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Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome Upon Oxidative Stress Albert Lee, Devin Miller, Roger Henry, Venkata Durga Prasad Paruchuri, Robert N. O'Meally, Tatiana Boronina, Robert N. Cole, and Natasha E. Zachara J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00369 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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Journal of Proteome Research

Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome Upon Oxidative Stress Albert Lee1, Devin Miller1‡, Roger Henry1‡, Venkata D. P. Paruchuri1, Robert N. O’Meally2, Tatiana Boronina2, Robert N. Cole1,2, and Natasha E. Zachara1*. ‡ These authors contributed equally 1. Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St, Baltimore, MD 21205-2185, USA. 2. Mass Spectrometry and Proteomics Facility, Johns Hopkins School of Medicine, 733 N. Broadway Street, Baltimore, MD 21205-2185, USA. KEYWORDS: O-GlcNAc, Glycosylation, Glycoproteins, Signal transduction

1 ACS Paragon Plus Environment


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ABSTRACT

O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3,000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or OGlcNAcylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcNAcylated upon acute oxidative stress (H2O2) to provide insight into the mechanisms by which O-GlcNAc promotes survival. We achieved this goal by employing Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and a novel “G5-lectibody” immunoprecipitation strategy that combines four O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39, and HGAC85) and the lectin WGA. Using the G5-lectibody column in combination with basic Reversed Phase (bRP) chromatography and C18 RPLC-MS/MS, 990 proteins were identified and quantified. Hundreds of proteins identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcNAcylation status of 24 proteins. Analysis of proteins with altered glycosylation suggest that stress-induced changes in O-GlcNAcylation cluster into pathways known to regulate the cells response to injury and include protein folding, transcriptional regulation, epigenetics, and proteins involved in RNA biogenesis. Together, these data suggest that stress-induced O-GlcNAcylation regulates numerous and diverse cellular pathways to promote cell and tissue survival.


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INTRODUCTION The dynamic modification of Ser/Thr residues by O-linked N-acetyl-β-D-glucosamine (OGlcNAc) has been implicated in the regulation of over 3,000 nuclear, cytoplasmic and mitochondrial proteins 1. Unlike the more commonly studied ER/Golgi glycosylation pathways (N-linked, O-GalNAc, GPI-anchored), O-GlcNAc is a monosaccharide that is rarely extended into more complex structures. The addition and removal of O-GlcNAc is catalyzed by the uridine diphospho-N-acetylglucosamine: peptide N-acetyl-β-D-glucosaminyltransferase (OGT; EC 2.4.1.255) 3.2.1.52)

2,3

4,5

and a neutral O-GlcNAc-specific hexosaminidase (O-GlcNAcase; OGA; EC

, respectively. Deletion of OGT is lethal in animals, tissues, and single cells,

highlighting the key role of O-GlcNAc in regulating cellular function and biological processes 68

. O-GlcNAc regulates proteins in a manner analogous to other post-translational modifications

such as protein phosphorylation regulating phosphorylation

10-17

1,9

, and in some instances modulates protein function by

. This regulation can be direct, by competing with

phosphorylation for Ser/Thr residues on proteins such as c-Myc 10, RNA polymerase II 13,14 and endothelial nitric oxide synthase

16

. Consistent with a model in which residues are cycled

between O-GlcNAc-modified and phosphorylated, OGT and O-GlcNAcase are found in complexes that also contain phosphatases 18 and kinases 19. O-GlcNAc may also regulate protein phosphorylation indirectly, by altering the activity of protein kinases

20

. In a recent study, OGT

was demonstrated to modify 42 kinases on a functional kinase array and to alter the activity of Ca2+/calmodulin-dependent proteinKinase IV 20,21. In response to stress and injury, cells and tissues remodel the cellular environment to repair damaged structures and if necessary to undergo apoptosis 22. This process, known as the cellular



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stress response, includes robust and dynamic changes in the O-GlcNAc modification 8. OGlcNAcylation of numerous proteins increases in a dose-dependent manner in response to diverse forms of cellular injury 8; whereas O-GlcNAc levels decline in some models of apoptosis 23

. Suggesting that stress-induced O-GlcNAcylation promotes a survival program, elevating O-

GlcNAc levels before or immediately after injury reduces cell and tissue death in numerous stress models including: heat stress 8, oxidative stress 23, hypoxia, ischemia reperfusion injury 2327

, and trauma hemorrhage

28,29

. One mechanism by which O-GlcNAc may promote survival is

by suppressing the generation of reactive oxygen species (ROS), a common characteristic of these models of cell and tissue injury 30. To gain insight into the proteins and pathways regulated by O-GlcNAc in cells experiencing oxidative stress, we sought to define the differential O-GlcNAc sub-proteome of mouse embryonic fibroblasts (MEF) oxidatively stressed with hydrogen peroxide (H2O2). Our experimental approach relied on a SILAC-based strategy that we used previously to identify proteins O-GlcNAc-modified in response to heat stress

31

. Combining this approach with an

improved enrichment strategy we have identified 990 putatively O-GlcNAc modified proteins and 60 O-GlcNAc modified peptides. Hundreds of proteins identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcNAcylation status of 24 proteins. These proteins fall into diverse pathways, and suggest that O-GlcNAc regulates numerous proteins and pathways to reduce cytotoxicity.


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METHODS Reagents The following O-GlcNAc-specific antibodies were used in this study: CTD110.6 (Gift: Core C4), RL2 (Gift: Core C4), HGAC 39, HGAC 85 (Gift: Neil Greenspan). Antibodies used in this study include: OGT (DM-17), heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) and actin (Sigma-Aldrich); Carm1 (coactivator-associated arginine methyltransferase 1), Caprin and FUS RNA binding protein (Fus; Bethyl Laboratories); heat shock cognate 71 kDa (Hsc70), Protein Arginine Methyltransferase 1 (PRMT1), Voltage-dependent anion channels (VDAC), Protein Arginine Methyltransferase 5 (PRMT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and Casein kinase 2 (CK2; Santa Cruz Biotechnology); AKT phospho-substrate, 14-3-3 substrate, 14-3-3γ, 14-3-3η (Cell Signaling); and mAb414 (Abcam). Wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP), Vicia villosa-HRP (VVA-HRP) and Concanavalin A-HRP (Con A-HRP) were obtained from EY laboratories. Ovalbumin and 4hydroxytamoxifen

(4OHT)

were

purchased

from

Sigma-Aldrich.

PNGase

F

and

Endoglycosidase E (Endo H) were obtained from New England Biolabs. BSA-glycoconjugates were purchased from Vector Laboratories. All other chemicals were of the highest grade.

Cell lines and treatment Inducible OGT null mouse embryonic fibroblasts (MEFs) stably transduced with either green fluorescent protein (GFP) or a mutated estrogen receptor-GFP (mER-Cre-2A-GFP) fusion protein were described previously

17,32

. MEFs were cultured in Dulbecco's modified Eagle's

medium (DMEM; 1 g/liter glucose; Mediatech, Manassas, VA), 10% v/v fetal bovine serum (FBS), 1% v/v penicillin/streptomycin at 37 °C in a water-jacketed, humidified CO2 (5%)



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incubator. Typically, cells were plated at 5 × 105 cells/100-mm plate (Corning Inc., Corning, NY), 46 h prior to the initiation of experiments. Unless otherwise noted, Cre-recombinase was activated using 0.5 µM 4OHT 8 h post-plating (vehicle control: ethanol) resulting in the deletion of OGT. For cells treated ±4OHT, medium containing the drug was removed 14 h prior to the experiment, the cells were washed with phosphate-buffered saline pH 7.4 (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), and the cells were re-fed complete media. MEFs were treated with hydrogen peroxide (2.5 mM) and incubated in a humidified water-jacketed CO2 incubator at 37 °C for the indicated lengths of time (1, 2, 3, or 4 h).

Lactate Dehydrogenase Assay The Lactate Dehydrogenase (LDH) assay was performed as per the manufacturer’s instructions (Roche). Briefly, MEFs were plated in a 96 well plate (~2500 cells/well) and grown as described above. The cells were treated with H2O2 (2.5 mM; 1, 2, 3, or 4 h) and each supernatant (100 µl) was transferred to a new plate. The remaining cells were lysed with 2% v/v Nonidet P-40 (NP40) (100 µl). The cytotoxicity assay reagent mixture was added to all wells containing supernatants and lysed cells, and incubated at room temperature for up to 30 min. LDH release and retention was measured by the absorbance of the reaction mixture at 492 nm and the total LDH release was calculated (% release = [released LDH]/[released + retained LDH] x 100). Experiments included no less than 6 biological replicates and were repeated at least three times. Error bars represent one standard error and p-values are the result of a Students T-test.

Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)


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MEFs were passaged 9 times in complete DMEM (1 g/l glucose) supplemented with Larginine/lysine (light), 13C6 L-arginine/lysine-D4 (medium), or 13C6 15 N4 L-arginine/13C4 15 N2 Llysine (heavy). Cells (1 × 106) were seeded in 150 mm (Corning) dishes 48 h prior to treatments. Medium and heavy-labeled MEFs were treated with 2.5 mM H2O2 for 1 and 2 h respectively and harvested.

Total Nuclear/Cytoplasmic Extraction and O-GlcNAc Immunoprecipitation Cells were washed with ice-cold PBS (3x) and removed from plates by scraping. Cell pellets were stored at -80°C until required. Total nuclear and cytoplasmic proteins were extracted by swelling cell pellets on ice in low salt buffer (20mM HEPES pH 7.3, 1mM EDTA, 1mM EGTA, and the protease/phosphatase/glycosidase inhibitors 0.5 mM PMSF, PIC1, PIC2, 500nM Thiamet-G, 10 mM β-hexosaminidase inhibitor (Calbiochem), 5 mM NaF, and 5 mM βglycerophosphate) for 10 min. The cytosoplasmic fraction was released by dounce homogenization (5x). Subsequently, high salt buffer (1/3 of low salt volume) containing 20mM HEPES pH 7.3, 1mM EDTA, 1mM EGTA, 420mM NaCl, 20% v/v glycerol and protease/phosphatase/glycosidase inhibitors was added to the extract. The cell extract was dounce homogenized (5x) and incubated at 4oC (30 min) with mixing. Following incubation, the cell extract was probe sonicated (10 s, Setting 3, Fisher 550 Sonic dismembrator) and cellular debris were pelleted by high-speed centrifugation (18,000 xg, 30 min, 4oC). The resulting supernatant containing cytoplasmic and nuclear proteins was used for O-GlcNAc immunoprecipitations. Equal protein (3.5 mg) from each sample (control, 1 and 2 h H2O2 treatment) was combined (total protein 10.5 mg) and split into two parts (5.25 mg each). O-GlcNAc-modified proteins



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were purified using CTD110.6 (1.5 mg), RL2 (1 mg), HGAC39 (0.25 mg) and HGAC85 (0.25 mg) and WGA (0.5 mg) coupled to Cyanogen bromide-activated sepharose (total bed volume 3 ml). As a control, a column containing sepharose modified by non-specific IgG or IgM antibodies was used. The columns were equilibrated with 5 column volumes of Nuc/Cyt buffer (20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 105 mM NaCl, 5% v/v glycerol) containing 0.01% v/v NP-40. Protein extracts were applied to the column and incubated with mixing at 4oC overnight. Coupled anti-O-GlcNAc antibodies (and controls) bound to proteins were packed on top of a trap column containing sepharose-coupled protein A/G and anti-mouse IgM in a PolyPrep gravity flow column (Bio-Rad; Figure 1 A and B). Unbound proteins were eluted and the resin was washed with 5 column volumes of Nuc/Cyt buffer containing 0.01% v/v NP-40 followed by 5 column volumes of 1 M galactose (Gal) in Nuc/Cyt buffer. Bound proteins were eluted with 1 M free N-acetylglucosamine (GlcNAc) in Nuc/Cyt buffer and 400 µL fractions were collected. Each fraction was precipitated with chloroform/methanol (2x) to remove free sugar and resuspended in 4x LDS sample buffer (Invitrogen) with 50 mM Dithiothreitol (DTT), or in 50 mM ammonium bicarbonate.

Deglycosylation Reactions Ovalbumin (50µg) was treated with Endo H (250 000 units) according to the manufacturers instructions for 2h at 37oC (New England Biolabs). 50% of the reaction was subsequently treated with PNGase F (125 000 units) for 2h at 37oC according to the manufacturers instructions (New England Biolabs).

Immunoprecipitation


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Cells were lysed and total protein was extracted with sonication (10 s, Setting 3, Fisher 550 Sonic dismembrator) in extraction buffer (1% v/v NP-40 in Tris-buffered saline (TBS; 50 mm Tris-HCl, pH 7.5, 150 mm NaCl)) with 0.5 mM PMSF, PIC1, PIC2, 500nM Thiamet-G, 10 mM β-hexosaminidase inhibitor, 5 mM NaF, and 5 mM β-glycerophosphate). Cellular debris were pelleted at 18,000 ×g (30 min at 4 °C). Protein concentration was estimated using the Pierce 600nm Protein Assay Reagent (Pierce Biotechnology). Typically, 1 µg of antibody per 500 µg of protein extract was used for immunoprecipitations. Protein A/G magnetic beads (GE Healthcare) were used to capture the antibody:protein complex. Immunoprecipitates were washed with TBS + 1% v/v NP-40 (3x) to remove non-specifically bound proteins, and then resuspended in 1x LDS buffer with 50 mM DTT, and heated at 85oC for 10 min.

1D SDS PAGE and Western blot Denatured proteins (20 µg) were separated on 8% or 4-12% Bis-Tris pre-cast gels using a 3(N-morpholino) propane sulfonic acid (MOPS) running buffer (180 V, 125 mA). Proteins were transferred onto either nitrocellulose or polyvinylidene difluoride membranes (50 V, 400 mA for 60 min). For whole protein staining, the gels were fixed in 7% v/v acetic acid and 10% v/v methanol and stained overnight with Sypro Ruby (Invitrogen, San Diego, CA). Gels were destained in 7 % v/v acetic acid and 10 % v/v methanol and imaged using the Typhoon Trio Variable Mode Imager (GE Healthcare, Uppsala, Sweden). Non-saturating western blots from independent experiments (n=3) were scanned in and analyzed by Image J. Error bars represent standard error and p-values are the result of a Students T-test.

Liquid Chromatography Tandem Mass Spectrometry



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Immunoprecipitated proteins were resuspended in 50 mM NH4HCO3 pH 7.8, reduced and alkylated with 10 mM DTT and 55 mM Iodoacetamide (IAA) respectively, and digested with trypsin. The tryptic peptides were dried down by vacuum centrifugation, and resuspended in 50 µL of 10mM triethylammonium bicarbonate (TEAB) for subsequent basic reversed phase (bRP) micro scale fractionation. bRP fractionation was performed using an Agilent 1200 capLC system with multiwavelength detector at 5 µL/min and fractions were collected at 2 min intervals with a Probot (LC Packings) fraction collector. Fractionation was performed with a 300 µm I.D. fused silica column self-packed with Waters XBridge BEH130 C18 (3.5 µm, 130A) reversed phase resin. After loading for 12 min onto the column a gradient from 0-30% v/v mobile phase A (10mM TEAB) to B (90% v/v ACN, 10mM TEAB) was performed over a 50 min time interval before ramping to 100% v/v B over 10 min and holding for an additional 10 min. Fractions were concatenated into 12 fractions for subsequent LC/MS/MS analysis. Peptide fractions from bRP chromatography were injected onto a 2 cm desalting trap column packed with YMC C18 material (75 µm ID, 5–15 µm, 120 A) at 5 µl/min for 6 min before being eluted onto an analytical column packed with Michrom Magic C18 (75 µm × 15 cm, 5 µm, 120 A) using a nanoAquity nanoLC system (Waters, MA) with a nanoflow solvent delivery of 300 nl/min. Each sample was separated on a 90 min gradient (5%–90% v/v acetonitrile, 0.1% v/v formic acid) with a flow rate of 300 nl/min. The peptides were eluted and ionized into an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). The electrospray source was fitted with an emitter tip 10 µm (New Objective, Woburn, MA) and maintained at 2.0 kV electrospray voltage. Precursor ions were selected for MS/MS fragmentation using a data-dependent “Top 8” method operating in FT-FT acquisition mode with HCD fragmentation. The survey scan range was between m/z 350 – 1700 Da at 30,000 resolution with a target of 1 x 106 ions; and MS/MS


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scans were carried out at 7500 resolution with a target of 3 x 104 ions. Maximum injection times were set to 100 and 300 milliseconds respectively. The ion selection threshold for triggering MS/MS fragmentation was set to 2,000 counts and an isolation width of 1.9 Da was used to perform HCD fragmentation with a normalized collision energy of 35%. Ambient polysiloxane produced a background peak at 371.101230 m/z was used as a lock mass for each scan.

Mass Spectrometry Data Analysis Spectra files (*.RAW) were processed using the Proteome Discoverer 1.3 software (Thermo Scientific, CA, USA) incorporating the Mascot search algorithm (V2.1, Matrix Sciences, UK). Peptide identifications were determined using a 30-ppm precursor ion tolerance, a 0.05 Da MS/MS fragment ion tolerance and a maximum of 2 missed cleavages. Methylthio modification of cysteines was considered a static modification while oxidation of methionine, deamidation of asparagine and glutamine, and N-acetylhexosamine (HexNAc) modification on serine and threonine residues were set as variable modifications. modifications included lysine (2H4 and

13

For the SILAC labeling variable

C615N2) and arginine (13C6 and

13

C615N4) (Medium:

Arg6 Lys4; Heavy: Arg10 Lys8; Light: No labels). Neutral loss of HexNAc (m/z 203.0794) and diagnostic ions (m/z 204.0866) were used to predict potential O-GlcNAc peptides for manual validation. MS/MS spectra were searched through Proteome Discoverer (1.3; Thermo Scientific) using the Mascot algorithm against the RefSeq 2012 Mus musculus database (sequences 15742024, May 7 2012). The data was processed through the Xtract and MS2 Processor nodes together with a direct search and the combined searches were sent to Percolator (Department of Genome Sciences, University of Washington) for estimation of false discovery rates. Protein identifications were validated employing a q-value of 0.01 (1% false discovery rate (FDR))



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within the Proteome Discoverer software, and a minimum of 2 unique peptides (Peptide cutoff score: 10) and 3 Peptide Spectral Matches (PSMs) per identification. Protein quantification was generated in Proteome Discoverer 1.3 on the identified proteins in which the SILAC label was detected in all three treatments. The threshold for proteins differentially associated with the lectibody column was determined by calculating the fold change Z-score based on the distribution of proteins 33. Proteins whose Zscore falls outside one standard deviation are identified as either increased or decreased (Figure S1). While these data suggest the limits of 0.79-fold (decreased) and 1.26-fold (increased), we used more stringent limits of 1.3 fold that we, and others, have used in the past 17,31,34,35

. The mass spectrometry proteomics data have been deposited to the ProteomeXchange

Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000849

36

. To access the data, please visit http://tinyurl.com/pbe3c2p

(User: [email protected] Pwd: FnR16lAR). Further evaluation of O-GlcNAcylated peptides was carried out using the OScore scoring algorithm to search the list of filtered peptides (q-value 2 peptides and > 3 peptide spectral matches (PSMs) per identification. Of these proteins, 990 were identified and quantified in all three treatments (Table 1, Supplemental Table 1). 318 proteins identified and quantified in this study have previously been annotated as O-GlcNAc modified (Supplemental Table


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111,12,19,31,43,45-132). Moreover, only 18 of the identified proteins are predicted to be membrane associated or secreted, suggesting that the majority of the proteins are likely modified by OGlcNAc or associated with an O-GlcNAc modified protein (Table S1). Of the 990 proteins, 258 (26.1%) and 259 (26.2%) proteins demonstrated increased affinity for the lectibody column by > 1.3 fold at 1 and 2 h respectively with 180 commonly increased proteins at both time points (Figure 4A). In addition, 114 (11.5%) and 168 (17%) proteins demonstrated decreased affinity for the lectibody column by < 0.7 fold at 1 and 2 h respectively with 69 common decreased proteins at both time points (Figure 4B). Many of the proteins determined to be differentially increased upon oxidative stress are known to regulate the cellular stress response including: stress granules components, proteins of the mediator complex, chaperonins, heat shock proteins (HSPs), and the ubiquitin protein family. Interestingly, the family of Nucleoporin (Nup) proteins was observed to be decreased at both the 1 and 2 h time point. These proteins, many of which are known to be O-GlcNAc modified (bold) 12,43,133, include: Nup50 43, Nup62 53, Nup88 43, Nup93, Nup98 43, Nup153 12, Nup188, Nup205, and Nup214 12. These

data

were

interrogated

using

the

DAVID

Bioinformatics

Resource

6.7

(http://david.abcc.ncifcrf.gov/) gene ontology function. The classes of proteins in our dataset included: nucleic acid/nucleotide binding (49%)(e.g. Dead Box proteins and TATA box binding proteins), protein binding (25%) (e.g. DnaJ and Sec24 protein families) and hydrolase activity (10%)(e.g. ATPases and Eukaryotic Translation Initiation Factors) (Figure 4C). Examination of the dataset suggested that key survival proteins were enriched in proteins displaying enhanced affinity for the G5-lectibody column, including Heat Shock Proteins, Chaperonins (TCP1, CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8, HSPH1, HSPB1, and HSPA1A/1B) and proteins



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involved in the 14-3-3/PI3K signaling pathway (AHSA1, SRPK1, SRPK2, YWHAB, YWHAE, YWHAG, YWHAH, YWHAQ, and YWHAZ).

Validation of the O-GlcNAc sub-proteome As O-GlcNAc modified proteins were enriched under non-denaturing conditions, changes in the quantification of proteins in the G5-lectibody immunoprecipitation could be due to changes in: i) the O-GlcNAcylation status of identified proteins; ii) the association with an O-GlcNAcmodified protein; and/or iii) the expression of a protein upon oxidative stress. Therefore, we validated a list of 12 proteins that were identified in our mass spectrometry screen and either increased (> 1.3 fold change), decreased (< 0.7 fold change) or did not change in response to oxidative stress. The proteins selected for analysis included: OGT, HSC70, hnRNP-U, Caprin, Carm1, PRMT1, VDAC, PRMT5, Actin (negative control), Nup p62, NFκB (p65, p100) and Fus. In addition, we tested the substrates of 14-3-3 proteins and 14-3-3 proteins γ and η, which appeared enriched in stressed samples (Supplemental table 1). Total Nuc/Cyt extracts of OGT wild-type (WT) MEFs oxidatively stressed (2.5 mM H2O2) for 1, 2 and 3 h, and OGT null (negative control) were immunoprecipitated using the G5-lectibody column as described above. The presence of target proteins in the immunoprecipitates was assessed by SDS-PAGE (Figure 5A). Oxidative stress did not change the expression of OGT, HSC70, O-GlcNAcase, hnRNPU, Caprin, Carm1, PRMT1, PRMT5, 14-3-3 γ and η, Nup62, Fus, and NFκB (p65, p100), suggesting that any differences determined by SILAC and mass spectrometry was either due to changes in the O-GlcNAcylation state of the protein or their association with an O-GlcNAc-modified protein. Analysis of the substrates of 14-3-3 proteins,


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demonstrated complex changes in phosphorylation; with some proteins displaying enhanced phosphorylation (~50kDa), whereas others exhibited reduced phosphorylation (>70kDa). Analysis of the G5-lectibody immunoprecipitates by western blotting demonstrated similar data to that quantified by mass spectrometry (Figure 5A and B). Proteins such as 14-3-3 γ and η, Caprin, PRMT5 and NFκB (p65, p100) exhibiting increased association with the G5 lectibody column post-stress; whereas Nup62 displayed decreased association. To assess whether proteins identified in the screen were being directly modified in response to oxidative stress, a subset of these proteins were immunoprecipitated and the O-GlcNAc modification was assessed by immunoblotting. As shown in Figure 5C, OGT is O-GlcNAc modified, however the OGlcNAcylation status does not change in response to oxidative stress. For proteins such as Fus, hnRNP U and Carm1, O-GlcNAcylation appeared to decrease at 1h before increasing at 2 or 3h. To assess the enrichment of 14-3-3 substrate proteins, we used an antibody raised against the 14-3-3 protein-binding motif: a phospho-serine with proline at +2 and arginine and lysine at -3. While the signals for many of these proteins decrease upon stress, they are enriched in the G5lectibody immunoprecipitate. These data suggest that 14-3-3 proteins or their substrates are OGlcNAc modified in response to oxidative stress. While the immunoprecipitation with the 14-33 substrate antibody was not optimal, we were able to confirm a subset of 14-3-3 proteins were O-GlcNAc modified in response to oxidative stress (Figure 5C).

Numerous O-GlcNAc-modified peptides and sites were identified In addition to confirming that proteins identified in the screen were O-GlcNAc modified by immunoprecipitation and western blot, we were able to identify glycopeptides and glycosylation sites for a subset of proteins. The peaks algorithm identified 502 HexNAc containing spectra at a



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false discovery rate of 1% from 26, 721 spectra. Each HexNAc containing spectra was manually validated, yielding 55 glycopeptides (Supplemental Table 2, Supplemental Spectra). An additional 5 glycopeptides were identified using the OScore algorithm yielding a total of 60 OHexNAcylated peptides in 18 proteins. Notably, none of the identified proteins are predicted to be synthesized in the ER. Combined with the specificity of the G5-lectibody column (discussed above), we conclude that these peptides are modified by O-GlcNAc. Of the 60 O-GlcNAcylatedmodified peptides identified, 22 are putatively unique O-GlcNAc-modified sites/peptides and the remaining 40 have been identified and catalogued in the dbOGAP Technology

databases

(PhosphoSitePlus®,

www.phosphosite.org)48.

47

and Cell Signaling Of

the

confirmed

glycopeptides, 19 were isolated from 10 proteins that displayed differential affinity for the G5lectibody column by 1.3 during acute H2O2 treatment (1 or 2 h; Table 2). Of the glycopeptides identified, a subset contained quantitative information (Supplemental Table 2). In general, peptide quantification reflects that of the total protein and as a result information about site-specific cycling cannot be inferred.

The stress-induced changes glycosylation of an

additional 5 proteins were confirmed by immunoprecipitation and western blot (Figure 5, discussed above). Not surprisingly, 32 peptides (with multiple O-GlcNAc sites) corresponded to the highly O-GlcNAcylated protein Host Cell Factor 1, which has been characterized to contain > 30 O-GlcNAcylation sites among different cells and tissue types 134.


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DISCUSSION In this study we defined the oxidative stress-induced changes to the O-GlcNAc sub-proteome to gain insight into the mechanisms by which O-GlcNAc protects cells and tissues from cellular stress and injury. To accomplish this goal we have improved methodologies for enriching OGlcNAc-modified proteins in which we used a combination of O-GlcNAc- and GlcNAc-specific antibodies and lectins (G5-lectibody column) in combination with SILAC labeling. Using this approach we identified and quantified 990 proteins in all three treatments (control, 1 and 2 h) and 60 glycopeptides of which 3 sites of glycosylation are novel. Many of the proteins identified exhibited either increased or decreased binding to the G5-lectibody column in response to oxidative stress, suggesting that their O-GlcNAcylation status, expression, or protein-protein interactions with an O-GlcNAc modified protein changed in a stress-dependent manner. Sitemapping and biochemical analysis confirmed the identity of 24 glycoproteins, of which OGlcNAc appears to cycle in a stress-dependent manner on 15.

A novel strategy for enriching O-GlcNAc-modified proteins using the G5-column. A small cohort of studies have employed O-GlcNAc-specific antibodies or WGA as singular entities for immunoprecipitation to identify differentially expressed O-GlcNAc-modified proteins in response to cellular stress and tissue injury

31,43

isotopically labeled with SILAC media (13C6 L-arginine and

. Previously, we used Cos-7 cells 13

C615N4 L-arginine) and enriched

O-GlcNAc-modified proteins by immunoprecipitation with the CTD110.6 antibody. 15 proteins were identified with differential O-GlcNAcylation and 7 were confirmed to be directly OGlcNAc modified

31

. In order to improve coverage, we altered this strategy (Figure 1) and this

enabled us to identify and quantify 990 proteins (versus 30 in our previous study) and to



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Page 24 of 61

characterize a number of O-GlcNAc modified peptides (60) and O-GlcNAc modification sites (5). O-GlcNAc is often more labile than the peptide bond during CID/HCD fragmentation and the ionization of O-GlcNAc modified peptides is suppressed during mass spectrometry

135,136

.

Thus, the identification of 60 glycopeptides was unexpected and is likely the result of the improved fractionation (discussed below). We expect that combining this approach with Electron Transfer Dissociation (ETD) mass spectrometry, in which the O-GlcNAc-peptide bond is more stable 134, would further improve the number of sites mapped. We attribute the improvements in the number of proteins identified and quantified, and the increase in the number of O-GlcNAc modification sites mapped to three changes made in our approach. Firstly, O-GlcNAc modified proteins were enriched with a combination of OGlcNAc/GlcNAc specific antibodies and lectins: CTD110.6 138

137

, RL2 42, HGAC39

138

, HGAC85

and the lectin WGA. Our studies suggest that this approach is likely to give a less biased

enrichment, as many of these antibodies/lectins appeared to have different affinities for OGlcNAc modified proteins. Secondly, we fractionated peptides into 12 fractions by bRP chromatography, before analysis by reversed phase chromatography and mass spectrometry. bRP demonstrates improved resolution of peptides over strong cation exchange fractionation, while reducing sample complexity and allowing detection of lower abundance peptides and glycopeptides by mass spectrometry

139

. Finally, we extrapolated information from the tandem

mass spectra using the Oscore algorithm to discriminate O-GlcNAc-modified peptide spectra from spectra of unmodified peptides based on a set of O-GlcNAc-specific fragment and neutral loss ions 38,39. One disadvantage of the approach utilized in this study is that we immunoprecipitate proteins and not glycopeptides, and thus validation is required to confirm that proteins are modified by O-


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GlcNAc rather than associated with an O-GlcNAc-modified protein. However, because >50% of the identified proteins have >10% coverage, there is greater confidence in the identification and quantification. Moreover, O-GlcNAc/GlcNAc specific antibodies do not immunoprecipitate peptides well. Some researchers have used lectin weak affinity chromatography to enrich glycopeptides

74,140

, and in future studies we will investigate this approach using the G5-

lectibody column.

Effects of O-GlcNAc on cellular pathways in response to oxidative stress. Recent work has demonstrated that dynamic O-GlcNAcylation is one component of the cellular stress response that is relevant to a variety of models of injury in several cell lines and tissue types

1,141

. In this study we have identified numerous proteins whose O-GlcNAcylation

state changes in response to oxidative stress. Interestingly, while there appears to be a global increase in the O-GlcNAc modification at the 2h time-point (Figure 2A) numerous proteins appear to have reduced O-GlcNAcylation based on their reduced affinity for the G5-lectibody column. Supporting these data, immunoprecipitation and Western blotting studies (Figure 5C) demonstrate that the O-GlcNAc modification state of proteins such as hnRNPU and Caprin decrease at acute time points (1 h) before increasing (2 and 3 h). These previously unappreciated dynamics in the O-GlcNAc-modification in models of cellular stress may highlight proteins on which a decrease in O-GlcNAcylation promotes cellular processes that lead to apoptosis and necrosis. Our data suggest a number of pathways that are targeted by O-GlcNAcylation during cellular injury, including: the chromatin remodeling, transcription, chaperone biology and 14-3-3 biology (Figure 4, Supplemental table 1). As discussed below, these data implicate that O-GlcNAc in



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Page 26 of 61

mediating transcription and translation as well as protein folding. The induction of cellular injury results in widespread changes in transcriptional patterns, many of which require ATP-dependent chromatin remodelers that act both as transcription co-repressors and activators

142-145

.

Highlighting the importance of chromatin remodelers, deletion of one or more components of these complexes can sensitize cells to injury 143 and can impact the ability of heat shock factor 1 (HSF1) to induce the expression of the canonical chaperones or heat shock proteins

142

.

Numerous proteins that are components of the BAF and pBAF SWI/SNF chromatin remodeling complexes were identified in the G5-lectibody Immunoprecipitate including: SmarcA5/snf2h, Smarcb1/Baf47,

Smarcc2/Baf170,

SmarcD3/Baf60c, Dmap1/Mmtr, Trrap

Smarce1/Baf57, 146-148

.

Smarcc1/Baf155, Arid1b/Baf250a,

SmarcD1/Baf60a, Pbrm1/Baf180,

SmarcD2/Baf60b, ep400/Domino,

AT-rich interactive domain-containing protein 1A (Arid1A)

demonstrated a robust change in O-GlcNAcylation, with a 2.15 and 2.52 fold increase after 1 and 2h of oxidative stress respectively. In addition to the SWI/SNF protein members, we identified numerous subunits of the Mediator complex, further suggesting that dynamic changes in O-GlcNAcylation fine tune the changes in transcription that occur in response to injury. The Mediator complex binds to the C-terminus of RNA pol II, and is involved in control of activator-independent transcription though pre- and post-initiation events acting as the scaffold between RNA pol II and transcription factors

149

. Of

the 31 proteins which comprise the Mediator Complex, 13 were identified and quantified in the G5-lectibody immunoprecipitate: MED1, MED4, MED10, MED12, MED13, MED14, MED15, MED16, MED17, MED20, MED23, MED24 and MED27. Stress is known to alter mediator function; in models of mitochondrial and heat stress, MED12 promotes the association of mediator with heat shock protein promoters

150

. Notably, in models of osmotic stress,


Page 27 of 61

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dephosphorylation of mediator complex subunits is associated with the activation of stress related genes

151

. In contrast, phosphorylation is associated with transcriptional suppression of

stress-related genes under basal conditions

152

. These data lead to the attractive hypothesis that

O-GlcNAc may promote the stress-associated functions of mediator. Numerous proteins involved in the biogenesis of RNA were identified in this screen and suggest roles for O-GlcNAc in mediating RNA degradation, stalling translational machinery and controlling the localization, storage, degradation, stability, and re-initiation of translation of RNA. Stress granules contain stalled or abortive translation initiation complexes that accumulate after stress-induced translational arrest

153

. Overexpression of proteins that promote stress

granule formation, such as polyadenylate binding protein 1 (PABP-1), promote cell survival in a model of oxidative stress

154

. Previous studies have demonstrated that OGT is necessary for the

formation of stress granules

155

, although the mechanisms underlying the phenomena remain

elusive. In this study we have demonstrated that a number of proteins known to be involved in the formation of stress granule production are differentially O-GlcNAc modified, including: CCR4-NOT complex proteins, 40S ribosomal proteins, fragile X mental retardation protein, eIF3, eIF4B, eIF4E, Fus, Caprin, hnRNPU, PRMT1, Ataxin 2, PABP-1. CCR4-NOT, Fus, Caprin, hnRNPU, Ataxin 2 were confirmed to be O-GlcNAc modified by site-mapping or immunoprecipitation/western blot. In addition to these proteins, many subunits of the CCR4-Not complex were identified and a site of glycosylation was detected on CCR4-NOT transcription complex subunit 2. Finally, our data suggest that O-GlcNAc may impact stress-signaling or protein folding through the 14-3-3 family of proteins. 14-3-3 proteins are a class of highly conserved proteins that play multiple roles in regulating apoptosis, adhesion, cellular proliferation, differentiation,



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survival, and signal transduction pathways proteins regulating their localization

156

156

Page 28 of 61

. While 14-3-3 are known to bind phosphorylated

, recent data suggests that 14-3-3 proteins can act as a

molecular chaperones that protect against stress-induced apoptosis

157-159

. Consistent with these

data, in a model of diabetic cardiomyopathy overexpression of a dominant negative 14-3-3 protein exacerbates oxidative stress and inflammation

160

. One mechanism by which 14-3-3

confers resistance to stress is through its chaperonin-type ability to assist in maintaining physiologically relevant structure of damaged proteins and by binding to and inhibiting proapoptotic proteins

157

. For example, dephosphorylated Bcl-associated death promoter (Bad)

forms a heterodimer with and inactivates Bcl2/Bcl-xl, releasing Bax followed by release of cytochrome c and activation of caspases 161,162 . While phosphorylated Bad forms a heterodimer with 14-3-3 leaving Bcl-2/Bcl-xl to inhibit Bax-dependent apoptosis GlcNAcylated (Figure 5)

73

163

. 14-3-3 itself is O-

, but the consequence of this modification on its functional role

within the context of cell survival and oxidative stress is not yet known. The increase of the 143-3 complex and phosphorylated substrates of 14-3-3 in our O-GlcNAc immunoprecipitation indicates that their O-GlcNAcylation status and/or its association with O-GlcNAcylated proteins are elevated in response to oxidative stress. The concomitant elevation of global O-GlcNAc levels, O-GlcNAcylation status of the 14-3-3 complex, and the 14-3-3 canonical pathway including chaperonins 164 in this study suggests that these constituents work in concert within the cellular milieu in responding to acute oxidative stress damage; and could potentially be one mechanism by which O-GlcNAc promotes survival. In summary, the data presented suggest that the G5-lectibody enrichment provides greater coverage of the O-GlcNAc sub-proteome. When combined with basic reversed phase chromatography, the enhanced separation enabled the identification and quantification of 990


Page 29 of 61

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proteins and surprisingly provided some O-GlcNAc modification site information. This study of the O-GlcNAc sub-proteome highlights the importance of O-GlcNAc in regulating diverse pathways to promote survival under conditions of oxidative stress.



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FIGURES


Page 30 of 61

Page 31 of 61

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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

1.

Workflow

for

enriching

O-GlcNAc-modified

Page 32 of 61

Proteins

by

G5-

Immunoprecipitation. (A) Mouse embryonic fibroblasts (MEFs) were labeled with medium and heavy isotopes of arginine and lysine, and treated with 2.5 mM H2O2 for 1 and 2 h respectively. Equal amounts of protein were combined and O-GlcNAc-modified proteins were purified using a combination of immobilized O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39 and HGAC89) and WGA referred to as the G5-lectibody resin/column. Eluted proteins were trypsin digested, separated into 12 fractions by basic reversed phase (bRP) chromatography and subjected to LC-MS/MS. (B) To reduce antibody contamination of the eluent, the G5-lectibody resin was packed on top of a trap column containing equal parts Protein A, Protein G and antiIgM sepharose. (C) O-GlcNAc modified proteins were enriched from Nuc/Cyt lysates using sepharose-4B covalently coupled to the indicated antibodies and lectins at 2mg/ml. Immunoprecipitates were separated by SDS-PAGE and OGT, OGA, Carm1, Nup62, Caprin, hnRNP-U and Casein Kinase 2 α (CK2α) were detected by immunoblot. (D) O-GlcNAc modified proteins were enriched from wildtype (WT) or OGT null (KO) Nuc/Cyt lysates (400 µg) or buffer alone (B) using WGA sepharose (moles GlcNAc binding) and the G5-lectibody column (3 nmoles GlcNAc-binding). Input (5µg, 1.4%), unbound (5µg, 1.4%), and immunoprecipitated (5.25%) proteins were separated by SDS-PAGE. OGT, Carm1, and Caprin were detected by immunoblot (n=3).


Page 33 of 61

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Page 34 of 61

Figure 2. O-GlcNAc levels change in response to oxidative stress. (A) Mouse embryonic fibroblasts (MEFs) were treated with 2.5mM H2O2 for 1, 2 and 4 h and the levels of O-GlcNAc, OGT, Actin, PARP and Caspase 3 were assessed by SDS PAGE and immunoblot (n=3). Arrows indicate the cleavage product of Caspase 3 and PARP in cells treated with Staurosporin (2 µM, 4h) to induce apoptosis. (B) The activation of the AKT signaling pathway was assessed in MEFs treated with 2.5mM H2O2 for up to 60 minutes by immunoblot using an antibody specific for phosphorylated substrates of AKT. As a control, the expression of OGT and Actin were also determined. (C) Densitometry of O-GlcNAc is normalized to actin (n=3 *p

GlcNAc Sub-proteome upon - ACS Publications - American Chemical

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