Covalent disulfide binding of human IL-1β to α2-macroglobulin: Inhibition by d-penicillamine

Abstract

Secreted human IL-1β is known to have two free SH groups due to unpaired cysteines (positions 8 and 71). α 2-Macroglobulin ( α 2M) has internal thioester bonds between cysteine and glutamate residues. Free SH groups may be generated at these α 2M residues through the action of proteinases, amines such as methylamine, or at a slow rate, by H 2O (“aging” of α 2M). Thus, the possibility that IL-1β forms a disulfide bond with α 2M was investigated. 125I-Labeled human rIL-1β (15 kDa) was incubated with fresh normal human serum or with purified α 2M, treated or not with methylamine. The mixtures were submitted to nondenaturing and denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. IL-1β bound to commercially purified “aged” α 2M and to α 2M in methylamine-treated serum but not to native serum α 2M. It did not bind detectably to any other serum proteins. The addition of d-penicillamine ( d-pen) during the reaction of [ 125I]rIL-1β with serum or purified α 2M blocked the covalent binding of rIL-1β to α 2M. [ 125I]rIL-1β was removed from α 2M by 2-mercaptoethanol in SDS. Thus, disulfide bonds were formed between the free SH groups on [ 125I]rIL-1β and those resulting from the cleavage of the internal thioester bonds of α 2M. “Cold” rIL-1β and a Cys 71 → Ser 71 rIL-1β mutant effectively competed with [ 125I]rIL.1β for binding sites on α 2M. When complexes of rIL-1β or the mutant rIL-1β and α 2M were subjected to nonreducing SDS-PAGE and subsequent Western blot analysis, the rIL-1β molecules were found to be present in the α 2M bands in a dose-dependent manner. rIL-1β attached to α 2M in the presence or absence of d-pen showed similar biological activity in the mouse thymocyteassay. Thus, rIL-1β attached noncovalently to α 2M is biologically active. The lack of inhibition of rIL-1β activity by binding to methylamine-treated α 2M in the absence of d-pen suggests, but does not prove, that the covalently bound rIL-1β is also active. We concluded that human rIL-1β binds to α 2M through the Cys at position 8 and that d-pen inhibits this binding. We speculate that this inhibitory effect may contribute to the therapeutic benefits of d-pen in patients with rheumatoid arthritis.