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Targeted Metabolic Labeling of Yeast N-Glycans with Unnatural Sugars

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Targeted Metabolic Labeling of Yeast N-Glycans with Unnatural Sugars Targeted metabolic labeling of yeast N-glycans with unnatural sugars Mark A. Breidenbacha, Jennifer E. G. Gallagherb, David S. Kingc, Brian P. Smarta, Peng Wua,2, and Carolyn R. Bertozzia,b,c,d,1 aDepartments of Chemistry; bMolecular and Cell Biology; and cHoward Hughes Medical Institute, University of California, Berkeley, CA 94720; and dThe Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved December 23, 2009 (received for review September 30, 2009) Metabolic labeling of glycans with synthetic sugar analogs has and mass spectrometric analyses of proteins displaying the nonna- emerged as an attractive means for introducing nonnatural chemi- tural chemical functionality (5, 6). cal functionality into glycoproteins. However, the complexities of While existing glycan labeling methodology is suitable for stud- glycan biosynthesis prevent the installation of nonnatural moieties ies that require only stochastic insertion of analogs at low levels, at defined, predictable locations within glycoproteins at high levels the technique is inadequate for applications in which specific of incorporation. Here, we demonstrate that the conserved monosaccharides must be reliably targeted for metabolic replace- N-acetyglucosamine (GlcNAc) residues within chitobiose cores of ment. For example, high-efficiency and predictable installation of N-glycans in the model organism Saccharomyces cerevisiae can sugar analogs could dramatically facilitate biophysical studies of be specifically targeted for metabolic replacement by unnatural glycoprotein structure and function via site-specific introduction sugars. We introduced an exogenous GlcNAc salvage pathway into of fluorophores and heavy atoms. Unfortunately, site-specific me- yeast, allowing cells to metabolize GlcNAc provided as a supple- tabolic labeling of glycans is hindered by multiple factors. First, ment to the culture medium. We then rendered the yeast auxo- nucleotide-sugar analogs are necessarily in direct competition ’ trophic for production of the donor nucleotide-sugar uridine- with a cell s endogenous pool of donor sugars, often leading to diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential poor levels of metabolic incorporation of the synthetic analog (7). gene GNA1. We demonstrate that gna1Δ strains require a GlcNAc Further, many eukaryotes possess epimerase activities that can CHEMISTRY supplement and that expression plasmids containing both exoge- interconvert nucleotide-sugar stereochemistries and thereby alter nous components of the salvage pathway, GlcNAc transporter the final metabolic destination of a given monosaccharide (8, 9). NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo Finally, because glycan assembly is not genetically encoded and is sapiens, are required for rescue in this context. Further, we show therefore inherently prone to microheterogeneity, targeting ana- that cells successfully incorporate synthetic GlcNAc analogs logs to specific positions within a defined class of glycoconjugates N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosa- is untenable. Even though glycan biosynthesis is not template-driven, some mine (GlcNAl) into cell-surface glycans and secreted glycoproteins. GENETICS glycans share conserved structural features as a result of the pro- To verify incorporation of the nonnatural sugars at N-glycan core cess by which they are assembled. Asparagine-linked glycans positions, endoglycosidase H (endoH)-digested peptides from a (N-glycans) are an excellent example; while antennary regions purified secretory glycoprotein, Ygp1, were analyzed by mass of N-glycans are ultimately edited to diverse compositions and spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, structures, N-glycan cores are all derived from a conserved isotopically labeled GlcNAc, or GlcNAz were identified; these mod- tetradecasaccharide precursor featuring a β1,4-linked GlcNAc ifications were dependent on the supplement added to the culture chitobiose unit at the site of polypeptide attachment (10). Both medium. This system enables the production of glycoproteins that GlcNAc monomers of the chitobiose unit, donated by UDP- are functionalized for specific chemical modifications at their GlcNAc, persist throughout the lifespan of the N-glycan. Here, glycosylation sites. we target the conserved core GlcNAc residues for metabolic re- placement, effectively sidestepping the issue of glycan structural ∣ ∣ ∣ ∣ click chemistry GlcNAc metabolic engineering N-glycosylation GNA1 heterogeneity (Fig. 1A). N-glycans are also uniquely suited for targeted metabolic labeling because they can profoundly influ- he introduction of unnatural chemical moieties into proteins ence a protein’s folding, trafficking, solubility, and stability (11); Thas enormous potential to facilitate both in vivo and in vitro more so than many other types of glycosylation, N-glycan occu- studies of protein function, mechanism, and structure. For exam- pancy can be mandatory for function. Indeed, occupancy of many ple, genetically encoded and residue-specific approaches for N-glycosylation sites is sufficiently high to observe core carbohy- introduction of unique amino acids have enabled integration of drate residues crystallographically (12). several functionalities such as spectroscopic probes, bioorthogonal Targeted glycan labeling can also be dramatically simplified chemical reporters, metal chelators, and photoaffinity labels by working in an organism with relatively well-characterized directly into recombinant proteins (1, 2). As an alternative to glycan biosynthesis. In this study, we utilize the model eukaryote the insertion of nonnatural amino acids into a polypeptide chain, S. cerevisiae for several reasons including the relatively simple unique chemical functionality can also be introduced into a protein’s posttranslational modifications as exemplified by the Author contributions: M.A.B. designed research; M.A.B., J.E.G.G., and D.S.K. performed technique of glycan metabolic labeling (3). In this approach, research; M.A.B., J.E.G.G., and B.P.S. analyzed data; M.A.B., J.E.G.G., and C.R.B. wrote synthetic analogs of natural monosaccharides are introduced into the paper; P.W. contributed new reagents/analytic tools. cells where they are processed by a series of enzymes to generate The authors declare no conflict of interest. activated nucleotide-sugar analogs. The sugar analogs, bearing This article is a PNAS Direct Submission. unique chemical functionality, are subsequently utilized in the 1To whom correspondence should be addressed. E-mail: [email protected]. biosynthesis of various cellular glycoconjugates; this technique 2Present address: Department of Biochemistry, Albert Einstein College of Medicine, has been vividly illustrated in recent glycan imaging studies (4). Yeshiva University, Bronx, NY 10461. Additionally, the introduction of synthetic sugars into glycans This article contains supporting information online at www.pnas.org/cgi/content/full/ via metabolic labeling has been exploited for affinity capture 0911247107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0911247107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 30, 2021 We demonstrate that synthetic, azide- and alkyne-bearing GlcNAc analogs as well as isotopically labeled GlcNAc can be introduced into yeast N-glycans via this unique salvage pathway. Azide and alkyne functional groups are known to be versatile chemical handles for bioconjugation of proteins, cells, and living organisms (20). Here, we utilize phosphine (21), alkyne (22), and azide (23) probes in conjunction with mass spectrometry to verify that GlcNAc analogs are successfully metabolized and integrated into yeast cell surfaces and secreted glycoproteins. Results and Discussion Design of gna1Δ Strains. The four-step de novo UDP-GlcNAc bio- synthetic pathway (Fig. 1B) is responsible for metabolic conver- sion of fructose-6-phosphate, an intermediate of glycolysis, to UDP-GlcNAc. The highly conserved enzymes in this pathway are essential for yeast viability under normal growth conditions (24). An interruption to the UDP-GlcNAc biosynthetic pathway can be rescued if an appropriate downstream metabolite is delivered into cells. For example, gfa1Δ strains of S. cerevisiae are capable of internalizing and phosphorylating extracellular glucosamine, effectively bypassing the GFA1 deletion (25). Some organisms, such as C. albicans, have functional salvage pathways allowing Fig. 1. Targeting GlcNAc for metabolic replacement. (A) The structurally them to internalize and phosphorylate GlcNAc (18, 19, 26), conserved GlcNAc2Man8 core region of S. cerevisiae N-glycan is shown at- but S. cerevisiae lacks these activities and gna1Δ strains cannot tached to a hypothetical integral membrane protein. N-glycan cores are sus- be rescued by extracellular GlcNAc (Fig. 2). Because GlcNAc ceptible to specific cleavage by endoH and PNGaseF as indicated. Synthetic analogs with modifications to the C-2 acetamido substituent GlcNAc analogs bearing bioorthogonal chemical groups such as azides and alkynes allow for bioconjugation of diverse probes and cargos directly to are known to be tolerated by downstream eukaryotic biosynthetic glycans. (B) A strategy for bypassing de novo UDP-GlcNAc biosynthesis (black machinery (27), we chose to interrupt UDP-GlcNAc production arrows) is shown. An exogenous salvage pathway (blue arrows) allows extra-
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