Proteinase 3 is an IL-32 binding protein
| Autor: | Menachem Rubinstein, Aharon Rabinkov, Soohyun Kim, Daniela Novick, Tania Azam, Charles A. Dinarello |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Neutrophils
Myeloblastin medicine.medical_treatment Plasma protein binding Mass Spectrometry Cell Line Proinflammatory cytokine Mice Affinity chromatography Proteinase 3 medicine Animals Humans cardiovascular diseases Receptor Serine protease Multidisciplinary biology Chemistry Interleukins Binding protein Serine Endopeptidases Biological Sciences Molecular biology Kinetics Cytokine Biochemistry biology.protein Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 103:3316-3321 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.0511206103 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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