The staggering complexity of glycans renders their analysis extraordinarily difficult, particularly in living systems. A recently developed technology, termed metabolic oligosaccharide engineering, enables glycan labeling with probes for visualization in cells and living animals, and enrichment of specific glycoconjugate types for proteomic analysis. This technology involves metabolic labeling of glycans with a specifically reactive, abiotic functional group, the azide. Azido sugars are fed to cells and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged, either ex vivo or in vivo, using one of two azide-specific chemistries: the Staudinger ligation, or the strain-promoted [3+2] cycloaddition. These reactions can be used to tag glycans with imaging probes or epitope tags, thus enabling the visualization or enrichment of glycoconjugates. Applications to noninvasive imaging and glycoproteomic analyses are discussed.
"In a separate experiment, we demonstrated that the tagged Neu1 is enzymatically active and associates with CathA (Supplementary Fig. 3A). The biological activity of hRluc-tagged IRK was demonstrated previously (30). Confocal immunomicroscopy showed that tagged IRK and Neu1 colocalized at the cell surface (Fig. 4A), indicating that the tag did not interfere with the plasma membrane targeting of both proteins. "
[Show abstract][Hide abstract] ABSTRACT: Neuraminidases (sialidases) catalyze the removal of sialic acid residues from sialylated glycoconjugates. We now report that mammalian neuraminidase 1 (Neu1), in addition to its catabolic function in lysosomes, is transported to the cell surface where it is involved in regulation of insulin signaling. Insulin binding to its receptor rapidly induces interaction of the receptor with Neu1, which hydrolyzes sialic acid residues in the glycan chains of the receptor and, consequently, induces its activation. Cells from sialidosis patients with a genetic deficiency of Neu1 show impairment of insulin-induced phosphorylation of downstream protein kinase AKT, while treatment of these cells with purified Neu1 restores signaling. Genetically-modified mice with ∼10% of the normal Neu1 activity exposed to a high-fat diet develop hyperglycemia and insulin resistance twice as fast as their wild type counterparts. Together, these studies identify Neu1 as a novel component of the signaling pathways of energy metabolism and glucose uptake.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: An important frontier in glycoproteomics is the discovery of proteins with post-translational glycan modifications. The first step in glycoprotein identification is the isolation of glycosylated proteins from the remainder of the proteome. New enzymatic and metabolic methods are being used to chemically tag proteins to enable their isolation. Once isolated, glycoproteins can be identified by mass spectrometry. Additional information can be obtained by using either enzymatic or chemoselective reactions to incorporate isotope labels at specific sites of glycosylation. Isotopic labeling facilitates mass spectrometry-based confirmation of glycoprotein identity, identification of glycosylation sites, and quantification of the extent of modification. By combining chemical tagging for isolation and isotope labeling for mass spectrometry analysis, researchers are developing highly effective strategies for glycoproteomics. These techniques are enabling cancer biologists to identify biomarkers whose glycosylation state correlates with disease states, and developmental biologists to characterize stage-specific changes in glycoprotein expression. Next-generation methods will make functional analyses of the glycoproteome possible, including the discovery of glycoprotein interaction partners and the identification of enzymes responsible for synthesis of particular glycan structures.
Current Opinion in Chemical Biology 03/2007; 11(1):52-8. DOI:10.1016/j.cbpa.2006.11.032 · 6.81 Impact Factor
Note: This list is based on the publications in our database and might not be exhaustive.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.