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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.

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Inhibition of ncOGT activity by compounds 4 and 5. Increasing amounts of inhibitors 4 (A) and 5 (B) were added to the Ni-NTA PlateOGT Assay (as indicated).Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in triplicate(n = 3) and quantified using the Odyssey InfraredImagining System at 800 nm. IC50 values were calculatedusing SigmaPlot 12.5 software employing “Four parameter logisticequation in the standard curve analysis”. Error bars representstandard deviations.
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fig5: Inhibition of ncOGT activity by compounds 4 and 5. Increasing amounts of inhibitors 4 (A) and 5 (B) were added to the Ni-NTA PlateOGT Assay (as indicated).Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in triplicate(n = 3) and quantified using the Odyssey InfraredImagining System at 800 nm. IC50 values were calculatedusing SigmaPlot 12.5 software employing “Four parameter logisticequation in the standard curve analysis”. Error bars representstandard deviations.

Mentions: To validate the assay’s ability to identify known inhibitorsof OGT, we tested OGT inhibitors (4) and (5), which were reported to have IC50 values of 53 μM(±7 μM) and 10 μM (±1 μM),27 respectively. Prior to the inhibition assays,we examined the amount of OGT-containing lysate and reaction timerequired for linear enzyme activity [1.4 μL (equivalent to 56μg of a total protein) of ncOGT-containing lysate and 25 minof reaction time; see SI Figure S5] when40 μM of UDP-GlcNAz and 0.15 μg of Nup62 were used. 20mM OGT inhibitors 4 and 5 were preparedin DMSO and serially diluted, and equal volumes were added to eachreaction well containing bound Nup62 and UDP-GlcNAz. Enzymatic reactionswere initiated by adding 1.4 μL of ncOGT-containing lysate andincubated at 37 °C with gentle shaking for 25 min, followed byquenching by removal of reaction mixtures and PBST washes. Enzymeactivity was normalized to a well containing only DMSO but no inhibitor.As shown in Figure 5, compounds 4 and 5 inhibited ncOGT glycosyltransferase activitywith IC50 values for 4 and 5 tobe 19.7 (±1.4) μM and 6.0 (±0.8) μM, respectively(Figure 5B). Although our results differ fromthose previously reported,27 these valuesare well within the expected range. Variations may be caused by differencesin the relative sensitivity of methods used for quantification (radiometricassay vs Ni-NTA OGT Assay) and in the choice of OGT substrates (UDP-GlcNAcvs UDP-GlcNAz and peptide vs Nup62).


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)

Inhibition of ncOGT activity by compounds 4 and 5. Increasing amounts of inhibitors 4 (A) and 5 (B) were added to the Ni-NTA PlateOGT Assay (as indicated).Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in triplicate(n = 3) and quantified using the Odyssey InfraredImagining System at 800 nm. IC50 values were calculatedusing SigmaPlot 12.5 software employing “Four parameter logisticequation in the standard curve analysis”. Error bars representstandard deviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215860&req=5

fig5: Inhibition of ncOGT activity by compounds 4 and 5. Increasing amounts of inhibitors 4 (A) and 5 (B) were added to the Ni-NTA PlateOGT Assay (as indicated).Glycosyltransferase activity was detected using Staudinger ligationwith Biotin-Phosphine reagent. Experiments were performed in triplicate(n = 3) and quantified using the Odyssey InfraredImagining System at 800 nm. IC50 values were calculatedusing SigmaPlot 12.5 software employing “Four parameter logisticequation in the standard curve analysis”. Error bars representstandard deviations.
Mentions: To validate the assay’s ability to identify known inhibitorsof OGT, we tested OGT inhibitors (4) and (5), which were reported to have IC50 values of 53 μM(±7 μM) and 10 μM (±1 μM),27 respectively. Prior to the inhibition assays,we examined the amount of OGT-containing lysate and reaction timerequired for linear enzyme activity [1.4 μL (equivalent to 56μg of a total protein) of ncOGT-containing lysate and 25 minof reaction time; see SI Figure S5] when40 μM of UDP-GlcNAz and 0.15 μg of Nup62 were used. 20mM OGT inhibitors 4 and 5 were preparedin DMSO and serially diluted, and equal volumes were added to eachreaction well containing bound Nup62 and UDP-GlcNAz. Enzymatic reactionswere initiated by adding 1.4 μL of ncOGT-containing lysate andincubated at 37 °C with gentle shaking for 25 min, followed byquenching by removal of reaction mixtures and PBST washes. Enzymeactivity was normalized to a well containing only DMSO but no inhibitor.As shown in Figure 5, compounds 4 and 5 inhibited ncOGT glycosyltransferase activitywith IC50 values for 4 and 5 tobe 19.7 (±1.4) μM and 6.0 (±0.8) μM, respectively(Figure 5B). Although our results differ fromthose previously reported,27 these valuesare well within the expected range. Variations may be caused by differencesin the relative sensitivity of methods used for quantification (radiometricassay vs Ni-NTA OGT Assay) and in the choice of OGT substrates (UDP-GlcNAcvs UDP-GlcNAz and peptide vs Nup62).

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