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Detecting mucins in vivo A method for labeling O-linked glycoproteins in cultured cells is adapted for use in living animals.
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with a fluorescent dye conjugated to an analysis of metabolic labeling would proells often change their sugary coats anti-FLAG antibody and detected by vide a new understanding of how differwhen they become malignant, so flow cytometry. ent tissues and cells differently metabodetecting such changes might help cliniTo adapt this assay for use in vivo, lize precursor sugars and differently react cians diagnose cancer at an early stage. the researchers injected mice every day with the Staudinger reagent.” But sugary proteins called O-linked glyfor a week with peracetylated GalNAz Finally, Bertozzi’s group showed that coproteins have been difficult to assay in the Staudinger ligation also vivo with sufficient speed OR’ can be performed in vivo. for clinical work. Now, HO O HO OR’ O FLAG O After the 7-day Ac4GalNAz Carolyn Bertozzi and colRO H CO 3 AcO OAc RO HN O HN O DYKDDDDK Ph2P leagues at the University regimen, they gave mice a O Cell O AcO OAc O Phos-FLAG NH N O of California, Berkeley, and bolus of phos-FLAG, harvestH O= Staudinger FLAG PPh2 Metabolic N3 the Lawrence Berkeley Naed splenocytes 90 min later, N3 O ligation labeling tional Laboratory have deand detected FLAG by fluoGalNAz-modified Labeled Ac4 GalNAz protein glycoprotein vised a high-throughput rescent labeling and flow method for detecting Ocytometry. Cells from mice linked glycoproteins in liv- O-linked glycoproteins are detected in living animals via Ac4GalNAz treated with both Ac4GalNAz ing animals (Proc. Natl. and phos-FLAG were more labeling. The Staudinger ligation is then used to probe the label with Acad. Sci. U.S.A. 2006, intensely fluorescent than cells phos-FLAG. R and R´ are oligosaccharide chains or branches attached 103, 4819– 4824). The ap- to a GalNAc residue. (Adapted with permission. Copyright 2006 National from mice treated with either proach “is based on metamolecule alone. B cells were Academy of Sciences, U.S.A.) bolic flux and, therefore, strongly labeled by phos(Ac4GalNAz), which has better pharma- FLAG, whereas T cells were not, sugmight provide biomarkers that would be very difficult to discern using convengesting that the in vivo Staudinger ligacokinetic properties than GalNAz. On tional proteomics,” Bertozzi says. day 8, they harvested splenocytes, which tion mirrors Ac4GalNAz labeling. “Since The most common O-linked glycocontain mucin glycoproteins and are we can chemically label these cells in vivo protein, the mucin type, has N-acetyleasily isolated in large quantities from with the phosphine probe, there is pogalactosamine (GalNAc) at the base of mice. The researchers then treated the tentially the opportunity for noninvasive each oligosaccharide branch. In 2003, splenocytes with phos-FLAG and a fluo- imaging of changes in O-linked glycosylBertozzi and colleagues developed an rescent label and analyzed the cells with ation,” Bertozzi says. assay for these O-linked glycoproteins flow cytometry. Compared with cells More immediately, however, Bertozzi in cultured cells (Proc. Natl. Acad. Sci. from control mice, the cells from the wants to adapt the assay for screening U.S.A. 2003, 100, 14,846 –14,851). mice treated with Ac4GalNAz exhibited serum. She points out that the assay First, they demonstrated that an azide might be useful in early cancer detection more fluorescence, and the fluorescence analog of GalNAc, N-azidoacetylgalacbecause it might detect O-glycoproteins was dose-dependent and specific for tosamine (GalNAz), is incorporated into O-linked glycoproteins. that are rapidly turned over rather than O-linked glycoproteins as if it were Galthose that are more abundant at steady The researchers studied the different NAc. Second, they showed that molestate. “This method allows one to focus types of cells that constitute splenocytes cules that incorporate GalNAz can be on a small subset of information-rich and were surprised to find that Ac4Galdetected or purified by probing them glycoproteins that are undergoing NAz strongly labels B cells and macrowith reagents that have phosphine changes in tumors, without the abunphages in vivo but only weakly labels groups. Bertozzi’s laboratory developed dant yet irrelevant proteins obscuring T cells, which also have O-linked glycoa specific process, called the Staudinger the analysis,” she says. Yingming Zhao proteins on their surfaces. Although ligation, to react a GalNAz-labeled glyof the University of Texas Southwestern Bertozzi and colleagues can’t explain coprotein with a phosphine attached to this result yet, they suspect that O-linked Medical Center says, “The method a FLAG (phos-FLAG) peptide. Because glycoproteins might turn over faster in B should be able to be combined with exthe phosphines react only with azides, isting proteomics technologies for dycells than in T cells and that Ac4GalNAz phos-FLAG peptides bound only to namic analysis of the glycosylation under labels targets that are undergoing de those glycoproteins containing GalNAz. novo synthesis. Minoru Fukuda at the diverse physiological conditions.” a The phos-FLAG probe was then labeled Burnham Institute says, “An in-depth —Linda Sage 4246
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