HUMAN PRO-TUMOR NECROSIS FACTOR IS A HOMOTRIMER

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-I and TNF cDNA-transfected COS-7), a tri mer 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 pr o-TNF monomers by cleaving the cross-linkers. The trimeric structure w as 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 trimeri c structure and the biological activity of pro-TNF, we characterized s everal noncleavable pro-TNF deletion mutants. We observed a correlatio n between expression of TNF cytotoxicity in a juxtacrine fashion and d etection of trimer. Thus, human pro-TNF-alpha, like the secreted matur e TNF-alpha, has trimeric structure which is assembled intracellularly before transport. to the cell surface and is apparently required for mediating its biologic activity.