The Metabolic Chemical Reporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O-Glucose

Abstract

Metabolic chemical reporters of glycosylation in combination with bioorthogonal reactions have been known for two decades and have been used by many different research laboratories for the identification and visualization of glycoconjugates. More recently, however, they have begun to see utility for the investigation of cellular metabolism and the tolerance of biosynthetic enzymes and glycosyltransferases to different sugars. Here, we take this concept one step further by using the metabolic chemical reporter 6-azido-6-deoxy-glucose (6AzGlc). We show that treatment of mammalian cells with the per- O-acetylated version of 6AzGlc results in robust labeling of a variety of proteins. Notably, the pattern of this labeling was consistent with O-GlcNAc modifications, suggesting that the enzyme O-GlcNAc transferase is quite promiscuous for its donor sugar substrates. To confirm this possibility, we show that 6AzGlc-treatment results in the labeling of known O-GlcNAcylated proteins, that the UDP-6AzGlc donor sugar is indeed produced in living cells, and that recombinant OGT will accept UDP-6AzGlc as a substrate in vitro. Finally, we use proteomics to first identify several bona fide 6AzGlc-modifications in mammalian cells and then an endogenous O-glucose modification on host cell factor. These results support the conclusion that OGT can endogenously modify proteins with both N-acetyl-glucosamine and glucose, raising the possibility that intracellular O-glucose modification may be a widespread modification under certain conditions or in particular tissues.

Azide‐ and Alkyne‐Bearing Metabolic Chemical Reporters of Glycosylation Show Structure‐Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway

“…Furthermore, this suggests that treating cells with these MCRs will have little effect on the endogenous biosynthesis of UDP‐GlcNAc through the HBP. Finally, we tested the most recent MCRs developed by our lab: UDP‐6AzGlcNAc and UDP‐2AzGlc, which are selective for labeling O‐GlcNAc modifications, and UDP‐6AzGlc, which we recently showed might read out on intracellular O‐glucose modifications (Figure B) . In the case of UDP‐6AzGlcNAc, we saw inhibition in a similar range as UDP‐GlcNAc, thus suggesting that the 6‐azido‐6‐deoxy substitution does not interfere with binding of the UDP‐sugar to the allosteric site.…”

“…Using synthetic sugar donors and recombinant human GFAT, we show that several of the most commonly used MCRs, modified at their 2‐ N ‐acetyl position by larger substituents (UDP‐GlcNAz, ‐GalNAz, ‐GlcNAlk, and ‐GalNAlk), do not inhibit GFAT. In contrast, placement of an azide at either the 6‐ or 2‐position of glucose (UDP‐6AzGlc and 2AzGlc) results in GFAT inhibition, and 6‐azido‐6‐deoxy‐GlcNAc (UDP‐6AzGlcNAc) might be a slightly more potent inhibitor than UDP‐GlcNAc.…”

Dimethylaminomicheliolide (DMAMCL) Suppresses the Proliferation of Glioblastoma Cells via Targeting Pyruvate Kinase 2 (PKM2) and Rewiring Aerobic Glycolysis

“…Tumor cells mainly rely on aerobic glycolysis (high rates of glucose uptake and lactate production) to produce energy even in the presence of oxygen (10, 37–39). Aerobic glycolysis contributes to cancer cell proliferation by providing the raw components required for to synthesis of cellular materials (40). Increasing evidences have shown that PKM2 plays a critical function in the maintenance of aerobic glycolysis, with decreased PK activity favoring cancer cell proliferation (15, 41, 42).…”

O-Acetylated Chemical Reporters of Glycosylation Can Display Metabolism-Dependent Background Labeling of Proteins but Are Generally Reliable Tools for the Identification of Glycoproteins

“…A second bioorthogonal reaction step is then exploited to attached visualization or affinity tags for analysis. More recently, we and others have taken a broader approach to glycoprotein-MCR discovery through the synthesis and characterization of monosaccharide analogs that may not transit well-characterized biosynthetic pathways (Zaro et al, 2011(Zaro et al, , 2017Chuh et al, 2014Chuh et al, , 2017Li et al, 2016;Shen et al, 2017;Darabedian et al, 2018). For instance, we demonstrated that 6-azido-6-deoxy-N-acetylglucosamine (6AzGlcNAc) can bypass the traditional GlcNAc-salvage pathway to generate uridine diphosphate sugar (UDP-6AzGlcNAc) (Chuh et al, 2014), resulting in labeling of O-GlcNAcylated proteins and suggesting that cellular metabolism is more accommodating to MCRs than previously appreciated.…”