An OGA-Resistant Probe Allows Specific Visualization and Accurate

Subscriber access provided by SUNY DOWNSTATE

Letter

An OGA-resistant Probe Allow Specific Visualization and Accurate Identification of O-GlcNAc-modified Proteins in Cells Jing Li, Jiajia Wang, Liuqing Wen, He Zhu, Shanshan Li, Kenneth Huang, Kuan Jiang, Xu Li, Cheng Ma, Jingyao Qu, Aishwarya Parameswaran, Jing Song, Wei Zhao, and Peng George Wang ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00678 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

An OGA-resistant Probe Allow Specific Visualization and Accurate Identification of O-GlcNAc-modified Proteins in Cells Jing Li,*, †, ‡, # Jiajia Wang, †, §, # Liuqing Wen,‡ He Zhu,‡ Shanshan Li,‡ Kenneth Huang,‡ Kuan Jiang,† Xu Li,‡ Cheng Ma,‡ Jingyao Qu,‡ Aishwarya Parameswaran,‡ Jing Song,‡ Wei Zhao,† Peng George Wang*, †, ‡ †State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic of China ‡Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, 50 Decatur St SE, Atlanta, GA 30303, United States §School of Food and Drug, Luoyang Normal University, Jiqing Road, Luoyang, Henan, 471934B

Supporting Information ABSTRACT: O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modifications. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2azidoacetamido-2,4-dideoxy-D-glucopyranose (Ac34dGlcNAz), that can be processed by the GalNAc salvage pathway, and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of OGlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac34dGlcNAz allowed the selective visualization of O-GlcNAc in cells, and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

O-linked β-N-acetyl-glucosamine glycosylation (OGlcNAcylation) is a post-translational, monosaccharide modification on proteins in multicellular eukaryotes. This modification is involved in nutrient sensing, gene expression, protein degradation and other essential cell processes. Contrast to its indispensable role, only one pair of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) maintain the appropriate level of celluar OGlcNAcylation.1, 2 Altered O-GlcNAcylation has been linked to multiple human diseases, including diabetes, Alzheimer’s disease and cancer.3-5 However, the regulatory nature of the modification (i.e., substoichiometric, labile, and low cellular abundance) makes detection and identification of O-GlcNAc-modified proteins challenging.5-8

endogenous OGA, which attenuates the labeling efficiency. For these reasons, considerable efforts have been dedicated to developing effective and selective probes to monitor and manipulate OGlcNAcylation in vitro and in vivo.6 Herein, we report a selective and potent metabolic label for O-GlcNAcylation.

Small molecular probes, via metabolic labeling, are becoming crucial research tools in visualizing and identifying O-GlcNAcmodified proteins.6, 9 Currently, four common metabolic labels (Figure 1, 1–4) have been used for studying O-GlcNAc.10-13 Of these, probe 1 (Ac4GlcNAz), 2 (Ac4GalNAz) and 3 (Ac4GlcNAlk) are prone to be incorporated into multiple glycoconjugates such as N-/O-glycans, proteoglycans and glycolipids,6, 9, 13 raising concerns of labeling specificity. 4 (Ac36AzGlcNAc), has been proposed as a selective probe for O-GlcNAcylation, but cannot be processed by the canonical GlcNAc salvage pathway, resulting in low labeling efficiency.12 Additionally, O-GlcNAcylation is a dynamic process –metabolic labels on proteins can be cleaved by

Our previous work on screening sugar-nucleotide substrates of OGT 14 has shown that UDP-GlcNAc derivatives with modifications at C4 or C2, such as UDP-4deoxyGlcNAc (UDP-4dGlcNAc) and UDP-GlcNAz, were well tolerated by OGT. Given the majority of glycosidic linkages to GlcNAc or GalNAc in glycoconjugates (N-/O-glycans, proteoglycans and glycolipids) are to the C4-OH group of the sugar,15 a 4-deoxyacetylglucosamine (4dGlcNAc) derivative would lead to chain termination and decrease the incorporation in these glycoconjugates. Additionally, it has been reported that 4dGlcNAc was not accepted by polypeptide GalNAc transferase T1 to produce O-glycan proteins,16 and it was not incorporated into heparin sulfate.17 Therefore, usage of a 4dGlcNAc derivative in metabolic labeling is likely to enhance

Figure 1. Metabolic labels for O-GlcNAcylation.

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the specificity and fidelity for protein O-GlcNAcylation (Scheme 1). Furthermore, using the peptide (FITC-YAVVPVSK) as a substrate, UDP-GlcNAc and UDP-4dGlcNAz as donors, we prepared glycopeptides and examined the hydrolysis profile of OGA. The products were analyzed by HPLC and indicated that O4dGlcNAc-modified peptide was a 43-fold worse substrate (2% hydrolysis) than O-GlcNAc-modified peptide (87% hydrolysis) (Figure S1). Previous computational analysis of human OGA structure with O-(2-acetamido-2-deoxy-d-glucopyranosy-lidene amino N-phenylcarba-mate) (PUGNAc) also revealed that a hydrogen bond between C4-OH of PUGNAc and D285 of OGA might be the key factor in the formation of the 4E envelope conformation of the pyranose ring in reaction.18 Additionally, we constructed and expressed human OGA mutant D285A and tested its enzymatic characteristics (Figure S2). Michaelis-Menten kinetics indicated that D285A mutant (kcat/Km=5 s-1 M-1) had only 0.7% catalytic efficiency of WT (kcat/Km =606 s-1 M-1), consistent with the role of C4-OH of O-GlcNAc in polarization (Table S1). From these rationales, we designed and synthesized compound 5, 1,3,6tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-D-glucopyra -nose (Ac34dGlcNAz) as a potent and selective label for protein OGlcNAcylation (Scheme 1).

Page 2 of 7

mine the kinetics of protein labeling of 5, PC3 cells were incubated with 5 at 100 μM concentration for different lengths of time. Modified proteins can be clearly visualized against the background in 2 to 4 h and culminating in clarity in 18h (Figure S5). Hence, 100 μM concentration and 18h incubation was the optimized condition for the following cell experiments. To evaluate the potency of 5 for labeling, probe 1, a popular probe for labelling and isolating intra-celluar O-GlcNAc-modified proteins,20-22 and probe 2 were chosen as the positive controls. PC3 cells were metabolically labeled with 1, 2, 5 or DMSO at 100 μM for 18h, and then a copper-free click reaction was performed with biotin-dibenzocyclooctyne-alkyne (DIBO-biotin). After that, cells were fixed, permeabilized, and stained with Alexa Fluor 488 conjugated streptavidin. No fluorescence was observed in DMSO treated cells, and weak signals were seen in 1 and 2 treated. Due to the metabolic ‘bottleneck’ created by the rate-limiting AGX1, the incorporation of 1 was lowest among 3 treatments.6, 10 In contrast, strong fluorescence signaling was observed in cells treated with 5, indicating the high potency of 5 in metabolic labeling (Figure 2A). Similarly, cellular protein labeling demonstrated that 5 had a stronger signal than 1 and 2, confirming 5 was a more robust metabolic probe in cells (Figure 2B). To verify that 5 can be used to track O-GlcNAc-modified proteins, nucleoporin 62 (NUP62), a well-known O-GlcNAc protein, was immunoprecipitated and then detected through the incorporation of 5 in PC3 cells. Western blot, developed with the antiNUP62 mAb, showed that NUP62 was present in comparable amounts from cells cultured in the presence or absence of 5, 1 and 2. The immunoprecipitated samples were then either treated with OGA or buffer at 37 ℃ overnight. Samples were performed click reaction and analyzed by western blot with streptavidin-HRP. As shown in Figure 2 C, only cells incubated with 5, 1 and 2 produced azide-bearing NUP62. Interestingly, OGA treatment did not abrogate the signal arising from treatment with 5, whereas the signal from treatment with 1 and 2 decreased significantly (Figure 2C). In accordance with our prior detection on the OGA hydrolysis of 4dGlcNAc-modified peptide, 5 prove to be a novel OGAresistant probe for O-GlcNAcylation. To exclude the possibility that the observed effects were restricted to PC3 cells, nine other cell lines including HEK293, human erythromyeloblastoid leukemia cells (K562), Hela, mouse leukaemic monocyte macrophage cells (Raw), chinese hamster ovary cells (CHO-K1), human hepatocellular liver carcinoma cells (HepG2), mouse embryonic fibroblast cells (NIH3T3), human non-small cell lung carcinoma cells (H1299), human breast adenocarcinoma cells (MCF7), human lung adenocarcinoma epithelial cells (A549) were also treated with 5 at 100 μM for 18 h. All cell lines exhibited similar labeling efficiency with 5 as PC3. Altogether, the data showed that 5 was a potent and general metabolic probe for O-GlcNAcylation (Figure 2D).

Scheme 1. Rationale for Ac34dGlcNAz probe.

To assess the feasibility of the proposal we first synthesized 4dGlcNAz (Scheme S1) and evaluated whether it could be transformed into UDP-4dGlcNAz by human GalNAc salvage pathway.19 Two enzymes of this pathway: GalNAc kinase 2 (GK2) and UDP-GalNAc pyrophosphorylase (AGX1) were expressed and purified (Figure S3B). A one-pot reaction containing 4dGlcNAz, GK2, AGX1, ATP and UTP was applied to prepare UDP-4dGlcNAz. After Bio-Gel P-2 column purification, UDP4dGlcNAz was detected by TLC (Figure S3C) and confirmed by LC-MS analysis (Figure S3D), which was in accordance with previous reports regarding the substrate specificities of enzymes in the mammalian GalNAc salvage pathway.16 Next, we examined the toxicity of 5 and determined whether 5 could be incorporated into cellular proteins. Human, embryonic kidney 293 cells (HEK293), prostate cancer 3 cells (PC3) and human cervical cancer cells (Hela) were incubated with 5 at various concentrations. After 48 h, the viability of cells were measured. No cytotoxicity was observed up to concentrations at 100 μM (Table S2). Simultaneously, PC3 cellular proteins were extracted and clicked with diazo-biotin-alkyne under coppercatalyzed azide-alkyne cycloaddition (CuAAC) conditions. After normalization by bicinchoninic acid (BCA), equal amounts of proteins were subjected to western blot and developed with streptavidin-HRP for biotin detection. The labeling of a wide-range of proteins in concentrations as low as 25 μM and maximal labeling achieved at approximately 100 μM (Figure S4), which is low to the effective concentration of compound 4 (200 μM).12 To deter-

2


Page 3 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. The potency of 5 on O-GlcNAcylation. (A) PC3 cells were labeled with 5, 1, 2 or DMSO (D), and then incubated with DIBO-biotin followed by stained with streptavidin Alexa Fluor 488 for biotin imaging. (B) Western blot detection of cell lysates after incubated with D, 5, 1 and 2 and reacted with click reaction. The blot was developed with streptavidin-HRP for biotin detection. (C) Western blot detection of the incorporation and cleavage of 1, 5, and 2 on NUP62. Immunoprecipitated NUP62 from PC3 cells cultured in the presence or absence of probes were treated or untreated by OGA, then samples were subsequently reacted with click reaction and performed western blot with anti-NUP62 and streptavidinHRP. (D) Indicated cell lines were treated with 100 μM 5 for 18 h and then subjected to CuAAC with diazo-biotin-alkyne and analyzed by western blot.

Figure 3. The selectivity of 5 on protein O-GlcNAcylation. (A) Hela cells were labeled with 5, 1, 2 or DMSO (D) and incubated with DIBO-biotin and streptavidin Alexa Fluor 488 (green), followed by staining with Alexa Fluor 594 conjugated WGA (red) for plasma membrane and Hoechst 33342 (blue) for nucleus. The overlay (orange) indicates the colocalization of plasma membrane and the metabolic probe labeling. (B) Western blot detection of membrane, nucleic and cytoplasmic fractions of cells incubated with D, 5, 1 and 2 and reacted with click reaction. The blots were developed with streptavidin-HRP for biotin detection. Same samples were detected with anti-EGFR to show the cell membrane fraction, anti-HSP 90 to show the cell cytoplasm fraction, and anti-H3 to show the cell nucleus fraction. (C) SDS-PAGE and western blot detection of the incorporation of probes on GlyCAM-IgG. GlyCAM-IgG immunoprecipitated from NIH3T3 cells cultured in the presence of D, 5, 1 and 2 were reacted with DIBO-biotin, and then performed SDS-PAGE or western blot with streptavidin-HRP.

Unlike intracellular O-GlcNAc modification, most glycoconjugates (N-/O-glycans, proteoglycans and glycolipids) are synthesized via secretory pathways and present on the cell surface. To test the selectivity of 5 towards O-GlcNAcylation, multiple fluorescence imaging was used to determine the distribution of a metabolic probe in cells. Hela cells were cultured with 5, 1, 2 or DMSO at 100 μM for 18 h. The cells were then subjected to DIBO-biotin and incubated with streptavidin Alexa Fluor 488 (green) for localization of probes, Alexa Fluor 594 conjugated WGA (red) for plasma membrane and Hoechst 33342 (blue) for the nucleus. The overlaid images demonstrated that little or no colocalization of 5 labeling with the plasma membrane, indicating 5 was mainly metabolized into intracellular compartments. In contrast, colocalization was apparent in 1 and 2 treatment, especially 2, suggesting significant amounts of 2 was incorporated in membrane-bound glycoconjugates, consistent with previous reports (Figure 3A).12 For protein detection, cellular fractions from membrane, nucleus and cytoplasm were extracted respectively (cell fraction are confirmed by western blot with anti-EGFR indicating cell membrane, anti-HSP 90 indicating cell cytoplasm, and anti-H3 indicating cell nucleus), treated by CuAAC and followed by western blot analysis. As expected, metabolic labeling by 5 mainly existed in nucleic and cytoplasmic extractions and less in the membrane, whereas 1, and especially 2, were primarily in the membrane extraction (Figure 3B). Since O-GlcNAcylation is predominantly a nucleic and cytoplasmic modification, these results revealed that 5 was a more selective probe for OGlcNAcylation.

To determine whether 5 could label N-linked or O-linked glycans, we used the chimeric glycoprotein, GlyCAM-IgG, which contains both N- and mucin-type O-glycans.12 Mouse embryonic fibroblast cells (NIH3T3) that stably expressed GlyCAM-IgG were treated with 1, 2, 5 or DMSO. Then, GlyCAM-IgG was isolated using protein A-Sepharose, reacted with CuAAC, and followed by SDS-PAGE and western blot analysis. As shown in Figure 3C, with equal amounts of immunoprecipitated GlyCAMIgG, no signal was seen in samples treated with 5 and DMSO, whereas strong signals appeared in 1 or 2 treatments. These data indicated that 5 was not incorporated into N-linked or mucin-type O-linked glycans and represented a more selective probe for OGlcNAcylation. Finally, we examined the capacity of this probe for proteomewide analysis of O-GlcNAc-modified proteins from cell lysates. HEK293 cells were treated in triplicate with 5, 1, 2 or Ac4GlcNAc (negative control) respectively. Whole proteins were extracted and subjected to CuAAC.23 Equivalent amounts of the labeled proteins were reduced, alkylated, and subjected to biotinenrichment using streptavidin-conjugated beads. Proteins enriched on beads were digested and analyzed by LC-MS/MS. Identified proteins were quantified by spectral counting and threshold criteria screening.12 By these criteria, probe 5 identified 507 putative

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O-GlcNAc-modified proteins (Table S3), including 281 previously known and 226 novel, whereas probe 1 identified 369 proteins (Table S4) with 153 known and 216 unknown, probe 2 identified 377 proteins (Table S5) with 219 known and 158 new, confirming that 5 has a higher potential for identifying O-GlcNAc-modified proteins (Figure 4A). We next annotated cellular localization of all proteins identified (Figure 4B). Treatment with 5 enriched 483 (95%) of intracellular proteins (i.e., nucleic, cytoplasmic and mitochondrial), whereas 1 and 2 identified 311 (84%) and 316 (83.8%) respectively, underscoring the validity of 5 for O-GlcNAcylation. The non-selectivity of 1 and 2 was further evidenced by identification 41 (11%) and 52 (13.7%) proteins localized exclusively at extracellular regions or the lumen of the secretory pathway and lysosome. Interestingly, probe 1 has long been presumed the ubiquitously incorporation in intracellular proteins but also displayed a considerate distribution in membrane proteins, indicating the unspecific labelling for OGlcNAc-modified proteins. In addition, within the existing lists of O-GlcNAcylated proteins, nucleoporins (NUPs) are highly represented. To date, 19 NUPs have been identified as O-GlcNAc modified proteins including NUP62, NUP153, NUP214/CAN, NUP358, POM121, NUP98, NUP155, p58, p54, p45, NUP93, NUP210, NUP205, NUP160, NUP107, NUP188, NUP88, NUP85 and NUP35.24 Notably, from our proteomics analysis with 5, 11 NUPs were identified, while probe 1 identified and 2 identified 5 and 6 NUPs respectively, and the selective probe, Ac36AzGlcNAc (4), identified only 1 NUP12 (Table S6). These results highlight the improved accuracy and selectivity of probe 5 in the identification of O-GlcNAc-modified proteins.

Page 4 of 7

This material is available free of charge via the Internet at http://pubs.acs.org. Details of synthetic procedures, cell culture conditions, cyto-toxicity assay and LC-MS/MS analysis (PDF)

■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

Author Contributions #

These authors contributed equally.

Funding This work was supported by grants from the National Institutes of Health NIGMS (U01GM116263), National Natural Science Foundation of China (NO. 31000371, NO. 21372130) and Natural Science Foundation of Tianjin City (15JCYBJC29000).

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS We thank S. Walker from Harvard Medical School for providing the vector pET24b-ncOGT and G.W. Hart from Medicine School of Johns Hopkins University for providing the vector pET28aOGA and M.R. Pratt from University of Southern California for providing NIH3T3 cells with GlyCAM-IgG transfection.

REFERENCES Figure 4. Identification of O-GlcNAc-modified proteins using 5. (A) Overlap between proteins identified using 5, 1 and 2. (B) Cellular localization of identified proteins by 1, 2 and 5. Graphical representation of enriched proteins based on whether their localization is exclusively intracellular (i.e., cytoplasmic, nucleic, or mitochondrial), exclusively extracellular or lumenal (i.e., ER, Golgi, lysosome), or have domains in both (e.g., transmembrane protein).

(1) Hanover, J.A. (2010) Epigenetics gets sweeter: O-GlcNAc joins the "histone code". Chem. Biol. 17, 1272-1274. (2) Wells, L., Vosseller, K. and Hart, G.W. (2001) Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 291, 2376-2378. (3) Banerjee, P.S., Ma, J. & Hart, G.W. Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria. Proc. Natl. Acad. Sci. U. S. A. 112, 6050-6055 (2015). (4) Slawson, C. & Hart, G.W. (2011) O-GlcNAc signalling: implications for cancer cell biology. Nat. Rev. Cancer 11, 678684. (5) Yuzwa, S.A. (2012) Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat. Chem. Biol. 8, 393-399. (6) Cecioni, S. and Vocadlo, D.J. (2013) Tools for probing and perturbing O-GlcNAc in cells and in vivo. Curr. Opin. Chem. Biol. 17, 719-728. (7) Gross, B.J., Kraybill, B.C. and Walker, S. (2005) Discovery of O-GlcNAc transferase inhibitors. J. Am. Chem. Soc. 127, 1458814589. (8) Khidekel, N. (2003) A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J. Am. Chem. Soc. 125, 16162-16163.

In summary, we reported herein the development of Ac34dGlcNAz as a novel, potent and selective tool for detecting and identifying O-GlcNAc-modified proteins. The lack of hydroxyl at C4 allowed this probe to be less incorporated into cell surface glycoconjugates, offering a higher selectivity over previous probes for O-GlcNAcylation. Additionaly, O-4dGlcNAz modification was resistant to the hydrolysis of OGA, providing a more potency for labeling. Combined with click chemistry, this probe can be used in a variety of applications, including direct live cell imaging, affinity enrichment, and proteomics, all which will accelerate the discovery of new O-GlcNAc modified proteins and provide insights into the function of O-GlcNAcylation.

■ ASSOCIATED CONTENT Supporting Information

4


Page 5 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) Yu, S.H. et al. (2012) Metabolic labeling enables selective photocrosslinking O-GlcNAc-modified proteins to their binding partners. Proc. Natl. Acad. Sci. U. S. A. 109, 4834-8439. (10) Boyce, M. (2011) Metabolic cross-talk allows labeling of Olinked beta-N-acetylglucosamine-modified proteins via the Nacetylgalactosamine salvage pathway. Proc. Natl. Acad. Sci. U. S. A. 108, 3141-3146. (11) Vocadlo, D.J., Hang, H.C., Kim, E.J., Hanover, J.A. and Bertozzi, C.R. A chemical approach for identifying O-GlcNAcmodified proteins in cells. Proc. Natl. Acad. Sci. U. S. A. 100, 9116-9121 (2003). (12) Chuh, K.N., Zaro, B.W., Piller, F., Piller, V. and Pratt, M.R. (2014) Changes in metabolic chemical reporter structure yield a selective probe of O-GlcNAc modification. J. Am. Chem. Soc. 136, 12283-12295. (13) Zaro, B.W., Yang, Y.Y., Hang, H.C. and Pratt, M.R. (2011) Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc. Natl. Acad. Sci. U. S. A. 108, 8146-8151. (14) Ma, X. (2013) Substrate specificity provides insights into the sugar donor recognition mechanism of O-GlcNAc transferase (OGT). PLoS One 8, e63452. (15) Stanley, P. and Cummings, R.D. (2009) in Essentials of Glycobiology (eds. Varki, A. et al.) (Cold Spring Harbor (NY),. (16) Pouilly, S., Bourgeaux, V., Piller, F. and Piller, V. (2012) Evaluation of analogues of GalNAc as substrates for enzymes of

the mammalian GalNAc salvage pathway. ACS Chem. Biol. 7, 753-760. (17) van Wijk, X.M. (2013) Interfering with UDP-GlcNAc metabolism and heparan sulfate expression using a sugar analogue reduces angiogenesis. ACS Chem. Biol. 8, 2331-2338. (18) de Alencar, N.A. (2012) Computational analysis of human OGA structure in complex with PUGNAc and NAG-thiazoline derivatives. J. Chem. Inf. Model. 52, 2775-2783. (19) Pouilly, S., Piller, V. and Piller, F. (2012) Metabolic glycoengineering through the mammalian GalNAc salvage pathway. FEBS J. 279, 586-598. (20) Luchansky, S.J. (2003) Constructing azide-labeled cell surfaces using polysaccharide biosynthetic pathways. Methods in enzymology 362, 249-272. (21) Nandi, A. (2006) Global identification of O-GlcNAcmodified proteins. Anal. Chem. 78, 452-458. (22) Sprung, R. (2005) Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J. Proteome Re.s 4, 950-957. (23) Li, S., Zhu, H., Wang J. (2016) Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strainpromoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics. Electrophoresis 1, 1-6 (24) Li, B. and Kohler, J.J. (2014) Glycosylation of the nuclear pore. Traffic 15, 347-361.

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure legends

Figure 1. Metabolic labels for O-GlcNAcylation.

Scheme 1. Rationale for Ac34dGlcNAz probe.

Figure 2. The potency of 5 on O-GlcNAcylation. (A) PC3 cells were labeled with 5, 1, 2 or DMSO (D), and then incubated with DIBO-biotin followed by stained with streptavidin Alexa Fluor 488 for biotin imaging. (B) Western blot detection of cell lysates after incubated with D, 5, 1 and 2 and reacted with click reaction. The blot was developed with streptavidin-HRP for biotin detection. (C) Western blot detection of the incorporation and cleavage of 1, 5, and 2 on NUP62. Immunoprecipitated NUP62 from PC3 cells cultured in the presence or absence of probes were treated or untreated by OGA, then samples were subsequently reacted with click


Page 6 of 7

Page 7 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reaction and performed western blot with anti-NUP62 and streptavidin-HRP. (D) Indicated cell lines were treated with 100 μM 5 for 18 h and then subjected to CuAAC with diazo-biotin-alkyne and analyzed by western blot.

Figure 3. The selectivity of 5 on protein O-GlcNAcylation. (A) Hela cells were labeled with 5, 1, 2 or DMSO (D) and incubated with DIBO-biotin and streptavidin Alexa Fluor 488 (green), followed by staining with Alexa Fluor 594 conjugated WGA (red) for plasma membrane and Hoechst 33342 (blue) for nucleus. The overlay (orange) indicates the colocalization of plasma membrane and the metabolic probe labeling. (B) Western blot detection of membrane, nucleic and cytoplasmic fractions of cells incubated with D, 5, 1 and 2 and reacted with click reaction. The blots were developed with streptavidin-HRP for biotin detection. Same samples were detected with anti-EGFR to show the cell membrane fraction, anti-HSP 90 to show the cell cytoplasm fraction, and anti-H3 to show the cell nucleus fraction. (C) SDS-PAGE and western blot detection of the incorporation of probes on GlyCAM-IgG. GlyCAM-IgG immunoprecipitated from NIH3T3 cells cultured in the presence of D, 5, 1 and 2 were reacted with DIBO-biotin, and then performed SDS-PAGE or western blot with streptavidin-HRP.

Figure 4. Identification of O-GlcNAc-modified proteins using 5. (A) Overlap between proteins identified using 5, 1 and 2. (B) Cellular localization of identified proteins by 1, 2 and 5. Graphical representation of enriched proteins based on whether their localization is exclusively intracellular (i.e., cytoplasmic, nucleic, or mitochondrial), exclusively extracellular or lumenal (i.e., ER, Golgi, lysosome), or have domains in both (e.g., transmembrane protein).


An OGA-Resistant Probe Allows Specific Visualization and Accurate

Recommend Documents