4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Emma G Jackson,Ganka Bineva-Todd,Hazel M Holden,Mary W N Burns,Chloë Roustan,Toin H Van Kuppevelt,Christina M Woo,Jennifer J Kohler,James B Thoden,Matthew R Pratt,Benjamin Schumann,Giuliano Cutolo,Bo Yang,Halley M Lin-Jones,Nageswari Yarravarapu

doi:10.1021/acschembio.1c00818

Abstract

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

Highlights

Metabolic chemical reporters (MCRs) of protein glycosylation are powerful chemical tools that have been used for over a decade to identify and characterize different types of glycans (Figure 1a).[1,2] MCRs are typically analogues of naturally occurring monosaccharides that contain bio-orthogonal functionalities at different positions of the sugar ring
With IsoTaG, we identified 67 GalNAz-modified proteins significantly enriched over the DMSO control, and we localized GalNAz to 147 unique peptides (122 sites at S/T and 29 sites at C after filtering for peptide spectral matches ≥2; corresponds to 34 unambiguous glycosites identified by EThcD and delta Mod ≥10, Tables S2 and S3), representing the expected mixture of cell-surface and intracellular glycoproteins
O-GlcNAc transferase (OGT) substrate.[24−26] We found that UDP-4FGalNAz was a substrate accepted about 2.5 times better than UDPGalNAc but less efficiently than UDP-GlcNAc or UDPGlcNAz

Summary

Introduction

Metabolic chemical reporters (MCRs) of protein glycosylation are powerful chemical tools that have been used for over a decade to identify and characterize different types of glycans (Figure 1a).[1,2] MCRs are typically analogues of naturally occurring monosaccharides that contain bio-orthogonal functionalities at different positions of the sugar ring. If these chemical modifications are relatively small, MCRs can take advantage of carbohydrate salvage pathway enzymes with different levels of substrate tolerance to yield the corresponding nucleotide sugar donors for subsequent transfer onto proteins by glycosyltransferases.

Methods

Results

Conclusion