Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

Abstract

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-␤ superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (ϳ60 kDa) is processed into active BMP10 (ϳ14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR 316 2 site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin-and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/ Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.

Proprotein convertase furin regulates osteocalcin and bone endocrine function

“…That Dec-RVKR-CMK treatment and the ALAA mutation induced an identical migration shift by SDS-PAGE suggests that the RLRR motif is the main, if not only, sequence present in pro-OCN that is recognized and cleaved by a PC ( Figure 1E). Last, treatment of osteoblasts with hexa-Darginine (D6R), a nonpermeable inhibitor that blocks only extracellular or cell-surface PCs (20), had no impact ( Figure 1E). Altogether, these results suggest that an intracellular PC in osteoblasts contributes to the conversion of pro-OCN to OCN.…”

“…Importantly, mutation of the RLRR motif to RLAA completely results, we next tested the capacity of furin, PC5A, PC7, and PACE4 to cleave pro-OCN in vitro. Recombinant GST-pro-OCN protein produced in bacteria was incubated with the conditioned media of HEK293 cells transfected with either an empty vector or a vector expressing the soluble extracellular enzymatic domains of furin, PC5A, or PC7, or with recombinant soluble PACE4 expressed and purified from insect cells (20,21). The PC activity of each conditioned media or recombinant PC was measured using an artificial tetrapeptide substrate prior to the assay to ensure that an equal number of enzymatic units was used (see Methods and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI93437DS1).…”

Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

“…Thus, E64 was found to preserve the N terminus so that it can in principle be targeted by other noncysteine proteinase enzymes. In experiments using PC-specific inhibitors, 25 M cell-permeable decanoyl-RVKR-chloromethylketone (cmk) (RVKR-cmk; Bachem Bioscience) and 10 M nonpermeable hexa-D-arginine (D6R; EMD Chemicals) were added at this point with fresh serum-free medium (38). After 20 h, the cells were washed three times with PBS and then fixed in 3.7% formaldehyde with 0.01 mM sucrose for 15 min at room temperature.…”

“…Peptide bonds were detected at 214 nm, and Tyr-containing peptides were detected at 280 nm. By comparing the RP-HPLC profiles of trypsin-and PC-digested hPAR1 [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] , it was determined that PACE4 cleaves the 19-mer at the predicted site R 41 SFLLR 46 2. The percent cleavage was calculated as the ratio of the normalized peak area (peak area/ number of peptide bonds) of the C-terminal fragment NPNDKYE to that of the intact 19-mer peptide (at time zero).…”

“…The 19-mer synthetic peptide mimicking the tethered ligand and putative PC cleavage site (in boldface) of human PAR1, NATLDPR 41 SFLLR 46 2NPNDKYE (hPAR1 [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] ), was obtained from GenScript. To assess the in vitro cleavage of hPAR1 [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] by PCs, the synthetic peptide was incubated at 37°C in vitro (100 M peptide in 100 l of buffer consisting of 25 mM Tris-morpholineethanesulfonic acid [MES], 1 mM CaCl 2 , pH 7) with 90 nM active purified PCs (18 h) or with 2 g/ml trypsin (5 min and 1 h). Trypsin cleaves at Arg 41 2 (preferentially) and at Arg 46 2 and was used as a reference for the reverse-phase high-pressure liquid chromatography (RP-HPLC) profile of the resulting peptide fragments; a 5-min digestion results in cleavage at Arg 41 2 and a 1-h digestion leads to cleavage at both Arg 41 2 and Arg 46 2.…”