Caught in the act: precision tools to unravel and diagnose glycoprotein misfolding

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Reporters of cellular glycosylation: Engineering N-acetylglucosaminyl (GlcNAc) transferases- One of the major types of protein glycosylation, Asn(N)-linked glycosylation, undergoes extensive maturation in the secretory pathway in an assembly line principle. Crucial bifurcation points in glycan maturation are generated through introduction of GlcNAc at defined positions (https://www.ncbi.nlm.nih.gov/books/NBK453020/).- Dysfunctional N-glycosylation is a major cause of rare congenital disorders of glycosylation (CDGs) that are often complex and present with severe developmental/neurological symptoms.- We have the capacity to bump-and-hole engineer the GlcNAc transferases MGAT1, MGAT2 and MGAT5 to accept bioorthogonally labeled, bumped nucleotide-sugar substrates (https://doi.org/10.1016/j.cbpa.2020.09.001) to allow identification of CDG-relevant substrates and create reporter systems for glycoprotein maturation at different stages of the secretory pathway.- Objectives:o Engineering MGAT1, MGAT2 and/or MGAT5 to accept bumped versions of the substrate UDP-GlcNAc.o Synthesis of bumped UDP-sugars using chemoenzymatic synthesis.o Cellular delivery of the substrate, click/enrichment/proteomics of model glycoproteins to benchmark the approach.o Application to profile protein trafficking by imaging (e.g. through bioorthogonal ligation with cell-permeable turn-on fluorophores).o Establishment of a toolbox to probe protein maturation/recycling and shed light on CDGs, e.g. by