alpha 1-antitrypsin portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway

We studied the extent of cellular inhibitory activity of alpha 1-antitrypsin Portland (alpha 1-PDX), a patent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of alpha 1-PDX on the intracellular processing of two model precursors (pro ?Ba and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PCB-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 alpha 1-PDX in AtT20 cells. Results revealed that alpha 1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that alpha 1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length alpha 1-PDX (64 kDa), the cleaved (56 kDa) secreted product; does not significantly inhibit furin activity in vitro, Cellular expression of alpha 1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/alpha 1-PDX cells demonstrated that alpha 1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.