GENERATION AND BIOLOGICAL CHARACTERIZATION OF MEMBRANE-BOUND, UNCLEAVABLE MURINE TUMOR-NECROSIS-FACTOR

Tumor necrosis factor (TNF) is produced as a membrane-bound, 26-kDa pr eform from which the mature, 17-kDa TNF subunit is released by proteol ytic cleavage. In order to compare the biological activity of membrane -bound versus soluble TNF, mutational analysis of potential cleavage s ites in murine TNF was carried out. The biological activity was assess ed after transfection in L929 cells. Deletion of the first nine codons of the mature part of the murine TNF gene still led to the production of secretable TNF, indicating alternative cleavage sites separate fro m the -1/+1junction. However, an additional deletion of 3 amino acids, generating TNF Delta 1-12, resulted in a membrane-bound form of TNF. Site-directed mutagenesis revealed Lys(11) as the critical residue for alternative cleavage. Mutation of this residue to Glu in a TNF Delta 1-9 mutant gave rise to uncleavable, membrane-bound TNF with biologica l activities similar to wild-type TNF. Induction of apoptosis, prolife ration, or cytokine production by triggering of either 55-kDa or 75-kD a TNF receptors in appropriate cell lines occurred efficiently both wi th soluble and with membrane-bound TNF. The latter was, however, less active in the cytotoxic assays on U937 cells in which the 75-kDa TNF r eceptor is not signaling, but contributes to maximal TNF activity by l igand passing. This indicates that membrane-bound TNF cannot be passed from the 75-kDa to the 55-kDa TNF receptor.