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Optimization of metabolic oligosaccharide engineering with Ac4GalNAlk and Ac4GlcNAlk by an engineered pyrophosphorylase.

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journal contribution
posted on 03.11.2021, 10:43 by Anna Cioce, Ganka Bineva-Todd, Anthony J Agbay, Junwon Choi, Thomas M Wood, Marjoke F Debets, William M Browne, Holly L Douglas, Chloe Roustan, Omur Y Tastan, Svend Kjaer, Jacob T Bush, Carolyn R 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. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

Funding

Crick (Grant ID: 10749, Grant title: Schumann FC001749) Crick (Grant ID: 10015, Grant title: STP Structural Biology)

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