Glycan structures are established from building blocks termed monosaccharides. Unlike the biosynthesis of nucleic acids and proteins that are directly commanded by genes, formation of a particular glycan relies on an interplay of hundreds of enzymes called glycosyltransferases (GTs) and glycosidases. 

Addressing which enzymes are present in a specific type of cell at a specific time poses a challenge but is still only part of the truth. Further complication is added by the interplay between these enzymes – they can compensate for each other, compete with each other, and rely on each other’s function. Technical difficulty in glycan analysis by mass spectrometry is often attributed to their low ionisation efficiency and complex branching structures, which comprise various linkage isomers. Microheterogeneity, which refers to different glycoforms found at the same glycosylation site, adds extra complexity.

To overcome these challenges, our lab has developed GT bump-and-hole engineering, where a single enzyme is engineered to contain an enlarged active site, a “hole”, that can accommodate a substrate analogue bearing a complementary sterically-congested group, a “bump”, so as to create reporters for the activity of the glycosyltransferase of interest. This technology has been implemented in vitro, in cellulo, and in vivo, enabling us to study a single enzyme in the presence of the whole glycan biosynthetic machinery.

Our “workhorse” family of GTs are the polypeptide GalNAc transferases, or GalNAc-Ts, that initiate mucin-type O-GalNAc glycosylation. The human genome encodes approximately 20 GalNAc-Ts, multiple of which can be present in the secretory pathway of a given cell at any point in time. O-GalNAc glycosylation is thus riddled by biosynthetic complexity that is being slowly untangled. Complementing existing approaches in molecular cell biology, bump-and-hole engineering will enhance our understanding of O-glycans. For more information, see our review paper in Current Opinion in Chemical Biology here (link: https://www.sciencedirect.com/science/article/pii/S1367593120301228)

Further references:

Gonzalez-Rodriguez et al., ACS Cent. Sci. 9, 2023 (insert link)

Calle et al., STAR Protoc. 4, 2023 (insert link)

Scott et al., Oncogene 42, 2023 (insert link)

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

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

Choi, Wagner et al., J. Am. Chem. Soc. 141, 2019 (insert link)