Glycobiology and Extracellular Matrices
Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation
Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells.O-GlcNAcylation of Thr12/Ser56 in short-form O-GlcNAc transferase (sOGT) regulates its substrate selectivity
O-GlcNAcylation is a ubiquitous protein glycosylation playing different roles on variant proteins. O-GlcNAc transferase (OGT) is the unique enzyme responsible for the sugar addition to nucleocytoplasmic proteins. Recently, multiple O-GlcNAc sites have been observed on short-form OGT (sOGT) and nucleocytoplasmic OGT (ncOGT), both of which locate in the nucleus and cytoplasm in cell. Moreover, O-GlcNAcylation of Ser389 in ncOGT (1036 amino acids) affects its nuclear translocation in HeLa cells. To date, the major O-GlcNAcylation sites and their roles in sOGT remain unknown.eIF4G1 and carboxypeptidase E axis dysregulation in O-GlcNAc transferase–deficient pancreatic β-cells contributes to hyperproinsulinemia in mice
An early hallmark of type 2 diabetes is a failure of proinsulin-to-insulin processing in pancreatic β-cells, resulting in hyperproinsulinemia. Proinsulin processing is quite sensitive to nutrient flux, and β-cell–specific deletion of the nutrient-sensing protein modifier OGlcNAc transferase (βOGTKO) causes β-cell failure and diabetes, including early development of hyperproinsulinemia. The mechanisms underlying this latter defect are unknown. Here, using several approaches, including site-directed mutagenesis, Click O-GlcNAc labeling, immunoblotting, and immunofluorescence and EM imaging, we provide the first evidence for a relationship between the O-GlcNAcylation of eukaryotic translation initiation factor 4γ1 (eIF4G1) and carboxypeptidase E (CPE)-dependent proinsulin processing in βOGTKO mice.O-GlcNAcylation regulates integrin-mediated cell adhesion and migration via formation of focal adhesion complexes
O-GlcNAcylation is a post-translational modification of a protein serine or threonine residue catalyzed by O-GlcNAc transferase (OGT) in the nucleus and cytoplasm. O-GlcNAcylation plays important roles in the cellular signaling that affect the different biological functions of cells, depending upon cell type. However, whether or not O-GlcNAcylation regulates cell adhesion and migration remains unclear. Here, we used the doxycycline-inducible short hairpin RNA (shRNA) system to establish an OGT knockdown (KD) HeLa cell line and found that O-GlcNAcylation is a key regulator for cell adhesion, migration, and focal adhesion (FA) complex formation.Nutrient regulation of signaling and transcription
In the early 1980s, while using purified glycosyltransferases to probe glycan structures on surfaces of living cells in the murine immune system, we discovered a novel form of serine/threonine protein glycosylation (O-linked β-GlcNAc; O-GlcNAc) that occurs on thousands of proteins within the nucleus, cytoplasm, and mitochondria. Prior to this discovery, it was dogma that protein glycosylation was restricted to the luminal compartments of the secretory pathway and on extracellular domains of membrane and secretory proteins.O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis
The addition of a single β-d-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of O-GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1–ER) and therefore undergo erythropoiesis after β-estradiol (E2) addition.O-GlcNAc transferase missense mutations linked to X-linked intellectual disability deregulate genes involved in cell fate determination and signaling
It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families.Effects of hypo-O-GlcNAcylation on Drosophila development
Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc (O-GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila, null mutants of the Polycomb gene O-GlcNAc transferase (OGT; also known as super sex combs (sxc)) display homeotic phenotypes. To dissect the requirement for O-GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants.Checkpoint kinase 1–induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis
Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1’s pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O-linked β-N-acetylglucosamine (O-GlcNAc)-transferase (OGT) as one of Chk1’s substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis.Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability
N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans.Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells
We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) under various glucose conditions in macrophage cells. At normal glucose concentrations, GlcN dose dependently increased LPS-stimulated production of NO/iNOS. However, GlcN suppressed NO/iNOS production under high glucose culture conditions. Moreover, GlcN suppressed LPS-induced up-regulation of COX-2, IL-6, and TNF-α mRNAs under 25 mm glucose conditions yet did not inhibit up-regulation under 5 mm glucose conditions.Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells
O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function.Human RNA Polymerase II Promoter Recruitment in Vitro Is Regulated by O-Linked N-Acetylglucosaminyltransferase (OGT)
Although the O-linked N-acetylglucosamine (O-GlcNAc) modification of the RNA polymerase II C-terminal domain was described 20 years ago, the function of this RNA polymerase II (pol II) species is not known. We show here that an O-GlcNAcylated pol II species (pol IIγ) exists on promoters in vitro. Inhibition of O-GlcNAc-transferase activity and O-GlcNAcylation prevents pol II entry into the promoter, and O-GlcNAc removal from pol II is an ATP-dependent step during initiation. These data indicate that O-GlcNAc-transferase activity is essential for RNA pol II promoter recruitment and that pol II goes through a cycling of O-GlcNAcylation at the promoter.Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation
Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2–5% of the total glucose, generating UDP-GlcNAc as the end product.O-GlcNAcylation Antagonizes Phosphorylation of CDH1 (CDC20 Homologue 1)
The anaphase promoting complex/cyclosome (APC/C) orchestrates various aspects of the eukaryotic cell cycle. One of its co-activators, Cdh1, is subject to myriad post-translational modifications, such as phosphorylation and ubiquitination. Herein we identify the O-linked N-acetylglucosamine (O-GlcNAc) modification that occurs on Cdh1. Cdh1 is O-GlcNAcylated in cultured cells and mouse brain extracts. Mass spectrometry identifies an O-GlcNAcylated peptide that neighbors a known phosphorylation site.E2F1 Transcription Factor Regulates O-linked N-acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Expression
Protein O-GlcNAcylation, which is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), has emerged as an important posttranslational modification that may factor in multiple diseases. Until recently, it was assumed that OGT/OGA protein expression was relatively constant. Several groups, including ours, have shown that OGT and/or OGA expression changes in several pathologic contexts, yet the cis and trans elements that regulate the expression of these enzymes remain essentially unexplored.Enhanced Transfer of a Photocross-linking N-Acetylglucosamine (GlcNAc) Analog by an O-GlcNAc Transferase Mutant with Converted Substrate Specificity
Background: Photocross-linking O-GlcNAc (O-GlcNDAz) can be used to capture O-GlcNAc-mediated protein-protein interactions.Results: Mutagenesis of the UDP-GlcNAc binding pocket of OGT enhances transfer of a photoreactive GlcNAc analog (GlcNDAz).Conclusion: OGT(C917A) catalyzes increased enzymatic incorporation of GlcNDAz at sites of protein O-GlcNAc modification in vitro and in cells.Significance: Enabling identification of O-GlcNAc-mediated protein interactions will provide insights into molecular mechanisms of O-GlcNAc function.
