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Gene expression of SKOV3 cells after no treatment or treatment with 50 microM peracetylated GlcNAc or peracetylated 4-deoxy-GlcNAc for three days

ABSTRACT: Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analog of the HS constituent GlcNAc and studied the compound’s metabolic fate and its effect on angiogenesis. The 4-deoxy analog was activated intracellularly into UDP-4-deoxy-GlcNAc and HS expression was inhibited up to ~96% (IC50 = 16 µM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analog, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Micro-injection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis which hampers pro-angiogenic signaling and neo-vessel formation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE48284 | GEO | 2013/08/23

SECONDARY ACCESSION(S): PRJNA209515

REPOSITORIES: GEO

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Gene expression of SKOV3 cells after no treatment or treatment with 50 microM peracetylated GlcNAc or peracetylated 4-deoxy-GlcNAc for three days

Project description:Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analog of the HS constituent GlcNAc and studied the compoundM-bM-^@M-^Ys metabolic fate and its effect on angiogenesis. The 4-deoxy analog was activated intracellularly into UDP-4-deoxy-GlcNAc and HS expression was inhibited up to ~96% (IC50 = 16 M-BM-5M). HS chain size was reduced, without detectable incorporation of the 4-deoxy analog, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Micro-injection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis which hampers pro-angiogenic signaling and neo-vessel formation. 9 samples were analyzed: 3 biological replicates from untreated SKOV3 cells, 3 biological replicates from SKOV3 cells treated with peracetylated GlcNAc, 3 biological replicates from SKOV3 cells treated with peracetylated 4-deoxy-GlcNAc

2013-08-23 | E-GEOD-48284 | biostudies-arrayexpress

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is an inducer of apoptotic processes in Capan-1 pancreatic carcinoma cells

Project description:This SuperSeries is composed of the following subset Series: GSE22334: Induction of apoptotic processes in Capan-1 pancreatic carcinoma cells by restoration of p16INK4a expression GSE22336: UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is an inducer of apoptotic processes in Capan-1 pancreatic carcinoma cells: GNE silencing Refer to individual Series

2010-08-01 | E-GEOD-22337 | biostudies-arrayexpress

Plant UDP-N-acetylglucosamine (UDP-GlcNAc) transporter (UGNT1) glycopeptide analysis

Project description:Glycosylation reactions require activated glycosyl donors in form of nucleotide sugars to drive processes such as post-translational protein modifications, glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi requiring cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters. Here we present the identification of the plant UDP-N-acetylglucosamine (UDP-GlcNAc) transporter (UGNT1) indispensable for the delivery of a substrate for maturation of N-glycans and glycosyl inositol phosphorylceramides (GIPCs). Profiles of N-glycopeptides revealed that UGNT1 loss-of-function mutants are devoid of complex and hybrid N-glycans. Instead, most of the glycol-N-peptide population contained high mannose structures, representing the structure prior to the addition of the first GlcNAc in the Golgi. Our findings emphasize that the reference plant Arabidopsis contains a single UDP-GlcNAc transporter responsible for the maturation of complex N-glycans in the Golgi lumen.

2018-09-24 | PXD006635 | Pride

O-glcnAc reprograms cellular energetics

Project description:Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O- glcnAcylation) via overexpression of the O-glcnAc–regulating enzymes O- glcnAc transferase (OGT) or O- glcnAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O- glcnAcylation either by pharmacological or genetic manipulation also alters metabolic function. Sustained O-glcnAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-glcnAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-Seq in SH-SY5Y cells indicated transcriptome reprogramming and down regulation of the NRF2-mediated antioxidant response. Sustained O-glcnAcylation in mice brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-glcnAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-glcnAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

2017-07-20 | GSE101642 | GEO

Transcriptional profiing of cultured adherent and non-adherent monocytes

Project description:To determine the effect of adherence on the metabolic and functional response of human monocytes. Monocytes were stimulated with lipopolysaccharide (LPS) under non-adherent and adherent conditions. To determine the role of glycolysis in LPS-induced immune responses, this pathway was inhibited by glucose deprivation or the glucose analog 2-deoxy-D-glucose (2DG).

2020-12-08 | GSE161839 | GEO

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

Project description: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. 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 Arv-1 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.

2021-02-26 | PXD022979 | Pride

O-GlcNAc Homeostasis Controls Cell Fate Decisions During Hematopoiesis

Project description:The addition of O-GlcNAc (a single β-D-N-acetylglucosamine sugar at serine and threonine residues) by O-GlcNAc transferase (OGT) and removal by O-GlcNAcase (OGA) maintains homeostatic levels of O-GlcNAc. We investigated the role of OGlcNAc homeostasis in hematopoiesis utilizing G1E-ER4 cells carrying a GATA-1 transcription factor fused to the estrogen receptor (GATA-1ER) that undergo erythropoiesis following the addition of β-estradiol (E2) and myeloid leukemia cells that differentiate into neutrophils in the presence of all-trans retinoic acid. During G1E-ER4 differentiation, a decrease in overall O-GlcNAc levels and an increase in GATA-1 interactions with OGT and OGA were observed. Transcriptome analysis on G1E-ER4 cells differentiated in the presence of Thiamet-G (TMG), an OGA inhibitor, identified expression changes in 433 GATA-1 target genes. Chromatin immunoprecipitation demonstrated that the occupancy of GATA-1, OGT, and OGA at Laptm5 gene GATA site was decreased with TMG. Myeloid leukemia cells showed a decline in O-GlcNAc levels during differentiation and TMG reduced the expression of genes involved in differentiation. Sustained treatment with TMG in G1E-ER4 cells prior to differentiation caused a reduction of hemoglobin positive cells during differentiation. Our results show that alterations in O-GlcNAc homeostasis disrupt transcriptional programs causing differentiation errors suggesting a vital role of O-GlcNAcylation in control of cell fate.

2018-11-06 | GSE122104 | GEO

O-GlcNAc expression levels epigenetically regulate colon cancer tumorigenesis by affecting the cancer stem cell compartment via modulating expression of transcriptional factor MYBL1

Project description:To study the regulation of colorectal adenocarcinoma progression by O-GlcNAc, we have focused on the O-GlcNAc-mediated epigenetic regulation of human colon cancer stem cells (CCSC). Xenograft tumors from colon tumor cells with OGT knockdown grew significantly slower than those formed from control cells, indicating a reduced proliferation of tumor cells due to inhibition of OGT expression. Significant reduction of CCSC population was observed in the tumor cells after OGT knockdown, while tumor cells treated with O-GlcNAcase inhibitor showed an increased CCSC population, indicating that O-GlcNAc levels regulated the CCSC compartment. When grown in suspension, tumor cells with OGT knockdown showed a reduced ability to form tumorspheres, indicating a reduced self-renewal of CCSC due to reduced levels of O-GlcNAc. ChIP-seq experiments using an anti-O-GlcNAc antibody revealed significant chromatin enrichment of O-GlcNAc modified proteins at the promoter of the transcription factor MYBL1, which was also characterized by the presence of H3K27me3. RNA-seq analysis showed an increased expression of MYBL1 in tumor cells with OGT knockdown. Forced overexpression of MYBL1 led to a reduced population of CCSC and tumor growth in vivo, similar to the effects of OGT silencing. Moreover, two CpG islands near the TSS site of MYBL1 were identified, and O-GlcNAc levels regulated their methylation status. These results strongly argue that O-GlcNAc epigenetically regulates MYBL1, functioning similarly to H3K27me3. The aberrant CCSC compartment observed after modulating O-GlcNAc levels, therefore, is likely to result, at least in part, from the epigenetic regulation of MYBL1 expression by O-GlcNAc, thereby significantly affecting tumor progression.

2017-08-25 | GSE93656 | GEO

Genome-wide maps of GlcNAc bound proteins and Pho ChIP-seq in Drosophila S2 cells and pupae.

Project description:We developed a strategy to map GlcNAc modified proteins based on a chemical chromatin precipitation strategy. Drosophila S2 cells, as well as wild type (wt) and sxc null pupae, were fed with 100 uM Ac4GalNAz and the resulting DNA was cross-linked, sonicated, and purified. DNA strands cross-linked to GlcNAz proteins were isolated by congugating with alkyn-biotin by Staudinger ligation followed by streptavidin purification. The resulting DNA was constructed into libraries for sequencing. To asses the robustness of our strategy, we compared GalNAz ChIP-seq results in S2 cells with two other GlcNAc ChIP-seq strategies, using a mutant β-1,4-galactosyltransferase (GalT) and the lecting wheat germ agglutinin (WGA). Briefly, GalT was incubated with cross-linked, sonicated and purified DNA along with UDP-GalNAz. Ligation to biotin with click chemistry followed by streptavidin purification resulted in library ready material. For WGA and Pho ChIP-seq, cross-linked and purified DNA was incubated with pho antibody or WGA resin and purified followed by library preperation.

2017-07-18 | GSE84502 | GEO

Identification of LGALS1 binding partners in T84 cells by proteomics using a photocrosslinking GlcNAc analog

Project description:Surface glycoproteins from T84 cells were metabolically incorporated with a diazirine functionalized GlcNAc analog and crosslinked to biotinylated LGALS1 on intact cells. The crosslinked glycoproteins were enriched by streptavidin beads and separated by SDS PAGE. Crosslinked bands identified by silver stain were analyzed by proteomics. Regions at the same molecular weight from the silver stained gel but from a sample that was not incorporated with the diazirine functionalized GlcNAc analog and not crosslinked to biotinylated LGALS1 were used as negative controls.

2021-06-16 | MSV000087640 | MassIVE

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