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.
Highlights
To shed further light on the biological function of pro-domain-complexed bone morphogenic protein 9 (BMP9), we investigated the dynamics of mature BMP9 or BMP91⁄7pro-domain complex interactions with the major BMP9 receptor activin receptor-like kinase 1 (ALK1), type II receptors BMP receptor II (BMPRII), ActRIIA, ActRIIB, co-receptor ENG, or mature BMP9-domain targeting antibodies by real-time surface plasmon resonance (SPR)
BMP91⁄7Pro-domain Binding to ALK1, Type II Receptors, ENG, or Anti-mature BMP9 Antibodies Led to Complete Displacement of Pro-domains—To investigate binding kinetics of the different BMP9 variants, Biacore experiments were performed using the ECD of ALK1 linked to a Fc domain of human IgG (ALK1-Fc)
Like BMP9, other TGF family members were shown to remain associated with their pro-domains after secretion, including TGF, BMP2, BMP4, BMP5, BMP7, BMP10, GDF5, and myostatin (GDF8) [26]
Summary
Blood Donors—Human plasma from healthy individuals of Western European descent (n ϭ 17) was obtained from Indivumed (Hamburg, Germany). BMP91⁄7Pro-domain Binding to ALK1, Type II Receptors, ENG, or Anti-mature BMP9 Antibodies Led to Complete Displacement of Pro-domains—To investigate binding kinetics of the different BMP9 variants, Biacore experiments were performed using the ECD of ALK1 linked to a Fc domain of human IgG (ALK1-Fc). After the association of the ligand, detection antibodies recognizing either mature BMP9 (MAB3209) or the pro-domain (AF3879) were administered in a subsequent injection leading to a ternary complex in case of successful binding (Fig. 7A). Mature and precursor BMP9 were directly detectable by ELISA, BMP91⁄7pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and prodomains after antibody binding (Fig. 8A). Our results establish a model of complete displacement of the non-covalently bound pro-domains after binding of BMP91⁄7pro-domain to ALK1 and type II receptors (or anti-mature BMP9 antibodies) leading to rapid activation of the signaling process (Fig. 12)
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