Fig 2: Homozygous GDF2 truncation mutations lead to an absence of circulating BMP9 and pBMP10. (a,b) Levels of (a) BMP9 and (b) pBMP10 in plasma were measured with specific ELISAs. (a) Samples were assayed as 25% EDTA‐plasma, 0.2% goat‐serum, and 0.5% Triton X‐100 in BioRad ELISA buffer. (b) Samples were assayed as BMP9, but with inclusion of 4.5 mM EDTA. All samples were assayed in duplicate, each point represents a different individual. The dotted lines indicate the lower limits of quantification and dashed lines indicate the limits of detection for these assays. (c‐e) Endothelial serum BMP activity, representing BMP9 and BMP10, was assayed in the stable HMEC1‐BRE reporter line. Serum‐starved cells were incubated with 2%‐10% serum for 6 hr and luciferase activity then measured. Samples were measured in duplicate, and the assay was repeated on three separate occasions. Data are presented as mean of the averaged luciferase values for each individual experiment ± SEM. The dotted line indicates the mean SFM response. Individual graphs show: (c) control adult female samples from three separate individuals (FC1‐3), (d) p.[Gln26Ter]:[Gln26Ter] patient sample and control male samples from four separate individuals (MC1‐4), and (e) p.[Glu279Ter]:[Glu279Ter] patient sample and samples from unaffected p.[Glu279Ter] heterozygous parents. Data from all individuals were compared by a multiple comparisons two‐way ANOVA using GraphPad Prism 9. p < 0.001 is relative to all individual controls

Fig 3: M-form BMP9 can bind to ALK1 and prodomain as the D-form. A, M- and D- forms of HBMP9 co-migrate on native PAGE, and both can form complexes with ALK1 ECD. B, M- and D-forms of BMP9 co-migrate, both as free form and as prodomain-bound form. In A and B, bands 1 to 6 from native PAGE were cut out, boiled in 2× SDS-loading buffer for 20 min before loading onto a non-reducing SDS-PAGE to confirm the identities. For SDS-PAGE in A, two parts of the same gel are shown.

Fig 4: Intermolecular disulfide bond is not required for BMP9 signaling activity. A, conditioned media (6 μl) from HEK-EBNA cells transfected with transfection reagent alone (lane 1), empty vector (lane 2), pro-BMP9 (lane 3), pro-HBMP9 (lane 4), or pro-HBMP9 C73S (lane 5) were fractionated on a 12% SDS-PAGE and probed with anti-BMP9 prodomain antibody. The intensities of bands were quantified using ImageJ, and the ratio of HBMP9 C73S to HBMP9 obtained. HBMP9 C73S concentration in the conditioned medium was normalized to HBMP9 using the above ratio. B, left: hPAECs were serum-restricted in EGM-2, 0.1% FBS overnight, and stimulated with HBMP9 or HBMP9 C73S (both 0.25–3 ng/ml) for 1 h. Cells were harvested, and total cell protein was immunoblotted with anti-pSmad1/5/8 antibody. Band intensities of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the α-tubulin blot, and normalized to a 0.25 ng/ml wild type sample. Data of the mean ± S.E. from three repeats are shown on the right. C, after quiescence overnight in EGM-2, 0.1% FBS, hPAECs were treated with 1 ng/ml of HBMP9 or HBMP9 C73S. Samples were harvested at 1, 2, 6, and 24 h, and immunoblotting was carried out as described in B. Band intensity of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the α-tubulin blot and normalized to a wild type 1-h treatment sample. Data of the mean ± S.E. from three repeats are shown on the right. NS, not significant.

Fig 5: Expression, activity, and purification of recombinant BMP9. A, schematic diagram of BMP9 production and processing. B, schematic diagram of the generation of HBMP9. A His tag (filled star) is introduced at the beginning of the mature BMP9. C, conditioned medium from HEK-EBNA cells transfected with prepro-BMP9 (lane 1) or prepro-HBMP9 (lane 2) were separated on a non-reducing SDS-PAGE and probed with anti-BMP9 antibody (MAB3209). The diagram on the right shows the schematic drawing of the BMP9 molecules. D denotes dimer on non-reducing SDS-PAGE, and M denotes monomer on SDS-PAGE. D- and M-forms migrate slower in the HBMP9 due to the addition of the His6 tag at the N terminus of the mature ligand as depicted in B. D, HEK cell-produced BMP9 and HBMP9 have comparable activity with BMP9 from R&D Systems. Conditioned media containing pBMP9 (because it is very likely to be present as a prodomain bound complex in the conditioned media) or pHBMP9 were quantified by ELISA using R&D BMP9 as a standard and subjected to signaling assay using C2C12 cells transfected with ALK1. Plasmid containing Renilla was co-transfected with reporter plasmid containing BMP response element-luciferase, and the luciferase activity induced by BMP9 signaling was read using the Promega Dual-Luciferase system. E, purified pBMP9 and HBMP9 were fractionated on a 12% non-reducing SDS-PAGE and stained by Coomassie Blue. F, identical samples of R&D Systems BMP9, pBMP9, and HBMP9 were run in parallel on three 12% non-reducing SDS-PAGE, then blotted separately against the following: anti-BMP9 antibody (left), anti-BMP9 prodomain antibody (middle), and anti-His tag antibody (right). A single asterisk indicates a minor band that could not be seen on the SDS-PAGE in E but reacted very strongly with anti-BMP9 antibody. This may be a species of partially processed BMP9. Double asterisks indicate nonspecific carrier protein from R&D Systems BMP9. Pro, prodomain.