RT Journal Article SR Electronic T1 Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.04.23.057208 DO 10.1101/2020.04.23.057208 A1 Marjoke F. Debets A1 Omur Y. Tastan A1 Simon P. Wisnovsky A1 Stacy A. Malaker A1 Nikolaos Angelis A1 Leonhard K. R. Moeckl A1 Junwon Choi A1 Helen Flynn A1 Lauren J. S. Wagner A1 Ganka Bineva-Todd A1 Aristotelis Antononopoulos A1 Anna Cioce A1 William M. Browne A1 Zhen Li A1 David C. Briggs A1 Holly L. Douglas A1 Gaelen T. Hess A1 Anthony J. Agbay A1 Chloe Roustan A1 Svend Kjaer A1 Stuart M. Haslam A1 Ambrosius P. Snijders A1 Michael C. Bassik A1 W. E. Moerner A1 Vivian S. W. Li A1 Carolyn R. Bertozzi A1 Benjamin Schumann YR 2020 UL http://biorxiv.org/content/early/2020/04/25/2020.04.23.057208.abstract AB Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn (N)-linked glycans. We equip cells with the capacity to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a caged precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.Significance statement A large portion of all secreted and cell surface proteins in humans are modified by Ser/Thr(O)-linked glycosylation with N-acetylgalactosamine (GalNAc). While of fundamental importance in health and disease, O-GalNAc glycosylation is technically challenging to study because of a lack of specific tools to be used in biological assays. Here, we design an O-GalNAc specific reporter molecule termed GalNAzMe to selectively label O-GalNAc glycoproteins in living human cells. GalNAzMe is compatible with a range of experiments in quantitative biology to broaden our understanding of glycosylation. We further demonstrate that labeling is genetically programmable by expression of a mutant glycosyltransferase, allowing application even to experiments with low inherent sensitivity.Competing Interest StatementThe authors have declared no competing interest.