title: Inactivation of interleukin-6 in vitro by monoblastic U937 cell plasma membranes involves both protease and peptidyl-transferase activities.
abstract: Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N-terminal amino acid proteases as well as associated enzymes including elastase and cathepsin G. In this study, purified plasma membranes from U937 cells are reported to degrade the recombinant 21-kDa 125I-interleukin-6 (125I-IL-6) into 8-kDa products with loss of biological activity, as monitored by polyacrylamide gel electrophoresis and a cell-proliferation bioassay. Degradation of 125I-IL-6 by plasma membranes was completely prevented by the serine-protease inhibitor diisopropyl fluorophosphate, but was only partially impaired by alpha 1-protease inhibitor and antibody against cathepsin G. A similar incubation of 125I-IL-6 with cathepsin G purified from U937 cells caused hydrolysis of the cytokine into similar inactive 8-kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These findings indicate that U937 cells hydrolyze IL-6 using cell-associated serine-protease activity and that cathepsin G partially participates in this degradation. Prolonged incubation of 8-kDa 125I-IL-6 fragments with purified U937 plasma membranes, led to a complete loss of IL-6 activity related to the transformation of the 8-kDa forms into a higher-molecular-mass complex (16 kDa). This complex was stable in SDS and 2-mercaptoethanol at 100 degrees C and was not dissociated by hydroxylamine treatment, indicating the formation of a covalent non-ester bond between the 8-kDa 125I-IL-6-derived peptide and an undetermined acceptor. An initial oxidative treatment of 125I-IL-6 partially prevented complex formation, suggesting the presence of one or more oxidizable methionine residues at the binding site of 8-kDa 125I-IL-6 peptide. The kinetics of complex formation (time dependence and plasma-membrane-concentration dependence), as well as its inhibition by a specific inhibitor of N-amino-peptidase activity, bestatin, suggest the participation of peptidyl-transferase activity in complex formation. Finally, a plasma-membrane fraction, corresponding to a molecular mass > or = 30 kDa, was able to convert the 8-kDa 125I-IL-6 forms into the 125I-labeled 16-kDa complex, suggesting that a > or = 30-kDa peptidyl-transferase enzyme catalyzes the reaction and provides the 125I-labeled 16-kDa peptide by dimerization of 8-kDa 125I-IL-6-derived intermediates. Further identification of the plasma-membrane-associated peptidyl transferase as a regulator of IL-6 proteolysis may be of physiological relevance for the control of IL-6 biological activity.
authors.affiliation: Unité 365 INSERM, Institut Curie, Paris, France.
authors.forename: A
authors.initials: A
authors.lastname: Laouar
authors.forename: C
authors.initials: C
authors.lastname: Villiers
authors.forename: J
authors.initials: J
authors.lastname: Sancéau
authors.forename: C
authors.initials: C
authors.lastname: Maison
authors.forename: M
authors.initials: M
authors.lastname: Colomb
authors.forename: J
authors.initials: J
authors.lastname: Wietzerbin
authors.forename: B
authors.initials: B
authors.lastname: Bauvois
mesh_terms: D000626:Aminopeptidases / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D006801:Humans; D006868:Hydrolysis; D015850:Interleukin-6 / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012697:Serine Endopeptidases / Q000502:physiology*; D000093242:Interleukin-6 Inhibitors
chemical_list: D000626:Aminopeptidases; D015850:Interleukin-6; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000093242:Interleukin-6 Inhibitors