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• W2022674779 abstract "Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR316↓ 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. Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR316↓ 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. The heart is the first organ to be formed during embryogenesis and it develops via a complex and sequential process that involves simultaneous morphogenesis and differentiation. Cardiac morphogenesis is under the control of a large variety of regulatory proteins, including TGF-β-related family members (1Srivastava D. Olson E.N. Nature. 2000; 407: 221-226Crossref PubMed Scopus (530) Google Scholar). TGF-β-like proteins and other substrates like neuropeptides, receptors, or viral glycoproteins are known targets for members of the secretory basic amino acid (aa) 2The abbreviations used are: aaamino acidBMP10bone morphogenetic protein 10D6Rhexa d-arginineEndo Hendonuclease HKOknockoutLAleft atriaPCsproprotein convertasesPC5/6 and PC7proprotein convertases 5/6 and 7PNGase FN-glycosidase FRAright atriaTGNtrans Golgi networkMCA7-amido-4-methylcoumarinQPCRquantitative PCRGdf11growth differentiating factor 11AbantibodyTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinesFurinsoluble furincmkchloromethyl ketoneEembryonic dayProtCProtein C tag.-specific proprotein convertase(s) (PCs) (2Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar, 3Seidah N.G. Mayer G. Zaid A. Rousselet E. Nassoury N. Poirier S. Essalmani R. Prat A. Int. J. Biochem. Cell. Biol. 2008; 40: 1111-1125Crossref PubMed Scopus (280) Google Scholar). In mammals, seven members of the basic aa-specific PC family have been characterized (mouse genes Pcsk1 to Pcsk7) that cleave various precursors at the consensus motif K/R-Xn-K/R↓ (n = 0, 2, 4, or 6 and X is any aa, except Cys) (2Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar). Four of them, furin, PC5/6, PACE4, and PC7, are ubiquitously or widely expressed and are responsible for the majority of processing events occurring in the constitutive secretory pathway, at the cell surface and/or in the extracellular matrix (3Seidah N.G. Mayer G. Zaid A. Rousselet E. Nassoury N. Poirier S. Essalmani R. Prat A. Int. J. Biochem. Cell. Biol. 2008; 40: 1111-1125Crossref PubMed Scopus (280) Google Scholar). amino acid bone morphogenetic protein 10 hexa d-arginine endonuclease H knockout left atria proprotein convertases proprotein convertases 5/6 and 7 N-glycosidase F right atria trans Golgi network 7-amido-4-methylcoumarin quantitative PCR growth differentiating factor 11 antibody N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine soluble furin chloromethyl ketone embryonic day Protein C tag. Despite their functional redundancy ex vivo, these PCs fulfill key processing events in vivo, as revealed by the specific phenotypes resulting from their respective gene inactivation in mice. Pcsk3 (furin gene) knock-out (KO) in mice causes early death by embryonic day 10.5 (E10.5) due to hemodynamic insufficiency and cardiac ventral closure defects translated into failure of the heart tube to fuse and undergo looping morphogenesis (4Roebroek A.J. Umans L. Pauli I.G. Robertson E.J. van Leuven F. Van de Ven W.J. Constam D.B. Development. 1998; 125: 4863-4876Crossref PubMed Google Scholar). These phenotypes emphasize the critical involvement of furin in cardiac development. Pcsk5 (PC5/6 gene) KO leads to death at birth with an altered antero-posterior patterning, including extra vertebrae, lack of tail, kidney agenesis, hemorrhages, collapsed alveoli, and retarded ossification, as well as heart ventricular-septal defects (5Essalmani R. Zaid A. Marcinkiewicz J. Chamberland A. Pasquato A. Seidah N.G. Prat A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5750-5755Crossref PubMed Scopus (93) Google Scholar, 6Szumska D. Pieles G. Essalmani R. Bilski M. Mesnard D. Kaur K. Franklyn A. El Omari K. Jefferis J. Bentham J. Taylor J.M. Schneider J.E. Arnold S.J. Johnson P. Tymowska-Lalanne Z. Stammers D. Clarke K. Neubauer S. Morris A. Brown S.D. Shaw-Smith C. Cama A. Capra V. Ragoussis J. Constam D. Seidah N.G. Prat A. Bhattacharya S. Genes Dev. 2008; 22: 1465-1477Crossref PubMed Scopus (109) Google Scholar). Mice lacking Pcsk6 (PACE4 gene) KO survive to adulthood, and some develop incompletely penetrant left-right patterning defects combined with cyclopia, craniofacial, and cardiac malformations (7Constam D.B. Robertson E.J. Genes Dev. 2000; 14: 1146-1155PubMed Google Scholar, 8Constam D.B. Robertson E.J. Development. 2000; 127: 245-254Crossref PubMed Google Scholar). Pcsk7 (PC7 gene) KO mice exhibit no overt abnormalities (9Villeneuve P. Feliciangeli S. Croissandeau G. Seidah N.G. Mbikay M. Kitabgi P. Beaudet A. J. Neurochem. 2002; 82: 783-793Crossref PubMed Scopus (44) Google Scholar). 3D. Constam, personal communications. Thus, heart defects are a common phenotype associated with the single KO of the mouse genes coding for furin, PC5/6, or PACE4, but not PC7. Bone morphogenetic protein 10 (BMP10) is a newly identified cardiac-specific growth factor that is a member of the TGF-β superfamily and is known to play a critical role in heart development. BMP10 expression is most abundant in the developing and postnatal heart and weaker in the adult liver and lung (10Neuhaus H. Rosen V. Thies R.S. Mech. Dev. 1999; 80: 181-184Crossref PubMed Scopus (121) Google Scholar). During mouse cardiogenesis, after completion of embryonic cardiac patterning and looping and at the onset of trabeculation and chamber maturation, BMP10 is transiently expressed in the ventricular trabecular myocardium (E9–E13.5). By E16.5–E18.5, BMP-10 is only expressed in the atria, and is restricted to the right atrium (RA) in the postnatal heart, where it promotes increased cardiomyocyte and heart size (10Neuhaus H. Rosen V. Thies R.S. Mech. Dev. 1999; 80: 181-184Crossref PubMed Scopus (121) Google Scholar, 11Chen H. Shi S. Acosta L. Li W. Lu J. Bao S. Chen Z. Yang Z. Schneider M.D. Chien K.R. Conway S.J. Yoder M.C. Haneline L.S. Franco D. Shou W. Development. 2004; 131: 2219-2231Crossref PubMed Scopus (385) Google Scholar, 12Chen H. Yong W. Ren S. Shen W. He Y. Cox K.A. Zhu W. Li W. Soonpaa M. Payne R.M. Franco D. Field L.J. Rosen V. Wang Y. Shou W. J. Biol. Chem. 2006; 281: 27481-27491Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Homozygous BMP10 KO embryos die in utero between E10 and E10.5 due to arrested cardiac development. Compared with wild type (WT) embryos, KO embryos appear normal at E8.5, but display cardiac dysgenesis at E9–E9.5 with profound hypoplastic ventricular walls, absence of ventricular trabeculae, and a significantly lower heart rate (11Chen H. Shi S. Acosta L. Li W. Lu J. Bao S. Chen Z. Yang Z. Schneider M.D. Chien K.R. Conway S.J. Yoder M.C. Haneline L.S. Franco D. Shou W. Development. 2004; 131: 2219-2231Crossref PubMed Scopus (385) Google Scholar). As with all members of the TGF-β superfamily, BMP10 is synthesized as an inactive precursor protein (pro-BMP10, ∼60 kDa) that is presumably activated by proteolytic cleavage, likely at the motif RIRR313↓ (mouse nomenclature) releasing the secreted non-glycosylated C-terminal mature peptide of 108 aa (∼14 kDa; BMP10) and an N-terminal prosegment of ∼50 kDa (supplemental Fig. S1). Mature BMP10 exhibits a conserved pattern of 7 cysteines, one of which is thought to be engaged in an interchain disulfide bond (13Mazerbourg S. Sangkuhl K. Luo C.W. Sudo S. Klein C. Hsueh A.J. J. Biol. Chem. 2005; 280: 32122-32132Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Bessa P.C. Cerqueira M.T. Rada T. Gomes M.E. Neves N.M. Nobre A. Reis R.L. Casal M. Protein Expr. Purif. 2009; 63: 89-94Crossref PubMed Scopus (40) Google Scholar) (supplemental Fig. S1). BMP10 shares >98% aa sequence identity among human, mouse, and rat orthologs. Cleavage of pro-BMP10 at the motif RXRR↓ suggests the involvement of basic aa-specific PCs (2Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar). The restricted expression of BMP10 mRNA to the RA of the heart in adult mice, and heart-specific phenotypes observed in mice lacking furin, PC5/6, or PACE4, led us to test herein the implication of these convertases in the generation of mature BMP10. We identified all human and mouse protein sequences with signal peptides in ENSEMBL and screened them for the consensus sequence (R/K)-Xn-R↓[N] (where X = 0, 2, 4, 6 aa) using the FuzzPro program (EMBOSS). Positive hits, where the potential cleavage motif was present in both human and mouse proteins, were screened against the Mouse Genome Informatics data base to identify the proteins where there is also genetic evidence for their relevance in heart development. Data were integrated and queried using a relational data base. Enzymatic in vitro activities of the purified furin, PC5/6, PACE4, and PC7 (15Fugère M. Limperis P.C. Beaulieu-Audy V. Gagnon F. Lavigne P. Klarskov K. Leduc R. Day R. J. Biol. Chem. 2002; 277: 7648-7656Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) were measured at 37 °C in 100 μl of buffer (25 mm Tris-MES, 2 mm CaCl2, pH 7) in the presence of 100 μm of the fluorogenic substrate pyroglutamic acid-RTKR-7-amido-4-methylcoumarin (Pyr-RTKR-MCA; Peptide International). The release of free 7-amino-4-methylcoumarin (AMC) was detected with a Spectra MAX GEMINI EM microplate spectrofluorimeter (Molecular Devices) (excitation, 370 nm; emission, 460 nm; emission cutoff, 435 nm). 1 unit of enzymatic activity was defined as 1 pmol of AMC released from 100 μm Pyr-RTKR-MCA/min at 37 °C. The 12-mer synthetic peptides encompassing the predicted cleavage site of mouse pro-BMP10, DSSARIRR313↓NAKG (WT-mBMP10), and DSSARIRR313↓DAKG (N314D-mBMP10, in which the Asn at the P1′ position was mutated to Asp), were obtained from GenScript (NJ). To determine the specificity of the PCs for the processing of the above synthetic substrates, peptides were individually incubated at 37 °C for 2 h in vitro (200 μm peptide in 100 μl of buffer: 25 mm Tris-MES, 2 mm CaCl2, pH 7) with 2 units of purified PCs (15Fugère M. Limperis P.C. Beaulieu-Audy V. Gagnon F. Lavigne P. Klarskov K. Leduc R. Day R. J. Biol. Chem. 2002; 277: 7648-7656Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). For measurement of the time dependence of in vitro cleavage of WT-mBMP10 peptide by furin, PC5/6, or PACE4, the incubations were carried out as above for 0, 10, 20, 30, and 40 min. For each incubation mixture the cleavage products were separated by reversed-phase HPLC (RP-HPLC) on a Varian C18 column (5 μm, 100 Å, 4.6 × 250 mm) using a 0–50% acetonitrile (+0.1% TFA) gradient over 50 min, at 1 ml/min flow rate. Peptide bonds were detected at 214 nm. The cleavage site RIRR313↓ was confirmed by mass spectrometry using MS/MS. For the determination of kinetic parameters Vmax(app) and Km(app), furin samples (2 units) were incubated at 37 °C with increasing concentrations of WT-mBMP10 peptide ranging from 0 to 200 μm in a total volume of 200 μl of buffer as described above. From each reaction mixture, corresponding to one peptide concentration, 25 μl were subjected to RP-HPLC analysis per time point (see above) and 2–3 time points were assayed, varying from 0 to 15 min. The initial rate of substrate cleavage was obtained from the increase over time of the normalized peak area (peak area/number of peptide bonds) corresponding to the C-terminal fragment NAKG. The values were transformed into micromolar/h by a standard curve generated from variable concentrations of intact WT-mBMP10 peptide for which the peak area was normalized to the number of peptide bonds. Vmax(app) and Km(app) were calculated from a fit to Michaelis-Menten equation (vi = (Vmax(app) × [S])/(Km(app) + [S])) of plots of initial rates of cleavage (vi) against the peptide concentration ([S]) using TableCurve2D software (TableCurve2D version 5.01, SYSTAT Software Inc.). The cDNA encoding for human BMP10 (pCMV6 × 15 vector) was obtained from OriGene (MD). Human BMP10 and its mutant cDNAs (R313A, R316A, and R313A/R316A) were subcloned, with a ProtC tag at the N terminus, into pIRES2-EGFP vector (Clontech). Human furin, human PACE4, mouse PC5A, and their mutant cDNAs were cloned without a tag into pIRES-EGFP vector, as previously described (16Ozden S. Lucas-Hourani M. Ceccaldi P.E. Basak A. Valentine M. Benjannet S. Hamelin J. Jacob Y. Mamchaoui K. Mouly V. Desprès P. Gessain A. Butler-Browne G. Chrétien M. Tangy F. Vidalain P.O. Seidah N.G. J. Biol. Chem. 2008; 283: 21899-21908Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). All cDNAs were verified by DNA sequencing. HEK293 and COS-1 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen), whereas CHO-K1, furin-deficient CHO-FD11 (17Gordon V.M. Rehemtulla A. Leppla S.H. Infect. Immun. 1997; 65: 3370-3375Crossref PubMed Google Scholar), and furin-deficient LoVo (18Takahashi S. Kasai K. Hatsuzawa K. Kitamura N. Misumi Y. Ikehara Y. Murakami K. Nakayama K. Biochem. Biophys. Res. Commun. 1993; 195: 1019-1026Crossref PubMed Scopus (117) Google Scholar, 19Takahashi S. Nakagawa T. Kasai K. Banno T. Duguay S.J. Van de Ven W.J. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 26565-26569Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) cells were grown in Ham's F-12 medium with 10% (v/v) FBS. All cells were maintained at 37 °C under 5% CO2. In a typical transfection experiment, at about 80–90% confluence, cells were transiently transfected as follows: COS-1 (total of 4 μg of cDNA), CHO-K1 (total of 3 μg of cDNA), and CHO-FD11 (total of 3 μg of cDNA) cells with Lipofectamine 2000 (Invitrogen); HEK293 cells (total of 0.6 μg of cDNA) with Effectene (Qiagen); LoVo cells (total of 2 μg of cDNA) with FuGENE HD (Roche Diagnostics). In co-transfection experiments with cDNAs coding for (ProtC)-BMP10 and for the prosegment of furin (ppFurin) (20Zhong M. Munzer J.S. Basak A. Benjannet S. Mowla S.J. Decroly E. Chrétien M. Seidah N.G. J. Biol. Chem. 1999; 274: 33913-33920Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) or PACE4 (ppPACE4) (21Mayer G. Hamelin J. Asselin M.C. Pasquato A. Marcinkiewicz E. Tang M. Tabibzadeh S. Seidah N.G. J. Biol. Chem. 2008; 283: 2373-2384Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), the cDNA ratio of BMP10 to inhibitor was 1:2. The transfection efficiencies were as follows: COS-1, 40–42%; CHO-K1, 27–30%; CHO-FD11, 30–40%; HEK293, 75–80%; LoVo, 10–11%. At 24 h post-transfection, cells were washed for 1 h (at 37 °C) in serum-free medium followed by incubation for an additional 20 h in serum-free medium alone or in combination with 10 μm hexa-d-arginine (D6R; EMD Chemicals), or 25 μm decanoyl-RVKR-chloromethyl ketone (RVKR-cmk; Bachem), respectively, as indicated in the figure legends. Following treatments, cells and/or conditioned media were collected for Western blot analyses (see below). Proteins from conditioned media and cell lysates were incubated with endoglycosidase H (Endo H) or N-glycosidase F (PNGase F) for 90 min at 37 °C. Products were separated on 8% Tris glycine SDS-PAGE gels, transferred to a PVDF membrane (0.45 μm; PerkinElmer Life Sciences), and revealed by Western blot (see below). Mouse primary hepatocytes were isolated from 8–10-week-old male livers using a two-step collagenase perfusion method, as previously described (22Essalmani R. Susan-Resiga D. Chamberland A. Abifadel M. Creemers J.W. Boileau C. Seidah N.G. Prat A. J. Biol. Chem. 2011; 286: 4257-4263Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In 3.5-cm Petri dishes coated with fibronectin (0.5 mg/ml, Sigma), 5 × 105 cells were seeded in Williams' medium E supplemented with 10% (v/v) FBS (Invitrogen). After 2 h, the medium was replaced with hepatozyme medium (Invitrogen) for 12 h prior to transfection. Transfections were performed with Effectene (Qiagen) using a total of 5 μg of cDNA, following the manufacturer's instructions (transfection efficiencies of 10–15%). The medium was collected 48 h post-transfection and subjected to immunoprecipitation followed by Western blot analysis (see below). RNA from isolated mouse atria (right and left) were extracted using TRIzol, as recommended by the manufacturer (Invitrogen). Primers from neighboring exons were used to measure BMP10, furin, PACE4, PC5/6, and mouse TATA-box binding protein. cDNA synthesis and QPCR were performed as described previously (23Dubuc G. Chamberland A. Wassef H. Davignon J. Seidah N.G. Bernier L. Prat A. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1454-1459Crossref PubMed Scopus (526) Google Scholar). The sets of primers were as follows: BMP10, 5′-CATCATCCGGAGCTTCAAGAAC versus 5′-TCCGGAGCCCATTAAAAGTG; furin, 5′-CATGACTACTCTGCTGATGG versus 5′-GAACGAGAGTGAACTTGGTC; PACE4, 5′-GCTGGCTAAACAAGCTTTCGA versus 5′-CAAAAATGGAGCCCAGACCTT; PC5/6, 5′-ACTCTTCAGAGGGTGGCTA versus 5′-GCTGGAACAGTTCTTGAATC; TATA-box binding protein, 5′-GCTGAATATAATCCCAAGCGATTT versus 5′-GCAGTTGTCCGTGGCTCTCT. Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 μg) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2-μm PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen). Proteins from medium of cultured cells (transfected with ProtC-tagged pro-BMP10) were analyzed by 8% Tris glycine SDS-PAGE, followed by Western blotting using a ProtC-Ab (1:1000; GenScript) and a rabbit HRP-conjugated secondary Ab (1:10,000; Invitrogen). Pro-BMP10 and its prosegment tagged with ProtC at the N terminus were immunoprecipitated from mouse primary hepatocytes culture medium (200 μl) using a ProtC-Ab (2 μg; Abgent), resolved by 8% Tris glycine SDS-PAGE gels, and immunodetected using the same ProtC-Ab (1:1,000) and anti-rabbit IgG-HRP (1:2,000, TrueBlot). The antigen-antibody complexes were visualized using the enhanced chemiluminescence kit (ECL; Pierce). Quantitation was performed using ImageJ software (National Institutes of Health), and normalization was reported to β-actin. When media were analyzed by Western blotting, the same volumes were loaded on SDS-PAGE gels. HEK293 cells (1 × 106 cells) were transiently transfected in 60-mm dishes using Effectene (Qiagen) and a total of 1 μg of DNA. Two days post-transfection, the cells were washed and pulse-labeled with 250 μCi/ml of [32S]Met/Cys for either 15 min (pulse-chase experiments) or 2 h. When pulse-labeled for 2 h, the cells were also incubated with brefeldin A (2.5 μg/ml), tunicamycin (5 μg/ml), D6R (10 μm), cell permeable RVKR-CMK (50 μm), or dimethyl sulfoxide. For the pulse-chase experiments, following the 15-min pulse, the cells were chased for various periods in a medium containing excess unlabeled Met/Cys. The cells were lysed in modified radioimmune precipitation assay buffer (150 mm NaCl and 50 mm Tris-HCl, pH 7.5), 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and proteinase inhibitor mixture (Mixture Set I; Calbiochem). Cell lysates and media were immunoprecipitated with BMP10 Ab (2 μg) recognizing the mature form. Immunoprecipitates were resolved by SDS-PAGE on 8% Tricine gels and autoradiographed. Genetic evidence indicated that the proprotein convertases play key roles in cardiogenesis and left-right (furin and PACE4) or antero-posterior patterning (PC5/6) (5Essalmani R. Zaid A. Marcinkiewicz J. Chamberland A. Pasquato A. Seidah N.G. Prat A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5750-5755Crossref PubMed Scopus (93) Google Scholar, 6Szumska D. Pieles G. Essalmani R. Bilski M. Mesnard D. Kaur K. Franklyn A. El Omari K. Jefferis J. Bentham J. Taylor J.M. Schneider J.E. Arnold S.J. Johnson P. Tymowska-Lalanne Z. Stammers D. Clarke K. Neubauer S. Morris A. Brown S.D. Shaw-Smith C. Cama A. Capra V. Ragoussis J. Constam D. Seidah N.G. Prat A. Bhattacharya S. Genes Dev. 2008; 22: 1465-1477Crossref PubMed Scopus (109) Google Scholar, 7Constam D.B. Robertson E.J. Genes Dev. 2000; 14: 1146-1155PubMed Google Scholar, 8Constam D.B. Robertson E.J. Development. 2000; 127: 245-254Crossref PubMed Google Scholar). Indeed, deletion of the PC5/6 gene (Pcsk5) led to death at birth, with mice exhibiting multiple bone morphogenic defects and heart abnormalities, including ventricular-septal defects (5Essalmani R. Zaid A. Marcinkiewicz J. Chamberland A. Pasquato A. Seidah N.G. Prat A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5750-5755Crossref PubMed Scopus (93) Google Scholar, 6Szumska D. Pieles G. Essalmani R. Bilski M. Mesnard D. Kaur K. Franklyn A. El Omari K. Jefferis J. Bentham J. Taylor J.M. Schneider J.E. Arnold S.J. Johnson P. Tymowska-Lalanne Z. Stammers D. Clarke K. Neubauer S. Morris A. Brown S.D. Shaw-Smith C. Cama A. Capra V. Ragoussis J. Constam D. Seidah N.G. Prat A. Bhattacharya S. Genes Dev. 2008; 22: 1465-1477Crossref PubMed Scopus (109) Google Scholar). One of the in vivo specific PC5/6 substrates associated with patterning defects was identified as growth differentiating factor 11 (Gdf11) (5Essalmani R. Zaid A. Marcinkiewicz J. Chamberland A. Pasquato A. Seidah N.G. Prat A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5750-5755Crossref PubMed Scopus (93) Google Scholar, 6Szumska D. Pieles G. Essalmani R. Bilski M. Mesnard D. Kaur K. Franklyn A. El Omari K. Jefferis J. Bentham J. Taylor J.M. Schneider J.E. Arnold S.J. Johnson P. Tymowska-Lalanne Z. Stammers D. Clarke K. Neubauer S. Morris A. Brown S.D. Shaw-Smith C. Cama A. Capra V. Ragoussis J. Constam D. Seidah N.G. Prat A. Bhattacharya S. Genes Dev. 2008; 22: 1465-1477Crossref PubMed Scopus (109) Google Scholar). The PC5/6-specific processing of NTKRSRR295↓NLGLD in pro-Gdf11 required the presence of Asn at the P1′ position, as its replacement by Asp led to the loss of PC5/6 specificity (5Essalmani R. Zaid A. Marcinkiewicz J. Chamberland A. Pasquato A. Seidah N.G. Prat A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5750-5755Crossref PubMed Scopus (93) Google Scholar). We therefore hypothesized that the motifs (R/K)X(R/K)R↓N, (R/K)XXR↓N, or (R/K)XXXXR↓N may represent specific PC5/6 recognition sequences, as they fit the general basic aa-specific PC consensus motif (R/K)XnR↓, where Xn = 0, 2, or 4 aa (2Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar). Consequently, we undertook a bioinformatic analysis of all the genes coding for secretory proteins containing a similar luminal motif, and whose gene deletion results in heart defects. Interestingly, the only substrate matching these criteria and exhibiting a paired basic aa motif was BMP10 (supplemental Table S1). All the other hits contained a single Arg at the P1 cleavage site. We chose to synthesize five 12–13-aa peptides (shown in bold in supplemental Table S1) and tested their cleavage in vitro by furin, PC5/6, PACE4, or PC7. This prospective screening revealed that only BMP10 was cleaved to any significant extent by the PCs (not shown), making it the focus of the present study. Analysis of the BMP10 precursor (pro-BMP10) sequence (supplemental Fig. S1) strongly suggested that cleavage of the N-terminal prosegment at DSSARIRR313↓NAKG (mouse nomenclature) would release active BMP10. The prosegment contains two potential N-glycosylation sites at Asn67 and Asn131 and its size is predicted to be ∼50 kDa (supplemental Fig. S1). It was reported that, under non-reducing conditions, both purified human BMP10 precursor (pro-BMP10) and mature BMP10 migrate as a mixture of monomers and disulfide-linked homodimers (13Mazerbourg S. Sangkuhl K. Luo C.W. Sudo S. Klein C. Hsueh A.J. J. Biol. Chem. 2005; 280: 32122-32132Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Accordingly, we first expressed a recombinant human pro-BMP10 in COS-1 cells and analyzed the secreted products by SDS-PAGE under non-reducing conditions, followed by Western blotting using an antibody that only recognizes the native precursor and its mature BMP10 form (nonreducing conditions are recommended by the R&D manufacturer of the BMP10 antibody) (Fig. 1A, left panel). The data showed that endogenous enzymes in COS-1 cells are able to only partially process pro-BMP10 (present as monomer and dimer) into mature BMP10. However, co-expression of furin completely transformed both monomeric and dimeric forms of the precursor into the mature ∼17-kDa BMP10, which in our hands migrates as a monomer even under non-reducing, but denaturing conditions. Using the same antibody, Western blot analysis of left (LA) and right (RA) mouse heart atria extracts revealed the presence of mature ∼17-kDa BMP10 only in extracts of the RA (Fig. 1A, right panel). Because this antibody was raised in mouse, we could not identify with certainty the pro-BMP10 form, as it migrates close to non-reduced mouse IgGs recognized by the secondary anti-mouse HRP-labeled antibody (Fig. 1A, right panel). Nevertheless, the presence of the ∼17-kDa BMP10 only in heart RA extracts agrees with our QPCR analysis (Fig. 1B) and the previously reported in situ hybridization and QPCR analyses (11Chen H. Shi S. Acosta L. Li W. Lu J. Bao S. Chen Z. Yang Z. Schneider M.D. Chien K.R. Conway S.J. Yoder M.C. Haneline L.S. Franco D. Shou W. Development. 2004; 131: 2219-2231Crossref PubMed Scopus (385) Google Scholar), all of which revealed the restricted expression of BMP10 mRNA in adult mouse RA. In conclusion, mature BMP10 (∼17 kDa) is detected in mouse RA extracts and the cognate processing enzyme(s) of pro-BMP10 in the RA is/are yet to be defined. Thus, we first measured by QPCR the mRNA levels of the three plausible processing enzymes furin, PC5/6, and PACE4 in RA and LA of adult mice. Transcripts of the above three convertases were similarly expressed in both RA and LA (Fig. 1C), with furin and PACE4 as the major convertases found in this tissue. To define the best processing enzyme(s) of pro-BMP10, we first undertook an in vitro kinetic analysis of the cleavage of a 12-mer peptide mimicking the processing site of wild type (WT) mouse pro-BMP10: DSSARIRR313↓NAKG. This peptide was digested for 2 h with equal activity units of furin, PACE4, PC5/6, or PC7 and the products were separated by RP-HPLC and identified by mass spectroscopy (Fig. 2A). The data showed that 86% of the 12-mer peptide was cleaved by furin at the predicted Arg313↓ site, as compared with 67, 68, and 4% by PACE4, PC5/6, and PC7, respectively (Fig. 2B and supplemental Table S2A). Kinetic analyses revealed that furin is ∼3-fold more efficient in processing the 12-mer peptide as compared with P" @default.
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• W2022674779 title "Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10" @default.
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