Metabolic precision labeling enables selective probing of O-linked <i>N</i> -acetylgalactosamine glycosylation
Marjoke F. Debets, Ömür Y. Tastan, Simon Wisnovsky, Stacy A. Malaker, Nikolaos Angelis, Leonhard Moeckl, Junwon Choi, Helen R. Flynn, Lauren J. S. Wagner, Ganka Bineva‐Todd, Aristotelis Antonopoulos, Anna Cioce, William M. Browne, Zhen Li, David C. Briggs, Holly Douglas, Gaelen T. Hess, Anthony J. Agbay, Chloë Roustan, Svend Kjær, Stuart M. Haslam, Ambrosius P. Snijders, Michael C. Bassik, W. E. Moerner, Vivian Li, Carolyn R. Bertozzi, Benjamin Schumann
Abstract
-acetylgalactosamine-4-epimerase (GALE) 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 transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.