Oligosaccharide Structure and Biological Activity of Erythropoietin

促红细胞生成素的寡糖结构和生物活性

• 批准号: 63480504
• 负责人: TAKAHASHI Noriko
• 金额: $ 0.58万
• 依托单位: Nagoya City University College of Nursing
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480504/
• 关键词: Erythropoietin; recombinant EPO; BHK cell; Asn-linked oligosaccnarides; エリス ポエチン; アスパラギン結合オリゴ糖; シアル酸

The structures of the N-linked oligosaccharides of the urinary erythropoietin (u-EPO) purified from urine of aplastic anemic patients were analyzed and compared with those for recombinant erythropoietin (r-EPO) prepared with Baby Hamster Kidney (BHK) cells. Asparagine-linked neutral oligosaccharides were related from each EPO protein by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by High-Performance Liquid Chromatography (HPLC) on an ODS silica column. More than 8 and 13 kinds of oligosaccharide fractions for u-EPO and r-EPO (BHK), respectively, were completely separated by the one-step HPLC procedure. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amide-silica column. Furthermore, ^IH NMR spectroscopy and methylation analyses were carried out in the case of r-EPO (BHK). It has been found that (1) significantly different distributions of oligosaccharide components exist in u-EPO preparations isolated from different individuals, whereas the distribution of oligosaccharide components is quite similar for different r-EPO (BHK) preparations, (2) in r-EPO (BHK) a tetraantennary oligosaccharide with a fucose residue is predominant and various higher molecular weight tetraantennary oligosaccharides with one to three Gal( 1-4) GlcNAc repeating units exist, (3) all of the eight kinds of oligosaccharides of u-EPO also exist in r-EPO (BHK), although the distribution of the oligosaccharides is different for the two proteins, and (4) oligosaccharides with unusualstructures containing the Gal (1-3) GlcNAc group exist in both u- and r-EPO (BHK).

本文分析了从再生障碍性贫血患者尿中纯化的尿促红细胞生成素（u-EPO）的N-连接寡糖的结构，并与用婴儿肾（BHK）细胞制备的重组促红细胞生成素（r-EPO）的结构进行了比较。天冬酰胺连接的中性低聚糖相关的每个EPO蛋白的N-寡糖糖肽酶（杏仁）消化。将由此获得的寡糖链的还原末端用荧光试剂2-氨基吡啶胺化，并将寡糖的吡啶基氨基衍生物的混合物通过高效液相色谱法（HPLC）在ODS硅胶柱上分离。一步高效液相色谱法可将u-EPO和r-EPO（BHK）分别分离出8种和13种以上的寡糖组分。通过连续外切糖苷酶消化和另一种具有酰胺-硅胶柱的HPLC的组合来分析由此分离的每种寡糖的结构。此外，在r-EPO（BHK）的情况下进行1H NMR光谱和甲基化分析。已经发现（1）在分离自不同个体的u-EPO制剂中存在显著不同的寡糖组分分布，而对于不同的r-EPO（BHK）制剂，寡糖组分的分布非常相似，（2）在r-EPO（BHK）中，具有岩藻糖残基的四触角低聚糖占主导地位，各种具有一到三个Gal的较高分子量四触角低聚糖（（3）r-EPO（BHK）中也存在u-EPO的8种寡糖，但分布不同;（4）u-EPO和r-EPO（BHK）中均存在含有Gal（1-3）GlcNAc基团的特殊结构的寡糖。

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