Protease-protection strategy combined with the SERS tags for detection of O-GlcNAc transferase activity

The medial prefrontal cortex (mPFC), a key part of the brain networks that are closely related to the regulation of behavior, acts as a key regulator in emotion, social cognition, and decision making. Astrocytes are the majority cell type of glial cells, which play a significant role in a number of processes and establish a suitable environment for the functioning of neurons, including the brain energy metabolism. Astrocyte’s dysfunction in the mPFC has been implicated in various neuropsychiatric disorders. Glucose is a major energy source in the brain. In glucose metabolism, part of glucose is used to convert UDP-GlcNAc as a donor molecule for O-GlcNAcylation, which is controlled by a group of enzymes, O-GlcNAc transferase enzyme (OGT), and O-GlcNAcase (OGA). However, the role of O-GlcNAcylation in astrocytes is almost completely unknown. Our research showed that astrocytic OGT could influence the expression of proteins in the mPFC. Most of these altered proteins participate in metabolic processes, transferase activity, and biosynthetic processes. GFAP, an astrocyte maker, was increased after OGT deletion. These results provide a framework for further study on the role of astrocytic OGT/O-GlcNAcylation in the mPFC.

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Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

eLife ◽

10.7554/elife.31807 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 26

Author(s):

Heather J Tarbet ◽

Lee Dolat ◽

Timothy J Smith ◽

Brett M Condon ◽

E Timothy O'Brien ◽

...

Keyword(s):

Protein Interactions ◽

Transferase Activity ◽

Protein Protein Interactions ◽

Site Specific ◽

Form And Function ◽

Glycosylation Sites ◽

Wide Range ◽

Dynamics And Function ◽

Glcnac Transferase ◽

And Function

Intermediate filaments (IF) are a major component of the metazoan cytoskeleton and are essential for normal cell morphology, motility, and signal transduction. Dysregulation of IFs causes a wide range of human diseases, including skin disorders, cardiomyopathies, lipodystrophy, and neuropathy. Despite this pathophysiological significance, how cells regulate IF structure, dynamics, and function remains poorly understood. Here, we show that site-specific modification of the prototypical IF protein vimentin with O-linked β-N-acetylglucosamine (O-GlcNAc) mediates its homotypic protein-protein interactions and is required in human cells for IF morphology and cell migration. In addition, we show that the intracellular pathogen Chlamydia trachomatis, which remodels the host IF cytoskeleton during infection, requires specific vimentin glycosylation sites and O-GlcNAc transferase activity to maintain its replicative niche. Our results provide new insight into the biochemical and cell biological functions of vimentin O-GlcNAcylation, and may have broad implications for our understanding of the regulation of IF proteins in general.

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Altered expression of leucocyte sialoglycoprotein in Wiskott-Aldrich syndrome is associated with a specific defect in O-glycosylation

Biochemistry and Cell Biology ◽

10.1139/o89-081 ◽

1989 ◽

Vol 67 (9) ◽

pp. 503-509 ◽

Cited By ~ 30

Author(s):

Wenda L. Greer ◽

Elizabeth Higgins ◽

D. Robert Sutherland ◽

Abraham Novogrodsky ◽

Inka Brockhausen ◽

...

Keyword(s):

Immune Deficiency ◽

T Cell Activation ◽

Cell Activation ◽

Galactose Oxidase ◽

Cell Surface Expression ◽

Transferase Activity ◽

Membrane Glycoproteins ◽

Altered Expression ◽

Wiskott Aldrich Syndrome ◽

Glcnac Transferase

The Wiskott-Aldrich syndrome (WAS) is an X-linked immune deficiency disorder characterized clinically by both lymphocyte and platelet dysfunction. Studies of WAS T lymphocytes have revealed deficient or defective cell surface expression of the highly O-glycosylated leucocyte sialoglycoprotein CD43. To further elucidate the basis for, and functional relevance of, CD43 modifications on WAS lymphocytes, we have studied lymphocytes from two WAS patients with regard to membrane glycoprotein profile and mitogen-induced proliferative responses. CD43 was found to be either absent or altered in size on peripheral blood lymphocytes and lectin-stimulated T cells from both patients. Compared with control cells, the WAS lymphocytes displayed reduced, but measurable proliferative responses to lectins and neuraminidase/galactose oxidase, and virtually no response to periodate, a mitogenic agent which targets sialic acid residues on membrane glycoproteins such as CD43. Analysis of activities of three glycosyltransferases involved in O-glycosylation revealed marked reduction in the level of activity of UDP-N-acetylglucosamine: Galβ1-3GalNAc-R β-1,6-N-acetylglucosamine (β-1,6-GlcNAc) transferase in one WAS patient and no detectable activity of this enzyme in a second. β-1,6-GlcNAc transferase activity has recently been shown to increase during T cell activation coincident with changes in the O-linked glycans on CD43. A selective reduction of this glycosyltransferase in WAS lymphocytes suggests that O-linked oligosaccharides may be important to the structure of membrane glycoproteins involved in lymphocyte activation.Key words: Wiskott-Aldrich syndrome, immune deficiency, O-glycosylation, glycosyltransferase, lymphocyte activation.

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New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro

Preparative Biochemistry & Biotechnology ◽

10.1080/10826068.2017.1303614 ◽

2017 ◽

Vol 47 (7) ◽

pp. 699-702 ◽

Cited By ~ 2

Author(s):

Jieqiong Qi ◽

Ruihong Wang ◽

Yazhen Zeng ◽

Wengong Yu ◽

Yuchao Gu

Keyword(s):

Transferase Activity ◽

Glcnac Transferase

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Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei

10.1101/2021.03.18.436003 ◽

2021 ◽

Author(s):

Aurelio Jenni ◽

Sebastian Knüsel ◽

Rupa Nagar ◽

Mattias Benninger ◽

Robert Häner ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Trypanosoma Brucei ◽

Model Organism ◽

Surface Proteins ◽

Transferase Activity ◽

A Subunit ◽

Major Surface Proteins ◽

Major Surface ◽

Glcnac Transferase

AbstractThe biosynthesis of glycosylphosphatidylinositol (GPI) membrane protein anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from the sugar nucleotide UDP-GlcNAc to phosphatidylinositol. The reaction is catalyzed by GPI GlcNAc transferase, a multi-subunit complex comprising the catalytic subunit Gpi3/PIG-A, as well as at least five other subunits including the hydrophobic protein Gpi2 which is essential for activity in yeast and mammals, but whose function is not known. Here we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism, to investigate the function of Gpi2. We generated trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites (i) GPI GlcNAc transferase activity is reduced but not lost, in contrast with the situation in yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of the major surface proteins are underglycosylated when compared with their wild-type counterparts, indicating the importance of TbGPI2 for reactions that are expected to occur in the Golgi apparatus. Additionally, TbGPI2-null parasites were unable to perform social motility, a form of collective migration on agarose plates. Immunofluorescence microscopy localized TbGPI2 to the endoplasmic reticulum as expected, but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic non-canonical role in Golgi-localized GPI anchor modification in trypanosomes.

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The β1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone

FEBS Letters ◽

10.1016/s0014-5793(97)00818-1 ◽

1997 ◽

Vol 412 (3) ◽

pp. 637-642 ◽

Cited By ~ 9

Author(s):

J Helin ◽

L Penttilä ◽

A Leppänen ◽

H Maaheimo ◽

S Lauri ◽

...

Keyword(s):

Transferase Activity ◽

Site Specific ◽

Branch Formation ◽

Glcnac Transferase

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Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity

Analytical Chemistry ◽

10.1021/acs.analchem.7b04065 ◽

2017 ◽

Vol 89 (23) ◽

pp. 12992-12999 ◽

Cited By ~ 26

Author(s):

Juan Hu ◽

Yueying Li ◽

Ying Li ◽

Bo Tang ◽

Chun-yang Zhang

Keyword(s):

Quantum Dot ◽

Sensitive Detection ◽

Transferase Activity ◽

Single Quantum ◽

Single Quantum Dot ◽

Glcnac Transferase

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Gpi19, the Saccharomyces cerevisiae Homologue of Mammalian PIG-P, Is a Subunit of the Initial Enzyme for Glycosylphosphatidylinositol Anchor Biosynthesis

Eukaryotic Cell ◽

10.1128/ec.4.11.1801-1807.2005 ◽

2005 ◽

Vol 4 (11) ◽

pp. 1801-1807 ◽

Cited By ~ 19

Author(s):

Heather A. Newman ◽

Martin J. Romeo ◽

Sarah E. Lewis ◽

Benjamin C. Yan ◽

Peter Orlean ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Sequence Similarity ◽

Secretory Proteins ◽

Temperature Sensitive ◽

Transferase Activity ◽

Its Gene ◽

A Subunit ◽

Glcnac Transferase

ABSTRACT Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-N-acetylglucosaminyltransferase (GPI-GlcNAc transferase). The human enzyme has six known subunits, at least four of which, GPI1, PIG-A, PIG-C, and PIG-H, have functional homologs in the budding yeast Saccharomyces cerevisiae. The uncharacterized yeast gene YDR437w encodes a protein with some sequence similarity to human PIG-P, a fifth subunit of the GPI-GlcNAc transferase. Here we show that Ydr437w is a small but essential subunit of the yeast GPI-GlcNAc transferase, and we designate its gene GPI19. Similar to other mutants in the yeast enzyme, temperature-sensitive gpi19 mutants display cell wall defects and hyperactive Ras phenotypes. The Gpi19 protein associates with the yeast GPI-GlcNAc transferase in vivo, as judged by coimmuneprecipitation with the Gpi2 subunit. Moreover, conditional gpi19 mutants are defective for GPI-GlcNAc transferase activity in vitro. Finally, we present evidence for the topology of Gpi19 within the ER membrane.

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