Jenny Hayes

CASE – Metabolic oligosaccharide engineering (MOE) using Sortases

About me

Jenny Hayes completed an integrated Master’s in Biochemistry at the University of Oxford in 2012 and spent a few years working in industry before starting a PhD in Biological Chemistry at the University of York in 2016 supervised by Dr Martin Fascione. Her research interests include glycoprotein modification using unnatural sugars, bioorthogonal chemistry, and protein engineering.

My project

Metabolic oligosaccharide engineering (MOE) involves the exploitation of carbohydratebiosynthetic pathways in bacterial and mammalian cells with the aim of reengineering cell surfaceglycoproteins. Classically, cells are fed sugar precursors bearing latently reactive functionalgroups, these are almost always azides which can be modified with fluorescent/or purification tagsusing staudinger ligations, Cu-catalysed click reactions, or strain promoted click reactions. Thistechnique has facilitated fluorescent imaging inside live cells, and even dynamically insidezebrafish embryos. MOE has also been used to map, detect and enrich glycoproteins, specificallyGlcNAc modified and sialic acid capped glycoproteins. ManAc derivatives bearing azide groupsare metabolised by the mammalian biosynthetic machinery and incorporated into glycoproteins asmodified sialic acids. Ac4Glc/GalNAz can be similarly metabolised and incorporated as O-GlcNAcsugars via salvage pathways, however the levels of incorporation are low (due to “biologicalcrosstalk”) and the Glc/GalNAz moieties are also incorporated into N-glycans and O-mucinspreventing the specific detection of O-GlcNAcylated proteins, which are increasingly importantbiological targets, and play prominent roles in disease etiology. In this project we willreinvigorate glycoprotein detection by using sortase enzymes for MOE and use acombination of directed evolution and mining of gram-positive bacterial metogenomes todevelop a universal suite of Sortase A/B variants capable of specific detection of distinctsugars in live cells. Class A/B Sortases are transpeptidase enzymes from gram-positive bacteriathat attach virulence factors to cells, by ligating the N-terminal residue of peptidoglycan onto thethreonine of C-terminal sortase recognition sequences in virulence proteins (LPXTG for SrtA fromS. aureus, LPXTA for SrtA from S. pyogenes, or NPQTN for SrtB for example) with the expulsionof a C-terminal residue.


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