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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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Determination of ncOGTactivity. Four concentrations of the 6 ×His-tagged Nup62 substrate (as indicated) were used to detect activityof either unpurified or partially purified (p-ncOGT) enzyme as indicated.Glycosyltransferase activity was detected using copper-catalyzed “click”reaction with TAMRA-Alkyne.
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fig3: Determination of ncOGTactivity. Four concentrations of the 6 ×His-tagged Nup62 substrate (as indicated) were used to detect activityof either unpurified or partially purified (p-ncOGT) enzyme as indicated.Glycosyltransferase activity was detected using copper-catalyzed “click”reaction with TAMRA-Alkyne.

Mentions: As shownin Figure 3, OGT enzymatic reactionswere efficiently detected using an anti-TAMRA antibody, indicatingnickel ion precoated on the strip does not interfere with the copper-catalyzed“click” reaction. In addition, the RL2 antibody, whichrecognizes a number of O-GlcNAc-modified nuclearpore complex proteins, was successfully used to determine OGT activity(see SI Figure S2). These data indicatethat, while convenient, a chemoselective reaction to detect O-GlcNAzylated Nup62 is not required when Nup62 is usedas the OGT substrate. Importantly, results of OGT assays using partiallypurified ncOGT (lower panel in Figure 3) weresimilar to those obtained with bacterial lysate containing ncOGT (upperpanel in Figure 3), indicating that OGT purificationis not necessary in the Ni-NTA Plate OGT Assay.


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)

Determination of ncOGTactivity. Four concentrations of the 6 ×His-tagged Nup62 substrate (as indicated) were used to detect activityof either unpurified or partially purified (p-ncOGT) enzyme as indicated.Glycosyltransferase activity was detected using copper-catalyzed “click”reaction with TAMRA-Alkyne.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Determination of ncOGTactivity. Four concentrations of the 6 ×His-tagged Nup62 substrate (as indicated) were used to detect activityof either unpurified or partially purified (p-ncOGT) enzyme as indicated.Glycosyltransferase activity was detected using copper-catalyzed “click”reaction with TAMRA-Alkyne.
Mentions: As shownin Figure 3, OGT enzymatic reactionswere efficiently detected using an anti-TAMRA antibody, indicatingnickel ion precoated on the strip does not interfere with the copper-catalyzed“click” reaction. In addition, the RL2 antibody, whichrecognizes a number of O-GlcNAc-modified nuclearpore complex proteins, was successfully used to determine OGT activity(see SI Figure S2). These data indicatethat, while convenient, a chemoselective reaction to detect O-GlcNAzylated Nup62 is not required when Nup62 is usedas the OGT substrate. Importantly, results of OGT assays using partiallypurified ncOGT (lower panel in Figure 3) weresimilar to those obtained with bacterial lysate containing ncOGT (upperpanel in Figure 3), indicating that OGT purificationis not necessary in the Ni-NTA Plate OGT Assay.

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