Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL.
Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL.
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发表时间：
1994-10
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通讯作者：
Urban Gullberg;A. Lindmark;Eva Nilsson;A. Persson;Inge Olsson
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作者：
Urban Gullberg;A. Lindmark;Eva Nilsson;A. Persson;Inge Olsson
中性粒细胞的嗜天青颗粒含有中性蛋白酶，如白细胞弹性蛋白酶和组织蛋白酶G。这些被合成为无活性的前体，但在蛋白水解加工后，它们作为活性酶储存在颗粒中。我们描述了一种表达人髓样丝氨酸蛋白酶组织蛋白酶G的转基因细胞模型的建立。在大鼠嗜碱性/肥大细胞系RBL-1和翻译产物的cDNA的稳定表达，其特征在于通过使用生物合成标记，然后通过免疫沉淀，SDS-聚丙烯酰胺凝胶电泳，荧光。通过用内切糖苷酶H和N-聚糖酶处理后获得的产物判断，显示出转化为约3.5 kDa的天冬酰胺连接的碳水化合物单元的复合物形式。通过脉冲追踪实验证明了在合成后1-2小时内将32.5-kDa的组织蛋白原G蛋白水解加工成31-kDa的形式。进一步加工成30-kDa形式也发生在较小程度上。处理后的形式是酶活性的，判断的丝氨酸蛋白酶抑制剂二异丙基氟磷酸和抑肽酶的亲和力。亚细胞分级分离证明了组织蛋白酶G的加工形式向高密度组分的易位，表明蛋白酶向颗粒的靶向。弱碱NH 4Cl被证明延迟组织蛋白酶G的加工和酶促激活，而单价离子载体莫能菌素完全抑制这两个事件。我们的数据表明，人组织蛋白酶G转染到大鼠RBL-1细胞，是蛋白水解加工成酶活性的形式和亚细胞转移到颗粒细胞器发生。由于转基因人组织蛋白酶G的加工对应于髓样细胞的内源性蛋白酶的加工，该模型应该提供新的独特的可能性，以进一步表征髓样丝氨酸蛋白酶的活化和颗粒靶向。
The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.