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 Gal133/4GlcNAc, 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 Gal133/ 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...
