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 proVEGF-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 107QQVVKKRTKR116 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 (IC50) and only 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 proVEGF-C with either PC5A or Furin showed them to be as good inhibitors of PC5A as the serpin 1-antitrypsin Portland ( 1-PDX), ppFurin, or ppPACE4 but less potent toward overexpressed Furin. In conclusion, cleavages of the prosegment of PC5A at both Arg116 and Arg84 are required for PC5A cellular activity, and ppPC5 is a very potent but modestly selective cellular inhibitor of PC5A.