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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
ncOGT activity is linearly dependent on theconcentration of Nup62.Increasing amounts of the 6 × His-tagged Nup62 substrate wereused as indicated. ncOGT containing lysates were used for these experiments.Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in duplicateand quantified using the Odyssey Infrared Imaging System at 800 nm.Values were normalized such that no substrate was set to 0. Errorbars represent range.
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fig4: ncOGT activity is linearly dependent on theconcentration of Nup62.Increasing amounts of the 6 × His-tagged Nup62 substrate wereused as indicated. ncOGT containing lysates were used for these experiments.Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in duplicateand quantified using the Odyssey Infrared Imaging System at 800 nm.Values were normalized such that no substrate was set to 0. Errorbars represent range.

Mentions: Next, we evaluated the minimal amount of Nup62 necessaryin orderto obtain the maximal IR-signal intensity for this assay. Here, Biotin-Phosphine(3, Figure 1) was chemoselectivelyreacted with the incorporated azido-moiety and relative IR-signalintensity was determined, which reflects the enzymatic activity ofthe ncOGT. As shown in Figure 4, IR-signalintensity reached the maximum (wells 4 and 5) when the amount of Nup62was over 0.14 μg.


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)

ncOGT activity is linearly dependent on theconcentration of Nup62.Increasing amounts of the 6 × His-tagged Nup62 substrate wereused as indicated. ncOGT containing lysates were used for these experiments.Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in duplicateand quantified using the Odyssey Infrared Imaging System at 800 nm.Values were normalized such that no substrate was set to 0. Errorbars represent range.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: ncOGT activity is linearly dependent on theconcentration of Nup62.Increasing amounts of the 6 × His-tagged Nup62 substrate wereused as indicated. ncOGT containing lysates were used for these experiments.Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in duplicateand quantified using the Odyssey Infrared Imaging System at 800 nm.Values were normalized such that no substrate was set to 0. Errorbars represent range.
Mentions: Next, we evaluated the minimal amount of Nup62 necessaryin orderto obtain the maximal IR-signal intensity for this assay. Here, Biotin-Phosphine(3, Figure 1) was chemoselectivelyreacted with the incorporated azido-moiety and relative IR-signalintensity was determined, which reflects the enzymatic activity ofthe ncOGT. As shown in Figure 4, IR-signalintensity reached the maximum (wells 4 and 5) when the amount of Nup62was over 0.14 μg.

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