Formation of active monomers from tetrameric human β-tryptase
Formation of active monomers from tetrameric human β-tryptase
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DOI：
10.1042/bj20021418
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发表时间：
2003-02-01
影响因子：
4.1
通讯作者：
Pejler, G
中科院分区：
文献类型：
--
作者：
Fajardo, I;Pejler, G
类胰蛋白酶是一种丝氨酸蛋白酶，在低pH下与肝素蛋白聚糖复合储存在肥大细胞分泌颗粒中。当肥大细胞被激活时，例如在过敏反应期间，类胰蛋白酶/肝素复合物与多种其他预先形成的炎症介质一起释放。先前的人β-类胰蛋白酶的结晶揭示了独特的四聚体结构，所有的活性位点都面向一个中心孔，该中心孔对于潜在的底物以及蛋白酶抑制剂都具有有限的可及性。在这项研究中，我们检查了人类β-类胰蛋白酶是否可以形成活性单体。在中性pH和37 ℃下孵育重组四聚体人β-类胰蛋白酶，然后使用含有猪粘膜肝素的运行缓冲液进行凝胶过滤分析，导致形成酶活性化合物，其大小与类胰蛋白酶单体与肝素复合物相容。与四聚体形式的类胰蛋白酶相反，单体被牛胰蛋白酶抑制剂抑制。此外，单体而不是四聚体降解纤连蛋白。活性单体的形成在pH 7.5下比在pH 6.0下更明显，并且在室温下或在高肝素/类胰蛋白酶比率下未检测到。因此，本研究结果引入了这样的可能性，即在肥大细胞脱粒并暴露于组织中的中性pH后，人β-类胰蛋白酶可能解离成具有不同于四聚体对应物的性质的活性单体。人类类胰蛋白酶的某些生物活性可能是由于其单体形式而不是四聚体形式的活性类胰蛋白酶。
Tryptase is a serine protease that is stored at low pH in the mast cell secretory granules in complex with heparin proteoglycan. When mast cells are activated, e.g. during allergic responses, the tryptase/heparin complexes are released together with a variety of other preformed inflammatory mediators. Previous crystallization of human beta-tryptase revealed a unique tetrameric structure with all of the active sites facing a central pore that has a limited accessibility both for potential substrates as well as for protease inhibitors. In this study we examined whether human,beta-tryptase, in addition, could form active monomers. Incubation of recombinant tetrameric human beta-tryptase at neutral pH and 37 degreesC, followed by gel-filtration analysis using a running buffer containing pig mucosal heparin, led to the formation of enzymically active compounds that were of a size compatible with tryptase monomers in complex with heparin. The monomers were, in contrast to tryptase in the tetrameric form, inhibited by bovine pancreatic trypsin inhibitor. Further, the monomers, but not the tetramers, degraded fibronectin. Formation of active monomers was more pronounced at pH 7.5 than at pH 6.0 and was not detected at room temperature or at high heparin/tryptase ratios. The present findings thus introduce the possibility that human beta-tryptase, after mast cell degranulation and exposure to neutral pH in the tissue, may dissociate into active monomers with properties that are distinct from the tetrameric counterpart. Possibly, some of the biological activities of human tryptase may be attributable to active tryptase in its monomeric rather than tetrameric form.