A suite of tools for the study of tryptophan C-mannosylation - additional point mutants

Tryptophan C-mannosylation is an unusual co-translational protein modification performed by metazoans and apicomplexan parasites. The prevalence of this modification and its biological function is poorly understood, with progress being hampered by a dearth of tools for its study. Here, we engineer of a simple yeast system to enable the production of a diverse array of proteins with and without the modification to interrogate its effect on protein stability and function. This system also facilitated mutagenesis studies, to elucidate what features of the glycosyltransferase and its substrates are crucial for catalysis, and expedited the generation and thorough characterisation of monoclonal antibodies against the protein modification. These antibodies empowered proteomic analyses of the brain C-glycome by enriching for peptides possessing tryptophan C-mannosylation, which revealed new modification sites on difficult-to-study proteins throughout the secretory pathway on both conventional and non-canonical consensus sequences.