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Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms

机译：氨和氨基葡萄糖对促红细胞生成素糖型异质性的影响

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Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent from mammalian cell cultures for the treatment of anemia associated with severe kidney damage. The EPO structure has a high glycan content which is essential for bioactivity but shows considerable molecular heterogeneity. The cell culture conditions that affect the heterogeneity of the glycoforms of EPO are not well understood. However, the accumulation of ammonia in culture is one factor that has been associated with an enhanced heterogeneity of glycoforms. In this report we investigate the metabolic perturbations associated with ammonia and glucosamine that may give rise to an altered pattern of EPO glycosylation. Recombinant human erythropoietin was synthesized in serum-free cultures of transfected Chinese hamster ovary (CHO) cells. The molecular heterogeneity of erythropoietin was increased by supplementation of cultures with either ammonia or glucosamine. The enhanced molecular heterogeneity was shown to be due to variable glycosylation that resulted in EPO with an enhanced molecular weight and isoelectric point range. Enzymatic removal of the glycan moiety of EPO in all cases resulted in a single molecular form with a molecular weight of 18 000, which corresponded to non-glycosylated EPO. The variable glycosylation was consistent with reduced sialylation and antennarity of the carbohydrate structures present on the three N-linked sites of EPO. In the presence of ammonia (>30 mM) the proportion of tetrasialylated and tetraantennary glycan structures were reduced by 73% and 57%, respectively, as determined by HPLC analysis. Such changes were also observed, although to a lesser extent (41% and 37%), by an increase in the glucosamine concentration (> 10 mM) in the medium. The enhanced heterogeneity of the glycan structures coincided with a significant increase in the intracellular UDP-N-acetylhexosamine (UDP-GNAc) pool. The measured UDP-GNAc level was up to 2 orders of magnitude higher in the presence of either glucosamine or ammonia. However, the changes in the glycosylation profiles induced by either glucosamine or ammonia were significantly different even at the same intracellular 'UDP-GNAc concentration. This suggests that them enhanced EPO heterogeneity could not be mediated solely by the increased UDP-GNAc level. Glucosamine (but not ammonia) was shown to cause significant inhibition of glucose transport, into the cells, which could induce a different pattern of primary metabolism.
机译：重组人促红细胞生成素（EPO）是一种糖蛋白，是一种从哺乳动物细胞培养物中产生的治疗剂，用于治疗与严重肾脏损害相关的贫血。 EPO结构具有高聚糖含量，这对生物活性至关重要，但显示出相当大的分子异质性。尚未完全了解影响EPO糖型异质性的细胞培养条件。但是，培养物中氨的积累是与糖型异质性增强相关的因素之一。在本报告中，我们研究了与氨和葡萄糖胺有关的代谢扰动，这些扰动可能引起EPO糖基化模式的改变。在转染的中国仓鼠卵巢（CHO）细胞的无血清培养物中合成重组人促红细胞生成素。促红细胞生成素的分子异质性通过向培养物中添加氨或葡萄糖胺来增加。分子异质性增强表明是由于可变的糖基化作用导致EPO的分子量和等电点范围增加。在所有情况下，酶促除去EPO的聚糖部分均导致分子量为18 000的单一分子形式，这相当于未糖基化的EPO。可变的糖基化与存在于EPO的三个N-连接位点上的碳水化合物结构的唾液酸化和触角降低有关。如通过HPLC分析所确定的，在氨（> 30 mM）存在下，四唾液酸化和四触角聚糖结构的比例分别降低了73％和57％。还观察到了这种变化，尽管程度较小（41％和37％），这是由于培养基中葡萄糖胺浓度（> 10 mM）的增加所致。聚糖结构异质性的增强与细胞内UDP-N-乙酰己糖胺（UDP-GNAc）库的显着增加相吻合。在存在葡萄糖胺或氨气的情况下，测得的UDP-GNAc水平高出2个数量级。然而，即使在相同的细胞内UDP-GNAc浓度下，由葡糖胺或氨诱导的糖基化谱的变化也显着不同。这表明它们增强的EPO异质性不能仅通过增加的UDP-GNAc水平来介导。葡萄糖胺（而非氨水）显示出对进入细胞的葡萄糖转运有显着抑制作用，这可以诱导不同的初级代谢模式。

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