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J. Biol. Chem., Vol. 280, Issue 6, 4730-4737, February 11, 2005

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Galectin-4 Binds to Sulfated Glycosphingolipids and Carcinoembryonic Antigen in Patches on the Cell Surface of Human Colon Adenocarcinoma Cells^*

Hiroko Ideo, Akira Seko, and Katsuko Yamashita¶

From the Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan

Received for publication, September 9, 2004 , and in revised form, October 25, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Galectin-4, a member of the galectin family, is expressed in the epithelium of the alimentary tract. It has two tandemly repeated carbohydrate recognition domains and specifically binds to an pyranoside with high affinity (Ideo, H., Seko, A., Ohkura, T., Matta, K. L., and Yamashita, K. (2002) Glycobiology 12, 199–208). In this study, we found that galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, such as SB1a, SM3, SM4s, SB2, SM2a, and GM1, but not to glycosphingolipids with 3-O-sialylated Gal, such as sLc4Cer, snLc4Cer, GM3, GM2, and GM4, using both an enzyme-linked immunosorbent assay and a surface plasmon resonance assay. A confocal immunocytochemical assay showed that galectin-4 was colocalized with SB1a, GM1, and carcinoembryonic antigen (CEA) in the patches on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells. This localization was distinct from caveolin/VIP21 localization. Furthermore, immobilized galectin-4 promoted adhesion of CCK-81 cells through the sulfated glycosphingolipid, SB1a. CEA also bound to galectin-4 with K_D value of 2 x 10^-8 M by surface plasmon resonance and coimmunoprecipitated with galectin-4 in LS174T cell lysates. These findings suggest that SB1a and CEA in the patches on the cell surface of human colon adenocarcinoma cells could be biologically important ligands for galectin-4.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Galectins are a family of animal lectins defined by their affinity for -galactoside-containing saccharides and by common amino acid sequence elements. They are involved in regulating diverse biological phenomena, including proliferation, apoptosis, and cell-cell, or cell-matrix interactions (1, 2). Although galectins do not have any secretion signal peptide, they are often found outside of cells (1, 3). Galectin-4 has two carbohydrate recognition domains (CRDs)¹ and is expressed in the epithelium of oral mucosa, esophagus, and intestinal and colonic mucosa (4, 5). It has been reported that galectin-4 is present in certain fractions of rafts in the brush border membrane of pig intestine (6, 7). However, it is not clear how the soluble galectin-4 could be a component of rafts and with which molecules galectin-4 would interact.

We previously reported that galectin-4 binds to pyranoside with high affinity (8), and this structure occurs not only in O-linked oligosaccharides but also in certain glycosphingolipids (9). In further investigations of endogenous ligands of galectin-4, we were interested in determining whether glycosphingolipids carrying the 3-O-sulfated galactose residues bind to galectin-4, because galectin-8, which also has two conserved CRDs and is 34% identical to galectin-4 at amino acid levels, binds to glycosphingolipids carrying residues (10). Kopitz et al. (11, 12) reported that antibodies against GM1 inhibited galectin-1 binding to neuroblastoma cells, suggesting that galectin-1 binds to GM1 or to glycoprotein receptors in close proximity to GM1 on the cell surface. These reports suggested that glycosphingolipids could also be biologically important ligands for some members of the galectin family.

We also examined whether galectin-4 binds to carcinoembryonic antigen (CEA), and both molecules are colocalized in the cell surface of human colon adenocarcinoma, because CEA is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein (13), which carries 25–27 mol of N-glycans per molecules (14), and is produced by gastrointestinal tumor cells.

We show in this study that galectin-4 binds to glycosphingolipids carrying residues and CEA, using an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR) assay. Furthermore, galectin-4 on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells is colocalized in the patches with SB1a, GM1, and CEA, and this localization differed from caveolin/VIP21 staining on the cell surface, as determined by a confocal immunocytochemical assay.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Gal13GalNAc1-O-benzyl (core 1-O-Bn), TRITC-anti-rabbit IgG, and fluorescein isothiocyanate-anti-mouse IgG were purchased from Sigma Co. Lactose, GM1, GM2, GM3, SM4s, GM4, sLc4Cer, snLc4Cer, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The structures of these glycolipids are shown in Table I. GalNAc13(Fuc12)Gal14Glc (A-tetra), Fuc12Gal13GlcNAc13Gal14Glc (LNF-I), and Gal13GalNAc (core 1) were purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Fluorescein isothiocyanate-anti-mouse IgM was from Chemicon International Inc. (Temecula, CA). Lactose-O-p-nitrophenyl (Lac-O-pNP) and anti-SB1a and anti-GM1 antibodies were purchased from Seikagaku Co. (Tokyo, Japan). Anti-caveolin-1 antibody was purchased from BD Biosciences, and anti-CEA antibody was from Nichirei Co. (Tokyo, Japan). CEA was kindly provided by Dr. Kuroki (Fukuoka University, Fukuoka, Japan). was the kind gift of Dr. Matta (Roswell Park Memorial Cancer Institute). SM2a, SB2, and SB1a were purified from rat kidney as described previously (10). SM3 was kindly provided by Dr. Ishizuka (Teikyo University School of Medicine). was prepared as reported previously (8). A pGEX-6P-1 plasmid, Escherichia coli BL21 strain, glutathione-Sepharose, and PreScission protease were from Amersham Biosciences.

Preparation of Antibody—Antiserum against human galectin-4 was raised in rabbit according to standard procedures. Purified rhgalectin-4 (8) (1–1.5 mg) was injected into a rabbit (initial and two boosts) at intervals of 3 weeks.

Preparation of Sulfoglycosphingolipids—Crude lipids of homogenized CCK-81 cells (5 x 10⁷ cells) were extracted once with 19 volumes of chloroform/methanol (2:1, v/v) and then with 20 volumes of chloroform/methanol/water (1:2:0.8, v/v). The pooled extracts (the total lipid extracts) were dried and dissolved in chloroform/methanol (1:1, v/v). The solution was spotted on a high-performance thin layer chromatography plate (10 x 10 cm, Kieselgel 60, Merck, Darmstadt, Germany) and developed using chloroform/methanol/0.2% CaCl₂ (60:35:7) as the solvent. Lipids were detected by using primuline (5 mg in 100 ml of acetone/water (80/20, v/v)) and visualized with LAS1000 (Fujifilm, Japan). Sulfated glycosphingolipids were detected by Azure A (15). Phospholipids are detected with Dittmer-Lester reagent (16) and ninhydrin reagent for amino groups.

Estimation of Kinetic Constants Based on SPR—The dissociation constants between each of the CRDs of galectin-4 and various carbohydrates were measured using a BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden) as described previously (8).

ELISA for Binding of Galectin-4 to Various Glycosphingolipids— ELISAs for binding of galectin-4 to various glycosphingolipids using recombinant galectin-4 and anti-galectin-4 were performed in the same manner as described previously (10).

Binding of Galectin-4 to Glycosphingolipids Immobilized on the Surface of BIAcore Sensor Chip—Glycosphingolipids were hydrophobically immobilized onto the CM5 sensor chip according to Catimel et al. (17). Glycosphingolipids (1 mg/ml) were dissolved in EtOH/MeOH (9:1, v/v), diluted with HBS buffer (10 mM HEPES (pH 7.4), 3.4 mM EDTA, 150 mM NaCl), and injected (80 µl) at a flow rate of 5 µl/min over the unmodified surface.

Purified rhgalectin-4 in HBS buffer was introduced onto the surface at a flow rate of 20 µl/min. The interaction between rhgalectin-4 and glycosphingolipids was monitored at 25 °C, and the kinetic constants were calculated using BIAevaluation 3.0 software.

Immunofluorescence Analysis—Human colon adenocarcinoma CCK-81 and LS174T cells grown on coverslips were washed with PBS and fixed in 4% paraformaldehyde for 15 min, followed by washing and incubation with 1% BSA for 1 h at room temperature. For double labeling, cells were incubated at 4 °C overnight with anti-galectin-4 (rabbit IgG) and anti-SB1a (mouse IgM), anti-GM1 (mouse IgM), anti-CEA (mouse IgG), or anti-caveolin-1/VIP21 (mouse IgG) antibodies in 1% BSA. Cells were then rinsed several times and incubated for 1 h with TRITC-conjugated anti-rabbit IgG and fluorescein isothiocyanate-conjugated anti-mouse IgM or IgG in PBS containing 0.1% BSA. After extensive washing, the coverslips were mounted on slides. Fluorescent images were obtained using a confocal laser scanning microscope (LSM 5 PASCAL, Carl Zeiss, Germany). Images were acquired using the multitrack mode to avoid signal cross-talk. The specificity of labeling was assessed by incubation with control primary antibodies.

Cell Adhesion Assay—ELISA plates were pre-coated with rhgalectin-4 in PBS for 16 h at 4 °C, followed by blocking with 1% BSA for 2 h at 37 °C. CCK-81 cells and Chinese hamster ovary-K1 cells on tissue culture plates were detached with 0.25% trypsin and 0.02% EDTA in PBS. After washing with culture medium, cells were resuspended in serum-free medium containing 10 mM EDTA and re-seeded onto the galectin-4-coated plates. After incubating at 37 °C for 30 min, the plates were washed three times with PBS, and the adherent cells were stained with 0.2% crystal violet in PBS containing 20% methanol for 15 min at 22 °C. Excess dye was washed with water, and the bound cells were solubilized in 1% SDS for 1 h at 22 °C and quantified by measuring the absorbance at 595 nm with a spectrophotometer. All assays were performed in triplicate.

Binding of CEA to Galectin-4 Immobilized on the Surface of BIAcore Sensor Chip—The affinity of CEA to galectin-4 was measured by SPR assay using BIAcore 2000 instrument. Galectin-4 was immobilized on a CM5 sensor chip by the amine-coupling method. The coupling density was 7200 RU. Various concentrations of CEA were diluted in HBS buffer (10 mM HEPES, pH 7.4, 3.4 mM EDTA, 150 mM NaCl 150 mM NaCl, 0.005% (v/v) surfactant P-20) and injected onto the sensor chip at 20 µl/min. The sensor surface was regenerated by 0.1 M lactose.

Immunoprecipitation—1 x 10⁷ of LS174T cells were solubilized in lysis buffer (0.5% Triton X-100, 25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 2 mM EDTA, 1/100 (v/v) Halt protease inhibitor mixture (Pierce)) for 1 min at 4 °C and harvested. The postnuclear supernatants were collected following centrifugation at 15,000 rpm for 10 min. Aliquots of the supernatants were incubated with 60 µl of an anti-galectin-4 conjugated-Sepharose 4B (5.5 mg of IgG/ml gel). Following immunoprecipitation, the beads were washed five times with lysis buffer and boiled with Laemmli sample buffer for 5 min. Samples were subjected to SDS-PAGE (12. 5% acrylamide) followed by blotting onto a nitrocellulose membrane. The blots were incubated with anti-CEA and anti-galectin-4 antibodies followed by peroxidase-conjugated specific secondary antibodies (Amersham Biosciences), visualized by using the ECL system (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Carbohydrate Binding Specificities of Galectin-4 N-domain and C-domain to Oligosaccharides—Galectin-4 has two conserved CRDs, which share 34% amino acid identity. Because we found that N- and C-domains of galectin-8 have distinct carbohydrate-binding specificities (10), we prepared GST-fused recombinant proteins corresponding to the N-domain and the C-domain of galectin-4 (GST-N-domain and GST-C-domain, respectively) and compared their binding affinities toward oligosaccharides that had showed some binding ability to the full-length galectin-4 in our previous study (8). Binding of various oligosaccharides to the immobilized recombinant proteins was measured by SPR. As summarized in Table II, the GST-N-domain and the GST-C-domain showed similar affinity toward three oligosaccharides, , A-tetra, and LNF-I. On the other hand, the GST-C-domain showed a 12-fold higher affinity than the GST-N-domain for the 1-O-Bn oligosaccharide. These results indicated that the two domains of galectin-4 have different carbohydrate-binding specificities with respect to 1-O-Bn, which is a better ligand for the GST-C-domain than for the GST-N-domain.

View larger version (21K): [in this window] [in a new window]   FIG. 1. ELISA for binding of galectin-4 to various glycosphingolipids. The relative binding of galectin-4 (250 nM) to different concentrations of coated glycosphingolipids. The amount of bound galectin-4 was measured using an anti-galectin-4 antibody as described under "Experimental Procedures." SB1a (), GD1a (), SM3 (), SM4s (•), SM2a (), GM1 (), SB2 (), and GalCer (). Galectin-4 also did not bind to LacCer, GM2, GM3, GM4, sLc4Cer, and snLc4Cer same as GalCer ().

View larger version (29K): [in this window] [in a new window]   FIG. 2. The binding abilities of rhgalectin-4, GST-N- and C-domains (250 nM) to various glycosphingolipids. Relative binding to various glycosphingolipids was measured by ELISA using a concentration of 100 pmol of glycosphingolipids/well.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Sensorgrams of SPR for galectin-4 binding to glycosphingolipids. Various concentrations of galectin-4 were introduced onto the glycosphingolipid-immobilized surface for 180 s at a flow rate of 20 µl/min. The relative response was determined by subtracting the blank values on the non-immobilized surface from the values on the glycosphingolipids-immobilized surface. The immobilized glycosphingolipids were: A, SB1a (55 RU); B, GM1 (175 RU); and C, GM3 (162 RU).

We first examined what kinds of glycosphingolipids exist in human colon adenocarcinoma CCK-81 cells. Chloroform-methanol extracts were separated by TLC and visualized with both primuline (Fig. 4A, lane 1) and Azure A (Fig. 4A, lane 2). Mobilities of primuline staining-positive bands a, b, c, d, e, f, and g (Fig. 4, lane 1) corresponded to those of authentic phosphatidylethanolamine (and SM4), SM3, phosphatidylinositol, phosphatidylserine (and SM2), GM3, SB1a, and GM1, respectively, and bands a, b, c, d, and f were also stained with Azure A reagent (Fig. 4, lane 2). Faint or no bands were detected by primuline staining at the positions for authentic GM1 and GD1a. When the bands a to g were extracted and individually coated on ELISA plates, galectin-4 bound to each of these glycosphingolipids a, b, and f, which correspond to authentic SM4, SM3, and SB1a, respectively, and did not bind to bands c, d, e, and g (Fig. 4B). When we detected the phospholipids with Dittmer-Lester reagent, bands a, c, and d were stained (data not shown) and bands a and d were also stained with ninhydrin reagent. Sufficient amounts of authentic phosphatidylinositol and phosphatidylserine were Azure A-positive, suggesting that primuline-positive bands a, c, and d contain phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine, respectively. Galectin-4-binding component in band a should be SM4, because galectin-4 did not bind to phosphatidylethanolamine (data not shown). These results indicated that galectin-4 reactive-sulfated glycosphingolipids are prominent in human colon adenocarcinoma CCK-81 cells, in comparison with sialylated glycosphingolipids on the basis of comparative primuline staining. The similar results for lipid compositions were also obtained in LS174T cells (data not shown).

View larger version (39K): [in this window] [in a new window]   FIG. 4. Separation of sulfated glycosphingolipids from CCK-81 cells on TLC plates and relative binding ability to galectin-4. A, lane 1, primuline staining of lipids from CCK-81 cells; lane 2, azure A staining of lipids from CCK-81 cells. Positions of authentic phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), GM3, GM1, GD1a, SM4, SM3, SM2, and SB1a are indicated by arrows. The solvent system for TLC was chloroform/methanol/0.2% CaCl2 (60:35:7). B, relative binding ability of bands a–g to galectin-4. Lipids of bands a–g were extracted from silica gels with chloroform/methanol (1:1) and were evaporated and dissolved in an equal volume of methanol (100 µl). Ten microliters were coated on ELISA plate. Relative binding ability was measured using 50 µl of rhgalectin-4 (50 nM) and anti-galectin-4 antibody according to the "Experimental Procedures."

View larger version (55K): [in this window] [in a new window]   FIG. 5. Double immunofluorescence labeling of the cell surface. CCK-81 and/or LS174T cells washed with PBS were fixed with 4% paraformaldehyde for 15 min, stained with anti-galectin-4 (A–E), anti-SB1a (A and D), anti-GM1 (B), anti-caveolin-1 (C), and anti-CEA (E), followed by appropriate secondary antibodies, and examined by confocal laser microscopy. The merged confocal images are shown in the third column. Corresponding differential interference contrast images are shown in the fourth column.

Anti-GM1 antibody also stained the similar area as anti-galectin-4 (Fig. 5B), although the relative content of GM1 was less than that of SB1a (Fig. 4). However, the localization of caveolin-1/VIP21 was different from that of galectin-4 (Fig. 5C). CEA was localized in the similar area as anti-galectin-4 (Fig. 5E). These results suggest that galectin-4 is localized in patches, which involve SB1a, GM1, and CEA on the cell surface of CCK-81 and/or LS174T cells.

Immobilized Galectin-4 Bound to CCK-81 Cells through 3-O-Sulfated Glycosphingolipids—Sequentially, we investigated whether the galectin-4 on the cell surface of CCK-81 cells is related to cell adhesion. Galectin-4 immobilized on plates promoted the adhesion of CCK-81 cells in a dose-dependent manner in Ca²⁺-free condition (Fig. 6A), whereas plates without galectin-4 failed to support cell adhesion (Fig. 6B). Galectin-4 did not bind to Chinese hamster ovary cells (Fig. 6A), which contain LacCer and GM3 as glycosphingolipids (19) and glycoproteins with Neu5Ac23Gal residues at the non-reducing termini (20), consistent with the fact that galectin-4 has no affinity toward Neu5Ac23Gal residues (8). Anti-SB1a effectively inhibited the binding of CCK-81 cells to galectin-4-coated plates (Fig. 6B), suggesting that galectin-4 binds to CCK-81 cells through 3-O-sulfated glycosphingolipids without Ca²⁺ and stimulates adhesion of CCK-81 cells.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Cell adhesion to plates coated with galectin-4. A, 96-well ELISA plates were precoated with 0.1 ml of the indicated concentrations of galectin-4 at 4 °C overnight. CCK-81 cells (•) and Chinese hamster ovary cells () were detached from culture plates with 0.25% trypsin and 0.02% EDTA, washed with PBS, and seeded (7 x 105 each) in serum-free medium with 10 mM EDTA onto the coated wells. Following incubation at 37 °C for 30 min, bound cells were measured according to the "Experimental Procedures." B, 96-well ELISA plates were pre-coated with 0.1 ml of 10 µg/ml of galectin-4 at 4 °C overnight. CCK-81 cells detached from culture plates (3 x 105 each) were preincubated with serum-free medium with 10 mM EDTA and anti-SB1a antibody (20 µg/ml) or anti-galectin-4 antibody (30 µg/ml) at 22 °C for 30 min. Then cells were seeded on galectin-4-coated wells. Following incubation at 37 °C for 30 min, wells were washed, and the amount of adherent cells was measured according to the "Experimental Procedures."

View larger version (20K): [in this window] [in a new window]   FIG. 7. Binding of CEA to galectin-4 and cell adhesion assay in LS174T cells. A, various concentrations of CEA were introduced onto the galectin-4-immobilized (7200 RU) surface for 180 s at a flow rate of 20 µl/min. The relative response was determined by subtracting the blank values on the non-immobilized surface from the values on the galectin-4-immobilized surface. B, coimmunoprecipitation of CEA with galectin-4. LS174T cell lysates were immunoprecipitated with the anti-galectin-4 antibody. The immunoprecipitate was subjected to SDS-PAGE, blotted, and stained with anti-CEA and anti-galectin-4 antibody. C, 96-well ELISA plates were precoated with 0.1 ml of the indicated concentrations of galectin-4 at 4 °C overnight. The detached LS174T cells were incubated with anti-SB1a antibody (20 µg/ml) at 22 °C for 30 min () or without antibody (•) and seeded onto the galectin-4-coated wells (3 x 105 each). Following incubation at 37 °C for 30 min, the bound cells were measured according to the "Experimental Procedures." D, 96-well ELISA plates were precoated with 0.1 ml of 10 µg/ml of galectin-4 at 4 °C overnight and sequentially incubated with 0.1 ml of the indicated concentrations of CEA at 37 °C for 2 h, followed by blocking with 1% BSA for 2 h at 37 °C. The detached LS174T cells were seeded on CEA-galectin-4-coated wells. Following incubation at 37 °C for 30 min, wells were washed and the amount of adherent cells was measured according to the "Experimental Procedures."

Binding of LS174T Cells to Galectin-4 was Inhibited by CEA—Sequentially, it was examined whether the expression of CEA on LS174T cells influences the cell adhesion toward galectin-4. LS174T cells also bound to the galectin-4-coated plate dose-dependently, but the bound cells were much lower than those of CCK-81 cells (Fig. 7C, filled circles). Anti-SB1a antibody effectively inhibited the binding of LS174T cells to galectin-4-coated plates (Fig. 7C), suggesting that galectin-4 also binds to LS174T cells through 3-O-sulfated glycosphingolipids without Ca²⁺. After the galectin-4-immobilized plates were pre-incubated with CEA at 37 °C for 2 h, LS174T cells diminished the binding ability via sulfated glycosphingolipids to the galectin-4-coated plates (Fig. 7D), suggesting that the exogenous CEA suppresses the binding of galectin-4 molecule to sulfated glycosphingolipids. These results suggest that galectin-4 binds to both SB1a and CEA in the specific patches on the cell surface of CCK-81 and/or LS174T cells and modulates their adhesion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have clearly demonstrated in this study that galectin-4 binds strongly to glycosphingolipids carrying , including SM4, SM3, and SB1a, but not to glycosphingolipids carrying Sia23Gal, including sLc4Cer, snLc4Cer, GM3, GM2, and GM4, and that these sulfated glycosphingolipids exist in human colon adenocarcinoma cells (Fig. 4), and mouse intestine and human colon epithelium (data not shown). We also found that (sulfated)-glycosphingolipids and galectin-4 are colocalized on the cell surface of human colon adenocarcinoma cells by an immunocytochemical assay (Fig. 5, A and D). The galectin-4/SB1a immunoreactive area is distinct from the anti-caveolin/VIP21 immunoreactive area (Fig. 5C), but similar to anti-GM1 immunoreactive area (Fig. 5B), suggesting that galectin-4 is localized in a kind of glycosphingolipids-rich patches on the cell surface of CCK-81 cells and LS174T cells. CEA was also stained in a galectin-4-rich area (Fig. 5E). It is known that GPI-anchored proteins are specifically targeted to apical membranes of polarized epithelial cells (22), and many GPI-anchored proteins are sorted to glycolipid-enriched membrane subdomains (23). Accordingly, this galectin-4-SB1a-rich domain may be a kind of platform for GPI-anchored glycoproteins in colon adenocarcinoma cells.

Moreover, galectin-4 immobilized on plates promoted adhesion of CCK-81 cells through sulfated glycosphingolipids (SB1a) (Fig. 6), suggesting that secreted galectin-4 modulates cell-cell adhesion via sulfated glycosphingolipids on the cell surface of CCK-81 cells. Sulfated glycosphingolipids are known to bind specifically to diverse adhesion proteins such as laminin, selectins, and anti-coagulant factors in vitro (9). For example, Suzuki et al. (24) reported that L-selectin binds to SM4, SM3, SB2, and SB1a. These facts imply that secreted or cell surface galectin-4 may also modulate the interaction of these proteins with sulfated glycosphingolipids, because galectin-4 interacts with the membrane through adhesion to sulfated glycosphingolipids.

Galectin-4 also binds to LS174T cells through sulfated glycosphingolipids (Fig. 7C), and CEA suppressed galectin-4 binding to sulfated glycosphingolipids on LS174T cells (Fig. 7D). Because galectin-4 has much higher affinity for the residue of SB1a than for Gal14(3)GlcNAc and Fuc12Gal13(4)GlcNAc residues in glycoproteins (Table II), the binding between galectin-4 and sulfated glycosphingolipids may function for the cell adhesion of epithelial cells in the gastrointestinal tract. It has been reported that CEA mediates homotypic as well as heterotypic intercellular adhesion (25). However, the expression patterns of CEA in malignant cells suggested that CEA functions as a cell-contact inhibitory molecule rather than as an adhesion molecule (26). Accordingly, the overexpression of CEA on the surface of tumor cells suppresses the galectin-4 binding to the sulfated glycosphingolipids and may facilitate migration and motility of tumor cells, i.e. metastases formation.

Binding of galectins-1 and -3 to CEA in cell lysates of adenocarcinoma cells has been reported (27, 28). However, galectin-1 is abundant in muscles, neurons, thymus, kidney, and placenta (1), and galectin-3 is abundant in lung, artery, thymus, and spleen (29); expression of these proteins in intestine and colon is quite low in comparison with galectin-4. In fact, we could not detect galectin-3 on the cell surface of LS174T cells by immunofluorescence analysis. Because Danielsen et al. (30) showed that galectin-4 is present in the detergent-insoluble complex of pig intestine, galectin-4 may be one of the major ligands for CEA in the epithelium of alimentary tracts.

On the other hand, there are several reports that sulfated glycosphingolipids serve as receptors for bacterial or viral toxins. A human respiratory pathogen, Bordetella pertussis, binds to SM4 and glycosphingolipids containing a GalNAc14Gal sequence (31). Influenza A viruses bind to sulfatides (32), and neutrophil-activating protein of Helicobacter pylori binds to SM4 and (33). SM4 and some sulfated glycosphingolipids are expressed in mammal gastrointestinal tract (9). We also found that SB1a exists in mouse intestine and human colon epithelium (data not shown). Because galectin-4 is specifically expressed in the gastrointestinal tract, galectin-4 may play a role in host defense by masking the sulfated glycosphingolipids from bacterial or viral toxins.

The anti-SB1a antibody was originally obtained using the acidic glycolipid mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as immunogens. Although the SB1a antigen is a relatively minor glycolipid in hepatic cells, the anti-SB1a antibody strongly stained the surface of PLC/PRF/5 cells (18). Analysis of the glycolipids extracted from hepatocellular carcinoma tissues and cirrhotic livers of patients and from a normal liver revealed that SB1a is expressed in some hepatocellular carcinoma tissues (5 of 17 cases) but not expressed in the cirrhotic liver and normal liver (34). We found that SB1a was expressed on the surface of human colon adenocarcinoma cells (CCK-81 and LS174T cells) (Fig. 5) but not expressed on the surface of another type of human colon adenocarcinoma cell, M7609 (data not shown). These data suggest that the surface expression or disappearance of SB1a might be related to the respective characters of carcinoma.

Plant-derived lectins have been used for as lymphocyte-stimulating reagents, and some endogenous mammalian lectins also play an important role in immune responses by cross-linking certain cell surface glycoreceptors (35, 36). Furthermore, it is thought that glycosphingolipids, which are abundant in detergent-resistant membranes, influence signal transduction by modulating the binding of exogenous effectors to cell surface receptors (37–40). Accordingly, secreted galectin-4 may be able to cross-link cell surface ligands and thereby may mediate or modulate cell responses. It has been recently reported that galectin-4 stimulates interleukin-6 production by intestinal CD4⁺ T-cells under inflammatory conditions (41).

Galectin-4 bound to sulfated glycosphingolipids and promoted cell adhesion, whereas CEA modulated this interaction by binding to galectin-4 on the cell surface of colon adenocarcinoma cells. Whether these glycoconjugates are involved in galectin-4-dependent cell responses and/or cell anoikis should be resolved in the next step.

	FOOTNOTES

 
^* This work was supported by a grant-in-aid for Scientific Research on Priority Area(s), 14082208, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 81-3-3294-3286; Fax: 81-3-3294-2656; E-mail: yamashita{at}sasaki.or.jp.

¹ The abbreviations used are: CRD, carbohydrate recognition domain; CEA, carcinoembryonic antigen; rhgalectin-4, recombinant human galectin-4; N-domain, N-terminal carbohydrate recognition domain; C-domain, C-terminal carbohydrate recognition domain; TRITC, tetramethylrhodamine isothiocyanate; GPI, glycosylphosphatidylinositol; Man, mannose; pNP, p-nitrophenyl; SPR, surface plasmon resonance; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; core 1, Gal13GalNAc; lactose, Gal14Glc; A-tetra, GalNAc13(Fuc12)Gal14Glc; LNF-I, Fuc12Gal13GlcNAc13Gal14Glc; RU, resonance units; Cer, ceramide; GM1, Gal13Gal-NAc14(Neu5Ac23)Gal14Glc-Cer; GM2, GalNAc14(Neu5Ac23)Gal14Glc-Cer; GM3, Neu5Ac23Gal14GlcCer; GM4, Neu5Ac23Gal-Cer; SB1a, ; SB2, ; SM2a, ; SM3, ; SM4, .

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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