In vitro processing of human tumor necrosis factor-alpha.
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.
Robache-Gallea
S
S
Domaine Thérapeutique Immunologie, Roussel Uclaf, Romainville, France.
Morand
V
V
Bruneau
J M
JM
Schoot
B
B
Tagat
E
E
Réalo
E
E
Chouaib
S
S
Roman-Roman
S
S
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D014890:Granulomatosis with Polyangiitis / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D001323:Autoantibodies; D018076:DNA, Complementary; D007074:Immunoglobulin G; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011498:Protein Precursors; D014409:Tumor Necrosis Factor-alpha; D012697:Serine Endopeptidases