Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells
Hermann Walzel1,
Matthias Blach,
Jun Hirabayashi2,
Ken-Ichi Kasai2 and
Josef Brock
Institute of Medical Biochemistry and Molecular Biology, Schillingallee 70, University of Rostock, D-18057 Rostock, Germany and 2Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 19901, Japan
Received on April 8, 1999; revised on August 2, 1999; accepted on August 18, 1999.
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Abstract
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Galectin-1 (gal-1) a member of the mammalian ß-galactoside-binding proteins recognizes preferentially Galß14GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45+ Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca2+]i) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca2+]i is completely inhibited by lactose at 10 mM and was absent in CD45 Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca2+]i stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca2+]i. Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase C
1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.
Key words: galectin-1/T-cell signaling/inositol-1,4,5-trisphosphate/intracytoplasmic free calcium
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Introduction
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Galectins are a family of closely related ß-galactoside-binding proteins widely distributed in the animal kingdom containing a highly conserved amino acid sequence motif in the carbohydrate recognition domain (CRD), (Barondes et al., 1994a
,b; Kasai and Hirabayashi, 1996
). As yet, 10 members of the galectin family have been identified. From the viewpoint of their protein architecture, they have been classified into the proto-, chimera-, and tandem-repeat types (Hirabayashi, 1993
). The key structure preferentially recognized by galectins is the disaccharide unit N-acetyllactosamine (Galß14GlucNAc) included in a variety of glycoconjugates (Abbott et al., 1988
; Lee et al., 1990
). All galectins studied so far have the characteristics of cytoplasmic proteins such as acetylated N-termini (Hirabayashi et al., 1987
; Hirabayashi and Kasai, 1988
), the absence of a disulfide bond in spite of the presence of several cysteine residues, and a lack of glycosylation although a potential glycosylation site is available (Drickamer, 1993
; Hirabayashi, 1993
). In general, galectins apparently are synthesized without any signal peptide sequence (Ohyama et al., 1986
; Couraud et al., 1989
) suggesting that they are designed as intracellular proteins. Therefore, the mechanisms involved in lectin secretion are not understood. There is evidence that some members are externalized to the cell surface and extracellular matrix by an atypical secretion mechanism (Cooper and Barondes, 1990
; Lindstedt et al., 1993
). The growing progress in the identification, cloning, and sequencing of new galectins shows that the biological phenomena in which they are involved are very diverse (Barondes et al., 1994a
). Based on the specific recognition of glycoconjugates, galectins mediate cellular functions such as cell adhesion (Hughes, 1992
; Hafer-Macko et al., 1996
), metastasis (Raz and Lotan, 1987
; Lotan et al., 1994
), cell growth regulation (Wells and Mallucci, 1991
; Adams et al., 1996
), immunosuppression (Levi et al., 1983
; Offner et al., 1990
), and T-cell maturation (Perillo et al., 1995
, 1997). The most common mammalian galectin having a single CRD is the prototype galectin-1 (gal-1), which forms a noncovalently associated homodimer under physiological conditions. Therefore, the lectin can serve as a homobifunctional cross-linker for cell surface glycoconjugates containing the structural motif Galß14GlcNAc. Although the binding of gal-1 to lysosome-associated membrane glycoproteins and laminin has been demonstrated (Do et al., 1990
; Gu et al., 1994
), the recognition of membrane-associated glycoproteins such as the antigens CD45, CD43, CD3, and CD4 has been reported (Perillo et al., 1995
; Pace and Baum, 1997
). Therefore, by direct interaction with membrane glycoproteins relevant to several T-cell functions galectins may have the potency to induce cellular signaling (Walzel et al., 1996
; Dong and Hughes, 1996
). To understand the extracellular roles of gal-1 in T-cell activation it is necessary to identify its interacting glycoconjugates and to study the signaling events.
In this article, we have identified the CD2 and the CD3 antigen as recognition molecules for gal-1. The lectin induces in human Jurkat T-cells signaling events comparable with that induced by CD2 and CD3 monoclonal antibodies (mAbs) such as Ca2+-mobilization, inositol-1,4,5-trisphosphate (InsP3) generation, and the tyrosine phosphorylation of phospholipase C
-1 (PLC
-1), suggesting that CD2 and CD3 are involved.
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Results
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The CD2 antigen is a target for galectin-1
After immunoprecipitation of CD2 from Jurkat T-cell lysates with a CD2 mAb and adsorption on protein G agarose, the immunoprecipitates were resolved by SDSPAGE under non-dissociating conditions and electrophoretically transferred to nitrocellulose membranes. The binding pattern of biotinylated gal-1 is shown in Figure 1A. The lectin generates two main protein bands one in the 50 kDa molecular mass range and an additional one derived from the CD2 mAb (IgG1 isotype) applied for immunoprecipitation (lane 2). Inhibition experiments in the presence of asialofetuin at 4 mg/ml reduced the binding of gal-1 markedly (Figure 1, lane 3). Furthermore, Jurkat T-cell lysates were separated on gal-1 agarose and the binding material was eluted with 0.1 M lactose. On the blots the CD2 antigen was detected in the gal-1 agarose bound fraction by use of a biotinylated CD2 mAb recognizing the T11.1 epitope. Beside a faint protein band, the CD2 mAb binds preferentially to CD2 as detected by the main band (Figure 1B, lane 2). This band was also generated when CD2 immunoprecipitates from Jurkat T-cell lysates were probed with the biotinylated CD2 mAb (not shown).

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Fig. 1. Recognition of the CD2 antigen in CD2 immunoprecipitates from Jurkat E6.1 cell lysates by biotinylated gal-1 (A) and in the gal-1 agarose bound fraction of the cell lysates by a biotinylated CD2 mAb on blots (B). SDSPAGE was carried out on 12% gels under nonreducing (A) and reducing conditions (B). (A) Lane 1, biotinylated molecular mass markers; lanes 2 and 3, incubation of CD2 immunoprecipitates derived from 2.8 107 cells with 2 µg/ml biotinylated gal-1 in the absence (lane 2) and in the presence of 4 mg/ml asialofetuin (lane 3). (B) Lane 1, biotinylated molecular mass markers; lane 2, incubation of the gal-1 agarose bound fraction obtained from 2.9 107 cells with 0.5 µg/ml biotinylated CD2 mAb, The blots were incubated with streptavidin-HRP (1:1000 dilution) and developed by use of 3-amino-9-ethylcarbazole.
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Recognition of CD3 by galectin-1 on blots
From Jurkat E6.1 cell lysates the CD3 antigen was immunoprecipitated with a CD3 mAb (clone UCHT1), resolved by SDSPAGE under non-dissociating conditions and blotted on nitrocellulose membranes. The binding pattern of biotinylated gal-1 is shown in Figure 2A. The lectin recognizes in CD3 immunoprecipitates two bands in the 2226 kDa molecular mass range (lane 2). Gal-1 binding was not found to be inhibited by lactose at 30 mM (lane 3). However, in the presence of asialofetuin containing a triantennary oligosaccharide with three Galß14GlcNAc sequences and terminal nonreducing Gal residues (Leffler and Barondes, 1986
) lectin binding was completely inhibited (lane 4). By increasing the amount of the CD3 immunoprecipitate gal-1 recognizes an additional band in the 2226 kDa molecular mass range (Figure 2B, lane 3). Although the mAb (clone UCHT1) reacts with the
chain of the CD3 complex (Hannet et al., 1992
), two chains of the CD3 complex were found to be coimmunoprecipitated. In contrast to gal-1, the biotinylated mAb specifically reacts with the
chain of the CD3 complex (lane 2).

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Fig. 2. Binding of biotinylated gal-1 to CD3 immunoprecipitates from Jurkat E6.1 cell lysates on blots. SDSPAGE was performed on 12% gels under nonreducing conditions. (A) Lane 1, biotinylated molecular mass markers; lanes 24, incubation of CD3 immunoprecipitates obtained from 2.2 107 cells with 3 µg/ml biotinylated gal-1 (lane 2), in the presence of 30 mM lactose (lane 3), and in the presence of 2 mg/ml asialofetuin (lane 4). (B) Lane 1, low molecular mass markers (Pharmacia) stained with Ponceau S solution; lanes 2 and 3, incubation of CD3 immunoprecipitates from 4.8 107 cells with 2 µg/ml biotinylated CD3 mAb (clone UCHT1, lane 2) and with 4.5 µg/ml biotinylated gal-1 (lane 3). Then the blots were incubated with streptavidin-HRP (1:1000 dilution) and developed applying 3-amino-9-ethylcarbazole (lanes 2 and 3).
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Characterization of the increase in [Ca2+]i induced by gal-1
Figure 3 shows the effect of gal-1 on intracytoplasmic free calcium in fura-2 loaded CD45+ Jurkat E6.1 and in CD45J45.01 cells. In the presence of external Ca2+ the basal [Ca2+]i in Jurkat E6.1 cells was 80 ± 21 nM. The addition of gal-1 at 15 µg/ml induced a response after a lag of about 25 s resulting in a peak of 580 ± 43 nM which then decreased to a sustained level of around 500 nM after about 4 min (Figure 3a). Preincubation of fura-2 loaded cells in the presence of 10 mM lactose at 37°C for 100 s reduced the calcium response induced by 15 µg/ml gal-1 to basal levels (c). However, in contrast to E6.1 cells gal-1 has no effect on [Ca2+]i in CD45-deficient Jurkat J45.01 cells (d). Furthermore, stimulation of fura-2 loaded CD2+ CD3 3113 Jurkat T-cells with CD2 mAbs, CD3 mAbs, and with gal-1 did not elicit an elevation in [Ca2+]i (not shown). As illustrated in Figure 3b gal-1 induces in Jurkat E6.1 cells in calcium-free medium a transient calcium signal with peak values of approximately twice of the basal levels caused by the release of Ca2+ from intracellular stores. Repletion of the medium with CaCl2 (1 mM) results in a second phase of a sustained increase in [Ca2+]i originated by an influx of calcium from the extracellular space.

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Fig. 3. Gal-1 induced calcium signaling in Jurkat E6.1 and J45.01 cells. [Ca2+]i levels were measured in a suspension of fura-2 loaded cells in HEPES-buffer containing 1 mM CaCl2 as described in the Materials and methods section. Lactose was added to a final concentration of 10 mM at time 0 (c) and gal-1 was added as indicated by the arrow at 15 µg/ml to CD45+ Jurkat E6.1 cells in the absence (a), in the presence of 10 mM lactose (c), and to CD45 J45.01 cells (d). The [Ca2+]i -response induced by 15 µg/ml gal-1 in Jurkat E6.1 cells in a calcium free medium is demonstrated by b. EGTA (1.5 mM) was added at time 0. The addition CaCl2 (1.5 mM) is indicated by the arrow.
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Effects of protein kinase inhibitors on [Ca2+]i increases stimulated by gal-1, CD2, and CD3 mAbs
We next examined whether tyrosine kinases are involved in the gal-1 induced calcium response. Jurkat E6.1 cells were cultured with the tyrosine kinase inhibitor herbimycin A under conditions that abrogate TCR-mediated IL-2 secretion (June et al., 1990
). Then the increases in [Ca2+]i were measured after stimulation with gal-1 as well as with CD3 and CD2 mAbs in the presence of external Ca2+. Compared with nontreated cells (Figure 4B, a, b), herbimycin A pretreatment prevented the elevation in [Ca2+]i induced by CD3 mAb stimulation and that elicited by costimulation with two CD2 mAbs recognizing different epitopes (c, d). When gal-1 was applied for cell stimulation, the [Ca2+]i response after herbimycin A treatment was found only partially reduced (Figure 4A, b) compared to nontreated cells (a). The effect of preincubation (3 min) with the cell-permeable inhibitor of protein kinases staurosporine on the subsequent increase in [Ca2+]i stimulated by gal-1 in the presence of external calcium is shown in Figure 4A, c. When compared with nontreated cells, preincubation with staurosporine at 1 µM completely inhibited the subsequent gal-1 stimulated increase in [Ca2+]i. Pretreatment with staurosporine also abrogated the cellular calcium response induced by recombinant gal-1 (rec gal-1), by CD3 mAb (clone UCHT1) stimulation, and that elicited by costimulation with two CD2 mAbs (clone 39C1.5, clone 6F10.3), (results not shown).

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Fig. 4. [Ca2+]i response of non-treated, herbimycin A treated, and staurosporine-treated Jurkat E6.1 cells following gal-1, CD3 mAb, and CD2 mAb stimulation in the presence of external calcium. The cells were pretreated at 37°C with 3 µM herbimycin A (1 106 cells/2 ml) in supplemented cell culture medium for 21 h or with 1 µM staurosporine for 3 min. (A) Increase in [Ca2+]i induced by 15 µg/ml gal-1 in non-treated (a), herbimycin A treated (b), and staurosporine treated cells (c). (B) Increase in [Ca2+]i induced by 2 µg/ml CD3 mAb (clone X35) in nontreated (a) and herbimycin A treated cells (c) as well as [Ca2+]i responses induced by costimulation with two CD2 mAbs (clone 39C1.5, clone 6F10.3) both at 3.3 µg/ml in nontreated (b) and herbimycin A treated cells (d).
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Interference of cholera toxin with the increase in cytoplasmic free calcium
Elevation of intracellular calcium has been shown to be a crucial event in regulating IL-2 receptor expression during T-cell activation (Berry et al., 1990
). Therefore, it was obvious to investigate the effects of cholera toxin (CTX) on cell calcium signaling in Jurkat E6.1 cells induced by rec gal-1. As shown in Figure 5A, a, the elevation of the intracytoplasmic calcium concentration induced by rec gal-1 at 20 µg/ml to 674 nM which represent a 7.7-fold increase over the basal [Ca2+]i levels was found to be reduced to 325 nM after CTX-treatment of the cells (c). Compared with nontreated cells (b), both phases of the rec gal-1 stimulated [Ca2+]i elevation, the release of Ca2+ from intracellular stores and the uptake from the extracellular medium, were markedly reduced by CTX-treatment of the cells (d). Comparable patterns of calcium mobilization in CTX-treated E6.1 cells were recorded following cell stimulation with native gal-1 (not shown). However, in contrast to lectin stimulation pretreatment of the cells with cholera toxin abrogated the calcium response induced by CD3 mAb stimulation (Figure 5B, c) or by costimulation with two CD2 mAbs recognizing different epitopes (Figure 5B, d).

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Fig. 5. Inhibition by cholera toxin of rec gal-1, CD2, and CD3 mAb induced elevation of [Ca2+]i in E6.1 cells. The cells were cultured at 1.5 106 /ml in supplemented cell culture medium with 0.5 µg/ml cholera toxin for 2 h at 37°C. (A) Increase in [Ca2+]i of nontreated (a) and cholera toxin treated E6.1 cells (c) stimulated by 20 µg/ml rec gal-1 in the presence of external calcium as well as of nontreated (b) and cholera toxin treated cells (d) in the absence of external calcium stimulated by 20 µg/ml rec gal-1. EGTA was added at time 0. The addition of rec gal-1 and CaCl2 (1.5 mM) is indicated by arrows. (B) [Ca2+]i responses induced by a CD3 mAb (2.6 µg/ml, clone X35) in nontreated (a) and cholera toxin treated cells (c) as well as responses in nontreated (b) and cholera toxin treated cells (d) induced by CD2 mAb costimulation (clones 6F10.3 and 39C1.5; both at 3.3 µg/ml).
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The gal-1 stimulated elevation in [Ca2+]i is decreased by membrane depolarization
The initial transient gal-1 induced [Ca2+]i peak originated by the depletion of intracellular stores is insufficient to account for the sustained elevation of [Ca2+]i. Therefore, an altered flux across the plasma membrane is a second mechanism for Ca2+ accumulation. In order to characterize the gal-1 mediated Ca2+ influx pathway the effects of membrane depolarization by gramicidin and by a high-potassium buffer were studied. The gal-1 induced change in intracytoplasmic free calcium is dependent on the membrane potential. Membrane depolarization by gramicidin, a ionophore which allows the permeation of monovalent cations such as Na+ and K+, selectively inhibits the rec gal-1 stimulated receptor-operated calcium influx after repletion of the medium with CaCl2 (Figure 6A). When gramicidin was added 100 s before the addition of rec gal-1 in the absence of external calcium, the lectin-induced release from internal stores was comparable to that seen in control cells. Practically identical pattern of Ca2+ mobilization were recorded when gal-1 was applied for cell stimulation (not shown). Furthermore, depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on the basal [Ca2+]i levels (Figure 6B, b). In a high potassium medium in the absence of external calcium the gal-1 mediated release from internal stores was practically to that seen in control cells (a), while the calcium influx after Ca2+ repletion was strongly reduced (b).

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Fig. 6. Effect of membrane depolarization by gramicidin (A) and by a high-potassium buffer (110 mM K+, B) on the [Ca2+]i response of Jurkat E6.1 cells induced by rec gal-1 and gal-1. (A) EGTA (1.5 mM) and gramicidin (4.5 µg/ml) were added at time 0. The addition of rec gal-1 (13.3 µg/ml) and CaCl2 (1.5 mM) is indicated by arrows. (B) Gal-1 induced calcium transients in 5 mM K+-containing HEPES-buffer (a) and in a high-potassium buffer (b). EGTA (1.5 mM) was added at time 0. The addition of gal-1 (16 µg/ml) and CaCl2 (1.5 mM) is indicated by arrows.
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Gal-1 induces InsP3 generation and tyrosine phosphorylation of PLC
-1
Activation of T-cells by various T-cell receptor/CD3 (TCR/CD3) complex ligands including presented antigens (Shapiro et al., 1985
), monoclonal antibodies to TCR or to CD3 (Weiss et al., 1984a
,b) or mitogenic lectins (Tsien et al., 1982
) stimulates by phosphoinositide hydrolysis, the generation of the second messengers InsP3 and diacylglycerol as well as Ca2+ mobilization. By TCR/CD3 ligation the
-type isozymes of phospholipase C (PLC) are activated as a result of phosphorylation by tyrosine kinases (Mustelin et al., 1990
). Monoclonal antibodies to other surface molecules, especially to CD2 induce the same events as those stimulated by TCR/CD3 ligands, such as increased [Ca2+]i and the production of phosphatidylinositol pathway-related messengers (Alcover et al., 1986
; Pantaleo et al., 1987
). Therefore, it was obvious to investigate the effect of gal-1 on the generation of InsP3 and on the tyrosine phosphorylation of PLC
-1 in Jurkat E6.1 cells. Treatment of the cells with gal-1 stimulates the generation of InsP3. The kinetics is shown in Figure 7A. When compared with control incubations (C, 3.6 ± 0.3 pM/tube), the lectin at 10 µg did not induce significant changes of InsP3. However, when 45 µg gal-1 were applied for cell stimulation, a transient increase of InsP3 was recorded with peak values of 22.0 ± 2.1 pM/tube after 3 min. In these experiments the mutant lectin protein C2S was included because this derivative has considerably more stable ß-galactoside binding activity in the absence of a thiol-reducing reagent (Hirabayashi and Kasai, 1991
). As shown in Figure 7B, cell stimulation with C2S induces also transient, but lower InsP3 levels compared to the native lectin.

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Fig. 7. Kinetics of the generation of InsP3 in Jurkat E6.1 cells induced by gal-1 (A) and C2S (B). The cells at 1 107/0.5 ml in RPMI 1640 medium were treated at 37°C with gal-1 or C2S for the indicated periods of time. Control incubations (c) received an equal volume PBS, pH 7.4. After perchloric acid extraction and neutralization, InsP3 was measured by use of the BiotrakTM competitive [3H]assay system. (A) Kinetics of the generation of InsP3 induced by 10 µg (open bars) and 45 µg gal-1 (solid bars), B: kinetics of the generation of InsP3 induced by 40 µg C2S, Statistical significances were calculated using t-test. Error bars indicate the SD of four determinations. The differences between control incubations and gal-1 treated probes are statistically significant with P < 0.01 as indicated by asterisks.
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In order to investigate tyrosine phosphorylation of PLC, cell stimulation with gal-1 as well as with the mutant lectin protein C2S was performed for various periods of time as indicated in Figure 8. From the cell lysates the enzyme was immunoprecipitated with a PLC
-1 specific mAb. As evidenced by Western blot analysis with an anti-phosphotyrosine-HRP conjugate, lectin stimulation triggered in E6.1 cells the rapid tyrosine phosphorylation of PLC
-1.
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Discussion
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Various ligands that bind the TCR/CD3 complex including appropriately presented antigens, anti-CD3 mAbs, and mitogenic lectins induce phosphoinositide hydrolysis and calcium mobilization. In addition, T-cell activation via the CD2 pathway induces the same events as those stimulated by TCR/CD3 ligands such as increase in [Ca2+]i (Alcover et al., 1986
), generation of the phosphoinositol pathway-related second messengers (Pantaleo et al., 1987
), opening of voltage-insensitive calcium channels (Gardner et al., 1989
), and tyrosine phosphorylation of the CD3
chain (Monostori et al., 1990
). The results of the present study show that gal-1 recognizes appropriately glycosylated cell surface glycoproteins of the human lymphoblastoid T-cell line Jurkat. Among them, the T-cell activation antigens CD2 and CD3 have been identified to be a target for gal-1 as detected by the binding pattern of the biotinylated lectin to CD2 and CD3 immunoprecipitates from Jurkat T-cell lysates on blots. Binding of the lectin to CD2 and CD3 was efficiently blocked by asialofetuin, a glycoprotein carrying a triantennary oligosaccharide with three Galß14GlcNAc sequences and terminal non-reducing Gal residues (Leffler and Barondes, 1986
). The inhibition experiments clearly show that disaccharidic epitopes are available on CD2 and CD3 complementary for gal-1 recognition. Analysis of the gal-1 agarose bound fraction of the Jurkat cell lysate by immunoblotting applying a biotinylated CD2 mAb, revealed CD2 as the main protein band. According to the homodimeric structure with two CRDs, gal-1 may crosslink cell surface glycoconjugates and can modulate various cell response pathways such as growth regulation, adhesion, and activation (Bourne et al., 1994
). In Jurkat E6.1 cells the lectin induces the sustained increase in [Ca2+]i consisting of both an initial transient release from intracellular stores and an altered influx across the plasma membrane. Lactose at 10 mM reduced the gal-1 stimulated calcium response to basal levels. Studies with permeabilized Jurkat cells exposed to purified InsP3 suggest that the initial transient Ca2+ peak is due to InsP3-mediated release from intracellular stores (Imboden and Stobo, 1985
). In E6.1 cells gal-1 generates the transient production of InsP3 with peak values of 22 ± 2.0 pM after 3 min of stimulation which represent a 7.1-fold increase over the basal InsP3 levels. In this T cell model, InsP3 is mainly produced as a result of the rapid tyrosine phosphorylation of PLC
-1, an isoform that is highly expressed in T lymphocytes (Goldfien et al., 1991
). We suppose that such a mechanism is also involved in the Ca2+ response induced by stimulation of Jurkat T-cells with galectin-1 because we observed an increase of tyrosine phosphorylation of PLC
-1. As previously shown, gal-1 mediated changes in [Ca2+]i are not affected by organic Ca2+ channel blockers at doses known to inhibit voltage-operated calcium channels (Walzel et al., 1996
). Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on basal [Ca2+]i values (Figure 6B, b). However, membrane depolarization by gramicidin or by a high-potassium buffer was without effect on the calcium release from intracellular stores but abrogated the gal-1 mediated receptor-operated calcium influx. To determine whether differences exist between cell calcium signaling stimulated by gal-1 or that elicited by CD2 and CD3 mAbs the inhibitors of protein kinases staurosporine and herbimycin A as well as cholera toxin were included. Pretreatment of E6.1 cells with staurosporine, a cell permeable broad spectrum inhibitor of protein kinases, abrogated the cellular calcium response induced by gal-1, CD3 mAb, and CD2 mAb stimulation. When the potent protein tyrosine kinase inhibitor herbimycin A was applied no Ca2+-signal was recorded after CD2 and CD3 mAb stimulation. However, the gal-1 mediated Ca2+-response in the presence of herbimycin A was found to be reduced only to about one-third of the controls. T-Cell antigen receptor-activated GTP binding to CD3-
is involved in coupling the receptor to intracellular signaling pathways (Sancho et al., 1993
). Inhibition of GTP binding to the
-chain by cholera toxin uncouples the receptor from further signaling steps (Böl et al., 1993
).
Pretreatment of Jurkat T-cells with cholera toxin abrogated the CD2 and CD3 mAb stimulated Ca2+-response whereas the Ca2+-signal generated by the recombinant lectin was reduced to about 50% of the controls. It seems likely that in addition to CD2 and CD3 further membrane glycoconjugates and their ligation by the lectin may be sufficient to induce cell calcium signaling. Recently, ganglioside GM1 has been identified as a major receptor for galectin-1 (Kopitz et al., 1998
). In human Jurkat T-cells surface gangliosides GM1 may linked to a cell calcium activation pathway. The presence of functional TCR/CD3 molecules is not essential for GM1-induced cell calcium response (Gouy et al., 1994
). However, ganglioside GM1 seems not to be involved because the lectin was inefficient to induce a Ca2+ response in CD2+ CD3 Jurkat 3113 T-cells. The expression of the receptor-type protein tyrosine phosphatase CD45 was found to be essential for the calcium response elicited by gal-1. Treatment of CD45-deficient J45.01 Jurkat T-cells with the lectin was without an effect on [Ca2+]i. This result is in accordance with studies demonstrating a loss of protein tyrosine phosphorylation, a completely abrogated PIP2 hydrolysis, and a decreased TCR-induced Ca2+-signal in CD45-deficient T-cell lines in response to antigen or anti-CD3 antibody (Koretzky et al., 1990
, 1991; Weaver et al., 1991
). Thus galectin-1 mediated ligation of the CD3-complex can mimic antigen-induced TCR signaling. In T-cells, TCR/CD3 signaling involving elevated [Ca2+]i results in cellular activation, a process marked by proliferation, production of interleukin-2 and expression of other immune function such as cytotoxicity (Crabtree, 1989
). On the other hand, elevated [Ca2+]i is also associated with negative selection of immature thymic T-cells involving apoptosis (Mountz et al., 1995
). There is evidence that galectin-1 modulates TCR signals leading to apoptosis, inhibition of IL-2 production, and proliferation inhibition (Vespa et al., 1999
). The expression of CD45 was found to be required for the induction of apoptosis in activated human T-cells and human leukemic T-cell lines by galectin-1 (Perillo et al., 1995
, Walzel et al., 1999
).
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Materials and methods
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Materials
Asialofetuin, 3-amino-9-ethylcarbazole, aprotinin, divinyl sulfone, ethylene-diaminetetraacetic acid, ethylene glycol-bis(aminoethyl ether)-N,N,N",N"-tetraacetic acid, fura-2 AM,
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, lactose, leupeptin, phenylmethylsulfonyl fluoride, pepstatin, protein A agarose, protein G agarose, sodium deoxycholate, sodium orthovanadate, and sodium dodecyl sulfate were purchased from Sigma (Deisenhofen, Germany).
Kanamycin, fetal calf serum, and RPMI 1640 medium were from GIBCO-BRL (Eggenstein, Germany), CNBr-activated Sepharose 4B, electrophoresis low molecular weight marker, and the Resource Q column from Pharmacia (Uppsala, Sweden). The Biotrak IP3[3H]assay system, Hybond ECL nitrocellulose membranes, and ECL detection reagents were obtained from Amersham-Buchler (Braunschweig, Germany). Affi gel 15, Bio-gel P-6, and the biotinylated molecular weight standards were from Bio-Rad (Munich, Germany), biotinyl-N-hydroxysuccinimide, streptavidin-HRP, and the anti-phosphotyrosine HRP-conjugate from Boehringer (Mannheim, Germany). The CD2 mAbs (clone 39C1.5, IgG 2a isotype; clone 6F10.3, IgG1 isotype) and CD3 mAbs (clone UCHT1, IgG1 isotype; clone X35, IgG2a isotype) were obtained from Coulter-Immunotech (Marseille, France). The phospholipase C
-1 mAb was from BIOMOL (Hamburg, Germany).
Cells
The human leukemic T cell lines Jurkat, clone E6.1 (European Collection of Animal Cell Cultures, Salisbury, UK), the CD45-deficient cell line (clone J45.01, a gift from G.A.Koretzky, University of Iowa), and the CD2+CD3 3113 cell clone (kindly given by A.Alcover, Institut Pasteur, Paris, France) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 10 µg/ml kanamycin.
Purification of gal-1 from human placenta
Gal-1 was obtained from term placentas by lactose extraction with EDTA-MePBS (20 mM sodium phosphate, pH 7.2, 150 mM NaCl, 4 mM 2-mercaptoethanol, 2 mM EDTA) and purified by sequential affinity chromatography on asialofetuin Sepharose 4B (Hirabayashi and Kasai, 1984
) followed by lactosyl agarose. Then the protein was purified to homogeneity by anion exchange chromatography on a Resource Q column.
Production of the recombinant and mutant human 14 kDa galectins
The recombinant lectin (rec gal-1) was produced by insertion of the cDNA for a 14 kDa ß-galactoside-binding lectin into a plasmid carrying a Taq promoter and expressed in E.coli as previously described (Hirabayashi et al., 1989
). The production of the 14 kDa mutant lectin protein C2S by site-directed mutagenesis experiments is described in detail (Hirabayashi and Kasai, 1991
).
Gal-1 immobilization
The lectin (6.0 mg/ml 20 mM HEPES buffer, pH 8.0) was coupled to 400 µl Affi Gel 15 according to the manufacturers instructions. After blocking the N-hydroxysuccinimide ester groups by adding of 0.1 ml 1 M ethanolamine-HCl (pH 8.0), the affinity support was stored in EDTA-MePBS at 4°C.
Biotinylation of gal-1
The lectin (1 mg/ml) was biotinylated in PBS, pH 8.0, by the addition of 60 µl 10 mM biotinyl-N-hydroxysuccinimide in DMSO (Boorsma et al., 1986
). After incubation at room temperature for 1 h, buffer change was performed on a Bio-Gel P6 column equilibrated with PBS, pH 7.4.
Measurements of intracellular free calcium [Ca2+]i
Measurements of intracytoplasmic free Ca2+ were performed applying the fluorescent Ca2+-indicator fura-2 AM. The cells (2 106) were loaded with 5 µM fura-2 AM in 1 ml RPMI 1640 medium for 30 min at 37°C. After the addition of 3 ml RPMI 1640 medium, the incubation at 37°C was proceeded for 20 min. Then the fura-2 loaded cells were washed three times with 10 mM Na-HEPES buffer, pH 7.4, supplemented with 137 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 1 mM Na2HPO4, and 1 g BSA/l. For depolarization the fura-2 loaded cells were washed three times with a high-potassium medium (10 mM Na-HEPES, pH 7.4, 32 mM NaCl, 110 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 1 mM Na2HPO4, and 1 g BSA/l). Viability of cells as detected by trypan blue exclusion was higher than 94%. The cell suspension was excited alternatively at 339 and 380 nm and the fluorescence was monitored at 490 nm with a Shimadzu RF 5001 PC spectrofluorimeter. Graphic representations of [Ca2+]i were calculated by converting the ratio of 339/380 to [Ca2+]i using a Kd of 224 nM according to the equation [Ca2+]i = 224 (R - Rmin)/(Rmax - R) Sf 380/Sb 380 (Grynkiewicz et al., 1985
). Rmax and Rmin was evaluated in 1 mM Ca2+-containing medium by lysing the cells with 0.5% Triton X-100 for Rmax followed by the addition of an excess of EGTA for Rmin.
SDSPAGE and Western blot analysis
The SDSPAGE was performed in 1 mm slab gels (Laemmli, 1970
). Separated proteins were electrophoretically transferred to Hybond ECL nitrocellulose membranes and blocked in 20 mM TrisHCl, pH 7.2, 1 M NaCl, 1% Tween 20. Then the blots were probed with 2 µg/ml of biotinylated gal-1 in TBS/Tw (20 mM TrisHCl, pH 7.2, 1 M NaCl, 0,05% Tween 20) containing 4 mM 2-mercaptoethanol, or with biotinylated CD2 or CD3 mAbs at 1 µg/ml in TBS/Tw for 16 h at 4°C. After washing in incubation buffer, the blots were incubated with a streptavidin-HRP conjugate (1:1000 dilution) for 1 h at room temperature. The blots were washed four times in TBS/Tw and once with 0.05 M acetate buffer, pH 5.0, for 1 min. Staining was performed with a mixture of 10 mg 3-amino-9-ethylcarbazole in 1 ml acetone, 25 ml 0.05 M acetate buffer, and 15 µl H2O2 (30%). Washing of the blots completed the procedure.
Measurement of inositol-1,4,5-trisphosphate (InsP3) generation
Jurkat T-cells at 1 107 cells/0.5 ml RPMI 1640 medium containing 15 mM LiCl were treated with the lectin at 37°C for the periods of time as indicated in the figures. Then the stimulation was terminated on ice by the addition of 100 µl ice-cold 20% perchloric acid. After 20 min, the proteins were sedimented by centrifugation at 2000 g for 15 min and the supernatant was adjusted to pH 7.5 by titrating with ice-cold 10 M KOH. The supernatant obtained by centrifugation at 2000 g for 15 min was applied for the measurement of InsP3 using the Biotrak competitive IP3 [3H]assay system applying a bovine adrenal InsP3 binding protein. By use of this system, InsP3 may be measured in the range from 0.19 to 25 pM per tube.
Separation of Jurkat T-cell lysates on gal-1 agarose
The cells (5.8108) were lysed in 2 ml EDTA-MePBS, pH 7.2, supplemented with 1% NP 40, 10 µg/ml aprotinin, 10µg/ml leupeptin, and 1 mM PMSF on ice for 1 h. Then the lysate was centrifuged at 5000 g for 10 min at 4°C. After incubation of the supernatant end over end with 300 µl gal-1 agarose, the gel suspension was transferred to a 0.22 µm centrifuge filter unit, washed twice in cell lysis buffer and finally five times in 0.2% NP 40 containing lysis buffer. The gel was eluted with 300 µl 0.1 M lactose in washing buffer. For SDSPAGE, the gal-1 agarose bound fraction was treated with 100 µl of 4-fold concentrated electrophoresis sample buffer for 5 min at 100°C.
Immunoprecipitation of CD2 and CD3 from Jurkat T-cell lysates
The cells (3 108) were lysed in 1 ml cell lysis buffer (20 mM TrisHCl, pH 7.5, 1% NP 40, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin) on ice for 1 h. The supernatant obtained by centrifugation at 5000 g and 4°C for 15 min was incubated with 100 µg of a CD3 or 120 µg of a CD2 mAb on ice for 1 h. After incubation of the supernatant with 150 µl protein G agarose, the beads were collected by centrifugation and washed five times with cell lysis buffer on a 0.22 µm centrifuge filter unit. Then the beads were treated with 180 µl of double concentrated nonreducing sample buffer for 5 min at 100°C.
Analysis of tyrosine phosphorylation of phospholipase C
-1
Jurkat T-cells at 1 107/0.5 ml RPMI 1640 medium were treated with gal-1 or C2S for the periods of time as indicated in the figures. Control incubations received an equal volume PBS, pH 7.4. After termination on ice, the cells were collected by centrifugation at 300 g and the cell pellets were treated at 0°C with 300 µl lysis buffer (10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 1% NP 40, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM PMSF,10 µg/ml aprotinin, 2 µg/ml antipain, 2 µg/ml pepstatin, 2 µg/ml leupeptin) for 30 min. The supernatant obtained by centrifugation at 5000 g and 4°C for 10 min was incubated with 5 µg of a PLC
-1 mAb for 2 h on ice. After addition of 50 µl protein A agarose, the incubation on ice was continued for 1 h. Then the beads were collected by centrifugation, washed four times with cell lysis buffer, and were treated with 100 µl electrophoresis sample buffer for 5 min at 100°C. The immunoprecipitates were resolved by SDSPAGE on a 7.5% separation gel and transferred to Hybond ECL membranes. The membranes were blocked and incubated with an anti-phosphotyrosine HRP-conjugate (1:12,500 dilution) for 16 h at 4°C. Then the blots were washed and developed by enhanced chemiluminescence.
 |
Abbreviations
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BSA, bovine serum albumin; CRD, carbohydrate recognition domain; [Ca2+]i, concentration of intracytoplasmic free calcium; C2S, mutant lectin protein in which Cys 2 was replaced by serine; CTX, cholera toxin; DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; EDTA, ethylene-diaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N",N"-tetraacetic acid; gal-1, galectin-1; HEPES, N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid; HRP, horseradish peroxidase; InsP3, inositol-1,4,5-trisphosphate; NP 40, Nonidet P 40; PLC, phospholipase C; PBS, phosphate buffered saline; rec H, recombinant human 14 kDa lectin; SDSPAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; SD, standard deviation; TBS, tris-buffered saline; TCR, T-cell receptor for antigen; Tris, tris(hydroxymethyl)aminomethane.
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Footnotes
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1 To whom correspondence should be addressed 
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References
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