Method for determining activity of O-linked N-acetylglucosamine transferase and application of method

A technology of glucose transferase and acetylamino, which is applied in the field of biological analysis and detection, can solve the problems of inability to measure the activity of OGT, a large amount, etc., and achieves the effect of solving the problem of low catalytic activity and convenient operation.

Inactive Publication Date: 2020-11-13
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the FP-based substrate displacement method has made some breakthroughs in the study of OGT activity, this method requires a large number of enzymes and can only screen out inhibitors that are competitive with UDP-GlcNAc. More importantly, this method only Can measure inhibitor and enzyme binding ability, but cannot be used to measure OGT activity

Method used

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  • Method for determining activity of O-linked N-acetylglucosamine transferase and application of method
  • Method for determining activity of O-linked N-acetylglucosamine transferase and application of method
  • Method for determining activity of O-linked N-acetylglucosamine transferase and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Establishment of OGT activity assay method

[0042] Such as figure 2 As shown, the OGT activity assay method includes the following steps:

[0043] (1) Configure the OGT enzyme reaction system: containing 1 μM FAM-CKII peptide (its amino acid sequence is KKKYPGGSTPVSSANMM), 0.125mM UDP-GlcNAcN 3 , 50ng / μL ncOGT enzyme protein (no ncOGT enzyme protein was added to the control), buffer (150mM NaCl, 20mM Tris-HCl, 12.5mM MgCl 2 , 1 mM EDTA, pH 7.4).

[0044] (2) After incubating the above reaction system at 37°C in the dark for 6 hours, UDP with a final concentration of 1mM was added to terminate the reaction to obtain FAM-CKII-GlcNacN 3 .

[0045] (3) Mix bovine serum albumin (BSA) and dibenzocyclooctyne-active lipid-modified active lipid (DBCO-NHSester) at a molar ratio of 1:10, incubate at 37°C for 1 hour, and ultrafilter the unreacted DBCO-NHS ester is removed to generate DBCO-BSA complex;

[0046] (4) The reaction system (FAM-CKII-GlcNacN 3 ) was dil...

Embodiment 2

[0048] Example 2 Application of OGT activity assay method in high-throughput screening of OGT inhibitors

[0049] About 300 compounds in the marine natural product compound library preserved in the laboratory were screened for inhibitory activity. All reactions were carried out in a black 96-well plate (Cat.No.3694, Corning, USA). The specific screening method included the following steps:

[0050] (1) 10 mM natural product compound dissolved in DMSO (dimethyl sulfoxide) was diluted 40 times with 5% DMSO to obtain a sub-compound library.

[0051] (2) Add 30uL premix solution to the black 96-well plate, containing 50ng / μL ncOGT enzyme protein, 1μM FAM-CKII peptide (its amino acid sequence is KKKYPGGSTPVSSANMM), buffer (150mM NaCl, 20mM Tris-HCl, 12.5mMMgCl 2 , 1mM EDTA, pH 7.4), add 10μL of the compound of the sub-compound library (the compound of the sub-compound library in the OGT reaction system of the positive control), incubate at 37°C for 1 hour, then add 10uL of 0.125mM ...

Embodiment 3

[0058] Example 3 Application of OGT Activity Determination Method in OGT Inhibitor Half Inhibitory Concentration Determination

[0059] Half-inhibitory concentration (IC 50 ) determination, the determination method may further comprise the steps:

[0060] (1) Dilute the lead compound to 0, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 2000 μM with 10% DMSO, respectively.

[0061] (2) Add 30uL premix solution to the black 96-well plate, containing 50ng / μL ncOGT enzyme protein, 1μM FAM-CKII peptide (its amino acid sequence is KKKYPGGSTPVSSANMM), buffer (150mM NaCl, 20mM Tris-HCl, 12.5mMMgCl 2 , 1mM EDTA, pH 7.4), add 10μL of lead compounds diluted to different concentrations, incubate at 37℃ for 1 hour, then add 10uL 0.125mM (final concentration) UDP-GlcNAcN 3 start reacting.

[0062] (3) After incubating the above reaction system at 37°C in the dark for 1 hour, UDP with a final concentration of 1 mM was added to terminate the reaction to obtain FAM-CKII-GlcNacN 3 .

[0063] ...

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Abstract

The invention relates to the technical field of biological analysis and detection, in particular to a method for determining the activity of O-linked N-acetylglucosamine transferase and application ofthe method. The method comprises the following steps: firstly, modifying a glycosyl receptor of OGT by utilizing a fluorescent group, transferring azide-modified glycosyl to polypeptide through an enzyme reaction catalyzed by OGT, and then connecting the glycosyl receptor to macromolecular bovine serum albumin modified by dibenzocyclooctyne through a click reaction. The property that bovine serumalbumin is a macromolecule is utilized, and the bovine serum albumin is used as an 'FP tag 'capable of causing polarization signal enhancement, so that the enzyme activity of OGT is visually displayed. According to the invention, the problem of low catalytic activity of OGT on a complex modified substrate is solved, meanwhile, the correlation between the enzyme activity and the fluorescence polarization signal size is realized, and the method can be used for determining the enzyme activity of OGT, and can also be used for high-throughput screening of an OGT inhibitor and determination of theinhibitory activity of the OGT inhibitor.

Description

technical field [0001] The invention relates to the technical field of biological analysis and detection, in particular to a method for measuring the activity of O-linked N-acetylglucosamine transferase and the application of the method. Background technique [0002] O-linked N-acetylglucosamine (O-linkedβ-N-acetylglucosamine, O-GlcNAc) glycosylation is a common protein post-translational modification (Post-Translational Modifications, PTMs), which refers to the O-GlcNAc through O -The process of attaching glycosidic bonds to serines and threonines of proteins. Cytoskeletal proteins, transcription factors, epigenetic factors, kinases and phosphatases of eukaryotic cells all have different degrees of O-GlcNAc glycosylation. This post-translational modification plays an important role in cell signal transduction, transcription and translation, gene expression, growth and differentiation, immune protection and other processes. Similar to phosphorylation, O-GlcNAc glycosylatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48G01N21/64
CPCC12Q1/48G01N21/6428G01N21/6445G01N2333/91102
Inventor 高志增尹新坚李佳欣刘岚
Owner SUN YAT SEN UNIV
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