Achieving cell-type selectivity in metabolic oligosaccharide engineering

Abstract

Metabolic oligosaccharide engineering (MOE) is a transformative technology, enabling the chemical labeling and subsequent analysis of glycans. Central to this method are monosaccharide analogs, termed metabolic chemical reporters (MCRs), that contain abiotic functional groups that can undergo an increasing number of bioorthogonal reactions. Typically, these abiotic groups were designed to be as small as possible, allowing them to be tolerated by metabolic enzymes and glycosyltransferases that transform MCRs into donor sugars and add them into glycans, respectively. This generality allows MCRs to be used by a variety of cells and tissues but can also be a limitation in their application to investigate glycosylation of specific cell-types in multicellular systems. Here, we review different methods that are beginning to transition MCRs into cell selective tools, with the potential to increase the already large impact these compounds have had on glycoscience.

Fig. 1. Protein glycosylation and metabolic oligosaccharide engineering. (a) Glycosylation occurs on cell surface and secreted proteins, as well as intracellular proteins. Metabolic oligosaccharide engineering (MOE) takes advantage of metabolic chemical reporters (MCRs) bearing bioorthogonal handles, like Ac₄GlcNAz, that can be fed to cells where they are metabolized to donor sugars and incorporated into glycosylation.

Fig. 2. Liposome delivery of MCRs. (a) Liposome features including PEG-chains for improved stability and targeting ligands for cell-selective delivery. (b) Liposomes show inherent selectivity for tumors based on the enhanced permeability and retention (EPR) effect. (c) Targeting of liposomes to folate-receptor expressing cells as an example of cell-selective MCR delivery.

Fig. 3. Caged MCRs and monosaccharides. A caged group can be added at a position that prevents a downstream metabolic transformation. Various triggers can be used to remove the cage to enable glycan labeling. Examples of caged MCRs and GlcNAc covered in this review.

Fig. 4. Chemical genetics through bump-and-hole. Native enzymes have active sites evolved to bind their native substrates. Engineering selectivity into enzymes can be accomplished by using chemistry to introduce a physical “bump” onto the substrate molecule. The bump prevents native enzyme from using the new substrate. Genetics can then be exploited to introduce a complementary “hole” into the enzyme active site that can accommodate the new substrate, yielding selectivity for the engineered substrate/enzyme pair.

Fig. 5. Monosaccharide pathways engineered for MCR selectivity. (a) Schematic of the GlcNAc and GalNAc salvage pathways (black) and enzymatic steps that have been engineered by bump-and-hole or bypass. (b) Examples of MCRs used in combination with the engineered metabolic pathways.

Fig. 6. MCR incorporation into glycans is in competition with background cysteine labeling. If the 1-(anomeric) hydroxyl is de-esterified before others, the MCR sugar-ring can open revealing an aldehyde. Subsequent elimination of the 3-ester results in a Micheal acceptor that can react with cysteine residues, resulting in background chemical-labeling.