Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth - PubMed (original) (raw)
. 2011 Aug 26;286(34):30034-46.
doi: 10.1074/jbc.M111.260133. Epub 2011 Jul 7.
Eric D Werner, Robert G Matthews, Eleonora Presman, Aaron W Mulivor, Nicolas Solban, Dianne Sako, R Scott Pearsall, Kathryn W Underwood, Jasbir Seehra, Ravindra Kumar, Asya V Grinberg
Affiliations
- PMID: 21737454
- PMCID: PMC3191044
- DOI: 10.1074/jbc.M111.260133
Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth
Roselyne Castonguay et al. J Biol Chem. 2011.
Abstract
Endoglin (CD105), a transmembrane protein of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.
Figures
FIGURE 1.
Cloning, expression, and purification of mEngECD-mFc 27–581 and hEngECD-hFc 26–586. The mEngECD-mFc 27–581 (A–C) and hEngECD-hFc 26–586 (D–F) were cloned in pAID4 vector, expressed, and purified as described under “Experimental Procedures.” A and D, shown is a schematic representation of the mouse (A) and human (D) endoglin ECD fusion constructs with the boundaries of each domain proposed by the electron microscopy model for the human ECD construct (31) or by primary sequence alignment for the mouse ECD construct. B and E, shown are silver-stained SDS-PAGE gels of the purified mouse (B) and human (E) fusion proteins in CHO cells. Lane 1, reduced with β-mercaptoethanol (R); lane 2, non-reduced (NR). The molecular weight markers are indicated on the left of the gel. C and F, analytical SEC of the purified mouse (C) and human (F) fusion proteins expressed in CHO cells on a TSK G3000 column markers is shown. ZP-Cterm, C-terminal portion of the ZP domain; ZP-Nterm, N-terminal portion of the ZP domain.
FIGURE 2.
Kinetic analysis of BMP9 and BMP10 binding to mEngECD-mFc 27–581 and hEngECD-hFc 26–586 performed on Biacore 3000 and T100 at 20 °C. mEngECD-mFc 27–581 (A and B) and hEngECD-hFc 26–586 (C and D) produced in CHO cells were captured on an anti-mFc or anti-hFc IgG CM5 chip, and different concentrations of BMP9 (0.0195–0.625 n
m
(A); 0.0391–0.625 n
m
(C)) and BMP10 (0.0195–1.25 n
m
(B); 0.00977–1.25 n
m
(D)) were injected in duplicate over captured endoglin proteins. The raw data (black lines) are overlaid with a global fit to a 1:1 model with mass transport limitations (red lines) obtained by BIAevaluation software. RU, resonance units. E, summary of kinetic constants obtained for mEngECD-mFc 27–581 and hEngECD-hFc 26–586 expressed in CHO cells binding to BMP9 and BMP10 ligands, and relative potency of endoglin fusion proteins to antagonize BMP9/BMP10 induced signaling in A204 cells.
FIGURE 3.
Effect of truncations in the ECD on hEngECD-hFc binding to BMP9 and BMP10 determined by Biacore. A, hEngECD-hFc 26–359 secondary structure prediction, starting at amino acid 240, generated by Psipred and Jalview software (32, 33), and location of truncations performed at the C terminus of the orphan domain are shown. The α-helix is represented by a cylinder, and the β-sheet is represented by an arrow. B, shown is a schematic representation of hEngECD-hFc truncations with their constituent domains. Each fusion protein was purified from HEK293-transfected conditioned media by affinity chromatography and evaluated for binding to BMP9 and BMP10 by SPR. The endoglin fusion proteins were captured at high density (∼500–600 resonance units) on an anti-hFc IgG chip. BMP9 and BMP10 were injected at a concentration of 10 n
m
over each construct for detection of any significant binding. For those constructs showing binding activity with BMP9 and BMP10, a high resolution kinetic analysis was performed (Table 1). ZP-Cterm, C-terminal portion of the ZP domain; ZP-Nterm, N-terminal portion of the ZP domain.
FIGURE 4.
Truncated hEngECD-hFc 26–359 expressed in CHO cells retains high affinity binding to BMP9 and BMP10 in the Biacore assay and is a potent inhibitor of BMP9- and BMP10-induced signaling in A204 cells. A, silver-stained SDS-PAGE analysis of the purified hEngECD-hFc 26–359 protein under reduced (first lane, R) and non-reduced (second lane, NR) conditions is shown. The molecular mass markers are shown on the left of the gel. B, analytical SEC of the purified hEngECD-hFc 26–359 protein is shown. C and D, shown is kinetic characterization of BMP9 (C) and BMP10 (D) binding to hEngECD-hFc 26–359 measured by SPR (Biacore T100). Raw data (black lines) are overlaid with global fits to a 1:1 model with mass transport term (red lines) obtained by BIAevaluation software. RU, resonance units. E, kinetic rate constants and affinities determined for the BMP9 and BMP10 binding to hEngECD-hFc 26–359 and relative potency of hEngECD-hFc 26–359 in A204 cell-based assay are shown.
FIGURE 5.
hEngECD-hFc 26–359 and type II receptors bind to overlapping sites on BMP9. The hEngECD-hFc 26–359 (CHO-produced) was captured onto an anti-hFc IgG chip, then a mixture of BMP9 with buffer, mAlk1-mFc (2.5 or 50 n
m
) (A) or mActRIIB-mFc (1, 2.5 n
m
) (C) was injected in a solution inhibition assay. In a Biacore epitope exclusion assay, the hEngECD-hFc 26–359 was captured, then subsequent injections of BMP9 and buffer, mAlk1-mFc, or hAlk1-His (100 n
m
) (B) or mActRIIB-mFc (100 n
m
) (D) were performed. The inset is a magnification of the type I and II receptor injections. RU, resonance units. E, shown is a proposed model for the mode of action of endoglin ECD. The hEngECD-hFc 26–359 binds to the type II receptor binding site on BMP9, blocking access of a type II receptor to the ligand and impairing the formation of the ternary complex necessary for signaling.
FIGURE 6.
Endoglin ECD is able to inhibit angiogenesis in vivo in the CAM assay and in angioreactors as well as decrease tumor burden in a colon-26 cancer model. A, shown is blood vessel count with VEGF (50 ng) as the angiogenic factor and 3 doses of 14 μg of mEngECD-hFc 27–581 (NS0-produced; R&D Systems). B, shown is blood vessel count with VEGF (50 ng) as the angiogenic factor and 3 doses (20 μg each) of CHO-produced hEngECD-hFc 26–359. C, silicone angioreactors were removed from the mice after 11 days of treatment with mEngECD-mFc 27–581 (from CHO cells). Blood vessel sprouting into the angioreactors appears dark red. D, quantification of vessel formation in the angioreactors was done by injecting FITC-dextran into the animals before euthanasia. The amount of fluorescence in the angioreactor correlates to the amount of blood vessels present. In the presence of angiogenic growth factors, angioreactors from animals treated with mEngECD-mFc 27–581 had less relative fluorescence than the vehicle-treated controls. E, tumor volume, measured by digital calipers, in a mouse model of colon-26 cancer treated with modified TBS or mEngECD-mFc 27–581 is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
References
- ten Dijke P., Goumans M. J., Pardali E. (2008) Angiogenesis 11, 79–89 - PubMed
- López-Novoa J. M., Bernabeu C. (2010) Am. J. Physiol. Heart Circ Physiol 299, H959–H974 - PubMed
- McAllister K. A., Grogg K. M., Johnson D. W., Gallione C. J., Baldwin M. A., Jackson C. E., Helmbold E. A., Markel D. S., McKinnon W. C., Murrell J., McCormick M. K., Pericak-Vance M. A., Heutink P., Oostra B. A., Haitjema T., Westerman C. J. J., Porteous M. E., Guttmacher A. E., Letarte M., Marchuk D. A. (1994) Nat. Genet. 8, 345–351 - PubMed
- Lux A., Gallione C. J., Marchuk D. A. (2000) Hum. Mol. Genet. 9, 745–755 - PubMed
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