GLYCOSIDASE DIGESTION, ELECTROPHORESIS AND CHROMATOGRAPHIC ANALYSIS OF RECOMBINANT HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR GLYCOFORMS PRODUCED IN CHINESE-HAMSTER OVARY CELLS
GLYCOSIDASE DIGESTION, ELECTROPHORESIS AND CHROMATOGRAPHIC ANALYSIS OF RECOMBINANT HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR GLYCOFORMS PRODUCED IN CHINESE-HAMSTER OVARY CELLS
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DOI：
10.1016/0021-9673(93)83098-d
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发表时间：
1993-05-07
期刊：
影响因子：
--
通讯作者：
LU, HS
中科院分区：
文献类型：
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作者：
CLOGSTON, CL;HU, S;LU, HS
重组人粒细胞集落刺激因子（G-CSF）在中国仓鼠卵巢细胞中被糖基化。碳水化合物组成分析表明，G-CSF分子中含有唾液酸、半乳糖和半乳糖胺。通过对金黄色葡萄球菌V-8蛋白酶和溴化氰裂解得到的纯化糖肽的分离和鉴定，证实了o链糖基化位点为133位的苏氨酸残基。神经氨酸酶和o -甘聚糖酶酶切后，十二烷基硫酸钠聚丙烯酰胺和等电聚焦凝胶电泳区分出两种可能的碳水化合物结构：结构A， neunac - gal - β (1,3)-GalNAc-O-Thr；结构B为NeuNAc- gal - β (1,3)-[NeuNAc]- galnac - o - thr。不同的糖形式，未消化或经过糖苷酶消化，也可以通过离子交换或反相高效液相色谱分离。本报告中描述的方法提供了一种简单而有价值的方法来表征含有简单碳水化合物部分的糖蛋白结构。
Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.