Kurokawa Tomofumi, Wuhrer Manfred, Lochnit Günter, Geyer Hildegard, Markl Jürgen, Geyer Rudolf
Pharmaceutical Discovery Center, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd, Osaka, Japan.
Eur J Biochem. 2002 Nov;269(22):5459-73. doi: 10.1046/j.1432-1033.2002.03244.x.
Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(beta1-6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.
钥孔帽贝（Megathura crenulata）血蓝蛋白（KLH）是一种细胞外呼吸蛋白，被广泛用作半抗原载体和免疫刺激剂。尽管人们普遍认为这种糖蛋白的糖成分可能与KLH的抗原性和生物医学特性有关，但其碳水化合物结构的知识仍然有限。因此，我们研究了KLH的N-连接寡糖。聚糖链通过酶法从胰蛋白酶糖肽中释放出来，进行吡啶基胺化并通过二维高效液相色谱分离。仅获得了中性寡糖，并通过碳水化合物成分分析、甲基化分析、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）、电喷雾离子阱质谱（ESI-ion trap-MS）和外切糖苷酶顺序消化对其进行了表征。结果表明，KLH携带高甘露糖型聚糖及其衍生的截短糖链。作为一个特征，许多研究的N-聚糖含有一个Gal(β1-6)Man单元，该单元迄今为止尚未在糖蛋白-N-聚糖中发现。因此，我们的研究表明，这种海洋软体动物糖蛋白的特征在于独特的寡糖模式，部分包含新的结构元件。
