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

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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Inhibition of Trypanosoma brucei brucei peptidyl transferase activity by sparsomycin analogs and effects on trypanosome protein synthesis and proliferation

Biochemical Pharmacology ◽

10.1016/0006-2952(85)90147-9 ◽

1985 ◽

Vol 34 (17) ◽

pp. 3055-3060 ◽

Cited By ~ 1

Author(s):

Alan J. Bitonti ◽

Susan E. Kelly ◽

Gary A. Flynn ◽

Peter P. McCann

Keyword(s):

Protein Synthesis ◽

Trypanosoma Brucei ◽

Transferase Activity ◽

Peptidyl Transferase ◽

Trypanosoma Brucei Brucei

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A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification.

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.101153 ◽

2021 ◽

pp. 101153

Author(s):

Samuel M. Duncan ◽

Rupa Nagar ◽

Manuela Damerow ◽

Dmitry V. Yashunsky ◽

Benedetta Buzzi ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Gpi Anchor ◽

Glcnac Transferase

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Proteomic Profiling of Astrocytic O-GlcNAc Transferase-Related Proteins in the Medial Prefrontal Cortex

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.729975 ◽

2021 ◽

Vol 14 ◽

Author(s):

Jun Fan ◽

Qiu-Ling Zhong ◽

Ran Mo ◽

Cheng-Lin Lu ◽

Jing Ren ◽

...

Keyword(s):

Prefrontal Cortex ◽

Medial Prefrontal Cortex ◽

Transferase Activity ◽

Proteomic Profiling ◽

Major Energy Source ◽

Altered Proteins ◽

Glcnac Transferase ◽

Related Proteins ◽

The Brain

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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A UDP-GlcNAc : βGal β1-6 GlcNAc transferase involved in bloodstream form N-glycan and procyclic form GPI anchor elaboration in Trypanosoma brucei.

10.1101/2021.06.21.449264 ◽

2021 ◽

Author(s):

Samuel Martin Duncan ◽

Rupa Nagar ◽

Manuela Damerow ◽

Dmitry V. Yashunsky ◽

Benedetta Buzzi ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Bloodstream Form ◽

Wild Type ◽

Dual Roles ◽

Gpi Anchor ◽

Procyclic Form ◽

Life Cycle Stages ◽

Glcnac Transferase

Trypanosoma brucei has large carbohydrate extensions on its N-linked glycans and glycosylphosphatidylinositol (GPI) anchors in its bloodstream form (BSF) and procyclic form (PCF), respectively. The parasites glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are unknown. Here, we probe the function(s) of a putative β3GT gene, TbGT10. The BSF null mutant is viable in vitro and in vivo and can differentiate into PCF, demonstrating non-essentiality. However, the absence of TbGT10 led to impaired elaboration of N-glycans and GPI anchor sidechains in BSF and PCF parasites, respectively. Glycosylation defects include reduced BSF glycoprotein binding to ricin and to monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope of lysosomal glycoprotein p67 that we show here, using synthetic glycans, consists of (-6Gal1-4GlcNAc1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase glycopeptides isolated from BSF wild-type and TbGT10 null parasites show a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. Together, these data suggest that TbGT10 encodes a UDP-GlcNAc : βGal β1-6 GlcNAc-transferase active in both BSF and PCF life-cycle stages elaborating complex N-glycans and GPI sidechains, respectively. The β1-6 specificity of this β3GT gene product and its dual roles in N-glycan and GPI glycan elaboration are notable.

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

10.1101/2020.12.16.423025 ◽

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.

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