Every cell in the body decorates its proteins with sugar chains called glycans, yet no one knows exactly how the cellular machinery decides which sugars to add and when. This project builds chemical tools that act as trackable reporters for individual enzymes—specifically the GalNAc transferase family—so researchers can watch, in real time, which enzyme adds a sugar to a protein as it moves through the cell’s secretory pathway. The problem is that glycan biosynthesis is controlled by around 250 enzymes whose actions cannot be predicted from DNA sequence alone. Current methods cannot distinguish which enzyme does what, leaving a fundamental gap in cell biology. The researcher has discovered a way to engineer GalNAc transferases to accept traceable sugar analogs that wildtype enzymes ignore. By combining mass spectrometry, super-resolution microscopy, and correlative light-electron microscopy, these tools will reveal the timing and location of each enzymatic step. This is fundamental science. If it works, it will provide the first direct view of how protein maturation is choreographed inside living cells. Past fundamental work on glycosylation has underpinned everything from cancer biomarker discovery to the production of therapeutic antibodies. A clearer picture of this process could eventually improve how we manufacture biologics or understand diseases where glycosylation goes wrong.
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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.
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