Deciphering the causes and effects of O-GlcNAc Transferase nucleotide variants in neurodevelopmental disorders

  • Veronica Maria Pravata

    Student thesis: Doctoral ThesisDoctor of Philosophy


    Glycosylation is an essential cellular process where individual monosaccharides are arranged to form the most diverse type of protein or lipid modification known. While half of all cellular proteins are directly glycosylated in some form, it has been estimated that ~2% of all genes in the human genome encode proteins involved in various aspects of glycosylation. Among the several forms of glycosylation, the addition of a single moiety of O-linked N-acetylglucosamine (O-GlcNAc) stands out for its uniqueness. This highly dynamic modification, termed O-GlcNAcylation, is mediated by a single pair of enzyme, namely the O-GlcNAc Transferase (OGT) and the hydrolase O-GlcNAcase (OGA). The addition of O-GlcNAc onto serine and threonine of 4000 intracellular proteins plays a key role in regulating stress response, differentiation, nutrient sensing, and autophagy. Additionally, OGT is known to be essential for normal development of the vertebrate embryo, as ablation of the gene is incompatible to life.

    Recent studies have suggested that missense mutations distal to the OGT catalytic domain led to Intellectual Disability (ID), a generalized neurodevelopmental disorder characterized by significantly impaired intellectual and adaptive functioning. Biochemical and structural investigations tried to identify how missense mutations in the Tetratricopeptide (TPR) domain of OGT associated to ID. However, it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein-protein interactions or OGlcNAc-mediated misprocessing of the Host Cell Factor 1 (HCF1).

    The work described in this thesis was conducted with the aim of understanding how OGT missense mutations link to neurodevelopmental delay, with a particular focus on the underpinning biological mechanism.

    In the second chapter of this thesis, I describe the characterization of the first missense mutation identified in the OGT catalytic domain of patients suffering from Congenital Disorders of Glycosylation (CDG), an umbrella term that embraces inborn defects of metabolism caused by inherited defects in the synthesis of glycans. Molecular analyses revealed decreased OGT activity and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into mouse embryonic stem cells cause delayed differentiation down the neuronal lineage. These data are the first direct link between OGT mutations and developmental delay in patients.

    In chapter three, I further investigate the role of OGT-CDG mutations characterizing a novel missense mutation identified in the catalytic domain of a patient suffering of CDG. Using a combination of biochemical analyses and gene editing in mouse embryonic stem cells, I demonstrated that catalytic deficiency of OGT could contribute to the CDG phenotype. Notably, this novel missense mutation shows for the first time a potential link between global changes in the O-GlcNAc proteome and OGTCDG mutations.

    Lastly, in chapter four, I investigate the mechanistic causes of OGT-CDG mutations through proteomic studies. To achieve that, I engineer mouse embryonic stem cells carrying two different OGT-CDG mutations, which were chosen because of their severity. Using Tandem Mass Tag (TMT) proteomics, I show that OGT-CDG mouse embryonic stem cells increased their levels of OGT and several proteins linked to neurodevelopmental delay. Among these, I show the potential effects of β-TrCP, the substrate recognition component of a SCF E3 ubiquitin-protein ligase complex, and Zscan4, an early marker of 2-cell stage and totipotency. In cellulo analyses demonstrate that OGT upregulation is associated with increased formation of the Ogt-Tet1/2 complex, which may explain the deregulated levels of Zscan4, and may cause changes in the epigenetic landscape of OGT-CDG mESCs and the reduced neurite outgrowth phenotype.

    Overall, this work provides initial progress towards understanding the effect of ID-associated mutations on OGT. Through this work, we redefined these mutations as OGT-linked Congenital Disorders of Glycosylation (OGT-CDG), an umbrella term for a rapidly expanding group of rare genetic, metabolic disorders. Importantly, this work established a useful reductionist model to investigate OGT-CDG mutations, and model neurodevelopmental delay identified in patients. Finally, this thesis lays the foundations for the identifications of the underlying biological causes of OGT-CDG neurodevelopmental defects.
    Date of Award2021
    Original languageEnglish
    SponsorsChief Scientist Office
    SupervisorDaan van Aalten (Supervisor)

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