Limits...
Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.

Kim EJ, Abramowitz LK, Bond MR, Love DC, Kang DW, Leucke HF, Kang DW, Ahn JS, Hanover JA - Bioconjug. Chem. (2014)

Bottom Line: The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes.The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors.The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Science Education-Chemistry Major, Daegu University , Gyeongbuk 712-714, South Korea.

ABSTRACT
The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.

Show MeSH
Strategy for Ni-NTA PlateOGT Assay. The high-throughput Ni-NTAOGT assay described herein consists of simple steps as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215860&req=5

fig2: Strategy for Ni-NTA PlateOGT Assay. The high-throughput Ni-NTAOGT assay described herein consists of simple steps as indicated.

Mentions: In this Communication,we present a direct and efficient OGT microplateassay in which purification of the enzyme is unnecessary: both bacterialand eukaryotic cell extracts can be readily used as sources of OGT.In addition, because bacterial extracts expressing recombinant OGTisoforms can be employed, the method is applicable to identifyingnew enzyme variants. This method exploits one of the most widely usedaffinity tags, a six consecutive histidine (6 × His) tag, andnickel ions immobilized on a chelating microplate. The 6 × His-tagcan be genetically engineered at either the amino- or carboxy-terminusof recombinant proteins. The method we developed here uses UDP-GlcNAz(1, Figure 1) as a sugar donorand 6 × His-tagged protein substrates as the sugar acceptors.All reactions are carried out in a microplate coated with Ni2+ ions through coupling with the chelating moiety of nitrilotriaceticacid (NTA). As depicted in Figure 2, the strategyof our OGT assay involves five steps starting with attachment of the6 × His-tagged OGT substrate to the plate through Ni-NTA chelation,followed by the blocking of unoccupied nickel ion. Protein substratebound to the plate is then subjected to OGT enzymatic catalysis usingUDP-GlcNAz as the sugar donor. Next, a chemoselective reaction, suchas the copper-catalyzed “click” reaction, or Staudingerligation, is performed to label the azido-functionality. Finally,an immunoassay is used to detect the labeled proteins.


Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.

Kim EJ, Abramowitz LK, Bond MR, Love DC, Kang DW, Leucke HF, Kang DW, Ahn JS, Hanover JA - Bioconjug. Chem. (2014)

Strategy for Ni-NTA PlateOGT Assay. The high-throughput Ni-NTAOGT assay described herein consists of simple steps as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215860&req=5

fig2: Strategy for Ni-NTA PlateOGT Assay. The high-throughput Ni-NTAOGT assay described herein consists of simple steps as indicated.
Mentions: In this Communication,we present a direct and efficient OGT microplateassay in which purification of the enzyme is unnecessary: both bacterialand eukaryotic cell extracts can be readily used as sources of OGT.In addition, because bacterial extracts expressing recombinant OGTisoforms can be employed, the method is applicable to identifyingnew enzyme variants. This method exploits one of the most widely usedaffinity tags, a six consecutive histidine (6 × His) tag, andnickel ions immobilized on a chelating microplate. The 6 × His-tagcan be genetically engineered at either the amino- or carboxy-terminusof recombinant proteins. The method we developed here uses UDP-GlcNAz(1, Figure 1) as a sugar donorand 6 × His-tagged protein substrates as the sugar acceptors.All reactions are carried out in a microplate coated with Ni2+ ions through coupling with the chelating moiety of nitrilotriaceticacid (NTA). As depicted in Figure 2, the strategyof our OGT assay involves five steps starting with attachment of the6 × His-tagged OGT substrate to the plate through Ni-NTA chelation,followed by the blocking of unoccupied nickel ion. Protein substratebound to the plate is then subjected to OGT enzymatic catalysis usingUDP-GlcNAz as the sugar donor. Next, a chemoselective reaction, suchas the copper-catalyzed “click” reaction, or Staudingerligation, is performed to label the azido-functionality. Finally,an immunoassay is used to detect the labeled proteins.

Bottom Line: The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes.The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors.The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Science Education-Chemistry Major, Daegu University , Gyeongbuk 712-714, South Korea.

ABSTRACT
The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.

Show MeSH