LENGTH OF THE LINKING DOMAIN OF HUMAN PRO-TUMOR NECROSIS FACTOR DETERMINES THE CLEAVAGE PROCESSING

Several studies have indicated that only one cleavage site (Ala-1/Val1) is involved in the release of mature TNF from human pro-TNF, wherea s others have suggested that the linking sequence (residues -20 to -1) may be important. We previously demonstrated that a pro-TNF deletion mutant, Delta-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 regio n. Three 15-residue deletion mutants, Delta -20- -6, Delta -15- -1 and Delta -20- -16, -10- -1, were noncleavable, although able to form a t rimer and to mediate cytotoxicity through fell-to-cell contact. Three five- or ten-residue deletion mutants, Delta -20- -16, Delta -10- -1, and Delta -5- -1, behaved like the wild-type TNF; all formed a trimer and released mature TNF. These results suggested that in pro-TNF (I) t he number of residues between the base of the trimer and the plasma me mbrane determines accessibility of the cleavage site to the pro-TNF pr ocessing enzyme(s) since small deletions did not block cleavage wherea s large ones did regardless of the presence of the native cleavage sit e (-1/+1), (2) the native cleavage sire is not sufficient for releasin g mature TNF because mutant Delta -20- -6, in which the native cleavag e site was intact, was noncleavable, and (3) alternative cleavage site (s) may exist since mutants Delta -10- -1 and Delta -5- -1, which lack the native cleavage site, were cleavable.