The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.

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 QQVVKKRTKR116 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 ϳ 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 ␣1-antitrypsin Portland (␣1-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.Numerous secretory proteins and hormones are initially synthesized as inactive precursors that undergo post-translational processing into one or more biologically active polypeptide(s). The mammalian proprotein convertases (PCs) 1 of the secretory pathway are calcium-dependent serine proteinases related to bacterial subtilisin. The PCs recognize various precursors and cleave at the general consensus motif (K/R)X n (K/R)2, where n ϭ 0, 2, 4, or 6, and X is any amino acid (1-3). The PC family counts eight known members; that is, seven dibasic-specific kexin-like convertases, Furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7/LPC (4), and the recently discovered pyrolysinlike SKI-1/S1P, which cleaves at the consensus motif (R/K)X-(hydrophobic)Z2, where Z is variable (5-7).PCs contain an N-terminal signal sequence followed by a prosegment, a catalytic domain, and a P domain. In addition, PCs possess a C-terminal segment that varies between the different members. Although analysis of the tissue and cellular distribution revealed that PC5 is widely expressed but enriched in certain areas such as in brain, cardiovascular system, endothelial cells, and Sertoli cells (8 -11), it is one of the least understood enzymes of the convertase family. Its levels are up-regulated in proliferating vascular smooth muscle cells (12) as well as during embryo implantation (13). PC5 exists in two different isoforms, a soluble PC5A sorted to regulated secretory granules (8, 14) and a membrane-bound PC5B cycling between the trans-Golgi network and the cell surface (14, 15). Active PC5A can cleave a variety of se...