Golgi UDP-GlcNAc:Polypeptide
            <i>O</i>
            -α-
            <i>N</i>
            -Acetyl-
            <scp>d</scp>
            -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

Koeller, Carolina M.; Wel, Hanke van der; Feasley, Christa L.; Abreu, Fernanda; Rocha, Juliana Dutra B.; Montalvão, Fabricio; Fampa, Patrı́cia; Reis, Flavia C G; Atella, Geórgia C.; Souto-Padrón, Thaïs; West, Christopher M.; Heise, Norton

doi:10.1128/ec.00165-14

Cited by 13 publications

(13 citation statements)

References 89 publications

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“…GPI-anchored proteins were enriched from delipidated pellets by partitioning in butan-1-ol (n-BuOH, 99%; Alfa Aesar, Tewksbury, MA) as described (103,104). Briefly, 20 mg of protein powder was extracted three times with 4 ml of H 2 O saturated with n-BuOH (ϳ9%) for 4 h at room temperature.…”

Section: Enrichment In Gpi-anchored Proteins and Gpi-glycan Core Releasementioning

confidence: 99%

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Gas-Pascual

Ichikawa

Sheikh

et al. 2019

Journal of Biological Chemistry

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Infection with the protozoan parasite Toxoplasma gondii is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, Toxoplasma propagates in intracellular parasitophorous vacuoles, but like nearly all other eukaryotes, Toxoplasma glycosylates many cellular proteins and lipids and assembles polysaccharides. Toxoplasma glycans resemble those of other eukaryotes, but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The Toxoplasma genome encodes a suite of likely glycogenes expected to assemble N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in Toxoplasma, here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc 0-2-Man 6-GlcNAc 2-type N-glycans, a novel HexNAc-GalNAc-mucin-type O-glycan, and Tn-antigen; identified the glycosyltransferases for assembling novel nuclear O-Fuc-type and cell surface Glc-Fuc-type O-glycans; and showed that they are important for in vitro growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes. Toxoplasma gondii is a worldwide, obligate intracellular apicomplexan parasite that can infect most nucleated cells of warm-blooded animals (1), with up to 80% of some human populations being seropositive (2). Toxoplasmosis, the disease caused by Toxoplasma, is associated with encephalitis and blindness in individuals whose parasites are reactivated, as can occur in AIDS and other immunosuppressed patients (3). In utero infections can cause mental retardation, blindness, and death (4). Toxoplasma is transmitted by digesting parasites from feline feces (as oocysts) or undercooked meat (as tissue cysts). Once in the host, parasites convert to the tachyzoite form that disseminates to peripheral tissues (e.g. brain, retina, and muscle). The resulting immune response and/or drugs can control tachyzoite replication, but the parasite survives by encysting into slowly growing bradyzoites. Sporadically, burst of cysts allows the parasites to convert to tachyzoites, whose unchecked growth results in cell and tissue damage (5, 6). Currently, no Toxoplasma vaccine exists, anti-toxoplasmosis drugs have severe side effects, and resistance is developing to these drugs (7-11). As individuals remain infected for life, new anti-Toxoplasma drugs a...

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Section: Enrichment In Gpi-anchored Proteins and Gpi-glycan Core Releasementioning

confidence: 99%

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Gas-Pascual

Ichikawa

Sheikh

et al. 2019

Journal of Biological Chemistry

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“…This intracellular localization suggests that TcNST2 is, at least in part, responsible for providing the pool of UDP-Gal inside the Golgi apparatus. In T. cruzi mucins, the O-linked glycans are attached to the polypeptide backbone via a GlcNAc residue, whose addition to threonine residues is catalyzed by a Golgi-localized O-GlcNAc transferase (Previato et al ., 1998; Heise et al ., 2009; Koeller et al ., 2014). Galactose residues inside the Golgi lumen are then added to GlcNAc by specific galactosyl transferases.…”

Section: Discussionmentioning

confidence: 99%

TcNST2encodes a Golgi-localized UDP-galactose transporter inTrypanosoma cruzi

et al. 2019

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Survival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) from Trypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line. TcNST2 mRNA levels were detected in all T. cruzi life-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

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“…In T. brucei , in which the synthesis of UDP-GlcNAc is essential for parasite survival, the pool of UDP-GlcNAc inside the Golgi lumen is necessary for the synthesis of N -acetyllactosamine [ 31 ]. In T. cruzi , the Golgi content of UDP-GlcNAc is important for the synthesis of O-linked glycans in mucins, which starts with the attachment of GlcNAc, from UDP-GlcNAc, to a Ser or Thr residue via an α- N -acetylglucosaminyltransferase [ 32 – 34 ]. Therefore, the transport of UDP-GlcNAc across the Golgi membrane is essential for the proper synthesis of T. cruzi mucins .…”

Section: Discussionmentioning

confidence: 99%

Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi

et al. 2015

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BackgroundNucleotide sugar transporters (NSTs) play an essential role in translocating nucleotide sugars into the lumen of the endoplasmic reticulum and Golgi apparatus to be used as substrates in glycosylation reactions. This intracellular transport is an essential step in the biosynthesis of glycoconjugates.ResultsWe have identified a family of 11 putative NSTs in Trypanosoma cruzi, the etiological agent of Chagas’ disease. A UDP-N-acetylglucosamine transporter, TcNST1, was identified by a yeast complementation approach. Based on a phylogenetic analysis four candidate genes were selected and used for complementation assays in a Kluyveromyces lactis mutant strain. The transporter is likely expressed in all stages of the parasite life cycle and during differentiation of epimastigotes to infective metacyclics. Immunofluorescence analyses of a GFP-TcNST1 fusion protein indicate that the transporter is localized to the Golgi apparatus. As many NSTs are multisubstrate transporters, we also tested the capacity of TcNST1 to transport GDP-Man.ConclusionsWe have identified a UDP-N-acetylglucosamine transporter in T. cruzi, which is specifically localized to the Golgi apparatus and seems to be expressed, at the mRNA level, throughout the parasite life cycle. Functional studies of TcNST1 will be important to unravel the role of NSTs and specific glycoconjugates in T. cruzi survival and infectivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0601-7) contains supplementary material, which is available to authorized users.

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Golgi UDP-GlcNAc:Polypeptide O -α- N -Acetyl- d -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

Cited by 13 publications

References 89 publications

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

TcNST2encodes a Golgi-localized UDP-galactose transporter inTrypanosoma cruzi

Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi

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