Length of the Linking Domain of Human pro-Tumor Necrosis Factor Determines the Cleavage Processing

Abstract

Several studies have indicated that only one cleavage site (Ala−1/Val+1) is involved in the release of mature TNF from human pro-TNF, whereas others have suggested that the linking sequence (residues −20 to −1) may be important. We previously demonstrated that a pro-TNF deletion mutant, Δ −20- −1, was able to form a trimeric structure and mediate TNF cytotoxicity in a juxtacrine fashion without releasing mature TNF. We constructed seven mutants with smaller deletions within this region. Three 15-residue deletion mutants, Δ −20- −6, Δ −15- −1 and Δ −20- −16, −10- −1, were noncleavable, although able to form a trimer and to mediate cytotoxicity through cell-to-cell contact. Three five- or ten-residue deletion mutants, Δ −20- −16, Δ −10- −1, and Δ −5- −1, behaved like the wild-type TNF; all formed a trimer and released mature TNF. These results suggested that in pro-TNF (1) the number of residues between the base of the trimer and the plasma membrane determines accessibility of the cleavage site to the pro-TNF processing enzyme(s) since small deletions did not block cleavage whereas large ones did regardless of the presence of the native cleavage site (−1/+1), (2) the native cleavage site is not sufficient for releasing mature TNF because mutant Δ −20- −6, in which the native cleavage site was intact, was noncleavable, and (3) alternative cleavage site(s) may exist since mutants Δ −10- −1 and Δ −5- −1, which lack the native cleavage site, were cleavable.