GlycOTag - Precision Tools to unravel the fundamentals of O-glycan biosynthesis

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Glycans are essential for life and important mediators of biological processes. Despite slowly advancing from an enigma to scientific mainstream, their biosynthesis is one of the least understood issues in cell biology. In this multidisciplinary proposal, I will develop 'precision tools' that are customized to image and map the complex processes of adding glycans to proteins in the living cell.Innovations in quantitative biology often fall short when enzymatic reactions are involved that cannot be predicted from nucleic acid sequence alone. This is a particular challenge for the addition of glycans to proteins in the secretory pathway that is mediated by the combinatorial interplay of 250 glycosyltransferases. To delineate how glycan biosynthesis is coordinated, methods are needed to inform on the functional interplay between glycosyltransferases.To address this unmet need, I will develop chemical tools as traceable reporters for the activities of individual glycosyltransferases in the living cell. These tools will allow direct insight into the large GalNAc transferase enzyme family that introduces O-linked glycans as one of the most abundant and complex protein modifications. Cutting-edge methods of mass spectrometry, super-resolution microscopy and correlative light and electron microscopy will collectively reveal which enzyme acts when and where on glycoproteins trafficking through the secretory pathway. This paradigm-shifting work is only possible through my discovery of a tactic to engineer GalNAc transferases to selectively accommodate traceable sugar analogs as substrates that are not used by wildtype transferases. The sugars proposed here are armed with chemical tags that can be traced with fluorophores. My work will thus provide unprecedented insight into the details of protein maturation in the secretory pathway, a major process in cell biology with many unanswered questions.