title: Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains.
abstract: By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany, yvonne.kienast@roche.com.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Kienast
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Jucknischke
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Scheiblich
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Translational Technologies and Bioinformatics, Roche Innovation Center, Basel, 4070 Basel, Switzerland, and.
authors.forename: Martina
authors.initials: M
authors.lastname: Thier
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Translational Technologies and Bioinformatics, Roche Innovation Center, Basel, 4070 Basel, Switzerland, and.
authors.forename: Mariana
authors.initials: M
authors.lastname: de Wouters
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Large Molecule Research, Roche Innovation Center Penzberg, 82377 Penzberg Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Haas
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Lehmann
authors.affiliation: From the Roche Pharma Research and Early Development (pRED).
authors.forename: Verena
authors.initials: V
authors.lastname: Brand
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Bernicke
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany.
authors.forename: Konrad
authors.initials: K
authors.lastname: Honold
authors.affiliation: From the Roche Pharma Research and Early Development (pRED), Large Molecule Research, Roche Innovation Center Penzberg, 82377 Penzberg Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Lorenz
mesh_terms: D030301:Activin Receptors, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052006:Bone Morphogenetic Protein Receptors, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019281:Dimerization; D000071063:Endoglin; D005260:Female; D055427:Growth Differentiation Factor 2 / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D055412:Growth Differentiation Factors / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D010446:Peptide Fragments / Q000819:agonists / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011498:Protein Precursors / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013047:Specific Pathogen-Free Organisms
chemical_list: D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; C525887:GDF2 protein, human; C526108:Gdf2 protein, mouse; D055427:Growth Differentiation Factor 2; D055412:Growth Differentiation Factors; D010446:Peptide Fragments; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C579679:ACVR2B protein, human; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II; C498218:BMPR2 protein, human; D052006:Bone Morphogenetic Protein Receptors, Type II; C425585:activin receptor type II-A
keywords: bone morphogenetic protein (BMP); homeostasis; protein complex; surface plasmon resonance (SPR); transforming growth factor β (TGF-B)