2 Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 Japan and 3 Department of Biochemistry, Teikyo University School of Medicine, Tokyo 173-8605 Japan
Received on May 6, 2003; revised on June 17, 2003; accepted on June 19, 2003
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Abstract |
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Key words: galectin-8 / ganglioside / GM3 / sulfoglycosphingolipid / surface plasmon resonance
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Introduction |
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Galectin-8 is expressed on several types of tumor. For example, human prostate carcinoma tumor antigen, which is selectively expressed in prostate carcinoma cells but not in normal prostate or benign prostate hypertrophy, has been identified as galectin-8 (Su et al., 1996; Gopalkrishnan et al., 2000
). Po66, a mouse IgG1 monoclonal antibody produced by immunization of squamous cancer cells, recognizes a carbohydrate-binding protein (Po66-CBP), which has also been identified as galectin-8 (Bidon et al., 2001
). Furthermore, Lahm et al. (2001)
, who studied the expression of human galectins-1, -2, -3, -4, -7, -8, and -9 in 61 tumor cell lines of various origins using reverse transcription polymerase chain reaction (PCR), found that galectin-8 was the most abundantly expressed galectin in 59 of these cell lines. These reports suggest that galectin-8 plays a key role in regulating cell carcinogenesis or metastasis.
We previously reported the unique carbohydrate binding specificity of galectin-4 in comparison to galectin-3 (Ideo et al., 2002). Galectin-4 did not recognize Galß1
3GlcNAc (type 1) and Galß1
4GlcNAc (type 2), for which most of galectins have high affinity, but instead bound specifically to 3'-O-sulfated Galß1
3GalNAc (core 1). Although galectin-8 is 34% homologous to galectin-4 at the amino acid level, their tissue distributions are quite different (Hadari et al., 1995
). Expression of galectin-8 is high in rat liver, muscle, and kidney and is low or barely detectable in intestine, testis, fat, thymus, and lung (Hadari et al., 2000
), whereas galectin-4 is specifically expressed in intestine, colon, and stomach (Chiu et al., 1994
), suggesting that both galectins have distinct functional roles and carbohydrate binding specificities.
Here, we investigate the precise carbohydrate binding specificities of galectin-8 and its two CRDs using an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR) assay. We showed that the N-terminal CRD of galectin-8 binds to glycosphingolipids carrying SO3-3Gal/Sialyl
2 residues. We further found that galectin-8 binds to Chinese hamster ovary (CHO) cells not only through glycoproteins but also through GM3.
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Results |
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Discussion |
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The recognition of lectins for carbohydrate ligands should be affected by not only the nonreducing terminal side of carbohydrates but also their structures at the reducing terminal side and aglycons, such as peptides or hydrophobic lipid moieties of glycoconjugates. In fact, we had previously reported that galectin-4 recognizes SO3-3Galß1
3GalNAc pyranoside but not SO3-
3Galß1
3GalNAcOH (Ideo et al., 2002
). Because galectin-8 is 34% homologous to galectin-4 at amino acid sequence level, we had an interest in whether galectin-8 also recognizes sulfated glycans. Hirabayashi et al. (2002)
already reported that galectin-8 recognizes pyridylaminated 3'SL with high affinity using frontal affinity chromatography, which is effective to survey the carbohydrate binding specificity by using a set of pyridylaminated oligosaccharides. However, in preliminary experiments, we noticed that galectin-8 recognizes SO3-
3Galß1
4Glc-O-pNP and lactose but not lactitol using the SPR assay. Accordingly, we extensively investigated the carbohydrate binding specificity of galectin-8 for nonlabeled oligosaccharides and glycosphingolipids using the ELISA and SPR assays. KD values of pyridylaminated lactose, 2'FL, A-tetra, 3'SL, LNT, LNnT, LNF-I, LNF-II, and LNF-III to immobilized galectin-8 using frontal affinity chromatography were the same levels as those of the respective nonlabeled oligosaccharide pyranosides using the SPR assay as shown in Table I. However, the KD values of GM3 and SM3 to GST-N-domain were 225 and 268 times lower than those of 3'SL and SO3-
3Galß1
4Glc-O-pNP, suggesting that the clustering of glycosphingolipids or their hydrophobic lipid moieties enhances the binding ability to galectin-8. These results so far described show that the ELISA method is convenient to survey the interaction between galectins and various glycosphingolipids, and the SPR assay is very effective to analyze the kinetical characters.
Furthermore, we found that this unique carbohydrate binding specificity is attributed to the Gln47 residue on the S3-ß-sheet of the N-domain of galectin-8. This indicates that the amino acids on the S3 ß-sheet regulate the distinct carbohydrate binding specificity of each galectin family member. Both galectins-4 and -8 have high affinity for SO3-3Gal, but they exhibit completely different affinity for Neu5Ac
2
3Gal. Galectin-4 does not recognize GM3 but does bind sulfated glycosphingolipids, including SM4, SM3, SB2, and SB1a (unpublished data), whereas galectin-8 recognizes glycosphingolipids bearing the Neu5Ac
2
3Gal residue as well as SO3-
3Gal. Kopitz et al. (1996
, 1998)
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. From these results, it can be suggested that glycosphingolipids are biologically important ligands for some members of the galectin family.
It is accepted that glycosphingolipids are abundant in detergent-insoluble microdomains (rafts), and that they influence signal transduction by modulating binding of exogenous effectors to cell surface receptors, which are also enriched in rafts (Hakomori, 1990, 2002
; Simons and Ikonen, 1993
; Miljan et al., 2002
). GM3 in B16 melanoma cells colocalizes with transducer molecules, such as c-Src, Rho, Ras, and FAK (Yamamura et al., 1997
; Iwabuchi et al., 1998
). Sulfated glycosphingolipids accumulated in human hepatocellular carcinoma cells and tissues compared to normal liver (Hiraiwa et al., 1990
). Considering our evidence that galectin-8 binds to SM3 and GM3, it is possible that a part of galectin-8 localizes to rafts by binding to specific glycosphingolipids and thereby modulates the biological function of them. In fact, it has been shown that galectin-4 is present in certain fractions of rafts in the brush border membrane of enterocytes (Hansen et al., 2001
; Braccia et al., 2003
). Such a glycosphingolipid binding property of galectin-8 might give hints for the further elucidation of functional roles of this protein.
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Materials and methods |
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Preparation of sulfoglycosphingolipids, SM2a, SB2 and SB1a
Sulfoglycosphingolipids were purified from rat kidney according to the methods of Tadano et al. (1982) and Magnani et al. (1982)
. Briefly, 200 g of rat kidney (Wistar) was homogenized in 0.4 L water, and 1.44 L methanol and 0.72 L chloroform were added. The mixture was stirred for 2 h at room temperature and centrifuged. The precipitate was extracted once with 0.4 L water and 1.6 L chloroform/methanol (1:2). The two supernatant fractions were combined, 0.78 L water was added, and the solution was left to stand for 16 h at room temperature. The lower layer was extracted once with 0.47 L methanol and 0.31 L 10 mM KCl. The two upper layers were combined, evaporated, and dialyzed against water. The dialysate was adjusted to chloroform/methanol/water (30:60:8) and applied on a DEAE-Sephadex A-25 column (2.3 x 17.5 cm; acetate form; equilibrated with chloroform/methanol/water [30:60:8]).
After washing, glycosphingolipids were eluted with a linear gradient of ammonium acetate in methanol. The two fractions (eluted in 0.070.13 M [SM2a] and 0.51 M [SB2 and SB1a] of ammonium acetate) were individually collected, evaporated, and dialyzed. Aliquots of the two fractions were spotted on a high-performance thin-layer chromatography plate (10 x 10 cm, Kieselgel 60 F254, Merck, Darmstadt, Germany) and developed using a solvent, chloroform/methanol/0.2% CaCl2 (60:35:7). Glycosphingolipids and sulfoglycosphingolipids were detected by 10% H2SO4/ethanol (heated at 100120°C) and Azure A staining (Iida et al., 1989), respectively. Sulfoglycosphingolipids were extracted from silica gels with chloroform/methanol (1:1), and used for experiments.
Confirmation of these carbohydrate structures was performed as follows. Aliquots of the sulfoglycosphingolipids were digested with endoglycoceramidase II (10 mU, in 20 mM sodium acetate, pH 5.2, 0.4% Triton X-100 at 37°C for 16 h) (Takara Shuzo, Kyoto, Japan) and the released oligosaccharides were [3H]-labeled with NaB3H4. All [3H]oligosaccharides were Arthrobacter sialidaseresistant and mild methanolysissusceptible (Yamashita et al., 1983), indicating that these oligosaccharides contained sulfate residues but not sialic acids. The [3H]oligosaccharide derived from SM2a bound to a Wistaria floribunda agglutinin (WFA)-agarose column and was eluted with 10 mM GalNAc, suggesting the presence of nonsubstituted ß-GalNAc, because WFA binds to ß-GalNAc residues at the nonreducing termini (Smith and Torres, 1989
). The [3H]oligosaccharide derived from SB2 flowed through a WFA-agarose column but bound to it after mild methanolysis, suggesting the presence of a SO3-
GalNAcß1
moiety. The [3H]oligosaccharide derived from SB1a flowed through a WFA-agarose column regardless of mild methanolysis, but after mild methanolysis, bound to a peanut agglutininagarose column (4.5 mg/ml gel, E-Y Laboratories, San Mateo, CA) and was eluted with 0.3 M lactose, suggesting the presence of a SO3-
Galß1
3GalNAc moiety, because peanut agglutinin binds Galß1
3GalNAc residues (Pereira and Kabat, 1976
).
Preparation of GST-galectin-8, GST-N-domain, and GST-C-domain
A cDNA that contained the entire open reading frame for galectin-8 was obtained using a human testis cDNA library by PCR (30 cycles of 95°C for 0.5 min, 50°C for 1 min, and 72°C for 2 min). The primers used were 5'-atcgtcgactCATGATGTTGTCCTTAAAC-3' (forward primer) and 5'-atcgcggccgcCTACCAGCTCCTTACTTC-3' (reverse primer) for GST-galectin-8, 5'-atcgtcgactcATGATGTTGTC-CTTAAAC-3' (forward primer) and 5'-atcgcggccgcAAG CTGGGGCGTGCCAGA-3' (reverse primer) for GST-N-domain, and 5'-atcgtcgacTGCCATTCGCTGCAAGG-3' (forward primer) and 5'-atcgcggccgcCTACCAGCTCCTTACTTC-3' (reverse primer) for GST-C-domain. After the sequence of the amplified fragment was confirmed using ABI PRISM 310 Genetic Analyzer (PE Biosystems), the fragments were inserted into the pGEX-6P-1 plasmid between the Sal I and the Not I sites. Plasmids were transformed with E. coli BL21 strain. Luria-Bertani broth with 100 µg/ml ampicillin was inoculated with the overnight culture of the transformed E. coli. When the absorbance at 600 nm reached a value of 0.7, ispropylthiogalactoside was added to a final concentration of 0.5 mM and the cultures were allowed to stand for 1.5 h. The bacteria were harvested by centrifugation at 5000 x g for 5 min. Cell pellets were resuspended and disrupted using a sonicator in phosphate buffered saline (PBS) containing 4 mM 2-mercaptoethanol (2-ME). After addition of Triton X-100 to a final concentration of 1%, the suspensions were mixed gently for 30 min to solubilize the fusion protein. After centrifugation at 12,000 x g for 20 min, the supernatant was applied to a glutathione-Sepharose column. After washing the column with PBS-2ME, GST-galectin-8 and the two domains were eluted with 10 mM glutathione in 50 mM TrisHCl (pH 8.0). The purified GST-galectin-8 and the two domains were dialyzed against PBS-2ME and concentrated by filtration with an ULTRAFREE centrifugal filter device (Millipore, Bedford, MA) with PBS-2ME or PreScission cleavage buffer (50 mM TrisHCl, pH 7.0, 150 mM NaCl, 1 mM ethylenediamine tetra-acetic acid [EDTA], and 1 mM dithiothreitol). Protein concentration was determined using a Bio-Rad Protein Assay dye reagent and bovine serum albumin as a standard.
Site-directed mutagenesis of GST-N-domain
Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Strategene, La Jolla, CA) according to the manufacturer's instructions. Mutagenic primers used were 5'-GTGACGCAGACAGATTCgcGGTGGATCTGCAGAATGGC-3' (sense primer for galectin-8-N-domain) and 5'-GCCATTCTGCAGATCCACCgcGAATCTGTCTGCGTCAC-3' (antisense primer for galectin-8-N-domain). Sequences in lowercase indicate mismatch bases for the desired mutation. Nucleic acid sequences were analyzed using an Applied Biosystems PRISM 310 Genetic Analyzer.
Preparation of GST-tag-free forms of galectin-8, N-domain and C-domain, and domain-specific antibodies
Two units of PreScission protease and 100 µg of each GST-fused protein in PreScission cleavage buffer were incubated at 4°C for 4 h or overnight. The samples were applied to a glutathione-Sepharose column to remove the free GST moieties.
Antisera against the N- and C-domains were raised in rabbit according to standard procedures. Purified GST-tag-free domains (11.5 mg) were injected into a rabbit (initial and two boosts) with intervals of 3 weeks.
Estimation of kinetic constants based on SPR
The dissociation constants between the full length or the two domains of galectin-8 and various carbohydrates were measured using a BIAcore 2000 instrument as described previously (Ideo et al., 2002). The purified GST, GST-galectin-8, and the GST domains were immobilized on the CM5 sensor surface at pH 5 according to the manufacturer's instructions. Various carbohydrates in HBS-EP [0.01 M HEPES-NaOH (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20] buffer were introduced onto the surface at a flow rate of 20 µl/min. The interaction was monitored at 25°C by subtracting the signal obtained from GST-immobilized surface, and the dissociation constants were calculated using BIA evaluation 3.0 software.
Inhibition assay based on SPR
The inhibition effects of various carbohydrates on binding of the GST-N- and -C-domains to immobilized asialofetuin were measured using a BIAcore 2000 instrument as described previously (Ideo et al., 2002). Five µg/ml of the N-domain or 25 µg/ml of the C-domain, and various concentrations of carbohydrates were mixed in HBS-EP buffer and introduced onto the asialofetuin-immobilized surface at a flow rate of 20 µl/min at 25°C. For removing the bound proteins on the surface, 0.1 M lactose in HBS-EP buffer was used.
ELISA for binding of galectin-8 to various glycosphingolipids
Five microliters of various concentrations of glycosphingolipids in MeOH were added to each well of a 96-well microtiter plate (Dynatech Laboratories). After evaporation of the solvent, 100 µl of 1 % BSA in PBS was added as a blocking solution and the plate was left overnight at 4°C. After washing with PBS, 50 µl of 250 nM GST-N- or -C-domains in the blocking solution were added to each well and the plate was left for 2 h at room temperature. The plate was washed several times with washing buffer (0.01% Tween 20 in PBS), and N- and C-domain-specific antibodies diluted in the washing buffer were added. After incubation for 2 h at room temperature, the plate was washed and goat anti-rabbit IgG conjugated with alkaline phosphatase secondary antibody was added (The Binding Site Ltd., Birmingham, UK). After 100 min at room temperature, the enzyme substrate, 6.7 µmole of p-nitrophenylphosphoric acid disodium salt in 0.1 M carbonate buffer (pH 9.6), was added. The reaction mixture was incubated at room temperature for 15 min. The released chromogen was measured with a photospectrometer (EIA Reader, Bio-Rad Model 3550). The amount of glycosphingolipids that were retained in plastic microwells was compared before and after the washing and blocking steps. After 300 pmoles of the respective glycosphingolipids were extracted with 1-butanol from the microwells, they were applied to thin layer chromatography (10 x 10 cm, silica gel 60, Merck, Darmstadt, Germany) and were developed using a solvent, chloroform/methanol/0.2% CaCl2 (60:35:7). The plates were soaked with 0.01% primulin in acetone/water (4:1) and were scanned with a lumino-image analyzer LAS-1000 (FUJIFILM). More than 30% of glycosphingolipids were retained on microwells and no significant variations in their amounts were not observed among the glycosphingolipids used.
Binding of galectin-8, and its N- and C-domains to glycosphingolipids immobilized on the surface of BIAcore sensor chip
Glycosphingolipids were hydrophobically immobilized onto the CM5 sensor chip according to Catimel et al. (1998). They were dissolved in EtOH/MeOH, 9:1 (v/v) (1 mg/ml), diluted in HBS buffer (10 mM HEPES buffer [pH 7.4] containing 3.4 mM EDTA and 150 mM NaCl), and injected (80 µl) at a flow rate of 5 µl/min over the unmodified surface.
Purified GST, GST-galectin-8, GST-N- and -C-domains in HBS buffer were introduced onto the surface at a flow rate of 20 µl/min. The interaction was monitored at 25°C, and the kinetic constants were calculated using BIA evaluation 3.0 software.
Cell adhesion assay
ELISA plates were precoated with the wild-type or mutated GST-N-domain in PBS for 16 h at 4°C, followed by blocking with 0.1% bovine serum albumin for 2 h at 37°C. CHO-K1 cells were grown on tissue culture plates in F12 medium containing 10% fetal calf serum and were detached from the plates with 0.25% trypsin and 0.02% EDTA in PBS. After washing with the culture medium and PBS, cells were resuspended in serum-free medium containing 10 mM EDTA and reseeded on the coated plates. After 30 min at 37°C, 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% sodium dodecyl sulfate for 1 h at 22°C and quantified by measuring the absorbance at 595 nm with a spectrophotometer. All assays were performed in triplicate.
Western blotting
Proteins of CHO-K1 cell extracts were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by transfer to nitrocellulose membranes using standard techniques. After blocking the membrane with 1% bovine serum albumin overnight at 4°C, it was treated with Arthrobacter sialidase (200 mU/200 µl) for 30 min at 37°C. After washing, the membrane was incubated with the GST-N-domain (30 µg/ml) for 1 h at room temperature. The bound GST-N-domain was detected by anti-N-domain followed by horseradish peroxidaseconjugated secondary antibody and visualized using an enhanced chemiluminescent peroxidase substrate (Amersham Pharmacia Biotech), according to the manufacturer's instructions.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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