Generation and Biological Characterization of Membrane-bound, Uncleavable Murine Tumor Necrosis Factor

Abstract

Tumor necrosis factor (TNF) is produced as a membrane-bound, 26-kDa proform from which the mature, 17-kDa TNF subunit is released by proteolytic cleavage. In order to compare the biological activity of membrane-bound versus soluble TNF, mutational analysis of potential cleavage sites in murine TNF was carried out. The biological activity was assessed 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 from the -1/+1 junction. 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 Lys11 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 biological activities similar to wild-type TNF. Induction of apoptosis, proliferation, or cytokine production by triggering of either 55-kDa or 75-kDa TNF receptors in appropriate cell lines occurred efficiently both with 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 receptor is not signaling, but contributes to maximal TNF activity by ligand passing. This indicates that membrane-bound TNF cannot be passed from the 75-kDa to the 55-kDa TNF receptor.

Transmembrane TNF protects mutant mice against intracellular bacterial infections, chronic inflammation and autoimmunity

“…The same mutant strain of mice was also reported to be partially protected from LM infection [58]. Interestingly, in the present study, despite the reported partial deficiency of the tmTNF D1-12 mutant in activation of the p55TNFR in cellular assays (as described in [36]), mutant Tnf tm/tm mice also appear to be significantly protected from LM infections, especially following more physiological doses of bacteria (e.g. 100 CFU per mouse) (Fig.…”

“…Potential differences between our study and that of Ruuls et al may be due to the different mutations introduced in the TNF protein. Indeed, earlier data using gene transfers in cultured cell lines have indicated that TNF carrying the D1-12 mutation is a weaker inducer of p55TNFR-dependent cytotoxicity in comparison to D1-9, K11E TNF [36]. We could confirm, however, that like to memTNF D1-9,K11E mutant mice, Tnf tm/tm mice could not support the development of B cell follicles and GC.…”

“…Notably, despite not being processed to the soluble form, expression levels of tmTNF protein on the cell surface were physiologically regulated and followed induction kinetics and cell surface accumulation similar to the WT transmembrane form (Fig. 2) [7,36,55]. This observation indicates that tight regulation of tmTNF levels must exist independently of metalloproteasedependent processing.…”

“…Generation, phenotypic and functional characterization of tmTNF mutant mice CCE (129Sv) ES cells were electroporated with a targeting vector containing a floxed neo cassette in the first intron of the murine TNF gene and the D1-12 mutation that encodes an uncleavable tmTNF protein [13,36] (Fig. 1A).…”

“…A relevant question that remains to be addressed is whether the TNF D1-12 mutant binds with the same affinity as the WT transmembrane TNF to TNFR1 and TNFR2. Decoster et al [36] previously addressed this issue in cellular assays in which the TNF D1-12 mutant was shown to be a less potent activator of the p55TNFR in comparisons to the WT or TNF D1-9,K11E mutant. However, comparison of the phenotypes of the TNF D1-12 (present study) or TNF D1-9,K11E mutant mice [17] did not reveal any major differences in p55TNFR-dependent phenomena, such as LPS-D-gal lethality, splenic structure or host defence (see also discussion below), indicating a similar in vivo function.…”