2. Academic Validation
  3. Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase

  • ACS Chem Biol. 2021 Oct 15;16(10):1961-1967. doi: 10.1021/acschembio.1c00034.
Anna Cioce 1 2,  Ganka Bineva-Todd 2,  Anthony J Agbay 3,  Junwon Choi 3,  Thomas M Wood 3,  Marjoke F Debets 3,  William M Browne 1 2,  Holly L Douglas 4,  Chloe Roustan 5,  Omur Y Tastan 2,  Svend Kjaer 5,  Jacob T Bush 6,  Carolyn R Bertozzi 3 7,  Benjamin Schumann 1 2
Affiliations

Affiliations

  • 1 Department of Chemistry, Imperial College London, 80 Wood Lane, W12 0BZ London, United Kingdom.
  • 2 The Chemical Glycobiology Laboratory, The Francis Crick Institute, 1 Midland Road, NW1 1AT London, United Kingdom.
  • 3 Department of Chemistry, Stanford University, Stanford, California 94305, United States.
  • 4 Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, 1 Midland Road, NW1 1AT London, United Kingdom.
  • 5 Structural Biology Science Technology Platform, The Francis Crick Institute, NW1 1AT London, United Kingdom.
  • 6 GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY United Kingdom.
  • 7 Howard Hughes Medical Institute, 380 Roth Way, Stanford, California 94305, United States.
PMID: 33835779 DOI: 10.1021/acschembio.1c00034
Abstract

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.

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