Ideo Hiroko, Seko Akira, Ishizuka Ineo, Yamashita Katsuko
Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Glycobiology. 2003 Oct;13(10):713-23. doi: 10.1093/glycob/cwg094. Epub 2003 Jul 8.
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.
半乳糖凝集素-8是半乳糖凝集素家族的成员，有两个串联重复的碳水化合物识别结构域（CRD）。我们使用酶联免疫吸附测定（ELISA）和表面等离子体共振分析，确定了半乳糖凝集素-8及其两个CRD对寡糖和糖鞘脂的结合特异性。与以Galβ1→3/4GlcNAc结尾的寡糖相比，半乳糖凝集素-8对3'-O-硫酸化或3'-O-唾液酸化乳糖以及含Lewis x的聚糖具有更高的亲和力。这种特异性主要归因于N端CRD（N结构域），而C端CRD（C结构域）对A血型聚糖只有较弱的亲和力。N结构域不仅与寡糖结合，还与包括硫苷脂（SM4 s）、SM3、唾液酸化Lc4Cer、SB1a、GD1a、GM3和唾液酸化nLc4Cer在内的糖鞘脂结合，表明N结构域识别3-O-硫酸化或3-O-唾液酸化的Gal残基。乳糖或N-乙酰乳糖胺中Gal残基的C-3位被硫酸取代，比被唾液酸取代更能增强半乳糖凝集素-8的识别程度。这是首次证明半乳糖凝集素-8以高亲和力（KD约为10-8-10-9 M）结合特定的硫酸化或唾液酸化糖鞘脂。当N结构域的Gln47残基转变为Ala47时，对硫酸化或唾液酸化聚糖的特异性亲和力选择性丧失，表明该Gln47在与Neu5Acα2→3Gal或SO3→3Gal残基结合中起重要作用。使用主要表达GM3的CHO细胞证实了半乳糖凝集素-8与膜相关GM3的结合能力。CHO细胞与突变蛋白的结合明显低于与N结构域的结合。
