Glycobiology Advance Access originally published online on August 17, 2006
Glycobiology 2006 16(12):1262-1271; doi:10.1093/glycob/cwl037
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Effects of N-glycan processing inhibitors on signaling events and induction of apoptosis in galectin-1-stimulated Jurkat T lymphocytes
2 Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Schillingallee 70, D-18057 Rostock, Germany;
3 Faculty of Science, Department of Chemistry, Cairo University, Gisa 12613, Cairo, Egypt; and
4 Faculty of Specific Education, Mansoura University, Mansoura, 35516, New Damietta City, Egypt
1 To whom correspondence should be addressed; e-mail: hermann.walzel{at}med.uni-rostock.de
Received on June 2, 2006; revised on July 12, 2006; accepted on August 4, 2006
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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To elucidate the role of N-linked glycans in triggering T-cell functions, the effects of the N-glycan processing inhibitors 1-deoxymannojirimycin (1-DMM) and swainsonine (SW) were investigated on signaling events and induction of apoptosis in galectin-1 (gal-1)-stimulated Jurkat T lymphocytes. The treatment of Jurkat E6.1 cells with 1-DMM and SW strongly reduced the cell binding of gal-1–biotin, conjugate binding to cell lysate glycoproteins, and to cluster of differentiation (CD) 3 immunoprecipitates on blots as well as the binding of CD2 and CD3 to immobilized gal-1. The mannosidase inhibitors efficiently decreased gal-1-induced calcium mobilization. Both phases originated from a transient Ca2+ release of internal stores, and the sustained influx across the plasma membrane was found to be involved. Both inhibitors suppressed in transiently transfected Jurkat T lymphocytes the gal-1-induced expression of the luciferase (luc) reporter gene constructs pNFAT-TA-Luc and pAP1(phorbol-12-myristate-13-acetate [PMA])-TA-Luc. The data provide evidence that gal-1 triggers through binding to N-linked glycans a Ca2+-sensitive apoptotic pathway.
Key words: apoptosis / galectin-1 / Jurkat T lymphocytes / N-glycan processing / signaling
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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Galectins are members of a family of endogenous carbohydrate-binding proteins defined by a conserved carbohydrate recognition domain (CRD) with an affinity for ß-galactosides (Barondes et al., 1994
). Fourteen mammalian galectins have been identified so far in a variety of tissues from several species (Cooper and Barondes, 1999; Dunphy et al., 2002). Although designed as an intracellular protein without signal peptide, galectin-1 (gal-1), a prototype family member, has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway and is then bound to appropriate cell-surface glycoconjugates in an autocrine manner (Lutomski et al., 1997; Schäfer et al., 2004). Extracellularly, gal-1 exerts immunoregulatory functions and induces apoptosis in immature thymocytes and activated T cells. Thus, gal-1 produced by thymic epithelial cells may be involved in regulation of programmed cell death in the thymus during selection and in the periphery following an immune response (Perillo et al., 1995, 1997; Walzel et al., 1999; He and Baum, 2004). Because gal-1 synthesis is strongly up-regulated after peptide antigen-induced activation of murine T cells and inhibits antigen-induced proliferation, the lectin can act by an autocrine negative loop to control T-cell reactivity (Blaser et al., 1998). The expression of gal-1 at sites of T-cell activation, its potential use to ameliorate graft-versus-host disease (Baum et al., 2003), and the ability for inhibition of the allogeneic T-cell response (Rabinovich et al., 2002) indicate the functional relevance for T-cell tolerance.
The bivalent nature of gal-1 facilitates cross-linking of cell-surface receptors initiating signaling events leading to cell death (Pace et al., 1999; Brewer, 2002). The functional outcome induced by gal-1 depends on the cellular synthesis and processing of glycan recognition structures and on appropriate presentation. According to the homodimeric structure with two CRDs, gal-1 can induce cellular signaling when receptors that are linked to downstream signal transduction pathways are involved (Walzel et al., 2000; Chung et al., 2000). Although N-acetyllactosamine (LacNAc) is the basic disaccharidic ligand, gal-1 binds with increased avidity to poly-LacNAc residues presented on branched N-linked or to repeating LacNAc units on N- and/or O-linked glycans. The synthesis of poly-LacNAc sequences is regulated in part by the family of core-2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) enzymes for O-glycans and ß-1,6-N-acetylglucosaminyltransferase V (GnT-V) enzyme for N-glycans (Yousefi et al., 1991; Bierhuizen et al., 1994). By contrast with naive and memory T cells and mature thymocytes, activated T cells and cortical thymocytes undergoing selection express both the C2GnT enzyme and core-2 O-glycans on cell-surface glycoproteins and are susceptible to gal-1-induced apoptosis (Baum et al., 1995; Harrington et al., 2000). Expression of the C2GnT-I enzyme in a C2GnT-negative gal-1-resistant T-cell line rendered the cells susceptible to gal-1-induced apoptosis (Galvan et al., 2000). Although GnT-V is expressed by resting peripheral T cells and up-regulated with activation (Demetriou et al., 2001; Daniels et al., 2002), the enzyme seems to be not essential for apoptosis because a GnT-V-deficient murine T-cell line was susceptible to gal-1-induced death (Galvan et al., 2000). Beside GnT-V, GnT-II, GnT-IV, and GnT-VI branching enzymes are also involved in the generation of N-linked complex-type oligosaccharides with multiple LacNAc sequences. A committed step in the synthesis of diverse complex-type oligosaccharides is initiated by Golgi 
-mannosidase II that removes the terminal 1,3- and 1,6-linked mannose residues on the 1,6-arm of GlcNAcMan5GlcNAc2, resulting in a GlcNAcMan3GlcNAc2 structure. This intermediate serves as the initial substrate for the generation of tri-, tetra-, and penta-antennary N-glycans by GnTs, followed by extension with LacNAc residues (Qasba, 2000; Moremen, 2002). Whereas Golgi mannosidase II is inhibited by swainsonine (SW), 1-deoxymannojirimycin (1-DMM) mimics sensitivity to inhibition by the pyranose substrate of class I mannosidases with specificity for cleaving 1,2-mannose linkages (Daniel et al., 1994). Glycoprotein maturation and catabolism by mannosidase I precedes hybrid- and complex-type N-glycan biosynthesis.
The aim of the study was to investigate the effects of the N-glycan processing inhibitors SW and 1-DMM on gal-1 binding to cell-surface glycoconjugates and on gal-1-induced signaling events in human Jurkat T lymphocytes. Our data provide evidence that N-glycans are essentially involved in gal-1 binding to cluster of differentiation (CD)2 and CD3, in cell calcium signaling, and stimulation of Ca2+-dependent downstream signaling pathways leading to activation of the activator protein 1 (AP-1) and the nuclear factor of activated T cells (NFAT).
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Results |
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SW and 1-DMM decrease the binding of gal-1 and phytohemagglutinin to cell-surface glycoproteins
The binding of gal-1–biotin and phytohemagglutinin (PHA)–biotin to Jurkat E6.1 cells as analyzed by flow cytometry is summarized in Figure 1. The fluorescence histograms clearly show that the cells express appropriately glycosylated cell-surface glycoconjugates for gal-1 and PHA recognition (Figure 1, peak b). In the presence of 10 mM lactose as a disaccharidic competitor, the binding of gal-1–biotin was strongly reduced (Figure 1A, peak a). Decreased binding reactions for gal-1 compared with nontreated cells (peak b) were observed when the cells were cultured with 1 µM SW (Figure 1A, peak d) and 20 µM SW (peak c) or with 0.5 mM 1-DMM (Figure 1B, peak d) and 2 mM 1-DMM (peak c) for 65 h. Although blocking of mannosidase I with 1-DMM acts upstream of mannosidase II, inhibition of Golgi mannosidase II by SW also induces the synthesis of hybrid types of oligosaccharides (Tulsiani and Touster, 1983
; Moremen and Touster, 1988). Therefore, we tested the combination of both inhibitors. Cells that were cultured with 0.5 mM 1-DMM plus 5 µM SW (Figure 1C, peak d) displayed further decreased fluorescence intensities compared with 1-DMM at 0.5 mM (Figure 1B, peak d). However, slightly reduced fluorescence intensities were induced with a combination of 2 mM 1-DMM and 5 µM SW (Figure 1C, peak c) when compared with the fluorescence histogram of 2 mM 1-DMM-treated cells (Figure 1B, peak c). To demonstrate specificity and efficacy of inhibitors, PHA was included having the necessary structural determinants for complex N-linked glycan recognition. In comparison with PHA–biotin binding to control cells (Figure 1D, peak b), decreased fluorescence intensities were recorded when mannose-rich N-linked glycan processing was inhibited with 5 µM SW (peak e) and with 2 mM 1-DMM (peak d). Treatment of cells with the combination of both inhibitors (peak c) further reduced PHA–biotin binding relative to the streptavidin–fluorescein isothiocyanate (FITC) conjugate control (peak a).
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The differences in gal-1–biotin binding measured by flow cytometry were further analyzed by comparing the binding reactions to cell-lysate glycoproteins on blots derived from nontreated control cells (Figure 2A, lane 1) and from E6.1 cells cultured with 2 mM 1-DMM plus 30 µM SW for 72 h (lane 2). The weaker binding to glycoproteins of inhibitor-treated cells in the molecular mass range between 23 and 50kDa, and the involvement of CD2 and CD3 in gal-1-induced Ca2+ mobilization of T cells (Pace et al., 1999
; Walzel et al., 1996, 2000) prompted us to study the binding reactions of gal-1 to both CD antigens.
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Equal amounts of cell lysate proteins from nontreated cells (Figure 2B, lane 1) and from cells cultured with 2 mM 1-DMM (lane 2) were separated on gal-1 agarose. The bound material was eluted with 0.2 M lactose and analyzed on blots by using a CD2 and a CD3 monoclonal antibody (mAb). As demonstrated, processing inhibition of mannose-rich N-linked glycans with 1-DMM (lane 2) strongly decreased CD2 and CD3 binding to gal-1 agarose when compared with the controls (lane 1). Furthermore, analysis of gal-1 agarose-bound fractions for CD2 by immunoblotting revealed strongly decreased CD2 binding to immobilized gal-1 relative to the control (lane 1) when N-glycan maturation was inhibited with SW (Figure 2C, lanes 2–4) or with a combination of 1-DMM and SW (lane 5). N-glycan processing on CD2 was efficiently inhibited already at 1 µM SW (lane 2), and increasing concentrations up to 20 µM (lanes 3 and 4) as well as 1 mM 1-DMM plus 5 µM SW (lane 5) induced comparable effects. As demonstrated (Figure 2, panel D), slightly decreased binding of gal-1–biotin to CD3 immunoprecipitated from equal amounts of lysate proteins was recorded when cells were cultured with 1 µM SW (lane 2), 10 µM SW (lane 3), and with 1 mM 1-DMM plus 10 µM SW (lane 4).
Inhibition of N-glycan processing decreases gal-1-induced calcium mobilization
We demonstrated that gal-1 caused Ca2+ mobilization in T cells (Walzel et al., 1996). However, no data are available demonstrating the involvement of N-linked glycans in gal-1-stimulated Ca2+ responses. In the next step, we analyzed the inhibitor effects on gal-1-induced Ca2+ mobilization in Jurkat T lymphocytes (Figure 3). In the presence of 1 mM external Ca2+ (panel A), the basal concentration of intracytoplasmic free calcium ([Ca2+]i) was in the range between 50 and 80 nM. The addition of gal-1 at a concentration of 13 µg/mL increased [Ca2+]i in nontreated cells to sustained levels of 
450 nM after about 4 min (curve a). Preincubation of fura-2-loaded cells in the presence of 10 mM lactose at 37°C for 1 min completely prevented the gal-1-induced calcium response (curve e). Compared with nontreated cells (curve a), SW at 1 µM induced a slightly decreased gal-1-induced calcium mobilization kinetics (not shown). Treatment of the cells with 5 µM SW (curve b) and 30 µM SW (curve c) decreased gal-1-induced Ca2+ mobilization to sustained levels of around 330 and 260 nM, and 1-DMM at 2 mM further reduced the calcium response to sustained levels of about 170 nM (curve d). The combination of 2 mM 1-DMM and 5 µM SW did not further reduce the Ca2+ response induced by 13-µg/mL gal-1 (not shown). Benzyl--GalNAc at concentrations that inhibit O-glycosylation (Kuan et al., 1989) was without effects on gal-1-induced Ca2+ mobilization (not shown) when compared with the control (curve a). As demonstrated in Figure 3 (panel B), gal-1 induced in Jurkat E6.1 cells in calcium-free medium a transient calcium signal with peak values of approximately twice of the basal Ca2+ levels released from internal stores. The addition of CaCl2 (1 mM) to the incubation buffer induced a second phase of a sustained increase in [Ca2+]i originated from an influx of Ca2+ across the plasma membrane (Figure 3B, curve a). Compared with nontreated control cells (Figure 3B, curve a), both phases of gal-1-induced calcium mobilization were found to be strongly reduced after treatment of cells with 2 mM 1-DMM (Figure 3B, curve b).
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N-glycan maturation inhibition decreases the induction of luciferase reporter activity of NFAT-TA-Luc and pAP1(PMA)-TA-Luc
A sustained increase in [Ca2+]i can stimulate the calcium/calmodulin-regulated Ser/Thr-phosphatase calcineurin (CN) leading to dephosphorylation and nuclear translocation of NFAT. Therefore, we analyzed by NFAT reporter gene assays whether inhibition of N-glycan maturation with SW and 1-DMM had effects on gal-1-induced activation of the reporter gene construct in transiently transfected Jurkat T lymphocytes (Figure 4, panel A). The pNFAT-TA-Luc cis-reporter vector containing three tandem copies of the NFAT-consensus sequence upstream of the minimal TA promoter is designed to measure the induction of NFAT-mediated signaling events by quantitative luciferase (luc) reporter activities. Stimulation of transiently transfected control cells with 21-µg/mL gal-1 resulted in a more than 50-fold increase of the reporter activity relative to the transfected but nonstimulated controls. The inhibitor effects on gal-1-induced x-fold increases of luc activity are expressed as the percentage relative to the control (100%). When compared with nonstimulated controls, about 10-fold lower luc activities were measured in cell lysates from E6.1 cells transfected with the pTAL-Luc construct (negative control). Gal-1 failed to induce this construct (data not shown). The gal-1-mediated induction of the reporter was inhibited by 1-DMM in a concentration-dependent manner, whereas SW at 1 and 10 µM induced comparable decreases of reporter activity. Inhibition of the calcium/calmodulin-regulated CN-phosphatase by pretreatment of transfected cells with the immunosuppressive drug cyclosporin A (CsA), (Wesselborg et al., 1996
) and inhibition of gal-1-induced luc reporter activity by the disaccharidic competitor lactose demonstrate specificity and efficacy of the responses.
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The reporter gene construct pAP1(phorbol-12-myristate-13-acetate [PMA])-TA-Luc, driven by six tandem copies of the AP-1 enhancer, is designed for monitoring the induction of the c-Jun N-terminal kinase (JNK)-c-Jun–AP-1 signaling pathway and specifically responds to phorbol ester treatment (Walzel et al., 2002
). The stimulation of transiently transfected control cells with 21-µg/mL gal-1 induced more than 100-fold increased luc reporter activities relative to the transfected but nonstimulated controls (not shown). Inhibition of N-glycan maturation by preincubation of the cells with 1-DMM and SW decreased the stimulating effect of gal-1 and is expressed as the percentage relative to the control (100%; Figure 4, panel B). Furthermore, preincubation of the cells with curcumin (0.5, 2 µM), an inhibitor of c-Jun expression and AP-1 activation by PMA and ceramide (Huang et al., 1991; Sawai et al., 1995), reduced the gal-1-stimulated expression of the reporter with an efficiency comparable with that mediated by 1-DMM or SW.
Inhibition of N-glycan processing decreases gal-1-induced DNA fragmentation
It has been demonstrated in previous studies that gal-1-induced apoptosis is preceded by an early activation of AP-1 (Walzel et al., 2002), and pretreatment of T cells with curcumin decreased gal-1-induced DNA fragmentation in a concentration-dependent manner (Rabinovich et al., 2000). The comparable efficiencies of curcumin, 1-DMM, and SW to inhibit the induction of pAP1(PMA)-TA-Luc (Figure 4, panel B) prompted us to study the effects of 1-DMM and SW on gal-1-stimulated DNA fragmentation in Jurkat T lymphocytes. As demonstrated in Figure 5, panel A, gal-1 stimulated DNA cleavage into oligonucleosomal-sized fragments of about 180–200bp when compared with control cells cultured in the absence of gal-1 (lane C). This effect could be detected after 6 h of incubation and increased further with exposure time. Preincubation of the cells with 2 mM 1-DMM for 64 h decreased gal-1-induced DNA fragmentation when compared with control cells without gal-1 (lane C), as demonstrated in panel B. As shown in panel C, gal-1-initiated DNA ladder formation was slightly decreased when Jurkat T lymphocytes were treated with SW at 1 µM (lane 2), 10 µM (lane 3), and 30 µM (lane 4) for 68 h when compared with the gal-1-induced DNA fragmentation in nontreated cells (lane 1).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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The fate and functional activity of T cells crucially depends on gene expression and protein synthesis but also on post-translational modifications of proteins such as phosphorylation and glycosylation that alter protein function. There is considerable evidence that differential glycosylation is involved in immunoregulation, affects cellular recognition and T-cell reactivity, and influences T-cell signaling sensitivity and survival (Demetriou et al., 2001
; Daniels et al., 2002). Cell-surface glycosylation changes with T-cell development as well as with T-cell activation. Endogenous carbohydrate-binding proteins recognize these changes by specific interactions and cross-linking of glycans on T-cell glycoproteins (Perillo et al., 1995; Walzel et al., 1999, 2000; Pace et al., 2000).
In this study, we provide evidence that gal-1 binding to N-linked glycans on T-cell glycoproteins is important for Ca2+ mobilization, gene transcription, and apoptosis. As an approach, N-glycan maturation was blocked by 1-DMM and SW treatment of human lymphoblastoid Jurkat T cells. SW blocks complex-type N-glycan formation with LacNAc residues, whereas 1-DMM inhibits events upstream of hybrid- and complex-type N-glycan biosynthesis (Qasba, 2000; Moremen, 2002). As demonstrated here, SW- and 1-DMM-induced inhibition of cellular N-glycan maturation was recorded by reduced cell binding as well as by decreased binding of gal-1 to CD2 and CD3. Gal-1 has been identified to be a ligand for the CD3 complex, for CD2, and for CD45, and these CD antigens are essentially involved in gal-1-induced Ca2+ mobilization of T cells (Walzel et al., 1999, 2000). Signaling through CD2 requires ligation of conventional CD2 epitopes of the activation-associated epitope, termed CD2R, and the presence of the CD3 
-chain (Moingeon et al., 1992). Consistent with decreased binding to CD2 and CD3 of inhibitor-treated cells, SW and 1-DMM reduced gal-1-induced Ca2+ mobilization. Although 1-DMM at 2 mM efficiently reduced the calcium response, there was still a modest Ca2+ flux. Because treatment of the cells with the O-glycosylation inhibitor benzyl--GalNAc (Kuan et al., 1989) did not change gal-1-induced Ca2+ mobilization (results not shown), we conclude that N-glycan processing is incompletely blocked by 1-DMM and SW. Furthermore, it seems likely that the ganglioside GM1 as a major receptor for gal-1 is involved (Kopitz et al., 1998). Ganglioside GM1 may be linked to a cell calcium activation pathway in human Jurkat T cells also in the absence of a functional CD3 complex (Gouy et al., 1994).
Using reporter gene constructs under transcriptional control of NFAT and AP-1, it was demonstrated that gal-1 induces gene-activating signals required for interleukin-2 (IL-2)-promoter activation and IL-2 gene expression. Gal-1 was unable to induce the construct pNFAT-TA-Luc in CD3-deficient J31–13 cells, and induction was significantly reduced in CD45-deficient J45.01 cells (Walzel et al., 2002). NFAT and AP-1 activation depend on stimulation of two signaling pathways, namely, a sustained increase in [Ca2+]i and the activation of protein kinase C (Masuda et al., 1998). Although gal-1 stimulates signaling pathways involved in IL-2 gene transcription, gal-1 failed to induce a processive signal transduction leading to IL-2 production and T-cell proliferation (Vespa et al., 1999). However, gal-1 induces partial T-cell receptor (TCR) -chain phosphorylation generating inhibitory pp21, limited receptor clustering at the TCR contact site (Chung et al., 2000) and promotes apoptosis. Among the Src family of protein tyrosine kinases, Lck presumably phosphorylates TCR -chain to create double-phosphorylated docking sites for the tandem SH2 domains of ZAP-70 kinase and its activation (Mustelin and Tasken, 2003). During partial tyrosine phosphorylation, several other signaling molecules bind to TCR and transduce signals insufficient for complete activation (Sloan-Lancaster et al., 1994). Deficiency in Lck and ZAP-70 abolishes gal-1-induced T-cell death, and restoration of enzyme expression restores apoptosis (Ion et al., 2005). The participation of the TCR/Lck/ZAP-70 pathway in gal-1-induced apoptosis suggests that decreased binding of gal-1 to CD2 and CD3 from 1-DMM- and SW-treated cells may further modulate downstream signaling events resulting in decreased Ca2+ mobilization, NFAT and AP-1 activation, and in decreased DNA degradation. The CsA and curcumin controls as well as the inhibitory effects of lactose on gal-1-induced NFAT and AP-1 activation in untreated cells demonstrate specificity of the responses.
Gal-1 also induces T-cell death in a caspase- and cytochrome c-independent manner by nuclear translocation of endonuclease G (Hahn et al., 2004). Furthermore, the intracellular pathway of gal-1-induced T-cell death also involves hyperpolarization of mitochondria (Matarrese et al., 2005), caspase activation, Bcl-2 down-regulation, and AP-1 activation (Rabinovich et al., 2000, 2002). In this study, we could show that SW and 1-DMM reduced gal-1-stimulated induction of the reporter gene construct pAP1(PMA)-TA-Luc to the level comparable with that induced by curcumin, an inhibitor of c-Jun expression and AP-1 activation (Huang et al., 1991; Sawai et al., 1995). The nuclear transcription factor AP-1, composed of dimers of the Jun, Fos, ATF, and MAF protein families, determines life or death cell fates in response to extracellular stimuli (Ameyar et al., 2003; Eferl and Wagner, 2003). Different dimer combinations recognize different sequence elements in the promoters and enhancers of target genes. The consequence of AP-1 activity for apoptosis is probably mediated by differential regulation of pro- and anti-apoptotic genes.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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Materials
Asialofetuin, bovine serum albumin (BSA), benzyl-
-GalNAc, 1-DMM, dithiothreitol (DTT), ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ß-lactose, goat anti-rabbit IgG-horseradish peroxidase (HRP), Nonidet P 40 (NP 40), N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid, PHA, protein G agarose, ribonuclease A, streptavidin–FITC, streptavidin–HRP, SW, and tris(hydroxymethyl) aminomethane (Tris) were from Sigma (Deisenhofen, Germany). Antipain, aprotinin, D-biotin-N-hydroxysuccinimide ester, DNA molecular weight marker 100bp ladder, leupeptin, pepstatin, pefabloc, and proteinase K were from Roche Molecular Biochemicals (Mannheim, Germany). Fetal calf serum, kanamycin, and RPMI 1640 medium were from Gibco-BRL (Eggenstein, Germany), and enhanced chemiluminescence (ECL) detection reagents, Hybond ECL nitrocellulose membranes, NHS-activated Sepharose 4 Fast Flow, the Resource Q anion-exchange column, and Sepharose 4B were from Amersham Pharmacia (Freiburg, Germany). The CD2 mAb (clone 39C1.5, IgG2a isotype) and the CD3 mAb (clone X35, IgG2a isotype) were from Coulter-Immunotech (Marseille, France). Curcumin, CsA, and fura-2AM were ordered from Calbiochem (Bad Soden, Germany), and the reporter gene constructs pNFAT-TA-Luc, pAP1(PMA)-TA-Luc, and pTAL-Luc were from Clontech (Heidelberg, Germany).
Cells
The human leukemic T-cell line Jurkat, clone E6.1, (European Collection of Animal Cell Cultures, Salisbury, UK) was grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 10 µg/mL of kanamycin. For inhibition of N-glycan processing, the cells were cultured in complete culture medium with 1-DMM and SW at different concentrations and periods of time as indicated in the legends to Figures 1 through 5. For inhibition of O-glycan elongation, the cells were cultured with 2 mM benzyl--GalNAc for 64 h. Compared with control cells in the absence of inhibitors, 1-DMM and SW did neither alter the proliferation nor cell viability as detected by cell counting or trypan blue exclusion (data not shown). Cell viability was always higher than 96%. For induction of apoptosis, nontreated, 1-DMM, and SW-treated E6.1 cells (2 x 106/2 mL RPMI 1640 medium) were stimulated with gal-1, as specified in the Results section.
Purification, biotinylation, and immobilization of gal-1
Gal-1 was isolated from human placenta and purified by affinity chromatography and by anion-exchange chromatography, as described in Walzel et al. (2000). Biotinylation of gal-1 and PHA by use of biotinyl-N-hydroxysuccinimide and immobilization of gal-1 to NHS-activated agarose were performed according to the manufacturer’s protocol.
Flow cytometric measurement of gal-1 binding to E6.1 cells
For gal-1–biotin and PHA–biotin binding, the cells (1 x 106/mL PBS, pH 7.4) were incubated with 2 µg of the conjugate for 20 min on ice. Then the cells were washed twice with ice-cold PBS (pH 7.4) and incubated with streptavidin–FITC (1:500 dilution) for 15 min. After washing twice, cell-bound fluorescence was analyzed by a FACSCalibur (BD Biosciences, San Jose, CA).
Immunoprecipitation of CD3 from Jurkat T-cell lysates
Nontreated cells (control) and cells cultured with 2 mM 1-DMM plus 30 µM SW for 72 h (2.7 x 107 cells) were incubated on ice for 1 h in 500 µL of cell lysis buffer (20 mM Tris/HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM Pefabloc, 10 µg/mL of aprotinin, 10 µg/mL of leupeptin, 10 µg/mL of pepstatin, and 10 µg/mL of antipain). The supernatant obtained by centrifugation at 10,000x g for 20 min at 4°C (40 µL) was mixed with 20 µL of 3-fold concentrated Laemmli sample buffer (Laemmli, 1970) and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS–PAGE) (5 µL/lane). For immunoprecipitation of CD3, cell lysates were prepared from E6.1 cells (3.6 x 107) cultured without and with 1 µM SW, 10 µM SW, and with 1 mM 1-DMM plus 10 µM SW for 66 h. The supernatants (4.64 mg of lysate protein) were incubated with 40 µg CD3 mAb (clone X35) for 1 h on ice followed by incubation with 100 µL of protein G agarose. The beads were washed five times with 400 µL of cell lysis buffer each, mixed with 200 µL of 3-fold concentrated nonreducing electrophoresis sample buffer and were treated for 5 min at 100°C. Samples (70 µL) were separated by SDS–PAGE.
Separation of Jurkat T-cell lysates on gal-1 agarose
Cell lysates from nontreated cells and from cells cultured with 2 mM 1-DMM for 70 h, with 1 µM SW, 5 µM SW, 20 µM SW, and with 1 mM 1-DMM plus 5 µM SW for 68 h (4 x 107 cells) were prepared in 400 µL of cell lysis buffer with 2 mM DTT, as described above. After incubation of the supernatants with 200 µL of gal-1 agarose for 1 h in a 0.22-µm centrifuge filter unit (Corning Costar, Cambridge, MA), the beads were washed twice in cell lysis buffer. Then the gel was eluted with 200 µL 0.2 M lactose in cell lysis buffer, and the released fraction was treated with 200 µL of 2-fold concentrated sample buffer for 5 min at 100°C.
SDS–PAGE and western blot analysis
Proteins were separated in 12% polyacrylamide gels and transferred to Hybond ECL nitrocellulose membranes. The membranes were blocked by incubation in tris buffered saline/Tween 20 (TBS/Tw) (20 mM Tris/HCl [pH 7.2], 1 M NaCl, 1% Tween 20) for 10 min. Then the blots were probed with 0.5 µg/mL of gal-1–biotin in TBS/Tw containing 0.05% Tween 20 and 2 mM DTT for 16 h at 6°C. After washing in incubation buffer, the blots were incubated with a streptavidin–HRP conjugate (1:5000 dilution) for 1 h at room temperature. Then the blots were washed in TBS/Tw with 0.05% Tween 20 and developed with the ECL kit.
For analysis of the gal-1 agarose-bound fractions for CD2 and CD3, the blots were blocked with 5% BSA in TBS/Tw (pH 7.4) for 1 h at room temperature followed by incubation with 0.5 µg/mL of CD2 mAb (clone 39C1.5) and with 1 µg/mL of CD3 mAb (clone X35) in TBS/Tw with 5% BSA for 16 h at 6°C. After washing in TBS/Tw, the blots were incubated with rabbit anti-mouse IgG-HRP (1:2500 dilution in TBS/Tw with 4% BSA) for 1 h. Then the bands were visualized by use of the ECL detection system.
Measurement of intracellular free calcium ([Ca2+]i)
For measurement of [Ca2+]i, the cells were loaded with fura-2AM, as described in Walzel et al. (2000). After loading, cell viability as detected by trypan blue exclusion was always higher than 95%. The fluorescence of the cellular suspension at 37°C was monitored with a Shimadzu RF-5001 PC spectrofluorimeter by exciting at 339 and 380 nm and by measuring the fluorescence at 490 nm. Graphic representations of [Ca2+]i were calculated by converting the ratio of 339/380 to [Ca2+]i using a Kd of 224 nM, as described in Walzel et al. (1996).
Transient transfection—NFAT and AP-1 reporter gene assays
Jurkat T lymphocytes (1 x 107/0.8 mL RPMI 1640 medium) were transfected with 25 µg of the reporter constructs pNFAT-TA-Luc, pAP1(PMA)-TA-Luc, or pTAL-Luc as a negative control by electroporation at 350V and 900 µF applying a Bio-Rad gene pulser. Then the cells were cultured at a density of 5 x 106/4 mL in RPMI 1640 medium for different time periods at 37°C in the presence of gal-1. For inhibition of gal-1-mediated induction of the reporter gene, cells were preincubated with curcumin, 1-DMM, and SW before transfection and with CsA after transfection, as indicated in the legend to Figure 4. Cell lysis and the measurement of luc activity were performed by the luc assay system according to the manufacturer’s protocol (Promega, Mannheim, Germany).
DNA extraction and analysis by agarose gel electrophoresis
Degraded low molecular weight DNA from apoptotic cells was selectively extracted with phosphate–citrate (PC) buffer (Gong et al., 1994). Untreated and gal-1-treated E6.1 cells were collected by centrifugation, fixed in 70% ethanol, and stored at –25°C generally for 16 h. The cells were then centrifuged at 800x g for 5 min, and ethanol was thoroughly removed. Cell pellets (2 x 106 cells) were resuspended in 40 µL of PC buffer (0.2 M Na2HPO4 adjusted with 0.1 M citric acid to pH 7.8) at room temperature for 30 min. After centrifugation at 1000x g for 5 min, the supernatants were transferred to new tubes and treated in a vacuum concentrator 5301 (Eppendorf, Hamburg, Germany) for 15 min. Then 3 µL 0.25% NP 40 was added followed by 3 µL of a solution of RNase A (1 mg/mL water). After 30-min incubation at 37°C, 3 µL of a solution of proteinase K (1 mg/mL water) was added, and the extract was incubated for additional 30 min at 37°C. The enzyme-treated extracts were mixed with 12 µL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol) and subjected to a 1.5% agarose gel. Electrophoresis was performed at 10V/cm for 2.5 h. DNA ladders were visualized by ethidium bromide staining under UV light.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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We gratefully acknowledge the excellent technical assistance of Mrs Gisela Gaede.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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None declared.
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Abbreviations |
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AP-1, activator protein 1; BSA, bovine serum albumin; [Ca2+]i, concentration of intracytoplasmic free calcium; C2GnT, core-2 ß-1,6-N-acetylglucosaminyltransferase; CD, cluster of differentiation; CsA, cyclosporin A; 1-DMM, 1-deoxymannojirimycin; DTT, dithiothreitol; ECL, enhanced chemiluminescence; FITC, fluorescein isothiocyanate; gal-1, galectin-1; GlcNAc, N-acetylglucosamine; GnT-V, ß-1,6-N-acetylglucosaminyltransferase V; HRP, horseradish peroxidase; IL-2, interleukin-2; LacNAc, N-acetyllactosamine; luc, luciferase; mAb, monoclonal antibody; Man, mannose; NFAT, nuclear factor of activated T cells; PBS, phosphate buffered saline; PHA, phytohemagglutinin; PMA, phorbol-12-myristate-13-acetate; RPMI, Roswell Park Memorial Institute; SDS–PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; SW, swainsonine; TBS, tris buffered saline; TCR, T-cell receptor; Tris, tris(hydroxymethyl) aminomethane; Tw, Tween 20
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