Targeted metabolic labeling of yeast N-glycans with unnatural sugars

Mark A. Breidenbach, Jennifer E G Gallagher, David S. King, Brian P. Smart, Peng Wu, Carolyn R. Bertozzi

Research output: Contribution to journalArticle

49 Citations (Scopus)

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 uridinediphosphate- GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1△ 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.

Original languageEnglish (US)
Pages (from-to)3988-3993
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number9
DOIs
StatePublished - Mar 2 2010

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Polysaccharides
Glycoproteins
Yeasts
N-acetylglucosamine kinase
Glycosylation
Culture Media
Glycoside Hydrolases
Glucosamine
Essential Genes
Candida albicans
Saccharomyces cerevisiae
Mass Spectrometry
Plasmids
Nucleotides
Peptides

Keywords

  • Click chemistry
  • GIcNAc
  • GNA1
  • Metabolic engineering
  • N-glycosylation

ASJC Scopus subject areas

  • General

Cite this

Breidenbach, M. A., Gallagher, J. E. G., King, D. S., Smart, B. P., Wu, P., & Bertozzi, C. R. (2010). Targeted metabolic labeling of yeast N-glycans with unnatural sugars. Proceedings of the National Academy of Sciences of the United States of America, 107(9), 3988-3993. https://doi.org/10.1073/pnas.0911247107

Targeted metabolic labeling of yeast N-glycans with unnatural sugars. / Breidenbach, Mark A.; Gallagher, Jennifer E G; King, David S.; Smart, Brian P.; Wu, Peng; Bertozzi, Carolyn R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 9, 02.03.2010, p. 3988-3993.

Research output: Contribution to journalArticle

Breidenbach, Mark A. ; Gallagher, Jennifer E G ; King, David S. ; Smart, Brian P. ; Wu, Peng ; Bertozzi, Carolyn R. / Targeted metabolic labeling of yeast N-glycans with unnatural sugars. In: Proceedings of the National Academy of Sciences of the United States of America. 2010 ; Vol. 107, No. 9. pp. 3988-3993.
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