Chemical reporters to study mammalian O-glycosylation

Abstract

Glycans play essential roles in a range of cellular processes and have been shown to contribute to various pathologies. The diversity and dynamic nature of glycan structures and the complexities of glycan biosynthetic pathways make it challenging to study the roles of specific glycans in normal cellular function and disease. Chemical reporters have emerged as powerful tools to characterise glycan structures and monitor dynamic changes in glycan levels in a native context. A variety of tags can be introduced onto specific monosaccharides via the chemical modification of endogenous glycan structures or by metabolic or enzymatic incorporation of unnatural monosaccharides into cellular glycans. These chemical reporter strategies offer unique opportunities to study and manipulate glycan functions in living cells or whole organisms. In this review, we discuss recent advances in metabolic oligosaccharide engineering and chemoenzymatic glycan labelling, focusing on their application to the study of mammalian O-linked glycans. We describe current barriers to achieving glycan labelling specificity and highlight innovations that have started to pave the way to overcome these challenges.

Keywords: chemical biology; chemical reporter; chemoenzymatic labelling; glycobiology; glycosylation; metabolic engineering.

Figure 1.. Chemical reporter strategies to study glycans.

(A) Chemical or enzymatic oxidation of glycans enables the labelling of specific monosaccharide residues by reaction with amine-functionalised reporter groups. (B) Metabolic oligosaccharide engineering makes use of the cell's endogenous glycan biosynthetic machinery to install unnatural monosaccharide derivatives into glycans. The introduced tags can be further labelled by bioorthogonal ‘click’ reactions. (C) Chemoenzymatic glycan labelling exploits the activity of recombinant glycosyltransferases to transfer unnatural monosaccharides onto specific glycans.

Figure 2.. Chemical oxidation of cell surface glycans.

Monosaccharides that carry a cis diol motif, such as sialic acids, are sensitive to oxidation by periodate treatment. The aldehyde generated upon oxidative cleavage can react with an amine nucleophile, such as hydrazide, for conjugation to a desired reporter group.

Figure 3.. Metabolic oligosaccharide engineering.

Cells are treated with unnatural, tagged derivatives of naturally occurring monosaccharides, such as the peracetylated azide-tagged derivatives of ManNAc and GlcNAc shown (top left). After uptake and deprotection of the acetyl groups (bottom left), the metabolic precursors enter the cellular pathways for conversion into the corresponding CMP- or UDP-activated donors, which are then used for glycan biosynthesis. ManNAc derivatives are incorporated as sialic acids into cell surface glycans (A) while GlcNAc derivatives label intracellular proteins that are targets for O-GlcNAc modification (B). The azide-tagged glycans can be labelled by bioorthogonal ligation reactions such as the strain-promoted (A) or the copper(I)-catalysed (B) azide-alkyne cycloaddition.

Figure 4.. Tandem chemoenzymatic glycan engineering.

In CeGL, recombinant glycosyltransferases are used to transfer unnatural monosaccharides from appropriate donors onto glycan acceptors. Wu and coworkers [73] designed a double labelling strategy with successive reactions catalysed by ST3Gal1 and ST6Gal1, installing the same alkyne-tagged CMP-sialic acid derivative onto unextended Gal-GalNAc disaccharides present on O-glycans or uncapped Gal-GlcNAc disaccharides at the non-reducing end of N-glycans, respectively. Tagged glycans were labelled via two separate azide-alkyne cycloaddition reactions for the simultaneous visualisation of both glycan types at the cell surface with different fluorophores.