Partial purification and characterization of a tumor necrosis factor-alpha converting activity.

Tumor necrosis factor (TNF)-alpha is initially synthesized as an extracellular membrane-associated 26-kDa protein that is further cleaved at Ala76-Val77 to yield the soluble 17-kDa form. Recently, peptide-hydroxamate metalloproteinase inhibitors have been reported to block the proteolytic processing of TNF-alpha, thus suggesting that the putative TNF-alpha converting enzyme (TACE) is a zinc-dependent metalloendopeptidase. In this report, we characterize a TNF-alpha converting activity (TACA) that cleaves in vitro the human 26-kDa TNF-alpha at the physiological processing site. The chromatography steps followed for purification and the use of a panel of proteinase inhibitors indicate that the enzyme responsible for TACA is a membrane glycosylated metalloendopeptidase which is most likely different from the matrix-degrading metalloproteinases. The failure of TACA to process a Val77-->Gly77 precursor mutant emphasizes the importance of hydrophobic residue at P1' position. In addition, TACA is not able to cleave the mouse pro-TNF-alpha and does not catalyze in vitro the processing of other transmembrane proteins susceptible to metalloproteinase-mediated shedding, such as interleukin-6 or TNF receptors. These studies suggest the existence of an enzyme specific for TNF-alpha within the metalloproteinases involved in the processing/shedding of a number of cytokines and cytokine receptors.