Covalent disulfide binding of human IL-1 beta to alpha 2-macroglobulin: inhibition by D-penicillamine.
Secreted human IL-1 beta is known to have two free SH groups due to unpaired cysteines (positions 8 and 71). Alpha 2-Macroglobulin (alpha 2-M) has internal thioester bonds between cysteine and glutamate residues. Free SH groups may be generated at these alpha 2M residues through the action of proteinases, amines such as methylamine, or at a slow rate, by H2O ("aging" of alpha 2M). Thus, the possibility that IL-1 beta forms a disulfide bond with alpha 2M was investigated. 125I-labeled human rIL-1 beta (15 kDa) was incubated with fresh normal human serum or with purified alpha 2M, treated or not with methylamine. The mixtures were submitted to nondenaturing and denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. IL-1 beta bound to commercially purified "aged" alpha 2M and to alpha 2M in methylamine-treated serum but not to native serum alpha 2M. It did not bind detectably to any other serum proteins. The addition of D-penicillamine (D-pen) during the reaction of [125I]rIL-1 beta with serum or purified alpha 2M blocked the covalent binding of rIL-1 beta to alpha 2M. [125I]rIL-1 beta was removed from alpha 2M by 2-mercaptoethanol in SDS. Thus, disulfide bonds were formed between the free SH groups on [125I]rIL-1 beta and those resulting from the cleavage of the internal thioester bonds of alpha 2M. "Cold" rIL-1 beta and a Cys71----Ser71 rIL-1 beta mutant effectively competed with [125I]rIL.1 beta for binding sites on alpha 2M. When complexes of rIL-1 beta or the mutant rIL-1 beta and alpha 2M were subjected to nonreducing SDS-PAGE and subsequent Western blot analysis, the rIL-1 beta molecules were found to be present in the alpha 2M bands in a dose-dependent manner. rIL-1 beta attached to alpha 2M in the presence or absence of D-pen showed similar biological activity in the mouse thymocyte-assay. Thus, rIL-1 beta attached noncovalently to alpha 2M is biologically active. The lack of inhibition of rIL-1 beta activity by binding to methylamine-treated alpha 2M in the absence of D-pen suggests, but does not prove, that the covalently bound rIL-1 beta is also active. We concluded that human rIL-1 beta binds to alpha 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.
Teodorescu
M
M
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612.
McAfee
M
M
Skosey
J L
JL
Wallman
J
J
Shaw
A
A
Hanly
W C
WC
D000818:Animals; D015153:Blotting, Western; D004220:Disulfides; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000737:chemistry* / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D010396:Penicillamine / Q000737:chemistry; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins / Q000737:chemistry*
D004220:Disulfides; D007375:Interleukin-1; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D010396:Penicillamine