Targeted metabolic labeling of yeast N-glycans with unnatural sugars
Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved December 23, 2009 (received for review September 30, 2009)
Abstract
Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1Δ strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.
Acknowledgments.
We wish to extend our gratitude toward Dr. T. Starr, J. Baskin, S. Hubbard, Dr. J. Seeliger, and Dr. M. Boyce for technical assistance, reagents, and helpful discussion. This work was supported by National Institutes of Health Grant GM066047 (to C.R.B.) . J.E.G.G was supported by NSF postdoctoral fellowship DBI-0511799.
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Cover image: Pictured are residential burglary hotspots in a portion of Los Angeles, California. Martin B. Short et al. report on reaction-diffusion models indicating that spatial crime patterns come in two fundamental types: supercritical hotspots, which emerge from small stochastic fluctuations in crime, and subcritical hotspots, which emerge from large spikes in crime. The authors suggest that policing tactics focused on supercritical hotspots can displace the hotspot into a ring structure, which then breaks up into new hotspots, whereas tactics focused on subcritical hotspots may result in lasting crime dissipation. See the article by Short et al. on pages 3961–3965. Image courtesy of P. Jeffrey Brantingham.
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Published online: February 8, 2010
Published in issue: March 2, 2010
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Acknowledgments
We wish to extend our gratitude toward Dr. T. Starr, J. Baskin, S. Hubbard, Dr. J. Seeliger, and Dr. M. Boyce for technical assistance, reagents, and helpful discussion. This work was supported by National Institutes of Health Grant GM066047 (to C.R.B.) . J.E.G.G was supported by NSF postdoctoral fellowship DBI-0511799.
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This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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