Research Highlights

Yeast N-glycans: Tagged for follow-up

An exogenous salvage pathway (blue arrows) allows extracellular GlcNAc or analogs to be internalized by the transporter Ngt1 from C. albicans. The intracellular GlcNAc (or analog) is phosphorylated at the 6 position via the activity of the human GlcNAc kinase, and de novo UDP-GlcNAc biosynthesis (black arrows) is bypassed. For full figure and legend see Breidenbach, M. A. et al. PNAS 107, 3988–3993 (2010) doi:10.1073/pnas.0911247107

Synthetic sugars can be incorporated into glycans as they are produced, without disrupting normal biology. The unnatural analogs are useful because they contain chemical groups not found in native molecules; the nonnative functionality enables imaging, isolation and analysis of the glycans that contain them. However, several factors hinder reliable metabolic labeling of glycoconjugates, including competition with endogenous sugars and the inherent microheterogeneity of glycan structures.

Now, Carolyn Bertozzi and colleagues demonstrate a method for the production of metabolically-labeled glycoproteins in yeast. They report in Proceedings of the National Academy of Science of the USA that by targeting the core N-glycan structure and genetically engineering the yeast to reduce competition from natural saccharides, glycoproteins can be efficiently functionalized for follow up studies.

All N-glycan cores contain two N-acetylglucosamine (GlcNAc) residues. In the yeast Saccharomyces cerevisiae, unlike in higher eukaryotes, these core residues are the only location of GlcNAc in mature glycans (except for chitin, a minor cell wall component important for cell division), and are not subject to epimerase activity. To replace them with synthetic analogs, it was first necessary to deplete the endogenous supply that would otherwise compete for incorporation.

Disrupting the biosynthesis of an essential substance is lethal, unless the organism can scavenge replacement molecules from its environment, and S. cerevisiae lacks a salvage pathway for GlcNAc. The donor molecule for glycan synthesis is UDP-GlcNAc, which is generated from the phosphorylated sugar GlcNAc-6-P. The authors deleted GNA1, the gene responsible for production of GlcNAc-6-P, thus cutting off the endogenous supply. To enable replacement with exogenous, unnatural sugars, the genes for two enzymes were expressed in the yeast cells: a GlcNAc transporter from Candida albicans, and human GlcNAc kinase. Both genes were necessary for survival of the GNA1-deleted cells, but the transporter was toxic unless expressed at low levels.

Azide- and Alkyne-bearing analogs, GlcNAz and GlcNAl respectively, were used to show that the new salvage pathway could incorporate unnatural sugars. The GNA1-deleted cells survived when supplied with GlcNAz or GlcNAl alone, but required at least some GlcNAc to proliferate. Therefore, they were transferred to medium containing GlcNAz alone after growth in the presence of GlcNAc. Azide-modified sugars can undergo a reaction called the Staudinger ligation, wherein phosphine, with any conjugated substance, is joined to the sugar. Using phosphine-FLAG and an anti-FLAG antibody, GlcNAz was detected on secreted glycoproteins. Specific enzymatic digestions showed that the modification was present on the first core GlcNAc residue of the glycans.

By copper-catalyzed cycloaddition, alkynes can be added to azides. The authors tagged the azido and alkynyl sugars for visualization by fluorescence microscopy, using fluorescent labels bearing complementary chemical functionality. Cells supplemented with GlcNAz and GlcNAl had strong fluorescence on their surfaces, as well as morphological abnormalities that the authors attribute to disrupted synthesis of chitin.

Finally, an isotope-labeled GlcNAc was used to supplement the culture medium, and its incorporation measured in a highly glycosylated secretory protein. The data indicated that global replacement of GlcNAc can be achieved in GNA1-deleted yeast.

This study describes a novel strategy to exploit the convenience of unnatural sugars for reliable labeling and analysis of glycoproteins. The future applications could be wide-ranging, and will contribute to understanding of glycoprotein structure and function.

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Author: Emma Leah