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Originally published online as doi:10.1189/jlb.1106660 on January 22, 2007

Published online before print January 22, 2007
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(Journal of Leukocyte Biology. 2007;81:1002-1011.)
© 2007 by Society for Leukocyte Biology

Glycosylation-dependent interaction of Jacalin with CD45 induces T lymphocyte activation and Th1/Th2 cytokine secretion

Makoto Baba*,{dagger},1, Bruce Yong Ma*,1,2, Motohiro Nonaka*,{dagger}, Yukari Matsuishi{ddagger}, Makoto Hirano*,{dagger}, Natsuko Nakamura*,{dagger}, Nana Kawasaki{ddagger}, Nobuko Kawasaki* and Toshisuke Kawasaki*,2

* Research Center for Glycobiotechnology, Ritsumeikan University, Shiga, Japan;
{dagger} Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; and
{ddagger} Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan

2 Correspondence: Research Center for Glycobiotechnology, Ritsumeikan University, Shiga 525-8577, Japan. E-mail: byma{at}fc.ritsumei.ac.jp; tkawasaki{at}fc.ritsumei.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Jacalin, an {alpha}-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (galactose ß1-3 N-acetylgalactosamine, T-antigen)-specific lectin from jackfruit seeds, has been shown to induce mitogenic responses and to block infection by HIV-1 in CD4+ T lymphocytes. The molecular mechanism underlying Jacalin-induced T cell activation has not been elucidated completely yet. In the present study, protein tyrosine phosphatase (PTPase) CD45 was isolated from a Jurkat T cell membrane fraction as a major receptor for Jacalin through affinity chromatography and mass spectrometry. CD45, which is highly glycosylated and expressed exclusively on the surface of lymphocytes, is a key regulator of lymphocyte signaling, playing a pivotal role in activation and development. We found that the lectin induced significant IL-2 production by a CD45-positive Jurkat T cell line (JE6.1) and primary T cells. However, this effect did not occur in a CD45-negative Jurkat T cell line (J45.01) and was blocked completely by a specific CD45 PTPase inhibitor in Jurkat T (JE6.1) and primary T cells. Furthermore, we also observed that Jacalin caused a marked increase in IL-2 secretion in response to TCR ligation and CD28 costimulation and contributed to Th1/Th2 cytokine production by activating CD45. Jacalin increased CD45 tyrosine phosphatase activity, which resulted in activation of the ERK1/2 and p38 MAPK cascades. Based on these findings, we propose a new, immunoregulatory model for Jacalin, wherein glycosylation-dependent interactions of Jacalin with CD45 on T cells elevate TCR-mediated signaling, which thereby up-regulate T cell activation thresholds and Th1/Th2 cytokine secretion.

Key Words: carbohydrate recognition • lectin-carbohydrate interaction • protein-tyrosine phosphatases • protein-tyrosine kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T lymphocyte activation events figure prominently in initiation of an adaptive immune response. Stimulation of the T cell antigen receptor complex causes a series of intracellular, biochemical events, which regulate the production of several cytokines, including IL-2, and promote T cell activation [1 ]. Glycosylation of cell surface proteins controls critical T cell processes, including lymphocyte homing, thymocyte selection, the amplitude of an immune response, T cell activation, and death [2 ]. The role of glycosylation in these functions is specific; i.e., different functions require specific sugars on specific glycoprotein acceptors [3 ]. Regulated glycosylation of specific acceptor substrates can affect immune functions by creating or masking ligands for endogenous and extraneous lectins [3 ]. Plant lectins have long been used as surrogates for authentic T cell activational stimuli. These carbohydrate-binding proteins activate T lymphocytes by cross-linking many T cell surface glycoproteins, including some that contribute to the immunological synapse [1 ]. Although a number of observations have suggested that protein-glycan interactions contribute to immune system functions, nonetheless, it is still difficult to attach any physiological significance and to propose any molecular basis to plant lectin-dependent activation events.

Jacalin, an {alpha}-O-glycoside of the disaccharide Thomsen-Friedenreich antigen [galactose ß1-3 N-acetylgalactosamine (Galß1-3GalNAc), T-antigen], and in its sialylated form specific lectin from jackfruit seeds, is a tetrameric, two-chain lectin (molecular mass, 66 kDa) comprising a heavy {alpha} chain of 133 amino acid residues and a light ß chain of 20 amino acid residues [4 ]. It was first described as a general T cell mitogen [5 ] without any specificity for CD4+ or CD8+ T cell subsets [5 ]; however, other studies revealed that Jacalin triggered only CD4+ T cell signaling by binding CD4 [6 , 7 ] and that Jacalin also interacted with CD8 [8 ]. These contradictory results led us to consider the possibility that the lectin may interact directly with another widespread membrane glycoprotein expressing CD4+ and CD8+ T cell subsets. We examined this possibility by isolating and identifying the novel receptor expressed on T cells through affinity chromatography and mass spectrometry (MS). The results presented in this study constitute credible evidence that CD45, a Type I transmembrane protein tyrosine phosphatase (PTPase) found on the surface of all nucleated, hematopoietic cells and their precursors, is one of the major Jacalin targets in CD4+ and CD8+ T cell subsets. CD45 is highly glycosylated and has been estimated to comprise up to 10% of the T lymphocyte surface area [9 ]. CD45 is a critical component of the TCR signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs), such as p56Lck, which is well known as one of the CD45 substrates. Indeed, dysregulation of CD45 activity in T cell developmental arrest impaired T cell activation or autoimmunity [10 ]. Activation of p56Lck results in a sequence of events starting with tyrosine phosphorylation of the TCR {zeta} chain, followed by binding and activation of Syk family kinase ZAP70 and phosphorylation and activation of the MAPK cascade, leading to activation of nuclear transcription factors regulating the induction of IL-2 gene expression [11 , 12 ]. Although the requirement of invariant cytoplasmic domains with intrinsic PTPase activity has been documented, no data are available concerning the function of the CD45 extracellular domain in lymphocyte signal transduction. The N-terminal heterogeneity of the extracellular domain is generated through alternative mRNA splicing of three exons encoded by a single gene and through different glycosylation, resulting in CD45 isoforms with molecular masses in the range of 180–220 kDa. How the PTPase activity is regulated is one of the crucial problems to be solved. As the carbohydrate-recognition profile of Jacalin consists of a unique specificity for terminal Galß1-3GalNAc epitopes [4 ], which are present on CD45 of human lymphocyte [13 ], we analyzed the lectin-glycan interaction between Jacalin and CD45 for answering the question. Consequently, we found that the lectin induced significant IL-2 production by a CD45-positive Jurkat T cell line (JE6.1) and primary T cells, and the effect was blocked completely by a specific CD45 PTPase inhibitor. However, Jacalin-induced IL-2 secretion did not occur in a CD45-negative Jurkat T cell line (J45.01). In parallel, we also observed that Jacalin caused a marked increase of IL-2 secretion in response to TCR ligation and CD28 costimulation and contributed to Th1/Th2 cytokine secretion. Moreover, we showed the ability of the lectin as a positive inducer of the TCR signaling threshold to activate PTPase CD45, which resulted in activation of the MAPK cascade leading to up-regulation of the T cell response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Jacalin, FITC-conjugated Jacalin, and agarose-conjugated Jacalin were purchased from US Biological (Swampscott, MA, USA). A PTPase CD45 inhibitor N-(9, 10-dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide was obtained from Calbiochem (La Jolla, CA, USA). Polyclonal antibodies against phospho-p38 MAPK (Thr180/Tyr182) and phospho-p44/42 MAPK (Thr202/Tyr204) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antihuman CD3 (OKT3) and a human Th1/Th2 ELISA kit were purchased from eBioscience (San Diego, CA, USA). PE-conjugated anti-CD4, PC5-conjugated anti-CD8, and PE-conjugated anti-CD45 mAb were obtained from Beckman Coulter (Fullerton, CA, USA). A human IL-2 ELISA kit was obtained from BD PharMingen (San Diego, CA, USA). FITC-conjugated anti-CD45 mAb and anti-CD28 mAb were obtained from Immunotech (Marseille, France) and Chemicon (Temecula, CA, USA), respectively. Alexa546-conjugated antimouse IgG1 secondary antibody was obtained from Molecular Probes (Eugene, OR, USA). PMA was purchased from Sigma-Aldrich (St. Louis, MO, USA). A protease inhibitor cocktail and ionomycin were obtained from Nacalai Tesque (Kyoto, Japan). All chemicals for gel electrophoresis and Western blotting were purchased from ATTO (Tokyo, Japan), Bio-Rad (Hercules, CA, USA), Pierce (Rockford, IL, USA), or Zymed (South San Francisco, CA, USA).

Cell lines, cell culture, and cell stimulations
The human CD45-positive Jurkat T cell line, Clone JE6.1, and the CD45-deficient variant of this clone, J45.01, were obtained from American Type Culture Collection (Manassas, VA, USA). All the cell lines were cultured at 37°C in 5% CO2 in RPMI 1640 containing 10% FCS, 2 mM glutamine, 50 mg/ml penicillin, and 50 mg/ml streptomycin. For T cell stimulation, cells were diluted to 1 x 106 cells/ml and then incubated in the medium alone or with the specified additions for the times indicated.

Preparation of membrane fractions, affinity chromatography, and MS
Jurkat (Clone JE6.1) T cells were homogenized in homogenization buffer [150 mM NaCl, 20 mM Tris/HCl (pH 7.5), 0.32 M sucrose, 1 mM EDTA, and protease inhibitor cocktail]. The homogenate was centrifuged at 1000 g for 10 min at 4°C twice to remove cell debris and nuclei. The supernatant was then centrifuged at 105,000 g for 60 min at 4°C. The resulting total membrane pellet was solubilized with lysis buffer [150 mM NaCl, 20 mM Tris/HCl (pH 7.5), 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail] for 60 min on a rotary shaker at 4°C and then centrifuged at 150,000 g for 60 min at 4°C. The supernatant was saved as the Jurkat T cell membrane proteins and was applied to a Jacalin-agarose affinity column. After washing the column with TBS buffer (pH 7.5) containing 20 mM CaCl2 and 0.1% Triton X-100, the proteins bound to the column were eluted with TBS buffer (pH 7.5) containing 100 mM methyl-{alpha}-Gal (Me-{alpha}-Gal) and 0.1% Triton X-100. The eluted proteins were resolved on a 5–20% Tris-HCl gradient gel (ATTO) and then stained with colloidal Coomassie blue (GelCode Blue, Pierce). Bands were excised from the gel and subjected to in-gel digestion. The peptides released from the gel were subjected to liquid chromatography (LC)/MS/MS analysis with a linear ion trap mass spectrometer (Finnigan LTQ, Thermo Electron Corp., San Jose, CA, USA) interfaced on-line with a capillary HPLC (Paradigm, Michrom BioResources, Auburn, CA, USA) equipped with a Magic C18 column (0.2x50 mm, 3 µ, Michrom BioResources). The eluents consisted of H2O containing 2% CH3CN and 0.1% formic acid (Pump A) and 90% CH3CN and 0.1% formic acid (Pump B), and the peptides were eluted with a linear gradient of 5–95% from Pump B in 20 min at a flow rate of 3 µl/min. Data-dependent MS/MS acquisitions were performed for the most intense ions as precursors. Proteins were identified by searching the MS Protein Sequence Database (MSDB) and NCBI database (human) using the Mascot search engine (Matrixscience, London, UK) and TurboSEQUEST search engine (Thermo Electron), respectively.

Separation and culture of human primary lymphocytes
PBL were isolated from healthy donors by Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ, USA) density gradient centrifugation at 3000 rpm for 25 min. CD3+ T cells were purified by positive selection with anti-CD3 antibody-coated magnetic beads, and CD4+ and CD8+ T cells were purified by negative selection with a magnetic cell isolation system, respectively, according to the manufacturer’s instructions (Miltenyi Biotec GmbH, Germany). Flow cytometry of the resulting cell population indicated that more than 90% of the remaining cells expressed CD3, CD4, and CD8, respectively. The separated cells were incubated in a similar medium to that for Jurkat T cell lines. For stimulation, cells were diluted to 1 x 106 cells/ml and then incubated in the medium alone or with the specified additions for the times indicated.

Flow cytometry
Cells were washed once in PBS and pelleted, and then viable cells were resuspended in FACS buffer (PBS containing 2% FCS). To assess CD3, CD4, CD8, CD45, and Jacalin receptor surface expression, 20 µl FITC/PE-conjugated antibodies specific to the respective CDs were added to 1.0 x 106 cells in a volume of 200 µl for 1 h, followed by three washes in FACS buffer. All incubations were conducted on ice to prevent receptor internalization. After 1 h on ice, cells were washed once with FACS buffer, pelleted by centrifugation, and finally suspended in 500 µl FACS buffer before analysis with a FACScan (Becton Dickinson, San Jose, CA, USA) flow cytometer equipped with the CellQuest software program. The double-receptor expression on the surface of CD4+ T and CD8+ T cells was determined by two-color flow cytometry using anti-CD4-PE antibody/anti-CD8-PC5 antibody and anti-CD45-FITC antibody; anti-CD4-PE antibody/anti-CD8-PC5 antibody and Jacalin-FITC; or anti-CD45-PE antibody and Jacalin-FITC.

Confocal laser-scan microscopy
This experiment was carried out on two-well chamber glass slides. The Jurkat T cells and human primary CD3+ T cells were subsequently incubated overnight at 37°C and monitored under a confocal microscope (BX50, Olympus, Tokyo, Japan). For double immunofluorescence, the T cells were stained with FITC-conjugated Jacalin and antihuman CD45 mAb, followed by Alexa546-conjugated antimouse IgG1 polyclonal antibody.

Cytokine ELISA assay
For cytokine production, 2 x 105 Jurkat T cells and the purified human CD3+ T, CD4+ T, or CD8+ T cells were placed in round-bottom, 96-well plates. The cells were stimulated with various doses of Jacalin, PMA (50 ng/ml)/ionomycin (1 µM), or anti-CD28 (1 µg/ml) antibody for 24 h or 72 h, and then culture supernatants were harvested and analyzed for IL-2 secretion using a commercial kit from Becton Dickinson and for Th1 and Th2 differentiation (Th1 cytokines IL-2 and IFN-{gamma} and Th2 cytokines IL-4 and IL-10), using a Th1/Th2 ELISA panel from eBioscience, following the manufacturer’s protocol. The standards and test samples were analyzed with a Multilabel Counter (PerkinElmer, Wellesley, MA, USA), in accordance with the manufacturer’s instructions. For TCR-CD3 cross-linking, the plates were coated with antihuman CD3 mAb (OKT3, eBioscience) at 1 µg/well and left overnight at 4°C and then washed three times before incubation with the T cells. All experiments were performed in triplicate and were repeated a minimum of three times.

Immunoblotting and lectin blotting
One percent Nonidet P-40 lysis buffer cell extracts were boiled with the sample buffer. Samples were resolved on a 4–20% gradient SDS-PAGE gel (ATTO) and then transferred to nitrocellulose membranes, followed by immunoblot or lectin-blot detection. For visualization, a SuperSignal West Pico chemiluminescent kit (Pierce) was used with HRP-conjugated antimouse IgG antibody (Zymed) or HRP-conjugated antirabbit IgG (Cell Signaling Technology).

Phosphorylation of MAPK family kinases
For signal transduction studies, Jurkat (JE6.1) T cells and human primary CD3+ T cells were serum-starved overnight and then stimulated with PMA plus ionomycin as a positive control for T cell activation and tyrosine phosphorylation of various kinases. Cells in the medium alone were used as a negative control. For stimulation with Jacalin, cells were washed and incubated with 50 µg/ml Jacalin at 37°C for different times, and the incubation was terminated with ice-cold PBS + EDTA + sodium orthovanadate. Whole cell lysates were analyzed by Western blotting with specific antibodies for phospho-p44/42 MAPK (Thr202/Tyr204) and phospho-p38 MAPK (Thr180/Tyr182) purchased from Cell Signaling Technology.

Statistical analysis
The results are expressed as the means ± SD of data obtained in three or four experiments performed in duplicate or triplicate. Statistical significance was determined by means of Student’s test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Jacalin receptors on human Jurkat T cells and peripheral T cells
To obtain an insight into the expression profile of Jacalin receptors on T lymphocytes, a human Jurkat T cell line (Fig. 1A ) and peripheral CD3+ T cells (Fig. 1B) were stained with FITC-conjugated Jacalin and then analyzed by flow cytometry. As shown in Figure 1 , Jurkat T cells and peripheral CD3+ T cells exhibited high levels of Jacalin receptor expression. Following up on these binding data, we investigated whether Jacalin binding to the surface of T cells is dependent on carbohydrate interactions. Thus, we examined the ability of several free monosaccharides to competitively block the interaction of Jacalin with T cells. The addition of 50 mM Me-{alpha}-Gal significantly abrogated Jacalin binding to Jurkat T cells as well as to PBL CD3+ T cells. In contrast, Glc and Gal, at the concentration of 50 mM, exhibited substantially less inhibition of the binding of Jacalin to T cells (Fig. 1) . This competitive inhibition with free monosaccharides is consistent with the carbohydrate recognition specificity of Jacalin reported previously [4 ]. The data suggest that the glycans expressed on T cells may mediate the biological function of Jacalin induction through the interaction between the glycans and Jacalin.


Figure 1
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  		Figure 1. Expression of Jacalin receptors on the surfaces of Jurkat T and primary CD3+ T cells. Jurkat (JE6.1) T cells (A) and the separated human CD3+ T cells (B) were stained with FITC-labeled Jacalin (5 µg/ml) and then analyzed by flow cytometry, respectively. The cells were prepared and labeled as described in Materials and Methods. As a negative control, autofluorescence of the cells was measured (purple area). As shown in each histogram, the sugar specificity of the binding of Jacalin to Jurkat T and primary CD3+ T cells was analyzed in the presence of 50 mM Gal (pink), glucose (Glc; blue), or Me-{alpha}-Gal (orange), respectively.

 
Purification and identification of novel Jacalin receptors on Jurkat T cells
To isolate the Jacalin receptors expressed on T cells, a Jurkat (JE6.1) membrane fraction was obtained as described in Materials and Methods. The Jurkat membrane proteins were applied to an affinity column of Jacalin-agarose, and the bound proteins were eluted with 100 mM Me-{alpha}-Gal. The eluted proteins were separated by SDS-PAGE and then detected by colloidal Coomassie blue staining (Fig. 2B ). The binding with Jacalin was confirmed by Jacalin blotting (Fig. 2A) . As shown in Figure 2 A and B , the Jacalin receptors appeared as three major bands in the molecular mass range of 180–240 kDa under reducing conditions. For identification of the purified and separated Jacalin receptors, the three major bands, indicated by the arrows in Figure 2B , were excised and in-gel-digested with trypsin. The digested proteins were analyzed by nano-LC/MS/MS, as described in Materials and Methods. The acquired fragmentation nano-LC/MS/MS spectra of peptides were searched for in the mouse protein MSDB using the Mascot search engine. In this way, the three major materials were identified positively as PTPase CD45 and isoforms of it, which arise through alternative mRNA splicing and differential glycosylation, and as novel Jacalin receptors. As shown in Figure 2C and Supplemental Figure 1, the matched peptides shown in red within the complete CD45 and its isoform precursor sequences were found in the fragments analyzed by MS. CD45 is a heavily glycosylated transmembrane, two-domain PTPase, expressed exclusively in nucleated, hematopoietic cells, and is a critical component of the TCR signaling pathway [10 ]. Next, we investigated whether CD45 can be stained directly by Jacalin on the T cell surface. When examined by confocal microscopy, we found CD45 was costained substantially with FITC-conjugated Jacalin (green) and anti-CD45 mAb followed by Alexa546-conjugated 2nd antibody (red) in Jurkat T cells and primary T cells (Fig. 2D) . Collectively, the lectin-blotting experiment and confocal microscopy confirmed that CD45 and its isoforms are Jacalin, major targets expressing on T cell surfaces.


Figure 2
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  		Figure 2. Purification and identification of novel Jacalin receptors on T cells. (A and B) Purification of Jacalin receptors by affinity chromatography. Jacalin receptors were purified from human Jurkat T cell membrane proteins on a Jacalin-agarose affinity column and fractionated on a 5–20% reducing gradient SDS-PAGE gel. (A) Jacalin blotting; (B) colloidal Coomassie blue staining. The arrows indicate the elution positions of the purified Jacalin receptors. The molecular weight markers are shown on the left. (C) Identification of Jacalin receptors by MS. The purified Jacalin receptor bands were excised and digested with trypsin, and the fragments were used for the identification of Jacalin receptors by MS, as described in Materials and Methods. (D) Costaing Jurkat T cells and primary CD3+ T cells with Jacalin-FITC and CD45-Alexa548. The CD45-positive Jurkat T cells (top panels), CD45-negative Jurkat T cells (middle panels), and human primary CD3+ T cells (bottom panels) were costained with FITC-conjugated Jacalin and antihuman CD45 mAb, followed by Alexa546-conjugated 2nd antibody and then monitored by confocal microscope. The left and middle panels show the fluorescence from FITC (green) and Alexa548 (red), respectively, and the right panels show the combined fluorescence (yellow).

 
Jacalin-stimulated IL-2 production through CD45 triggering in T cells
CD45 is the major PTPase on the surface of T cells, which functions as a positive and a negative regulator of TCR signaling. With the present finding that Jacalin binds CD45 on the T cell surface as described above, we next examined its functions, examining whether CD45 has a special role in mediation of the T cell activation initiated by Jacalin stimulation. To evaluate whether the Jacalin-CD45 interaction affected T cell activation, we compared the Jacalin-induced IL-2 production using Jurkat (JE6.1) T cells, which endogenously express CD45 and are used frequently to study TCR-mediated signaling, and J45.01, a CD45-deficient derivative cell line derived from JE6.1. Jurkat T cells stimulated with PMA plus ionomycin were used as a positive control, and there were no significant differences in the ability of IL-2 production stimulated with PMA plus ionomycin between JE6.1 and J45.01 Jurkat T cells (data not shown). Jurkat T cells, in the medium alone, were used as a negative control. It is interesting that as shown in Figure 3 , Jacalin induced IL-2 secretion in a dose-dependent manner in CD45+ Jurkat T cells (Fig. 3A) , and the effect was markedly inhibited in the presence of 100 mM Me-{alpha}-Gal, a strong inhibitor of Jacalin-glycan interactions (Fig. 3B) , whereas Jacalin could not induce any IL-2 production by CD45– Jurkat T cells. Similar results were obtained for human primary CD3+ T cells, in which IL-2 was produced in a Jacalin-dose-dependent manner (Fig. 3C) . In parallel, to further confirm the role of CD45 signaling in IL-2 production triggered by Jacalin induction, we incubated CD45+ Jurkat T cells with a specific CD45 PTPase inhibitor, N-(9,10-dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide [14 ], during the lectin stimulation. We found the IL-2 secretion was blocked effectively in a concentration-dependent manner (Fig. 3B) . Taken together, the data evidently indicate that Jacalin induces T cell activation in a carbohydrate-dependent manner through CD45 triggering, resulting from the CD45-Jacalin interaction.


Figure 3
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  		Figure 3. Determination of Jacalin-dependent IL-2 production on activation of CD45 in Jurkat T and primary CD3+ T cells. (A and B) CD45+ Jurkat (JE6.1) and CD45– Jurkat (J45.01) T cells were stimulated with Jacalin at various concentrations (A), and CD45+ Jurkat cells were incubated with Jacalin in the presence of the PTPase CD45 inhibitor at different concentrations or Me-{alpha}-Gal at 100 mM (B) for 24 h, and then culture supernatants were harvested and analyzed for IL-2 production using a Becton Dickinson ELISA kit, according to the manufacturer’s instructions. The results are representative of three independent experiments. (C) CD3+ T cells purified from healthy human PBL were stimulated with Jacalin at various concentrations as described above for 72 h. Culture supernatants were harvested and analyzed for IL-2 production using a Becton Dickinson ELISA kit. Data for one representative experiment of at least three independent experiments are shown.

 
Jacalin modulates Th1/Th2 cytokine production by activating CD45
To investigate the expression of Jacalin receptors and the Jacalin-mediated cytokine secretion in T cell subsets in detail, CD4+ and CD8+ T cells were isolated from human PBL by negative selection with specific antibody-coated magnetic beads and analyzed by dual-color flow cytometry. The purity of the resulting subsets was checked by flow cytometry and was higher than 90%. As shown in Figure 4A , there is a strong correlation between CD45 and Jacalin staining on CD4+ and CD8+ T cells (Fig. 4A 4e and 4f) . The dichotomy of Types 1 and 2 Th cells, which have differentiated from naïve CD4+ T cells, stands as a central paradigm in cellular immunology, which provides the framework for understanding the nature of an immune response that is beneficial or detrimental to the host [15 , 16 ]. Th1 and Th2 cells are characterized by their mutually exclusive expression patterns of cytokines. Th1 cells produce IL-2 and IFN-{gamma}, whereas Th2 cells produce IL-4 and IL-10 [16 ]. As shown in Figure 4B , IFN-{gamma} was produced at a greater level than IL-4, which is typical of CD4+ cells from many sources, and corresponds with previous reports that a low level of IL-4 is functionally significant, and the specific activity of IL-4 is much greater than that of IFN-{gamma} [17 , 18 ]. In contrast, IL-10 was produced only minimally (<5 pg/ml) by T cell subsets by Day 3. It is interesting that Jacalin also induces respectable IFN-{gamma} production by CD8+ T cells. The Jacalin-induced cytokine secretion was blocked effectively in the presence of the specific CD45 PTPase inhibitor (Fig. 4B) . These data suggest that the level of signaling achieved on activation of CD45 is intimately associated with the induction of distinct cytokine-secreting T cell subsets. The data also indicate that IFN-{gamma}-producing cells were induced by a lower level of Jacalin than IL-4-producing cells and imply that Jacalin may contribute to the Th1/Th2 cytokine balance, in which T cell subsets will predominate through CD45 activation.


Figure 4
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  		Figure 4. Determination of Jacalin-induced Th1/Th2 cytokine production in CD4+ and CD8+ T cells on CD45 activation. (A) Expression of Jacalin receptors on human CD4+ and CD8+ T cell subsets. CD4+ (a, c, and e) and CD8+ (b, d, and f) T cells from among healthy human PBL were separated with a QuadroMACS separation unit and were then analyzed by two-color flow cytometry using PE-labeled anti-CD4 and FITC-labeled anti-CD45 (a), PC5-labeled anti-CD8 and FITC-labeled anti-CD45 (b), PE-labeled anti-CD4 and FITC-labeled Jacalin (c), PC5-labeled anti-CD8 and FITC-labeled Jacalin (d), PE-labeled anti-CD45 and FITC-labeled Jacalin (e), or PE-labeled anti-CD45 and FITC-labeled Jacalin (f). The proportions of subsets gated for separated CD4+ (a, c, e) or CD8+ (b, d, f) are indicated in the upper portions of the histograms. The concentration of Jacalin-FITC is 5 µg/ml. The results are representative of three independent experiments. (B) Jacalin-induced Th1/Th2 cytokine production by CD4+ and CD8+ T cells on activation of CD45. The separated CD4+ and CD8+ T cells were stimulated in the absence or presence of the PTPase CD45 inhibitor (1 µM) for 72 h as described above. Culture supernatants were harvested and analyzed for IL-2 (a), IFN-{gamma} (b), IL-4 (c), and IL-10 (d) Th1/Th2 cytokine production using an eBioscience Th1/Th2 ELISA kit, according to the manufacturer’s instructions. The results are representative of four independent experiments.

 
Jacalin promotes TCR signaling via CD45 signal initiation
In an attempt to correlate Jacalin induction with TCR signaling, CD45+ Jurkat T cells, treated as described in Figure 5 , were cultured for 24 h, and culture supernatants were harvested and analyzed for IL-2 production. It is surprising that there is obviously increased IL-2 secretion following stimulation with CD3+CD28 plus Jacalin when compared with treatment with CD3+CD28 alone or Jacalin alone. In addition, the enhancement was reduced significantly with the PTPase CD45 inhibitor to the level with CD3+CD28 plus Jacalin stimulation (Fig. 5) . The data indicate a positive, concerted relationship or synergistic effects of TCR signaling and Jacalin-induced CD45 activation.


Figure 5
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  		Figure 5. Jacalin enhances IL-2 secretion in response to TCR ligation and CD28 costimulation in Jurkat T cells. CD45+ Jurkat (JE6.1) T cells were stimulated as described above in the absence or presence of the PTPase CD45 inhibitor (1 µM) for 24 h, and culture supernatants were harvested and analyzed for IL-2 production using a Becton Dickinson ELISA kit. There is a marked increase in IL-2 secretion following stimulation with anti-CD3/anti-CD28 (1 µg/ml) plus Jacalin (50 µg/ml) in CD45+ Jurkat cells when compared with treatment with anti-CD3/anti-CD28 alone or with Jacalin alone. The enhancement was blocked significantly by the PTPase CD45 inhibitor. The results are representative of three independent experiments.

 
CD45 regulates Jacalin-induced MAPK signaling cascades
Although CD45 is usually thought about in terms of its contributions to initial events in T cell activation, some have suggested, based on its phosphatase activity and expected steric effects, that sustained colocalization of CD45 with the TCR complex may in fact be stimulatory and inhibitory as to TCR signal transduction [19 ]. To further investigate CD45-regulated T cell activation after Jacalin stimulation, we determined the CD45-mediated MAPK signaling cascades upon Jacalin treatment in the CD45-positive JE6.1 Jurkat T cells. It has been shown that CD45 mediated the activation of Src kinases such as p56Lck, a member of the Src family of protein tyrosine kinases, and a physiological substrate of CD45 initiates the signal transduction cascade by phosphorylating downstream substrates such as CD3{zeta} and ZAP70, followed by induction of the MAPK cascades, leading to activation of nuclear transcription factors regulating the induction of IL-2 gene expression [12 , 20 ]. To determine whether Jacalin induces MAPK signaling cascades through binding CD45, we analyzed the kinetics of phosphorylation of p38 MAPK and p44/42 MAPK (ERK1/2) after Jacalin stimulation in the absence and presence of the PTPase CD45 inhibitor. As shown in Figure 6A , the phosphorylation of p38 MAPK peaked at 10 min, and incubation of cells with the PTPase CD45 inhibitor prior to Jacalin stimulation inhibited the phosphorylation of p38 MAPK significantly (Fig. 6A) . In addition, maximal p44/42 MAPK phosphorylation was observed at 60 min following stimulation with Jacalin (Fig. 6B) . However, when cells were incubated with the PTPase CD45 inhibitor before stimulation with Jacalin, phosphorylation of p44/42 MAPK was also inhibited significantly (Fig. 6B) . Equal amounts of p38 MAPK and p44/42 MAPK were present in the cell lysates. These results suggested that the intracellular signaling events associated with T cell activation were triggered by the CD45-Jacalin interaction.


Figure 6
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  		Figure 6. Kinetics of Jacalin-induced phosphorylation of MAPKs on activation of CD45 in Jurkat T cells. CD45+ Jurkat (JE6.1) T cells were stimulated with Jacalin (50 µg/ml) in the absence or presence of the PTPase CD45 inhibitor (1 µM), as described above, and were harvested at various time-points as indicated. Whole lysates were analyzed by SDS-PAGE and transblotted onto a membrane. The membrane was probed with phospho-p38 MAPK (A) or phospho-p44/42 MAPK (B) antibodies (Ab), as indicated on the left, followed by HRP-conjugated 2nd antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Glycosylation is a cell-specific process, depending not only on the cellular composition of glycosyltransferases and glycosidases genes but also on the activation and differentiation status of the cell. Glycans decorate the surfaces of all mammalian cells and the extracellular matrix with which they interact [2 ]. Recent evidence indicates that differential glycosylation of cell surface proteins can control critical immunological processes, including T cell activation, migration, and apoptosis [2 ], although single glycans can have highly specific functions. The mission of decoding glycan information is assigned in part to a great variety of mammalian glycan-binding proteins or lectins [21 ]. Jacalin is an O-glycan-binding lectin specific for galactosides, especially Galß1-3GalNAc, even in its sialylated form, and not requiring metal ions for binding activity. The lectin has been shown to induce mitogenic responses and to block infection by HIV-1 in CD4+ T lymphocytes by binding CD4 [22 , 23 ]. As Jacalin also binds CD8, its mitogenic specificity cannot be attributed exclusively to its interaction with CD4. We therefore hypothesized that the lectin may interact directly with other widespread membrane glycoproteins expressed on CD4+ and CD8+ T cell subsets.

In the present work, we have identified, by affinity chromatography and MS experiments, the receptor-type PTPase CD45 and its isoforms as the major Jacalin targets on the membranes of T lymphocytes. CD45 is a heavily glycosylated, haemopoietic, cell-specific, two-domain tyrosine phosphatase, essential for efficient T and B cell and other lymphocyte antigen receptor signal transduction, and is expressed as multiple isoforms, which are generated through complex alternative splicing of Exons 4, 5, and 6, as to the extracellular domain of the molecule [13 , 20 ]. The expression of different CD45 isoforms is cell type-specific and dependent on the stage of differentiation and state of activation of cells. In humans, naïve T cells express high molecular weight isoforms containing the A exon (CD45RA cells), but following activation, the low molecular weight isoform is expressed (CD45RO cells). Humans lacking CD45 expression suffer from severe combined immunodeficiency [24 , 25 ]. The difficulty of working with cells expressing many isoforms has led to efforts to detect convincing differences in the function of individual CD45 isoforms, and the altered combinations of isoforms might influence CD45 function by affecting segregation into lipid rafts, dimerization, or interactions with other molecules [20 ]. Moreover, the putative O-glycosylation sites are clustered in the domains encoded by Exons 4–6, and these domains contain more sialylated O-linked sugar chains with Core 2 structure [13 ]. As Jacalin predominantly binds O-linked glycoproteins containing carbohydrate moieties specific for galactosides, such as GalNAc, Galß1-3GalNAc, Galß1-3(Galß1-4GlcNAcß1-6)GalNAc, monosialyl tri- and pentasacharides, and disialyl tetrasaccharides [4 ], we therefore hypothesize that Jacalin may bind Galß1-3GalNAc epitopes, which present on CD45 extracellular domains encoded by Exons 4–6 [13 ]. In this study, our observations evidently indicate that Jacalin induces T cell activation in a carbohydrate-dependent manner through the CD45-Jacalin interaction.

Although it is clear that CD45 phosphatase plays a key role in the life and death of lymphocytes, the great conundrum regarding CD45 is what the extracellular domain does. Equally intriguing is what role the alternative splicing plays in regulation of phosphatase activity. Recent human and animal data have revealed extensive polymorphism in the extracellular domain, and some CD45 isoforms affect alternative splicing with different glycosylation, which changes during T cell differentiation and activation [13 , 26 ]. Thus, cell-specific glycosylation of CD45 could provide a mechanism for influencing various immunological pathways, including TCR and cytokine receptor signaling. Here, our results demonstrate that Jacalin binds CD45 isoforms with different affinities through lectin-glycan interactions and induces lymphocyte activation by activating CD45, which may provide an opportunity to investigate the effects of changes in the CD45 extracellular domain and to determine the importance of CD45 polymorphisms in immune function and disease. Given the potential for glycosylation changes to affect the binding of lectin ligands to CD45, dimerization, and other protein–protein interactions, we need to understand the glycobiology of CD45 much better.

Stimulation of the TCR results in the activation of a series of PTKs and culminates in a variety of distal events, which include transcriptional activation of the IL-2 gene [27 ]. Costimulation through T cell surface CD28 increases the stability of phosphorylated proteins [28 ], thereby amplifying TCR signaling and facilitating T cell activation. In light of our finding in this study that Jacalin enhances TCR stimulation and CD28-mediated costimulation by activating CD45, we suggest that Jacalin may function by promoting the activity of CD45 phosphatase, which results in enhancement of T cell activation. Desai et al. [29 ] have shown previously that CD45 is a transmembrane PTPase of special interest in this regard, given that it accounts for up to 80% of the tyrosine phosphatase activity in T cell membranes.

T cells secreting different cytokines elicited by different stimuli may induce different kinds of T cell differentiation at different targets. In the present study, the distinctive cytokine secretion pattern on Jacalin induction implies that Jacalin may contribute to the Th1/Th2 cytokine balance, in which T cell subsets will predominate through CD45 activation, and the level of signaling achieved on activation of CD45 is intimately associated with the induction of distinct cytokine-secreting T cell subsets. CD45 glycosylation changes during T cell development, peripheral activation, and aging [30 , 31 ]. Furthermore, different CD45 isoforms are glycosylated differently [13 ]. Therefore, differential glycosylation of CD45 provides a "scaffold" for Jacalin-based regulation of TCR-mediated responses. Apparently, CD45 on T cells has the appropriate glycan structures, because of cell-specific glycosylation, to ensure Jacalin recognition. Recently, two putative "counter-receptors" for human CD45, macrophage galactose-type lectin and galectin-1, have been identified, both of which decrease T cell proliferation and increase T cell death through glycosylation-dependent interactions [14 , 32 ]. Although there is some controversy regarding whether CD45 activity can be controlled by the receptor-mediated mechanisms [10 ], we have indicated here that Jacalin can induce T cell activation and modulates Th1/Th2 cytokine secretion through carbohydrate-dependent interaction with CD45. Further study is required to determine the role of Jacalin-CD45 isoform ligation in different populations of T lymphocytes, including naïve T cells, memory T cells, and effector T cells, which have different activation requirements in terms of the number of MHC-peptide complexes and the duration of signaling. Although the Th1/Th2 choice is determined by cytokines, the response of T cells to cytokines may be modulated by the ligation between Jacalin and CD45 isoforms, just as the response of T cells to galectins, a family of highly conserved ß-galactoside-binding lectins, is modulated by the ligation between lectin and glycans [33 ]. It would be useful to determine whether the alterations in CD45 and its isoforms lead to changes in glycosylation patterns, thus affecting Jacalin recognition and CD45 activity. In addition, our results are in agreement with previous findings of Tamma et al. [34 , 35 ]; i.e., they reported recently that CD28 cross-linking followed by stimulation with Jacalin resulted in enhanced phosphorylation of p38 MAPK and increased secretion of IL-4. Although the concept of Jacalin acting as a T cell mitogen has been around for many years, studies about molecular organization in immune systems are just beginning. It is likely that many surprises are in store.

It is clearly established that CD45 is required for TCR signaling, and this appears to be caused by the up-regulation of kinase activity in p56Lck (also probably p59Fyn) [35 ], and CD45 may be required to act dynamically during the early events of TCR signaling [36 ]. To date, the best-described bioaction for CD45 is as an activator of the Src family kinases Lck and Lyn in TCR and BCR complexes. In thymocytes lacking CD45, maturation from immature, double-positive CD4/CD8 cells to single-positive CD4 and CD8 cells is blocked [37 ]. In B cells, loss of CD45 inhibits proliferation induced by antigen-dependent cross-linking of surface IgM and IgD [38 ]. Our findings here demonstrate the nature of the lymphocyte cell surface receptor for Jacalin, tyrosine phosphatase CD45, which is responsible for initiating dephosphorylation of Src-family kinases such as Lck and for positively inducing T cell activation. At least two potential mechanisms may underlie Jacalin-induced CD45 regulation of immune function. The first is control of the threshold of signaling through the TCR. The critical role of CD45 phosphatase activity in TCR signal transduction is well established, and the Src family of kinases has been identified as its primary targets [9 , 20 ]. Our findings and other studies indicate that Jacalin-induced CD45 activation is required for T cell activation by constitutively dephosphorylating the negative regulatory tyrosine of Lck, Tyr505. This creates a "primed" Lck molecule, which can become activated on autophosphorylation at Tyr394, which then initiates the signal transduction cascade by phosphorylating downstream substrates such as CD3{zeta} and ZAP70, followed by induction of the MAPK cascades. The second possible mechanism is Jacalin-induced CD45 regulation of cytokine production and responses; supporting this idea is the finding that CD45 is required for chemokine and cytokine production by NK cells after stimulation via Fc or MHC-binding receptors [39 ]. Moreover, Lafont et al. [40 ] reported previously that Jacalin induced IL-2 gene transcription in primary T lymphocytes through activation of the Raf-1/MEK-1/ERK-2 pathway, and Tamma et al. [41 ] showed recently that Jacalin induced tyrosine phosphorylation of ERK and JNK and that the event was blocked by pretreatment with gp120.

Jacalin, as a glycan-binding protein with affinity for galactoside-containing oligosaccharides on CD45, may regulate the inflammatory response by influencing T cell survival, activation, and cytokine synthesis by activating CD45. The carbohydrate specificity of Jacalin raises the possibility that CD45 might represent a natural ligand for one or more members of the naturally occurring galectin family [42 , 43 ] and that such a lectin might have an important, functional effect through a similar mechanism. The results obtained for the Jacalin-CD45 interaction also suggest generally that protein-glycan interactions control critical immunological processes involved in T cell activation and homeostasis. Further studies are needed to elucidate the physiological significance and molecular basis of the Jacalin-CD45 interaction. Understanding the molecular mechanisms involved in the immunoregulatory functions of Jacalin might help to delineate novel, therapeutic targets based on lectin-glycan interactions for autoimmune diseases and chronic inflammatory disorders.

In conclusion, our observations here imply that T cell activation induced by Jacalin probably does not result from an exclusive interaction of the Jacalin with CD4 or CD8. It seems that Jacalin triggers a positive signal through CD45, which is required for T cell activation and Th1/Th2 cytokine secretion. Jacalin, which activates PTPase CD45 specifically, resulting in activation of MAPK cascades in CD45-positive human lymphocytes, appears to be a good, potential model for the study of CD45-mediated signaling mechanisms in this restricted population.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas A-14082203 to T. K., and a Grant-in-Aid for Scientific Research C-18590471 to B. Y. M. from the Japan Society for the Promotion of Science, Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors thank the Shiga Prefecture Red Cross Blood Center (Shiga, Japan) for kindly supplying the human PBMC from healthy donors and Ms. Tomoko Tominaga for the secretarial assistance.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received November 7, 2006; revised December 18, 2006; accepted December 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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