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

Fahie, Kamau; Narayanan, Bhargavi; Zahra, Fiddia; Reeves, Russell A.; Fernandes, Steve M.; Hart, Gerald W.; Zachara, Natasha E.

doi:10.1002/cpz1.129

Cited by 11 publications

(19 citation statements)

References 125 publications

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“…Because N-acetylneuraminic acid binds strongly to WGA through ionic interaction, succinylation of WGA abrogates the binding to N-acetylneuraminic acid but not to GlcNAc [ 6 ]. Therefore, succinylated WGA is also used to detect O-GlcNAcylated proteins specifically [ 7 ]. The binding force between WGA and GlcNAc is as weak as K a = 1.3 x 10 3 M -1 [ 20 ], which is considered to be the reason for its poor sensitivity ( Fig 1B ).…”

Section: Discussionmentioning

confidence: 99%

“…Because of such reason, glycoproteins without O-GlcNAc modification may be also detected and purified together. To increase the sensitivity of O-GlcNAcylated proteins, mutated β1,4-galactosyltransferase and azido-modified GalNAz as a substrate are used in a Click-iT O-GlcNAc enzymatic labeling system (Invitrogen) [ 7 ]. This system can be detected in a relatively short period, however there is a risk that glycoproteins other than O-GlcNAc modified proteins will be also detected because GalNAz is metabolized to GlcNAz and so on.…”

Section: Discussionmentioning

confidence: 99%

“…Such chemical modification leads to WGA binds only to GlcNAc [ 6 ]. Therefore, succinylated WGA is used to specifically detect O-GlcNAcylated proteins [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

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Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

et al. 2022

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O-GlcNAcylation is the only sugar modification for proteins present in the cytoplasm and nucleus and is thought to be involved in the regulation of protein function and localization. Currently, several methods are known for detecting O-GlcNAcylated proteins using monoclonal antibodies or wheat germ agglutinin, but these methods have some limitations in their sensitivity and quantitative comparison. We developed a new disaccharide-tag method to overcome these problems. This is a method in which a soluble GalNAc transferase is expressed intracellularly, extended to a disaccharide of GalNAc-GlcNAc, and detected using a Wisteria japonica agglutinin specific to this disaccharide. We verified the method using human c-Rel protein and also highly sensitively compared the difference in O-GlcNAc modification of intracellular proteins associated with differentiation from embryonic stem cell (ESC) to epiblast-like cells (EpiLC). As one example of such a modification, a novel O-GlcNAc modification was found in the transcription factor Sox2 at residue Ser263, and the modification site could be identified by nano liquid chromatography-mass spectrometry.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

et al. 2022

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“…BeWo cells were cultured at 37 °C, 5% CO 2 in medium (1:1 DMEM:Ham’s F12; Sigma) supplemented with 10% fetal bovine serum (Sigma), 2 mM l -glutamine, 100 µg/mL streptomycin, 100 IU/mL penicillin (Sigma). To simulate nutrient flux through the HBP, 2 mM or 2.5 mM glucosamine (Sigma) Glucosamine is a well well-established tool for activating the HBP 55 , as unlike glucose it is not metabolised by other pathways, enabling the HBP to be studied in isolation. Glucosamine enters the HBP downstream of the rate-limiting enzyme GFAT 56 , which is normally inhibited by raised UDP-GlcNAc levels.…”

Section: Methodsmentioning

confidence: 99%

Altered protein O-GlcNAcylation in placentas from mothers with diabetes causes aberrant endocytosis in placental trophoblast cells

Palin

Russell

Graham

et al. 2021

Sci Rep

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Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing.

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“…47 Due to the negative charge of the lectin, succinylated WGA (sWGA) hardly binds to sialoglycoproteins, 48 thereby improving the specicity of O-GlcNAc, although its affinity is not as good as that of WGA. 49 Lectins can be used for separation of O-GlcNAc peptides in liquid chromatography (LC). Vosseller et al demonstrated that lectin (WGA) weak affinity chromatography (LWAC) successfully separated O-GlcNAc peptides, which eluted later than peptides.…”

Section: 21mentioning

confidence: 99%

Recent development of analytical methods for disease-specific proteinO-GlcNAcylation

Zhang

Zhou

et al. 2023

RSC Adv.

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

Cited by 11 publications

References 125 publications

Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

Altered protein O-GlcNAcylation in placentas from mothers with diabetes causes aberrant endocytosis in placental trophoblast cells

Recent development of analytical methods for disease-specific proteinO-GlcNAcylation

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