Preparative purification of tetraantennary oligosaccharides from human asialyl orosomucoid.
Preparative purification of tetraantennary oligosaccharides from human asialyl orosomucoid.
复制标题
从人脱唾液酸类粘蛋白中制备纯化四触角寡糖。
DOI：
10.1006/abio.1997.2070
复制
发表时间：
1997
影响因子：
2.9
通讯作者：
Rice,KG
中科院分区：
文献类型：
--
作者：
Stubbs,HJ;Shia,MA;Rice,KG
提出了一种从人乳清类粘蛋白中分离微摩尔量的四触角寡糖的方法。将500 mg糖蛋白中的N-连接寡糖酶促释放、去唾液酸化，并使用离子交换色谱法分离不含蛋白质的N-连接寡糖。通过与碳酸氢铵反应将汇集的寡糖转化为寡糖糖基胺，然后与BOC-酪氨酸偶联以制备酪氨酸酰胺寡糖。这些在半制备型RP-HPLC上解析以回收微摩尔量的六种纯化的酪氨酸酰胺寡糖。通过高场质子NMR和基质辅助飞行时间质谱的组合解析寡糖结构，包括双触角、三触角、单岩藻糖基化三触角、四触角、单岩藻糖基化四触角和含有单个聚乳糖胺延伸的四触角。Edman降解被用于逆转酪氨酸酰胺寡糖衍生化，导致还原寡糖的产生。这些用于表征高pH阴离子交换色谱上的脱唾液酸乳清类粘蛋白低聚糖的洗脱曲线。酪氨酸酰胺衍生化的这种应用首次允许在单一色谱图中以半制备规模从orosomucoid中完全解析复杂的寡糖混合物，并提供了来自该底物的聚乳糖胺四触角寡糖的第一个NMR表征。这项研究表明，广泛的实用性的酪氨酸酰胺衍生开发寡糖库用于探测糖基化的生物学功能。
An approach to isolate micromole quantities of tetraantennary oligosaccharides from human orosomucoid is presented. The N-linked oligosaccharides from 500 mg of the glycoprotein were released enzymatically, desialylated, and isolated free of protein using ion exchange chromatography. The pooled oligosaccharides were converted into oligosaccharide glycosylamines by reaction with ammonium bicarbonate then coupled to BOC-tyrosine to prepare tyrosinamide oligosaccharides. These were resolved on semipreparative RP–HPLC to recover micromole quantities of six purified tyrosinamide oligosaccharides. The oligosaccharide structures were elucidated by a combination of high-field proton NMR and matrix-assisted time of flight mass spectrometry and included biantennary, triantennary, monofucosylated triantennary, tetraantennary, monofucosylated tetraantennary, and a tetraantennary containing a single polylactosamine extension. Edman degradation was utilized to reverse the tyrosinamide oligosaccharide derivatization leading to the generation of reducing oligosaccharides. These were used to characterize the elution profile of asialyl orosomucoid oligosaccharides on high pH anion exchange chromatography. This application of tyrosinamide derivatization has allowed for the first time the complete resolution of the complex oligosaccharide mixture from orosomucoid on a semipreparative scale in a single chromatogram and provide the first NMR characterization of polylactosamine tetraantennary oligosaccharide from this substrate. This study demonstrates the broad utility of the tyrosinamide derivatization to develop oligosaccharide libraries useful for probing the biological functions of glycosylation.