Glycobiology Advance Access originally published online on October 11, 2007
Glycobiology 2007 17(12):1357-1364; doi:10.1093/glycob/cwm105
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Modification of Topoisomerase I Activity by Glucose and by O-Glcnacylation of the Enzyme Protein
2 Shraga Segal Department of Microbiology and Immunology, Ben-Gurion University Cancer Research Center, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel
1 To whom correspondence should be addressed: Tel: 972-8-6479537; Fax: 972-8-6479579; e-mail: priel{at}bgu.ac.il
Received on May 29, 2007; revised on September 23, 2007; accepted on September 24, 2007
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Abstract |
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The regulation of topoisomerase I (topo I) activity is of prime importance for gene expression. It participates in DNA replication, transcription, recombination, and DNA repair, and serves as a target for anticancer drugs. Many proteins and enzymes are modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), which exerts profound effects on their function. However, the modification of topo I by O-GlcNAc and the effect on its activity has not been previously reported. Here, we show that topo I protein is modified by O-GlcNAc in vitro in the porcine proximal tubular epithelial cell line (LLPCK-1), and in vivo in the mouse kidney. The level of O-GlcNAcylation of topo I protein correlates well with the enzyme activity, namely, a decrease in O-GlcNAc results in a reduction in topo I activity, and vice versa. O-GlcNAc transferase (OGT) was coprecipitated with topo I protein, suggesting a possible interaction between both enzymes. In addition, treatment of cells with glucosamine increased topo I activity and O-GlcNAcylation. The results of this study provide a novel mechanism for the regulation of topo I activity. Topo I is important for DNA transcription, therefore, its regulation by GlcNAcylation contributes to the mechanism by which glucose levels affect gene expression, and may pave the way to the development of new drugs that could control topo I activity.
Key words: DNA relaxation / glucose / O-GlcNAc / O-GlcNAc transferase (OGT) / topoisomerase I
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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DNA topoisomerases (topo) are essential nuclear enzymes, which participate in determining the topological state of the DNA. These enzymes are involved in a variety of DNA transactions including replication, transcription, recombination, repair, and nucleosomal condensation (Champoux 2001
; Wang 2002). Topoisomerases are classified as either type I or type II, and members of each family of these enzymes are distinct in sequence, structure, and functions (Roca 1995; Wang 1996; Champoux 2001; Wang 2002; Forterre et al. 2007). The eukaryotic type I topoisomerase (topo IB) plays an important role in various DNA transactions and in the maintenance of genomic stability (Champoux 2001; Wang 2002). Its inactivation leads to embryonic death at the 4–16 cell stage gestation (Morham et al. 1996).
Topo IB can relax both positive and negative supercoiled DNA by the formation of a transient single-strand DNA break, in which the active site tyrosine becomes attached to the 3' phosphate end of the cleaved strand, followed by rotation of the DNA and religation process (Champoux 2001; Wang 2002). Mammalian DNA topoisomerases are the targets of several anticancer drugs currently in clinical use (Wang 1996; Li and Liu 2000; Wang 2002). The activity of topo IB is regulated by posttranslational modifications of the enzyme protein. In vitro phosphorylation of mammalian topo I, predominantly at serine, by casein kinase II and protein kinase C is necessary for its DNA relaxation activity while dephosphorylation decreased its activity (Pommier et al. 1998). However, phosphorylation of topo I by the c-Abl tyrosine kinase enhanced its activity (Yu et al. 2004). The topo I protein is subjected to polyadenosine diphosfate ribosylation by poly-ADP ribose polymerase (PARP), which downregulates its DNA relaxation activity (Pommier et al. 1998).
Recent studies have demonstrated that several proteins and enzymes are modified by O-GlcNAc including kinases, phosphatases, transcription factors, chaperons, cytoskeleton proteins, and metabolic enzymes (Zachara and Hart 2002, 2004; Love and Hanover 2005). Changes in the level of O-GlcNAc of specific enzymes or proteins affect their activities or regulation (Parker et al. 2003), protein–protein interactions (Roos et al. 1997; InnOc Han and Jeffrey 1998), DNA binding ability (Gao et al. 2003), subcellular localization, and the half-life and proteolytic processing of these proteins (Hanover et al. 1987; Datta et al. 1989; Ray et al. 1992). The regulation of O-GlcNAcylation of proteins is achieved by the concerted regulation of two enzymes, the O-GlcNAc transferase (OGT) and the O-GlcNAcase (Zachara and Hart 2002, 2004). OGT adds a single N-acetylglucoseamine sugar to either serine or threonine residues of proteins (Kreppel et al. 1997; Lubas et al. 1997; Lubas and Hanover 2000). O-GlcNAcase is a member of a family of 84 glycoside hydrolases that cleave O-GlcNAc from the modified serine and threonine residues of proteins (Gao et al. 2001; Macauley et al. 2005). The involvement of topo I in essential cellular processes led us to investigate the possibility that topo I protein is modified by O-GlcNAc and the influence of this modification on the enzyme activity. In this study, we found that topo I protein is modified in vitro and in vivo by O-GlcNAcylation, and the DNA relaxation activity of topo I is regulated by the level of O-GlcNAc in the enzyme protein.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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Topo I protein is recognized by anti-O-GlcNAc antibody
The activity of topo I is regulated by posttranslational modifications of the enzyme protein such as phosphorylation/ dephosphorylation of serine/threonine or tyrosine residues (Pommier et al. 1998
; Yu et al. 2004), or by ADP-ribosylation (Pommier et al. 1998). However, an O-GlcNAcylation of the topo I protein was not reported. Since several proteins were shown to be modified by O-GlcNAc, which influence their activity and substrate recognition (Zachara and Hart 2002, 2004), we investigated whether topo I is modified by O-GlcNAc using: (1) anti-O-GlcNAc monoclonal antibody, which were previously shown to react specifically with O-GlcNAc residues in proteins (Macauley et al. 2005), (2) wheat germ agglutinin (WGA), a lectin which particularly binds GlcNAc residues in proteins (Wu et al. 1998). Nuclear proteins (200 µg) extracted from the cells grown at physiological concentrations of glucose (5 mM) were subjected to immunoprecipitation with anti-topo I antibody (Figure 1A), which were previously shown to react with topo I protein derived from different origins (Bendetz-Nezer et al. 2004, Plaschkes et al. 2005). The immunocomplexes were analyzed by Western blot assay by using the anti-O-GlcNAc monoclonal antibody (Figure 1A, lane 1). A protein band of 100 kDa, which was immunoprecipitated by anti-topo I antibody, was recognized by anti-O-GlcNAc antibody. To verify whether this band indeed represent topo I protein, the membrane was stripped from the O-GlcNAc antibody and reprobed with anti-topo I antibody which, as expected, reacted with the 100 kDa protein (Figure 1A, lane 2). To substantiate these results, the extracted nuclear proteins (200 µg) from cells grown in a physiological glucose concentration were subjected to immunoprecipitation with the anti-O-GlcNAc antibody in the absence or presence of 200 mM GlcNAc, and the immunocomplexes were analyzed by the Western blotting using anti-topo I antibody (Figure 1A, lanes 3 and 4). The results show that a 100 kDa protein that was immunoprecipitated by the O-GlcNAc antibody is recognized by anti-topo I antibody (lane 3). The recognition of topo I protein by the anti-O-GlcNAc antibody is significantly reduced when GlcNAc molecules were present during the immunoprecipitation process (lane 4). These data may suggest that the topo I protein is modified by the O-GlcNAc in cells grown under a physiological glucose concentration.
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Topo I protein is recognized by WGA, a GlcNAc specific lectin
To further prove the O-GlcNAcylation of topo I protein, we used WGA, an N-acetylglucoseamine-specific lectin that binds to GlcNAcylated proteins (Wu et al. 1998
; Bonnin et al. 1999). Agarose-WGA was added to the extracted nuclear proteins and following incubation, and centrifugation, as described in the "Material and methods" section, the WGA/protein complexes were analyzed on 10% sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). The Western blot analysis was performed with specific anti-topo I or anti-OGT antibodies. OGT was shown to undergo autoglycosylation, and thus, serves here as a positive control (Lubas and Hanover 2000). The results depicted in Figure 1B show that topo I protein was precipitated with the agarose WGA (lane 1) as well as the OGT protein (lane 4), which is a GlcNAcylated protein (Lubas and Hanover 2000). To show that the agarose-WGA indeed binds GlcNAc residues in topo I protein, a solution of agarose WGA, which contains 20 mM GlcNAc was used for the precipitation of topo I. The results depicted in Figure 1B show that in the presence of GlcNAc, a faint band of topo I protein was precipitated by WGA (lane 2) while most of the topo I protein remained in the supernatant (lane 3). These results indicate that topo I protein is indeed modified by the GlcNAc residues.
Nuclear OGT protein is coprecipitated with topo I protein by anti-topo I antibody
Since OGT is the enzyme that catalyze the GlcNAcylation of proteins (Lubas and Hanover 2000), one may assume that topo I is also GlcNAcylated by this enzyme. We examined the possibility that OGT protein is coprecipitated with topo I protein. Anti-topo I antibody was added to the nuclear proteins derived from the cells grown at 5 mM glucose, and the immune complexes were analyzed by SDS-PAGE followed by Western blotting with anti-OGT antibody. The results depicted in (Figure 1B, lane 5) show that the OGT protein was coprecipitated with topo I protein in physiological level of glucose, suggesting a possible interaction between both enzymes.
Topo I protein derived from mouse kidney nuclear extracts is modified by O-GlcNAc
The aforementioned experiments were performed on a kidney cell line grown under cell culture conditions. To determine that topo I GlcNAcylation is not a phenomena of in vitro conditions, we examined topo I O-GlcNAcylation and the activity in mouse kidneys derived from Balb/C mice. Kidneys were removed from mice, and nuclear proteins were extracted from the kidney cells as described in "Materials and Methods."
Topo I activity (Figure 2A), topo I protein level (Figure 2B), and topo I O-GlcNAcylation (Figure 2C) were determined. As expected, a significant topo I activity was present in mouse kidney cells (Figure 2A, lanes 2–6). A 100 kDa protein present in the kidney nuclear extract was recognized by the anti-topo I antibody (Figure 2B). The immunoprecipitation analysis with anti-O-GlcNAc antibody followed by the Western blotting with either anti-topo I antibody or anti-O-GlcNAc antibody revealed that a 100 kDa protein precipitated by anti-O-GlcNAc antibody was recognized by anti-topo I antibody (Figure 2C, lane1) and anti-O-GlcNAc antibody (Figure 2C, lane 2). These results suggest that topo I protein is also modified in vivo by O-GlcNAcylation. Few additional proteins were precipitated from the kidney nuclear extracts, and were recognized by the anti-O-GlcNAc antibodies in Western blot analysis (lane 2) as expected, since the kidney nuclear extracts probably contains several O-GlcNAcylated proteins.
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Topo I activity and topo I O-GlcNAcylation is reduced in cells treated with Alloxan, an OGT inhibitor
The enzyme responsible for O-GlcNAcylation of proteins is OGT, which transfers an O-GlcNAc residue to proteins from uridine diphosphate (UDP)-GlcNAc (Kreppel et al. 1997
; Lubas et al. 1997; Lubas and Hanover 2000). To determine the effect of this modification on topo I activity, we treated cells with Alloxan, which was used in several studies as inhibitor of OGT (Konrad et al. 2002; Dauphinee et al. 2005; Liu et al. 2007). Cells (16 x 106 cells) grown at a physiological concentration of glucose were treated with 5 mM Alloxan (Konrad et al. 2002), for various intervals (0.5, 1, 2, 3, and 6 h). Different nuclear protein concentrations (0.2, 0.1, 0.05, and 0.025 µg) were added to a topo I specific reaction mixture, and the reaction products were analyzed by agarose gel electrophoresis. Topo I activity is measured by the conversion of supercoiled plasmid to its partially relaxed or fully relaxed forms. A representative picture is shown in Figure 3, and the results revealed that topo I activity is significantly reduced in Alloxan treated cells in a time dependent manner (Figure 3A, compare lane 3 to lanes 7, 11, 15, 19, and 23, and lane 4 to lanes 8, 12, 16, 20, and 24). The quantification of topo I activity (Bendetz-Nezer et al. 2004) from multiple experiments was performed for the results obtained with 50 ng of nuclear proteins. A reduction of 45–65% (p < 0.05) in topo I activity is observed in cells treated with Alloxan for 0.5–6 h (Figure 3B). The reduction in topo I activity following the Alloxan treatment might be due to: (1) a direct effect on topo I GlcNAcylation or (2) an indirect effect, which cause a reduction in topo I protein level or (3) an effect on other enzymes, which are involved on topo I posttranslational modifications.
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To examine the first possibility, namely, the effect of the Alloxan treatment on topo I GlycNacylation, the nuclear protein extracts from the aforementioned treatments were subjected to immunoprecipitation with anti-O-GlcNAc antibody, followed by Western blot analysis with anti-topo I antibody. The results depicted in Figure 3C (upper panel) revealed that the treatment of cells with Alloxan reduced the O-GlcNAcylation of topo I protein. Densitometric analysis of the results obtained from multiple experiments revealed a 50–60% reduction in the level of O-GlcNAcylation of topo I protein when the cells were treated with Alloxan for 0.5–6 h (Figure 3B). No effect on the levels of topo I protein or ß-actin protein (serves as a control) were observed in Alloxan treated cells, at the various examined intervals (Figure 3C, lower panel). The growth rate of the treated cells at the examined intervals was not affected (data not shown). These results indicate that the decrease in topo I activity following Alloxan treatment correlates with the reduction in topo I O-GlcNAcylation (Figure 3B).
Treatment of nuclear protein extracts with ß-N-Acetylglucosaminidase reduced topo I activity
The aforementioned experiments suggest that the O-GlcNAcylation of the topo I protein regulates its activity. To substantiate this assumption, we examined the effect of cleavage of GlcNAc residues from the topo I protein on its DNA relaxation activity. Nuclear proteins (100 ng) derived from LLPCK-1 cells, grown at 5 mM glucose, were treated with 0.01 units of ß-N-Acetylglucosaminidase for 15, 30, or 60 min, at room temperature, followed by the examination of topo I activity. Since topo I activity could be affected by the buffers and the reaction conditions used for the hexosaminidase activity, controls for each time point was performed as follows: the nuclear protein extracts in the above reaction buffers, without the addition of hexosaminidase, were kept at room temperature for the indicated intervals prior to the addition to the topo I reaction mixture. The results depicted in Figure 4 demonstrate a significant reduction in the DNA relaxation activity of topo I in the nuclear extract, which was pretreated with the hexosaminidase prior to the topo I assay (compare lanes 3–2 and 5–4). Under these reaction conditions, the DNA relaxation ability of topo I was not significantly affected (compare lanes 2, 4, and 6). Topo I activity was reduced by 50–85% of the control, depending on the time of the hexosaminidase reaction (4B) suggesting that the removal of GlcNAc residues from topo I protein caused a reduction in the enzyme activity.
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Exposure of cells to glucosamine for 6 h increased the activity and O-GlcNAcylation of topo I
Cells (LLC-PK1) were cultured in a medium containing 5 mM glucose or 5 mM glucose supplemented with 5 mM glucosamine for 6 h. Nuclear protein extracts were prepared and increasing nuclear proteins concentrations were added to a specific topo I reaction mixture. The results depicted in Figure 5A show a significant increase in topo I activity when the cells were exposed to glucosamine (compare lanes 2–4 to lanes 5–7). The same nuclear extracts were subjected to immunoprecipitation with anti-O-GlcNAc antibody followed by Western blotting with anti-topo I antibody. The results depicted in Figure 5B show an increase in the O-GlcNAcylation of topo I in cells exposed to 5 mM glucose + 5 mM glucosamine in comparison to cells cultured in 5 mM glucose only (compare lane 2 to lane 1).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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The DNA relaxation activity of mammalian topo I is required for various DNA transactions such as transcription, replication, recombination, and DNA repair (Wang 2002
; Leppard and Champoux 2005). The enzyme is preferentially involved in transcription processes, i.e., it is associated with actively transcribed regions of the chromatin (Brill DiNardo et al. 1987; Zhang et al. 1988; Khobta et al. 2005), it functions as a coactivator of transcription at the initiation phase and interacts with TFIID (Merino et al. 1993; Shykind et al.). Topo I plays an integral role in processes associated with ribosomal DNA (Leppard and Champoux 2005). Therefore, factors that affect the activity of this enzyme or/and posttranslational modifications of the enzyme protein that regulate its activity will influence essential cellular processes including gene expressions. The modification of more than 100 proteins and enzymes by O-GlcNAc have been reported (Vosseller et al. 2002; Zachara and Hart 2002, 2004; Love and Hanover 2005), and the levels of O-GlcNAc on key cellular proteins could be modulated by altering the extracellular glucose levels (Du Edelstein et al. 2001; Vosseller et al. 2002; Parker et al. 2003; Walgren et al. 2003; Zachara and Hart 2004). Topo I posttranslational modifications include phosphorylation/dephosphorylation on serine/threonine residues or on tyrosine residues and ADP-ribosylation (Pommier et al. 1998; Yu et al. 2004), but O-GlcNAc modifications of the topo I protein were not previously reported. In this study, we show that the topo I protein derived from porcine proximal tubular epithelial cells (LLC-PK1), grown at physiological glucose doses, is immunoprecipitated by anti-O-GlcNAc monoclonal antibodies and vice versa, topo I protein that was immunoprecipitated by topo I antibody reacted with anti-O-GlcNAc monoclonal antibody. To substantiate the notion that topo I is modified by O-GlcNAc, we showed that topo I protein was precipitated by agarose-WGA, which recognized O-GlcNAc residues on proteins. This topo I recognition by anti-O-GlcNAc antibody or by WGA was specific, since in the presence of free GlcNAc molecules, topo I was not precipitated either by the antibody or agarose-WGA. The results strongly indicate that topo I protein derived from LLC-PK1 cells is modified by O-GlcNAc. The coprecipitation of nuclear OGT (the enzyme responsible for O-GlcNAcylation of proteins) together with topo I (when anti-topo I antibody were used) strengthen our finding of topo I modification by GlcNAc. Furthermore, this posttranslational modification is not an in vitro phenomenon observed in cell culture, since we showed that the topo I protein derived from kidneys, which were removed from Balb/C mice, is also modified by O-GlcNAc.
To determine the effect of the modification of topo I protein by O-GlcNAc on its activity, we used three approaches: (1) treatment of the cells with Alloxan, a compound used by several investigators as an inhibitor of OGT, which as a consequence reduced the level of O-GlcNAcylation of proteins (Konrad et al. 2002; Dauphinee et al. 2005; Liu et al. 2007); (2) an in vitro assay in which the GlcNAc moiety is removed from nuclear proteins by the activity of Jack Bean hexosaminidase; (3) the effects of exposure of the cells to glucosamine at low glucose concentrations on the activity as well as on the O-GlcNAcylation level of topo I protein. A statistically significant reduction in topo I activity was observed in cells treated with Alloxan grown at physiological glucose concentrations. This reduction in the activity was accompanied by a significant decrease in the level of O-GlcNAcylated topo I but not in the level of topo I protein. Moreover, exposure of the cells to glucosamine increased topo I activity as well as topo I O-GlcNAcylation. In addition, removal of GlcNAc residues by hexosaminidase activity significantly reduced the topo I activity. Altogether, these results indicate that topo I activity is directly correlated with the O-GlcNAc level of topo I protein. An increase in topo I O-GlcNAcylation increased the activity of topo I and vice versa, a decrease in O-GlcNAcylation of topo I decreased the DNA relaxation activity of this enzyme. This phenomenon was observed with other nuclear proteins such as Sp1, in which the glycosylated form of Sp1 was more active than that of the nonglycosylated protein (Majumdar et al. 2003, 2004).
Phosphorylation of topo I protein on serine/threonine residues enhanced the activity of topo I and dephosphorylation decreased its activity (Pommier et al. 1998). Since O-GlcNAc modifications is a result of the addition of GlcNAc by OGT to serine or threonine residues, it is not yet clear how O-GlcNAcylation of the topo I protein affects its O-phosphorylation. However, phosphorylation and O-GlcNAc were shown to be reciprocal in some proteins such as the C-terminal domain of the large subunit of RNA polymerase II (Comer and Hart 2001), SV40 large T-antigen (Medina et al. 1998), Sp1, and other essential enzymes (Zachara and Hart 2002, 2004). What is the relationship between phosphorylation and O-GlcNAcylation of topo I, and how does it affects the enzyme activity and its regulation are still open questions.
Many cells have evolved mechanisms to sense glucose levels in their environment and to adapt the expression of genes to glucose availability (Towle 2005). Moreover, it was shown that the cellular O-GlcNAc level is influence by the extracellular concentrations of glucose (Vosseller et al. 2002). The O-GlcNAcylation of topo I and the influence of this modification on the enzyme activity suggest that the cellular topo I is regulated by the level of glucose and that hyperglycemic conditions may significantly affect the enzyme activity.
Topoisomerase I is the sole molecular target for the camptothecin class of anticancer drugs, which are used in the treatment of many types of cancer including colorectal and ovarian cancer (Garcia-Carbonero and Supko 2002). The efficacy of these anticancer drugs rely on the presence of active topo I since they exert their cytotoxic effects by binding to the transient covalent topo I-DNA complex (Pommier 1999). Therefore, the finding that topo I is modified by O-GlcNAc that affects the enzyme's activity is important for the development of new therapeutic strategies and possible new anti-topo I drugs.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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Cell culture
Porcine proximal tubular epithelial cells (LLC-PK1) (Takakura et al. 1995
) were grown in 5 mM (physiological value) glucose or in 5 mM glucose + 5 mM glucosamine in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal calf serum, streptomycin 10 µL /mL and 10 mM glutamine.
Compounds
A solution of Alloxan (Sigma–Eldrich) was freshly prepared at 10 mM in ddH2O. Glucosamine and GlcNAc were purchase from Sigma–Eldrich (Rehovot, Israel).
Preparation of nuclear extracts
The nuclear extracts from the various treated cells were prepared as described (Auer et al. 1982; Sambrook et al. 1989; Bendetz-Nezer et al. 2004) and a mixture of protease inhibitors (final concentrations: 2 µg/mL aprotinin, 2 µg/mL leupeptin, 1 µg/mL pepstatin A, 2 µg/mL antipain, and 100 µg/mL PMSF–phenyl-methylsulfonyl) were added to the extraction buffer. Total protein concentration was determined by using the BIO-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).
Preparation of nuclear extracts from mouse kidney
Kidneys from Balb/C mice were immersed in cold PBS (0.136 M NaCl, 2.68 mM KCl, 1.76 mM KH2PO4, 10.1 mM Na2HPO4, pH 7) and subjected to homogenization with a manual homogenizer, followed by centrifugation at 800 g in 4°C for 10 min. The resulting pellet was washed in 5 mL cold PBS and centrifuged again. To lyse erythrocytes, 1 mL of KCl (0.83%) was added to the pellet and sustained in room temperature for 6 min. The lysate was recentrifuged at 800 g in 4°C for 10 min, rewashed in 5 mL PBS, and centrifuged again. The preparation of nuclear extracts was made as described before.
Topo I assay
Topo I assay was performed as previously described (Bendetz-Nezer et al. 2004). Increasing (or, alternatively, specific) concentrations of nuclear proteins were added to a topo I reaction mixture containing, at a final volume of 25 µL, 20 mM Tris–HCl (pH 8.1), 1 mM dithiothreitol, 20 mM KCl, 10 mM MgCl2, 1 mM ethylene-diamine-tetra-acetic acid (EDTA), 30 µg/mL bovine serum albumin, and 250 ng pUC19 supercoiled DNA plasmid (MBI, Fermentas, Hanover, MD). Following incubation at 37°C for 30 min, the reaction was terminated by adding 5 µL of stopping buffer [final concentration; 1% sodium dodecyl sulfate, 15% glycerol, 0.5% bromophenol blue, and 50 mM EDTA (pH 8)]. The reaction products were analyzed by electrophoresis on a 1% agarose gel using a Tris/Borate/EDTA (TBE) buffer (89 mM Tris–HCl, 89 mM boric acid, and 62 mM EDTA) at 1 V/cm, stained by ethidium bromide (1 µg/mL), and photographed by using a short wavelength UV lamp (ChemiImagerTM 5500 equipment, Alpha Inotech Corporation, St. San Leandro, CA).
Densitometric analysis of the results were performed with the AlphaEasFC image processing and analysis software, and the percentage of topo I activity was calculated by using the following equation: [1–(sample/control)] x 100 (Bendetz-Nezer et al. 2004)
Treatment of the nuclear protein extracts with Jack Beans hexosaminidase
Nuclear protein extracts derived from LLCPK-1 cells grown at 5 mM glucose were diluted with the nuclear protein extraction buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 2 µg/mL aprotinin, 2 µg/mL leupeptin, 1 µg/mL pepstatin A, 2 µg/mL antipain, 100 µg/mL PMSF). The diluted nuclear proteins (100 ng) were incubated with or without 0.01 units of hexosaminidase from Jack Beans (Sigma–Eldrich, Rehovot, Israel) at room temperature for 15, 30, and 60 min. Topo I reaction mixture containing 250 ng supercoiled pUC 19 DNA was added and the reaction was carried out at 37°C for 30 min. The reaction products were analyzed by agarose gel electrophoresis as described earlier.
Determination of the level of topo I protein by Western blot analysis
Antibodies
Anti-topo I antibody (lot no. D101) were purchased from Santa Cruz Biotechnology Inc., Santa Curz, CA, and anti-ß actin antibody from ICN (lot no. 8739F).
Equal amounts (40 µg) of nuclear proteins derived from the various treated cells were analyzed by Western blot analysis, as previously described (Sambrook et al. 1989), using either an anti-topo I antibody (1: 2000), or an anti-ß actin antibody (1:1000). The immunocomplexes were detected by enhanced chemiluminescence (ECL) (Santa Cruz Biotechnology Inc., Santa Curz, CA). Densitometric analysis was performed as described earlier. The level of topo I protein was calculated by using the following equation: [topo I/ß actin] x 100.
Immunoprecipitation Assay
Antibody
Mouse anti-O-GlcNAc monoclonal IgM antibody (mAb CTD 110.6) were purchased from Covance (Denver, PA) (BabCo). Rabbit anti-OGT IgG antibody (H-300) were purchase from Santa Cruz Biotechnology Inc., CA.
The immunoprecipitation of topo I protein or O-GlcNAc modified proteins was performed as previously described (Bendetz-Nezer et al. 2004). Equal amounts of nuclear proteins (200 µg or as indicated) were subjected to immunoprecipitation with either 1 µL of anti-O-GlcNAc antibody solution (3–5 mg/mL) in the absence or presence of 200 mM free GlcNAc or with anti-topo I antibody at a final volume of 100 µL nuclear buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl and 1.5 mM MgCl2, and protease inhibitors: 2 µg/mL aprotinin, 2 µg/mL leupeptin, 1 µg/mL pepstatin A, 2 µg/mL antipain, 100 µg/mL PMSF). The mixture was rotated overnight at 4°C. Protein A-Sepharose (0.1 g/mL) in TE buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) was added for an additional 1 h. The samples were centrifuged at 10,000 x g for 2 min and the beads were washed up to five times with TE buffer. The pellet was resuspended in 50 µL of sample buffer (final concentration; 7.5% glycerol, 1% SDS, 50 mM Tris–HCl pH 6.8, 2.5% ß-mercaptoethanol and 0.025% bromophenol blue) boiled for 5 min and centrifuged. The samples were loaded on 10% SDS-polyacrylamide gel and Western blot analysis was performed using anti-topo I or anti-O-GlcNAc antibodies.
Stripping of antibodies from the nitrocellulose membrane
The membrane was immersed in a stripping buffer (100 mM 2-ß-mercaptoethanol, 2% SDS, and 62.5 mM Tris–HCl pH 6.7) followed by incubation at 50°C for 30 min with occasional agitation. The membrane was washed twice with TPBS (31.25 mM Na2HPO4, 12.5 mM Na2HPO4, 13.7 mM NaCl, 0.1% Tween) for 10 min at room temperature.
Precipitation of GlcNAcylated proteins by Agarose-WGA
The agarose WGA (Vector Laboratory, Burlingame, CA) was washed from the stabilizing sugar according to the instructions. The agarose WGA was resuspended in binding buffer (20 mM Tris–HCl pH 7.4, 1 mM MgCl2, 1 mM CaCl2). Nuclear proteins (200 µg) solution (after desalting) was added to 200 µL of washed agarose WGA solution (7 mg/mL) followed by overnight incubation at 4°C on a rotating wheel. When indicated, 20 mM of GlcNAc was added together with the agarose WGA solution. The complexes were harvested by centrifugation at 10,000 x g for 2 min. The supernatant was removed and kept for further analysis, and the precipitated agarose WGA-protein complexes were washed 5 times with binding buffer. The complexes were resuspended in 25 µL of binding buffer and protein sample buffer (final concentration; 7.5% glycerol, 1% SDS, 50 mM Tris–HCl pH 6.8, 2.5% ß-mercaptoethanol, and 0.025% bromophenol blue) was added to the complexes and to the supernatant solution, boiled for 5 min and centrifuged. The samples were loaded on 10%-SDS-polyacrylamide gel and Western blot analysis was performed using anti-topo I or anti-O-GlcNAc or anti-OGT antibodies.
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Statistical analysis |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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The t-test was used for the comparison of continuous variables. A p value of less than 0.05 was considered as significant. Means are given as ±SEM.
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Conflict of interest |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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None declared.
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Acknowlegements |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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This paper is dedicated to Prof. Shraga Segal, a wonderful human being and an outstanding and great scientist, who passed away during the preparation of this manuscript.
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EDTA, ethylene-diamine-tetra-acetic acid; GlcNAc, ß-N-acetylglucoseamine; LLPCK-1, porcine proximal tubular epithelial cell line; OGT, O-GlcNAc transferase; PMSF, phenyl-methylsulfonyl; SDS-PAGE, sodium dodecyl sulfate polyacrylamidegel electrophoresis; topo, topoisomerase; WGA, wheat germ agglutinin
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TopAbstractIntroductionResultsDiscussionMaterials and methodsStatistical analysisConflict of interestAcknowlegementsReferences |
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