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-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.
Tang
P
P
Department of Tumor Biology, University of Texas, M. D. Anderson Cancer Center, Houston, 77030, USA.
Hung M-C
Klostergaard
J
J
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D011498:Protein Precursors; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C061153:tumor necrosis factor precursor