Structures of Sialylated O-Linked Oligosaccharides of Bovine Peripheral Nerve α-Dystroglycan
Structures of Sialylated O-Linked Oligosaccharides of Bovine Peripheral Nerve α-Dystroglycan
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DOI：
10.1074/jbc.272.4.2156
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发表时间：
1997-01
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影响因子：
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通讯作者：
A. Chiba;K. Matsumura;H. Yamada;T. Inazu;Teruo Shimizu;S. Kusunoki;I. Kanazawa;A. Kobata;T. Endo
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文献类型：
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作者：
A. Chiba;K. Matsumura;H. Yamada;T. Inazu;Teruo Shimizu;S. Kusunoki;I. Kanazawa;A. Kobata;T. Endo
α-肌营养不良聚糖是一种高度糖基化的蛋白质，其定位于许旺细胞膜以及肌膜上，并将跨膜蛋白β-肌营养不良聚糖连接至细胞外基质中的层粘连蛋白。我们之前已经证明，牛外周神经α-肌营养不良蛋白聚糖的唾液酸酶处理（而非N-聚糖酶处理）大大降低了其与层粘连蛋白的结合活性，这表明O-糖苷连接寡糖的唾液酸可能是这种结合所必需的。本文采用两种方法分析了牛外周神经α-肌营养不良蛋白聚糖的唾液酸化O-连接寡糖的结构。O-糖苷键连接的寡糖通过碱-硼氢化钠处理或通过温和肼解随后2-氨基苯甲酰胺衍生化而释放。在对应于单唾液酸化寡糖的位置处洗脱的通过阴离子交换柱层析获得的酸性级分通过极限唾液酸酶消化转化为中性寡糖。产脲节杆菌和纽卡斯尔病病毒的唾液酸酶产生相同的水解度。由此获得的中性寡糖级分在凝胶过滤时产生迁移率为3.8-3.9葡萄糖单位的主峰，并且其还原末端被鉴定为甘露糖衍生物。基于连续的外切糖苷酶消化、凝集素柱层析和反相高效液相色谱的结果，我们得出结论，α-肌营养不良蛋白聚糖的主要唾液酸化O-糖苷连接寡糖是一种新型O-甘露糖基型寡糖，其结构为Siaα2-3Galβ1-4GlcNAcβ1- 2 Man-Ser/Thr（其中Sia是唾液酸）。该寡糖构成至少66%的唾液酸化O-连接糖链。此外，层粘连蛋白结合抑制研究表明，该糖链的唾液酸N-乙酰乳糖胺部分参与α-肌营养不良聚糖与层粘连蛋白的相互作用。
α-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein β-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve α-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve α-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the α-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaα2-3Galβ1-4GlcNAcβ1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the α-dystroglycan with laminin.