title: Engineering of α1-antitrypsin variants with improved specificity for the proprotein convertase furin using site-directed random mutagenesis.
abstract: Furin, PACE4, PC5/6 and PC7 are members of the subtilisin-like proprotein convertase (SPC) family. Although these enzymes are known to play critical roles in various physiological and pathological events including cell differentiation, tumor growth, virus replication and the activation of bacterial toxins, their distinct functions are yet to be fully delineated. α1-PDX is an engineered α1-antitrypsin variant carrying the RXXR consensus motif for furin within its reactive site loop. However, α1-PDX inhibits other SPCs in addition to furin. In this work, we prepared various rat α1-antitrypsin variants containing Arg at the P1 site within the reactive site loop, and examined their respective selectivity. The novel α1-antitrypsin variant AVNR (AVPM(352)/AVNR) was identified as a highly selective inhibitor of furin. This variant formed a sodium dodecyl sulfate- and heat-stable furin/α1-antitrypsin complex and inhibited furin activity ex vivo and in vitro. Other SPC members including PACE4, PC5/6 and PC7 were not inhibited by the AVNR variant. Furin-mediated maturation of bone morphogenetic protein-4 was completely inhibited by ectopic expression of the AVNR variant. The AVNR variant should prove to be a useful inhibitor in identifying the specific role of furin.
authors.affiliation: Department of Biological Science and Technology,  University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506,  Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Hada
authors.forename: Kinuka
authors.initials: K
authors.lastname: Isshiki
authors.forename: Shinya
authors.initials: S
authors.lastname: Matsuda
authors.forename: Keizo
authors.initials: K
authors.lastname: Yuasa
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tsuji
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D000990:Antithrombin III / Q000737:chemistry; D055415:Bone Morphogenetic Protein 4 / Q000737:chemistry; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D045683:Furin / Q000037:antagonists & inhibitors / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D045702:Proprotein Convertase 5 / Q000037:antagonists & inhibitors / Q000737:chemistry; D043484:Proprotein Convertases / Q000037:antagonists & inhibitors / Q000737:chemistry; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D059748:Proteolysis; D051381:Rats; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D011498:Protein Precursors; D000515:alpha 1-Antitrypsin; D000990:Antithrombin III; C080654:PCSK6 protein, human; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C468731:FURIN protein, human; D045683:Furin