Asn to Lys mutations at three sites which are N-glycosylated in the mammalian protein decrease the aggregation of Escherichia coli-derived erythropoietin
Asn to Lys mutations at three sites which are N-glycosylated in the mammalian protein decrease the aggregation of Escherichia coli-derived erythropoietin
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DOI：
10.1093/protein/14.2.135
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发表时间：
2001-02-01
期刊：
影响因子：
--
通讯作者：
Cheetham, J
中科院分区：
文献类型：
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作者：
Narhi, LO;Arakawa, T;Cheetham, J
来源于大肠杆菌的促红细胞生成素（EPO）对高温不稳定，并且倾向于随时间聚集，使得其不适合高分辨率结构分析。哺乳动物EPO含有约40%的碳水化合物，这使得这种蛋白质比非糖基化的大肠杆菌衍生的EPO更稳定且更不易聚集，但由于其大小和柔性而使其不适合高分辨率分析。为了减少大肠杆菌衍生的EPO的聚集，将24、38和83位的三个天冬酰胺残基突变为赖氨酸残基。在天然蛋白质中，这些残基是N-连接糖基化的位点，这表明它们应该位于蛋白质的表面上，并且不应该参与疏水蛋白质核心中的相互作用。因此，预计碱性氨基酸取代这些中性天冬酰胺残基不会影响蛋白质结构，但应增加蛋白质的等电点及其净正电荷，降低其在中性pH或低于中性pH时由于静电相互作用而聚集的趋势。通过细胞表面受体竞争试验和体外受体二聚化实验确定，当这些突变引入EPO序列时，未观察到受体结合的明显改变。然而，相对于野生型分子，该突变蛋白显示出对热处理和储存的稳定性的显著增加。这导致在2D NOESY实验中突变体EPO中可观察到的交叉峰的数量更大。然而，突变体在尿素作为变性剂时的稳定性与野生型相似。这表明引入的突变导致在加热或在环境温度下延长孵育时聚集减少，而不改变突变蛋白的构象稳定性或受体结合亲和力。这种将带电残基置于体内发生N-糖基化的位点的方法也可以应用于其他系统。
Erythropoietin (EPO) derived from Escherichia coli is unstable to elevated temperature and tends to aggregate with time, making it unsuitable for high-resolution structure analysis. The mammalian EPO contains about 40% carbohydrate, which makes this protein more stable and less prone to aggregate than non-glycosylated E.coli-derived EPO, but makes it unsuitable for high-resolution analysis owing to its size and flexibility. In an attempt to decrease the aggregation of E.coli-derived EPO, the three asparagine residues at positions 24, 38 and 83 were mutated to lysine residues. In the native protein, these residues are the sites of N-linked glycosylation, which suggests that they should be located on the surface of the protein and should not be involved in interactions in the hydrophobic protein core. Therefore, the substitution of basic amino acids for these neutral asparagine residues is not expected to affect the protein structure, but should increase the isoelectric point of the protein and its net positive charge, decreasing its tendency to aggregate at or below neutral pH due to electrostatic interactions. No apparent alterations in receptor binding, as determined by both cell-surface receptor competition assay and in vitro receptor dimerization experiments, were observed when these mutations were introduced into the EPO sequence. However, this mutant protein displayed a significant increase in stability to heat treatment and to storage, relative to the wild-type molecule. This resulted in a greater number of observable cross peaks in the mutant EPO in 2D NOESY experiments. However, the mutant was similar to the wild-type in stability when urea was used as a denaturant. This indicates that the introduced mutations resulted in a decrease in aggregation with heating or with prolonged incubation at ambient temperature, without changing the conformational stability or the receptor binding affinity of the mutant protein. This approach of placing charged residues at sites where N-glycosylation occurs in vivo could be applied to other systems as well.