Laboratory of Glycobiology and Marine Biochemistry, Department of Genome System Sciences, Graduate School of NanoBiosciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan.
Comp Biochem Physiol B Biochem Mol Biol. 2011 Apr;158(4):266-73. doi: 10.1016/j.cbpb.2010.12.004. Epub 2010 Dec 19.
A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galβ1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galβ1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes β1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.
本研究中，一种从棘皮动物羽腕海星 Oxycomanthus japonicus 中纯化出的凝集素 OXYL 被指定为可以强烈识别血清糖蛋白中复杂型寡糖的物质。OXYL 通过阴离子交换和亲和层析的组合，分别使用 Q 琼脂糖和胎球蛋白琼脂糖凝胶进行纯化。凝集素在还原条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中被确定为 14kDa 的多肽。然而，在非还原条件下，14kDa 和 28kDa 带以相同的比例出现。凝胶渗透色谱显示出 54kDa 的峰，表明凝集素由四个 14kDa 的亚基组成。二价阳离子不被指示，并且在 pH4-12 和低于 60°C 的温度下显示出稳定的血凝活性。OXYL 对胎球蛋白的表面等离子体共振分析显示 k(ass) 和 k(diss) 值分别为 1.4×10(-6)M(-1)s(-1)和 3.1×10(-3)s(-1)，表明它作为凝集素有很强的与糖蛋白的结合亲和力。使用 25 种 prydylamine 偶联聚糖的前沿亲和层析表明，OXYL 特异性识别含有型-2 N-乙酰乳糖胺（Galβ1-4GlcNAc）的多触角复杂型寡糖，如果非还原末端连接的是α2-3 连接的唾液酸。然而，OXYL 从未识别出型-1 N-乙酰乳糖胺（Galβ1-3GlcNAc）链和α2-6 连接的唾液酸。这项分析研究表明，OXYL 基本上识别 Gal 在 C-1 位置的β1-4 连接以及 C-6 位置的游离 OH 基团，此外还识别 GlcNAc 在 C-2 位置的 N-乙酰基团和 C-3 位置的游离 OH 基团的保守性。这是首次报道从属于 Pelmatozoa 亚门的棘皮动物中纯化的凝集素的糖组学。
