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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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Structuresof chemical compounds used in Ni-NTA Plate OGT Assays:UDP-GlcNAz (1), TAMRA-Alkyne (2), Biotin-Phosphine(3), and OGT inhibitors (4–6).
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fig1: Structuresof chemical compounds used in Ni-NTA Plate OGT Assays:UDP-GlcNAz (1), TAMRA-Alkyne (2), Biotin-Phosphine(3), and OGT inhibitors (4–6).

Mentions: While there are several specific OGA inhibitors,the complexity,high cost, and limited versatility of classical OGT assays, in additionto the enzyme’s instability during purification, have beenmajor obstacles in developing potent and selective OGT inhibitors.The conventional in vitro OGT activity assay26 utilizes a radiolabeled sugar substrate, suchas UDP-[14C] GlcNAc or UDP-[3H] GlcNAc. Thismethod measures OGT activity by quantifying radiolabeled GlcNAc incorporationinto a protein substrate, but is high in cost and produces radiochemicalwaste. As an alternative to using radiochemicals, the Walker groupintroduced a ligand displacement OGT assay27 and a protease-protection assay.28 Theformer method exploits fluorescence polarization using a fluorophore-containingUDP-GlcNAc analogue. The latter protease-protection assay exploitsthe fluorescence resonance energy transfer (FRET) phenomenon usingspecially designed, protease-specific peptide substrates. However,both assays indirectly measure the glycosyltransferase activity ofOGT, requiring a second OGT activity assay to validate positive hitcompounds. Finally, Leavy et al. introduced an azido-enzyme-linkedimmunosorbent assay (azido-ELISA) method using the unnatural sugardonor, UDP-N-azidoacetyl glucosamine (UDP-GlcNAz, 1, Figure 1) and biotinylated peptidesubstrates.29 In this method, a solution-phaseenzymatic reaction is transferred into a microplate for subsequentsolid-phase reactions, reducing the method’s efficiency.


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)

Structuresof chemical compounds used in Ni-NTA Plate OGT Assays:UDP-GlcNAz (1), TAMRA-Alkyne (2), Biotin-Phosphine(3), and OGT inhibitors (4–6).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Structuresof chemical compounds used in Ni-NTA Plate OGT Assays:UDP-GlcNAz (1), TAMRA-Alkyne (2), Biotin-Phosphine(3), and OGT inhibitors (4–6).
Mentions: While there are several specific OGA inhibitors,the complexity,high cost, and limited versatility of classical OGT assays, in additionto the enzyme’s instability during purification, have beenmajor obstacles in developing potent and selective OGT inhibitors.The conventional in vitro OGT activity assay26 utilizes a radiolabeled sugar substrate, suchas UDP-[14C] GlcNAc or UDP-[3H] GlcNAc. Thismethod measures OGT activity by quantifying radiolabeled GlcNAc incorporationinto a protein substrate, but is high in cost and produces radiochemicalwaste. As an alternative to using radiochemicals, the Walker groupintroduced a ligand displacement OGT assay27 and a protease-protection assay.28 Theformer method exploits fluorescence polarization using a fluorophore-containingUDP-GlcNAc analogue. The latter protease-protection assay exploitsthe fluorescence resonance energy transfer (FRET) phenomenon usingspecially designed, protease-specific peptide substrates. However,both assays indirectly measure the glycosyltransferase activity ofOGT, requiring a second OGT activity assay to validate positive hitcompounds. Finally, Leavy et al. introduced an azido-enzyme-linkedimmunosorbent assay (azido-ELISA) method using the unnatural sugardonor, UDP-N-azidoacetyl glucosamine (UDP-GlcNAz, 1, Figure 1) and biotinylated peptidesubstrates.29 In this method, a solution-phaseenzymatic reaction is transferred into a microplate for subsequentsolid-phase reactions, reducing the method’s efficiency.

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