Metabolic precision labeling enables selective probing of O-linked
| Autor: | Marjoke F, Debets, Omur Y, Tastan, Simon P, Wisnovsky, Stacy A, Malaker, Nikolaos, Angelis, Leonhard K R, Moeckl, Junwon, Choi, Helen, Flynn, Lauren J S, Wagner, Ganka, Bineva-Todd, Aristotelis, Antonopoulos, Anna, Cioce, William M, Browne, Zhen, Li, David C, Briggs, Holly L, Douglas, Gaelen T, Hess, Anthony J, Agbay, Chloe, Roustan, Svend, Kjaer, Stuart M, Haslam, Ambrosius P, Snijders, Michael C, Bassik, W E, Moerner, Vivian S W, Li, Carolyn R, Bertozzi, Benjamin, Schumann |
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| Rok vydání: | 2020 |
| Předmět: |
bioorthogonal
Acetylgalactosamine Glycosylation Racemases and Epimerases Cell Biology Biological Sciences glycosyltransferase Gene Expression Regulation Enzymologic Substrate Specificity carbohydrates (lipids) Chemistry mucin Uridine Diphosphate N-Acetylgalactosamine Physical Sciences Humans lipids (amino acids peptides and proteins) Glycoproteins |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 |
| Popis: | Significance Most human secreted and cell surface proteins are modified by Ser/Thr(O)-linked glycosylation with N-acetylgalactosamine (O-GalNAc). While of fundamental importance in health and disease, O-GalNAcglycosylation is technically challenging to study because of a lack of specific tools for biological assays. Here, we design an O-GalNAc–specific reporter molecule termed uridine diphosphate (UDP)–N-(S)-azidopropionylgalactosamine (GalNAzMe) to selectively label O-GalNAc glycoproteins in living human cells. UDP-GalNAzMe can be biosynthesized in cells by transfection with an engineered metabolic enzyme and is compatible with a range of experiments in quantitative biology to broaden our understanding of glycosylation. We demonstrate that labeling is genetically programmable by ectopic expression of a mutant glycosyltransferase, “bump-and-hole”–GalNAc-T2, allowing application to experiments with low inherent sensitivity. 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. |
| Databáze: | OpenAIRE |
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