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Cioce Bineva-Todd et al.pdf (2.11 MB)

Metabolic Engineering Optimizes Bioorthogonal Glycan Labeling in Living Cells

preprint
submitted on 02.01.2021, 17:49 and posted on 04.01.2021, 13:18 by Anna Cioce, Ganka Bineva-Todd, Anthony J. Agbay, Junwon Choi, Thomas M. Wood, Marjoke Debets, William Browne, Holly Douglas, Chloe Roustan, Omur Yilmaz Tastan, Svend Kjaer, Jacob T. Bush, Carolyn Bertozzi, Benjamin Schumann
Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. Comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

Funding

Cancer Research UK Grant FC001749

Medical Research Council Grant FC001749

Wellcome Trust Grant FC001749

NIH Grant R01 CA200423

EPSRC Centre for Doctoral Training in Chemical Biology – Innovation for the Life Sciences Grant EP/S023518/1

Wellcome Trust New Investigator Award 104785/B/14/Z

Dutch Research Council (NWO) Rubicon Postdoctoral Fellowship

GlaxoSmithKline

History

Email Address of Submitting Author

b.schumann@imperial.ac.uk

Institution

Imperial College London, The Francis Crick Institute

Country

United Kingdom

ORCID For Submitting Author

0000-0001-5504-0147

Declaration of Conflict of Interest

The authors declare no conflict of interest.

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