Proteinase 3 is an IL-32 binding protein.
IL-32, a recently discovered proinflammatory cytokine with four isoforms, induces IL-1beta, TNF-alpha, IL-6, and chemokines. Here, we used ligand (IL-32alpha) affinity chromatography in an attempt to isolate an IL-32alpha soluble receptor or binding protein. Recombinant IL-32alpha was covalently immobilized on agarose, and preparations of concentrated crude human urinary proteins were applied for chromatographic separation. A specific 30-kDa protein eluted from the column during acid washing and was identified by mass spectrometry as proteinase 3 (PR3) and confirmed by N-terminal microsequencing. PR3, a neutrophil granule serine protease, exists in a soluble or membrane form and is the major autoantigen for autoantibodies in the systemic vasculitic disease, Wegener's granulomatosis. The affinity of IL-32alpha to PR3 was determined by surface plasmon resonance. The dissociation constants were 2.65 +/- 0.4 nM for urinary PR3 and 1.2 +/- 0.05 nM for neutrophil-derived PR3. However, irreversible inactivation of PR3 enzymatic activity did not significantly change binding to the cytokine. Nevertheless, limited cleavage of IL-32 yielded products consistent with PR3 enzyme activity. Moreover, after limited cleavage by PR3, IL-32alpha was more active than intact IL-32alpha in inducing macrophage inflammatory protein-2 in mouse macrophages and IL-8 in human peripheral blood mononuclear cells. We suggest that PR3 is a specific IL-32alpha binding protein, independent of its enzymatic activity. However, limited cleavage of IL-32alpha by PR3 enhances activities of the cytokine. Therefore, specific inhibition of PR3 activity to process IL-32 or neutralization of IL-32 by inactive PR3 or its fragments may reduce the consequences of IL-32 in immune regulated diseases.
Novick
Daniela
D
Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel. daniela.novick@weizmann.ac.il
Rubinstein
Menachem
M
Azam
Tania
T
Rabinkov
Aharon
A
Dinarello
Charles A
CA
Kim
Soo-Hyun
SH
D000818:Animals; D002460:Cell Line; D006801:Humans; D007378:Interleukins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000652:urine; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology / Q000652:urine
C496971:IL32 protein, human; D007378:Interleukins; D012697:Serine Endopeptidases; D053146:Myeloblastin