Proteinase 3 is an IL-32 binding protein

Abstract

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.

Fig. 1.

Silver-stained SDS/PAGE (under nonreducing conditions) of elution fractions (60 μl) of urinary proteins from the IL-32α affinity chromatography column. Shown are the wash fraction (lane 1) representing crude urinary proteins and acid elution fractions 1–5 (lanes 2–6, respectively). Molecular mass (lane 7) is indicated on the right. The arrow indicates an IL-32 binding protein.

Fig. 2.

Kinetics of binding of urinary and neutrophil-derived PR3 to IL-32α. (A) Binding of elution fraction 3 from the IL-32α affinity column at concentrations of 10, 20, 30, 40 and 80 nM to IL-32α immobilized to a BIAcore chip and analysis by the BIAcore apparatus. (B) The same experiment as in A but after inactivation of the urinary PR3 aliquot by pretreatment with PMSF. (C) Same analysis as A but with neutrophil-derived PR3. (D) Same as C but with neutrophil-derived PR3 inactivated by PMSF.

Fig. 3.

Digestion of ¹²⁵I-IL-32α by urinary PR3. (A) Undigested ¹²⁵I-IL-32α (lane 1) and ¹²⁵I-IL-32α after incubation with PR3 for 1, 5, 15, 30, and 60 min at 37°C (lanes 2, 3, 4, 6, and 7, respectively). The molecular mass of cleaved products is shown by arrows on the left. Molecular mass (lane 5) is indicated on the right. (B) An overnight digestion of IL-32α by neutrophil-derived PR3 at 4°C. Intact IL-32α (lane 1) is compared with IL-32α incubated with neutrophil-derived PR3 (lane 2). Molecular mass is indicated on the left. Samples were resolved by 12% SDS/PAGE, and bands were visualized by silver staining.

Fig. 4.

Comparison of IL-32α cleavage by urinary PR3 and neutrophil-derived PR3. Urinary PR3 (lanes 1–3) or neutrophil-derived PR3 (lanes 5–7) was incubated with ¹²⁵I-IL-32α for 0, 1, and 5 min or undigested ¹²⁵I-IL-32 (lanes 1 and 5). The size of cleavage products is shown by arrows on the left. The molecular mass (lane 4) is shown on the right.

Fig. 5.

PMSF-treated PR3 does not cleave IL-32α. Undigested ¹²⁵I-IL-32α is shown in lane 6. Lanes 1–4 show ¹²⁵I-IL-32α after incubation with urinary PR3 for 1 and 5 min. Lanes 1 and 2 show PR3 not pretreated with PMSF, and lanes 3 and 4 show PR3 pretreated with PMSF. Lanes 7–10 show ¹²⁵I-IL-32α after incubation with neutrophil-derived PR3 for 1 and 5 min. Lanes 7 and 8 show PR3 pretreated with PMSF, and, in lanes 9 and 10, PR3 is not pretreated. The sizes of the cleavage products is shown by arrows on the left. Molecular mass markers in kDa (lane 5) are indicated at the right.

Fig. 6.

PR3 cleavage sites in IL-32 isoforms. The arrows indicate the positions of the corresponding valine for each isoform shown above each IL-32 isoform. IL-32 isoforms β, γ, and δ have an additional 57 aa in the C terminus (hatched area) because of the lack of splicing between exons 7 and 8. The hatched area in the N terminus of isoform γ indicates the insertion of 46 aa by a similar splicing event between exons 3 and 4 (1). The arrow at the bottom indicates the specific cleavage site by PR3 between threonine and valine (the sequences of 10 amino acids are shown).

Fig. 7.

Enhanced biological activity of IL-32α after incubation with PR3. (A) Induction of MIP-2 by mouse Raw 264.7 cells stimulated with intact recombinant IL-32α or IL-32α preincubated for 30 min with PR3. The basal level of MIP-2 (225 pg/ml) was subtracted. (B) Induction of IL-8 in PBMC incubated with IL-32α as described in A. The basal level of IL-8 (625 pg/ml) was subtracted.