Proteinase 3 is an IL-32 binding protein
Proteinase 3 is an IL-32 binding protein
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DOI：
10.1073/pnas.0511206103
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发表时间：
2006-02-28
影响因子：
11.1
通讯作者：
Kim, SH
中科院分区：
文献类型：
--
作者：
Novick, D;Rubinstein, M;Kim, SH
IL-32 是一种最近发现的促炎细胞因子，具有四种异构体，可诱导 IL-1β、TNF-α、IL-6 和趋化因子。在这里，我们使用配体 (IL-32 α) 亲和层析尝试分离 IL-32a 可溶性受体或结合蛋白。将重组IL-32a共价固定在琼脂糖上，并将浓缩的粗制人尿蛋白制剂用于色谱分离。在酸洗过程中，一种特定的 30 kDa 蛋白质从柱中洗脱下来，通过质谱分析鉴定为蛋白酶 3 (PR3)，并通过 N 端微测序确认。 PR3 是一种中性粒细胞颗粒丝氨酸蛋白酶，以可溶性或膜形式存在，是系统性血管炎、韦格纳肉芽肿病中自身抗体的主要自身抗原。 IL-32a对PR3的亲和力通过表面等离子共振测定。尿 PR3 的解离常数为 2.65 +/- 0.4 nM，中性粒细胞来源的 PR3 的解离常数为 1.2 +/- 0.05 nM。然而，PR3 酶活性的不可逆失活并没有显着改变与细胞因子的结合。然而，IL-32 的有限裂解产生了与 PR3 酶活性一致的产物。此外，在被 PR3 有限裂解后，IL-32a 在诱导小鼠巨噬细胞中的巨噬细胞炎症蛋白 2 和人外周血单核细胞中的 IL-8 方面比完整的 IL-32a 更活跃。我们认为 PR3 是一种特异性 IL-32a 结合蛋白，与其酶活性无关。然而，PR3 对 IL-32a 的有限裂解增强了细胞因子的活性。因此，特异性抑制PR3处理IL-32的活性或通过无活性的PR3或其片段中和IL-32可能会减少IL-32在免疫调节疾病中的后果。
IL-32, a recently discovered proinflammatory cytokine with four isoforms, induces IL-1 beta, TNF-alpha, IL-6, and chemokines. Here, we used ligand (IL-32 alpha) affinity chromatography in an attempt to isolate an IL-32a soluble receptor or binding protein. Recombinant IL-32a 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-32a 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-32a was more active than intact IL-32a 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-32a binding protein, independent of its enzymatic activity. However, limited cleavage of IL-32a 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.