Processing of tumor necrosis factor by the membrane-bound TNF-alpha-converting enzyme, but not its truncated soluble form

Tumour necrosis factor (TNF)-alpha -converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the preform of TNF-alpha. In this study, we constructed expression vectors for the membrane-bound fu ll-length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF-alpha expression vector was introduced into human 293 cells, processin g of TNF-alpha to its mature form was enhanced by coexpressing mTACE, and t his processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF-alpha, although the culture medium of sTACE-transfected cells could cleave a pepti de containing the TNF-alpha cleavage site. Fas ligand (FasL)-transfected 29 3 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and We stern blotting analysis with cells that were cotransfected with TACE and TN F-alpha indicated that both mTACE and sTACE could interact with the proform of TNF-alpha. In the same assay, neither mTACE nor sTACE interacted with F asL. The catalytic domain-lacking TACE mutant, which could also interact TN F-alpha, showed a dominant negative effect on not only TNF-alpha secretion but also FasL secretion. These results suggest that binding of the membrane -anchored but not the soluble form of TACE to TNF-alpha results in efficien t ectodomain shedding, and that FasL secretase is a metalloproteinase simil ar, but not identical, to TACE.