Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray

Abstract

O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity.

Crosstalk between phosphorylation and O‐GlcNAcylation: friend or foe

“…To fast track the identification of OGT substrates, peptide microarrays have been developed, whereby thousands of proteins can be screened simultaneously for their ability to be OGT substrates [120,121]. To fast track the identification of OGT substrates, peptide microarrays have been developed, whereby thousands of proteins can be screened simultaneously for their ability to be OGT substrates [120,121].…”

In Vitro Biochemical Assays for O‐GlcNAc‐Processing Enzymes

“…3.2.4. Peptide microarray‐based OGT assay : Recently, a peptide microarray‐based OGT assay (Figure ) was derived from a protein microarray OGT assay approach, which was originally designed to identify protein substrates for OGT isoforms by using an array of commercially available proteins bound to a chip. In this assay, proteins or peptides are subjected to OGT‐catalyzed glycosyl transfer, followed by fluorescent antibody detection of O‐GlcNAcylation.…”

“…In this method, a highly purified form of OGT is not necessary, and the kinetic activity of the enzyme can be measured by scanning the signal at regular intervals. Utilizing this peptide microarray‐based assay, retinoblastoma‐like 2 protein (RBL‐2), which is a key regulator of entry into cell division and proposed to be a tumor suppressor, was identified as an OGT substrate, and its O‐GlcNAc modification site (Ser420) was recapitulated . However, the sensitivity of this method depends on the anti‐O‐GlcNAc antibody used in this assay, which has relatively low detection sensitivity.…”

“…The chemical energy of luciferin oxidation is converted to light through an electron transition, giving oxyluciferin (Figure D) . The intensity of this resulting light signal correlates to the amount of UDP released by the glycosyltransferase, which is indicative of glycosyltransferase activity, and this method has been applied to the study of OGT activity and kinetic parameters . Ideally, the UDP‐Glo method requires purification of the glycosyltransferase enzyme and might be subject to interference due to the coupled assay method in which UDP is converted to another product.…”

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

“…Peptide microarrays were later used to examine the substrate specificity of different OGT isoforms. Using RL2 (an anti‐ O ‐GlcNAc antibody) for O ‐GlcNAc detection, one study found that a number of peptides were glycosylated at different levels by each of the three OGT isoforms, thus indicating differential substrate specificities . Besides applications in studying OGT, a microarray with synthetic glycopeptides was also developed for OGA, which could be used in the future to investigate the substrate preferences of this hydrolase.…”