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Originally published In Press as doi:10.1074/jbc.M407911200 on August 20, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45759-45765, October 29, 2004
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Neutrophils Exhibit Rapid Agonist-induced Increases in Protein-associated O-GlcNAc*

Zachary T. Kneass and Richard B. Marchase{ddagger}

From the Department of Cell Biology, University of Alabama, Birmingham, Alabama 35294

Received for publication, July 14, 2004 , and in revised form, August 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A variety of cytoplasmic and nuclear proteins can be modified on serine and threonine residues by O-linked {beta}-N-acetylglucosamine (O-GlcNAc), although the effects of this modification on protein and cellular functions are not completely defined. The sugar donor for the O-GlcNAc transferase that catalyzes this post-translational modification is UDP-N-acetylglucosamine (UDP-GlcNAc), a product of the hexosamine biosynthesis pathway (HBP). Here, the dynamics of the O-GlcNAc modification are examined in the physiological context of agonist-induced signal transduction using neutrophils. Formylated Met-Leu-Phe (fMLF) is shown to stimulate a rapid and transient increase in protein O-GlcNAcylation in both immunoblot and immunofluorescence imaging assays using O-GlcNAc-specific antibodies. In high performance liquid chromatography analyses of HBP metabolic activity, short term exposure to an exogenous substrate of the HBP, glucosamine (GlcNH2), leads to increased GlcNH2 6-phosphate and then UDP-GlcNAc levels. The GlcNH2 treatments also increase O-GlcNAcylation and augment the aforementioned fMLF-associated increase. In functional assays, GlcNH2 pre-treatment selectively augments fMLF-induced chemotaxis but has little effect on respiratory burst activity. Furthermore, augmenting levels of O-GlcNAc in the absence of agonist is sufficient to stimulate chemotaxis. These data demonstrate that neutrophils possess a functionally significant O-GlcNAcylation pathway that is robustly induced by stimulation with agonist. We propose that O-GlcNAcylation plays an important role in rapid and dynamic neutrophil signal transduction, especially with respect to chemotaxis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The addition of O-glycosidic GlcNAc in {beta} linkage to Ser/Thr residues (O-GlcNAc)1 is a dynamic post-translational modification that occurs on numerous cytoplasmic and nuclear proteins and is distinct from complex carbohydrates synthesized in the secretory pathway (1, 2). Proteins modified in this manner include nuclear pore components (2, 3), transcription factors (4, 5), cytoskeleton-associated proteins, and signaling enzymes (6, 7).

Early work on this modification suggested that it was dynamic and inducible and led to the prediction that O-GlcNAc would have a regulatory role analogous to phosphorylation (8–10). The cloning and characterization of enzymes responsible for the addition, O-GlcNAc transferase (OGT), and removal, neutral {beta}-N-acetylglucosaminidase, of O-GlcNAc moieties supported this idea (11–13). However, it has only been over the last few years that studies have begun to elucidate functional roles for O-GlcNAc and have uncovered mechanisms through which it may modulate cellular signaling pathways, and thereby cellular function, in health and disease. For example, altered O-GlcNAcylation has been extensively investigated with respect to its role in the pathogenesis of diabetes mellitus (6, 14–16) and may play a role in the etiology of certain cancers (17, 18) and Alzheimer disease (19, 20).

Many of the approaches used to investigate O-GlcNAc function have relied on the manipulation of hexosamine biosynthesis. Approximately 2–5% of the total glucose transported into cells feeds into the hexosamine biosynthesis pathway (HBP) to form glucosamine (GlcNH2) 6-phosphate (GlcNH2 6-P) and ultimately UDP-N-acetylglucosamine (UDP-GlcNAc), the sugar donor for OGT (21). OGT activity has been found to be sensitive to relatively small changes in substrate availability over a wide range of substrate concentrations and, additionally, recognizes different acceptor proteins at different concentrations of UDP-GlcNAc (22). Thus, as several studies have shown, when cells in culture (4, 14, 23) or tissues in vivo (15) are exposed to hyperglycemic conditions they exhibit enhanced levels of O-GlcNAc that can be blocked by HBP inhibitors. This indicates that elevated levels of glucose lead to enhanced HBP flux and UDP-GlcNAc formation and thereby enhanced O-GlcNAc. Exogenous GlcNH2, which is not normally present in the cellular environment at appreciable levels, is also transported into cells and preferentially metabolized through this pathway, resulting in enhanced O-GlcNAc (6, 21, 23).

Proponents of O-GlcNAc as an important protein modification theorize that O-GlcNAc regulates critical aspects of protein biology, including protein stability, subcellular localization, and protein-protein interactions. The current body of research in this area supports these ideas, and several reviews have recently emerged expounding their various facets (1, 7, 24). However, important questions remain. Foremost among these is whether O-GlcNAc can serve as a rapid, highly inducible signal transduction mechanism akin to phosphorylation, the archetypal regulatory mechanism for the coupling of extracellular signals to specific cell responses.

The bulk of the work on the functional significance of O-GlcNAc has relied on long term treatments, hours or days, with either high glucose (4, 14, 15) or GlcNH2 (6, 23), or the use of pharmacological agents (6, 25–27) that inhibit specific enzymes in the pathways affecting O-GlcNAcylation to manipulate this protein modification. Lymphocytes provided early evidence that O-GlcNAcylation can be a dynamic process, because pharmacological mitogens were shown to induce changes in cellular O-GlcNAc over times as short as 1 h (9). In addition, certain pharmacological agents that bypass physiologically relevant agonist- and receptor-specific signaling mechanisms induce relatively rapid changes in O-GlcNAc. In particular, pharmacological treatments with the calcium ionophore A23187 [GenBank] and the protein phosphatase inhibitor okadaic acid have been shown to induce changes in O-GlcNAc within 1 min, suggesting that O-GlcNAc responses may indeed proceed quite rapidly (28). These examples are often used as corroborative evidence of the potential of O-GlcNAc as a signaling mechanism that is analogous to phosphorylation. There is, however, only limited evidence of physiological agonist-induced, receptor-mediated changes in O-GlcNAcylation. Specifically, insulin infusions over several hours lead to increases in O-GlcNAc, although it is likely that these responses are due to an increase in glucose transport and a slowly progressive increase in HBP flux rather than a direct effect of insulin on the O-GlcNAcylation machinery (6, 29, 30).

Neutrophils (polymorphonuclear leukocytes or PMNs) respond to a large and diverse group of stimuli over a range of times, resulting in a variety of metabolic and functional responses. These responses, which include degranulation, phagocytosis, chemotaxis, a respiratory burst, and alterations in gene expression, ultimately lead to microbe killing. Within this context PMNs have served as useful models for studying the signal transduction mechanisms involved in cellular stimulation. One of the most commonly used PMN stimuli, the chemotactic tripeptide formyl-methionine-leucine-phenylalanine (fMLF), binds to cell surface receptors and induces protein phosphorylation within tens of seconds (31–33). These phosphorylation events are central to a diverse set of signaling pathways, leading, for example, to chemotaxis and the production of reactive oxygen species (34–38).

This study was initiated to examine the relevance of O-GlcNAc in cellular responsiveness to external stimuli using an established PMN model. We report here that PMNs possess a functionally significant HBP and associated O-GlcNAcylation mechanism. In doing so, we also provide evidence of rapid and robust agonist-induced changes in O-GlcNAcylation. We show that PMNs respond rapidly to both GlcNH2 and agonist, leading to similar and additive increases in protein O-GlcNAcylation. Our data support the premise that protein O-GlcNAcylation is a highly dynamic signaling mechanism capable of rapidly transducing receptor-associated signals to influence cellular function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
PMN Isolation—Whole blood from volunteers was obtained by venipuncture and layered onto a double discontinuous gradient formed with equal volumes of Histopaque-1077 (Sigma) over Histopaque-1119 (Sigma) (39). The blood was centrifuged at 700 x g for 30 min. Granulocytes were collected from the 1077/1119 interface and washed in Hanks' buffered salt solution buffered with 10 mM HEPES, pH 7.4 (HBSS). Contaminating red blood cells were lysed by incubation in 0.15 M NH4Cl/1 mM KHCO3/0.1 mM EDTA for 5 min at 37 °C. The granulocytes were then washed twice in HBSS and resuspended in HBSS. The resulting samples were >95% PMNs as indicated by the polymorphic nature of their nuclei (see "Immunofluorescence Microscopy" below). Cell viability was assessed through Trypan Blue (Sigma) exclusion.

Immunoblotting—2 x 106 PMNs were treated as indicated, directly lysed in 5x sample buffer (0.3 M Tris·HCl/5% SDS/50% glycerol/0.025% bromphenol blue/5% mercaptoethanol) and boiled for 5 min. 7.5 x 105 cell equivalents per lane (~60 µg of protein) were separated by SDS-PAGE (40) and transferred to Immobilon-P (Millipore). Immunoblotting was performed using a rapid immunodetection method for Immobilon-P (Millipore Tech Note TN051). Briefly, the membranes were equilibrated in methanol and air dried. The dry membrane was incubated with a 1:1000 dilution of anti-O-GlcNAc antibody CTD110.6 (41) (Covance) in 1% casein/phosphate-buffered saline (PBS) (Pierce) with 0.01% Tween 20 for 2 h and then washed three times in PBS. To demonstrate O-GlcNAc-specific immunoreactivity, 10 mM GlcNAc was added during the primary antibody incubation (41). The membrane was then incubated with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgM (Calbiochem) in 1% casein/PBS with 0.01% Tween 20 for 1 h. After further washing in PBS the immunoblots were developed with enhanced chemiluminescence (SuperSignal West Pico; Pierce).

Immunofluorescence Microscopy—PMNs at 1.25 x 106 cells/ml were treated as indicated. The cells were fixed by adding an equal volume of 6% formaldehyde (Tousimis)/PBS for 30 min and resuspended in PBS. The cells were cytospun onto glass coverslips at 500 x g for 5 min. The coverslips were post-fixed in 3% formaldehyde/PBS for 10 min and washed with PBS. The PMNs were permeabilized with methanol (–20 °C) for 2 min. After washing, the coverslips were blocked for 30 min at room temperature in 1% casein/PBS with 0.2% Tween 20 and then incubated with a 1:250 dilution of anti-O-GlcNAc antibody CTD110.6 in 1% casein/PBS with 0.2% Tween 20 for 60 min at 37 °C. To demonstrate O-GlcNAc-specific immunoreactivity, 100 mM GlcNAc was added during the primary antibody incubation. After washing, the coverslips were then blocked for 10 min at room temperature with 10% normal goat serum (Sigma)/PBS with 0.2% Tween 20 and then incubated with a 1:250 dilution of Alexa Fluor 594-conjugated goat antimouse IgM (Molecular Probes) in 10% normal goat serum/PBS with 0.2% Tween 20 for 45 min at room temperature. The coverslips were finally washed and mounted with 9:1 glycerol/PBS. Hoechst 33258 (Molecular Probes), 1:1000 dilution of 10 mg/ml stock, was used when indicated to counterstain nuclei (>95% of cells stained had polymorphic nuclei consistent with PMN nuclear morphology). Quantification of fMLF-induced O-GlcNAc label by fluorescence intensity was performed using Image J (National Institutes of Health).

GlcNH2 6-P and UDP-GlcNAc Determinations—5 x 106 PMNs were treated as indicated and analyzed for GlcNH2 6-P and UDP-GlcNAc as previously described (42). Briefly, the cells were centrifuged, and the pellet was extracted with 100 µl of 0.3 M perchloric acid. The extract was centrifuged for 10 min at 4 °C at 14,000 x g, and 200 µl of 1:4 trioctylamine:1,1,2-trichlorotrifluoroethane (freon) was mixed with the resulting supernatant. The mixture was centrifuged for 5 min at 4 °C at 14,000 x g, and the aqueous phase (top) was stored at –80 °C for up to 1 week. For HPLC determination of UDP-GlcNAc the aqueous phase was run on a strong anion exchange column (Partisil SAX; Thermo) with the detector set to 262 nm. The flow rate was 2 ml/min, with the mobile phases consisting of 5 mM NH4H2PO4, pH 2.8, and 750 mM NH4H2PO4, pH 3.7, for buffers A and B, respectively, with a linear gradient from 0% to 100% buffer B over 40 min. The retention time for UDP-GlcNAc was ~17 min. For the HPLC determination of GlcNH2 6-P 100 µl of the aqueous phase was mixed with 200 µl of 6 mM O-pthalaldehyde (OPA) (Sigma)/1% ethanol/0.2% mercaptoethanol/0.2 M boric acid, pH 9.7, for 1 min followed by neutralization with 400 µl of 100 mM NaH2PO4. The sample was then loaded onto a C-18 column (Ultrasphere; Beckman Coulter) with the detector set to 340 nm. The flow rate was 0.5 ml/min, with the mobile phases consisting of 90% 16.7 mM NaH2PO4, pH 7.2/5% iso-propanol/5% acetonitrile and 76% 19.7 mM NaH2PO4, pH 7.2/12% iso-propanol/12% acetonitrile for buffers A and B, respectively, with a linear gradient from 0% to 100% B over 15 min followed by a linear gradient of 100% to 0% buffer B over 10 min. The retention time for GlcNH2 6-P was ~9.5 min. The data were analyzed and quantified as area under the curve by System Gold Nouveau software (Beckman Coulter). For determination of nanomoles of UDP-GlcNAc/mg of protein and picomoles of GlcNH2 6-P/mg of protein, UDP-GlcNAc (Sigma) and GlcNH2 6-P (Sigma) standards were run to establish molar reference values. PMN cell equivalents lysed in 1% Nonidet P-40/0.5% sodium deoxycholate/50 mM Tris-HCl, pH 7.4/150 mM NaCl were assayed using the Lowry method (43) for protein concentration determination.

Chemotaxis Assays—2 x 105 PMNs were pre-treated as indicated, washed, resuspended in Dulbecco's modified Eagle's medium/5% bovine serum albumin and then placed in the upper filter plate of a Multi-Screen-MIC chemotaxis plate (Millipore) with 3-µm membrane pores. The lower receiver plate contained Dulbecco's modified Eagle's medium and 100 µM fMLF where indicated. Basal chemotaxis was assessed in the absence of fMLF. The chemotaxis chambers were placed in a 37 °C incubator for 45 min. The filter plate was then carefully removed, and migrating cells were counted by microscopic examination of receiver plates (44).

Respiratory Burst Assays—PMN respiratory burst activity was assessed by luminol-dependent chemiluminescence (45). The PMNs were pretreated where indicated, and all samples were rapidly washed before being assayed (the presence of free GlcNH2 in the chemiluminescence buffer was found to inhibit chemiluminescence readings). Samples were prepared by mixing 106 washed PMNs into HBSS/10 µM luminol/100 µM sodium azide/10 units/ml horseradish peroxidase. Chemiluminescence was monitored at room temperature in a tube-based luminometer (Optocomp 1; MGM Instruments) without stirring or shaking. Baseline readings were established over 30 s followed by stimulation with fMLF as indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PMNs Display Rapid Agonist-induced Changes in O-GlcNAc—The formylated peptide fMLF is a well established and widely used PMN agonist. It rapidly activates a diverse group of signal transduction pathways by engaging a specific G-protein-coupled receptor (33). We used fMLF to assess whether receptor-associated stimulation could signal protein O-GlcNAcylation. 2 min of stimulation with 100 nM fMLF led to increased protein-associated O-GlcNAc as determined by immunoblot analyses of whole cell extracts with the O-GlcNAc-specific antibody CTD110.6 (Fig. 1A). A variety of proteins with molecular weights above ~50 kDa were observed, with some variation in the banding pattern and/or intensity of some bands from experiment to experiment, as might be expected for primary PMN isolates. Additional analyses revealed that O-GlcNAcylation begins within 1 min of stimulation and progresses through 5 min (Fig. 1B). In each experiment the specificity of CTD110.6 immunoreactivity was established by competitively blocking antibody binding with free GlcNAc.



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  		FIG. 1.
PMNs display rapid agonist-induced protein O-GlcNAcylation as assessed by O-GlcNAc-specific immunoblots. Immunoblots were performed using the anti-O-GlcNAc antibody CTD110.6. The specificity of the antibody for protein O-GlcNAc was determined by adding 10 mM GlcNAc to the primary antibody dilution buffer (indicated as +GlcNAc). A, PMNs were left unstimulated (control) or stimulated with agonist, 100 nM fMLF, for 2 min. B, PMNs were left unstimulated (control) or stimulated with 100 nM fMLF for 1, 3, or 5 min.

 
Although the O-GlcNAc-specific antibody CTD110.6 has been successfully used previously in immunoblot and enzyme-linked immunosorbent assay-based assays (41), it has not been evaluated with respect to immunocytochemical microscopy. We developed a protocol using CTD110.6 in a fluorescence-based immunocytochemical assay as an alternate and complementary method of examining changes in O-GlcNAc modifications. 100 nM fMLF increased O-GlcNAc over 2 min of stimulation (Fig. 2A). We obtained low background, antibody-specific signal using formaldehyde fixation followed by methanol permeation. The permeation agent was a critical variable in developing this assay, as commonly used agents such as Triton and acetone yielded high nonspecific backgrounds (data not shown). The agonist-induced CTD110.6 signal was specific for protein-associated O-GlcNAc, because it was competitively blocked by free GlcNAc. These results paralleled our immunoblot data.



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  		FIG. 2.
Immunofluorescence microscopy reveals rapid agonist-induced protein O-GlcNAcylation. Immunofluorescence labeling was performed using the anti-O-GlcNAc antibody CTD110.6. The specificity of this antibody for protein O-GlcNAc was assessed by adding 100 mM GlcNAc to the primary antibody dilution buffer (indicated as +GlcNAc). A, PMNs were left unstimulated (control) or stimulated with 100 nM fMLF for 2 min. Two predominant labeling patterns were evident: a relatively intense punctate label and a less intense, diffuse and homogenous label. B, the more intense, punctate O-GlcNAc label was limited to the cytoplasm, as was evident when the cells were counterstained using the nuclear stain Hoechst 33258. PMNs were left unstimulated (control) or stimulated with 100 nM fMLF for 2 min. Higher magnification images of selected cells revealed a punctate label that was localized exclusively to the cytoplasm (the arrows in the low magnification images that are labeled as C and F, control, and fMLF, respectively, are the individual cells shown in the higher magnification views).

 
CTD110.6 labeled both the nucleus and cytoplasm in resting and stimulated cells. The cells displayed two predominant patterns of label: a less intense, diffuse, and relatively homogenous label and a comparatively intense, non-homogenous, punctate label that, in contrast to a Hoechst counterstain that clearly defined the polymorphic multilobed PMN nucleus, was restricted to the cytoplasm. The punctate cytoplasmic label displayed the major increase in fluorescence intensity upon agonist stimulation (Fig. 2B). Labeling of the nucleus was more diffuse and considerably less intense than the punctate cytoplasmic label, although it too increased with stimulation. This was somewhat unexpected, because a variety of O-GlcNAcylated proteins are known to be nuclear, and a more intense nuclear signal might be anticipated (7). It is not clear whether this was a property of the particular labeling protocol used, a feature of O-GlcNAc-modified proteins in PMNs, or a reflection of a specific property of the antibody and/or the cell.

Of further interest, this assay also proved to be a sensitive means of assessing the temporal dynamics of agonist-induced O-GlcNAcylation. Increases in protein O-GlcNAcylation were evident within 30 s of stimulation with fMLF and returned to near resting levels after 10 min (Fig. 3). This provided further support for the notion that O-GlcNAc is a rapidly inducible post-translation protein modification.



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  		FIG. 3.
Immunofluorescence microscopy is an effective means of determining the temporal dynamics of agonist-induced protein O-GlcNAcylation. Immunofluorescence labeling was performed using the anti-O-GlcNAc antibody CTD110.6, and Hoechst 33258 was used as a nuclear counterstain. fMLF induced robust cytoplasmic O-GlcNAcylation within 30 s that continued for at least 5 min, after which it appeared to decrease toward resting levels. PMNs were left unstimulated (C) or stimulated with 100 nM fMLF for various times ranging from 30 s (F 30 s) to 10 min (F 10min). The inset shows quantification of fMLF-induced O-GlcNAc label by fluorescence intensity (n = number of cells examined for each condition; *, indicates p < 0.05 in comparison to control as evaluated by Student's t test).

 
PMNs Possess a Functional HBP That Can Use Exogenous GlcNH2 to Form the Metabolic Precursors for Protein O-GlcNAcylation—The HBP plays an integral role in O-GlcNAc signaling, providing UDP-GlcNAc, the necessary substrate to drive OGT activity and O-GlcNAcylation (21). Having established that PMNs posses a robust and rapidly inducible O-GlcNAcylation mechanism, we next asked what the effects of increasing flux through the HBP would have in PMNs. GlcNH2 is an exogenous substrate for the HBP that is commonly used to drive HBP-dependent O-GlcNAcylation (6, 21, 23). On entering a cell it is phosphorylated by hexokinase to form GlcNH2 6-P, thus directly entering the HBP. Further processing yields UDP-GlcNAc, the HBP product necessary for O-GlcNAcylation.

We examined GlcNH2 6-P and UDP-GlcNAc levels in GlcNH2-treated PMNs by HPLC. Resting GlcNH2 6-P levels were about 20-fold less than UDP-GlcNAc levels, ~0.06 pmol/mg of protein versus ~1.4 nmol/mg of protein (Fig. 4). UDP-GlcNAc was the predominant sugar nucleotide in PMNs and was a major phospho-nucleotide, along with ADP, ATP, and UDP-glucose (data not shown). This suggests that a relatively abundant pool of UDP-GlcNAc is important for normal PMN physiology and function. Short duration treatments with 10 mM GlcNH2 led to the production of both GlcNH2 6-P and UDP-GlcNAc. GlcNH2 6-P levels rose following GlcNH2 treatment, doubling over 2 min and increasing ~8-fold by 60 min (Fig. 4A), indicating that GlcNH2 rapidly entered the cells and was quickly phosphorylated by hexokinase. The increase in UDP-GlcNAc levels clearly followed the rise in GlcNH2 6-P (Fig. 4B), as might be expected for HBP processing. Although the percent increase in UDP-GlcNAc was significantly smaller than that for GlcNH2 6-P at any given time (for example, 60% versus 700% after 60 min), the molar increase in UDP-GlcNAc was greater than that for GlcNH2 6-P (an increases of 0.83 versus 0.42 nmol/mg of protein after 60 min). No drop in cellular ATP levels was observed for GlcNH2 treatment (data not shown). In fMLF-treated cells there was no apparent change in GlcNH2 6-P or UDP-GlcNAc levels with stimulation (data not shown).



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  		FIG. 4.
PMNs metabolize exogenous GlcNH2 into metabolic precursors that are used in the pathway leading to protein O-GlcNAcylation. A, GlcNH2 6-P levels in PMNs treated with GlcNH2. PMNs were left untreated (C) or treated with 10 mM GlcNH2 for 2, 10, 30, or 60 min at 37 °C. OPA-conjugated acid extracts (n = 3 for each condition) were analyzed on a C-18 column using HPLC. B, UDP-GlcNAc levels in PMNs treated with GlcNH2. PMNs were left untreated (C) or treated with 10 mM GlcNH2 for 2, 10, 30, or 60 min at 37 °C. Acid extracts (n = 3 for each condition) were analyzed on a strong anion exchange column using HPLC.

 
GlcNH2 Increases Protein-associated O-GlcNAc under the Same Conditions That Increase UDP-GlcNAc—We next determined if the observed GlcNH2-induced changes in HBP flux were associated with alterations in O-GlcNAc. Short duration GlcNH2 treatment, 10 mM for 30 min, enhanced protein O-GlcNAc in whole cell extracts as determined by immunoblotting with the O-GlcNAc-specific antibody CTD110.6 (Fig. 5A). A group of proteins bearing O-GlcNAc with a wide range of molecular weights above ~50 kDa was observed, with some variation in the banding pattern from experiment to experiment. The overall banding pattern was similar to that induced by fMLF (refer to Fig. 1, A and B). The specificity of these immunoblots in identifying proteins modified through GlcNH2-specific metabolism was confirmed by pre-treatment with 10 mM galactosamine (GalNH2), a stereoisomer of GlcNH2, that did not lead to increased O-GlcNAc in the same time frame. The specificity of CTD110.6 for detecting O-GlcNAcylated proteins was again established through competitive binding assays using free GlcNAc.



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  		FIG. 5.
GlcNH2 increases protein O-GlcNAc under the same conditions that increase UDP-GlcNAc. A, immunoblots were performed using the anti-O-GlcNAc antibody CTD110.6. The specificity of the antibody for protein O-GlcNAc was assessed by adding 10 mM GlcNAc to the primary antibody dilution buffer (indicated as +GlcNAc). Substrate driven O-GlcNAcylation was assessed by adding 10 mM GlcNH2 or 10 mM GalNH2 for 30 min at 37 °C. Control PMNs were left untreated. B, single matched UDP-GlcNAc (HPLC) and O-GlcNAcylation (immunoblots with CTD110.6) analyses for PMNs treated with 0.1, 1, or 10 mM GlcNH2 for 10, 30, 60, and 120 min each at 37 °C. Control cells were left untreated. C, immunofluorescence labeling was performed using the anti-O-GlcNAc antibody CTD110.6. The specificity of this antibody for protein O-GlcNAc was assessed by adding 100 mM GlcNAc to the primary antibody dilution buffer (indicated as +GlcNAc). PMNs remained untreated (left) or were treated with 10 mM GlcNH2 for 30 min at 37 °C (right).

 
To investigate the relationship between increased UDP-GlcNAc and protein O-GlcNAc in our system, aliquots from the same PMN preparation were taken for each experimental condition (Fig. 5B). These experiments used PMNs from a single donor and cell isolation, eliminating day-to-day and inter-donor variation. We found that significant changes in GlcNH2-induced protein O-GlcNAcylation corresponded with only modest increases in UDP-GlcNAc. UDP-GlcNAc levels and O-GlcNAcylation were dependent on the GlcNH2 dose and pre-treatment duration: higher doses and longer pre-treatments enhanced UDP-GlcNAc and O-GlcNAc, whereas lower doses and shorter pre-treatments were either without effect or produced more modest increases. There was an apparent threshold for robust O-GlcNAcylation at 60 min of treatment for all concentrations tested, although this did not clearly reflect a defined level of UDP-GlcNAc. However, there was a trend for progressively elevated UDP-GlcNAc to correspond to progressively enhanced O-GlcNAc (for the representative experiment shown in Fig. 5B this was especially evident for the 10 and 1 mM GlcNH2 conditions).

We used our previously described fluorescence-based immunocytochemical assay again as an alternate and complementary method of evaluating GlcNH2-induced O-GlcNAcylation. Cells treated with 10 mM GlcNH2 for 30 min showed a significantly increased fluorescent signal versus untreated controls (Fig. 5C). The signal was specific for O-GlcNAc, because it was competitively blocked by free GlcNAc. These results paralleled and confirmed our immunoblot data. Interestingly, the GlcNH2-associated increase in CTD110.6 label was, as it was by immunoblot, grossly similar to that observed for fMLF (refer to Fig. 2, A and B). The effects of GlcNH2 were most readily apparent in the cytoplasm, with a relatively large increase in the punctate label. The more diffuse and homogenous labeling pattern, present in the cytosol and the nucleus, was also increased by GlcNH2 treatment.

GlcNH2 Treatment Amplifies fMLF-induced Protein O-GlcNAcylation—We next asked if GlcNH2 was effective in conjunction with fMLF as a means of manipulating agonist-associated O-GlcNAcylation. Combined GlcNH2 and agonist treatments were additive for O-GlcNAcylation, with short duration GlcNH2 pre-treatments, which do not significantly increase O-GlcNAc by themselves (refer to Fig. 5B), leading to noticeably increased fMLF-induced O-GlcNAc (Fig. 6). These additive effects were most clearly evident at higher GlcNH2 concentrations. These experiments also confirmed that the pattern of O-GlcNAcylation due to fMLF approximated that observed for GlcNH2.



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  		FIG. 6.
GlcNH2 treatment modifies fMLF-induced protein O-GlcNAcylation. Immunoblots were performed using the anti-O-GlcNAc antibody CTD110.6. PMNs were pretreated as indicated with 10, 1, or 0.1 mM GlcNH2 for 10, 30, 60, and 120 min at 37 °C. The cells were then stimulated with 100 nM fMLF for 2 min (indicated by F for the non-pretreated sample). The control sample, C, was not stimulated and was not pretreated.

 
GlcNH2 Selectively Modulates PMN Functional Outputs— Having shown that GlcNH2 pre-treatment can be used to modulate fMLF-induced O-GlcNAcylation, we attempted to elucidate what effect(s) this had on fMLF-associated function. This would provide an initial determination of whether changes in O-GlcNAc have a signaling role. We examined the effect of GlcNH2 treatment on agonist-induced respiratory burst (defined by the production of reactive oxygen species) and chemotactic activities. GlcNH2 pre-treatments of various durations, 10–120 min, and concentrations, 0.1–10 mM GlcNH2, had little effect (in all cases statistically non-significant, p > 0.05) on the respiratory burst elicited by 100 nM fMLF in luminol-dependent assays (Fig. 7A). There was a trend toward reduced and slower onset activity for short duration GlcNH2 pre-treatments, 10 and 30 min, and somewhat enhanced and faster onset activity for long duration pre-treatments, 120 min. The GlcNH2 treatments did not affect cell viability or morphology (data not shown).



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  		FIG. 7.
GlcNH2 modulates PMN chemotaxis but not respiratory burst activity. A, respiratory burst activity was assessed by luminol-dependent chemiluminescence. PMNs (n = 3 for all conditions) were pretreated with 0.1, 1, or 10 mM GlcNH2 for 10, 30, 60, and 120 min at 37 °C and then stimulated with 100 nM fMLF (open circles {circ}). Control samples were not pretreated with GlcNH2 but were stimulated with 100 nM fMLF (closed circles ). In all cases fMLF stimulation was preceded by 30 s of baseline recording. B, chemotactic activity was assessed in Boyden chamber-type migration assays (n = 3 for all conditions). PMNs did not receive a pretreatment, Con and F, or were pretreated with 10 mM GlcNH2 30 min at 37 °C, G and F+G. Basal levels of chemotaxis were monitored in the absence of chemotactic factor, Con and G, and fMLF-induced chemotaxis was monitored in the presence of 100 nM fMLF, F and F+G. Assays were carried out at 37 °C for 45 min. C, chemotactic activity was assessed in 96-well Boyden chamber-type migration assays (n = 3 for all conditions). PMNs were not pretreated, Con and 0, or pretreated with 0.1 mM (light gray bars ), 1 mM (white bars {square}), or 10 mM (dark gray bars {blacksquare}) GlcNH2 for 10, 30, 60, and 120 min at 37 °C as indicated. 100 nM fMLF was used to initiate chemotaxis in samples labeled 0, 10, 30, 60, and 120 min. The control sample, Con, was not stimulated with chemotactic factor (basal chemotaxis). Assays were carried out at 37 °C for 45 min.

 
An initial assessment of the effect of short duration GlcNH2 pre-treatment, 10 mM for 30 min, on chemotaxis revealed that GlcNH2 significantly increases both basal and fMLF-induced activity (60 and 63%, respectively) in simple 45-min Boyden chamber-type assays (Fig. 7B). These data supported the possibility that GlcNH2 selectively modulates only certain aspects of neutrophil physiology. We further defined GlcNH2-enhanced chemotaxis by altering both the duration of GlcNH2 pre-treatment and the concentration of GlcNH2 used (Fig. 7C). The chemotactic response was dependent on the GlcNH2 dose and pre-treatment duration: higher doses and longer pre-treatments clearly enhanced chemotactic activity toward fMLF, whereas lower doses and shorter pre-treatments were without significant effect. Significantly enhanced chemotaxis was observed for all GlcNH2 doses tested by 120 min of pre-treatment, with 10 mM GlcNH2 requiring as little as 10 min of pre-treatment and 1 mM requiring ~60 min.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Protein O-GlcNAcylation is emerging as an important posttranslational modification that regulates protein function. However, the physiological contexts in which rapid changes in the level of O-GlcNAc occur are only beginning to be elucidated. Changes in O-GlcNAcylation are implicated in the pathogenesis of diabetes mellitus, a disease characterized by chronic hyperglycemia (6, 14, 15), and have also been suggested to serve as a nutrient sensor, in which O-GlcNAc signals are modulated through nutrient availability (46, 47). Recently, work by Zhang has suggested that O-GlcNAc may couple proteasomes to the general metabolic state of the cell (48), and Zachara has provided evidence that O-GlcNAc modulates stress-activated signaling networks and may be integral to cellular survival responses (49). In addition, O-GlcNAcylation is also often compared with phosphorylation (8–10), which is fundamental in the regulation of a variety of signaling pathways. The central feature of many receptor-mediated signaling events, in which extracellular agonists engage cognate receptors to activate signaling pathways, involves rapid alterations in protein phosphorylation. As yet, there is only limited evidence to suggest such a role for O-GlcNAc (6, 29, 30). Using a PMN model and fMLF as an extracellular stimulus, we have found that O-GlcNAc can be rapidly induced in a receptor-dependent manner and consequently may act as a regulatory signal for downstream functional responses.

The receptor-derived signal for many functional responses is rapidly transduced, occurring over tens of seconds. Our data show changes in O-GlcNAc within this time frame. In conjunction with modified chemotactic responsiveness as a result of O-GlcNAc-inducing treatments, these data support a role for this post-translational modification in rapidly dynamic signal transduction. fMLF rapidly and robustly increased O-GlcNAc on a large number of proteins. The rate at which this modification was induced was evident in both immunofluorescence microscopy and immunoblots assays, in which fMLF caused an easily detected increase in CTD110.6 immunoreactivity within 30 s and 1–2 min, respectively. Furthermore, a number of proteins were modified by O-GlcNAc in response to fMLF. Our immunofluorescence analysis of the CTD110.6 label revealed that the immunoreactivity was predominantly cytoplasmic with the most concentrated labeling being associated with punctate structures in the cell. It is not clear whether this pattern is truly reflective of O-GlcNAc distribution or is in part a consequence of the methodology, because a variety of nucleus-associated proteins are known to be O-GlcNAcylated, including nuclear pore proteins and transcription factors (7). It may be that the large size of the CTD110.6 IgM prevents it from efficiently penetrating the nucleus, even in fixed cells. Nonetheless, the label we detected was shown to be specific for O-GlcNAc by competition with free GlcNAc, providing evidence for the validity of the assay in assessing the temporal dynamics of O-GlcNAcylation. In addition, GlcNH2-induced increases in O-GlcNAc, in both immunoblot and immunofluorescence analyses, revealed similar results to those observed for fMLF. As a whole, these data provide substantive evidence of agonist-induced O-GlcNAcylation and represent the first evidence that such changes occur over short intervals.

We felt it essential to establish that exogenous GlcNH2 would be metabolized by the HBP in a comparable time frame. Short term GlcNH2 treatments increased the levels of HBP metabolites, first GlcNH2 6-P and then UDP-GlcNAc. UDP-GlcNAc formation was dependent on the time and concentration of GlcNH2 treatment, with treatments at high GlcNH2 concentrations, 10 mM, significantly increasing UDP-GlcNAc within 30 min while lower concentrations required 60–120 min. Robust increases in O-GlcNAcylation occurred at 60-min GlcNH2 treatment for most concentrations tested, although 10 mM GlcNH2 increased O-GlcNAc within 30 min. Thus, when compared with the respective UDP-GlcNAc levels, there is a parallel between increases in UDP-GlcNAc and O-GlcNAcylation. Interestingly, at 100 µM GlcNH2 increases in O-GlcNAcylation correlated with increases in UDP-GlcNAc of only 20–25%. These modest increases are likely relevant, although, because the protein acceptor specificity of OGT has been shown to be sensitive to small changes in UDP-GlcNAc (22). In addition, the observed levels of UDP-GlcNAc reflect net changes and not just UDP-GlcNAc synthesis and, thus, do not take into account its utilization, which may be significant as the length of GlcNH2 treatment increases. Furthermore, the observed levels may not accurately reflect localized concentration changes that could occur in particular cellular compartments. This is relevant because cytosolic levels of UDP-GlcNAc are thought to be 10–20 times lower than endoplasmic reticulum/Golgi levels (50, 51).

We also combined GlcNH2 and agonist treatments and found additive increases in O-GlcNAc, in particular for several high molecular weight proteins in the short duration GlcNH2 pretreatments. These additive changes varied by protein, but clearly suggest that GlcNH2 might modify agonist-induced signal transduction by altering the level of O-GlcNAcylation induced by the agonist itself. This also suggests how GlcNH2 might alter agonist-induced functional responses such as chemotaxis.

By understanding the conditions under which UDP-GlcNAc and O-GlcNAc levels can be manipulated through HBP metabolism, we have begun to establish conditions for understanding how O-GlcNAc affects PMN function. The same conditions that altered HBP metabolism, UDP-GlcNAc formation and O-GlcNAcylation, also affected chemotactic but not respiratory burst activity. This suggests that protein O-GlcNAc modulates certain aspects of agonist-induced signaling. Furthermore, the diverse signaling pathways that lead from fMLF-associated receptor engagement to chemotactic and respiratory burst activity are different (34, 52–54).

In conclusion, our results support the theory that O-GlcNAc is a rapidly induced signal that operates in the context of physiological receptor-mediated signal transduction. The ubiquitous nature of this post-translational modification, the large numbers of target proteins that are either suggested or known to be modified by it, and its similarities to phosphorylation in modulating protein function suggest that it may play a general role in dynamic signaling in response to cellular stimulation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK55647 and the Juvenile Diabetes Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Cell Biology, University of Alabama, 1918 University Blvd., MCLM 690, Birmingham, AL 35294. Tel.: 205-934-1294; Fax: 205-934-0950; E-mail: marchase{at}uab.edu.

1 The abbreviations used are: O-GlcNAc, O-linked {beta}-N-acetylglucosamine; UDP-GlcNAc, UDP-N-acetylglucosamine; OGT, UDP-Glc-NAc, polypeptide O-{beta}-N-acetylglucosaminyltransferase (EC 2.4.1.94 [EC] ); GlcNH2, glucosamine; GalNH2, galactosamine; GlcNH2 6-P, glucosamine 6-phosphate; fMLF, formyl-methionine-leucine-phenylalanine tripeptide; PMN, neutrophil or polymorphonuclear leukocyte; HBP, hexosamine biosynthesis pathway; HBSS, Hanks' buffered salt solution; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; OPA, O-pthalaldehyde; DMEM, Dulbecco's modification of Eagle's medium. Back


   ACKNOWLEDGMENTS
 
We thank Sherry Johnson for administrative support and Pam Bounelis for insightful input. We additionally thank Mary Ann Accavitti and the University of Alabama at Birmingham (UAB) Epitope Recognition and Immunodetection Core for the production of the CTD110.6 monoclonal antibody and Albert Tousson and the UAB High Resolution Imaging Facility for technical expertise with respect to immunofluorescence microscopy.



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