Targeted metabolic labeling of yeast N-glycans with unnatural sugars

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 gna1Delta 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.

Fig. 1.

Targeting GlcNAc for metabolic replacement. (A) The structurally conserved GlcNAc₂Man₈ core region of S. cerevisiae N-glycan is shown attached to a hypothetical integral membrane protein. N-glycan cores are susceptible to specific cleavage by endoH and PNGaseF as indicated. Synthetic GlcNAc analogs bearing bioorthogonal chemical groups such as azides and alkynes allow for bioconjugation of diverse probes and cargos directly to glycans. (B) A strategy for bypassing de novo UDP-GlcNAc biosynthesis (black arrows) is shown. 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, NAGK (28). The 6-phosphorylated product is subsequently converted into an activated nucleotide-sugar via the mutase and pyrophosphorylase activities of Pcm1 and Qri1 respectively.

Fig. 2.

Verification of an exogenous GlcNAc salvage pathway in S. cerevisiae. Tenfold serial dilutions of cultures with either GNA1 or gna1Δ and carrying combinations of NGT and NAGK plasmids spotted onto solid media reveal the necessity of an extracellular GlcNAc source and that NGT1 and NAGK-encoding plasmids are essential for gna1Δ rescue. Haploid gna1Δ strains (rows 2, 4, and 6) grow on rich media only when supplemented with GlcNAc (YPD + GlcNAc) while wild-type cells showed no difference in growth (rows 1, 3, and 5). The requirement for NAGK and NGT1 encoded by pRS416 plasmids in gna1Δ yeast is demonstrated by growth on 5-FOA + GlcNAc media. Cells containing pRS413-GAL-NGT1 and pRS415-GPD-NAGK (rows 1-2) are insensitive to 5-FOA, but if either NGT1 or NAGK is introduced on a pRS416 plasmid, 5-FOA prevents growth of gna1Δ, but not GNA1 strains (rows 3-6).

Fig. 3.

Introduction of GlcNAc analogs into cell-surface and secreted glycoproteins. (A) Secreted glycoproteins (5 μg total protein load/lane) from gna1Δ cells expressed in medium supplemented with either GlcNAc or GlcNAz were labeled with phos-FLAG and analyzed by α-FLAG immunoblotting. PNGaseF treatment totally removes N-glycans and prevents phos-FLAG ligation (lanes 2,4). Samples treated with endoH (lanes 6, 8) remain reactive to phos-FLAG; proteins detected by α-FLAG blotting are collectively downshifted in molecular weight due to deglycosylation. Cell-surface azidosugar-bearing glycans can also be readily detected via fluorescence microscopy following chemospecific labeling with alk-AF488; GlcNAz-supplemented gna1Δ cells (B) show strong reactivity with alk-AF488 but display morphological abnormalities, neither of which are observed in the same gna1Δ strain supplemented with GlcNAc (C). Similarly, GlcNAl-supplemented gna1Δ cells (D) are strongly labeled by N₃-AF647, but GlcNAc-supplemented cells (E) are not. Nuclear and mitochondrial DNA are indicated with DAPI stain.

Fig. 4.

Analysis of GlcNAz incorporation into Ygp1. (A) Polyhis-tagged Ygp1 was overexpressed in GlcNAz-supplemented SD-URA medium and purified. Identical (1 nanogram) loads of Ygp1 were subjected to detection by silverstain (lanes 1&2), and immunoblotting with α-HIS (lanes 3&4) and α-FLAG (lanes 5&6). Film exposure times were varied to generate visibly comparable Western blots: lane 3, 1 hour; lane 4, 1 min, lanes 5&6, 1 second. The required differences may reflect variability in epitope accessibility. A molar excess of EndoH_f (silverstain band **) was used to remove N-glycans from the heavily glycosylated Ygp1 (silverstain band *) which otherwise migrates as a diffuse high-molecular weight smear (lanes 1 and 2). Samples to be probed with α-FLAG were subjected to chemospecific phos-FLAG ligation prior to blotting; immunodetection of FLAG peptide indicates presence of GlcNAz. (B) Ygp1 was expressed in culture medium supplemented with either GlcNAc or . Ygp1 was treated with endoH, trypsinized, and subjected to ESI-FTICR MS analysis. Masses for a representative glycopeptide, spanning Leu 98-Arg 115 and glycosylated at only one of two potential sites (indicated in green), are shown. Relative intensities of the GlcNAc- and -modified peptides have been normalized to each other. (C) Ygp1 was expressed in culture medium supplemented with GlcNAz and subjected to ESI-FTICR MS analysis. Masses corresponding to the same glycopeptide from (B) are shown.