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.

A prostate-specific antigen (PSA) is widely used as a diagnostic marker for prostate cancer (PC) because of its high specificity. However, elevated serum PSA does not occur only in PC but also in benign prostatic hyperplasia (BPH). Since the structural changes of N-glycans during carcinogenesis are common phenomena, we investigated whether PC-specific N-glycans are linked to PSA. We first analyzed the carbohydrate structures of PSA derived from seminal fluid, serum of BPH and PC patients, and PC cell line, namely, LNCaP using eight lectin-immobilized columns and then with enzyme-linked immunosorbent assay (ELISA). The fraction of serum PSA from PC patients bound to both Fucalpha1-2Gal and betaGalNAc binding Trichosanthes japonica agglutinin-II (TJA-II) column, while that from BPH patients did not exhibit this binding ability, thereby implying that there is elevated expression of alpha1,2-fucosylation and beta-N-acetylgalactosaminylation of PSA during carcinogenesis. We then performed a real-time polymerase chain reaction (PCR) and confirmed that these structural changes were responsible for the elevated expression of fucosyltransferase I (FUT1) and beta-N-acetylgalactosaminyltransferase 4(B4GALNT4). Second, we measured TJA-II-bound PSA contents and the binding ratios of TJA-II column chromatography in serum PSA samples from 40 patients of both PC and BPH. The results indicated that both TJA-II-bound PSA content and TJA-II binding ratios (%) could be used to discriminate between PC and BPH with more than 95% probability, and TJA-II-bound PSA can be regarded as a potential marker of PC.

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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.