Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway

Abstract

Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

Fig. 1.

(A) The GlcNAc and GalNAc salvage and O-GlcNAc signaling pathways. Enzyme names shown in red. (B) GalNAz and GlcNAz.

Fig. 2.

Metabolic labeling by GalNAz, but not GlcNAz, robustly mimics natural O-GlcNAc. 293T cells were mock-transfected or transfected with a construct expressing (A) OGT or (B) OGA and treated with vehicle or azidosugar for 24 h. Nuclear and cytoplasmic extracts were prepared, reacted with phosphine-Flag, and analyzed by immunoblot.

Fig. 3.

GlcNAz does not transit the UDP-GlcNAc pyrophosphorylase step in the GlcNAc salvage pathway. (A) Jurkat or 293T cells were treated with vehicle or 100 μM azidosugar for 24 h. Ethanol extracts were made and analyzed by HPAEC. Synthetic UDP-GlcNAz and UDP-GalNAz standards were included. Asterisk: an unknown species present in all cell-derived samples. (B) 293T cells were mock-transfected or transfected with the expression construct indicated and treated with vehicle or Ac₄GlcNAz for 24 h. Cell lysates were reacted with phosphine-Flag and analyzed by immunoblot. (C) 293T cells were mock-transfected or transfected with an AGX2-myc expression construct. After 21 h, all cells were treated with 100 μM Ac₄GlcNAz, samples were harvested by ethanol extraction after 3, 6, or 9 additional hours and analyzed by HPAEC. Asterisk: an unknown species present in all cell-derived samples. (D) Ethanol-insoluble protein fractions from the samples in C were resuspended in 8 M urea, reacted with phosphine-Flag, and analyzed by immunoblot.

Fig. 4.

Ac₄GalNAz treatment results in GALE-dependent UDP-GlcNAz biosynthesis and labeling of O-GlcNAcylated proteins. (A) CHO or ldlD CHO cells were treated with vehicle or Ac₄GalNAz for 24 h. Nuclear and cytoplasmic extracts were prepared, reacted with phosphine-Flag, and analyzed by immunoblot. Total protein was visualized by India ink staining. (B) CHO or ldlD mutant CHO cells were treated with vehicle or Ac₄GalNAz for 72 h. Cells were fixed and azidoglycans detected via reaction with phosphine-Flag and immunofluorescence microscopy using an anti-Flag-FITC antibody conjugate. A Hoechst 33258 stain was included to visualize nuclei. (Top) FITC only. (Bottom): FITC/Hoechst merge.