NMR investigations of the role of the sugar moiety in glycosylated recombinant human granulocyte-colony-stimulating factor
NMR investigations of the role of the sugar moiety in glycosylated recombinant human granulocyte-colony-stimulating factor
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DOI：
10.1111/j.1432-1033.1997.00386.x
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发表时间：
1997-07-01
期刊：
影响因子：
--
通讯作者：
Oschkinat, H
中科院分区：
文献类型：
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作者：
Gervais, V;Zerial, A;Oschkinat, H
人粒细胞集落刺激因子(G-CSF)是一种造血生长因子，在促进粒细胞增殖和成熟中发挥重要作用。近年来，随着对其体内生物学特性的深入了解，以及现有的重组人G-CSF制剂的应用，该生长因子已成为临床应用的重要药物。在133位的O-连接碳水化合物链的存在极大地提高了蛋白质的物理稳定性。为了阐明糖基对人粒细胞集落刺激因子(G-CSF)稳定作用的分子基础，我们用两次H-1-核磁共振波谱和两次H-1异核H-1-C-13实验研究了在CHO细胞中表达的全糖蛋白，并与非糖基化形式进行了比较。本文的核磁共振研究报告了结合糖链NeuNAc(Alpha2-3)Gal(beta 1-3)[NeuNAc(Alpha2-6)]GalNAc的H-1和C-13共振归属，其中NeuNAc代表N-乙酰神经氨酸，并证明了N-乙酰半乳糖胺-苏氨酸的α-异构体构型。它还提供了结果，表明碳水化合物部分降低了糖基化位点周围的局部迁移率。这可能是观察到的对糖蛋白稳定作用的原因。
Human granulocyte-colony-stimulating factor (G-CSF) is a hematopoietic growth factor that plays a major role in the stimulation of the proliferation and maturation of granulocyte neutrophil cells. With the recent increased understanding of its biological properties in vivo together with available preparations of recombinant human G-CSF, this growth factor has become an essential agent for clinical applications. The presence of an O-linked carbohydrate chain at position 133 greatly improves the physical stability of the protein. To clarify the molecular basis for the stabilisation effect of saccharide moieties on human G-CSF, the whole glycoprotein expressed in CHO cells has been investigated by means of two H-1-NMR-spectroscopy and two H-1-detected-heteronuclear H-1-C-13 experiments at natural abundance, and compared with the non-glycosylated form. The present NMR study reports assignments of H-1 and C-13 resonances of the bound saccharidic chain NeuNAc(alpha 2-3)Gal(beta 1-3)[NeuNAc(alpha 2-6)]GalNAc, where NeuNAc repre sents N-acetylneuraminic acid, and demonstrates the alpha-anomeric configuration of the N-acetylgalaclosamine-threonine linkage. It also provides results suggesting that the carbohydrate moiety reduces the local mobility around the glycosylation site. which could be responsible for the stabilising effect observed on the glycoprotein.