Optimization of Metabolic Oligosaccharide Engineering with Ac 4 GalNAlk and Ac 4 GlcNAlk by an Engineered Pyrophosphorylase

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 t...

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Bibliographic Details
Published in:ACS chemical biology 2021-10, Vol.16 (10), p.1961-1967
Main Authors: Cioce, Anna, Bineva-Todd, Ganka, Agbay, Anthony J, Choi, Junwon, Wood, Thomas M, Debets, Marjoke F, Browne, William M, Douglas, Holly L, Roustan, Chloe, Tastan, Omur Y, Kjaer, Svend, Bush, Jacob T, Bertozzi, Carolyn R, Schumann, Benjamin
Format: Article
Language:English
Subjects:
Alkynes - chemistry
Amino Acid Sequence
Animals
Azides - chemistry
Cell Line, Tumor
Click Chemistry
Fluorescent Dyes - chemistry
Galactosamine - analogs & derivatives
Galactosamine - metabolism
Galactosyltransferases - metabolism
Glucosamine - analogs & derivatives
Glucosamine - metabolism
Glycoproteins - chemistry
Glycoproteins - metabolism
Glycosylation
Humans
Metabolic Engineering - methods
Mice
Molecular Probes - chemistry
Oligosaccharides - biosynthesis
Polysaccharides - biosynthesis
Uridine Diphosphate Sugars - biosynthesis
Uridine Diphosphate Sugars - metabolism
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Summary: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 Ac GalNAlk and Ac GlcNAlk 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.
ISSN:1554-8929
1554-8937
DOI:10.1021/acschembio.1c00034