Proteomics

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4-deoxy-4-fluoro-GalNAz (4FGalNAz) is a metabolic chemical reporter of O-GlcNAc modifications, highlighting the notable substrate flexibility of O-GlcNAc transferase


ABSTRACT: Bioorthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogs of monosaccharides that contain bioorthogonal functionality, like azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bioorthogonal 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 disappointing from the standpoint of a mucin-selective MCR, 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christina Woo  

LAB HEAD: Christina Woo

PROVIDER: PXD027333 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

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

Jackson Emma G EG   Cutolo Giuliano G   Yang Bo B   Yarravarapu Nageswari N   Burns Mary W N MWN   Bineva-Todd Ganka G   Roustan Chloë C   Thoden James B JB   Lin-Jones Halley M HM   van Kuppevelt Toin H TH   Holden Hazel M HM   Schumann Benjamin B   Kohler Jennifer J JJ   Woo Christina M CM   Pratt Matthew R MR  

ACS chemical biology 20211221 1


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 v  ...[more]

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