Galectin-8 has much higher affinity for 3-O-sulfated or 3-Osialylated glycoconjugates and a Lewis X-containing glycan than for oligosaccharides terminating in Gal␤133/4GlcNAc, and this specificity is mainly attributed to the N-terminal carbohydrate recognition domain (N-domain, CRD) (Ideo, H., Seko, A., Ishizuka, I., and Yamashita, K. (2003) Glycobiology 13, 713-723). In this study, we elucidated the crystal structures of the human galectin-8-N-domain (-8N) in the absence or presence of 4 ligands. The apo molecule forms a dimer, which is different from the canonical 2-fold symmetric dimer observed for galectin-1 and -2. In a galectin-8N-lactose complex, the lactose-recognizing amino acids are highly conserved among the galectins. However, Arg 45 , Gln 47 , Arg 59 , and the long loop region between the S3 and S4 ␤-strands are unique to galectin-8N. These amino acids directly or indirectly interact with the sulfate or sialic acid moieties of 3-sialyl-and 3-sulfolactose complexed with galectin-8N. Furthermore, in the LNF-III-galectin-8N complex, van der Waals interactions occur between the ␣1-3-branched fucose and galactose and between galactose and Tyr 141 , and these interactions increase the affinity toward galectin-8N. Based on the findings of these x-ray crystallographic analyses, a mutagenesis study using surface plasmon resonance showed that Arg 45 , Gln 47 , and Arg 59 of galectin-8N are indispensable and coordinately contribute to the strong binding of galectins-8N to sialylated and sulfated oligosaccharides. Arg 59 is the most critical amino acid for binding in the S3-S4 loop region.Galectin-8 is a member of the galectin family, which share similar carbohydrate recognition domains (CRDs) 5 and affinity for ␤-galactosides (1, 2). Twelve galectins have been identified and characterized thus far. Each galectin has one (galectin-1, -2, -3, -5, -7, and -10) or two (galectin-4, -6, -8, -9, -11, and -12) conserved CRDs within a single molecule. According to the characteristics of their evolutionary conservation, wide tissue distribution, marked development-dependent expression, and abundance in specific tissues, galectins are presumed to function in various biological processes (3, 4). Galectin-8 is widely expressed and made up of two CRDs joined by a short linking peptide (1, 2). Recent studies have shown that galectin-8 modulates cell adhesion, spread, growth, and apoptosis (5-8). However, the precise recognition mechanisms and physiological roles of galectin-8 have not been fully elucidated.We determined the carbohydrate binding specificity of galectin-8 and found that it has much higher affinity for 3Ј-Osulfated or 3Ј-O-sialylated lactose and Lewis X-containing glycans than for oligosaccharides terminating in Gal␤133/ 4GlcNAc; this carbohydrate binding specificity was mainly attributed to the N-terminal CRD (N-domain) (9). The high affinity of galectin-8 for 3Ј-O-sialylated galactose is unique to this member of the galectin family (10); it is therefore of interest in determining its biological function via c...

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 Galbeta1-->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 approximately 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 Neu5Acalpha2-->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.

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 SO 3 ؊ 33Gal␤133GalNAc 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 ؋ 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.

Galectin-4 is a member of galectin family and has two carbohydrate recognition domains. Although galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of galectin-4 comparatively with that of galectin-3, using surface plasmon resonance, galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.