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

Transmembrane tumor necrosis factor alpha (tmTNF-alpha) has a variety of biological activities different from soluble TNF-alpha (sTNF-alpha), but the only difference in sequence is its leader sequence (LS). To investigate the effect of the LS on tmTNF-alpha 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-alpha, whilst their secretory form retained full activity compared to parental sTNF-alpha. The lost cytotoxicity of Met(-71) mutant tmTNF-alpha was partly due to a reduced receptor binding activity. In spite of full receptor binding activity, Cys(-28) mutant tmTNF-alpha failed to induce NO production and iNOS mRNA transcription via forward signaling, but synergized with sTNF-alpha in IL-8 mRNA transcription via reverse signaling. The Asp(143) mutant tmTNF-alpha lost the ability to bind TNFR and to kill MCF-7 cells, whilst its secretory form retained about 60% cytotoxicity of parental sTNF-alpha. Although the mutation at Phe(87) 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-alpha 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-alpha.