Henningsson Frida, Yamamoto Kenji, Saftig Paul, Reinheckel Thomas, Peters Christoph, Knight Stefan D, Pejler Gunnar
Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Centre, Box 575, 751 23 Uppsala, Sweden.
J Cell Sci. 2005 May 1;118(Pt 9):2035-42. doi: 10.1242/jcs.02333.
Mast-cell carboxypeptidase A is stored in the secretory granule and is released, together with a range of other inflammatory mediators, upon mast-cell degranulation. Carboxypeptidase A, like all mast-cell proteases, is stored in the granule as an active enzyme (i.e. with its propeptide removed). Although the processing mechanisms for the other classes of mast-cell proteases (in particular the chymases) have been clarified to some extent, the processing of procarboxypeptidase A is poorly characterized. Here, we show that mast cells from mice lacking the aspartic protease cathepsin E display an accumulation of procarboxypeptidase A, indicating a defect in carboxypeptidase-A processing. By contrast, mast cells lacking cathepsins B, L or D have normal carboxypeptidase-A processing. Furthermore, recombinant cathepsin E was found to process recombinant procarboxypeptidase A in vitro, under conditions resembling those found in mast-cell granules. Immunohistochemical analysis revealed staining for cathepsin E in mast cells from normal mice but not in mast cells from mice lacking heparin, indicating that cathepsin E is bound to heparin proteoglycan within mast-cell granules. In accordance with this notion, affinity chromatography showed that recombinant cathepsin E bound strongly to heparin under acidic conditions (the conditions prevailing in mast-cell granules) but not at neutral pH. Moreover, mast-cell degranulation resulted in the release of cathepsin E. Taken together, our results indicate that cathepsin E is located in mast-cell secretory granules in complex with heparin proteoglycans, and that it has a role in the processing of procarboxypeptidase A into active protease.
肥大细胞羧肽酶A储存于分泌颗粒中，在肥大细胞脱颗粒时与一系列其他炎症介质一起释放。羧肽酶A与所有肥大细胞蛋白酶一样，以活性酶的形式（即前肽已去除）储存于颗粒中。尽管其他类别的肥大细胞蛋白酶（特别是糜蛋白酶）的加工机制已在一定程度上得到阐明，但前羧肽酶A的加工过程却鲜为人知。在此，我们发现，缺乏天冬氨酸蛋白酶组织蛋白酶E的小鼠的肥大细胞中存在前羧肽酶A的积累，这表明羧肽酶A的加工存在缺陷。相比之下，缺乏组织蛋白酶B、L或D的肥大细胞对羧肽酶A的加工正常。此外，发现在类似于肥大细胞颗粒中的条件下，重组组织蛋白酶E在体外可加工重组前羧肽酶A。免疫组织化学分析显示，正常小鼠肥大细胞中有组织蛋白酶E染色，而缺乏肝素的小鼠肥大细胞中则无此染色，这表明组织蛋白酶E与肥大细胞颗粒内的肝素蛋白聚糖结合。与此观点一致，亲和层析显示，重组组织蛋白酶E在酸性条件下（肥大细胞颗粒中的主要条件）与肝素强烈结合，而在中性pH条件下则不然。此外，肥大细胞脱颗粒导致组织蛋白酶E的释放。综上所述，我们的结果表明，组织蛋白酶E与肝素蛋白聚糖结合存在于肥大细胞分泌颗粒中，并且它在将前羧肽酶A加工成活性蛋白酶的过程中发挥作用。
