Anna R. Batt

O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and biochemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lowering O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6-Alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modification affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly affected by O-GlcNAcylation as a potential antiapoptotic mechanism.

However, fundamental processes associated with glycans are not yet fully understood and the development of glycobiology is relatively recent compared to the study of genes or proteins. Approximately 25 years ago, the studies of Bertozzi's and Reutter's groups paved the way for metabolic oligosaccharide engineering (MOE), a strategy which consists in the use of modified sugar analogs which are taken up into the cells, metabolized, incorporated into glycoconjugates, and finally detected in a specific manner. This groundbreaking strategy has been widely used during the last few decades and the concomitant development of new bioorthogonal ligation reactions has allowed many advances in the field. Typically, MOE has been used to either visualize glycans or identify different classes of glycoproteins. The present review aims to highlight recent studies that lie somewhat outside of these more traditional approaches and that are pushing the boundaries of MOE applications.

Glycans can be directly labeled using unnatural monosaccharide analogs, termed metabolic chemical reporters (MCRs). These compounds enable the secondary visualization and identification of glycoproteins by taking advantage of bioorthogonal reactions. Most widely used MCRs have azides or alkynes at the 2-N-acetyl position but are not selective for one class of glycoprotein over others. To address this limitation, we are exploring additional MCRs that have bioorthogonal functionality at other positions. Here, we report the characterization of 2-azido-2-deoxy-glucose (2AzGlc). We find that 2AzGlc selectively labels intracellular O-GlcNAc modifications, which further supports a somewhat unexpected, structural flexibility in this pathway. In contrast to the endogenous modification N-acetyl-glucosamine (GlcNAc), we find that 2AzGlc is not dynamically removed from protein substrates and that treatment with higher concentrations of per-acetylated 2AzGlc is toxic to cells. Finally, we demonstrate that this toxicity is an inherent property of the small-molecule, as removal of the 6-acetyl-group renders the corresponding reporter nontoxic but still results in protein labeling.

Since the pioneering work by Reutter and co-workers that demonstrated structural flexibility in the carbohydrate biosynthesis and glycosylation pathways, many different labs have used unnatural monosaccharide analogues to perform glycan engineering on the surface of living cells. A subset of these unnatural monosaccharides contain bioorthogonal groups that enable the selective installation of visualization or enrichment tags. These metabolic chemical reporters (MCRs) have proven to be powerful for the unbiased identification of glycoproteins; however, they do have certain limitations. For example, they are incorporated substoichiometrically into glycans, and most MCRs are not selective for one class (e.g., O-GlcNAcylation) of glycoprotein. Here, we explore the relationship between the biosynthesis of MCR donor sugars in cells and the labeling levels of four different N-acetylglucosamine- and N-acetylgalactosamine-based MCRs. We found that the buildup of the different donor sugars correlated well with the overall labeling levels but less so with intracellular labeling of proteins by O-GlcNAcylation.