Tools and tactics to define specificity of metabolic chemical reporters

Mana Mohan Mukherjee; Michelle R Bond; Lara K Abramowitz; Devin Biesbrock; Carolyn C Woodroofe; Eun Ju Kim; Rolf E Swenson; John A Hanover

doi:10.3389/fmolb.2023.1286690

Tools and tactics to define specificity of metabolic chemical reporters

Front Mol Biosci. 2023 Dec 7:10:1286690. doi: 10.3389/fmolb.2023.1286690. eCollection 2023.

Authors

Mana Mohan Mukherjee¹, Michelle R Bond¹, Lara K Abramowitz¹, Devin Biesbrock¹, Carolyn C Woodroofe², Eun Ju Kim³, Rolf E Swenson³, John A Hanover¹

Affiliations

¹ Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States.
² Frederick National Laboratory for Cancer Research, National Cancer Institute, Fredrick, MD, United States.
³ Department of Chemistry Education, Daegu University, Gyeongsan-si, South Korea.

Abstract

Metabolic chemical reporters (MCRs) provide easily accessible means to study glycans in their native environments. However, because monosaccharide precursors are shared by many glycosylation pathways, selective incorporation has been difficult to attain. Here, a strategy for defining the selectivity and enzymatic incorporation of an MCR is presented. Performing β-elimination to interrogate O-linked sugars and using commercially available glycosidases and glycosyltransferase inhibitors, we probed the specificity of widely used azide (Ac₄GalNAz) and alkyne (Ac₄GalNAlk and Ac₄GlcNAlk) sugar derivatives. Following the outlined strategy, we provide a semiquantitative assessment of the specific and non-specific incorporation of this bioorthogonal sugar (Ac₄GalNAz) into numerous N- and O-linked glycosylation pathways. This approach should be generally applicable to other MCRs to define the extent of incorporation into the various glycan species.

Keywords: O- and N-glycans; bioorthogonal chemistry; hexosamine biosynthetic pathway; metabolic chemical reporters; tools and tactics.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by NIDDK, National Institutes of Health, intramural research program.