10779/crick.12472733.v1 Benjamin Schumann Benjamin Schumann Stacy Alyse Malaker Stacy Alyse Malaker Simon Peter Wisnovsky Simon Peter Wisnovsky Marjoke Froukje Debets Marjoke Froukje Debets Anthony John Agbay Anthony John Agbay Daniel Fernandez Daniel Fernandez Lauren Jan Sarbo Wagner Lauren Jan Sarbo Wagner Liang Lin Liang Lin Zhen Li Zhen Li Junwon Choi Junwon Choi Douglas Michael Fox Douglas Michael Fox Jessie Peh Jessie Peh Melissa Anne Gray Melissa Anne Gray Kayvon Pedram Kayvon Pedram Jennifer Jean Kohler Jennifer Jean Kohler Milan Mrksich Milan Mrksich Carolyn Ruth Bertozzi Carolyn Ruth Bertozzi Bump-and-hole engineering identifies specific substrates of glycosyltransferases in living cells. The Francis Crick Institute 2020 O-glycosylation bioorthogonal chemical proteomics glycosyltransferase isoenzyme mucin Schumann FC001749 CS-ack 06 Biological Sciences 11 Medical and Health Sciences Developmental Biology 2020-06-25 14:04:16 Journal contribution https://crick.figshare.com/articles/journal_contribution/Bump-and-hole_engineering_identifies_specific_substrates_of_glycosyltransferases_in_living_cells_/12472733 Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.