A Chemoenzymatic Histology Method for O‐GlcNAc Detection

Aguilar, Aime Lopez; Hou, Xiaomeng; Wen, Liuqing; Wang, Peng George; Wu, Peng

doi:10.1002/cbic.201700515

Cited by 25 publications

(17 citation statements)

References 48 publications

(74 reference statements)

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“…In the past, various methods have been explored for O-GlcNAc detection (Thompson et al, 2018). For examples, radioisotope methods for O-GlcNAc detection include incorporation of [ 3 H]-Gal by galactosyltransferase (Holt et al, 1987) and [ 35 S]-SO 3 by carbohydrate sulfotransferases, CHST2 and CHST4 (Wu et al, 2014); chemical methods for O-GlcNAc labeling include incorporation of modified Gal or GalNAc or GlcNAc using engineered mutant of b-1,4-galactosyltransferase (Aguilar et al, 2017;Clark et al, 2008;Khidekel et al, 2003) or through metabolic pathways (Vocadlo et al, 2003). Besides, O-GlcNAc antibody and O-GlcNAc binding protein (Mariappa et al, 2015) have been developed for identification of O-GlcNAc modified proteins.…”

Section: Introductionmentioning

confidence: 99%

Detecting and Imaging O-GlcNAc Sites Using Glycosyltransferases: A Systematic Approach to Study O-GlcNAc

Tatge

Grill

et al. 2018

Cell Chemical Biology

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O-GlcNAcylation is a reversible serine/threonine glycosylation for regulating protein activity and availability inside cells. In a given protein, O-GlcNAcylated and unoccupied O-linked β-N-acetylglucosamine (O-GlcNAc) sites are referred to as closed and open sites, respectively. The balance between open and closed sites is believed to be dynamically regulated. In this report, closed sites are detected using in vitro incorporation of GalNAz by B3GALNT2, and open sites are detected by in vitro incorporation of GlcNAz by O-GlcNAc transferase (OGT), via click chemistry. For assessing total O-GlcNAc sites, a sample is O-GlcNAcylated in vitro by OGT before detecting by B3GALNT2. The methods are demonstrated on purified recombinant proteins including CK2, AKT1, and PFKFB3, and cellular extracts of HEK cells. Through O-GlcNAc imaging, the modification degree of O-GlcNAc in nuclei of Chinese hamster ovary cells was estimated. The detection and imaging of both open and closed O-GlcNAc sites provide a systematic approach to study this important post-translational modification.

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Section: Introductionmentioning

confidence: 99%

Detecting and Imaging O-GlcNAc Sites Using Glycosyltransferases: A Systematic Approach to Study O-GlcNAc

Tatge

Grill

et al. 2018

Cell Chemical Biology

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“…For example, tissue-specific knockout of OGT in neurons or even specifically in the forebrain of mice results in neurodegeneration and neuron death (3,4). Measurement of O-GlcNAc levels in human brains has shown decreased modification in Alzheimer's disease compared with healthy controls (5,6). In mice, increasing the amounts of O-GlcNAcylation with a small-molecule inhibitor of OGA (7) slows neurodegeneration and the formation of tau aggregates in a model of Alzheimer's disease (8).…”

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confidence: 99%

α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease

Levine

Galesic

Balana

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

174

189

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A compelling link is emerging between the posttranslational modification O-GlcNAc and protein aggregation. A prime example is α-synuclein, which forms toxic aggregates that are associated with neurodegeneration in Parkinson’s and related diseases. α-Synuclein has been shown to be O-GlcNAcylated at nine different positions in in vivo proteomics experiments from mouse and human tissues. This raises the possibility that O-GlcNAc may alter the aggregation of this protein and could be both an important biological mediator of neurodegeneration and also a therapeutic target. Here, we expand upon our previous research in this area through the chemical synthesis of six site-specifically O-GlcNAcylated variants of α-synuclein. We then use a variety of biochemical experiments to show that O-GlcNAc in general inhibits the aggregation of α-synuclein but can also alter the structure of α-synuclein aggregates in site-specific ways. Additionally, an α-synuclein protein bearing three O-GlcNAc modifications can inhibit the aggregation of unmodified protein. Primary cell culture experiments also show that several of the O-GlcNAc sites inhibit the toxicity of extracellular α-synuclein fibers that are likely culprits in the spread of Parkinson’s disease. We also demonstrate that O-GlcNAcylation can inhibit the aggregation of an aggressive mutant of α-synuclein, indicating that therapies currently in development that increase this modification might be applied in animal models that rely on this mutant. Finally, we also show that the pan-selective antibody for O-GlcNAc does not generally recognize this modification on α-synuclein, potentially explaining why it remains understudied. These results support further development of O-GlcNAcylation tools and therapeutic strategies in neurodegenerative diseases.

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“…The combination of metabolic labeling and click chemistry has emerged as a powerful method to study proteins and protein modifications (Mahal et al, ; Hang et al, ; Bond and Kohler, ; Dieterich et al, ; Hanson et al, ; Hong et al, ; Smeekens et al, ; Chen et al, ; Cheng et al, ; Spiciarich et al, ; Xiao and Wu, ; Xiao et al, ). Metabolic labeling of glycans with unnatural sugar analogs is an excellent way to study glycans and protein glycosylation (Kayser et al, ; Saxon and Bertozzi, ; Feng et al, ; Aguilar et al, , ; Park et al, ; Qin et al, ). In the recent two decades, the Bertozzi group has performed pioneering work on using unnatural sugar analogs to label glycoproteins (Mahal et al, ; Saxon and Bertozzi, ; Hang et al, ; Breidenbach et al, ).…”

Section: Global Analysis Of Glycoproteinsmentioning

confidence: 99%

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

Xiao

Sun

Suttapitugsakul

et al. 2019

Mass Spectrometry Reviews

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Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site‐specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)‐based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS‐based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS‐based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX–XX, 2019.

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A Chemoenzymatic Histology Method for O‐GlcNAc Detection

Cited by 25 publications

References 48 publications

Detecting and Imaging O-GlcNAc Sites Using Glycosyltransferases: A Systematic Approach to Study O-GlcNAc

Detecting and Imaging O-GlcNAc Sites Using Glycosyltransferases: A Systematic Approach to Study O-GlcNAc

α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

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