title: Structure-function analysis of the prosegment of the proprotein convertase PC5A.
abstract: To investigate if some residues within the prosegment of PC5A are important for its optimal proteolytic function, various PC5A mutants were cellularly expressed, and their processing activities were compared using pro-vascular endothelial growth factor C (pro-VEGF-C) as a substrate. Although wild type PC5A almost completely processes pro-VEGF-C, a prosegment deletion as well as both P1 mutants of the primary (R116A) and secondary (R84A) autocatalytic cleavage sites are inactive. The in vitro inhibitory potency of various decapeptides mimicking the C-terminal sequence of PC5 prosegment (pPC5) revealed that the native (107)QQVVKKRTKR(116) peptide is a nanomolar inhibitor, whereas its P6 mutant K111H is more selective toward PC5A than Furin. In vitro activity assays using the bacterially expressed pPC5 and its mutants revealed them to be very potent nanomolar inhibitors (IC(50)) and only approximately 6-fold more selective inhibitors of PC5A versus Furin. Expression of the preprosegment of PC5 (ppPC5) and its mutants in Chinese hamster ovary FD11 cells overexpressing pro-VEGF-C with either PC5A or Furin showed them to be as good inhibitors of PC5A as the serpin alpha1-antitrypsin Portland (alpha1-PDX), ppFurin, or ppPACE4 but less potent toward overexpressed Furin. In conclusion, cleavages of the prosegment of PC5A at both Arg(116) and Arg(84) are required for PC5A cellular activity, and ppPC5 is a very potent but modestly selective cellular inhibitor of PC5A.
authors.affiliation: Laboratories of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada.
authors.forename: Nadia
authors.initials: N
authors.lastname: Nour
authors.forename: Ajoy
authors.initials: A
authors.lastname: Basak
authors.forename: Michel
authors.initials: M
authors.lastname: Chrétien
authors.forename: Nabil G
authors.initials: NG
authors.lastname: Seidah
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D045702:Proprotein Convertase 5; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042582:Vascular Endothelial Growth Factor C
chemical_list: D017931:DNA Primers; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D011994:Recombinant Proteins; D042582:Vascular Endothelial Growth Factor C; D045702:Proprotein Convertase 5; D012697:Serine Endopeptidases; C473807:propeptide convertase 5, rat