Studying the O-GlcNAcome of human placentas using banked tissue samples.

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 µg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.

The O-GlcNAc cycling in neurodevelopment and associated diseases.

Proper neuronal development is essential to growth and adult brain function. Alterations at any step of this highly organized sequence of events, due to genetic mutations or environmental factors, triggers brain malformations, which are leading causes of diseases including epilepsy, intellectual disabilities, and many others. The role of glycosylation in neuronal development has been emphasized for many years, notably in studying human congenital disorders of glycosylation (CDGs). These diseases highlight that genetic defects in glycosylation pathways are almost always associated with severe neurological abnormalities, suggesting that glycosylation plays an essential role in early brain development. Congenital disorders of O-GlcNAcylation are no exception, and all mutations of the O-GlcNAc transferase (OGT) are associated with X-linked intellectual disabilities (XLID). In addition, mouse models and in vitro mechanistic studies have reinforced the essential role of O-GlcNAcylation in neuronal development and signaling. In this review, we give an overview of the role of O-GlcNAcylation in this critical physiological process and emphasize the consequences of its dysregulation.

Consumption of non-nutritive sweeteners during pregnancy.

In an effort to reduce sugar consumption to prevent diabetes mellitus and cardiovascular diseases, "sugar-free" or "no added sugar" products that substitute sugar with non-nutritive sweeteners (NNSs) (eg, Splenda, Sweet'N Low, and Stevia) have become increasingly popular. The use of these products during pregnancy has also increased, with approximately 30% of pregnant women reporting intentional NNS consumption. In clinical studies with nonpregnant participants and animal models, NNSs were shown to alter gut hormonal secretion, glucose absorption, appetite, kidney function, in vitro insulin secretion, adipogenesis, and microbiome dysbiosis of gut bacteria. In pregnant animal models, NNS consumption has been associated with altered sweet taste preference later in life and metabolic dysregulations in the offspring (eg, elevated body mass index, increased risk of obesity, microbiome dysbiosis, and abnormal liver function tests). Despite the accumulating evidence, no specific guidelines for NNS consumption are available for pregnant women. Furthermore, there are limited clinical studies on the effects of NNS consumption during pregnancy and postpartum and long-term outcomes in the offspring.

Nutrient-driven O-linked N-acetylglucosamine (O-GlcNAc) cycling impacts neurodevelopmental timing and 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.

Nutrient-driven O-GlcNAc in proteostasis and neurodegeneration.

Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine (O-GlcNAc) regulates protein homeostasis. The O-GlcNAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-GlcNAc transferase- and O-GlcNAcase-knockout alleles have helped define the role O-GlcNAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an 'O-GlcNAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-GlcNAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-GlcNAc in the physiology of the brain and its susceptibility to neurodegenerative injury.

Glucokinase expression is regulated by glucose through O-GlcNAc glycosylation.

Blood glucose fluctuates with the fasting-feeding cycle. One of the liver's functions is to maintain blood glucose concentrations within a physiological range. Glucokinase (GCK) or hexokinase IV, is the main enzyme that regulates the flux and the use of glucose in the liver leading to a compensation of hyperglycemia. In hepatocytes, GCK catalyzes the phosphorylation of glucose into glucose-6-phosphate. This critical enzymatic reaction is determinant for the metabolism of glucose in the liver which includes glycogen synthesis, glycolysis, lipogenesis and gluconeogenesis. In liver, simultaneous increase of glucose and insulin enhances GCK activity and gene expression, changes its subcellular location and interaction with regulatory proteins. The post-translational O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) acts as a glucose-sensitive modification and is believed to take part in hepatic glucose sensing by modifying key regulatory proteins. Therefore, we aimed to determine whether GCK is modified by O-GlcNAcylation in the liver of mice and investigated the role that this modification plays in regulating GCK protein expression. We demonstrated that endogenous GCK expression correlated with O-GlcNAc levels in the pathophysiological model ob/ob mice. More specifically, in response to the pharmacological inhibition of O-GlcNAcase (OGA) contents of GCK increased. Using the GlcNAc specific lectin succinylated-WGA and click chemistry labeling approaches, we demonstrated that GCK is modified by O-GlcNAcylation. Further, we demonstrated that siRNA-mediated Ogt knock-down not only decreases O-GlcNAc content but also GCK protein level. Altogether, our in vivo and in vitro results demonstrate that GCK expression is regulated by nutrient-sensing O-GlcNAc cycling in liver.

You are what you eat: O-linked N-acetylglucosamine in disease, development and epigenetics.

PURPOSE OF REVIEW: The O-linked N-acetylglucosamine (O-GlcNAc) modification is both responsive to nutrient availability and capable of altering intracellular cellular signalling. We summarize data defining a role for O-GlcNAcylation in metabolic homeostasis and epigenetic regulation of development in the intrauterine environment. RECENT FINDINGS: O-GlcNAc transferase (OGT) catalyzes nutrient-driven O-GlcNAc addition and is subject to random X-inactivation. OGT plays key roles in growth factor signalling, stem cell biology, epigenetics and possibly imprinting. The O-GlcNAcase, which removes O-GlcNAc, is subject to tight regulation by higher order chromatin structure. O-GlcNAc cycling plays an important role in the intrauterine environment wherein OGT expression is an important biomarker of placental stress. SUMMARY: Regulation of O-GlcNAc cycling by X-inactivation, epigenetic regulation and nutrient-driven processes makes it an ideal candidate for a nutrient-dependent epigenetic regulator of human disease. In addition, O-GlcNAc cycling influences chromatin modifiers critical to the regulation and timing of normal development including the polycomb repression complex and the ten-eleven translocation proteins mediating DNA methyl cytosine demethylation. The pathway also impacts the hypothalamic-pituitary-adrenal axis critical to intrauterine programming influencing disease susceptibility in later life.

O-GlcNAcylation stabilizes ß-catenin through direct competition with phosphorylation at threonine 41.

Dysfunctions in Wnt signaling increase ß-catenin stability and are associated with cancers, including colorectal cancer. In addition, ß-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of ß-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 ß-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of ß-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of ß-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 ß-catenin stability. Analyses of ß-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the ß-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the ß-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 ß-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of ß-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.

X marks the spot: does it matter that O-GlcNAc transferase is an X-linked gene?

O-GlcNAcylation has emerged as a critical post-translational modification important for a wide array of cellular processes. This modification has been identified on a large pool of intracellular proteins that have wide-ranging roles, including transcriptional regulation, cell cycle progression, and signaling, among others. Interestingly, in mammals the single gene encoding O-GlcNAc Transferase (OGT) is located on the X-chromosome near the Xist locus suggesting that tight dosage regulation is necessary for normal development. Herein, we highlight the importance of OGT dosage and consider how its genomic location can contribute to a gender-specific increased risk for a number of diseases.

Insulin signaling controls the expression of O-GlcNAc transferase and its interaction with lipid microdomains.

Lipid microdomains (rafts) are cholesterol-enriched dynamic ordered lipid domains belonging to cell membranes involved in diverse cellular functions, including signal transduction, membrane trafficking, and infection. Many studies have reported relationships between insulin signaling and lipid rafts. Likewise, links between insulin signaling and O-GlcNAcylation have also been described. However, the potential connection between O-GlcNAc and raft dynamics remains unexplored. Here we show that O-GlcNAc and the enzyme that creates this modification, O-GlcNAc transferase (OGT), are localized in rafts. On insulin stimulation, we observe time-dependent increases in OGT expression and localization within rafts. We show that these processes depend on activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Inhibition of OGT does not significantly affect cholesterol synthesis and raft building but decreases insulin receptor expression and PI3K and mitogen-activated protein kinase pathway activation. Taken together, these findings indicate that O-GlcNAcylation, lipid rafts, and signaling pathways are spatiotemporally coordinated to enable fundamental cellular functions.

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition.

BACKGROUND: DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood. METHODS AND RESULTS: Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation. CONCLUSIONS: The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication. GENERAL SIGNIFICANCE: Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.

O-GlcNAc transferase contributes to sex-specific placental deregulation in gestational diabetes.

INTRODUCTION: Gestational diabetes (GDM) is traditionally thought to emerge from placental endocrine dysregulations, but recent evidence suggests that fetal sex can also impact GDM development. Understanding the molecular mechanisms through which sex modulates placenta physiology can help identify novel molecular targets for future clinical care. Thus, we investigated the nutrient-sensing O-GlcNAc pathway as a potential mediator of sex-specific placenta dysfunction in GDM. METHODS: Expression levels of O-GlcNAc enzymes were measured in male and female (n = 9+/gender) human placentas based on the maternal diagnosis of GDM. We then simulated the observed differences in both BeWo cells and human syncytiotrophoblasts primary cells (SCT) from male and female origins (n = 6/gender). RNA sequencing and targeted qPCR were performed to characterize the subsequent changes in the placenta transcriptome related to gestational diabetes. RESULTS: O-GlcNAc transferase (OGT) expression was significantly reduced only in male placenta collected from mothers with GDM compared to healthy controls. Similar downregulation of OGT in trophoblast-like BeWo male cells demonstrated significant gene expression deregulations that overlapped with known GDM-related genes. Notably, placental growth hormone (GH) production was significantly elevated, while compensatory factors against GH-related insulin resistance were diminished. Inflammatory and immunologic factors with toxic effects on pancreatic ß cell mass were also increased, altogether leaning toward a decompensatory diabetic profile. Similar changes in hormone expression were confirmed in male human primary SCTs transfected with siOGT. However, down-regulating OGT in female primary SCTs did not impact hormone production. CONCLUSION: Our study demonstrated the significant deregulation of placental OGT levels in mothers with GDM carrying a male fetus. When simulated in vitro, such deregulation impacted hormonal production in BeWo trophoblast cells and primary SCTs purified from male placentas. Interestingly, female placentas were only modestly impacted by OGT downregulation, suggesting that the sex-specific presentation observed in gestational diabetes could be related to O-GlcNAc-mediated regulation of placental hormone production.

Non-Nutritive Sweeteners Acesulfame Potassium and Sucralose Are Competitive Inhibitors of the Human P-glycoprotein/Multidrug Resistance Protein 1 (PGP/MDR1).

Non-nutritive sweeteners (NNS) are popular sugar replacements used in foods, beverages, and medications. Although NNS are considered safe by regulatory organizations, their effects on physiological processes such as detoxification are incompletely understood. Previous studies revealed that the NNS sucralose (Sucr) altered P-glycoprotein (PGP) expression in rat colon. We also demonstrated that early-life exposure to NNS Sucr and acesulfame potassium (AceK) compromises mouse liver detoxification. Building upon these initial discoveries, we investigated the impact of AceK and Sucr on the PGP transporter in human cells to assess whether NNS influence its key role in cellular detoxification and drug metabolism. We showed that AceK and Sucr acted as PGP inhibitors, competing for the natural substrate-binding pocket of PGP. Most importantly, this was observed after exposure to concentrations of NNS within expected levels from common foods and beverage consumption. This may suggest risks for NNS consumers, either when taking medications that require PGP as the primary detoxification transporter or during exposure to toxic compounds.

O-GlcNAcylation regulates OTX2's proteostasis.

O-GlcNAcylation is a key post-translational modification, playing a vital role in cell signaling during development, especially in the brain. In this study, we investigated the role of O-GlcNAcylation in regulating the homeobox protein OTX2, which contributes to various brain disorders, such as combined pituitary hormone deficiency, retinopathy, and medulloblastoma. Our research demonstrated that, under normal physiological conditions, the proteasome plays a pivotal role in breaking down endogenous OTX2. However, when the levels of OTX2 rise, it forms oligomers and/or aggregates that require macroautophagy for clearance. Intriguingly, we demonstrated that O-GlcNAcylation enhances the solubility of OTX2, thereby limiting the formation of these aggregates. Additionally, we unveiled an interaction between OTX2 and the chaperone protein CCT5 at the O-GlcNAc sites, suggesting a potential collaborative role in preventing OTX2 aggregation. Finally, our study demonstrated that while OTX2 physiologically promotes cell proliferation, an O-GlcNAc-depleted OTX2 is detrimental to cancer cells.

The hexosamine biosynthetic pathway and O-GlcNAcylation drive the expression of ß-catenin and cell proliferation.

The short half-life protooncogene ß-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 ß-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 ß-catenin and O-GlcNAcylation levels in the colon. MCF7 cells possessing intact Wnt/ß-catenin signaling heavily expressed ß-catenin when cultured in high glucose; this was reversed by the HBP inhibitor azaserine. HBP inhibition also decreased the expression of ß-catenin in HT29 and, to a lesser extent, HCT116 cells. The same observation was made with regard to the transcriptional activity of ß-catenin in HEK293 cells. Inhibition of HBP also blocked the glucose-mediated proliferation capacity of MCF7 cells, demonstrating that glucose affects both ß-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 ß-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 ß-catenin.

Automatization and self-maintenance of the O-GlcNAcome catalog: a smart scientific database.

Post-translational modifications (PTMs) are ubiquitous and essential for protein function and signaling, motivating the need for sustainable benefit and open models of web databases. Highly conserved O-GlcNAcylation is a case example of one of the most recently discovered PTMs, investigated by a growing community. Historically, details about O-GlcNAcylated proteins and sites were dispersed across literature and in non-O-GlcNAc-focused, rapidly outdated or now defunct web databases. In a first effort to fill the gap, we recently published a human O-GlcNAcome catalog with a basic web interface. Based on the enthusiasm generated by this first resource, we extended our O-GlcNAcome catalog to include data from 42 distinct organisms and released the O-GlcNAc Database v1.2. In this version, more than 14 500 O-GlcNAcylated proteins and 11 000 O-GlcNAcylation sites are referenced from the curation of 2200 publications. In this article, we also present the extensive features of the O-GlcNAc Database, including the user-friendly interface, back-end and client-server interactions. We particularly emphasized our workflow, involving a mostly automatized and self-maintained database, including machine learning approaches for text mining. We hope that this software model will be useful beyond the O-GlcNAc community, to set up new smart, scientific online databases, in a short period of time. Indeed, this database system can be administrated with little to no programming skills and is meant to be an example of a useful, sustainable and cost-efficient resource, which exclusively relies on free open-source software elements (www.oglcnac.mcw.edu).

O-GlcNAcylation Is Essential for Rapid Pomc Expression and Cell Proliferation in Corticotropic Tumor Cells.

Pituitary adenomas have a staggering 16.7% lifetime prevalence and can be devastating in many patients because of profound endocrine and neurologic dysfunction. To date, no clear genomic or epigenomic markers correlate with their onset or severity. Herein, we investigate the impact of the O-GlcNAc posttranslational modification in their etiology. Found in more than 7000 human proteins to date, O-GlcNAcylation dynamically regulates proteins in critical signaling pathways, and its deregulation is involved in cancer progression and endocrine diseases such as diabetes. In this study, we demonstrated that O-GlcNAc enzymes were upregulated, particularly in aggressive adrenocorticotropin (ACTH)-secreting tumors, suggesting a role for O-GlcNAcylation in pituitary adenoma etiology. In addition to the demonstration that O-GlcNAcylation was essential for their proliferation, we showed that the endocrine function of pituitary adenoma is also dependent on O-GlcNAcylation. In corticotropic tumors, hypersecretion of the proopiomelanocortin (POMC)-derived hormone ACTH leads to Cushing disease, materialized by severe endocrine disruption and increased mortality. We demonstrated that Pomc messenger RNA is stabilized in an O-GlcNAc-dependent manner in response to corticotrophin-releasing hormone (CRH). By affecting Pomc mRNA splicing and stability, O-GlcNAcylation contributes to this new mechanism of fast hormonal response in corticotropes. Thus, this study stresses the essential role of O-GlcNAcylation in ACTH-secreting adenomas' pathophysiology, including cellular proliferation and hypersecretion.

The human O-GlcNAcome database and meta-analysis.

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.