A NOVEL KERATAN SULFATE PROTEOGLYCAN FROM A HUMAN EMBRYONAL CARCINOMA CELL-LINE
A NOVEL KERATAN SULFATE PROTEOGLYCAN FROM A HUMAN EMBRYONAL CARCINOMA CELL-LINE
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DOI：
10.1042/bj2860959
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发表时间：
1992-09-15
影响因子：
4.1
通讯作者：
FINCH, JT
中科院分区：
文献类型：
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作者：
COOPER, S;PERA, MF;FINCH, JT
我们在这里描述的纯化和部分特性的200 kDa硫酸角质素蛋白聚糖中发现的人胚胎癌细胞的细胞周围基质。以前我们已经表明，这种分子是由单克隆抗体（GCTM-2）识别。使用离子交换色谱和凝胶过滤分离抗原，通过e.l.i.s.a.使用GCTM-2。用[S-35]硫酸钠对GCT 27 C-4胚胎癌细胞进行代谢标记，导致[S-35]硫酸盐掺入纯化的分子中。在整个纯化过程中，S-35放射性峰与免疫反应性峰一致。并且该标记通过用角质素酶和软骨素酶消化而释放，证实了抗原的蛋白聚糖性质。完整分子以200 kDa的单一宽带运行，其已通过凝胶电泳后的银染色和免疫印迹鉴定。纯化抗原的氨基酸分析表明丝氨酸、甘氨酸和天冬氨酸/天冬酰胺残基含量高。通过旋转阴影电子显微镜的可视化表明，纯化的材料形成大的聚集体，即使在变性条件下。用三氟甲磺酸对该制剂进行去糖基化，得到55 kDa的主带和48 kDa的次带。这里描述的分子的生化性质，沿着使用GCTM-2的组织分布研究。表明该抗原与先前描述的硫酸角质素蛋白聚糖无关。
We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [S-35]sulphate resulted in the incorporation of [S-35]sulphate into the purified molecule. Throughout the purification procedure, the peaks of S-35 radioactivity were coincident with the peaks of immunoreactivity. and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2. indicates that the antigen is not related to previously described keratan sulphate proteoglycans.