Tomlinson S, Wang Y, Ueda E, Esser A F
Department of Comparative and Experimental Pathology, University of Florida Health Science Center, Gainesville 32610, USA.
J Immunol. 1995 Jul 1;155(1):436-44.
Equine C9, in contrast to human C9, has extremely low hemolytic activity against most mammalian erythrocytes, although the amino acid sequences of both proteins show 77% identity. In an attempt to define the region of human C9 responsible for conferring its lytic activity, or conversely, the region of equine C9 responsible for its restriction, recombinant human and equine C9 and four chimeric human/equine C9 proteins were constructed and expressed in COS-7 cells. Recombinant human and equine C9 displayed hemolytic profiles similar to those of the purified native proteins. Exchange of a fragment extending from residues 145 to 290 in horse C9 with the corresponding one from human C9 created a fully hemolytic protein. This region contains the putative hinge region but not the membrane-interacting domain. Nonlytic chimeric C9 proteins inhibited hemolysis and binding of human C9 to EAC1-8 cells, indicating that they bind to their receptor, but subsequent unfolding or insertion into the membrane is impaired. These results suggest that restriction factors, such as glycophorin, CD59, or homologous restriction factor, on erythrocytes may limit the activity of horse C9 by interacting with its hinge region. In support of this conclusion direct binding of CD59 to immobilized horse C9 was detected by ligand blotting, and it was observed that a polyclonal anti-CD59 Ab enhanced human and horse C9-mediated hemolysis of human EAC1-7, but the increase in hemolytic activity of horse C9 by inhibition of CD59 was less than what could be achieved by insertion of the human C9 hinge region into horse C9.
与人类C9不同，马C9对大多数哺乳动物红细胞的溶血活性极低，尽管这两种蛋白质的氨基酸序列有77%的同一性。为了确定赋予人类C9溶血活性的区域，或者相反，确定限制马C9活性的区域，构建了重组人C9和马C9以及四种嵌合人/马C9蛋白，并在COS-7细胞中表达。重组人C9和马C9的溶血谱与纯化的天然蛋白相似。将马C9中从第145位氨基酸到第290位氨基酸的片段与人C9相应片段进行交换，产生了一种完全溶血的蛋白。该区域包含假定的铰链区，但不包含膜相互作用结构域。非溶血嵌合C9蛋白抑制人C9与EAC1-8细胞的溶血和结合，表明它们与受体结合，但随后的展开或插入膜的过程受到损害。这些结果表明，红细胞上的限制因子，如血型糖蛋白、CD59或同源限制因子，可能通过与其铰链区相互作用来限制马C9的活性。为支持这一结论，通过配体印迹法检测到CD59与固定化马C9的直接结合，并且观察到多克隆抗CD59抗体增强了人C9和马C9介导的人EAC1-7溶血，但通过抑制CD59增加马C9的溶血活性低于将人C9铰链区插入马C9所能达到的水平。
