.
date: 2022
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/32151/1/202205_PhDThesis_AC_pdfA.pdf
identifier: DOI:10.11588/heidok.00032151
identifier: urn:nbn:de:bsz:16-heidok-321511
identifier:   Cornean, Alex  (2022) The role of the medaka protein O-mannosyltransferase 2 across tissues, development and dystroglycanopathies.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/32151/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng