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 cellproliferation 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 α₁-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°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 plasmamembrane-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 ≥ 30 kDa, was able to convert the 8-kDa ¹²⁵I-IL-6 forms into the ¹²⁵I-labeled 16-kDa complex, suggesting that a ≥ 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 II-6 biological activity.