Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells.
Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells.
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中国仓鼠卵巢细胞表达的重组人促红细胞生成素完整主要唾液酸化寡糖链的结构测定。
DOI：
10.1093/glycob/4.2.227
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发表时间：
1994
期刊：
影响因子：
4.3
通讯作者：
vanHalbeek,H
中科院分区：
文献类型：
--
作者：
Watson,E;Bhide,A;vanHalbeek,H
重组人促红细胞生成素(RHuEPO)在临床上被广泛用于刺激贫血患者的红细胞生长。这种药物的疗效很大程度上取决于其碳水化合物的唾液酸化程度。在相互矛盾的文献报道的推动下，我们重新研究了在中国仓鼠卵巢(CHO)细胞中表达的完整的rHuEPO唾液酸化糖链的结构。用N-聚糖酶从rHuEPO中释放天冬酰胺连接的寡糖，并用阴离子交换层析进行分级。O-连接的低聚糖在碱性硼氢化物条件下释放。主要唾液酸化N-型和O型低聚糖的一级结构经500-MHz1H-核磁共振波谱确证，并得到组成分析、甲基化分析、低pH和高pH阴离子交换层析以及快原子轰击-质谱图的支持。CHO细胞来源的重组人促红细胞生成素含有丰富的N-连接低聚糖，包括二天线、2，4-分支三天线、2，6-分支三天线和四天线链(按7：6：5：82的比例)，后者在明确的分支中含有0到3个重复的N-乙酰乳糖胺单元。主要的(>；95%)二、三和四触角结构是完全唾液酸化的，即它们分别有两个、三个和四个唾液酸残基，专门连接α(2←3)和半乳糖残基。大多数(>；95%)N-连接结构在近端的GlcNAc残基上含有α(1-←-6)连接的岩藻糖。O型单糖和双唾液酸寡糖的结构分别为线形三糖和支链四糖。
Recombinant human erythropoietin (rHuEPO) is used abundantly in the clinic to stimulate red blood cell growth in anaemic patients. The efficacy of the drug depends strongly on the extent of sialylation of its carbohydrate moiety. Prompted by conflicting literature reports on the issue, we reinvestigated the structures of the intact sialylated carbohydrate chains of rHuEPO expressed in Chinese hamster ovary (CHO) cells. The asparagine-linked oligosaccharides were released from rHuEPO withN-glycanase and fractionated by anion-exchange chromatography. The O-linked oligosaccharides were released under alkaline borohydride conditions. The primary structures of the major sialylated N- and O-type oligosaccharides were identified by 500-MHz1H-NMR spectroscopy, supported by data from composition analysis, methylation analysis, low- and high-pH anion-exchange chromatography, and fast atom bombardment-mass spectrometry. The mod abundant N-linked oligosaccharides in CHO cell-derived rHuEPO were found to be di-antennary, 2,4-branched tri-antennary, 2,6-branched tri-antennary and tetra-antennary chains (in the ratio of 7:6:5:82), with the latter containing between zero and three repeatingN-acetyllactosamine units, in well-defined branches. The major (>95%) di-, tri- and tetra-antennary structures are fully sialylated, i.e. they have two, three and four sialic acid residues, respectively, Linked exclusively α(2←3) to galactose residues. The majority (>95%) of N-Linked structures contain α(1←6)-linked fucose at the proximal GlcNAc residue. The O-type mono- and disialyl oligosaccharides were characterized as a linear tri- and a branched tetra-saccharide, respectively.