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

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

Balyn W Zaro et al. Proc Natl Acad Sci U S A. 2011.

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 + 2] azide-alkyne 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 in-gel 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The HBP and chemical reporters of O-GlcNAc modification. (A) Glucose, glutamine, and acetyl-CoA are transformed by the metabolic enzymes of the HBP to UDP-GlcNAc, which can be used by OGT to modify protein substrates and reversed by OGA. Per-_O_-acetylated analogues (Ac4GlcNAz and Ac4GlcNAlk) are deacetylated by intracellular esterases, and then enter the GlcNAc salvage pathway. (B) GlcNAz and GlcNAlk bearing proteins can be covalently modified with detection tags using CuAAC.

Fig. 2.

Fig. 2.

Fluorescent detection of O-GlcNAc-modified proteins. (A) Fluorescent-tags Alk-Rho and Az-Rho for CuAAC-dependent detection of GlcNAz and GlcNAlk (B) HEK293 cells were treated with the indicated concentrations of Ac4GlcNAz or Ac4GlcNAlk for 16 h and analyzed by in-gel fluorescence scanning. Coomassie blue staining demonstrated equal protein loading. (C) Higher contrast of lanes corresponding to 0 μM in B to allow for comparison of background levels.

Fig. 3.

Fig. 3.

Characterizing GlcNAz and GlcNAlk protein labeling. (A) Monosaccharide chemical reports have several possible metabolic fates. GlcNAz and GlcNAlk can enter the glucosamine salvage pathway (blue pathway) and potentially label O-GlcNAc-modified proteins and N-linked glycans. They could also be reversibly converted to the corresponding GalNAc analogs, GalNAz and GalNAlk (red pathway), resulting in the potential labeling of mucin-type O-linked glycosylation. (B and C) COS-7 cells were transfected with a plasmid encoding GlyCAM-IgG, treated with the indicated chemical reporter, and analyzed by in-gel fluorescence scanning. PNGase-F treatment prior to CuAAC was performed to selectively remove N-linked glycans. (D and E) COS-7 cells were transfected with a plasmid encoding FoxO1A, treated with the indicated chemical reporter, and analyzed by in-gel fluorescence. For comparison experiments B and C, as well as experiments D and E, fluorescent levels were measured and normalized simultaneously.

Fig. 4.

Fig. 4.

Identification of O-GlcNAlk-modified proteins. (A) O-GlcNAlk-modified proteins were enriched from NIH3T3 cells treated with Ac4GlcNAlk (200 μM) using azido-azo-biotin and analyzed by Western blotting. (B) Known O-GlcNAcylated proteins p62 and α-B crystallin were immunoprecipitated from cells treated with Ac4GlcNAlk (200 μM) and analyzed by in-gel fluorescence. (C) NEDD4-1 was selectively enriched from cells treated with Ac4GlcNAlk (200 μM) and analyzed by in-gel fluorescence. (D) NEDD4-1 was immunoprecipitated from cells and analyzed by Western blotting.

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