Probing mucin-type O-linked glycosylation in living animals

Abstract

Changes in O-linked protein glycosylation are known to correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we report a technique for rapid profiling of O-linked glycoproteins in living animals by metabolic labeling with N-azidoacetylgalactosamine (GalNAz) followed by Staudinger ligation with phosphine probes. After injection of mice with a peracetylated form of GalNAz, azide-labeled glycoproteins were observed in a variety of tissues, including liver, kidney, and heart, in serum, and on isolated splenocytes. B cell glycoproteins were robustly labeled with GalNAz but T cell glycoproteins were not, suggesting fundamental differences in glycosylation machinery or metabolism. Furthermore, GalNAz-labeled B cells could be selectively targeted with a phosphine probe by Staudinger ligation within the living animal. Metabolic labeling with GalNAz followed by Staudinger ligation provides a means for proteomic analysis of this posttranslational modification and for identifying O-linked glycoprotein fingerprints associated with disease.azide ͉ Staudinger ligation ͉ glycan ͉ N-azidoacetylgalactosamine ͉ metabolic labeling

Imaging the glycome

“…This unnatural sugar is processed by the GalNAc salvage pathway to form the intermediate uridine diphospho (UDP)-GalNAz, which is recognized by a family of polypeptide GalNAc transferases in the Golgi compartment. Ac 4 GalNAz has been used to label mucin-type O-linked glycans in numerous cell lines, live mice (76), and zebrafish (73).…”

Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

“…These methods include different bioorthogonal reactions such as ketoneaminooxy/hydrazide ligation (12,13), Staudinger ligation (14), Michael addition (15), and the strain-promoted, and Cu(I)-catalyzed [3 ϩ 2] azide-alkyne cycloaddition (16)(17)(18)(19). Many sugar analogs with small functional handles, including ketones and azides, are tolerated and have been incorporated into glycoconjugates (14,20,21). These reporting sugars have been coupled with various tags such as FLAG peptides, biotin, and fluorescent or fluorogenic molecules.…”

Dual-sugar imaging using isonitrile and azido-based click chemistries

“…The azide group has been the most widely used reporter group due to its chemical stability, small size and bioorthogonal reactivity with alkynes and Staudinger phosphines. [12][13][14] Other chemical reporters used for metabolic labelling include: alkynes, 15,16 ketones, 17 thiols, 18 cyclopropenes 19,20 and alkenes. 21 We have recently shown that the isonitrile group can also be used as a novel chemical reporter group for glycan metabolic labelling.…”

“…19,21 In these two accounts of dual sugar labelling N-acetyl mannosamine was the monosaccharide bearing the cyclopropene 19 or alkene 21 tag. As with per-acetylated N-azidoacetyl mannosamine (Ac 4 ManNAz), 13 these non-natural mannosamine derivatives are incorporated into cell surface glycoproteins as modified terminal sialic acids. The modified sialic acids were then detected using a fluorescent tetrazine.…”

“…Dual sugar metabolic labelling was achieved by using these mannosamine derivatives in conjunction with per-acetylated Nazidoacetyl galactosamine (Ac 4 GalNAz). 13,19,21 We wanted to explore whether we could use our newly developed isonitrile-tetrazine metabolic labelling chemistry to perform dual-glycan labelling on cancer cells. We have demonstrated that primary isonitrile derivatives of both mannosamine (Ac 4 ManN-n-Iso) and glucosamine (Ac 4 GlcN-n-Iso) are incorporated into cell surface glycoproteins, making them valuable tools for dual metabolic labelling studies.…”