Endoglin Human, His

What is the molecular structure of human endoglin and how does it interact with ligands?

Human endoglin (CD105) is a 180 kDa homodimeric transmembrane glycoprotein that functions as an auxiliary receptor for the transforming growth factor β (TGF-β) superfamily. Crystal structure analysis reveals that endoglin contains an N-terminal orphan domain with a unique duplicated fold generated by circular permutation, which provides a hydrophobic surface for interaction with bone morphogenetic protein 9 (BMP9). The C-terminal zona pellucida module suggests that two copies of endoglin embrace homodimeric BMP9, allowing for ligand recognition by type I receptors but preventing interaction with type II receptors. This structural arrangement plays a crucial role in the BMP signaling cascade . When working with recombinant His-tagged endoglin, researchers should note that the tag generally doesn't interfere with this binding interface but may need validation in specific experimental contexts.

What role does endoglin play in TGF-β signaling pathways?

Endoglin serves as a critical determinant of TGF-β-induced Smad1/5 phosphorylation but not of TGF-β-induced Smad2 phosphorylation in endothelial cells. Experimental evidence shows that siRNA-mediated knockdown of endoglin blocks TGF-β-induced activity of BRE2-Luc (a specific reporter for Smad1/5 activation) and inhibits expression of ID1 and ID2 genes, which are downstream targets of the TGF-β/ALK1 pathway . Endothelial cells derived from endoglin heterozygous embryos exhibit reduced TGF-β signaling, with TGF-β-induced Smad1/5 phosphorylation being absent compared to wild-type cells . For researchers studying this pathway, generating reporter cell lines expressing His-tagged endoglin mutants can be valuable for mapping critical functional residues involved in signaling modulation.

How can researchers optimize purification protocols for recombinant human endoglin with His-tag?

For optimal purification of His-tagged human endoglin, researchers should employ a multi-step approach beginning with immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ matrices. Since endoglin forms homodimers and contains multiple glycosylation sites, expression in mammalian systems (HEK293 or CHO cells) rather than bacterial systems is recommended to ensure proper folding and post-translational modifications. After initial IMAC purification, size-exclusion chromatography should be employed to separate dimeric from monomeric forms and remove aggregates. For studying interactions with BMP9, researchers should verify that the His-tag doesn't interfere with the hydrophobic interaction surface in the N-terminal orphan domain . Quality control should include SDS-PAGE under reducing and non-reducing conditions to confirm homodimer formation, as well as functional binding assays with known ligands such as BMP9/10.

What are effective strategies for studying the proteolytic cleavage of endoglin to generate soluble endoglin?

To study proteolytic cleavage of endoglin, researchers should design experiments that monitor both membrane-bound and soluble forms simultaneously. Recent findings reveal that thrombin can cleave endoglin at specific sites, in addition to previously known metalloprotease cleavage mechanisms . For in vitro studies, researchers can use purified endoglin incubated with candidate proteases, followed by Western blot analysis to identify specific fragments. N- and C-terminal sequencing of these fragments can confirm predicted cleavage sites . For cellular models, treating endoglin-expressing cells with thrombin or other proteases and measuring released soluble endoglin by ELISA while simultaneously quantifying cell surface endoglin reduction by flow cytometry provides a comprehensive assessment of cleavage dynamics. Site-directed mutagenesis of predicted cleavage sites in His-tagged endoglin constructs allows for mechanistic validation of specific protease recognition sequences.

How should researchers design experiments to investigate endoglin's role in endothelial cell proliferation and angiogenesis?

When investigating endoglin's role in endothelial cell proliferation and angiogenesis, researchers should employ both loss-of-function and gain-of-function approaches. For loss-of-function, siRNA-mediated knockdown of endoglin has been successfully used to demonstrate its critical role in TGF-β/ALK1 signaling . Alternatively, CRISPR/Cas9 gene editing can generate stable endoglin-knockout cell lines, though complete knockout may affect cell survival as evidenced by the failure to establish endothelial cell lines from endoglin knockout embryos . For gain-of-function studies, overexpression of His-tagged endoglin allows for controlled experiments with pulldown capability. Functional assays should include endothelial tube formation on Matrigel, scratch wound healing assays, and BrdU incorporation for proliferation. For angiogenesis assessment, fetal bone metatarsal assays and developmental angiogenesis in zebrafish embryos have been effectively used to demonstrate the effects of combining endoglin and VEGF targeting .

How does endoglin modulate the crosstalk between TGF-β and other angiogenic pathways?

Substantial crosstalk exists between the endoglin and vascular endothelial growth factor (VEGF) pathways. To investigate this experimentally, researchers should examine the effects of endoglin manipulation on VEGF pathway components and vice versa. In vitro studies have shown that TRC105 (anti-endoglin antibody) increases VEGF-induced ERK1/2 phosphorylation in human umbilical vein endothelial cells (HUVECs) . Additionally, in colorectal cancer patient samples, endothelial Smad1 phosphorylation increased upon anti-VEGF therapy, suggesting compensatory activation . For comprehensive pathway analysis, researchers should employ phosphoproteomics approaches to identify changes in signaling networks when endoglin is manipulated. Reporter gene assays using pathway-specific response elements (like BRE-luc for BMP/Smad1/5 signaling) can quantitatively measure pathway modulation. RNA-seq analysis of endoglin-overexpressing or knockdown cells treated with different TGF-β family ligands and VEGF can reveal global transcriptional changes and pathway interactions.

What protein complexes does endoglin form, and how can they be effectively isolated and characterized?

Endoglin forms homodimers and associates with multiple TGF-β family receptors and ligands. To isolate and characterize these complexes, researchers should implement co-immunoprecipitation using antibodies against endoglin or its His-tag when using recombinant protein. Cross-linking approaches prior to complex isolation can stabilize transient interactions. For identifying novel binding partners, proximity labeling methods such as BioID or APEX2 fused to endoglin can be powerful, allowing for identification of proteins in close proximity under physiological conditions. Mass spectrometry analysis of isolated complexes can reveal both known and novel interaction partners. Structural characterization can be achieved through techniques like cryo-electron microscopy, particularly for larger complexes that may be challenging for X-ray crystallography. The structural data showing how two copies of endoglin embrace homodimeric BMP9 provides a foundation for designing experiments to investigate how these complexes form and function dynamically.

How does endoglin heterozygosity affect TGF-β signaling in endothelial cells?

Endoglin heterozygosity significantly impacts TGF-β signaling in endothelial cells. Experimental evidence shows that TGF-β-induced Smad1/5 phosphorylation is absent in endoglin heterozygous cell lines but occurs normally in cell lines derived from wild-type embryos. Consistently, TGF-β-induced expression of Id1, a specific downstream target gene of the TGF-β/ALK1 pathway, is induced in endoglin wild-type endothelial cells but not in endoglin heterozygous cell lines . Additionally, TGF-β-induced Smad2 phosphorylation is reduced by approximately half in endoglin heterozygous endothelial cells . To investigate this experimentally, researchers should utilize endothelial cells derived from endoglin heterozygous models, or employ precise gene dosage control using inducible expression systems. Comprehensive signaling analysis should include phosphorylation status of multiple Smads (Smad1/5/8 and Smad2/3), expression of pathway-specific target genes, and functional readouts like endothelial cell migration, proliferation, and tube formation.

How can researchers effectively model hereditary hemorrhagic telangiectasia type 1 (HHT1) using endoglin mutations?

To model hereditary hemorrhagic telangiectasia type 1 (HHT1), researchers should incorporate mutations identified in HHT1 patients into their experimental systems. The crystal structure of endoglin reveals that the BMP9 interaction interface involves residues mutated in HHT1 , providing structural insights for mutation selection. For cellular models, CRISPR/Cas9 gene editing can introduce specific patient mutations into endothelial cells. Induced pluripotent stem cells (iPSCs) derived from HHT1 patients or engineered with HHT1 mutations can be differentiated into endothelial cells to study developmental aspects. For in vivo models, endoglin heterozygous mice exhibit some HHT1 features, though complete knockout is embryonically lethal . Zebrafish models offer advantages for high-throughput screening of vascular phenotypes. When studying His-tagged endoglin variants carrying HHT1 mutations, researchers should assess their ability to form homodimers, bind BMP9/10, and participate in TGF-β receptor complexes, as these functions may be compromised by disease-causing mutations.

What are the optimized methodologies for measuring soluble endoglin as a biomarker in patient samples?

For clinical research using soluble endoglin (sEng) as a biomarker, standardized measurement protocols are essential. ELISA remains the gold standard for quantifying sEng in serum, plasma, or urine samples. When developing or selecting assays, researchers should consider antibody specificity for different sEng fragments, as thrombin and metalloproteases produce distinct cleavage products . Western blot analysis can identify specific endoglin fragments consistent with different protease-mediated cleavage events . For preeclampsia research, longitudinal sampling is valuable as sEng increases precede clinical manifestations. In diabetic adolescents, sEng concentrations increase in parallel with endothelial dysfunction before subclinical structural vascular alterations become evident, suggesting its value as an early biomarker . Researchers should correlate sEng levels with other markers of endothelial dysfunction, such as flow-mediated dilation (FMD) of the brachial artery and plasma nitric oxide concentrations , to establish its validity as part of a comprehensive biomarker panel.

How can researchers investigate endoglin's role in fibrosis progression in chronic kidney disease models?

To investigate endoglin's role in fibrosis progression, researchers should examine both human biopsy samples and experimental models. In human studies, measuring renal endoglin expression in biopsies from different types of chronic kidney disease (CKD) has revealed that endoglin is upregulated in chronic allograft dysfunction and diabetic nephropathy compared to control kidneys . Importantly, interstitial endoglin expression correlates with estimated glomerular filtration rate (eGFR) and the amount of interstitial fibrosis, independent of the specific diagnosis . For in vitro studies, overexpression of endoglin in TGF-β-stimulated human kidney fibroblasts enhances expression of fibrosis markers ACTA2, CCN2, and SERPINE1, as well as extracellular matrix components collagen type I (COL1A1) and fibronectin (FN1) . Researchers should employ both genetic approaches (siRNA knockdown, CRISPR/Cas9) and pharmacological interventions (anti-endoglin antibodies) to modulate endoglin function in fibrosis models. For in vivo studies, unilateral ureteral obstruction or adenine-induced kidney injury in endoglin heterozygous mice can provide insights into how endoglin levels affect fibrosis progression.

How should researchers address contradictory findings regarding endoglin's role in BMP signaling?

Contradictory findings regarding endoglin's role in BMP signaling require careful experimental design and interpretation. While high overexpression of endoglin and BMP receptors has suggested a functional link, siRNA-mediated knockdown of endoglin does not affect BMP-induced Smad1/5 phosphorylation, arguing against a physiological role for endoglin in BMP signaling . To resolve such contradictions, researchers should:

• Employ multiple complementary approaches (siRNA, CRISPR/Cas9, dominant-negative constructs)

• Use physiologically relevant expression levels and cell types

• Distinguish between direct effects on signaling and indirect effects on receptor availability or trafficking

• Consider cell-type specific cofactors that might influence endoglin's function

• Examine dose-dependent effects, as high concentrations may produce non-physiological interactions

A systematic approach comparing endoglin's effects on different BMP family members across multiple endothelial cell types can help resolve contradictory findings and establish context-dependent functions.

What technical challenges arise when studying membrane-bound versus soluble endoglin, and how can they be overcome?

Studying membrane-bound versus soluble endoglin presents several technical challenges. The structural heterogeneity of soluble endoglin suggests the involvement of multiple proteases beyond the well-characterized metalloproteases, including thrombin . To overcome these challenges:

• Employ detection methods that distinguish between different soluble endoglin fragments using domain-specific antibodies

• Use recombinant His-tagged endoglin with differential N- and C-terminal tags to track cleavage patterns

• Implement protease inhibitor panels to identify responsible proteases in different contexts

• Develop cell models with endogenous endoglin tagged with fluorescent proteins to monitor trafficking and release in real-time

• For clinical samples, use multiple antibodies targeting different endoglin epitopes to ensure comprehensive detection

When interpreting results, researchers should consider that different soluble endoglin fragments may have distinct biological activities, potentially explaining contradictory findings regarding soluble endoglin's effects on TGF-β signaling.

How can researchers distinguish between endoglin-specific effects and indirect effects through altered TGF-β signaling?

Distinguishing between direct endoglin-specific effects and indirect effects through altered TGF-β signaling requires sophisticated experimental approaches:

• Use receptor-binding deficient endoglin mutants that maintain structural integrity but cannot participate in TGF-β receptor complexes

• Employ endoglin constructs with mutations in specific domains to determine which interactions are required for observed phenotypes

• Implement rescue experiments in endoglin-depleted cells using endoglin variants with differential binding abilities to TGF-β family ligands

• Compare immediate (minutes to hours) versus delayed (hours to days) responses to identify direct signaling versus secondary transcriptional effects

• Use pathway-specific inhibitors to determine whether endoglin effects persist when downstream signaling is blocked

For comprehensive analysis, researchers should combine genetic approaches with phosphoproteomics and transcriptomics to map the immediate signaling events triggered by endoglin manipulation versus secondary pathway alterations. Time-course experiments are particularly valuable in distinguishing primary from secondary effects.