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@article{Debets:2020:10.1073/pnas.2007297117,
author = {Debets, MF and Tastan, OY and Wisnovsky, SP and Malaker, SA and Angelis, N and Moeckl, LKR and Choi, J and Flynn, H and Wagner, LJS and Bineva-Todd, G and Antonopoulos, A and Cioce, A and Browne, WM and Li, Z and Briggs, DC and Douglas, HL and Hess, GT and Agbay, AJ and Roustan, C and Kjaer, S and Haslam, S and Snijders, AP and Bassik, MC and Moerner, WE and Li, VSW and Bertozzi, CR and Schumann, B},
doi = {10.1073/pnas.2007297117},
journal = {Proceedings of the National Academy of Sciences of USA},
pages = {25293--25301},
title = {Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation},
url = {http://dx.doi.org/10.1073/pnas.2007297117},
volume = {117},
year = {2020}
}

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TY  - JOUR
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 by the 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 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 knock-out (KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, 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.
AU  - Debets,MF
AU  - Tastan,OY
AU  - Wisnovsky,SP
AU  - Malaker,SA
AU  - Angelis,N
AU  - Moeckl,LKR
AU  - Choi,J
AU  - Flynn,H
AU  - Wagner,LJS
AU  - Bineva-Todd,G
AU  - Antonopoulos,A
AU  - Cioce,A
AU  - Browne,WM
AU  - Li,Z
AU  - Briggs,DC
AU  - Douglas,HL
AU  - Hess,GT
AU  - Agbay,AJ
AU  - Roustan,C
AU  - Kjaer,S
AU  - Haslam,S
AU  - Snijders,AP
AU  - Bassik,MC
AU  - Moerner,WE
AU  - Li,VSW
AU  - Bertozzi,CR
AU  - Schumann,B
DO  - 10.1073/pnas.2007297117
EP  - 25301
PY  - 2020///
SN  - 0027-8424
SP  - 25293
TI  - Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation
T2  - Proceedings of the National Academy of Sciences of USA
UR  - http://dx.doi.org/10.1073/pnas.2007297117
UR  - https://www.pnas.org/content/117/41/25293
UR  - http://hdl.handle.net/10044/1/82802
VL  - 117
ER  -

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