Benjannet S, Savaria D, Laslop A, Munzer J S, Chrétien M, Marcinkiewicz M, Seidah N G
J. A. DeSève, Clinical Research Institute of Montreal and Protein Engineering Network of Centres of Excellence, Montreal, Quebec H2W 1R7, Canada.
J Biol Chem. 1997 Oct 17;272(42):26210-8. doi: 10.1074/jbc.272.42.26210.
We studied the extent of cellular inhibitory activity of alpha1-antitrypsin Portland (alpha1-PDX), a potent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of alpha1-PDX on the intracellular processing of two model precursors (pro-7B2 and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PC5-A, PC5-B, and PC7. The substrates selected were pro7B2, a precursor cleaved within the trans-Golgi network (TGN), and pro-opiomelanocortin, which is processed in the TGN and secretory granules. Biosynthetic analyses were performed using either vaccinia virus expression in BSC40, GH4C1, and AtT20 cells, or stable transfectants of alpha1-PDX in AtT20 cells. Results revealed that alpha1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that alpha1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length alpha1-PDX (64 kDa), the cleaved (56 kDa) secreted product does not significantly inhibit furin activity in vitro. Cellular expression of alpha1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/alpha1-PDX cells demonstrated that alpha1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.
我们研究了α1-抗胰蛋白酶波特兰型（α1-PDX）的细胞抑制活性程度，α1-PDX是枯草杆菌蛋白酶/kexin型前蛋白转化酶的一种有效抑制剂。我们比较了α1-PDX对由七种已知哺乳动物转化酶中的六种介导的两种模型前体（pro-7B2和阿黑皮素原）细胞内加工的抑制作用，这六种转化酶分别是弗林蛋白酶、PC1、PC2、PACE4、PC5-A、PC5-B和PC7。所选择的底物是pro7B2，一种在反式高尔基体网络（TGN）内被切割的前体，以及阿黑皮素原，它在TGN和分泌颗粒中进行加工。使用痘苗病毒在BSC40、GH4C1和AtT20细胞中表达，或在AtT20细胞中稳定转染α1-PDX进行生物合成分析。结果显示，α1-PDX主要在组成型分泌途径内抑制这些前体的加工，并且α1-PDX被一些转化酶切割成较短的形式。有证据表明，与全长α1-PDX（64 kDa）相比，切割后的（56 kDa）分泌产物在体外对弗林蛋白酶活性没有显著抑制作用。α1-PDX的细胞表达导致成熟分泌颗粒内容物发生改变，部分加工产物水平增加。对AtT20/α1-PDX细胞的生物合成和免疫细胞化学分析表明，α1-PDX主要定位于TGN内，并且一小部分进入分泌颗粒，在那里它大多以切割产物的形式储存。
