Global Identification of O-GlcNAc-Modified Proteins

Abstract

The O-linked N-acetylglucosamine (O-GlcNAc) modification of serine/threonine residues is an abundant posttranslational modification present in cytosolic and nuclear proteins. The functions and subproteome of O-GlcNAc modification remain largely undefined. Here we report the application of the tagging-via-substrate (TAS) approach for global identification of O-GlcNAc-modified proteins. The TAS method utilizes an O-GlcNAc azide analogue for metabolic labeling of O-GlcNAc-modified proteins, which can be chemoselectively conjugated for detection and enrichment of the proteins for proteomics studies. Our study led to the identification of 199 putative O-GlcNAcmodified proteins from HeLa cells, among which 23 were confirmed using reciprocal immunoprecipitation. Functional classification shows that proteins with diverse functions are modified by O-GlcNAc, implying that OGlcNAc might be involved in the regulation of multiple cellular pathways.The modification of nuclear and cytoplasmic proteins at serine and threonine residues with O-linked N-acetylglucosamine (OGlcNAc) was first described over two decades ago. 1 The modification was found in various classes of proteins including enzymes, transcription factors, cytoskeletal proteins, signaling proteins, receptors, nuclear pore complex proteins, and kinases. 2 Similar to phosphorylation, the O-GlcNAc modification is dynamic with a turnover rate faster than that of the proteins it modifies. 3 The O-GlcNAc modification has been shown to affect protein-protein interactions, protein-DNA interactions, protein stability and activity, and cell signaling cascades. 4 Disregulation of the OGlcNAc modification has been implicated in the development of disease states including diabetes, cancer, and Alzheimer's. 2,4 Given the potentially broad regulatory influence of the O-GlcNAc modification, a more comprehensive understanding of the targets of O-GlcNAc transferase is needed to elucidate its functional consequences.In this report, we describe the global detection and proteomic analysis of O-GlcNAc-modified proteins in HeLa cells. An affinitytagged version of the O-GlcNAc modification is metabolically incorporated onto proteins using an azide-tagged analogue of N-acetylglucosamine. The azido-GlcNAc-modified proteins thus contain an azide handle for chemoselective conjugation using a biotinylated phosphine reagent. The resulting conjugates were affinity-purified with streptavidin beads and subsequently digested with trypsin and analyzed by nano-HPLC-MS/MS. Using this strategy, we identified 199 azido-GlcNAc-modified proteins in HeLa cells. We subsequently validated the presence of this modification among 10 previously reported and 13 newly identified O-GlcNAcmodified proteins using specific antibodies. Our results reveal that proteins with a wide range of functions are modified by O-GlcNAc, implying its diverse cellular functions.

Chemical approaches to understanding O-GlcNAc glycosylation in the brain

“…Notably, many of the proteins identified participate in the regulation of gene expression (for example, CCR4-NOT, SOX2, SRC1, HCF and TLE4), neuronal signaling (WNK1, bassoon, PDZ-GEF) and synaptic plasticity (synGAP, synaptopodin), suggesting that O-GlcNAc may contribute to neuronal communication processes. In another approach, Nandi and co-workers metabolically labeled HeLa cells with N-(2-azido-acetyl) glucosamine 90 . Tryptic digestion of the captured proteins led to the identification of 199 putative O-GlcNAc-modified proteins.…”

Cancer Metabolism and Elevated O-GlcNAc in Oncogenic Signaling

“…Several studies now link hyper-O-GlcNAcylation to cancer-associated metabolic reprogramming. O-GlcNAc has been reported to modify a variety of glycolytic enzymes (33)(34)(35). In particular, a role for O-GlcNAc modification has been described at Ser-529 of PFK1 (phosphofructokinase 1), which catalyzes the rate-limiting step of glycolysis to generate fructose 1,6-bisphosphate from fructose 6-phosphate (36).…”

A high-throughput assay for O-GlcNAc transferase detects primary sequence preferences in peptide substrates

“…20 In cells, the azide-modified substrate appears to be transferred to the same spectrum of proteins as the natural sugar. 21,22 Here, we capitalize on this finding in the development of a high-throughput azido-ELISA for OGT and we employ the assay in a preliminary peptide substrate screen.A schematic of the azido-ELISA is shown in Figure 1. Biotinylated peptide substrates are captured onto NeutrAvidin-coated 96-well plates and are subsequently covalently tagged by treatment with phosphine-FLAG.…”

“…20 In cells, the azide-modified substrate appears to be transferred to the same spectrum of proteins as the natural sugar. 21,22 Here, we capitalize on this finding in the development of a high-throughput azido-ELISA for OGT and we employ the assay in a preliminary peptide substrate screen.…”