ABSTRACT
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.
Subject(s)
Metalloendopeptidases/isolation & purification , Tumor Necrosis Factor-alpha/metabolism , ADAM Proteins , ADAM17 Protein , Amino Acid Sequence , Animals , Cell Line , Humans , Hydroxamic Acids/pharmacology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/isolation & purification , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/chemistry , Mice , Molecular Sequence Data , Substrate Specificity , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
Tumor necrosis factor (TNF)-alpha is initially synthesized as a membrane-bound, cell-associated 26-kDa protein that is further cleaved to yield the soluble 17-kDa form. By using a radiolabeled in vitro translated TNF-alpha precursor we detected a serine proteinase processing activity present in crude membrane preparations of monocytic cells able to generate a 17-kDa active protein. A similar processing pattern was obtained using purified neutral serine proteinase proteinase-3 (PR-3). Moreover, while a secretory leukocyte proteinase inhibitor (a natural serine anti-proteinase) did not affect the in vitro TNF-alpha processing, IgG preparations containing high titers of anti-PR-3 autoantibodies completely blocked this activity. The NH2-terminal sequencing of the reaction products obtained with either membrane preparations or PR-3 showed that cleavage occurs in both cases between Val77 and Arg78. These results together with cellular expression and localization of PR-3 suggest a potential role for this enzyme as an accessory TNF-alpha processing enzyme.
