Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation

Abstract

Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic analysis are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approximately 1 week. A cell metabolic labeling experiment can be completed in approximately 4 d.

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

“…Chemical reporter strategies have been applied to visualize glycans in cells and living organisms with success (18)(19)(20)(21)(22)(23)(24)(25), however, insights into the functional relevance of glycans in normal and disease biology have yet to emerge from these applications. Our findings pave the way for the use of chemical reporter strategies to study the impact of altered glycosylation on the pathophysiology of human diseases.…”

“…The azide is the most versatile chemical reporter because of its small size, bio-orthogonality, and diverse mode of reactivity. It can be tagged by Staudinger ligation using modified phosphines, by a copper (I)-catalyzed cycloaddition with terminal alkynes (CuAAC), or by a strain-promoted alkyne-azide cycloaddition (SPAAC) using cyclooctynes (20,21). Such chemical reporter strategies can be used to monitor the trafficking of glycans within cells and to localize the expression of specific classes of glycans within living organisms, such as developing zebrafish embryos or Caenorhabditis elegans (21)(22)(23)(24)(25).…”

O-GlcNAcylation regulates EZH2 protein stability and function

“…Metabolic labeling was performed as previously described (42). Briefly, MCF7 cells were treated with 40 μM O-GlcNAz overnight at 37°C with 5% (vol/vol) CO 2 .…”

A Putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren Syndrome Chromosome Region 17 (WBSCR17) Regulates Lamellipodium Formation and Macropinocytosis

“…Metabolic Labeling of Glycoproteins with N-Azidoacetylgalactosamine (GalNAz)-GalNAz (Invitrogen) labeling of glycoproteins was performed as reported previously (21). Briefly, 10 l of 100 mM GalNAz (Invitrogen) in ethanol was added to a 10-cm culture dish, and the ethanol was evaporated at room temperature.…”