Targeted metabolic labeling of yeast N- with unnatural

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 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 . However, the complexities of While existing 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 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 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 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 (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 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. -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- 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 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 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 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 , 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- at the N-acetyltransferase step catalyzed by Gna1. Glucosamine, cellular GlcNAc or analogs to be internalized by the transporter Ngt1 from C. which can be produced by chemical or enzymatic deacetylation of albicans. The intracellular GlcNAc (or analog) is phosphorylated at the 6 GlcNAc, cannot rescue gna1Δ strains as it does in the case of position via the activity of the human GlcNAc kinase, NAGK (28). The GFA1 disruption. To bypass GNA1 disruption, it was first 6-phosphorylated product is subsequently converted into an activated necessary to bestow the cells with a functional alternative path- nucleotide-sugar via the mutase and pyrophosphorylase activities of Pcm1 and Qri1 respectively. way, as illustrated in Fig. 1B. We introduced expression plasmids bearing the human GlcNAc kinase NAGK into a heterozygous GNA1/gna1Δ strain of S. cerevisiae primarily because NAGK composition of its glycans and the ease with which yeast can be has previously been shown to tolerate GlcNAc analogs in vitro genetically modified. Importantly, UDP-GlcNAc is known to (28). This is capable of phosphorylating the six position provide GlcNAc for only two S. cerevisiae types of cytosolic GlcNAc (or analogs), thereby delivering the required besides the core chitobiose units of N-glycans. UDP-GlcNAc do- metabolite to compensate for GNA1 disruption. NAGK tran- nates GlcNAc into chitin, a linear polymer of β-1,4-GlcNAc (13) scription was placed under the control of the constitutive that is a minor component of the yeast and is deposited glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter in as a ring around the neck of the growing bud during cell division. pRS41X vector scaffolds with URA3, HIS3, and LEU2 selective Additionally, UDP-GlcNAc is the donor for the first step of GPI markers (29). anchor biosynthesis, but the GlcNAc residue is subsequently dea- To enable utilization of extracellular GlcNAc, we introduced cetylated (14). Unlike higher eukaryotes, S. cerevisiae does not plasmids carrying the recently characterized C. albicans GlcNAc incorporate GlcNAc into O-glycans (15) or antennary regions transporter NGT1 into the same GNA1/gna1Δ strain (19). While of N-glycans (16). Furthermore, S. cerevisiae lacks epimerases NGT1 was initially cloned into the pRS41X-GPD scaffolds, no that act on UDP-GlcNAc (17). Thus, the distribution of GlcNAc transformants could be obtained from plasmids containing in the S. cerevisiae glycoproteome is expected to be restricted to GPD promoters, suggesting S. cerevisiae may be sensitive to the chitobiose cores of N-glycans. NGT1 expression level. NGT1 was subsequently placed under the To address the issue of the competing endogenous UDP- -inducible GAL1 promoter which remains largely re- GlcNAc pool, we hypothesized that genetic disruption of de novo pressed in glucose-containing medium. The combinations of UDP-GlcNAc biosynthesis via removal of the essential glucos- NGT1 and NAGK plasmids transformed into GNA1/gna1Δ are amine 6-phosphate N-acetyltransferase (GNA1) gene would summarized in Table S1. To isolate gna1Δ haploids, we subjected force cells to scavenge environmental GlcNAc to survive. Disrup- heterozygous transformants to sporulation. Notably, only dually tion of S. cerevisiae GNA1 is possible only if a viable alternate transformed strains carrying both NGT1 and NAGK plasmids de- pathway is provided that can complement its loss (18). S. cerevi- veloped tetrads. The GNA1/gna1Δ heterozygotes sporulated siae does not have an endogenous GlcNAc salvage pathway, so we slowly and produced few tetrads after 7 d in sporulation media; equipped strains with a two-component salvage pathway bor- supplementation with GlcNAc made no difference. Dissections rowed from other organisms (Fig. 1B). Specifically, we employed of resulting tetrads demonstrated a high rate of spore death of the recently characterized GlcNAc transporter NGT1 from 25.5% 2.1% on YPD þ GlcNAc. Only 28.4% 2.3% of the Candida albicans (19) and human GlcNAc kinase NAGK. This tetrads contained four viable spores and half (49.8% 5%)of functional salvage pathway allows bypass of the otherwise lethal the tetrads had only two viable spores that contained wild-type GNA1 deletion so long as the cells are provided with a GlcNAc- GNA1. It is unclear why the gna1Δ segregants demonstrated a supplement in their growth medium. With these modifications in high rate of inviability. GNA1 disruption in surviving segregants place, we obtained viable gna1Δ GlcNAc auxotrophs. was confirmed by PCR (Fig. S1). Surviving gna1Δ segregants

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911247107 Breidenbach et al. Downloaded by guest on September 30, 2021 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 þ 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 þ 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).

demonstrated GlcNAc auxotrophy (Fig. 2) and had a doubling ment. The secreted proteins from both GlcNAc- and GlcNAz- time approximately twice that of wild-type haploids. supplemented cultures were then subjected to Staudinger ligation To verify that plasmids bearing NGT1 and NAGK were essen- (21) with phosphine-FLAG (phos-FLAG) to chemospecifically tial for GlcNAc salvage and gna1Δ rescue, a negative selection scheme was implemented. Haploid strains harboring either NAGK or NGT1 within the URA3 marked plasmid (pRS416) were isolated from dissected tetrads. To assess the growth re- quirements of the various gna1Δ haploids, our strains were spotted onto 5-fluoroorotic acid (5-FOA) media þ GlcNAc (Fig. 2). Cells containing a URA3 marked plasmid will convert 5-FOA to 5-fluorouracil and will not grow in the presence of this

compound, thus enabling negative selection against each indivi- CHEMISTRY dual component of the salvage pathway (30). Because gna1Δ cells could not grow on 5-FOA media when either NGT1 or NAGK was encoded on a pRS416 (URA3) plasmid, but could if the genes were encoded on pRS415 (LEU2) and pRS413 (HIS3), respec- tively, we conclude that gna1Δ yeast can only be rescued if both NAGK and NGT1 gene products are expressed and the growth medium is supplemented with GlcNAc. NGT1 transcription was controlled by the GAL1 promoter GENETICS which is repressed in glucose-containing media. Despite the pre- sence of glucose in rich medium and synthetic dropout (SD) plates, the GAL1 promoter was not fully repressed and we con- firmed low levels of both NGT1 and NAGK mRNA transcripts in cells by qPCR. If cells were grown in nonrepressive, galactose- containing media (YPGAL þ GlcNAc) we observed apparent toxicity of Ngt1 in gna1Δ cells (Fig. 2). Wild-type cells grown on YPGAL þ GlcNAc were not under selective pressure to retain the NGT1 plasmid and are therefore able to grow on YPGAL medium (Fig. 2). These observations suggest that a high level of NGT1 expression is toxic to S. cerevisiae.

Incorporation of Synthetic Sugars into Glycoproteins. Significantly, exogenous salvage pathways can serve as convenient routes to in- troduce nonnatural sugar analogs into glycans. For example, an artificial salvage pathway was recently exploited to install fucose analogs into cell-surface of E. coli in which de novo biosynthesis of the donor nucleotide-sugar GDP- fucose had been disrupted (31). We conducted several experi- Δ Fig. 3. Introduction of GlcNAc analogs into cell-surface and secreted glyco- ments to determine if the GlcNAc salvage pathway in gna1 yeast μ Δ could assimilate unnatural GlcNAc analogs into secreted glyco- proteins. (A) Secreted glycoproteins (5 g total protein load/lane) from gna1 cells expressed in medium supplemented with either GlcNAc or GlcNAz were proteins. First, we utilized the azide-bearing analog GlcNAz. We labeled with phos-FLAG and analyzed by α-FLAG immunoblotting. PNGaseF observed that cell proliferation in gna1Δ cultures administered treatment totally removes N-glycans and prevents phos-FLAG ligation (lanes with a pure unnatural sugar supplement was severely impaired 2,4). Samples treated with endoH (lanes 6, 8) remain reactive to phos-FLAG; but that the cells remained viable—they continued to produce proteins detected by α-FLAG blotting are collectively downshifted in molecu- protein and readily proliferated if switched to a GlcNAc supple- lar weight due to deglycosylation. Cell-surface azidosugar-bearing glycans ment, or if supplemented with a GlcNAc/analog mix. To increase can also be readily detected via fluorescence microscopy following chemos- Δ yields of secreted protein, we adopted the strategy of growing pecific labeling with alk-AF488; GlcNAz-supplemented gna1 cells (B) show strong reactivity with alk-AF488 but display morphological abnormalities, cells to midlog phase in the presence of GlcNAc before pellet- neither of which are observed in the same gna1Δ strain supplemented with ing, washing, and resuspending in analog-supplemented culture GlcNAc (C). Similarly, GlcNAl-supplemented gna1Δ cells (D) are strongly Δ medium. Conditioned medium from gna1 yeast cultures was labeled by N3-AF647, but GlcNAc-supplemented cells (E) are not. Nuclear collected and desalted to remove unmetabolized sugar supple- and mitochondrial DNA are indicated with DAPI stain.

Breidenbach et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 30, 2021 modify any resident azides (Fig. 3A). The labeled glycoproteins Characterization of Ygp1 N-glycans. A single, highly glycosylated were then analyzed by immunoblotting with an α-FLAG anti- protein was purified from conditioned growth medium to per- body. Notably, only secreted proteins from GlcNAz-supplemen- form a precise analysis of analog location as well as labeling ef- ted cultures showed immunoreactivity with α-FLAG, indicating ficiency. YGP1 encodes a secretory glycoprotein of unknown azides are indeed present in these glycoproteins. function containing 15 potential sites of N-glycosylation (36). To verify azide incorporation is limited to N-glycan cores, YGP1 was placed under the control of the GPD promoter in the we subjected the secreted protein fractions to digestion with multicopy pRS426 vector with a C-terminal polyhistidine tag, either peptide:N-glycosidase F (PNGaseF) or endoglycosidase transformed into gna1Δ yeast, and expressed in the presence of H (endoH), which specifically target N-glycans (33). Consistent GlcNAc, isotopically labeled GlcNAc, and GlcNAz supplements. with N-glycan-specific labeling, PNGaseF-digested proteins lost Ygp1 was affinity purified to near-homogeneity as assessed by reactivity with phos-FLAG (Fig. 3A). However, secreted glyco- silver-stained SDS-PAGE (Fig. 4A). The purified glycoprotein proteins still reacted with phos-FLAG following endoH diges- was subjected to endoH digestion to remove all but core tion. We observed an overall downshift in molecular weight GlcNAc (or GlcNAz) residues, causing a significant downshift in consistent with deglycosylation. These data confirm that the dis- molecular weight. The identity of Ygp1 was confirmed by immu- played azides are located on N-glycans of glycoproteins and that noblotting for its polyhistidine tag. Ygp1 expressed in GlcNAz- the first core GlcNAc residue in these glycans carries a phos- supplemented medium (Fig. 4A) was highly reactive with phos- phine-reactive azide modification. FLAG and both fully glycosylated and endoH-treated samples GlcNAc analogs displayed on cell-surface glycoproteins were could be readily detected by α-FLAG blotting. detected by fluorescence microscopy. In the case of GlcNAz- supplemented yeast, we utilized an alkyne-functionalized Alexa- fluor-488 (alk-488) dye to chemospecifically modify azides via Cu (I)-catalyzed azide-alkyne cycloaddition (CuAAC) (22). GlcNAz- supplemented gna1Δ yeast were reactive with alk-488, while GlcNAc-supplemented cells were not (Fig. 3B, C). Surfaces were not uniformly intense in fluorescence, possibly due to differences in cell age. In some cases, alk-488 strongly labeled bud-neck regions between cells. Notably, GlcNAz-supplemented cells appeared to display morphological and cytokinetic defects; branched, multicell chains were frequently observed in these cul- tures (Fig. 3B). The abnormalities we observe in cell shape, size, and bud-neck morphology are strikingly similar to those observed in strains in which chitin synthases CHS1 and CHS2 have been deleted (34). In contrast, GlcNAc-supplemented gna1Δ cells dis- played little reactivity with alk-488 and had comparatively normal morphology (Fig. 3C). In a small minority of GlcNAz-supplemen- ted/alk-488 labeled cells, we observed strongly fluorescent ring- like features on cell surfaces (Fig. S2) consistent with bud scars, the remnants of the primary septa seen on the surfaces of mother cells following division. Bud scars are the primary repository of chitin, and chitin-specific stain calcofluor white (CW) (35) colo- calized with alk-488 in bud scars, possibly indicating the presence of azide-bearing chitinous deposits. More frequently, however, we observed a notable decrease in CW fluorescence in GlcNAz- supplemented cells relative to those supplemented with GlcNAc. We speculate that the chitin synthases in S. cerevisiae are rela- tively intolerant of the GlcNAz analog and that defects in chitin production lead to the observed morphological abnormalities and decreased viability. Similarly, cultures supplemented with alkyne-bearing GlcNAl 647 Fig. 4. Analysis of GlcNAz incorporation into Ygp1. (A) Polyhis-tagged Ygp1 were probed with azide-functionalized Alexafluor-647 (N3- ). was overexpressed in GlcNAz-supplemented SD-URA medium and purified. GlcNAl-supplemented gna1Δ yeast were reactive with N3-647 Identical (1 nanogram) loads of Ygp1 were subjected to detection by silver- and displayed strong fluorescence on their surfaces, while stain (lanes 1&2), and immunoblotting with α-HIS (lanes 3&4) and α-FLAG GlcNAc-supplemented cells did not (Fig. 3D, E). GlcNAl-supple- (lanes 5&6). Film exposure times were varied to generate visibly comparable mented cells also demonstrated morphological abnormalities Western blots: lane 3, 1 hour; lane 4, 1 min, lanes 5&6, 1 second. The required such as increased size relative to those from GlcNAc-supplement differences may reflect variability in epitope accessibility. A molar excess of cultures. As with GlcNAz, an increased tendency for intercellular EndoHf (silverstain band **) was used to remove N-glycans from the heavily glycosylated Ygp1 (silverstain band *) which otherwise migrates as a diffuse connectivity was observed with the GlcNAl supplement; single high-molecular weight smear (lanes 1 and 2). Samples to be probed with cells were rarely observed in these cultures. α-FLAG were subjected to chemospecific phos-FLAG ligation prior to In S. cerevisiae, UDP-GlcNAc is also known to provide GlcNAc blotting; immunodetection of FLAG peptide indicates presence of GlcNAz. for GPI anchor biosynthesis. While GlcNAc is coupled to phos- (B) Ygp1 was expressed in culture medium supplemented with either 13 photidylinositol in the first step of GPI anchor biosynthesis, its GlcNAc or C6-GlcNAc. Ygp1 was treated with endoH, trypsinized, and sub- C-2 acetamido substituent is promptly removed by the essential jected to ESI-FTICR MS analysis. Masses for a representative , deacetylase activity of Gpi12 (14). While we would not expect to spanning Leu 98-Arg 115 and glycosylated at only one of two potential sites (indicated in green), are shown. Relative intensities of the GlcNAc- find the azide or alkyne functionality in the mature GPI anchors 13 and C6-GlcNAc-modified peptides have been normalized to each other. of analog-supplemented cells, we cannot explicitly discount the (C) Ygp1 was expressed in culture medium supplemented with GlcNAz possibility that GlcNAc analogs remain unprocessed by Gpi12 and subjected to ESI-FTICR MS analysis. Masses corresponding to the same activity. glycopeptide from (B) are shown.

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911247107 Breidenbach et al. Downloaded by guest on September 30, 2021 To further verify nonnatural sugar labeling at core N-glycan hydrogen bonds between the acetamido group and the transfer- positions, endoH-treated Ygp1 samples were trypsinized and ase may be essential for activity (39), but these features are pre- the resulting peptides were examined by served in GlcNAz and GlcNAl analogs. Importantly, our data do Fourier-transform ion cyclotron resonance mass spectrometry not explicitly confirm analog incorporation at the Asn-distal site (ESI-FTICR MS). Tryptic peptides covering 68% of the Ygp1 of the N-glycan core. Though available biochemical data suggest sequence were successfully identified in mass spectra from our modifications to the C-2 acetamido group would not interfere samples derived from cultures supplemented with GlcNAc, in either Alg13∕14 or OT activities, explicit confirmation of 13 13 N-acetyl-½ C6glucosamine ð C6-GlcNAcÞ,orGlcNAz(Ta b l e S 3 ). analog incorporation at the Asn-distal site is a subject of future Five of fifteen potential N-glycosylation sites were observed to interest. Qualitatively, we did not observe any reduction in the be occupied. Specifically, positions Asn 100, 106, 118, 239, and glycosylation level of Ygp1 when expressed in GlcNAz- 286 were found to be glycosylated, but tryptic peptides covering supplemented medium; one might expect decreased glycosylation the other ten potential sites in the Ygp1 sequence were not ob- if GlcNAz strongly inhibited the dolichol pathway. served. The tryptic peptide spanning Leu 98-Arg 115 contained two potential N-glycosylation sites and peptide masses corre- Conclusion sponding to both singly- and doubly glycosylated forms of this High-efficiency, site-specific replacement of GlcNAc with unna- peptide were detected, but no unglycosylated peptide was tural analogs in the chitobiose cores of N-glycans allows for reli- observed. Tryptic peptides spanning Val 116-Lys 124 and Glu able and predictable introduction of unique functionalities into 236-Lys 314 were observed to be fully glycosylated. N-glycans with little risk of perturbing glycoprotein structure A stable isotope of GlcNAc was used to unequivocally verify and function. We envision GlcNAc analogs as convenient means that the supplement added to the culture medium is metabolically for reliable introduction of fluorophores, anomalous x-ray scat- incorporated into N-glycan cores of secreted glycoproteins. Ygp1 terers, crosslinkers, and affinity tags directly onto glycoprotein 13 expressed in cultures supplemented with a C6-GlcNAc isotope surfaces. Larger cargos, including peptides, macromolecules, or yielded tryptic with the expected 6 Da shift per even cells, may also be tethered to N-glycosylated carrier proteins occupied glycosylation site (Fig. 4B, Table S3). The labeling at uniform, predictable sites of attachment. Given the utility of 13 efficiency with C6-GlcNAc appears to be extremely high; yeast as an expression platform for glycoprotein biotherapeutics, GlcNAc-modified peptides were not detectable in cultures this technique may find use in biotechnology applications as well. 13 supplemented with C6-GlcNAc. These data indicate that it is Although we tested only azide, alkyne, and isotopic GlcNAc possible to achieve global replacement of GlcNAc with unnatural analogs in this study, we anticipate that gna1Δ yeast will be able CHEMISTRY isotopes in gna1Δ strains. to assimilate additional nonnatural sugars into N-glycans, a sub- Similarly, Ygp1 expressed in the presence of GlcNAz was ana- ject of future interest. We believe targeted metabolic labeling of lyzed by ESI-FTICR MS to determine if high levels of this non- N-glycans with unnatural sugars will afford numerous unique natural GlcNAc analog occupy N-glycan core positions. In this applications that were not previously possible, ultimately facilitat- case, cultures were grown to midlog phase with a GlcNAc supple- ing studies of glycoprotein structure and mechanism. ment before switching to GlcNAz due to the slow doubling time Δ of gna1 cells in GlcNAz-supplemented medium. The resulting Materials and Methods GENETICS Ygp1 glycopeptide masses we observed were consistent with a Preparation of NAGK, NGT1, and YGP1 Expression Vectors. Details pertaining to mix of GlcNAc and GlcNAz occupying the core N-glycan posi- plasmid construction are included in SI Text. tions (Fig. 4C, Table S3). The mixture of GlcNAc and GlcNAz modifications likely results from a cytosolic reservoir of GlcNAc Isolation of gna1Δ Strains and Viability Assays. Standard rich and SD media that persists after cells are resuspended in GlcNAz-supplemented formulations and lithium acetate transformation protocols were used except gna1Δ cells were supplemented with 100 μM GlcNAc (or GlcNAz) in liquid culture medium. Unfortunately, it is not possible to draw quan- μ ∶ culture. Solid medium was supplemented with 200 M GlcNAc. A heterozy- titative conclusions about the GlcNAz GlcNAc ratio in the gous GNA1/gna1Δ strain (ATCC#4025635; MATa∕MATαhis3Δ∕his3Δleu2Δ∕ glycopeptides detected by mass spectrometry due to potential dif- leu2Δlys2Δ∕ þ met15Δ∕ þ ura3Δ∕ura3Δyfl017c::KanMX4∕þ) was trans- ferences in ionization efficiency. However, it is likely that expres- formed with GAL1-NGT1 and GPD-NAGK plasmids (Table S1) and then sporu- sion conditions can be optimized to more thoroughly deplete cells lated in 2% potassium acetate. Haploid segregants were grown overnight in of their internal GlcNAc reservoirs before switching to the non- media (SD-URA-HIS or SD-HIS-LEU) to select for both plasmids. Cells were diluted natural sugar supplement, thus enabling extremely high labeling to 107 per mL and serially diluted ten-fold. Approximately 5 μL was spotted efficiencies. onto solid media. Cells were allowed to grow for 2–4 d at 30 °C and then photographed. Implications for Promiscuity in the Dolichol Pathway. Collectively, our data indicate that enzymes involved in the early steps of yeast Microscopy. Procedures used for imaging fluorophore-labeled cells are described in SI Text. N-glycan biosynthesis can tolerate subtle chemical modifications of the C-2 acetamido moiety of GlcNAc. Here, we present strong Western Blots. Protein electrophoresis was performed with Criterion tris-HCl evidence that acetamido group of the Asn-proximal GlcNAc can 4–12% gels (BioRad). Polyhistidine-tagged proteins were detected with be modified and still correctly processed by phosphoglycosyl- α-5xHis-peroxidase conjugate kit (Qiagen) as specified by product literature. transferase Alg7 and later by the oligosaccharyl transferase as- Synthesis of detection reagent phos-FLAG has been described previously (40). sembly (OT). During the oligosaccharyl transfer, it is believed Fully secreted yeast glycoproteins were obtained from conditioned medium that the C-2 acetamido group of this GlcNAc acts to stabilize of small-scale (5 mL) cultures by centrifugation to remove cells. Proteins were the oxonium intermediate prior to nucleophilic attack by the simultaneously concentrated and buffer exchanged into PBS using Amicon receiving Asn residue (37); removal or substantial electronic per- 10 kDa net molecular weight cutoff (NMWCO) centrifugal filters (Millipore). turbation of this acetamido group, but not that of the Asn-distal Total protein concentration was measured with the colorimetric DC protein GlcNAc, effectively blocks OT activity (38). While azido and al- assay (BioRad). Glycoprotein samples were labeled via Staudinger ligation (21) over a 12 h, room temperature incubation with 500 μM phos-FLAG. kynyl additions to the C-2 acetamido group obviously increase FLAG-conjugated proteins were detected with α-FLAG M2-peroxidase anti- size, we believe that these modifications would not interfere with body (Sigma-Aldrich). Peroxidase-conjugated were detected by oxonium stabilization during transfer. Modifications to the C-2 chemiluminescence using the SuperSignal West Pico substrate (Pierce). acetamido group of the Asn-proximal GlcNAc may also impact the activity of the Alg13∕14 enzyme complex which transfers the Protein Expression and Purification. Procedures used for protein expression, Asn-distal GlcNAc onto the Asn-proximal GlcNAc. Specific purification and enzymatic deglycosylation are described in SI Text.

Breidenbach et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 30, 2021 Chemical Synthesis. Syntheses of GlcNAz and GlcNAl are described in ACKNOWLEDGMENTS. We wish to extend our gratitude toward Dr. T. Starr, SI Text. J. Baskin, S. Hubbard, Dr. J. Seeliger, and Dr. M. Boyce for technical assistance, reagents, and helpful discussion. This work was supported by National Insti- Mass Spectrometry. Highly purified Ygp1 was treated with endoH (New Eng- tutes of Health Grant GM066047 (to C.R.B.) . J.E.G.G was supported by NSF land Biolabs) and the deglycosylated products were separated by SDS-PAGE. postdoctoral fellowship DBI-0511799. Desalted, in-gel tryptic digests of Ygp1 samples were analyzed by ESI-FTICR MS (Bruker, 9.4 T magnet).

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