The N-terminal carbohydrate recognition domain of galectin-8 recognizes specific glycosphingolipids with high affinity

Hiroko Ideo2, Akira Seko2, Ineo Ishizuka3 and Katsuko Yamashita1,2

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Galectin-8 is a member of the galectin family and has two tandem repeated carbohydrate recognition domains (CRDs). We determined the binding specificities of galectin-8 and its two CRDs for oligosaccharides and glycosphingolipids using ELISA and surface plasmon resonance assays. Galectin-8 had much higher affinity for 3'-O-sulfated or 3'-O-sialylated lactose and a Lewis x–containing glycan than for oligosaccharides terminating in Galß1->3/4GlcNAc. This specificity was mainly attributed to the N-terminal CRD (N-domain), whereas the C-terminal CRD (C-domain) had only weak affinity for a blood group A glycan. The N-domain bound not only to oligosaccharides but also to glycosphingolipids including sulfatide (SM4 s), SM3, sialyl Lc4Cer, SB1a, GD1a, GM3, and sialyl nLc4Cer, suggesting that the N-domain recognizes a 3-O-sulfated or 3-O-sialylated Gal residue. The substitution of the C-3 of the Gal residue in lactose or N-acetyllactosamine with sulfate increased the degree of recognition by galectin-8 more potently than substitution with sialic acid. This is the first demonstration that galectin-8 binds to specific sulfated or sialylated glycosphingolipids with high affinity (KD~10-8–10-9 M). When the Gln47 residue of the N-domain was converted to Ala47, the specific affinity for sulfated or sialylated glycans was selectively lost, indicating that this Gln47 plays important roles for binding to Neu5Ac{alpha}2->3Gal or SO3-->3Gal residues. The binding ability of galectin-8 to membrane-associated GM3 was confirmed using CHO cells, which predominantly express GM3. Binding of CHO cells to the mutein was significantly lower than to the N-domain.

Key words: galectin-8 / ganglioside / GM3 / sulfoglycosphingolipid / surface plasmon resonance


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Galectin-8 was initially cloned from a rat liver cDNA library (Hadari et al., 1995Go) and exhibits affinity for ß-galactose-bearing carbohydrates, similar to other members of the galectin family (Barondes et al., 1994Go; Cooper and Barondes, 1999Go; Varki et al., 1999Go). Like galectins-4, -6, -9, -11, and -12, galectin-8 has two conserved carbohydrate recognition domains (CRDs) within a single molecule. Levy et al. (2001)Go showed that galectin-8 is involved in mediating cell–cell interactions through its binding to a subset of integrins. However, the biological functions of galectin-8 remain to be fully elucidated.

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., 1996Go; Gopalkrishnan et al., 2000Go). 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., 2001Go). Furthermore, Lahm et al. (2001)Go, 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., 2002Go). 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., 1995Go). 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., 2000Go), whereas galectin-4 is specifically expressed in intestine, colon, and stomach (Chiu et al., 1994Go), 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{alpha}2 residues. We further found that galectin-8 binds to Chinese hamster ovary (CHO) cells not only through glycoproteins but also through GM3.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Carbohydrate binding specificity of galectin-8 to oligosaccharides
Galectin-8 has two conserved CRDs, which share 38% amino acid identity. To investigate carbohydrate binding specificities of galectin-8 and these two domains, we prepared glutathione-S-transferase (GST)-fused recombinant proteins for full-length galectin-8, the N-domain, and the C-domain. First, binding of various oligosaccharides to the immobilized recombinant proteins was measured by surface plasmon resonance. As summarized in Table I, GST-galectin-8 strongly bound to SO3-->3Lac-O-p-nitrophenyl (pNP), 3'SL (Neu5Ac{alpha}2->3Galß1->4Glc), and LNF-III (Galß1->4[Fuc{alpha}1->3]GlcNAcß1->3Galß1->4Glc) with KD values of 1.7 x 10-6, 2.4 x 10-6, and 6.2 x 10-6 M, respectively. A-tetra (GalNAc{alpha}1->3[Fuc{alpha}1->2]Galß1->4Glc), LNF-II (Galß1->3[Fuc{alpha}1->4]GlcNAcß1->3Galß1->4Glc), and SO3-->3core 1-O-Bn had weaker affinity for GST-galectin-8 with KD values of ~2 x 10-5 M. Lactose, type 1, type 2, and core 1 were poor ligands for galectin-8. GST-N-domain showed high affinity for SO3-->3Lac-O-pNP, 3'SL, and LNF-III and weakly bound to LNF-II, LNnT (Galß1->4GlcNAcß1->3Galß1->4Glc), and SO3-->3core 1-O-Bn. In contrast, GST-C-domain had only weak affinity for A-tetra, LNF-I (Fuc{alpha}1->2Galß1->3GlcNAcß1->3Galß1->4Glc), and LNF-II. These results indicate that the binding of GST-galectin-8 to SO3-->3Lac-O-pNP, 3'SL, LNF-III, and SO3-->3core 1-O-Bn is mediated by the N-domain, and the binding to A-tetra is mediated by the C-domain. The carbohydrate binding specificity of GST-N-domain is characteristic for the following reasons: (1) SO3-->3Lac-O-pNP and 3'SL were the best ligands among the oligosaccharides examined. Hirabayashi et al. (2002)Go showed that pyridylaminated 3'SL is a good ligand for a mutated galectin-8, which was prepared by substitution of the conserved residue Arg233 with His. 6'SL did not bind to GST-N-domain, indicating that the substitution of anionic residues at the C-3 of Gal is important for the high affinity. (2) LNnT and LNF-III showed stronger affinity than LNT (Galß1-> 3GlcNAcß1->3Galß1->4Glc) and LNF-II, respectively, suggesting that the GST-N-domain prefers type 2 oligosaccharides to type 1. Moreover, fucosylation of the distal GlcNAc in LNT and LNnT increased the binding affinity.


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Table I. The KD values of GST-galectin-8, GST-N-domain, and GST-C-domain to various oligosaccharides

 
To further confirm the carbohydrate binding specificity of galectin-8, we examined inhibitory effects of various oligosaccharides on binding between GST-N- or C-domain and immobilized asialofetuin. As shown in Figure 1, 3'SL inhibited the binding of GST-N-domain to asialofetuin at low concentrations. The concentration of 3'SL giving 50% inhibition was 4.4 x 10-7 M, which is 180 times lower than that of lactose. On the other hand, the binding of GST-C-domain was efficiently inhibited by A-tetra but not 3'SL. These results are in good agreement with the data presented in Table I.



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Fig. 1. Inhibition curves of various oligosaccharides for binding of GST-N- and -C-domains to immobilized asialofetuin. (A) GST-N-domain; (B) GST-C-domain. Relative binding abilities in the presence of various oligosaccharides were measured using SPR methods. Lactose (open circles), 3'SL (closed circles), LNT (open squares), LNnT (closed squares), A-tetra (open triangles), and LNF-III (closed triangles).

 
Carbohydrate binding specificity of galectin-8 to glycosphingolipids
Because the oligosaccharides summarized in Table I could be found in vivo as glycosphingolipids, it was necessary to clarify whether galectin-8 binds to specific glycosphingolipids. To investigate the interaction of galectin-8 with various glycosphingolipids, we measured the binding of GST-N- and -C-domains to glycosphingolipid-coated plates using antibodies against the N- or C-domains. As shown in Figure 2, GST-N-domain strongly bound to sLc4Cer, SB1a, GD1a, SM3, snLc4Cer, and GM3 and bound weakly to SM4 s, SB2 and SM2a. GalCer, LacCer, GM1, GM2, GM4, and sLeX-Cer were not recognized by GST-N-domain, at least at the concentrations tested. GST-C-domain did not bind to most of glycosphingolipids examined, except slightly to GM1 and SB1a (data not shown). Several points emerged regarding the binding specificity of the N-domain to glycosphingolipids: (1) A 3-O-sulfated or -sialylated Gal residue is important for the binding to the GST-N-domain, because these moieties were common among sLc4Cer, SB1a, GD1a, snLc4Cer, GM3, and SM3. GM3 and SM3 were much better ligands for GST-N-domain than LacCer, suggesting the importance of anionic residues at the C-3 of Gal for good recognition. (2) SM3 and SM2a were better ligands than GM3 and GM2, respectively, suggesting that the N-domain prefers 3-O-sulfated Gal to 3-O-sialylated Gal. (3) The substitution of ß-GalNAc or Galß1->3GalNAcß1-> at the C-4 of Gal in SM3 and GM3 decreased the affinity to the N-domain, because GM3 and SM3 were better ligands than GM2 and GM1, and SM2a, respectively.



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Fig. 2. ELISA for binding of GST-N-domain to various glycosphingolipids. (A) The binding abilities of GST-N-domain (250 nM) for different concentrations of coated glycosphingolipids. The amount of bound GST-N-domain was measured using an anti-N-domain antibody as described in Materials and methods. SLc4Cer (closed circles), SB1a (open circles), GD1a (asterisks), SM3 (closed squares), snLc4Cer (open squares), GM3 (closed triangles), SM4 s (open triangles), SB2 (closed upside-down triangles), SM2a (open upside-down triangles), and GalCer (diamonds). (B) relative binding abilities of GST-N-domain to various glycosphingolipids were calculated at a concentration of 100 pmol glycosphingolipids/well.

 
The binding of GST-N-domain to immobilized glycosphingolipids was further examined using the SPR assay (Figure 3 and Table II). As shown in Figure 3, GST-N-domain bound to GM3 (Figure 3A), sLc4Cer (Figure 3B), snLc4Cer (Figure 3C), and SM3 (Figure 3F), and was released with 0.1 M lactose. On the other hand, GST-C-domain did not bind to GM3 (Figure 3D), sLc4Cer (Figure 3E), snLc4Cer, SM3, or GM1 (same as Figure 3D or 3E). The KD value (1.2 x 10-8 M) of GST-N-domain binding to GM3 was significantly lower than the KD value (2.7 x 10-6 M) for immobilized GST-N-domain binding to 3'SL. The KD value (7.1 x 10-9 M) of GST-N-domain binding to SM3 was also lower than that for GST-N-domain binding to SO3-->3Lac-O-pNP (1.9 x 10-6 M). These results implied that clustering of these carbohydrates enhances their affinity for the N-domain. A similar effect was also observed between galectin-4 and core 1 (Ideo et al., 2002Go). Otherwise, there may exist some hydrophobic interaction between GST-N-domain and lipid moieties of these glycosphingolipids. Furthermore, GST-N-domain bound more rapidly to SM3 than to GM3, sLc4Cer, and snLc4Cer, resulting in higher affinity toward SM3 (Figure 3 and Table II).



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Fig. 3. Sensorgrams of SPR for binding of GST-N-domain to glycosphingolipids. Various concentrations of GST-N-domain were introduced onto the glycosphingolipid-immobilized surface for 180 s at a flow rate of 20 µl/min. The relative response (RU) was indicated as subtraction of the blank values on the nonimmobilized surface from the values on the glycosphingolipids-immobilized surface. (A) GST-N-domain to GM3; (B) GST-N-domain to sLc4Cer; (C) GST-N-domain to snLc4Cer; (D) GST-C-domain to GM3; (E) GST-C-domain to sLc4Cer; (F) GST-N-domain to SM3.

 

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Table II. Kinetic data for the interaction between the GST-N-domain and glycosphingolipids immobilized on sensor chips

 
Gln47 residue of galectin-8 is important for recognizing sulfated or sialylated glycans
X-ray crystal structures of human galectins-2, -3, -7, and -10 (Lobsanov et al., 1993Go; Seetharaman et al., 1998Go; Leonidas et al., 1998Go; Swaminathan et al., 1999Go) showed that these galectins have very similar tertiary structures. Thus to investigate which amino acid(s) of galectin-8 interact with the Neu5Ac{alpha}2->3 or SO3-->3 residue, we created a structural model of the N-domain (Figure 4A, 4B, 4C, and 4D) using that of the galectin-3-CRD as a template (Peitsch, 1996Go; Guex and Peitsch, 1997Go). The X-ray crystal structure of human galectin-3-CRD in complex with N-acetyllactosamine (Figure 4E and 4F) showed that the Lac/LacNAc binding site, involving ß-sheets S4, S5, and S6, is a cleft. Amino acids critical for lactose binding (Hirabayashi and Kasai, 1994Go) in the S4, S5, and S6 ß-sheets are well conserved among galectins-1–9 (colored red in Figure 4G). The galactose-3-O-linked nonreducing terminal moiety was expected to interact with amino acids in the extended clefts formed by the S3 ß-sheet of galectins. Because the sequence and conformation of amino acids on the ß-sheets S4, S5, and S6 involved in the binding to the Lac/LacNAc moiety were conserved between the galectin-8-N-domain and galectin-3-CRD (as shown in Figure 4C, 4E, and 4G), it appeared that amino acids on the S3 sheet of galectin-8-N-domain are involved in binding to the Neu5Ac{alpha}2->3 or SO3-->3 residue. Because side chains of Arg45, Gln47, Asp49, and Gln51 on the S3 sheet of galectin-8-N-domain were exposed toward carbohydrate ligands in the structural model (Figure 4C, 4D) and were hydrophilic, they were considered prime candidates for binding to Neu5Ac{alpha}2->3 or SO3-->3 residues. The Arg45 residue is conserved in galectins-3, -4, -7, -8 and -9, and Asp49 is conserved in galectins-3 and -8. Gln51 is also conserved in galectins-3, -8, and -9, whereas Gln47 is the only amino acid that is distinct from other galectins. The binding abilities of galectins-1, -2, -3, -7, and -9 to 3'SL were similar to lactose (Hirabayashi et al., 2002Go), and galectin-4 did not show any detectable affinity for 3'SL (Ideo et al., 2002Go). Therefore, we constructed a mutated galectin-8-N-domain, which has Ala47 instead of Gln47 (mutein) and analyzed its carbohydrate-binding specificity.



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Fig. 4. Molecular models of the N-domain of galectin-8. (A) and (B) show the overall structure of the N-domain of galectin-8. (A) Ribbon diagram of galectin-8 is shown perpendicular to the ß-sheets. (B) 90° rotation of the A diagram around a vertical axis. Secondary structure elements were assigned using Swiss-Pdb Viewer (Peitsch, 1996Go; Guex and Peitsch, 1997Go). Strands belonging to the concave and convex ß-sheets are shown in red and blue, respectively. Strands that do not form part of the ß-sheets are shown in gray. (C) and (D) show the side chains of amino acids on the S3–S6 ß-sheets that face toward the outside of the galectin-8 molecule. (AD) were prepared using SWISS-MODEL (protein structure homology-modeling server) (Peitsch, 1996Go; Guex and Peitsch, 1997Go). (E) and (F) were drawn using Swiss-PdbViewer with the data of X-ray crystal structure of human galectin-3-CRD (PDB:1A3K) in complex with N-acetyllactosamine (shown in orange). Blue and red color in (CF) are nitrogen and oxygen atoms, respectively. (G) Amino acid alignment of the S3–S6 ß-sheets of galectins. Amino acids that face carbohydrate ligands are indicated by arrowheads. Residues that are common in all the sequences are shown in red. And Gln47 in galectin-8 is painted in green.

 
As shown in Table III, the mutein had similar KD values for Lac-O-pNP, LNT, LNnT, and LNF-III compared to those of the GST-N-domain, whereas, the KD values of the mutein for SO3-->3Lac-O-pNP and 3'SL were 8 and 21 times higher, respectively, than those of the GST-N-domain. The affinity of the mutein for the two anionic oligosaccharides was lower than that for LNnT or LNF-III, indicating that the Gln47 residue plays important roles in the binding of the N-domain to 3'-sulfated or sialylated lactose moieties. The polar side chain of Gln47 may be involved in the interaction for the sulfate or sialic acid but not affect the binding to the neutral oligosaccharides.


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Table III. The KD values of the GST-N-domain and the mutein (Gln47->Ala) for various oligosaccharides

 
The N-domain recognizes both GM3 and glycoproteins on the plasma membrane of CHO cells
Next we investigated whether galectin-8 can bind to the Neu5Ac{alpha}2->3Gal moiety present on the surface of cells under physiological conditions. It has been previously reported that N- and O-linked sugar chains of glycoproteins produced in CHO cells predominantly have Neu5Ac{alpha}2->3Gal residues at the nonreducing termini (Fukushima et al., 1993Go; Hokke et al., 1995Go) and that CHO cells contain rather simple patterns of glycosphingolipids, namely, LacCer and GM3 (Warnock et al., 1993Go). Accordingly, we compared the binding ability of the GST-N-domain and the mutein toward CHO cells. As shown in Figure 5, both bound to CHO cells, but the binding ability of CHO cells to the immobilized mutein was low to approximately 60% of that of the wild-type domain. Addition of anti-GM3 antibody decreased the binding ability of the GST-N-domain toward CHO cells to 70% (data not shown), suggesting that GM3 on the plasma membrane is one of the major ligands for galectin-8 in CHO cells. The GST-N-domain also bound to various glycoproteins on CHO cells (Figure 6, lane 1). Sialidase treatment before the addition of the GST-N-domain significantly reduced immunostaining for the N-domain (Figure 6, lane 2), indicating that galectin-8 binds to 3-O-sialylated glycoproteins in CHO cells. These results suggested that galectin-8 binds both to glycoproteins and GM3 on the plasma membrane of CHO cells.



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Fig. 5. Adhesion of CHO cells to plates coated with GST-N-domain and the mutein. 96-well ELISA plates were precoated with 0.1 ml of the indicated concentrations of GST-N-domain (filled circles) or the mutein (open circles) for 16 h at 4°C and then were incubated with 0.1% bovine serum albumin for 2 h at 37°C. CHO-K1 cells (1.6 x 105 each) were detached from culture plates with 0.25% trypsin and 0.02% EDTA in PBS, washed with PBS, and seeded in serum-free medium on the coated wells. After incubation for 30 min at 37°C, cells were washed and stained with crystal violet, and the amounts of adherent cells were determined as described in Materials and methods. Values were the mean ± SD of five experiments.

 


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Fig. 6. SDS–PAGE analysis of glycoproteins of CHO cells recognized by the N-domain. The proteins were incubated with (lanes 1 and 2) or without (lane 3) the GST-N-domain, and the bound GST-N-domain was detected with anti-N-domain antibody. The proteins were incubated with Arthrobacter sialidase before the incubation with the GST-N-domain (lane 2).

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this study, we clearly demonstrate that the N-domain of galectin-8 binds to immobilized glycosphingolipids carrying SO3-->3Gal/Sia{alpha}2->3Gal residues. In fact, galectin-8 bound not only to sialylated glycoproteins but also to GM3 on the plasma membrane of CHO cells. To our knowledge, this is the first report that has directly shown the binding of galectin-8 to glycosphingolipids. We also demonstrate that galectin-8 strongly recognizes 3'-O-sulfated/sialylated lactose/LacNAc and Lex, and this interaction is mediated by the N-domain.

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., 2002Go). 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)Go 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{alpha}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{alpha}2->3Gal residue as well as SO3-->3Gal. Kopitz et al. (1996Go, 1998)Go 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, 1990Go, 2002Go; Simons and Ikonen, 1993Go; Miljan et al., 2002Go). GM3 in B16 melanoma cells colocalizes with transducer molecules, such as c-Src, Rho, Ras, and FAK (Yamamura et al., 1997Go; Iwabuchi et al., 1998Go). Sulfated glycosphingolipids accumulated in human hepatocellular carcinoma cells and tissues compared to normal liver (Hiraiwa et al., 1990Go). 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., 2001Go; Braccia et al., 2003Go). Such a glycosphingolipid binding property of galectin-8 might give hints for the further elucidation of functional roles of this protein.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Bovine fetuin and core 1-O-Bn were purchased from Sigma (St. Louis, MO). Lactose, GM1, GM2, GM3, SM4 s, GM4, GD1a, sLc4Cer, snLc4Cer, and sLex-Cer (see Table IV) were purchased from Wako Pure Chemical (Osaka, Japan). 2'FL, 3FL (Galß1->4[Fuc{alpha}1->3]Glc), 3'SL, 6'SL (Neu5Ac{alpha}2->6Galß1->4Glc), Gal{alpha}1->4Galß1->4Glc, A-tetra, type 1, type 2, LNT, LNnT, LNF-I, LNF-II, LNF-III, core 1, core 1-O-pNP, and core 2-O-pNP were purchased from Funakoshi (Tokyo). Lactose-O-pNP and anti-GM3 were purchased from Seikagaku (Tokyo). SO3-->3Lac-O-pNP was a kind gift from Dr. Matta (Roswell Park Memorial Cancer Institute), and SO3-->3Galß1->3GalNAc{alpha}1-O-Bn was prepared as reported previously (Ideo et al., 2002Go). Lactitol was prepared from lactose by NaBH4 reduction. SM3 (see Table IV) was prepared as described previously (Ishizuka, 1997Go).


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Table IV. Glycolipid structures

 
Human testis cDNA library was purchased from Life Technologies (Rockville, MD). A pGEX-6P-1 plasmid, Escherichia coli BL21 strain, glutathione-Sepharose, and PreScission protease were from Amersham Pharmacia Biotech (England). Arthrobacter sialidase was from Nakarai Tesque (Kyoto, Japan).

Preparation of sulfoglycosphingolipids, SM2a, SB2 and SB1a
Sulfoglycosphingolipids were purified from rat kidney according to the methods of Tadano et al. (1982)Go and Magnani et al. (1982)Go. 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 0.07–0.13 M [SM2a] and 0.5–1 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 100–120°C) and Azure A staining (Iida et al., 1989Go), 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 sialidase–resistant and mild methanolysis–susceptible (Yamashita et al., 1983Go), 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, 1989Go). 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 agglutinin–agarose 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, 1976Go).

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 Tris–HCl (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 Tris–HCl, 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 (1~1.5 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., 2002Go). 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., 2002Go). 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)Go. 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 sulfate–polyacrylamide 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 peroxidase–conjugated secondary antibody and visualized using an enhanced chemiluminescent peroxidase substrate (Amersham Pharmacia Biotech), according to the manufacturer's instructions.


    Acknowledgements
 
This work was supported by the Grant-in-Aid for Scientific Research on Priority Area(s) No. 14082208 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Uehara Memorial Foundation.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: yamashita{at}sasaki.or.jp Back


    Abbreviations
 
2-ME, 2-mercaptoethanol; 3FL, Galß1->4(Fuc{alpha}1->3)Glc; 3'SL, Neu5Ac{alpha}2->3Galß1->4Glc; 6'SL, Neu5Ac{alpha}2->6Galß1->4Glc; A-tetra, GalNAc{alpha}1->3(Fuc{alpha}1->2)Galß1->4Glc; CHO, Chinese hamster ovary; CRD, carbohydrate recognition domain; rhgalectin-8, recombinant human galectin-8; EDTA, ethylenediamine tetra-acetic acid; ELISA, enzyme-linked immuno-absorbent assay; GST, glutathione-S-transferase; LNF-I, Fuc{alpha}1->2Galß1->3GlcNAcß1->3Galß1->4Glc; LNF-II, Galß1->3(Fuc{alpha}1->4)GlcNAcß1->3Galß1->4Glc; LNF-III, Galß1->4(Fuc{alpha}1->3)GlcNAcß1->3Galß1->4Glc; LNT, Galß1->3GlcNAcß1->3Galß1->4Glc; LNnT, Galß1->4GlcNAcß1->3Galß1->4Glc; PBS, phosphate buffered saline; PCR, polymerase chain reaction; pNP, p-nitrophenyl; SPR, surface plasmon resonance; WFA, Wistaria floribunda agglutinin


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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