The role of O-GlcNAcylation in RNA polymerase II transcription.

Eukaryotic RNA polymerase II (RNAPII) is responsible for the transcription of the protein-coding genes in the cell. Enormous progress has been made in discovering the protein activities that are required for transcription to occur, but the effects of post-translational modifications (PTMs) on RNAPII transcriptional regulation are much less understood. Most of our understanding relates to the cyclin-dependent kinases (CDKs), which appear to act relatively early in transcription. However, it is becoming apparent that other PTMs play a crucial role in the transcriptional cycle, and it is doubtful that any sort of complete understanding of this regulation is attainable without understanding the spectra of PTMs that occur on the transcriptional machinery. Among these is O-GlcNAcylation. Recent experiments have shown that the O-GlcNAc PTM likely has a prominent role in transcription. This review will cover the role of the O-GlcNAcylation in RNAPII transcription during initiation, pausing, and elongation, which will hopefully be of interest to both O-GlcNAc and RNAPII transcription researchers.

Engineering Corynebacterium glutamicum for the efficient production of N-acetylglucosamine.

N-acetylglucosamine (GlcNAc) is significant functional monosaccharides with diverse applications in medicine, food, and cosmetics. In this study, the GlcNAc synthesis pathway was constructed in Corynebacterium glutamicum and its reverse byproduct pathways were blocked. Simultaneously the driving force of GlcNAc synthesis was enhanced by screening key gene sources and inhibiting the GlcNAc consumption pathway. To maximize carbon flux, some competitive pathways (Pentose phosphate pathway, Glycolysis pathway and Mannose pathway) were weakened and the titer of GlcNAc reached 23.30 g/L in shake flasks. Through transcriptome analysis, it was found that dissolved oxygen was an important limiting factor, which was optimized in a 5 L bioreactor. Employing optimal fermentation conditions and feeding strategy, the titer of GlcNAc reached 138.9 g/L, with the yeild of 0.44 g/g glucose. This study significantly increased the yield and titer of GlcNAc, which lay a solid foundation for the industrial production of GlcNAc in C. glutamicum.

N-acetylglucosamine kinase, Hxk1 is a multifaceted metabolic enzyme in model pathogenic yeast Candida albicans.

The sensing of environmental conditions such as nutrient availability and the ability to adapt and respond to changing conditions are crucial for the survival of living organisms. Evidence from several organisms have revealed that some metabolic enzymes act as sensors of nutrient status and regulate the expression of sets of genes required for nutrients utilization and condition specific environmental adaptation. Thus metabolic enzymes regulate the signaling pathway by acting as transcriptional regulators and providing required metabolites. The commensal yeast, Candida albicans has recently emerged as a model system for understanding the N-acetylglucosamine (GlcNAc) signaling pathway in eukaryotes. GlcNAc kinase (Hxk1), the first enzyme of the catabolic cascade, has been shown to perform several functions such as regulation of gene expression and regulation of the metabolic status of the cell thereby resulting in a change in cell morphology (yeast-hyphal transition, white-opaque switching), metabolic gene expression, synthesis of metabolic precursors, induction of glycolytic flux rate and biofilm formation. Here, in this review we have discussed various roles of Hxk1that have not been reported in other organisms previously. The enzyme exhibits dynamic changes in subcellular localization consistent with its expanded functions inside the cell. Thus Hxk1 in C. albicans orchestrates several dynamic cellular processes and this signaling system can act as a paradigm to understand the cell fate and metabolic specialization in other eukaryotes too. Still, the molecular cues involved in Hxk1 mediating functions are yet to be unveiled; the relationship between Hxk1 sensing and its signaling effects is also not understood yet.

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.

4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

Peptide Sequence Mapping around Bisecting GlcNAc-Bearing N-Glycans in Mouse Brain.

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.

O-GlcNAcylation: Implications in normal and malignant hematopoiesis.

Posttranslational protein modification through addition of the O-linked ß-N-acetyl-D-glucosamine (O-GlcNAc) moiety to serine or threonine residues, termed O-GlcNAcylation, is a highly dynamic process conserved throughout eukaryotes. O-GlcNAcylation is reversibly catalyzed by a single pair of enzymes, O-GlcNAc transferase and O-GlcNAcase, and it acts as a fundamental regulator for a wide variety of biological processes including gene expression, cell cycle regulation, metabolism, stress response, cellular signaling, epigenetics, and proteostasis. O-GlcNAcylation is regulated by various intracellular or extracellular cues such as metabolic status, nutrient availability, and stress. Studies over decades have unveiled the profound biological significance of this unique protein modification in normal physiology and pathologic processes of diverse cell types or tissues. In hematopoiesis, recent studies have indicated the essential and pleiotropic roles of O-GlcNAcylation in differentiation, proliferation, and function of hematopoietic cells including T cells, B cells, myeloid progenitors, and hematopoietic stem and progenitor cells. Moreover, aberrant O-GlcNAcylation is implicated in the development of hematologic malignancies with dysregulated epigenetics, metabolism, and gene transcription. Thus, it is now recognized that O-GlcNAcylation is one of the key regulators of normal and malignant hematopoiesis.

O-GlcNAc modification of nuclear pore complexes accelerates bidirectional transport.

Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated transport of proteins in both directions, and decreasing modification slowed transport. Superresolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the nonspecific permeability of the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.

Bombyx mori ß1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis.

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcß1,2-R with ß1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of ß1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

MS-based proteomics for comprehensive investigation of protein O-GlcNAcylation.

Protein O-GlcNAcylation refers to the covalent binding of a single N-acetylglucosamine (GlcNAc) to the serine or threonine residue. This modification primarily occurs on proteins in the nucleus and the cytosol, and plays critical roles in many cellular events, including regulation of gene expression and signal transduction. Aberrant protein O-GlcNAcylation is directly related to human diseases such as cancers, diabetes and neurodegenerative diseases. In the past decades, considerable progress has been made for global and site-specific analysis of O-GlcNAcylation in complex biological samples using mass spectrometry (MS)-based proteomics. In this review, we summarized previous efforts on comprehensive investigation of protein O-GlcNAcylation by MS. Specifically, the review is focused on methods for enriching and site-specifically mapping O-GlcNAcylated peptides, and applications for quantifying protein O-GlcNAcylation in different biological systems. As O-GlcNAcylation is an important protein modification for cell survival, effective methods are essential for advancing our understanding of glycoprotein functions and cellular events.

JF1/B6F1 Ngly1-/- mouse as an isogenic animal model of NGLY1 deficiency.

N-Glycanase 1 (NGLY1) deficiency is a congenital disorder caused by mutations in the NGLY1 gene. Because systemic Ngly1-/- mice with a C57BL/6 (B6) background are embryonically lethal, studies on the mechanism of NGLY1 deficiency using mice have been problematic. In this study, B6-Ngly1-/+ mice were crossed with Japanese wild mice-originated Japanese fancy mouse 1 (JF1) mice to produce viable F2 Ngly1-/- mice from (JF1×B6)F1 Ngly1-/+ mice. Systemic Ngly1-/- mice with a JF1 mouse background were also embryonically lethal. Hybrid F1 Ngly1-/- (JF1/B6F1) mice, however, showed developmental delay and motor dysfunction, similar to that in human patients. JF1/B6F1 Ngly1-/- mice showed increased levels of plasma and urinary aspartylglycosamine, a potential biomarker for NGLY1 deficiency. JF1/B6F1 Ngly1-/- mice are a useful isogenic animal model for the preclinical testing of therapeutic options and understanding the precise pathogenic mechanisms responsible for NGLY1 deficiency.

N-acetylglucosamine-mediated morphological transition in Candida albicans and Candida tropicalis.

Morphological transitions in Candida species are key factors in facilitating invasion and adapting to environmental changes. N-acetylglucosamine (GlcNAc) is a monosaccharide signalling molecule that can regulate morphological transitions in Candida albicans and Candida tropicalis. Interestingly, although the uptake and metabolic pathways of GlcNAc and GlcNAc-mediated white-to-opaque cell switching are similar between the two Candida species, GlcNAc induces hyphal development in C. albicans, whereas it suppresses hyphal development in C. tropicalis. These findings indicate that the characteristics of C. albicans and C. tropicalis in response to GlcNAc are remarkably different. Here, we compare the conserved and divergent GlcNAc-mediated signalling pathways and catabolism between the two Candida species. Deletion of NGT1, a GlcNAc transportation gene, inhibited hyphal formation in C. albicans but promoted hyphal development in C. tropicalis. To further understand these opposite effects on filamentous growth in response to GlcNAc in the two Candida species, the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signalling pathways in both C. albicans and C. tropicalis were compared. Interestingly, GlcNAc activated the cAMP/PKA signalling pathway of the two Candida species, suggesting that the hyphal development-regulated circuit is remarkably diverse between the two species. Indeed, the Ndt80-like gene REP1, which is critical for regulating GlcNAc catabolism, exhibits distinct roles in the hyphal development of C. albicans and C. tropicalis. These data suggest possible reasons for the divergent hyphal growth response in C. albicans and C. tropicalis upon GlcNAc induction.

The elucidation of phosphosugar stress response in Bacillus subtilis guides strain engineering for high N-acetylglucosamine production.

Bacillus subtilis is a preferred microbial host for the industrial production of nutraceuticals and a promising candidate for the synthesis of functional sugars, such as N-acetylglucosamine (GlcNAc). Previously, a GlcNAc-overproducer B. subtilis SFMI was constructed using glmS ribozyme dual-regulatory tool. Herein, we further engineered to enhance carbon flux from glucose towards GlcNAc synthesis. As a result, the increased flux towards GlcNAc synthesis triggered phosphosugar stress response, which caused abnormal cell growth. Unfortunately, the mechanism of phosphosugar stress response had not been elucidated in B. subtilis. To reveal the stress mechanism and overcome its negative effect in bioproduction, we performed comparative transcriptome analysis. The results indicate that cells slow glucose utilization by repression of glucose import and accelerate catabolic reactions of phosphosugar. To verify these results, we overexpressed the phosphatase YwpJ, which relieved phosphosugar stress and allowed us to identify the enzyme responsible for GlcNAc synthesis from GlcNAc 6-phosphate. In addition, the deletion of nagBB and murQ, responsible for GlcNAc precursor degradation, further improved GlcNAc synthesis. The best engineered strain, B. subtilis FMIP34, increased GlcNAc titer from 11.5 to 26.1 g/L in shake flasks and produced 87.5 g/L GlcNAc in 30-L fed-batch bioreactor. Our results not only elucidate, for the first time, the phosphosugar stress response mechanism in B. subtilis, but also demonstrate how the combination of rational metabolic engineering with novel insights into physiology and metabolism allows the construction of highly efficient microbial cell factories for the production of high-value chemicals.

Role of O-Linked N-Acetylglucosamine Protein Modification in Cellular (Patho)Physiology.

In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N-acetylglucosamine moiety (O-GlcNAc) via an O-linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery, O-GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.

MYPT1 O-GlcNAc modification regulates sphingosine-1-phosphate mediated contraction.

Many intracellular proteins are modified by N-acetylglucosamine, a post-translational modification termed O-GlcNAc. This modification is found on serine and threonine side chains and has the potential to regulate signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. We find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P), culminating in the phosphorylation of myosin light chain (MLC) and cellular contraction. Specifically, O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen-matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing.

Potential Roles of O-GlcNAcylation in Primary Cilia- Mediated Energy Metabolism.

The primary cilium, an antenna-like structure on most eukaryotic cells, functions in transducing extracellular signals into intracellular responses via the receptors and ion channels distributed along it membrane. Dysfunction of this organelle causes an array of human diseases, known as ciliopathies, that often feature obesity and diabetes; this indicates the primary cilia's active role in energy metabolism, which it controls mainly through hypothalamic neurons, preadipocytes, and pancreatic ß-cells. The nutrient sensor, O-GlcNAc, is widely involved in the regulation of energy homeostasis. Not only does O-GlcNAc regulate ciliary length, but it also modifies many components of cilia-mediated metabolic signaling pathways. Therefore, it is likely that O-GlcNAcylation (OGN) plays an important role in regulating energy homeostasis in primary cilia. Abnormal OGN, as seen in cases of obesity and diabetes, may play an important role in primary cilia dysfunction mediated by these pathologies.

Enhancement of Production of D-Glucosamine in Escherichia coli by Blocking Three Pathways Involved in the Consumption of GlcN and GlcNAc.

D-Glucosamine is a commonly used dietary supplement that promotes cartilage health in humans. Metabolic flux analysis showed that D-glucosamine production could be increased by blocking three pathways involved in the consumption of glucosamine-6-phosphate and acetylglucosamine-6-phosphate. By homologous single-exchange, two key genes (nanE and murQ) of Escherichia coli BL21 were knocked out, respectively. The D-glucosamine yields of the engineered strains E. coli BL21ΔmurQ and E. coli BL21ΔnanE represented increases by factors of 2.14 and 1.79, respectively. Meanwhile, for bifunctional gene glmU, we only knocked out its glucosamine-1-phosphate acetyltransferase domain by 3D structural analysis to keep the engineered strain E. coli BL21glmU-Δgpa survival, which resulted in an increase in the production of D-glucosamine by a factor of 2.16. Moreover, for further increasing D-glucosamine production, two genes encoding rate-limiting enzymes, named glmS and gna1, were coexpressed by an RBS sequence in those engineered strains. The total concentrations of D-glucosamine in E. coli BL21 glmU-Δgpa', E. coli BL21ΔmurQ', and E. coli BL21ΔnanE' were 2.65 g/L, 1.73 g/L, and 1.38 g/L, which represented increases by factors of 8.83, 5.76, and 3.3, respectively.

Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis.

Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD600 in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation.

Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.

Mechanical stress alters protein O-GlcNAc in human periodontal ligament cells.

Protein O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of intracellular proteins that regulates several physiological and pathophysiological process, including response to various stressors. However, O-GlcNAc's response to mechanical stress has not been investigated yet. As human periodontal ligament (PDL) cells are stimulated by compression force during orthodontic tooth movement that results in structural remodelling, in this study we investigated whether mechanical stress induces any alteration in protein O-GlcNAc in PDL cells. In this study, PDL cells isolated from premolars extracted for orthodontic indications were exposed to 0, 1.5, 3, 7 and 14 g/cm2 compression forces for 12 hours. Cell viability was measured by flow cytometry, and protein O-GlcNAc was analysed by Western blot. Cellular structure and intracellular distribution of O-GlcNAc was studied by immunofluorescence microscopy. We found that between 1.5 and 3 g/cm2 mechanical compression, O-GlcNAc significantly elevated; however, at higher forces O-GlcNAc level was not increased. We also found that intracellular localization of O-GlcNAc proteins became more centralized under 2 g/cm2 compression force. Our results suggest that structural changes stimulated by compression forces have a significant effect on the regulation of O-GlcNAc; thus, it might play a role in the mechanical stress adaptation of PDL cells.