Ozawa M, Sato M, Muramatsu H, Hamada H, Muramatsu T
Exp Cell Res. 1985 May;158(1):127-43. doi: 10.1016/0014-4827(85)90438-0.
An antiserum was raised against glycoproteins isolated from teratocarcinoma OTT6050 by affinity chromatography on Ricinus communis agglutinin-1 (RCA-1). The antiserum inhibited attachment and spreading of the teratocarcinoma cells on plastic tissue culture plates. Embryonal carcinoma cells (F9 and N4-1) and PYS-2 parietal endodermal cells were also affected. The antiserum was also effective in preventing adhesion of trophoblasts to substratum. Furthermore, the antiserum caused rounding and detachment of the cells previously spread on the substrates, and the process could be reversed by removing the antiserum. Fab fragments isolated from the antiserum also inhibited cell-substratum adhesion, but not cell-cell adhesion, indicating that the factor involved is specific for cell-substratum adhesion. The antiserum inhibited adhesion of F9 and PYS-2 cells not only to plastic dishes but also to those coated with plasma fibronectin, laminin, and plant lectins such as concanavalin A (conA). Therefore, it is likely that the antiserum inhibited cell-substratum adhesion by blocking the function of an intrinsic component of cell-substratum adhesion rather than interfering with specific receptors for fibronectin or laminin. The glycoprotein with the antibody-blocking activity was partially purified from particulate fraction of teratocarcinoma OTT6050 by extraction with Triton X-100 and affinity chromatography on RCA-1-agarose and conA-agarose Upon Western blot analysis of the glycoprotein preparation, the antibody reacted only with a glycoprotein of the apparent molecular weight (MW), 125 000 (GP-125). An antibody preparation was isolated by affinity chromatography on Sepharose 4B coupled with the glycoprotein fraction and was shown to have all of the activities described above. Molecules resembling GP-125 could be also isolated from cultured F9 and PYS-2 cells by solubilization with Triton X-100 and indirect immunoprecipitation.
通过蓖麻凝集素-1（RCA-1）亲和层析从畸胎瘤OTT6050中分离出糖蛋白，并以此制备了抗血清。该抗血清可抑制畸胎瘤细胞在塑料组织培养板上的附着与铺展。胚胎癌细胞（F9和N4-1）以及PYS-2壁层内胚层细胞也受到影响。该抗血清在阻止滋养层细胞与基质黏附方面同样有效。此外，抗血清会使先前铺展在基质上的细胞变圆并脱离，去除抗血清后这一过程可逆转。从抗血清中分离出的Fab片段也能抑制细胞与基质的黏附，但不影响细胞与细胞之间的黏附，这表明所涉及的因子对细胞与基质的黏附具有特异性。该抗血清不仅抑制F9和PYS-2细胞与塑料培养皿的黏附，还抑制它们与包被有血浆纤连蛋白、层粘连蛋白以及植物凝集素（如伴刀豆球蛋白A（conA））的培养皿的黏附。因此，抗血清可能是通过阻断细胞与基质黏附的内在成分的功能来抑制细胞与基质的黏附，而非干扰纤连蛋白或层粘连蛋白的特异性受体。通过用Triton X-100提取以及在RCA-1琼脂糖和conA琼脂糖上进行亲和层析，从畸胎瘤OTT6050的颗粒部分中部分纯化出具有抗体阻断活性的糖蛋白。对该糖蛋白制剂进行蛋白质印迹分析时，抗体仅与表观分子量（MW）为125000的糖蛋白（GP-125）发生反应。通过在与糖蛋白部分偶联的琼脂糖4B上进行亲和层析，分离出一种抗体制剂，它具有上述所有活性。通过用Triton X-100溶解并进行间接免疫沉淀，也可从培养的F9和PYS-2细胞中分离出类似于GP-125的分子。
