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

Abstract

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 Binds to Sulfated Glycosphingolipids and Carcinoembryonic Antigen in Patches on the Cell Surface of Human Colon Adenocarcinoma Cells

“…2, both domains bound to glycosphingolipids tested; however, the affinity of GST-C-domain toward 3-O-sulfated glycosphingolipids SB1a and SM3 was higher than that of GST-N-domain. This characteristic of galectin-4 is different from galectin-8, of which only the Ndomain has high affinity for glycosphingolipids (10).…”

“…SO 3 Ϫ 33Gal␤134-Glc␤1-O-pNP (SO 3 Ϫ 33Lac-O-pNP) was the kind gift of Dr. Matta (Roswell Park Memorial Cancer Institute). SM2a, SB2, and SB1a were purified from rat kidney as described previously (10). SM3 was kindly provided by Dr. Ishizuka (Teikyo University School of Medicine).…”

“…ELISA for Binding of Galectin-4 to Various GlycosphingolipidsELISAs for binding of galectin-4 to various glycosphingolipids using recombinant galectin-4 and anti-galectin-4 were performed in the same manner as described previously (10).…”

Galectin-8 binds to LFA-1, blocks its interaction with ICAM-1 and is counteracted by anti-Gal-8 autoantibodies isolated from lupus patients

“…The CRDs of diff erent galectins and even the N-terminal or C-terminal CRDs of tandem repeat galectins display variations which serve to engage different subsets of functionally distinct glycoproteins (Hirabayashi et al, 2002;Ideo et al, 2003;Patnaik et al, 2006;Stillman et al, 2006;Carlsson et al, 2007;Stowell et al, 2008;Ideo et al, 2011). Thus, understanding the functions of a particular galectin requires defi ning the range of its interacting elements in diff erent cellular contexts.…”

Novel Role for Galectin-8 Protein as Mediator of Coagulation Factor V Endocytosis by Megakaryocytes

“…A different specificity for a particular glycan has also been demonstrated for the two CRDs within Gal8 itself (33). The N-terminal CRD has been reported to exhibit a high affinity for 3Ј-O-sulfated or 3Ј-O-sialylated glycoconjugates (34), whereas the C-terminal CRD domain preferentially binds poly-Nacetyl-lactosamine glycans (33). It therefore seems feasible that one CRD of Gal8 specifically binds FV, whereas the other CRD may have a preference for binding glycans expressed on the cell surface.…”