Oligosaccharide Structure and Biological Activity of Erythropoietin

分析了再生障碍性贫血患者尿液中纯化的尿促红细胞生成素（u-EPO）的n -链寡糖结构，并与用小鼠肾（BHK）细胞制备的重组红细胞生成素（r-EPO）的n -链寡糖结构进行了比较。通过n -寡糖糖肽酶（杏仁）酶切，将天冬酰胺连接的中性寡糖与EPO蛋白相关联。用荧光试剂2-氨基吡啶对得到的低聚糖链的还原端进行胺化，并在ODS硅胶柱上用高效液相色谱（HPLC）对低聚糖的吡啶胺衍生物混合物进行分离。通过一步高效液相色谱法，u-EPO和r-EPO （BHK）分别有8种和13种寡糖组分被完全分离。采用序列外糖苷酶切法和另一种酰胺-硅柱高效液相色谱法对分离得到的低聚糖进行结构分析。此外，对r-EPO （BHK）进行了^IH NMR波谱和甲基化分析。研究发现：(1)不同个体分离的u-EPO制剂中低聚糖组分的分布存在显著差异，而不同的r-EPO （BHK）制剂中低聚糖组分的分布非常相似；(2)在r-EPO （BHK）中，具有聚焦残基的四天线低聚糖占主导地位，存在各种具有1- 3个Gal(1-4) GlcNAc重复单元的高分子量四天线低聚糖。(3) u- epo的8种低聚糖均存在于r-EPO （BHK）中，尽管这两种蛋白的低聚糖分布不同；(4)含有Gal (1-3) GlcNAc基团的结构异常的低聚糖在u-和r-EPO （BHK）中均存在。

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).