Fig 1.
The schematic depiction of the peptide microarray process for discovering OGT substrates.
The microarray was blocked with BSA, and followed by the addition of OGT (s-OGT, m-OGT, and nc-OGT), UDP-GlcNAc, and a FITC conjugated antibody. The enzymatic reaction was run by pumping the mixture up and down through the porous Al2O3 chip material. Images of the fluorescent signals were generated every 10 minutes during 4 hours (kinetic readout). Images were quantified using Bionavigator 6 software.
Fig 2.
Identification of human OGT substrates from a kinase substrate and nuclear hormone receptor interaction peptide microarray.
The assay was performed using bacterial lysates containing s-OGT (7 μg/μL), m-OGT (7 μg/μL), or nc-OGT (7 μg/μL), respectively, and in all cases in the presence of UDP-GlcNAc (1 mM). A parallel reaction without UDP-GlcNAc was used as a negative control. Representative images from the kinase substrate peptide microarray (A) and nuclear receptor interaction peptide microarray (B) are shown. Reference spot is highlighted in gray and peptide O-GlcNAcylation by all three isoforms of OGT is highlighted in red. O-GlcNAcylation of each peptide by the three isoforms OGT was quantified and corrected for non-specific signal by subtracting the signal generated without UDP-GlcNAc (from signal with UDP-GlcNAc). Representative heat maps are shown for O-GlcNAcylation of kinase substrate peptides (C) and nuclear receptor interaction peptides (D).
Table 1.
A list of OGT substrates identified on kinase substrate peptide microarray.
Table 2.
A list of OGT substrates identified on nuclear hormone receptor interaction peptide microarray.
Fig 3.
Validation of RBL-2_410–422 O-GlcNAcylation.
A, O-GlcNAcylation of RBL-2_410–422 peptide by bacterial lysate containing m-OGT (7 μg/μL) with increasing concentration of UDP-GlcNAc (0–10 mM). B, O-GlcNAcylation of RBL-2_410–422 peptide dependency of increasing total protein concentrations of bacterial lysate containing m-OGT (0, 1.7, 3.5, 7 μg/μL) at 1 mM UDP-GlcNAc. C, O-GlcNAcylation of RBL-2_410–422 peptide by purified m-OGT (0.2 μg/μL) with 1 mM UDP-GlcNAc was inhibited by a known OGT inhibitor (ST045849, 0–200 μM). D, Km value for UDP-GlcNAc was determined with fixed saturating concentration of RBL-2_410–422 peptide, purified m-OGT (0.2 μg/μL) and varying concentration of UDP-GlcNAc (0–2 mM). The Km value derived from the fit to Michaelis-Menten model is 24 μM.
Fig 4.
S420A is a possible O-GlcNAc site in the RBL-2 protein.
A, peptide mutant used for an Ala scan. For immobilization purposes, peptides were prepared with an extra CG at the N-terminus and Cys 415 was replaced by Ala. B, OGT activity against RBL-2_410–422 peptide mutants was determined using peptide microarray analysis with 0.2 μg/μL purified m-OGT and 1 mM UDP-GlcNAc. C, UDP-Glo assay was used to measure O-GlcNAcylation of RBL-2_410–422 peptide mutants as well. D, kinetic signals from the same microarray experiment of panel B are shown.
Fig 5.
The RBL-2_S420A 410–422 peptide inhibited OGT activity.
The inhibitory effect of RBL-2_S420A 410–422 peptide on OGT activity was determined on the nuclear receptor interaction peptide microarray. The reaction was performed by incubating a mixture of purified m-OGT (0.2 μg/μL) and UDP-GlcNAc (0.5 mM) with or without the S420A RBL2 peptide (0.5 mM). The known OGT inhibitor (ST045849) and a no UDP-GlcNAc reaction were used for positive and negative control, respectively. O-GlcNAcylation of NCOA6_1479–1501 peptide (A) and WIPI_1313–318 peptide (B) are shown for the inhibitory effect of RBL-2_S420A 410–422 peptide on OGT activity.