In vitro characterisation of the A319T OGT XLID mutation (360G-Wellcome-207251_Z_17_Z)

Protein O-GlcNAcylation is an essential posttranslational modification regulated by two opposing highly conserved enzymes, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), influencing a wide range of biological processes. The dysregulation of the O-GlcNAc system is linked to neurodegeneration, cancer and diabetes. OGT consists of a catalytic domain, and an N-terminal domain comprising 13.5 tetratricopeptide repeats (TPRs). This TPR domain is believed to mediate protein-protein interactions. The OGT gene is located on the Xq13.1 band. Four XLID causing mutations were identified in the OGT gene so far. Residing in the TPR region, they lead to a decrease of steady-state global OGT protein levels and effects on the O-GlcNAc proteome. In my summer research project, I would like to focus on the A319T (c.1193G > A) missense mutation. Having been found in a French family with three XLID affected sons, this mutation is regarded as the possible pathogenic factor in this particular case. My key goals include the production of the A319T mutated OGT in E. coli, to be further on purified and characterised in vitro. The structural integrity will be checked and assays screening the enzymatic activity will be carried out. I hope to detect differences in enzyme kinetics caused by the structural changes.