Over the past 35 years, ~1700 articles have characterized protein O-GlcNAcylation. Found in almost all living organisms, this post-translational modification of serine and threonine residues is highly conserved and key to biological processes. With half of the primary research articles using human models, the O-GlcNAcome recently reached a milestone of 5000 human proteins identified. Herein, we provide an extensive inventory of human O-GlcNAcylated proteins, their O-GlcNAc sites, identification methods, and corresponding references (www.oglcnac.mcw.edu). In the absence of a comprehensive online resource for O-GlcNAcylated proteins, this list serves as the only database of O-GlcNAcylated proteins. Based on the thorough analysis of the amino acid sequence surrounding 7002 O-GlcNAc sites, we progress toward a more robust semi-consensus sequence for O-GlcNAcylation. Moreover, we offer a comprehensive meta-analysis of human O-GlcNAcylated proteins for protein domains, cellular and tissue distribution, and pathways in health and diseases, reinforcing that O-GlcNAcylation is a master regulator of cell signaling, equal to the widely studied phosphorylation.

Dysfunctions in Wnt signaling increase β‐catenin stability and are associated with cancers, including colorectal cancer. In addition, β‐catenin degradation is decreased by nutrient‐dependent O ‐GlcNAcylation. Human colon tumors and colons from mice fed high‐carbohydrate diets exhibited higher amounts of β‐catenin and O ‐GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O ‐GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β‐catenin. By ETD‐MS/MS, we identified 4 O ‐GlcNAcylation sites at the N terminus of β‐catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β‐catenin reduced O ‐GlcNAcylation by 75%. Interestingly, elevating O ‐GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β‐catenin stability. Analyses of β‐catenin O ‐GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β‐catenin degradation rate. Finally, inhibiting O ‐GlcNAcylation decreased the β‐catenin/α‐catenin interaction necessary for mucosa integrity, whereas O ‐GlcNAcase silencing improved this interaction. These results suggest that O ‐GlcNAcylation regulates not only the stability of β‐catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O ‐GlcNAcylation of β‐catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.—Olivier‐Van Stichelen, S., Dehennaut, V., Buzy, A., Zachayus, J.‐L., Guinez, C., Mir, A‐M., El Yazidi‐Belkoura, I., Copin, M.‐C., Boureme, D., Loyaux, D., Ferrara, P., Lefebvre, T. O ‐GlcNAcylation stabilizes β‐catenin through direct competition with phosphorylation at threonine 41. FASEB J. 28, 3325–3338 (2014). www.fasebj.org

Non-nutritive sweeteners (NNS) are marketed as sugar alternatives providing sweet taste with few or no calories. Yet their consumption has been linked to metabolic dysfunction and changes in the gut microbiome. NNS exposure mostly originates from diet beverages and sweetener packages in adults or breastmilk in infants. Consequences of early life exposure remain largely unknown. We exposed pregnant and lactating mice to NNS (sucralose, acesulfame-K) at doses relevant for human consumption. While the pups’ exposure was low, metabolic changes were drastic, indicating extensive downregulation of hepatic detoxification mechanisms and changes in bacterial metabolites. Microbiome profiling confirmed a significant increase in firmicutes and a striking decrease of Akkermansia muciniphila. Similar microbiome alterations in humans have been linked to metabolic disease and obesity. While our findings need to be reproduced in humans, they suggest that NNS consumption during pregnancy and lactation may have adverse effects on infant metabolism.

Nutrient-driven O-GlcNAcylation is strikingly abundant in the brain and has been linked to development and neurodegenerative disease. We selectively targeted the O-GlcNAcase (Oga) gene in the mouse brain to define the role of O-GlcNAc cycling in the central nervous system. Brain knockout animals exhibited dramatically increased brain O-GlcNAc levels and pleiotropic phenotypes, including early-onset obesity, growth defects, and metabolic dysregulation. Anatomical defects in the Oga knockout included delayed brain differentiation and neurogenesis as well as abnormal proliferation accompanying a developmental delay. The molecular basis for these defects included transcriptional changes accompanying differentiating embryonic stem cells. In Oga KO mouse ES cells, we observed pronounced changes in expression of pluripotency markers, including Sox2, Nanog, and Otx2. These findings link the O-GlcNAc modification to mammalian neurogenesis and highlight the role of this nutrient-sensing pathway in developmental plasticity and metabolic homeostasis.

The short half-life protooncogene β-catenin acquires a remarkable stability in a large subset of cancers, mainly from mutations affecting its proteasomal degradation. In this sense, colorectal cancers (CRC) form a group of pathologies in which early steps of development are characterized by an aberrant expression of β-catenin and an uncontrolled proliferation of epithelial cells. Diet has long been described as an influence in the emergence of CRC, but the molecular events that link metabolic disorders and CRC remain elusive. Part of the explanation may reside in hexosamine biosynthetic pathway (HBP) flux. We found that fasted mice being force-fed with glucose or glucosamine leads to an increase of β-catenin and O-GlcNAcylation levels in the colon. MCF7 cells possessing intact Wnt/β-catenin signaling heavily expressed β-catenin when cultured in high glucose; this was reversed by the HBP inhibitor azaserine. HBP inhibition also decreased the expression of β-catenin in HT29 and, to a lesser extent, HCT116 cells. The same observation was made with regard to the transcriptional activity of β-catenin in HEK293 cells. Inhibition of HBP also blocked the glucose-mediated proliferation capacity of MCF7 cells, demonstrating that glucose affects both β-catenin expression and cell proliferation through the HBP. The ultimate element conducting these events is the dynamic posttranslational modification O-GlcNAcylation, which is intimately linked to HBP; the modulation of its level affected the expression of β-catenin and cell proliferation. In accordance with our findings, we propose that metabolic disorders correlate to CRC via an upregulation of HBP that reverberates on high O-GlcNAcylation levels including modification of β-catenin.