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Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis Wei Qina,b, Pinou Lva,b, Xinqi Fana, Baiyi Quana, Yuntao Zhua, Ke Qina, Ying Chena, Chu Wanga,b,c,d,e,1, and Xing Chena,b,c,d,e,1 a College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China; bPeking–Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; cBeijing National Laboratory for Molecular Sciences, Peking University, Beijing 100871, China; dSynthetic and Functional Biomolecules Center, Peking University, Beijing 100871, China; and eKey Laboratory of Bioorganic Chemistry and Molecular Engineering of the Ministry of Education, Peking University, Beijing 100871, China
O-GlcNAcylation snoRNP
| metabolic labeling | proteomics | protein stability |
I
n mammalian cells, a large number of nuclear, cytoplasmic, and mitochondrial proteins are posttranslationally or cotranslationally modified with a GlcNAc monosaccharide β-linked to serine or threonine residues, which is termed as O-linked GlcNAcylation (O-GlcNAcylation) (1, 2). The O-GlcNAc modification has been implicated in a whole range of cellular processes, including transcriptional regulation (3), signal transduction (4), and stress response (5). At the protein level, O-GlcNAcylation was found to regulate protein stability (6–10) and activity (11–14). Remarkably, a sole known enzyme O-linked GlcNAc transferase (OGT) catalyzes all cellular O-GlcNAc modifications (15, 16). Another enzyme, O-linked GlcNAcase (OGA), can remove O-GlcNAc from modified proteins, making O-GlcNAcylation a reversible modification (17). To date, the number of candidate O-GlcNAcylated proteins in mammalian cells has accumulated to more than 1,000, mainly owing to recent MS-based proteomic profiling in various cell lines (18– 24). Such a broad scope of protein substrates raises several interesting questions about O-GlcNAcylation. Are all of the protein O-GlcNAcylation events reversible in cells? Do O-GlcNAcylated proteins turn over at different rates? Is the stability of O-GlcNAylated proteins differentially affected by the modification? To address these important questions, we sought to develop a quantitative time-resolved method for globally profiling the turnover dynamics of O-GlcNAcylated proteins in living cells. The MSbased O-GlcNAcome profiling has been largely facilitated by emerging methods for selective enrichment of O-GlcNAcylated proteins, such as affinity purification using O-GlcNAc–recognizing antibodies (19, 25) or lectins (20, 26), and chemoenzymatic (21, 27) www.pnas.org/cgi/doi/10.1073/pnas.1702688114
or metabolic labeling of O-GlcNAc with bioorthogonal chemical reporters followed by click labeling with affinity tags (23, 28–31). In contrast to most of the enrichment methods that are performed in vitro, metabolic incorporation of O-GlcNAc reporters enables chemical labeling of O-GlcNAc in living cells. Moreover, metabolic labeling is compatible with pulse-chase experiments (32–35), and therefore, chemical reporters of O-GlcNAc may potentially be exploited to measure the O-GlcNAcylation dynamics and turnover rates of O-GlcNAcylated proteins. However, proteomic studies based on O-GlcNAc chemical reporters have so far been performed using spectral counting, a semiquantitative method. Herein, we describe a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy for global analysis of the turnover dynamics of O-GlcNAcylated proteins in living cells. The qTOP approach combines the strengths of two protocols: (i) the metabolic pulse-chase labeling with an O-GlcNAc chemical reporter in living cells, which enables time-resolved enrichment of O-GlcNAcylated proteins, and (ii) the stable isotope labeling with amino acids in cell culture (SILAC), which permits quantitative analysis of the enriched O-GlcNAc proteomes. Applying qTOP, we successfully quantified the degradation rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells, which were categorized into three subgroups: “hyperstable,” “dynamic,” and “hyperdynamic,” The hyperstable population accounted for an unexpectedly high portion (∼14%) of Significance In mammalian cells, more than 1,000 intracellular proteins are posttranslationally modified with O-linked GlcNAc (O-GlcNAc), which regulates many important biological processes. The O-GlcNAc modification has been found to dynamically cycle on and off the modified proteins. How O-GlcNAc affects protein stability remains to be investigated at the proteome level. In this work, we developed a quantitative time-resolved proteomic strategy to analyze the turnover dynamics of O-GlcNAcylated proteins. We discovered that not all protein O-GlcNAcylation events were reversible and that a subset of O-GlcNAcylated proteins exhibited minimal removal of O-GlcNAc or degradation of protein backbones. Our work reveals stable O-GlcNAc as an important regulatory mechanism for stabilizing proteins, such as core proteins of box C/D small nucleolar ribonucleoprotein complexes. Author contributions: W.Q., C.W., and X.C. designed research; W.Q., P.L., X.F., B.Q., Y.Z., K.Q., and Y.C. performed research; W.Q., C.W., and X.C. analyzed data; and W.Q., C.W., and X.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence may be addressed. Email:
[email protected] or xingchen@ pku.edu.cn.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1702688114/-/DCSupplemental.
PNAS | Published online July 31, 2017 | E6749–E6758
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O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2′-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.
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Edited by Carolyn R. Bertozzi, Stanford University, Stanford, CA, and approved June 29, 2017 (received for review February 16, 2017)
the O-GlcNAcyated proteome that we quantified. More importantly, those hyperstable O-GlcNAc modifications had more profound effects on stabilizing the modified proteins, among which were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs)—fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). Box C/D snoRNPs consist of a box C/D small nucleolar RNA (snoRNA) and a set of four core proteins—FBL, NOP56, NOP58, and nonhistone chromosome protein 2-like 1 (NHP2L1; SNU13, 15.5K). They play an essential role in ribosome assembly by methylating rRNAs at the 2′-O-ribose with FBL as the methyltransferase (36). In this work, qTOP revealed that FBL, NOP56, and NOP58 were modified with stable O-GlcNAc. We further characterized the functional consequences of O-GlcNAcylation for box C/D snoRNPs and showed that it played an important role in snoRNP assembly and biogenesis via stabilizing the modified core proteins and regulating their subnuclear localization and interactions. Furthermore, we identified a major O-GlcNAcylation site on FBL and found that O-GlcNAcylation of FBL regulated rRNA methylation patterns and promoted tumorigenesis. Results SILAC-Based Quantification Improves O-GlcNAcylated Proteome Profiling. To establish the qTOP platform, we first implemented
SILAC into the chemical reporter-based O-GlcNAc proteomic identification. Several GlcNAc and GalNAc analogs containing an azide or alkyne have been developed as O-GlcNAc chemical reporters, which showed varied efficiencies and specificities for O-GlcNAc labeling (23, 24, 28–31) (Fig. S1). We chose peracetylated 6-azido-6-deoxy-GlcNAc (Ac36AzGlcNAc) for this work, because it was shown to exclusively label O-GlcNAc without entering other glycosylation pathways and enabled identification of 366 O-GlcNAcylated proteins in NIH 3T3 cells by spectral counting (23). NIH 3T3 cells grown in culture medium containing isotopically labeled “heavy” lysine and arginine or standard “light” medium were treated with 200 μM Ac36AzGlcNAc for 36 h (Fig. S2A). In-gel fluorescence scanning of the light and heavy cell lysates reacted with alkyne-Cy5 via Cu(I)-catalyzed azide-alkyne cycloaddition (i.e., click chemistry) exhibited identical labeling patterns and intensities, indicating that metabolic incorporation of 6-azido-6-deoxy-GlcNAc (6AzGlcNAc) does not differentiate between light and heavy cells (Fig. S2B). Furthermore, 6AzGlcNAc labeling was OGT-dependent (Fig. S2C). Because SILAC-based quantification has been previously used to improve the accuracy and sensitivity of palmitoylation proteomics using an alkyne-containing palmitic acid analog (33), we sought similar improvements for large-scale identification of O-GlcNAcylated proteins. We first tested the accuracy of SILAC quantification. The heavy and light isotope-labeled cell lysates were combined at a 1:1 ratio followed by reaction with alkynebiotin, streptavidin enrichment, and on-bead trypsin digestion. The resulting peptides were analyzed by liquid chromatography (LC)-MS/MS using the multidimensional protein identification technology (37) protocol (Fig. S2A). Peptides were identified by ProLuCID (38), and their light/heavy SILAC ratios were quantified using CIMAGE (39). The protein ratio was then quantified as the median ratio across all peptides assigned to a specific protein. The distribution of protein ratios had a mean of 0.99, accurately matching the dilution factor value (Fig. S2D). We then applied the SILAC-based quantitative chemoproteomics to identify O-GlcNAcylated proteins by performing the labeling experiments with Ac36AzGlcNAc as the chemical reporter (Fig. 1A and Fig. S2E). In the standard “forward” SILAC experiment, the light cells were treated with Ac36AzGlcNAc as a reporter, and the heavy cells were treated with peracetylated GlcNAc (Ac4GlcNAc) as a control. We also performed a replicate “reverse” SILAC experiment, where the labeling order was switched (i.e., heavy with the reporter and light as the control) to increase the robustness and E6750 | www.pnas.org/cgi/doi/10.1073/pnas.1702688114
accuracy for quantitation. The 6AzGlcNAc-incorporated proteins were enriched and quantified against the control, which identified 1,139 and 1,451 O-GlcNAcylated proteins in the forward and reverse labeling experiments, respectively (Fig. S2F); 896 proteins were reciprocally enriched and quantified in both experiments (Fig. 1B and Dataset S1). Reanalyzing the same datasets with the spectral counting method, we could identify 699 O-GlcNAcylated proteins, of which 634 overlapped with the SILAC quantification (Fig. 1C). Notably, by implementing the SILAC quantification method, the Ac36AzGlcNAc-based chemoproteomics identified an additional 262 O-GlcNAcylated proteins with low spectral counts in NIH 3T3 cells, such as AHNAK, GAK, and UBR4 (Fig. 1D). These results show that SILAC quantification can significantly improve the accuracy and sensitivity of the chemical reporter-based O-GlcNAc proteomics. Excluding those with uncharacterized cellular localizations, all of the O-GlcNAcylated proteins identified by Ac36AzGlcNAc labeling possess intracellular domains accessible by OGT, in agreement with the high specificity of Ac36AzGlcNAc for O-GlcNAc labeling (Fig. 1E). We further validated the identified O-GlcNAcylated proteins using a chemoenzymatic method based on a mutant galactosyltransferase (Y289L GalT), which recognizes terminal GlcNAc moieties and allows for labeling of endogenous and natural O-GlcNAc with uridine diphosphate N-azidoacetylgalactosamine (UDP-GalNAz) in cell lysates (40, 41). By preforming the forward and reverse Y289L GalT labeling followed by click reaction with alkyne-biotin, streptavidin enrichment, and LC-MS/MS analysis, a total of 1,272 O-GlcNAcylated proteins were identified (Fig. S2G); 754 proteins were identified in both Ac36AzGlcNAc and Y289L GalT labeling, which were classified as high-confidence O-GlcNAcylated proteins (Fig. 1F). Quantitative Analysis of the Dynamics of O-GlcNAcylated Proteins by qTOP. Having established that Ac36AzGlcNAc in combination
with SILAC could quantify more than 750 O-GlcNAcylated proteins in NIH 3T3 cells that could also be verified by Y289L GalT labeling, we next integrated the pulse-chase protocol to construct the qTOP platform for profiling the turnover dynamics of O-GlcNAc proteome (Fig. 2A and Fig. S3A). Light and heavy cells were pulse labeled with Ac36AzGlcNAc for 36 h to ensure sufficient metabolic incorporation of the chemical reporter (Fig. S3B). In a forward qTOP experiment, the heavy cells were immediately harvested after pulse labeling to serve as the reference at time of 0 h, and the light cells were chased with Ac4GlcNAc for another 12 h (Fig. 2A). This chase time was optimized to ensure that significant O-GlcNAc turnover events can be observed, and there are still enough chemical reporters remaining for detection (Fig. S3C). Similarly, a reverse replicate of qTOP was also performed, in which the pulse-labeled heavy cells were chased with Ac4GlcNAc (Fig. S3A). After mixing and lysing the cells, 6AzGlcNAc-labeled proteins were enriched, analyzed by LC-MS/MS, and quantified between the chased and reference samples. The residual amount of the pulse-labeled fraction of individual proteins after 12 h was revealed by the qTOP ratio (chased vs. reference), with smaller ratios corresponding to faster turnover rates of O-GlcNAcylated proteins. By isolating 6AzGlcNAc-incorporated proteins, qTOP quantified exclusively the O-GlcNAcylated proteoform, avoiding interferences imposed by the unmodified fraction. Moreover, the turnover of O-GlcNAcylated proteins measured in qTOP could result from protein degradation and/or OGA-catalyzed removal of 6AzGlcNAc from the pulse-labeled proteins (Fig. S3D). In three biological replicates, we acquired qTOP ratios for 533 of 754 high-confidence O-GlcNAcylated proteins (Fig. S3E and Dataset S2), which were normally distributed at a mean of 0.74 with an SD of 0.17 (Fig. 2B). The proteins were classified into three groups by ranking their qTOP ratios: hyperstable O-GlcNAcylated proteins with ratios > 0.91 (i.e., mean + SD), Qin et al.
Stable O-GlcNAc Modification Has a Profound Effect on Stabilizing Proteins. Given that a number of proteins have been found to
be stabilized by O-GlcNAc modification, such as SP1, p53, BMAL1/CLOCK, and nuclear pore complex protein 62 (NUP62) (2, 7–9), we asked whether the hyperstable O-GlcNAc revealed by qTOP contributes to the stability of those O-GlcNAcylated proteins. To answer this question, we used an OGT inhibitor, peracetylated 2-acetamido-2-deoxy-5-thio-D-glucopyranose (Ac45SGlcNAc) (46), to globally lower the cellular level of O-GlcNAcylation and then measured changes of protein abundance by SILAC-based quantitative proteomic analysis (Fig. 3A). Heavy NIH 3T3 cells were treated with 50 μM Ac45SGlcNAc for 48 h, and light cells Qin et al.
were treated with the DMSO control. After mixing and lysing the cells, 2,500 proteins were quantified with high confidence (i.e., at least twice in three biological replicates), and the heavy to light ratios indicated the changes of protein abundance on O-GlcNAcylation inhibition (Fig. 3B, Dataset S3, and Fig. S4A). The list of 2,500 quantified proteins covered 62 of 75 hyperstable O-GlcNAcylated proteins, 295 of 378 dynamic O-GlcNAcylated proteins, and 43 of 80 hyperdynamic O-GlcNAcylated proteins (Fig. 3C). On OGT inhibition, >80% of the hyperstable O-GlcNAcylated proteins exhibited SILAC ratios smaller than 1.0, indicating that their abundance was decreased when O-GlcNAcylation was inhibited, whereas the percentages were much lower (57 and 60%) in the dynamic and hyperdynamic populations, respectively (Fig. 3D). Furthermore, the extent of decrease was significantly greater for the hyperstable O-GlcNAcylated proteins than the dynamic and hyperdynamic populations (the mean ratio of 0.89 for the hyperstable group vs. 0.98 for the dynamic group and 0.99 for the highly dynamic group). Collectively, these results suggest that stable O-GlcNAc has more profound effects on stabilizing proteins. Three Box C/D snoRNP Core Proteins Are Modified with Stable O-GlcNAc.
The 75 hyperstable O-GlcNAcylated proteins are mainly associated with three biological processes, including transport, metabolic process, and RNA regulation (Fig. 2C). Among the list of transportassociated proteins, nucleoporin NUP214, a nuclear pore complex PNAS | Published online July 31, 2017 | E6751
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dynamic O-GlcNAcylated proteins with ratios ≤ 0.91 and ≥ 0.57 (i.e., mean – SD), and hyperdynamic O-GlcNAcylated proteins with ratios < 0.57 (Fig. 2B and Dataset S2). Consistent with the general view that O-GlcNAc modifications are often rapidly reversible and dynamic (42–45), 86% of the O-GlcNAcylated proteins quantified in the qTOP experiments showed a dynamic turnover in 12 h. Meanwhile, it is also intriguing to observe that quite a significant portion (∼14%) of the O-GlcNAcylated proteins (i.e., 75 proteins in the hyperstable group) showed not only minimal degradation of the protein backbone but also, minimal removal of O-GlcNAc within 12 h, reflecting their stable nature with slow turnover dynamics.
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Fig. 1. Quantitative identification of O-GlcNAcylated proteins by SILAC-based chemoproteomics. (A) Schematic of the workflow of SILAC-based quantitative O-GlcNAcylation proteomics using metabolic labeling of chemical reporters. In the forward SILAC, light and heavy cells were treated with 200 μM Ac36AzGlcNAc and Ac4GlcNAc for 36 h, respectively. The lysates were mixed at a 1:1 ratio, reacted with alkyne-biotin for enrichment, digested by trypsin, and analyzed by LC/LC-MS/MS. The ratio of each identified peptide was quantified by comparing areas under the light and heavy chromatographic peaks, and the ratio for each protein was calculated as the median of all of the relevant peptide ratios. In the reverse SILAC, light cells were treated with Ac4GlcNAc, and heavy cells were labeled with Ac36AzGlcNAc the scheme is shown in Fig. S2E. (B) Overlap of the identified O-GlcNAcylated proteins (enrichment ratios ≥ 2) between the forward and reverse SILAC experiments; 896 proteins were enriched in both experiments. (C) Overlap of the O-GlcNAcylated proteins assigned by SILAC and spectral counting. (D) MS1 chromatographic peaks of the representative peptides from AHNAK, cyclin G-associated kinase (GAK), and E3 ubiquitin-protein ligase UBR4, which are newly identified as O-GlcNAcylated proteins by SILAC-based chemoproteomics. The light and heavy chromatographic traces are colored in red and blue, respectively. Green solid lines delineate the chromatographic peak boundary for quantification. Black asterisks indicate the MS/MS event that supports the identification of the corresponding peptide. (E) Cellular localization analysis of 896 6AzGlcNAc-labeled proteins. Intracellular proteins include those with defined locations in the cytoplasm, nucleus, or mitochondria. Extracellular/luminal proteins include those with defined locations exclusively outside the plasma membrane or within the lumen. Dual proteins are those with both intracellular and extracellular/luminal domains. Unassigned proteins are those with no defined information on their subcellular locations. (F) Overlap of the O-GlcNAcylated proteins identified by Ac36AzGlcNAc and Y289L GalT labeling. The proteins identified by the two labeling methods were classified as high-confidence O-GlcNAcylated proteins.
Fig. 2. Turnover dynamics of O-GlcNAcylated proteins revealed by qTOP. (A) Schematic of the qTOP workflow. The light and heavy cells were pulse labeled with 200 μM Ac36AzGlcNAc for 36 h. In the forward qTOP experiment, the heavy cells are harvested immediately, and the light cells are chased with 200 μM Ac4GlcNAc for another 12 h. The cells were then mixed, lysed, and conjugated with alkyne-biotin for enrichment and quantitative proteomic analysis. The workflow of the reverse qTOP experiment, in which the heavy cells are chased instead, is shown in Fig. S3A. The SILAC ratio for each given protein (chased vs. reference) is defined as its qTOP ratio. (B) Histogram of the qTOP ratios for 533 high-confidence O-GlcNAcylated proteins quantified in three replicates of qTOP experiments (mean = 0.74 and SD = 0.17). Using means ± SD as cutoffs, these proteins were classified as hyperstable, dynamic, and hyperdynamic. (C) Gene ontology analysis of 75 hyperstable O-GlcNAcylated proteins regarding the biological processes in which they are involved. (D) qTOP ratios of FBL, NOP56, and NOP58. For each protein, the sequence of one representative peptide and its associated MS1 chromatographic peaks are shown in Upper, and its qTOP ratio quantified from three replicates of qTOP experiments (mean ± SD) is shown in Lower. N.S., not significant (one sample t test against 1.0, the value indicating no degradation).
component protein, exhibited no detectable protein degradation or removal of O-GlcNAc in the qTOP experiments (Fig. S3F). Consistent with our hypothesis that stable O-GlcNAc stabilizes proteins, NUP214 was indeed reported to be stabilized by OGlcNAcylation previously (10). When we looked into the stable OGlcNAcylated proteins that are associated with RNA regulation, we noticed that three of four core protein components of box C/D snoRNPs were among the list, which are FBL, NOP56, and NOP58 (Fig. 2D). Given the functional importance of these proteins in regulating snoRNP assembly and biogenesis, we continued to investigate the impact of O-GlcNAcylation on their protein stability as well as functional consequences. FBL, NOP56, and NOP58 all showed selective enrichment in SILAC-based identification of O-GlcNAcylated proteins in Ac36AzGlcNAc-labeled cells (Fig. 4A). To further validate that they are bona E6752 | www.pnas.org/cgi/doi/10.1073/pnas.1702688114
fide O-GlcNAcylated proteins, each of three proteins was recombinantly expressed with a C-terminal Flag-His tandem tag in HeLa cells treated with Ac4GalNAz and immunoprecipitated to measure its labeling by the chemical reporter. We chose Ac4GalNAz here as the chemical reporter, because it enters the GalNAc salvage pathway to form UDP-GalNAz, which is interconverted with UDP-N-azidoacetylglucosamine (UDP-GlcNAz), and provides a much better O-GlcNAc labeling efficiency (24, 29). Despite that Ac4GalNAz also labels cell surface glycans and therefore, complicates the global proteomic profiling, it would not interference with biochemical studies on individual immunoprecipitated O-GlcNAcylated proteins. The cell lysates were conjugated with alkyne-biotin and immunoprecipitated with an anti-His antibody. Western blotting with an antibiotin antibody showed that FBL, NOP56, and NOP58 were all modified with GlcNAz (Fig. 4B). Furthermore, Qin et al.
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FBL, NOP56, and NOP58 were all identified in the Y289L GalTbased proteomics, confirming their modifications with nature GlcNAc on the endogenous proteins (Fig. S5A). We adapted a previously developed method based on a resolvable mass tag to quantify the O-GlcNAcylation stoichiometry on the endogenous FBL, NOP56, and NOP58 (47). Cell lysates were treated with Y289L GalT and UDP-GalNAz to tag O-GlcNAc with azides, which were subsequently click labeled with an alkyne-functionalized PEG mass tag with the molecular weight of about 2,000 or 5,000 (alkyne-PEG2k or alkyne-PEG5k) and resolved by SDS/PAGE. Western blotting with antibodies against FBL, NOP56, or NOP58 exhibited a shift of molecular mass attributed to O-GlcNAc moieties chemoenzymatically labeled with the PEG mass tag (Fig. 4C). The O-GlcNAcylation stoichiometries were estimated by quantifying the relative band intensities of modified and unmodified proteins. Using the alkyne-PEG2K, the stoichiometry of FBL O-GlcNAcylation was estimated to be 40%. The alkyne-PEG5K resulted in a lower labeling of FBL O-GlcNAc, which was probably caused by higher steric hindrance of the larger Qin et al.
PEG chain (Fig. S5B). Because of the higher molecular weights, NOP56 and NOP58 are better separated in SDS/PAGE with the alkyne-PEG5K tag, giving the estimated O-GlcNAcylation stoichiometries as 49 and 45%, respectively (Fig. 4C). Because it is possible that the PEGylation reactions did not proceed to completion, the O-GlcNAcylation stoichiometries could be underestimated to some extent. Our O-GlcNAc proteomic profiling identified FBL, NOP56, and NOP58, leaving NHP2L1 as the only box C/D snoRNP protein component that is not O-GlcNAcylated. To confirm this result, we further examined the O-GlcNAcylation state of NHP2L1 by using the Ac 4GalNAz labeling and the PEG mass tag-based assays, both of which exhibited no detectable O-GlcNAc modification on NHP2L1 (Fig. S5C). We next attempted to identify the exact sites of O-GlcNAcylation on these proteins. Flag/His-tagged proteins were purified from HeLa cells and subjected to analysis by tandem MS with electron transfer dissociation (ETD) fragmentation to preserve the labile O-GlcNAc glycosidic linkage. The tandem ETD spectrum PNAS | Published online July 31, 2017 | E6753
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Fig. 3. Quantification of the changes of protein abundance on OGT inhibition by SILAC-based quantitative proteomics. (A) Workflow of quantitative proteomics for cells treated with an OGT inhibitor. Equal numbers of heavy and light cells are treated with 50 μM Ac45SGlcNAc and DMSO, respectively, for 48 h. After mixing and lysing the cells, the whole proteomes are digested by trypsin. The peptides were fractionated by high pH reversed-phase liquid chromatography (RPLC) and analyzed by SILAC-based quantitative proteomics. (B) Overlap of the proteins quantified in three replicate experiments. A total of 2,500 proteins were quantified in at least two of three replicates and selected for analysis of protein abundance change on OGT inhibition. (C) Overlap of 2,500 proteins quantified on OGT inhibition with the hyperstable, dynamic, and hyperdynamic O-GlcNAcylated proteins from the qTOP experiments. (D) Abundance changes of O-GlcNAcylated proteins on OGT inhibition. For each of the hyperstable, dynamic, and hyperdynamic populations of O-GlcNAcylated proteins quantified by qTOP, a boxplot was drawn showing the distribution of protein abundance change on OGT inhibition. The SILAC ratios (OGT inhibition vs. control) of each protein are shown as gray diamonds, with those of FBL, NOP56, and NOP58 highlighted in blue. Box limits represent 25th percentiles, medians, and 75th percentiles. Whiskers extend to nonoutliers. ***P < 0.001 (one-sided Wilcoxon rank sum test).
Fig. 4. Core proteins of box C/D snoRNP are O-GlcNAcylated. (A) MS1 chromatographic peaks of representative peptides of FBL, NOP56, and NOP58 from the SILAC-based identification of O-GlcNAcylated proteins showing the selective enrichment of these proteins through 6AzGlcNAc labeling. (B) Detection of O-GlcNAcylation on immunoprecipitated FBL, NOP56, and NOP58. HeLa cells transiently expressing Flag/His-tagged FBL, NOP56, or NOP58 were treated with 100 μM Ac4GalNAz or Ac4GlcNAc as a negative control for 36 h. The cells were lysed and reacted with 100 μM alkyne-biotin followed by immunoprecipitation (IP) using an anti-His antibody. With similar amounts of the immunoprecipitated proteins, the antibiotin blotting showed the specific incorporation of the GlcNAz chemical reporter in FBL, NOP56, and NOP58. (C) Detection of O-GlcNAcylation on endogenous FBL, NOP56, and NOP58 using chemoenzymatic labeling with a resolvable mass tag. The solid and dashed arrows indicate untagged proteins and proteins modified with the PEG mass tag, respectively. (D) Identification of Ser142 on FBL as the O-GlcNAcylation site by ETD. Prominent c/z fragment ions used to identify the O-GlcNAcylated peptides from FBL were shown in red, and the raw ETD MS/MS spectrum is shown in Fig. S5D. (E) Verification of O-GlcNAcylation of Ser142 on immunoprecipitated FBL by chemical reporter. Antibiotin Western blot on FBLWT-Flag/His and FBLS142A-Flag/His immunoprecipitated from cells treated with Ac4GalNAz or Ac4GlcNAc. Anti-His blot indicated the loading of the immunoprecipitated proteins. (F) Verification of O-GlcNAcylation of Ser142 on immunoprecipitated FBL by O-GlcNAc antibody. Western blot detection of O-GlcNAc by using the RL2 antibody on isolated FBLWT-Flag/His and FBLS142A-Flag/His. Anti-His blot indicated the loading of the immunoprecipitated proteins.
unambiguously identified serine-142 (S142) of FBL as a site of O-GlcNAcylation (Fig. 4D and Fig. S5D). To further verify the O-GlcNAcylation on S142 of FBL, an S142A mutant of FBL, FBLS142A-Flag/His, was overexpressed in HeLa cells treated with Ac4GalNAz. The lysates were reacted with alkyne-biotin and immunoprecipitated with the anti-His antibody, and Western blotting with the antibiotin antibody showed significantly reduced GlcNAz labeling on FBLS142A (Fig. 4E). Moreover, Western blotting with an O-GlcNAc–recognizing antibody RL2 detected a strong O-GlcNAylation signal on purified Flag/His-FBL in contrast to Flag/His-FBLS142A, confirming S142 as the major O-GlcNAc modification site of FBL (Fig. 4F). Our attempts to identify O-GlcNAc modification sites on NOP56 and NOP58 using ETD MS were not successful. O-GlcNAcylation Stabilizes Box C/D snoRNP Core Proteins. Next, we assessed whether O-GlcNAcylation regulates the expression levels of FBL, NOP56, and NOP58. The overall cellular level of O-GlcNAc was inhibited by the OGT inhibitor Ac45SGlcNAc. Consistent with our proteomic quantification (Fig. 3D and Fig. S4B), the abundance of all of these three proteins, as detected by Western blot analysis, was significantly diminished on deprivation of O-GlcNAc in a time-dependent manner (Fig. 5A and Fig. S6 A–C). Quantitative RT-PCR (qRT-PCR) analysis showed that the mRNA levels of FBL, NOP56, and NOP58 were not lowered on OGT inhibition (Fig. S6 E–G), excluding the possibility of transcriptional E6754 | www.pnas.org/cgi/doi/10.1073/pnas.1702688114
regulation. By knocking down OGT with siRNA to reduce O-GlcNAcylation (Fig. S7A), we also observed that the protein levels of FBL, NOP56, and NOP58 were greatly reduced with no apparent decrease in their mRNA levels (Fig. S7 B–D and F). To test whether the reduced protein level was owing to decreased protein stability on O-GlcNAc deprivation, the cells were treated with cycloheximide (CHX) to stop protein synthesis. The immunoblotting of FBL, NOP56, and NOP58 showed that protein degradation was significantly accelerated by inhibiting OGT either genetically (Fig. S7G) or pharmacologically (Fig. 5B and Fig. S8 A–C). Interestingly, the protein level of NHP2L1 was also diminished by OGT inhibition (Fig. 5A and Fig. S6D) and knockdown (Fig. S7F), whereas its mRNA level was not lowered (Figs. S6H and S7E). Correlatively, the degradation of NHP2L1 was accelerated by OGT knockdown (Fig. S7G) and inhibition (Fig. 5B and Fig. S8D). Because NHP2L1 was not O-GlcNAc modified, we suspected that the whole box C/D snoRNP complex might be destabilized when one of the essential components was disrupted. We therefore knocked down FBL by siRNA, which as expected, resulted in down-regulation of NHP2L1 expression as well as the expression of NOP56 and NOP58 (Fig. S9). Similar results were observed for siRNA knockdown of NOP56 or NOP58. Taken together, these results show that O-GlcNAc regulates the stability and integrity of box C/D snoRNP by stabilizing FBL, NOP56, and NOP58. Qin et al.
PNAS PLUS CHEMISTRY BIOCHEMISTRY
Fig. 5. O-GlcNAcylation stabilizes box C/D core proteins and regulates the assembly of box C/D snoRNPs and rRNA methylation. (A) Time-dependent loss of box C/D core proteins on OGT inhibition. HeLa cells were treated with 50 μM Ac45SGlcNAc for up to 72 h, and the protein level was monitored by immunoblotting. RL2 was used to monitor the cellular level of O-GlcNAc. Actin was used as the loading control. The relative protein level was shown under each band. Statistical analysis is shown in Fig. S6 A–D. (B) Inhibition of O-GlcNAcylation expedites degradation of box C/D core proteins. HeLa cells were treated with DMSO or 50 μM Ac45SGlcNAc for 48 h followed by treatment with 50 μM CHX for up to 8 h and monitoring of the protein level. Actin was used as the loading control. The relative protein level is shown under the bands. Statistical analysis is shown in Fig. S8. (C) O-GlcNAcylation of Ser142 is critical for maintaining the stability of FBL. HeLa cells expressing FBLWT-Flag/His and FBLS142A-Flag/His were treated with 50 μM CHX for 12 h. The protein level was monitored by immunoblotting with an anti-His antibody. Actin was used as the loading control. The relative protein level is shown under each band. Statistical analysis is shown in Fig. S10D. (D) Fluorescence imaging of HeLa cells expressing GFP-FBLWT or GFP-FBLS142A, which were costained with antiNOP58 followed by an Alexa Fluor 555-conjugated secondary antibody (nucleolus; red signal) and Hoechst 33342 (nuclei; blue signal). (Scale bar: 1 μm.) (E) Boxplots of the nucleolar proportions of GFP-FBLWT and GFP-FBLS142A. The nucleolar proportion was estimated as the ratio of nucleolar fluorescence intensity to nuclear fluorescence intensity of individual cells (n = 50). Box limits represent 25th percentiles, medians, and 75th percentiles. Whiskers extend to the maximum and minimum. **P < 0.01 (Student’s t test). (F) O-GlcNAcylation of Ser142 on FBL regulates the assembly of box C/D snoRNPs. Coimmunoprecipitation (Co-IP) of NOP56, NOP58, and NHP2L1 with FBLWT-Flag/His and FBLS142A-Flag/His. Anti-His blotting was used to adjust the loading of precipitated FBL. Values under each band indicate relative protein levels coimmunoprecipitated with FBL. In the bar graph, values represent means ± SD from three replicate experiments. ***P < 0.001 (Student’s t test). (G) OGT inhibition reduces the overall methylation level on rRNAs. The relative amounts of Am and Gm were quantified by LC-MS/MS. ***P < 0.001 (Student’s t test). (H) OGT inhibition reduces the relative methylation level at five sites of rRNA measured by qRTPCR. Values represent means ± SD from three replicate experiments. *P < 0.05 (Student’s t test); **P < 0.01 (Student’s t test); ***P < 0.001 (Student’s t test).
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proteins were coimmunoprecipitated, and their abundance was compared by Western blot analysis. Starting with the same amount of FBLS142A-Flag/His and FBLWT-Flag/His, much less NOP56, NOP58, and NHP2L1 were coimmunoprecipitated with the mutant FBL than with the WT, indicating that loss of O-GlcNAcylation at S142 of FBL disrupted its interactions with core proteins of box C/D snoRNPs and the assembly of the mature complex (Fig. 5F).
Fig. 6. O-GlcNAcylation of FBL contributes to cell proliferation and tumor growth. (A–C) Colony formation (A), cell proliferation (B), and cell growth with doxorubicin (C) of MCF7 cells expressing GFP, FBLWT, or FBLS142A. Values represent means ± SD from three replicate experiments. N.S., not significant (one-way ANOVA). ***P < 0.001. (D) Tumor formation in nude mice injected with MCF7 cells expressing FBLWT or FBLS142A. Shown in Upper are representative photographs of dissected tumors after 1 wk of growth in mice. Shown in Lower are masses of the dissected tumors. Values represent means ± SD from three replicate experiments. ***P < 0.001 (Student’s t test).
As exemplified by NHP2L1, it is possible that global deprivation of O-GlcNAc may indirectly affect the stability of certain proteins. To rule out such an indirect effect for FBL, we used its S142A mutant to specifically assay the functional role of O-GlcNAylation on its stability without interference by O-GlcNAc on other proteins. Computational modeling by Rosetta (48) indicated that the S142A mutation did not cause significant perturbation to the protein structural stability (Fig. S10A). Furthermore, FBLWT and FBLS142A were expressed in Escherichia coli, which does not have O-GlcNAc modification. The thermal shift assay on cell lysates showed that FBLS142A exhibited unaltered thermal stability compared with FBLWT, suggesting that S142 mutation itself does not cause protein instability (Fig. S10B). We then transfected HeLa cells with equal amounts of plasmids of FBLS142A-Flag/His and FBLWT-Flag/His, respectively. Comparable mRNA levels were confirmed by qRTPCR (Fig. S10C). As expected, Western blot analysis showed that the protein expression level of FBLS142A-Flag/His was significantly lower and that the degradation rate was much faster than that of the WT FBL-Flag/His (Fig. 5C and Fig. S10D). Furthermore, confocal fluorescence microscopy confirmed that the S142A mutation resulted in a decreased protein level of GFP-FBL in HeLa cells (Fig. S10 E and F). Collectively, these results show that the stability of FBL was directly regulated by O-GlcNAcylation at S142. O-GlcNAc Is Essential for Subcellular Location of FBL and Its Interactions with Other Core Proteins. The box C/D snoRNP proteins are syn-
thesized in the cytosol, imported into the nucleoplasm and Cajal bodies for assembly with box C/D RNAs, and then, transported to the nucleolus as the mature snoRNPs (49). To investigate the effects of O-GlcNAcylation on the subcellular location of FBL, HeLa cells transiently expressing GFP-FBL or GFF-FBLS142A were visualized by immunofluorescence microscopy. Hoechst staining and immunofluorescence staining of endogenous NOP58 were used to delineate the boundaries of nucleus and nucleolus, respectively (Fig. 5D). The distribution of FBL or FBLS142A in each subcellular region was quantified by GFP fluorescence intensity, which showed that both the overall expression level and the nucleolar/nuclear percentage of GFP-FBLS142A were significantly lower than those of GFP-FBL (Fig. 5E). These results indicate that O-GlcNAc is important in regulating the subnuclear localization of FBL. To assay how O-GlcNAcylation of FBL affects its interactions with other box C/D snoRNP core proteins, FBLS142A-Flag/His and FBLWT-Flag/His were expressed in HeLa cells and immunoprecipitated using the anti-Flag antibody. The FBL-interacting E6756 | www.pnas.org/cgi/doi/10.1073/pnas.1702688114
O-GlcNAc–Mediated Alteration of rRNA Methylation. Given that O-GlcNAcylation regulates the stability of C/D snoRNP core proteins, we assessed whether changes in the O-GlcNAc level may alter rRNA methylation, one of the key functions of the box C/D snoRNPs. The 5S, 18S, and 28S rRNAs from Ac45SGlcNAc- and DMSO-treated cells were isolated and digested into nucleosides. The relative amounts of methylated adenosine (Am) and methylated guanine (Gm) were quantified by LC-MS/MS (50). Our results showed that O-GlcNAcylation inhibition reduced methylation globally in rRNAs (Fig. 5G). In addition, we analyzed the methylation level of five known methylation sites located in the 5S, 18S, and 28S rRNAs using a site-specific semiquantitative qRT-PCR–based method (51, 52). Consistent with the global reduction of rRNA methylation, all of five sites were significantly less methylated in the Ac45SGlcNAc-treated cells, where the overall O-GlcNAc level was decreased and the expression of FBL was repressed (Fig. 5H). These results indicate that aberrant O-GlcNAcylation alters the rRNA methylation pattern, presumably through destabilizing core proteins and regulating the assembly and biogenesis of box C/D snoRNPs. O-GlcNAcylation of FBL at S142 Facilitates Tumorigenesis. FBL overexpression has been implicated in tumorigenesis, probably through modulation of ribosome activity by altering rRNA methylation (52–54). To investigate whether O-GlcNAcylation of FBL contributes to tumorigenesis, we stably expressed FBL or FBLS142A in MCF7 cancer cells, and in agreement with the previous results (52), overexpression of FBL in MCF7 cells significantly increased colony formation and cell proliferation (Fig. 6 A and B). Mutation of the O-GlcNAcylation site at S142 completely abolished these effects, suggesting that O-GlcNAc modification is essential for the FBLpromoted colony formation and cell proliferation. In addition, overexpression of FBL but not FBLS142A protected MCF7 cells against the drug doxorubicin (Fig. 6C). The impact of FBL O-GlcNAcylation on tumor growth was further investigated in vivo by using a xenograft mouse model. MCF7 cells stably expressing FBL or FBLS142A were injected into nude mice. Decreased tumor mass was observed in mice injected with FBLS142A-expressing cells compared with mice injected with FBLWT-expressing cells (Fig. 6D). Taken together, these in vitro and in vivo results show that the hyperstable O-GlcNAcylation of FBL at Ser142, through maintaining the protein stability, promotes cancer cell proliferation and tumor formation.
Discussion It is intriguing that over 1,000 proteins are O-GlcNAcylated by a single glycosyltransferase OGT. In this regard, oligosaccharyltransferase, an endoplasmic reticulum (ER) membrane protein complex that catalyzes the first step of protein N-linked glycosylation, is one of the few enzymes that have comparably broad protein substrates (55). Although N-glycosylation is generally regarded as an irreversible modification for membrane-associated and -secreted proteins, O-GlcNAcylation is thought to be dynamically regulated by OGA in concerted with OGT. The level of O-GlcNAcylation fluctuates in response to the nutrient condition and cellular stress (5, 56–58). However, whether OGA can remove O-GlcNAc from all modified proteins and whether the turnover dynamics is differentially regulated among O-GlcNAcylated proteins remain largely uninvestigated. Qin et al.
3. Yang X, Zhang F, Kudlow JE (2002) Recruitment of O-GlcNAc transferase to promoters by corepressor mSin3A: Coupling protein O-GlcNAcylation to transcriptional re-
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pression. Cell 110:69–80. 4. Wells L, Vosseller K, Hart GW (2001) Glycosylation of nucleocytoplasmic proteins: Signal transduction and O-GlcNAc. Science 291:2376–2378.
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ACKNOWLEDGMENTS. We thank Dr. Guifang Jia and Ye Wang for help with LC-MS/MS quantification of rRNA methylation, Dr. Yuan Liu for help with Rosetta simulation, the MS facility of the National Center for Protein Sciences at Peking University for assistance with ETD MS, and the computing platform of the Center for Life Science for supporting the LC-MS/MS data analysis. This work is supported by National Natural Science Foundation of China Grants 21472008 (to C.W.), 81490740 (to C.W.), 21425204 (to X.C.), 21521003 (to X.C.), and 21672013 (to X.C.); National Key Research and Development Projects Grant 2016YFA0501500 (to C.W. and X.C.); and a “1000 Talents Plan” Young Investigator Award (to C.W.).
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1. Hart GW, Slawson C, Ramirez-Correa G, Lagerlof O (2011) Cross talk between O-GlcNAcylation and phosphorylation: Roles in signaling, transcription, and chronic disease. Annu Rev Biochem
Materials and Methods Details are in SI Materials and Methods, which includes detailed methods for quantitative chemoproteomic profiling, fluorescence imaging, rRNA methylation quantification, quantitative real-time PCR, in-gel fluorescence, cell proliferation assay, and in vivo tumor xenografts. All animal experiments were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee of Peking University accredited by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.
CHEMISTRY
O-GlcNAc dynamics and the effects on protein stability quantified by qTOP are validated by SILAC-based proteomic quantification of the protein abundance on OGT inhibition. In addition, functional studies on individual proteins were cross-validated by using methods detecting natural O-GlcNAc, such as chemoenzymatic labeling with Y289L GalT and an anti–O-GlcNAc antibody, in addition to the chemical reporter-based labeling. Other than the cytoplasmic and nuclear O-GlcNAc modification, an EGF-specific O-GlcNAcylation was recently discovered to occur in the ER for several secreted proteins containing EGF domains (69, 70). The modification is catalyzed by an ERlocalized EGF-specific O-GlcNAc transferase (EOGT). In this work, none of these EOGT-modified proteins were quantified in qTOP. Interestingly, a recent study detected and identified a GlcNAc modification on cysteine of a few proteins, suggesting the possible occurrence of S-GlcNAcylation (71). Because it was shown that S-GlcNAc was not cleaved by OGA, the fact that the Ac36AzGlcNAc labeling is sensitive to OGA suggests that this particular chemical reporter mainly labels O-GlcNAc. Furthermore, we performed quantitative proteomics with the Y289L GalT labeling to ensure that those proteins analyzed with qTOP are indeed O-GlcNAcylated. In conclusion, we present a chemoproteomic strategy for selective enrichment and quantitative analysis of O-GlcNAcylated proteins as well as their turnover profiles in living cells. The large number of O-GlcNAcylated proteins categorized with distinct turnover profiles provides a rich and valuable resource for better understanding the O-GlcNAc biology. However, it should be noted that the qTOP methodology currently does not differentiate the removal of O-GlcNAc modification from the degradation of modified proteins, and we, therefore, chose to focus on the hyperstable population where both the protein and the modification appear to be highly stable with slow turnover kinetics. For the dynamic and hyperdynamic populations, it is possible that O-GlcNAc turns over more rapidly than protein or vice versa. To further distinguish between those situations, we envision expansion of qTOP with multiplexed quantitative proteomics. Equally important is to develop effective methods for site-specific profiling of O-GlcNAcylation that can be integrated into qTOP, and this integration would ultimately enable detailed analysis for proteins bearing multiple O-GlcNAc modifications with different turnover kinetics.
BIOCHEMISTRY
In this study, we developed the qTOP platform that enables quantitative analysis of the turnover rates of O-GlcNAcylated proteins. Our analysis revealed a population of hyperstable O-GlcNAcylated proteins, with O-GlcNAcylation that is minimally perturbed within a 12-h timeframe. Moreover, we found that the abundance of those proteins carrying hyperstable O-GlcNAc is more susceptible to OGT inhibition than those in the dynamic and hyperdynamic populations. We, therefore, propose that the stable O-GlcNAc with slow turnover kinetics has important functional implications in regulating the stability of modified proteins. Among the hyperstable O-GlcNAcylated proteins identified in this study are FBL, NOP56, and NOP58, three of the box C/D snoRNP core proteins. The assembly pathway of box C/D snoRNPs has been extensively studied, and various assembly factors, such as the HSP90/R2TP chaperone/cochaperone system, have been characterized (49). However, posttranslational modifications (PTMs) of the core proteins and their functional roles in snoRNP biogenesis are rarely studied. Recent proteomic profiling identified NOP58 as a substrate of small ubiquitin-related modifier (SUMO), which promotes its high-affinity binding with snoRNAs (59, 60). Moreover, SUMO modification of NOP58 seems to be regulated by flanking serine phosphorylation (60). Our study reveals O-GlcNAcylation as another PTM that regulates snoRNP assembly and biogenesis through regulating the stability, subcellular location, and interactions of the core proteins. The extensive interplays between those PTMs are very possible and remain to be investigated. Alteration of O-GlcNAcylation impacts the functions of box C/D snoRNPs. For example, we show that inhibition of O-GlcNAcylation impairs the rRNA methylation level and pattern. Altered rRNA methylation was reported to affect ribosome production and translation (52, 61). Correlatively, ribosome proteins were also found to be modified with O-GlcNAc (62, 63). Together, O-GlcNAc has proven an important PTM for regulating ribosome biogenesis and functions. Elevated O-GlcNAcylation has been found in various cancer types and emerged as a general feature of cancer (64). Many of the O-GlcNAcylated proteins in cancer cells are associated with tumorigenesis. One mechanism through which O-GlcNAc contributes to tumorigenesis is to regulate activities of those proteins. For example, O-GlcNAcylation of phosphofructokinase 1 was found to block its kinase activity and increase glucose flux through the pentose phosphate pathway (PPP), which promoted cancer cell proliferation and tumor formation (12). Conversely, O-GlcNAc modification could activate the rate-limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase, thus increasing glucose flux through the PPP (14). Alternatively, O-GlcNAcylation may stabilize proteins promoting tumorigenesis. The examples include c-MYC (65), FoxM1 (66), and Snail1 (67). In this work, we show that O-GlcNAcylation on box C/D snoRNP core proteins, particularly FBL, enhances the stability of box C/D snoRNPs, which impacts rRNA methylation and ribosome biogenesis. The dysregulation of ribosome biogenesis in cancer cells plays key roles in oncogenesis (68). Therefore, our work provides another link between O-GlcNAc and tumorigenesis. Metabolic labeling with chemical reporters, such as 6AzGlcNAc, enables the pulse-chase experiments for dynamic profiling of O-GlcNAc, which is difficult to achieve by using other labeling methods. Notably, 6AzGlcNAc incorporation results in an unnatural alteration on O-GlcNAc. Although we show that the resulting O-6AzGlcNAc can be removed by OGA, it might be possible that it is removed at a different rate. Nevertheless, the
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