Detection and Analysis of Proteins Modified by O‐Linked <i>N</i>‐Acetylglucosamine

Zachara, Natasha E.; Vosseller, Keith; Hart, Gerald W.

doi:10.1002/0471140864.ps1208s66

Cited by 53 publications

(34 citation statements)

References 86 publications

(82 reference statements)

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“…Using these methods, many S. elongatus proteins were detected, but wild-type and mutant samples showed the same labeling patterns (data not shown). Several antibodies are currently available to detect the O-GlcNAc modification (18). Western blotting using these antibodies detected many proteins but did not reveal differences between the mutant and wild type (data not shown).…”

Section: Discussionmentioning

confidence: 96%

“…Several methods are currently used to identify O-GlcNAcylated proteins, including transfer of labeled galactose to a known terminal O-GlcNAc, the use of antibodies against the OGlcNAc modification, and chemi-enzymatic methods (18). The GalT assay uses the enzyme ␤-1,4-galactosyl transferase, which adds a labeled galactose to proteins with glycosylations terminating in GlcNAc.…”

Section: Discussionmentioning

confidence: 99%

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The Putative Eukaryote-LikeO-GlcNAc Transferase of the Cyanobacterium Synechococcus elongatus PCC 7942 Hydrolyzes UDP-GlcNAc and Is Involved in Multiple Cellular Processes

Sokol

Olszewski

2014

J. Bacteriol.

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The posttranslational addition of a single O-linked ␤-N-acetylglucosamine (O-GlcNAc) to serine or threonine residues regulates numerous metazoan cellular processes. The enzyme responsible for this modification, O-GlcNAc transferase (OGT), is conserved among a wide variety of organisms and is critical for the viability of many eukaryotes. Although OGTs with domain structures similar to those of eukaryotic OGTs are predicted for many bacterial species, the cellular roles of these OGTs are unknown. We have identified a putative OGT in the cyanobacterium Synechococcus elongatus PCC 7942 that shows active-site homology and similar domain structure to eukaryotic OGTs. An OGT deletion mutant was created and found to exhibit several phenotypes. Without agitation, mutant cells aggregate and settle out of the medium. The mutant cells have higher free inorganic phosphate levels, wider thylakoid lumen, and differential accumulation of electron-dense inclusion bodies. These phenotypes are rescued by reintroduction of the wild-type OGT but are not fully rescued by OGTs with single amino acid substitutions corresponding to mutations that reduce eukaryotic OGT activity. S. elongatus OGT purified from Escherichia coli hydrolyzed the sugar donor, UDP-GlcNAc, while the mutant OGTs that did not fully rescue the deletion mutant phenotypes had reduced or no activity. These results suggest that bacterial eukaryote-like OGTs, like their eukaryotic counterparts, influence multiple processes.A lthough long believed to be solely attributes of eukaryotes, many glycosylation pathways are now evident in prokaryotes. Bacterial proteins can be modified with a variety of N-linked (1) and O-linked glycans (2). Posttranslational modification of serine or threonine residues with single O-linked ␤-N-acetylglucosamine (GlcNAc) by O-GlcNAc transferases (OGTs) is common in eukaryotes. All eukaryotic OGTs share a domain structure consisting of tetratricopeptide repeats (TPRs) that are involved in protein-protein interactions (3) followed by the glycosyltransferase catalytic region (Fig. 1A). The catalytic region is composed of two highly conserved domains (4), which are linked by a variable-length insertion. OGT homologs occur across prokaryotic phyla (4), and a number of these OGTs are eukaryote-like (Fig. 1B); this is best illustrated by the similarities in the crystal structures of the Xanthomonas campestris (5, 6) and human (7,8) OGTs. In addition to sharing similar domain structures, key amino acids in and around the active site are conserved in the eukaryotic and bacterial enzymes (see Fig. S1 in the supplemental material).Unlike other eukaryotic glycosylations, O-GlcNAcylation occurs in the nucleus and cytosol and is dynamically added and removed from proteins. O-GlcNAcylation can regulate a cellular process directly or by acting in competition with phosphorylation (9, 10). Notably, and unlike phosphorylation, which employs a plethora of site-specific kinases and phosphatases, the cycling of O-GlcNAcylation relies on two highly conserved enzyme...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

The Putative Eukaryote-LikeO-GlcNAc Transferase of the Cyanobacterium Synechococcus elongatus PCC 7942 Hydrolyzes UDP-GlcNAc and Is Involved in Multiple Cellular Processes

Sokol

Olszewski

2014

J. Bacteriol.

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“…Detection of PilA was performed as previously described (2), while detection of HmpD followed the same protocol (using a 1:10,000 dilution of primary antibody), with the exception that a 10% SDS-PAGE gel was used. Detection of O-GlcNAcylated proteins using the anti-OGlcNAc monoclonal antibody CTD110.6 (Enzo Life Sciences, Inc.) in conjunction with goat anti-mouse IgG-horseradish-conjugated peroxidase (HRP) (sc2005; Santa Cruz Biotechnology), or biotinylated succinylated wheat germ agglutinin (sWGA) (Vector Laboratories) in conjunction with the avidin-HRP conjugate Vectastain ABC reagent, was performed as previously described (2,41). Detection and quantification of soluble hormogonium polysaccharide in the culture medium by lectin blotting with biotinylated Ulex europaeus agglutinin (UEA) were performed as previously described (3).…”

Section: Methodsmentioning

confidence: 99%

A Putative O-Linked β- N -Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme

et al. 2017

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Most species of filamentous cyanobacteria are capable of gliding motility, likely via a conserved type IV pilus-like system that may also secrete a motilityassociated polysaccharide. In a subset of these organisms, motility is achieved only after the transient differentiation of hormogonia, which are specialized filaments that enter a nongrowth state dedicated to motility. Despite the fundamental importance of hormogonia to the life cycles of many filamentous cyanobacteria, the molecular regulation of hormogonium development is largely undefined. To systematically identify genes essential for hormogonium development and motility in the model heterocyst-forming filamentous cyanobacterium Nostoc punctiforme, a forward genetic screen was employed. The first gene identified using this screen, designated ogtA, encodes a putative O-linked ␤-N-acetylglucosamine transferase (OGT). The deletion of ogtA abolished motility, while ectopic expression of ogtA induced hormogonium development even under hormogonium-repressing conditions. Transcription of ogtA is rapidly upregulated (1 h) following hormogonium induction, and an OgtA-GFPuv fusion protein localized to the cytoplasm. In developing hormogonia, accumulation of PilA but not HmpD is dependent on ogtA. Reverse transcriptionquantitative PCR (RT-qPCR) analysis indicated equivalent levels of pilA transcript in the wild-type and ΔogtA mutant strains, while a reporter construct consisting of the intergenic region in the 5= direction of pilA fused to gfp produced lower levels of fluorescence in the ΔogtA mutant strain than in the wild type. The production of hormogonium polysaccharide in the ΔogtA mutant strain is reduced compared to that in the wild type but comparable to that in a pilA deletion strain. Collectively, these results imply that O-GlcNAc protein modification regulates the accumulation of PilA via a posttranscriptional mechanism in developing hormogonia.IMPORTANCE Filamentous cyanobacteria are among the most developmentally complex prokaryotes. Species such as Nostoc punctiforme develop an array of cell types, including nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogonia, that function in dispersal as well as the establishment of nitrogen-fixing symbioses with plants and fungi. These symbioses are major contributors to global nitrogen fixation. Despite the fundamental importance of hormogonia to the life cycle of filamentous cyanobacteria and the establishment of symbioses, the molecular regulation of hormogonium development is largely undefined. We employed a genetic screen to identify genes essential for hormogonium development and motility in Nostoc punctiforme. The first gene identified using this screen encodes a eukaryotic-like O-linked ␤-N-acetylglucosamine transferase that is required for accumulation of PilA in hormogonia.

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“…These antibodies fall into two classes: those that can be considered pan-specific and others that are site specific (Zachara et al 2011c). The two most commonly used pan-specific antibodies for detecting O-GlcNAc-modified proteins are CTD110.6 , a mouse IgM antibody raised against the C-terminus of RNA Pol II, and RL2 (Snow et al 1987), a mouse IgG antibody raised against O-GlcNAc-modified components of the nuclear pore complex.…”

Section: Immunopurification With Antibodies and Lectinsmentioning

confidence: 99%

“…As such, proteins that interact with O-GlcNAc-modified proteins are also identified, and in most cases O-GlcNAc-modified peptides are suppressed in the MS making O-GlcNAc modification sites difficult to map. Nonetheless, a number of studies have been performed highlighting proteins that are potentially OGlcNAc-modified in response to cellular stress and tissue injury Teo et al 2010;Zachara et al 2011c), and the O-GlcNAcylation status of a number of proteins has been confirmed by independent techniques (Table 1). One recent study used 1F5.D6(14), 18B10.C7(3), and 9D1.E3(10) to immunoprecipitate O-GlcNAc-modified proteins followed by large-scale shotgun proteomics to identify more than 200 differentially expressed glycoproteins from HEK293 cells and rat livers responding to trauma hemorrhage and resuscitation (Teo et al 2010).…”

Section: Immunopurification With Antibodies and Lectinsmentioning

confidence: 99%

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

Groves

Lee

Yildirir

et al. 2013

Cell Stress and Chaperones

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120

112

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O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a ubiquitous and dynamic post-translational modification known to modify over 3,000 nuclear, cytoplasmic, and mitochondrial eukaryotic proteins. Addition of O-GlcNAc to proteins is catalyzed by the O-GlcNAc transferase and is removed by a neutral-N-acetyl-β-glucosaminidase (OGlcNAcase). O-GlcNAc is thought to regulate proteins in a manner analogous to protein phosphorylation, and the cycling of this carbohydrate modification regulates many cellular functions such as the cellular stress response. Diverse forms of cellular stress and tissue injury result in enhanced O-GlcNAc modification, or O-GlcNAcylation, of numerous intracellular proteins. Stress-induced OGlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, calcium homeostasis, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation. Here, we will discuss the regulation of these processes by O-GlcNAc and the impact of such regulation on survival in models of ischemia reperfusion injury and trauma hemorrhage. We will also discuss the misregulation of O-GlcNAc in diseases commonly associated with the stress response, namely Alzheimer's and Parkinson's diseases. Finally, we will highlight recent advancements in the tools and technologies used to study the O-GlcNAc modification.

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Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine

Cited by 53 publications

References 86 publications

The Putative Eukaryote-LikeO-GlcNAc Transferase of the Cyanobacterium Synechococcus elongatus PCC 7942 Hydrolyzes UDP-GlcNAc and Is Involved in Multiple Cellular Processes

The Putative Eukaryote-LikeO-GlcNAc Transferase of the Cyanobacterium Synechococcus elongatus PCC 7942 Hydrolyzes UDP-GlcNAc and Is Involved in Multiple Cellular Processes

A Putative O-Linked β- N -Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

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