Clogston C L, Hu S, Boone T C, Lu H S
Amgen Inc., Amgen Center, Thousand Oaks, CA 91320.
J Chromatogr. 1993 May 7;637(1):55-62. doi: 10.1016/0021-9673(93)83098-d.
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.
在中国仓鼠卵巢细胞中产生的重组人粒细胞集落刺激因子（G-CSF）是糖基化的。碳水化合物组成分析表明，G-CSF分子含有唾液酸、半乳糖和氨基半乳糖。通过对金黄色葡萄球菌V-8蛋白酶和溴化氰切割得到的纯化糖肽进行分离和表征，确认O-连接糖基化位点是第133位的苏氨酸残基。神经氨酸酶和O-聚糖酶消化后，进行十二烷基硫酸钠聚丙烯酰胺和等电聚焦凝胶电泳，区分了连接在苏氨酸-133上的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，NeuNAc-Gal-β(1,3)-[NeuNAc]-GalNAc-O-Thr。未消化或糖苷酶消化后的不同糖型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为表征含有简单碳水化合物部分的糖蛋白结构提供了一种简单而有价值的程序。
