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 proform of TNF-alpha. In this study, we constructed expression vectors for the membrane-bound full-length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF-alpha expression vector was introduced into human 293 cells, processing of TNF-alpha to its mature form was enhanced by coexpressing mTACE, and this 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 peptide containing the TNF-alpha cleavage site. Fas ligand (FasL)-transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF-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 FasL. The catalytic domain-lacking TACE mutant, which could also interact TNF-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 efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.
Department of Genetics, Osaka University Medical School, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka, Japan. titai@schering.co.jp
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D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D053222:Fas Ligand Protein; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D011498:Protein Precursors; D014409:Tumor Necrosis Factor-alpha; C061153:tumor necrosis factor precursor; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human