IN-VITRO PROCESSING OF HUMAN TUMOR-NECROSIS-FACTOR-ALPHA

Tumor necrosis factor (TNF)-alpha is initially synthesized as a membra ne-bound, cell-associated 26 kDa protein that is further cleaved to yi eld the soluble 17-kDa form. By using a radiolabeled in vitro translat ed TNF-alpha precursor we detected a serine proteinase processing acti vity present in crude membrane preparations of monocytic cells able to generate a 17-kDa active protein. A similar processing pattern was ob tained using purified neutral serine proteinase proteinase 3 (PR-3). M oreover, while a secretory leukocyte proteinase inhibitor (a natural s erine anti-proteinase) did not affect the in vitro TNF-alpha processin g, 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 s howed that cleavage occurs in both cases between Val(77) and Arg(78). 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.