Leader sequence is required for activity of transmembrane tumor necrosis factor-α

Transmembrane tumor necrosis factor α (tmTNF-α) has a variety of biological activities different from soluble TNF-α (sTNF-α), but the only difference in sequence is its leader sequence (LS). To investigate the effect of the LS on tmTNF-α activity, single amino acid substitutions in the LS and its linked extracellular mature domain were made in an in vitro translation system and in an intact cell system. Mutations at Met−71 and Cys−28 in the LS obliterated cytotoxicity of tmTNF-α, whilst their secretory form retained full activity compared to parental sTNF-α. The lost cytotoxicity of Met−71 mutant tmTNF-α was partly due to a reduced receptor binding activity. In spite of full receptor binding activity, Cys−28 mutant tmTNF-α failed to induce NO production and iNOS mRNA transcription via forward signaling, but synergized with sTNF-α in IL-8 mRNA transcription via reverse signaling. The Asp143 mutant tmTNF-α lost the ability to bind TNFR and to kill MCF-7 cells, whilst its secretory form retained about 60% cytotoxicity of parental sTNF-α. Although the mutation at Phe87 had full activity in both forms, its membrane form induced a change in cell death mode from apoptosis to necrosis, in contrast to wild-type TNF-α whose membrane molecule chiefly induced apoptosis and secretory molecule mainly caused necrosis in MCF-7, respectively. The data suggest that the LS may be required for maintaining the correct structure and the bioactivity of tmTNF-α.