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Expression and purification of OGT or CKII. (a) The pGEX-2T-OGT plasmid was transformed into BL21 (DE3) cells, and the transformants were induced by IPTG. GST-OGT was purified as described. Proteins were analyzed by SDS-PAGE and Coomassie Blue-stained. Line 1 is the whole protein of negative control which did not express GST-OGT, the soluble supernatant containing GST-OGT is shown in line 2, and line 3 is the purified GST-OGT. (b) BL21 (DE3) cells containing pET30a-CKII plasmid was induced by IPTG. CKII-His was obtained as described. Proteins were analyzed by SDS-PAGE and Coomassie Blue-stained. The whole protein of negative control which did not express CKII is shown in line 1, line 2 shows the whole protein containing CKII, the soluble supernatant is shown in line 3 and the insoluble substance in line 4, line 5 is the purified CKII-His protein. Note: OGT, O-GlcNAc transferase; IPTG, isopropyl-β-D-thiogalactoside; SDS-PAGE, SDS-polyacrylamidegel electrophoresis.
Source publication
O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from UDP-GlcNAc to selected Ser/Thr residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with a number of diseases, suggesting...
Contexts in source publication
Context 1
... the recombinant protein was purified with Glutathione Sepharose column as described. SDS-PAGE analysis indicated that OGT was expressed and purified effectively (Figure 1a), the yields was 58% by one-step affinity chromatography. At last, the concentration of GST- OGT was determined by BCA Protein Quantification Kit, and the protein was diluted to 0.5 mg/mL. ...
Context 2
... pET30a-CKII expression vector was used to express His-tagged CKII in BL21 (DE3), then the protein was obtained as described. SDS-PAGE showed that CKII was expressed and purified effectively (Figure 1b), the yields were 87% by one-step affinity chromatography. Then, the concentration of CKII was determined by BCA Protein Quantification Kit, and the protein was diluted to 0.2 mg/mL. ...
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Citations
... In 2007, Leavy et al. reported the first high throughput azido-ELISA for measuring the activity of OGT on small peptide substrates, laying the foundation for HTS of small molecule inhibitors or larger peptide libraries (Fig. 10) [63]. In 2017, Qi et al. reported a new ELISA-based method for the detection of OGT activity in vitro [64]. This method uses recombinant casein kinase II (CKII) as the substrate for OGT, with UDP-GlcNAc as the sugar donor. ...
Glycosyltransferases (GTs) are a diverse group of enzymes widely distributed in nature that catalyze the transfer of glycosyl moieties from activated sugar donors to a diverse range of acceptor molecules. GTs play important roles in various biological processes, such as cellular signal transduction, cell–cell interactions, immune response and disease. In addition, the utilization of glycosyltransferases to synthesize glycosides has become a major area of pharmaceutical research. The construction of a rapid and efficient screening method for glycosyltransferases is the key to the successful discovery, identification, and modification of new enzymes. In the past few decades, a series of screening methods have been developed to assess GT activity. This review focuses on various methods for rapid screening of GTs, outlining their advantages and disadvantages.
Graphical Abstract
... New England Biolabs, Thermo Fisher Scientific, R&D Systems, Enzo Life Sciences, and Sino Biological). Examples of its bacterial expression and purification also exist in the literature.4 UDP-GlcNAc standards: Dissolve 25 mg UDP-GlcNAc sodium salt in 384 mL water to obtain a 100 mM stock solution. ...
Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is the end product of the hexosamine biosynthetic pathway and the substrate for protein O-linked N-acetylglucosaminylation (O-GlcNAcylation). Here, we present a protocol for the quantification of UDP-GlcNAc using an enzymatic microplate assay. We also detail procedures for the extraction of polar metabolites and total protein fraction for the parallel quantification of UDP-GlcNAc and the western blot analysis of O-GlcNAcylated proteins, O-linked N-acetylglucosamine transferase, and O-GlcNAcase from the same sample.
For complete details on the use and execution of this protocol, please refer to Sunden et al. (2023).¹ In addition, a preview article by Chatham et al. provides a useful summary of the method.²
... In 2007, Leavy et al. reported the first high throughput azido-ELISA assay for measuring the activity of OGT on small peptide substrates, laying the foundation for HTS of small molecule inhibitors or larger peptide libraries (Fig.10) 63 . In 2017, Qi et al. reported a new ELISA-based method for the detection of OGT activity in vitro 64 . This method uses recombinant casein kinase II (CKII) as the substrate for OGT, with UDP-GlcNAc as the sugar donor. ...
Glycosyltransferases (GTs) are a diverse group of transferases widely distributed in microorganisms and plants which catalyze the transfer of glycosyl moieties from activated sugar donors to a diverse range of acceptor molecules. GTs play important roles in biological processes, such as cell signal transduction, cell-cell interactions, immune response and diseases. In addition, the utilization of glycosyltransferases to synthesize glycosides has become a major area of pharmaceutical research. The construction of a rapid and efficient screening method for glycosyltransferases is the key to the successful discovery, identification, and modification of new enzymes. In the past few decades, a series of screening methods have been developed to assess activity. This review focuses on various methods for rapid screening of GTs, outlining their advantages and disadvantages.
... After unsuccessful attempts to quantify UDP-GlcNAc with the previously reported enzymatic assay 18 ( Figure S1), we looked into methods to measure glycosyltransferase activity of OGT 19,20 and whether these methods could be converted for the measurement of UDP-GlcNAc. Given the reported 11 high affinity of OGT for UDP-GlcNAc and multiple options to detect O-GlcNAcylated proteins, we reasoned that OGT-mediated O-GlcNAcylation could, in theory, be used to develop an assay for UDP-GlcNAc. ...
... This is our experience and is also reported in the literature. 19,23 The initial assay concept was successful with purified UDP-GlcNAc standards and, importantly, with liver tissue extract ( Figures 1B-1E). In the absence of OGT, the liver extract did not give any measurable signal, verifying complete removal of endogenous O-GlcNAcylated proteins ( Figure 1B). ...
O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).
... After unsuccessful attempts to quantify UDP-GlcNAc with the previously reported enzymatic assay 18 ( Supplementary Fig. 1) and competitive enzyme-linked lectin-binding approach (Supplementary Materials and Methods), we looked into methods to measure glycosyltransferase activity of OGT, 19,20 and whether these methods could be converted for the measurement of UDP-GlcNAc. Given the reported high affinity of OGT for UDP-GlcNAc and multiple options to detect O-GlcNAcylated proteins, we reasoned that OGT-mediated O-GlcNAcylation could, in theory, be utilized to develop an assay for UDP-GlcNAc. ...
... This is our experience and also reported in the literature. 19,23 The initial assay concept was successful with purified UDP-GlcNAc standards and, importantly, with liver tissue extract ( Figure 1B-E). In the absence of OGT, the liver extract did not give any measurable signal, verifying complete removal of endogenous O-GlcNAcylated proteins ( Figure 1B). ...
O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic yet still relatively poorly understood non-canonical glycosylation of intracellular proteins. Several vital branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of this metabolite is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it restrict to specialized chromatographic procedures. To overcome this limitation, we introduce here an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by recombinant O-linked N-acetylglucosamine transferase (OGT) and detection of the modification with a specific antibody. The assay can be performed in dot blot or microplate formats. The key to successful assay was the removal of strong inhibition of OGT by the reaction side product, uridine diphosphate (UDP). We applied the assay to provide the first systematic report of UDP-GlcNAc concentrations in mouse tissues and cultured cells. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).
... The recombinant plasmid pET-28a-OGT was preserved by our laboratory. The expression and purification experiments were carried out as descripted previously (24). Briefly, the plasmid DNA was transformed into E. coli BL21 (DE3) cells and induced with isopropylb-D-thiogalactoside (IPTG). ...
Introduction
O-GlcNAcylation is a type of reversible post-translational modification on Ser/Thr residues of intracellular proteins in eukaryotic cells, which is generated by the sole O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Thousands of proteins, that are involved in various physiological and pathological processes, have been found to be O-GlcNAcylated. However, due to the lack of favorable tools, studies of the O-GlcNAcylation and OGT were impeded. Immunoglobulin new antigen receptor (IgNAR) derived from shark is attractive to research tools, diagnosis and therapeutics. The variable domain of IgNARs (VNARs) have several advantages, such as small size, good stability, low-cost manufacture, and peculiar paratope structure.
Methods
We obtained shark single domain antibodies targeting OGT by shark immunization, phage display library construction and panning. ELISA and BIACORE were used to assess the affinity of the antibodies to the antigen and three shark single-domain antibodies with high affinity were successfully screened. The three antibodies were assessed for intracellular function by flow cytometry and immunofluorescence co-localization.
Results
In this study, three anti-OGT VNARs (2D9, 3F7 and 4G2) were obtained by phage display panning. The affinity values were measured using surface plasmon resonance (SPR) that 2D9, 3F7 and 4G2 bound to OGT with KD values of 35.5 nM, 53.4 nM and 89.7 nM, respectively. Then, the VNARs were biotinylated and used for the detection and localization of OGT by ELISA, flow cytometry and immunofluorescence. 2D9, 3F7 and 4G2 were exhibited the EC50 values of 102.1 nM, 40.75 nM and 120.7 nM respectively. VNAR 3F7 was predicted to bind the amino acid residues of Ser375, Phe377, Cys379 and Tyr 380 on OGT.
Discussion
Our results show that shark single-domain antibodies targeting OGT can be used for in vitro detection and intracellular co-localization of OGT, providing a powerful tool for the study of OGT and O-GlcNAcylation.
... Actually, it is possible to measure OGT activity with natural donor substrate UDP-GlcNAc and substrate proteins/peptides (e.g., coated in an ELISA plate), in which the pan GlcNAc-specific antibody (e.g., CTD 110.6 and RL2) followed by fluorescence or horseradish peroxidase (HRP) conjugated secondary antibody is utilized for detection. 265 Since the sensitivity depends on the anti-O-GlcNAc antibody used, this approach may only be applicable to purified OGT rather than OGT in crude samples. ...
Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a unique monosaccharide modification discovered in the early 1980s. With the technological advances in the past several decades, great progress has been made to reveal the biochemistry of O-GlcNAcylation, the substrates of O-GlcNAcylation, and the functional importance of protein O-GlcNAcylation. As a nutrient sensor, protein O-GlcNAcylation plays important roles in almost all biochemical processes examined. Although the functional importance of O-GlcNAcylation of proteins has been extensively reviewed previously, the chemical and biochemical aspects have not been fully addressed. In this review, by critically evaluating key publications in the past 35 years, we aim to provide a comprehensive understanding of this important post-translational modification (PTM) from analytical and biochemical perspectives. Specifically, we will cover (1) multiple analytical advances in the characterization of O-GlcNAc cycling components (i.e., the substrate donor UDP-GlcNAc, the two key enzymes O-GlcNAc transferase and O-GlcNAcase, and O-GlcNAc substrate proteins), (2) the biochemical characterization of the enzymes with a variety of chemical tools, and (3) exploration of O-GlcNAc cycling and its modulating chemicals as potential biomarkers and therapeutic drugs for diseases. Last but not least, we will discuss the challenges and possible solutions for basic and translational research of protein O-GlcNAcylation in the future.
... [12,13] ELISA-based luminescent approaches have also been developed, whereby the transfer of am onosaccharide unit is detected by at agged pan-specific antibody. [14] Furthermore,acoupled fucosyltransferase assay,i n which the transfer of afucose to afluorogenic oligosaccharide prevents ag lycoside hydrolase from acting to release the fluorophore reporter was developed recently. [15] Va rious fluorescence polarization (FP) assays have also been advanced. ...
... Despite its importance in contributing to cellular physiology,and the complexity of the mechanisms by which OGT function must accordingly be regulated, studying this enzyme remains challenging in part because of al ack of convenient assays and chemical biology tools. [14,36] Herein, we describe the development of asimple and sensitive fluorescence-based assay that directly and precisely measures glycosyl transfer catalyzed by OGT.W ev alidate this assay and illustrate its amenability to HTS in amicroplate format. After implementing the assay in at rial screen, we identify ak nown bioactive inhibitor that also antagonizes OGT and confirm its ability to directly inhibit OGT. ...
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.
... [12,13] ELISA-based luminescent approaches have also been developed, whereby the transfer of am onosaccharide unit is detected by at agged pan-specific antibody. [14] Furthermore,acoupled fucosyltransferase assay,i n which the transfer of afucose to afluorogenic oligosaccharide prevents ag lycoside hydrolase from acting to release the fluorophore reporter was developed recently. [15] Va rious fluorescence polarization (FP) assays have also been advanced. ...
... Despite its importance in contributing to cellular physiology,and the complexity of the mechanisms by which OGT function must accordingly be regulated, studying this enzyme remains challenging in part because of al ack of convenient assays and chemical biology tools. [14,36] Herein, we describe the development of asimple and sensitive fluorescence-based assay that directly and precisely measures glycosyl transfer catalyzed by OGT.W ev alidate this assay and illustrate its amenability to HTS in amicroplate format. After implementing the assay in at rial screen, we identify ak nown bioactive inhibitor that also antagonizes OGT and confirm its ability to directly inhibit OGT. ...
Within mammals there are often several functionally related glycoside hydrolases, which makes monitoring their activities problematic. This problem is particularly acute for the enzyme β-glucocerebrosidase (GCase), malfunction of which is a key driver of Gaucher Disease (GD) and a major risk factor for Parkinson Disease (PD). Humans harbour two other functionally related β-glucosidases known as GBA2 and GBA3 and currently used fluorogenic substrates are not selective, which has driven the use of complicated subtractive assays involving the use of detergents and inhibitors. Here we describe the preparation of fluorogenic substrates based upon the widely used non-selective substrate resorufin β-D-glucopyranoside. Using recombinant enzymes, we show these substrates are highly selective for GCase. We also demonstrate their value through analysis of GCase activity in brain tissue homogenates from transgenic mice expressing mutant human GCase and patient fibroblasts expressing mutant GCase. This approach simplifies analysis of cell and tissue homogenates and should facilitate analysis of clinical and laboratory tissues and samples.
... [12,13] ELISA-based luminescent approaches have also been developed, whereby the transfer of am onosaccharide unit is detected by at agged pan-specific antibody. [14] Furthermore,acoupled fucosyltransferase assay,i n which the transfer of afucose to afluorogenic oligosaccharide prevents ag lycoside hydrolase from acting to release the fluorophore reporter was developed recently. [15] Va rious fluorescence polarization (FP) assays have also been advanced. ...
... Despite its importance in contributing to cellular physiology,and the complexity of the mechanisms by which OGT function must accordingly be regulated, studying this enzyme remains challenging in part because of al ack of convenient assays and chemical biology tools. [14,36] Herein, we describe the development of asimple and sensitive fluorescence-based assay that directly and precisely measures glycosyl transfer catalyzed by OGT.W ev alidate this assay and illustrate its amenability to HTS in amicroplate format. After implementing the assay in at rial screen, we identify ak nown bioactive inhibitor that also antagonizes OGT and confirm its ability to directly inhibit OGT. ...
A robust fluorescence-based assay accurately reports on the sugar-transfer activity of glycosyltransferases. This assay is amenable to high-throughput screening and should be applicable to a diverse set of group-transfer enzymes. This assay is validated by screening of a library of known bioactive molecules to identify a new O-GlcNAc transferase antagonist.
Abstract
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O-GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group-transfer enzymes.
