Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Studying posttranslationalmodifications classically relies on experimental strategies that oversimplify the complex biosyntheticmachineries 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 anduse 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 polypeptideN-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionalitymodification on cells, where the products of individual glycosyltransferases can be selectively characterizedormanipulated to understandglycan contributiontomajorphysiological processes.