Building on the foundational work by Werner Reutter, the Nobel Prize in 2022 to Carolyn R. Bertozzi has recognised the importance of Chemistry to understanding glycans. Building on the contributions by many great chemists in the field, we are creating tools for metabolic oligosaccharide engineering (MOE) approaches that probe glycans in a variety of biological settings.

MOE reagents are synthetically modified monosaccharide analogues with functional groups amenable to biorthogonal ligation and consequent tracking of glycoconjugates. To enter the quantitative glycosciences, we develop such tools to report on the presence of specific subtypes of glycans, or of glycoconjugates made by individual cell types. Our work demonstrates that tailoring either the chemical tag or the metabolic processing provides an entry into such biological specificity.

Selective chemical tools are achieved by designing "bulky" GalNAc analogues that do not show promiscuity toward certain endogenous enzymes involved in biosynthetic pathways. We recently found that introducing additional steric bulk on the promiscuous probe UDP-GalNAz leads to specific labelling of O-GalNAc glycans. The resulting probe UDP-GalNAz(Me) is resistant to epimerisation to the corresponding UDP-GlcNAc analogue by the enzyme GALE (UDP-glucose-4-epimerase) that would otherwise lead to “dilution” of cell-surface labelling signal to several glycan subtypes.

Key enzymes in the GalNAc salvage pathway (in human cells, GalK2 and AGX1) are inactive towards GalNAc derivatives containing a large acylamide modification. Initially, we bypassed the second step of the GalNAc salvage pathway by using an engineered version of AGX1, based on the groundbreaking work by Kohler and colleagues (Yu et al., PNAS 2021). We then also bypassed the first step, a phosphorylation event, by expressing the bacterial kinase NahK in human cells. Our strategy turned the apparent bottleneck of bulky MOE reagents into an advantage, as an artificial metabolic pathway conferred cell specificity: Using Bio-Orthogonal Cell-specific Tagging of Glycoproteins (BOCTAG), we created a tool to selectively profile glycoproteins of a given cell type (BOCTAG)  in complex co-culture systems. We are currently applying the technology to diverse cellular and in vivo model systems.

We are also developing tools to improve the analysis of glycoconjugates. For instance, we found that the analysis of glycopeptides by mass spectrometry is enhanced when clickable tags are used that introduce a positive charge into the glycopeptide of interest.

Further references:

Ciocce et al., Nat. Commun. 13, 2022 (insert link)

Cioce, Bineva-Todd et al., ACS Chem. BIol. 16 (2021) (insert link).

Calle et al., J. Am. Soc. Mass Spectrom. 32 (2021) (insert link)

Cioce et al., Cur.. Opin. Chem. Biol. 60, 2021 (insert link)

Debets, Tastan et al., Proc. Natl Acad. Sci. USA 117, 2020 (insert link)

Zol-Hanlon and Schumann, Commun. Chem. 3 (2020) (insert link)

Schumann et al., Mol. Cell 78, 2020 (insert link)