Mechanism for activation of mouse mast cell tryptase: Dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6
Mechanism for activation of mouse mast cell tryptase: Dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6
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DOI：
10.1021/bi000973b
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发表时间：
2000-10-24
期刊：
影响因子：
2.9
通讯作者：
Pejler, G
中科院分区：
文献类型：
--
作者：
Hallgren, J;Karlson, U;Pejler, G
类胰蛋白酶是一种丝氨酸蛋白酶，具有类胰蛋白酶底物的切割特性，是变态反应性炎症过程中的关键效应分子之一。它大量储存在肥大细胞分泌颗粒中，与肝素蛋白多糖形成复合体，并在肥大细胞脱颗粒过程中释放出来。在本论文中，我们研究了类胰蛋白酶的激活机制。在哺乳动物表达系统中表达重组小鼠类胰蛋白酶--小鼠肥大细胞蛋白酶6(mMCP-6)。MMCP-6融合蛋白含有一个N端6xHis标签，其后是一个取代天然激活肽的肠激酶(EK)位点(6xHis-EK-mMCP-6)。在无肝素的情况下，6×His-EK-mMCP-6在pH为5.5-7.5的范围内被肠激酶酶切，几乎检测不到酶的活性。但在肝素存在的情况下，6×His-EK-mMCP-6在pH 5.5~6.0范围内进行肠激酶切割时产生活性酶，而在中性pH下则不产生活性酶。亲和层析分析表明，在pH为6.0时，mMCP-6与肝素-琼脂糖强结合，而在中性pH条件下，mMCP-6不结合。样品在pH 6.0下裂解后，Superdex 200柱上的FPLC分析表明，mMCP-6以不活跃的单体形式存在。当肝素加入pH为6.0时，形成具有酶活性的高分子量复合体，例如一个类似于200 kDa的显性复合体，可能对应于类胰蛋白酶四聚体。在中性pH条件下，未观察到活性四聚体的形成。腹腔注射mMCP-6和肝素可引起中性粒细胞内流，但无肝素时未见炎症迹象。因此，本寻呼机表明肝素在肥大细胞类胰蛋白酶活性的形成中起着至关重要的作用。
Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6 x His-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6 x His-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6 x His-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant similar to 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present pager thus indicates a crucial role for heparin in the formation of active mast cell tryptase.