A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to <i>O</i>‐GlcNAc Transferase

Alteen, Matthew G.; Gros, Christina; Meek, Richard W.; Cardoso, David A.; Busmann, Jil A.; Sangouard, Gontran; Deen, Matthew C.; Tan, Hong; Shen, David L.; Russell, Cecilia C.; Davies, G.J.; Robinson, Phillip J.; McCluskey, Adam; Vocadlo, David J.

doi:10.1002/anie.202000621

Cited by 20 publications

(25 citation statements)

References 57 publications

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“…Many studies have contributed to the current knowledge of glycosylation of cells in TGF-β signaling. To determine the activity of glycosyltransferases and glycosidases in vitro , researchers have developed, and continue to improve, chromatographic, radiochemical or spectrophotometric techniques to follow the loss of substrates or the formation of the reaction products (Laughlin and Bertozzi, 2009 ; van Kooyk et al, 2013 ; Alteen et al, 2020 ). The lectin microarray (Zhang et al, 2016 ) and mass spectrometry (Couto et al, 2018 ; Gargano et al, 2020 ) are used to check glycosylation profiles and to discover new glycan structures.…”

Section: Discussionmentioning

confidence: 99%

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

et al. 2020

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Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions. Aberrant glycosylation can lead to uncontrolled cell proliferation, cell-matrix interactions, migration and differentiation, and has been shown to be involved in cancer and other diseases. The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream. This cellular transformation process, which is associated by morphological change, loss of epithelial traits and gain of mesenchymal markers, is triggered by the secreted cytokine transforming growth factor-β (TGF-β). TGF-β bioactivity is carefully regulated, and its effects on cells are mediated by its receptors on the cell surface. In this review, we first provide a brief overview of major types of glycans, namely, N-glycans, O-glycans, glycosphingolipids and glycosaminoglycans that are involved in cancer progression. Thereafter, we summarize studies on how the glycosylation of TGF-β signaling components regulates TGF-β secretion, bioavailability and TGF-β receptor function. Then, we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer. Identifying and understanding the mechanisms by which glycosylation affects TGF-β signaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.

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Section: Discussionmentioning

confidence: 99%

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

et al. 2020

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“…The Vocadlo group published a new bioassay for screening OGT inhibitors in HTS based on a chemically modified UDP-GlcNAc [ 39 ]. The modified GlcNAc was prepared by extending the amino group of the corresponding glucosamine precursor with a Bodipy fluorescent group ( Figure 11 ).…”

Section: Assays For Evaluation Of Glcnac-transferase Activitymentioning

confidence: 99%

“…This assay was used to perform a high-throughput screening on a library of known bioactive compounds [ 39 ]. The screening led to the discovery of Dingo-4a as a potent OGT inhibitor with IC 50 of 6 µM ( Figure 13 ), which was originally developed as a target for dynamic GTPase (Dynl).…”

Section: Assays For Evaluation Of Glcnac-transferase Activitymentioning

confidence: 99%

Overview of the Assays to Probe O-Linked β-N-Acetylglucosamine Transferase Binding and Activity

2021

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O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked β-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer’s disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.

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“…[6] The Vocadlo group recently reported a novel, highthroughput, direct fluorescent activity assay for OGT based on streptavidin-coated microplates or streptavidin-coated magnetic beads. [7] However, the assay requires washing steps and those microplates or magnetic beads are expensive for large scale HTS campaigns.…”

mentioning

confidence: 99%

An Economical High‐Throughput “FP‐Tag” Assay for Screening Glycosyltransferase Inhibitors**

Yin

Chen

et al. 2021

ChemBioChem

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O-GlcNAc transferase (OGT) is involved in many cellular processes, and selective OGT inhibitors are valuable tools to investigate O-GlcNAcylation functions, and could potentially lead to therapeutics. However, high-throughput OGT assays that are suitable for large-scale HTS and can identify inhibitors targeting both acceptor, donor sites, and allosteric binding-sites are still lacking. Here, we report the development of a highthroughput "FP-Tag" OGT assay with bovine serum albumin (BSA) as a low-cost and superior "FP-Tag". With this assay, 2methyleurotinone was identified as a low-micromolar OGT inhibitor. This type of assay with BSA as "FP-Tag" would find more applications with other glycosyltransferases.

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A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Cited by 20 publications

References 57 publications

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

Overview of the Assays to Probe O-Linked β-N-Acetylglucosamine Transferase Binding and Activity

An Economical High‐Throughput “FP‐Tag” Assay for Screening Glycosyltransferase Inhibitors**

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