跨膜型TNF-α生物学活性的膜依赖性

Dependence of Transmembrane TNF-α Bioactivity on Biomembrane

跨膜型TNF-α(TM-TNF-α)是激活的单核/巨噬细胞表达在胞膜上的完整蛋白分子。为了确定TM-TNF与细胞膜的关系,本文借助体外转录翻译系统及微粒体膜结构,建立了表达在微粒体膜上的TM-TNF以及无膜的裸26kD TNF。比较这两种体外表达产物对靶细胞的细胞毒效应,结果表明,只有在微粒体存在时所合成的26kD TNF具有细胞毒活性;而无微粒体存在所合成的裸26kD TNF不显示杀瘤效应。进一步用间接免疫荧光技术观察它们与HL60靶细胞胞膜上TNFR的结合情况,结果显示,结合在微粒体膜上的TM-TNF能与TNFR有效结合,而裸26kD TNF与TNFR则不能有效结合。提示TM-TNF只有“锚定”在细胞膜上,才能利于其空间构型的展示,发挥生物学效应;而一旦与膜分离,则不能与TNFR有效地结合,故丧失了其生物学活性。

Transmembrane TNF-α is an integral protein expressed on the surface of avtivated monocytes/macrophages. In order to define the interrelation between TM-TNF and biomembrane, we used an in vitro translation system and microsomal membranes to engender expression models of two types of TM-TNF; TM-TNF anchored to microsomes and naked 26kD TNF without membranes. When the cytotoxic activity of these two types of TNF was compared, the synthesized 26kD TNF was found to exert its effects only in the presence of microsomal membranes. With the use of indirect immunofluorescence technique, we further studied the binding of the in vitro translated 26kD TNF with TNFR on the membrane of HL60 cells. It was found that only TM-TNF attached to the microsomes could bind effectively with TNFR, while naked 26kD TNF could not. These results suggest that binding with biomembrane may be the prerequisite for 26kD TNF to fold properly for displaying its biological effects. In case 26kD TNF is freed from membrane, it can no more bind with TNFR, there by leading to loss of its cytotoxic effect.