Tang P, Klostergaard J
Department of Tumor Biology, University of Texas, M. D. Anderson Cancer Center, Houston, 77030, USA.
Biochemistry. 1996 Jun 25;35(25):8216-25. doi: 10.1021/bi952182t.
The structure of human transmembrane pro-TNF-alpha was studied both in intact cell systems and in an in vitro translation system. In intact cell systems (LPS-induced THP-1 and TNF cDNA-transfected COS-7), a trimer of pro-TNF was detected after chemical cross-linking based on its molecular weight in Western blotting analysis. The trimer was shown to be a TNF-specific protein and could be partially cleaved to 26-kDa pro-TNF monomers by cleaving the cross-linkers. The trimeric structure was assembled intracellularly, because it could be detected in both the in vitro microsomal translation system and in THP-1 cells coincident with the appearance of pro-TNF in the cell lysate, prior to secretion of mature TNF. To further analyze the relationship between the trimeric structure and the biological activity of pro-TNF, we characterized several noncleavable pro-TNF deletion mutants. We observed a correlation between expression of TNF cytotoxicity in a juxtacrine fashion and detection of trimer. Thus, human pro-TNF-alpha, like the secreted mature TNF-alpha, has trimeric structure which is assembled intracellularly before transport to the cell surface and is apparently required for mediating its biologic activity.
在完整细胞系统和体外翻译系统中研究了人跨膜前肿瘤坏死因子-α（pro-TNF-α）的结构。在完整细胞系统（脂多糖诱导的THP-1细胞和转染了肿瘤坏死因子cDNA的COS-7细胞）中，基于蛋白质印迹分析中的分子量，化学交联后检测到前肿瘤坏死因子的三聚体。该三聚体被证明是一种肿瘤坏死因子特异性蛋白，通过切割交联剂可部分切割为26 kDa的前肿瘤坏死因子单体。三聚体结构在细胞内组装，因为在体外微粒体翻译系统和THP-1细胞中均能检测到，且与细胞裂解物中前肿瘤坏死因子的出现一致，此时成熟肿瘤坏死因子尚未分泌。为了进一步分析三聚体结构与前肿瘤坏死因子生物学活性之间的关系，我们对几种不可切割的前肿瘤坏死因子缺失突变体进行了表征。我们观察到以旁分泌方式表达肿瘤坏死因子细胞毒性与检测到三聚体之间存在相关性。因此，人前肿瘤坏死因子-α与分泌的成熟肿瘤坏死因子-α一样，具有三聚体结构，该结构在转运至细胞表面之前在细胞内组装，显然是介导其生物学活性所必需的。
