Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1

Abstract

The dynamic modification of nuclear and cytoplasmic proteins by the monosaccharide N-acetyl-glucosamine (GlcNAc) continues to emerge as an important regulator of many biological processes. Herein we describe the development of an alkynyl-modified GlcNAc analog (GlcNAlk) as a new chemical reporter of O-GlcNAc modification in living cells. This strategy is based on metabolic incorporation of reactive functionality into the GlcNAc biosynthetic pathway. When combined with the Cu(I)-catalyzed [3 þ 2] azidealkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent visualization of O-GlcNAc and affinity enrichment and identification of O-GlcNAc-modified proteins. Using ingel fluorescence detection, we characterized the metabolic fates of GlcNAlk and the previously reported azido analog, GlcNAz. We confirmed previous results that GlcNAz can be metabolically interconverted to GalNAz, whereas GlcNAlk does not, thereby yielding a more specific metabolic reporter of O-GlcNAc modification. We also used GlcNAlk, in combination with a biotin affinity tag, to identify 374 proteins, 279 of which were not previously reported, and we subsequently confirmed the enrichment of three previously uncharacterized proteins. Finally we confirmed the O-GlcNAc modification of the ubiquitin ligase NEDD4-1, the first reported glycosylation of this protein.click chemistry | bioorthogonal labeling | proteomics

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

“…These O-GlcNAc sites (S539 and T577) with proximal known phosphorylation sites (S541, S545, and T552) (33) on TNIK are all located within its predicted (34) binding region to NEDD4-1, an E3 ubiquitin ligase. O-GlcNAcylation of human NEDD4-1 was recently reported (17), but with no site localization.…”

“…A recent metabolic labeling study that used alkyne-modified GlcNAc incorporated into OGT substrates and Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) to a chemically cleavable biotin-azide probe, enabled identification of 374 putative O-GlcNAc modified proteins, but yielded no information on sites of O-GlcNAc modification (17). In addition to limited coverage of O-GlcNAcylation sites, a drawback to using these approaches is that they typically require milligram quantities of protein (4,10,13,14,16) or are limited to cultured cells (17), which makes them generally ill-suited for clinically derived tissue samples often available in small amounts.…”

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

“…Metabolic sugar engineering, which inserts 'tags' into cellular glycoconjugates, has proven to be a powerful tool for the glycoproteomic analysis of a specific sugar modification [1][2][3] . This technique involves the incorporation of chemically modified sugars, such as azide-or alkyne-modified sugars, into cellular glycans via a normal biosynthetic pathway, and its subsequent detection with a probe, such as a FLAG-tag, biotin or fluorescent molecules [4][5][6] .…”

Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates

“…Ac 4 GlcNAz enters the cell metabolism after cleavage of the acetyl groups by esterases and is metabolized in various pathways. On the one hand, it can be incorporated into cell-surface N-and O-linked glycans as Neu5Az or directly as GlcNAz [4,29,31,33]. On the other hand, many cyctosolic O-linked glycans can incorporate GlcNAz residues [34,35].…”

“…The tagged carbohydrates are partly incorporated into cell-surface glycan structures and present the bioorthogonal side chain that can be reacted with the appropriate counterpart (alkyne) in a [3 + 2]-cycloaddition by copper catalyzed azide-alkyne cycloaddition (CuAAc) [4,31,33,[36][37][38].…”