title: Alpha1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway.
abstract: 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.
authors.affiliation: J. A. DeSève, Clinical Research Institute of Montreal and Protein Engineering Network of Centres of Excellence, Montreal, Quebec H2W 1R7, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Benjannet
authors.forename: D
authors.initials: D
authors.lastname: Savaria
authors.forename: A
authors.initials: A
authors.lastname: Laslop
authors.forename: J S
authors.initials: JS
authors.lastname: Munzer
authors.forename: M
authors.initials: M
authors.lastname: Chrétien
authors.forename: M
authors.initials: M
authors.lastname: Marcinkiewicz
authors.forename: N G
authors.initials: NG
authors.lastname: Seidah
mesh_terms: D000818:Animals; D002460:Cell Line; D007150:Immunohistochemistry; D016296:Mutagenesis; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014162:Transfection; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D011498:Protein Precursors; D000515:alpha 1-Antitrypsin; D012697:Serine Endopeptidases