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In vitro processing of human tumor necrosis factor-alpha
S Robache-Gallea1, V Morand, J M Bruneau
1Domaine Thérapeutique Immunologie, Roussel Uclaf, Romainville, France.
Abstract:
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.
Insights
Researchers identified a specific enzyme, proteinase-3 (PR-3), involved in processing tumor necrosis factor-alpha (TNF-alpha). This finding sheds light on the biological mechanisms regulating this important inflammatory cytokine.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory cytokine.
- TNF-alpha is initially synthesized as a 26-kDa membrane-bound precursor.
- This precursor is cleaved to release a soluble 17-kDa active form.
Purpose of the Study:
- To identify the specific enzyme responsible for processing the TNF-alpha precursor.
- To investigate the role of serine proteinases in TNF-alpha maturation.
Main Methods:
- In vitro translation of radiolabeled TNF-alpha precursor.
- Analysis of processing activity in monocytic cell membrane preparations.
- Use of purified proteinase-3 (PR-3) and inhibitors.
- NH2-terminal sequencing of reaction products.
Main Results:
- A serine proteinase activity in monocytic cells processed TNF-alpha precursor to the 17-kDa form.
- Purified proteinase-3 (PR-3) exhibited similar processing activity.
- A specific serine proteinase inhibitor did not block processing, but anti-PR-3 antibodies did.
- Cleavage occurred between Val77 and Arg78, consistent for both membrane preparations and PR-3.
Conclusions:
- Proteinase-3 (PR-3) is a likely candidate enzyme for processing TNF-alpha.
- PR-3 may function as an accessory enzyme in TNF-alpha maturation.
- These findings contribute to understanding TNF-alpha regulation.