The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

2016 ◽  
Vol 85 (1) ◽  
pp. 631-657 ◽  
Author(s):  
Zebulon G. Levine ◽  
Suzanne Walker
Keyword(s):  
Mammalian Cells ◽  
Glcnac Transferase

scholarly journals The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila

2020 ◽  
Vol 117 (4) ◽  
pp. 2004-2013 ◽  
Author(s):  
Kenneth Kin Lam Wong ◽  
Ta-Wei Liu ◽  
Jessica M. Parker ◽  
Donald A. R. Sinclair ◽  
Yi-Yun Chen ◽  
...  
Keyword(s):  
Protein Stability ◽  
Mammalian Cells ◽  
Normal Diet ◽  
Cancer Proliferation ◽  
Hexosamine Biosynthetic Pathway ◽  
Interacting Protein ◽  
Molecular Sensors ◽  
Cellular Behaviors ◽  
Glcnac Transferase ◽  
Necessary And Sufficient

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.

scholarly journals The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress

2020 ◽  
Author(s):  
Sarel J. Urso ◽  
Marcella Comly ◽  
John A. Hanover ◽  
Todd Lamitina
Keyword(s):  
Stress Response ◽  
Cell Volume ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Physiology ◽  
Hypertonic Stress ◽  
C Elegans ◽  
Intracellular Proteins ◽  
Molecular Functions ◽  
Glcnac Transferase

AbstractThe conserved O-GlcNAc transferase OGT O-GlcNAcylates serine and threonine residues of intracellular proteins to regulate their function. OGT is required for viability in mammalian cells, but its specific roles in cellular physiology are poorly understood. Here we describe a conserved requirement for OGT in an essential aspect of cell physiology: the hypertonic stress response. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, mutations that ablate O-GlcNAcylation activity in either human or C. elegans OGT rescue the hypertonic stress response phenotype. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins.Author SummaryThe ability to sense and adapt to changes in the environment is an essential feature of cellular life. Changes in environmental salt and water concentrations can rapidly cause cell volume swelling or shrinkage and, if left unchecked, will lead to cell and organismal death. All organisms have developed similar physiological strategies for maintaining cell volume. However, the molecular mechanisms that control these physiological outputs are not well understood in animals. Using unbiased genetic screening in C. elegans, we discovered that a highly conserved enzyme called O-GlcNAc transferase (OGT) is essential for regulating physiological responses to increased environmental solute levels. A human form of OGT can functionally substitute for worm OGT, showing that this role is conserved across evolution. Surprisingly, the only known enzymatic activity of OGT was not required for this role, suggesting this enzyme has important undescribed molecular functions. Our studies reveal a new animal-specific role for OGT in the response to osmotic stress and show that C. elegans is an important model for defining the conserved molecular mechanisms that respond to alterations in cell volume.

scholarly journals Proteomic identification of the UDP-GlcNAc : PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

2020 ◽  
Author(s):  
Zhe Ji ◽  
Michele Tinti ◽  
Michael A.J. Ferguson
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Label Free ◽  
Proteomics Data ◽  
Protein Database ◽  
Gpi Anchor ◽  
C Terminus ◽  
Significant Enrichment ◽  
Glcnac Transferase

AbstractThe first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc : PI α1-6 GlcNAc-transferase. This enzyme has been shown to be a complex of at least seven subunits in mammalian cells and a similar complex of homologous subunits has been postulated in yeast. Homologs of most of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and partner proteins TbGPI15, TbGPI9, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc : PI α1-6 GlcNAc-transferase complex.Graphical abstractFirst step of GPI anchor biosynthesis pathway in T.brucei BSF is catalysed by TbGPI3 complex.

Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi

Parasitology ◽  
2001 ◽  
Vol 123 (1) ◽  
pp. 33-43 ◽  
Author(s):  
J. A. MORGADO-DÍAZ ◽  
C. V. NAKAMURA ◽  
O. A. AGRELLOS ◽  
W. B. DIAS ◽  
J. O. PREVIATO ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Trypanosoma Cruzi ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Sucrose Density Gradient ◽  
Isolation And Characterization ◽  
Golgi Region ◽  
Electron Microscopy Analysis ◽  
Glcnac Transferase

In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the β-galactosyltransferase (β-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-α-N-acetyl-glucosaminyltransferase (O-α-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the β-Gal transferase, the O-α-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.

scholarly journals Genetically encoded green fluorescent biosensors for monitoring UDP-GlcNAc in live cells

2021 ◽  
Author(s):  
Zefan Li ◽  
Jing Zhang ◽  
Huiwang Ai
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Fluorescent Protein ◽  
Culture Conditions ◽  
Live Cells ◽  
Uridine Diphosphate ◽  
Fluorescent Biosensors ◽  
Nucleotide Sugars ◽  
Green Fluorescent ◽  
Glcnac Transferase

Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a nucleotide sugar used by glycosyltransferases to synthesize glycoproteins, glycosaminoglycans, glycolipids, and glycoRNA. UDP-GlcNAc also serves as the donor substrate for the formation of O-GlcNAc, a dynamic intracellular protein modification involved in diverse signaling and disease processes. UDP-GlcNAc is thus a central metabolite connecting nutrition, metabolism, signaling, and disease. There is a great interest in monitoring UDP-GlcNAc in biological systems. Here, we present the first genetically encoded, green fluorescent UDP-GlcNAc sensor (UGAcS), an optimized insertion of a circularly permuted green fluorescent protein (cpGFP) into an inactive mutant of an E. coli UDP-GlcNAc transferase, for ratiometric monitoring of UDP-GlcNAc dynamics in live mammalian cells. Although UGAcS responds to UDP-GlcNAc quite selectively among various nucleotide sugars, UDP and UTP interfere with the response. We thus developed another biosensor named UXPS, which is responsive to UDP and UTP but not UDP-GlcNAc. We demonstrated the use of the biosensors to follow UDP-GlcNAc levels in cultured mammalian cells perturbed with nutritional changes, pharmacological inhibition, and knockdown or overexpression of key enzymes in the UDP-GlcNAc synthesis pathway. We further utilized the biosensors to monitor UDP-GlcNAc concentrations in pancreatic MIN6 β-cells under various culture conditions.

scholarly journals Point mutations that inactivate MGAT4D-L, an inhibitor of MGAT1 and complex N-glycan synthesis

2020 ◽  
Vol 295 (41) ◽  
pp. 14053-14064
Author(s):  
Ayodele Akintayo ◽  
Joshua Mayoral ◽  
Masahiro Asada ◽  
Jian Tang ◽  
Subha Sundaram ◽  
...  
Keyword(s):  
Amino Acids ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Mechanism Of Inhibition ◽  
C Terminus ◽  
Membrane Bound ◽  
Galanthus Nivalis ◽  
Glcnac Transferase

The membrane-bound, long form of MGAT4D, termed MGAT4D-L, inhibits MGAT1 activity in transfected cells and reduces the generation of complex N-glycans. MGAT1 is the GlcNAc-transferase that initiates complex and hybrid N-glycan synthesis. We show here that Drosophila MGAT1 was also inhibited by MGAT4D-L in S2 cells. In mammalian cells, expression of MGAT4D-L causes the substrate of MGAT1 (Man5GlcNAc2Asn) to accumulate on glycoproteins, a change that is detected by the lectin Galanthus nivalis agglutinin (GNA). Using GNA binding as an assay for the inhibition of MGAT1 in MGAT4D-L transfectants, we performed site-directed mutagenesis to determine requirements for MGAT1 inhibition. Deletion of 25 amino acids (aa) from the C terminus inactivated MGAT4D-L, but deletion of 20 aa did not. Conversion of the five key amino acids (PSLFQ) to Ala, or deletion of PSLFQ in the context of full-length MGAT4D-L, also inactivated MGAT1 inhibitory activity. Nevertheless, mutant, inactive MGAT4D-L interacted with MGAT1 in co-immuno-precipitation experiments. The PSLFQ sequence also occurs in MGAT4A and MGAT4B GlcNAc-transferases. However, neither inhibited MGAT1 in transfected CHO cells. MGAT4D-L inhibitory activity could be partially transferred by attaching PSLFQ or the 25-aa C terminus of MGAT4D-L to the C terminus of MGAT1. Mutation of each amino acid in PSLFQ to Ala identified both Leu and Phe as independently essential for MGAT4D-L activity. Thus, replacement of either Leu-395 or Phe-396 with Ala led to inactivation of MGAT4D-L inhibitory activity. These findings provide new insights into the mechanism of inhibition of MGAT1 by MGAT4D-L, and for the development of small molecule inhibitors of MGAT1.

scholarly journals Proteomic identification of the UDP-GlcNAc: PI α1–6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0244699
Author(s):  
Zhe Ji ◽  
Michele Tinti ◽  
Michael A. J. Ferguson
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Label Free ◽  
Proteomics Data ◽  
Protein Database ◽  
C Terminus ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Significant Enrichment ◽  
Glcnac Transferase

The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc: PI α1–6 GlcNAc-transferase, also known as GPI GnT. This enzyme has been shown to be a complex of seven subunits in mammalian cells and a similar complex of six homologous subunits has been postulated in yeast. Homologs of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and the expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc: PI α1–6 GlcNAc-transferase complex.

Ultrastructural Alterations in Hela Cells Induced by Spider Venom

1967 ◽  
Vol 25 ◽  
pp. 20-21
Author(s):  
Dale E. McClendon ◽  
Paul N. Morgan ◽  
Bernard L. Soloff
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Carcinoma Cells ◽  
Venom Protein ◽  
Sterile Saline ◽  
Carbon Coated ◽  
Available Information ◽  
Loxosceles Reclusa ◽  
Essential Growth ◽  
Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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