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Structural and functional insights into endoglin ligand recognition and binding.

Alt A, Miguel-Romero L, Donderis J, Aristorena M, Blanco FJ, Round A, Rubio V, Bernabeu C, Marina A - PLoS ONE (2012)

Bottom Line: The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition.These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin.In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, Valencia, Spain.

ABSTRACT
Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

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Functional interactions between endoglin, ALK1 and BMP-9.The interactions of (A) EndoEC, (B) ALK1EC, (E) Endo338 and (F) Endo362 with BMP-9 were investigated by SPR. While HG-EndoEC and Endo362 dissociated slowly, Endo338 dissociated much faster, indicating a rigid body type of binding as opposed to an induced fit mechanism. For affinity measurements, the indicated recombinant proteins were injected at six concentrations ranging from 12.5 to 400 nM over BMP-9 (which was immobilized on a CM5 sensor chip by amine coupling) to generate sensorgrams (colored curves). When testing competition between HG-EndoEC and HG-ALK1EC (C and D) the chip was first pre-equilibrated with 750 mM of either HG-EndoEC (C, left) or HG-ALK1EC (D, left) before injecting the various concentration of the second ligand, showing the curve for the highest concentrations of the 2nd ligand. Both HG-ALK1EC (C, right) and HG-EndoEC (D, right) yielded, after subtracting the background, similar results to those in runs in which no first ligand was preequilibrated before injecting the second ligand (D right vs. E; C right vs. B). This leads to the conclusion that endoglin and ALK1 bind independently to different sites on BMP-9. The kinetic parameters for the interaction were determined by global fitting (curves in black) of the 1∶1 Langmuir binding model to these data, providing values for the association (ka) and dissociation (kd) rate constants and the dissociation affinity constant (KD).
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pone-0029948-g002: Functional interactions between endoglin, ALK1 and BMP-9.The interactions of (A) EndoEC, (B) ALK1EC, (E) Endo338 and (F) Endo362 with BMP-9 were investigated by SPR. While HG-EndoEC and Endo362 dissociated slowly, Endo338 dissociated much faster, indicating a rigid body type of binding as opposed to an induced fit mechanism. For affinity measurements, the indicated recombinant proteins were injected at six concentrations ranging from 12.5 to 400 nM over BMP-9 (which was immobilized on a CM5 sensor chip by amine coupling) to generate sensorgrams (colored curves). When testing competition between HG-EndoEC and HG-ALK1EC (C and D) the chip was first pre-equilibrated with 750 mM of either HG-EndoEC (C, left) or HG-ALK1EC (D, left) before injecting the various concentration of the second ligand, showing the curve for the highest concentrations of the 2nd ligand. Both HG-ALK1EC (C, right) and HG-EndoEC (D, right) yielded, after subtracting the background, similar results to those in runs in which no first ligand was preequilibrated before injecting the second ligand (D right vs. E; C right vs. B). This leads to the conclusion that endoglin and ALK1 bind independently to different sites on BMP-9. The kinetic parameters for the interaction were determined by global fitting (curves in black) of the 1∶1 Langmuir binding model to these data, providing values for the association (ka) and dissociation (kd) rate constants and the dissociation affinity constant (KD).

Mentions: BMP-9 has been described as the ligand of ALK1 [18], [36] and endoglin [28]. In order to evaluate the importance of the glycosylation of these receptors for binding to BMP-9, we carried out surface plasmon resonance (SPR) binding assays with EndoEC protein produced in both HEK293S GnTI-, which incorporates only short, well defined sugars rendering low glycosylated proteins (LG-EndoEC), and HEK293 FreeStyle, which produce mature, branched sugars and proteins with high glycosylation (HG-EndoEC) (Figure 2A). Sensor chip-immobilized BMP-9 bound LG-EndoEC and HG-EndoEC with apparent equilibrium dissociation constants (KD) of 2 nM and 10 nM respectively (Table 1, rows 1–2), indicating that recruitment of endoglin is mainly independent of the glycosylation state. The somewhat lower KD value of the glycosylated endoglin form comes from both a lower avidity (ka∼0.5 times lower) and a faster dissociation (kd∼2.2-fold higher) (Table 1), suggesting that the sugar impairs to some extent the interaction of endoglin with BMP-9. This result also demonstrates that endoglin recognizes and binds BMP-9 in a highly efficient and selective manner on its own, at variance with other TGF-β family members that require the assistance of a type I TGF-β receptor [37], [38].


Structural and functional insights into endoglin ligand recognition and binding.

Alt A, Miguel-Romero L, Donderis J, Aristorena M, Blanco FJ, Round A, Rubio V, Bernabeu C, Marina A - PLoS ONE (2012)

Functional interactions between endoglin, ALK1 and BMP-9.The interactions of (A) EndoEC, (B) ALK1EC, (E) Endo338 and (F) Endo362 with BMP-9 were investigated by SPR. While HG-EndoEC and Endo362 dissociated slowly, Endo338 dissociated much faster, indicating a rigid body type of binding as opposed to an induced fit mechanism. For affinity measurements, the indicated recombinant proteins were injected at six concentrations ranging from 12.5 to 400 nM over BMP-9 (which was immobilized on a CM5 sensor chip by amine coupling) to generate sensorgrams (colored curves). When testing competition between HG-EndoEC and HG-ALK1EC (C and D) the chip was first pre-equilibrated with 750 mM of either HG-EndoEC (C, left) or HG-ALK1EC (D, left) before injecting the various concentration of the second ligand, showing the curve for the highest concentrations of the 2nd ligand. Both HG-ALK1EC (C, right) and HG-EndoEC (D, right) yielded, after subtracting the background, similar results to those in runs in which no first ligand was preequilibrated before injecting the second ligand (D right vs. E; C right vs. B). This leads to the conclusion that endoglin and ALK1 bind independently to different sites on BMP-9. The kinetic parameters for the interaction were determined by global fitting (curves in black) of the 1∶1 Langmuir binding model to these data, providing values for the association (ka) and dissociation (kd) rate constants and the dissociation affinity constant (KD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275592&req=5

pone-0029948-g002: Functional interactions between endoglin, ALK1 and BMP-9.The interactions of (A) EndoEC, (B) ALK1EC, (E) Endo338 and (F) Endo362 with BMP-9 were investigated by SPR. While HG-EndoEC and Endo362 dissociated slowly, Endo338 dissociated much faster, indicating a rigid body type of binding as opposed to an induced fit mechanism. For affinity measurements, the indicated recombinant proteins were injected at six concentrations ranging from 12.5 to 400 nM over BMP-9 (which was immobilized on a CM5 sensor chip by amine coupling) to generate sensorgrams (colored curves). When testing competition between HG-EndoEC and HG-ALK1EC (C and D) the chip was first pre-equilibrated with 750 mM of either HG-EndoEC (C, left) or HG-ALK1EC (D, left) before injecting the various concentration of the second ligand, showing the curve for the highest concentrations of the 2nd ligand. Both HG-ALK1EC (C, right) and HG-EndoEC (D, right) yielded, after subtracting the background, similar results to those in runs in which no first ligand was preequilibrated before injecting the second ligand (D right vs. E; C right vs. B). This leads to the conclusion that endoglin and ALK1 bind independently to different sites on BMP-9. The kinetic parameters for the interaction were determined by global fitting (curves in black) of the 1∶1 Langmuir binding model to these data, providing values for the association (ka) and dissociation (kd) rate constants and the dissociation affinity constant (KD).
Mentions: BMP-9 has been described as the ligand of ALK1 [18], [36] and endoglin [28]. In order to evaluate the importance of the glycosylation of these receptors for binding to BMP-9, we carried out surface plasmon resonance (SPR) binding assays with EndoEC protein produced in both HEK293S GnTI-, which incorporates only short, well defined sugars rendering low glycosylated proteins (LG-EndoEC), and HEK293 FreeStyle, which produce mature, branched sugars and proteins with high glycosylation (HG-EndoEC) (Figure 2A). Sensor chip-immobilized BMP-9 bound LG-EndoEC and HG-EndoEC with apparent equilibrium dissociation constants (KD) of 2 nM and 10 nM respectively (Table 1, rows 1–2), indicating that recruitment of endoglin is mainly independent of the glycosylation state. The somewhat lower KD value of the glycosylated endoglin form comes from both a lower avidity (ka∼0.5 times lower) and a faster dissociation (kd∼2.2-fold higher) (Table 1), suggesting that the sugar impairs to some extent the interaction of endoglin with BMP-9. This result also demonstrates that endoglin recognizes and binds BMP-9 in a highly efficient and selective manner on its own, at variance with other TGF-β family members that require the assistance of a type I TGF-β receptor [37], [38].

Bottom Line: The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition.These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin.In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, Valencia, Spain.

ABSTRACT
Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

Show MeSH
Related in: MedlinePlus