Developmental Regulation of Protein O-GlcNAcylation, O-GlcNAc Transferase, and O-GlcNAcase in Mammalian Brain

O-GlcNAcylation is a common posttranslational modification of nucleocytoplasmic proteins by β-N-acetylglucosamine (GlcNAc). The dynamic addition and removal of O-GlcNAc groups to and from proteins are catalyzed by O-linked N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA), respectively. O-GlcNAcylation often modulates protein phosphorylation and regulates several cellular signaling and functions, especially in the brain. However, its developmental regulation is not well known. Here, we studied protein O-GlcNAcylation, OGT, and OGA in the rat brain at various ages from embryonic day 15 to the age of 2 years. We found a gradual decline of global protein O-GlcNAcylation during developmental stages and adulthood. This decline correlated positively to the total protein phosphorylation at serine residues, but not at threonine residues. The expression of OGT and OGA isoforms was regulated differently at various ages. Immunohistochemical studies revealed ubiquitous distribution of O-GlcNAcylation at all ages. Strong immunostaining of O-GlcNAc, OGT, and OGA was observed mostly in neuronal cell bodies and processes, further suggesting the role of O-GlcNAc modification of neuronal proteins in the brain. These studies provide fundamental knowledge of age-dependent protein modification by O-GlcNAc and will help guide future studies on the role of O-GlcNAcylation in the mammalian brain.     

N-乙酰氨基葡萄糖转移酶（OGT）研究进展

O-GlcNAc是一种广泛存在于蛋白质丝/苏氨酸残基上的动态、可逆的蛋白翻译后修饰,它广泛分布在细胞浆和细胞核中,参与调节多种细胞途径。研究表明蛋白的O-GlcNAc糖基化与神经退行性疾病、糖尿病和癌症等疾病相关。在体内,O-GlcNAc动态修饰由N-乙酰氨基葡萄糖转移酶（OGT）和N-乙酰氨基葡萄糖苷酶（OGA）协同完成。近年来,OGT逐渐成为糖生物学领域的研究热点,在其结构、作用机制及晶体学方面取得了快速发展。     

Increased O-GlcNAc levels correlate with decreased O-GlcNAcase levels in Alzheimer disease brain

The potential role of the posttranslational modification of proteins with O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) in the pathogenesis of Alzheimer disease (AD) has been studied extensively, yet the exact function of O-GlcNAc in AD remains elusive. O-GlcNAc cycling is facilitated by only two highly conserved enzymes: O-GlcNAc transferase (OGT) catalyzes the addition, while O-GlcNAcase (OGA) catalyzes the removal of GlcNAc from proteins. Studies analyzing global O-GlcNAc levels in AD brain have produced inconsistent results and the reasons for altered O-GlcNAcylation in AD are still poorly understood. In this study, we show a 1.2-fold increase in cytosolic protein O-GlcNAc modification in AD brain when compared to age-matched controls. Interestingly, O-GlcNAc changes seem to be attributable to differential modification of a few individual proteins. While our finding of augmented O-GlcNAcylation concurs with some reports, it is contrary to others demonstrating decreased O-GlcNAc levels in AD brain. These conflicting results emphasize the need for further studies providing conclusive evidence on the subject of O-GlcNAcylation in AD. We further demonstrate that, while OGT protein levels are unaffected in AD, OGA protein levels are significantly decreased to 75% of those in control samples. In addition, augmented protein O-GlcNAc modification correlates to decreased OGA protein levels in AD subjects. While OGA inhibitors are already being tested for AD treatment, our results provide a strong indication that the general subject of O-GlcNAcylation and specifically its regulation by OGA and OGT in AD need further investigation to conclusively elucidate its potential role in AD pathogenesis and treatment.     

Site-specific interplay between O-GlcNAcylation and phosphorylation in cellular regulation

Ser(Thr)-O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous modification of nucleocytoplasmic proteins. Extensive crosstalk exists between O-GlcNAcylation and phosphorylation, which regulates signaling in response to nutrients/stress. The development of novel O-GlcNAc detection and enrichment methods has improved our understanding of O-GlcNAc functions. Mass spectrometry has revealed O-GlcNAc’s many interactions with phosphorylation-mediated signaling. However, mechanisms regulating O-GlcNAcylation and phosphorylation are quite different. Phosphorylation is catalyzed by hundreds of distinct kinases. In contrast, in mammals, uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyl transferase (OGT) and β-D-N-acetylglucosaminidase (OGA) are encoded by single highly conserved genes. Both OGT’s and OGA’s specificities are determined by their transient associations with many other proteins to create a multitude of specific holoenzymes. The extensive crosstalk between O-GlcNAcylation and phosphorylation represents a new paradigm for cellular signaling.     

O-GlcNAc cycling in the developing,adult and geriatric brain

Hundreds of proteins in the nervous system are modified by the monosaccharide O-GlcNAc. A single protein is often O-GlcNAcylated on several amino acids and the modification of a single site can play a crucial role for the function of the protein. Despite its complexity, only two enzymes add and remove O-GlcNAc from proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Global and local regulation of these enzymes make it possible for O-GlcNAc to coordinate multiple cellular functions at the same time as regulating specific pathways independently from each other. If O-GlcNAcylation is disrupted, metabolic disorder or intellectual disability may ensue, depending on what neurons are affected. O-GlcNAc's promise as a clinical target for developing drugs against neurodegenerative diseases has been recognized for many years. Recent literature puts O-GlcNAc in the forefront among mechanisms that can help us better understand how neuronal circuits integrate diverse incoming stimuli such as fluctuations in nutrient supply, metabolic hormones, neuronal activity and cellular stress. Here the functions of O-GlcNAc in the nervous system are reviewed.     

Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG.     

Molecular mechanisms of O-GlcNAcylation

Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of serines/threonines on metazoan proteins and occurring with similar time scales, dynamics and stoichiometry as protein phosphorylation. Levels of this modification are regulated by two enzymes-O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Although the biochemistry of these enzymes and functional implications of O-GlcNAc have been studied extensively, until recently the structures and molecular mechanisms of OGT/OGA were not understood. This review covers a body of recent work that has led to an understanding of the structure of OGA, its catalytic mechanism and the development of a plethora of different inhibitors that are finding their use in cell biological studies towards the functional implications of O-GlcNAc. Furthermore, the very recent structure determination of a bacterial OGT orthologue has given the first insights into the contribution of the tetratricopeptide repeats (TPRs) to the active site and the role of some residues in catalysis and substrate binding.     

O-GlcNAcase is essential for embryonic development and maintenance of genomic stability

Dysregulation of O-GlcNAc modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer's disease. Here we found that natural aging in wild-type mice was marked by a decrease in OGA and OGT protein levels and an increase in O-GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O-GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum-stimulated cell cycle entry induced increased O-GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O-GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O-GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O-GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.     

The potential role of O-GlcNAc modification in cancer epigenetics

There is no doubt that cancer is not only a genetic disease but that it can also occur due to epigenetic abnormalities. Diet and environmental factors can alter the scope of epigenetic regulation. The results of recent studies suggest that O-GlcNAcylation, which involves the addition of N-acetylglucosamine on the serine or threonine residues of proteins, may play a key role in the regulation of the epigenome in response to the metabolic status of the cell. Two enzymes are responsible for cyclic O-GlcNAcylation: O-GlcNAc transferase (OGT), which catalyzes the addition of the GlcNAc moiety to target proteins; and O-GlcNAcase (OGA), which removes the sugar moiety from proteins. Aberrant expression of O-GlcNAc cycling enzymes, especially OGT, has been found in all studied human cancers. OGT can link the cellular metabolic state and the epigenetic status of cancer cells by interacting with and modifying many epigenetic factors, such as HCF-1, TET, mSin3A, HDAC, and BAP1. A growing body of evidence from animal model systems also suggests an important role for OGT in polycomb-dependent repression of genes activity. Moreover, O-GlcNAcylation may be a part of the histone code: O-GlcNAc residues are found on all core histones.     

A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin

O-linked β-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.     

Functional O-GlcNAc modifications: Implications in molecular regulation and pathophysiology

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of intracellular proteins. The dynamic and inducible cycling of the modification is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in response to UDP-GlcNAc levels in the hexosamine biosynthetic pathway (HBP). Due to its reliance on glucose flux and substrate availability, a major focus in the field has been on how O-GlcNAc contributes to metabolic disease. For years this post-translational modification has been known to modify thousands of proteins implicated in various disorders, but direct functional connections have until recently remained elusive. New research is beginning to reveal the specific mechanisms through which O-GlcNAc influences cell dynamics and disease pathology including clear examples of O-GlcNAc modification at a specific site on a given protein altering its biological functions. The following review intends to focus primarily on studies in the last half decade linking O-GlcNAc modification of proteins with chromatin-directed gene regulation, developmental processes, and several metabolically related disorders including Alzheimer’s, heart disease and cancer. These studies illustrate the emerging importance of this post-translational modification in biological processes and multiple pathophysiologies.     

O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy

O-GlcNAc is a monosaccharide attached to serine or threonine hydroxyl moieties on numerous nuclear and cytoplasmic proteins; O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Although recent studies have shown that O-GlcNAcylation plays essential roles in breast cancer progression, it is also necessary to know whether O-GlcNAcylation is involved in other types of human cancer. In this study, O-GlcNAcylation levels and the expressions of OGT and OGA in human lung and colon cancer tissues were examined by immunohistochemistry analysis. We found that O-GlcNAcylation as well as OGT expression was significantly elevated in the cancer tissues compared with that in the corresponding adjacent tissues. Additionally, the roles of O-GlcNAcylation in the malignancy of lung and colon cancer were investigated in vitro. The results showed that O-GlcNAcylation markedly enhanced the anchorage-independent growth of lung and colon cancer cells; O-GlcNAcylation could also enhance lung and colon cancer invasion in a context-dependent manner. All together, this study suggests that O-GlcNAcylation might play important roles in lung and colon cancer formation and progression, and may be a valuable target for diagnosis and therapy of cancer.     

BLT1-mediated O-GlcNAcylation is required for NOX2-dependent migration,exocytotic degranulation and IL-8 release of human mast cell induced by Trichomonas vaginalis-secreted LTB4

Trichomonas vaginalis is a sexually-transmitted protozoan parasite that causes vaginitis and cervicitis. Although mast cell activation is important for provoking tissue inflammation during infection with parasites, information regarding the signaling mechanisms in mast cell activation and T. vaginalis infection is limited. O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine and threonine residues that functions as a critical regulator of intracellular signaling, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). We investigated if O-GlcNAcylation was associated with mast cell activation induced by T. vaginalis-derived secretory products (TvSP). Modified TvSP collected from live trichomonads treated with the 5-lipooxygenase inhibitor AA861 inhibited migration of mast cells. This result suggested that mast cell migration was caused by stimulation of T. vaginalis-secreted leukotrienes. Using the BLT1 antagonist U75302 or BLT1 siRNA, we found that migration of mast cells was evoked via LTB₄ receptor (BLT1). Furthermore, TvSP induced protein O-GlcNAcylation and OGT expression in HMC-1 cells, which was prevented by transfection with BLT1 siRNA. TvSP-induced migration, ROS generation, CD63 expression and IL-8 release were significantly suppressed by pretreatment with OGT inhibitor ST045849 or OGT siRNA. These results suggested that BLT1-mediated OGlcNAcylation was important for mast cell activation during trichomoniasis.