Cellular distribution of a GPI-anchored complement regulatory protein CD59: homodimerization on the surface of HeLa and CD59-transfected CHO cells.
Cellular distribution of a GPI-anchored complement regulatory protein CD59: homodimerization on the surface of HeLa and CD59-transfected CHO cells.
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GPI 锚定补体调节蛋白 CD59 的细胞分布：HeLa 和 CD59 转染的 CHO 细胞表面上的同二聚化。
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
A. Shimizu
中科院分区：
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作者：
M. Hatanaka;T. Seya;S. Miyagawa;M. Matsumoto;T. Hara;K. Tanaka;A. Shimizu
人糖基磷脂酰肌醇锚定蛋白CD 59溶解在洗涤剂不溶性复合物（DIC），并在核后丸通过两步溶解程序，使用Triton X-100和辛基葡糖苷。CD 59分子在两种级分中回收，在所有测试的细胞类型中，后一级分中的量更大。表面CD 59分子的特异性标记显示，在DIC中检测到的CD 59来自细胞内的隔间，而在核后颗粒部分来自细胞表面。用化学交联剂交联表面蛋白，然后用抗CD 59抗体进行Western印迹，结果显示在HeLa和人CD 59 cDNA转染的CHO细胞上的交联产物分子量为28-36 kDa; CD 59相关分子的大小估计为13-18 kDa。核后颗粒中提取出交联产物，CD 59主要以交联形式存在于细胞表面。交联产物的二维电泳显示除了CD 59之外没有分子的痕迹。交联产物显示与CD 59相同的N-末端序列和与CD 59惊人相似的氨基酸组成。因此，最有可能的是，交联产物是CD 59二聚体。CD 59定位于外膜上均为二聚体形式的发现表明二聚化对于CD 59功能的重要性。
Human glycosyl phosphatidylinositol-anchored protein CD59 was solubilized in detergent-insoluble complexes (DICs) and in post-nuclear pellets by a two-step solubilization procedure using Triton X-100 and octylglucoside. CD59 molecules are recovered in both fractions, the amount being greater in the latter fraction in all cell types tested. Specific labeling of surface CD59 molecules revealed that the CD59 detected in DICs originated from intracellular compartments, whereas that in post-nuclear pellets was in part derived from the cell surface. Cross-linking of surface proteins with chemical cross-linker followed by Western blotting with anti-CD59 antibody revealed cross-linked products with molecular masses of 28-36 kDa on HeLa and human CD59 cDNA-transfected CHO cells; the CD59-associating molecules were estimated to be 13-18 kDa in size. The cross-linked products were extracted in the post nuclear pellets, and CD59 existed mainly as a cross-linked form on the cell surface. Two-dimensional electrophoresis of the cross-linked products revealed no trace of molecules other than CD59. The cross-linked products showed the same N-terminal sequences as CD59 and a strikingly similar amino acid composition to that of CD59. Thus, most likely, the cross-linked products are CD59 dimers. The finding that CD59 localized on outer membranes is all in the form of dimers suggests the importance of dimerization for CD59 functioning.