Exploring novel chemical and enzymatic labelling approaches in metabolic oligosaccharide engineering of mammalian cells

Glycoproteins have many important roles in cell signalling and diseases including cancer, neurodegeneration, and pathogenic infections. Metabolic oligosaccharide engineering has previously been used to incorporate unnatural sugar derivatives into glycoproteins and detect them with bioorthogonal labelling. Additional methods of labelling that work orthogonally to current methods, and are reversible, would allow the simultaneous investigation of multiple glycoproteins and their properties. Sortase is a transpeptidase found in Gram-positive bacteria that has been previously engineered to perform ligations of glycine- and LPXTG- bearing biomolecules. This project sought to use sortase to specifically label functional groups carried by sugar derivatives incorporated into glycoproteins. Initial incorporation of sugar derivatives bearing glycine-azide showed successful labelling with click chemistry. Subsequent attempts to label incorporated sugar derivatives bearing glycine-amine with the commercial sortase Srt5M failed. An alternative mannosamine-thiazolidine sugar was tolerated in HEK cells up to 300 μM. Live cells that had incorporated this sugar were successfully labelled using the organocatalyst-mediated protein aldol ligation. Labelling of intracellular glycoproteins was also tested but requires further optimisation. To expand the currently available sortases, 22 novel sortases were cloned and expressed from a metagenome database at the industrial partner Prozomix. Testing for the ability to ligate a range of peptides in vitro revealed two functioning enzymes, which were named Srt021 (class C) and Srt025 (class D). The two sortases were shown to function well at physiological temperature and pH, and in a range of buffers including without calcium. This represents the second class C sortase made to function in vitro, and the first class D sortase in vitro. Further characterisation and directed evolution of these two sortases should result in a wider range of tools for specific labelling of biomolecules.