Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation
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Marjoke F Debets Omur Yilmaz Tastan Simon P Wisnovsky Stacy A Malaker Nikos Angelis Leonhard KR Moeckl Junwon Choi Helen Flynn Lauren JS Wagner Ganka Bineva-Todd Aristotelis Antonopoulos Anna Cioce William Browne Zhen Li David Briggs Holly Douglas Gaelen T Hess Anthony J Agbay Chloe Roustan Svend Kjaer Stuart M Haslam Bram Snijders Michael C Bassik WE Moerner Vivian Li Carolyn R Bertozzi Ben Schumann Toggle all authors (27)
Abstract
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 N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-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.
Journal details
Volume 117
Issue number 41
Pages 25293-25301
Available online
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Publisher website (DOI) 10.1073/pnas.2007297117
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Europe PubMed Central 32989128
Pubmed 32989128
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- Tumour Biology
- Synthetic Biology
- Structural Biology & Biophysics
- Stem Cells
- Signalling & Oncogenes
- Metabolism
- Infectious Disease
- Human Biology & Physiology
- Genetics & Genomics
- Developmental Biology
- Computational & Systems Biology
- Chemical Biology & High Throughput
- Cell Biology
- Biochemistry & Proteomics
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