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Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein.

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cam.issuedOnline2021-10-19
dc.contributor.authorRuiz-Llorente, Lidia
dc.contributor.authorVega, M Cristina
dc.contributor.authorFernández, Francisco J
dc.contributor.authorLanga, Carmen
dc.contributor.authorMorrell, Nicholas
dc.contributor.authorUpton, Paul
dc.contributor.authorBernabeu, Carmelo
dc.contributor.orcidMorrell, Nicholas [0000-0001-5700-9792]
dc.contributor.orcidUpton, Paul [0000-0003-2716-4921]
dc.contributor.orcidBernabeu, Carmelo [0000-0002-1563-6162]
dc.date.accessioned2021-10-21T02:30:16Z
dc.date.available2021-10-21T02:30:16Z
dc.date.issued2021-10-19
dc.date.updated2021-10-21T02:30:16Z
dc.description.abstractEndoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin.
dc.identifier.doi10.17863/CAM.77141
dc.identifier.eissn1422-0067
dc.identifier.issn1661-6596
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/329695
dc.languageen
dc.publisherMDPI AG
dc.subjectendoglin
dc.subjectGFP
dc.subjectsoluble endoglin
dc.subjectfusion protein
dc.subjectrecombinant protein
dc.subjectfluorescence
dc.subjectBMP
dc.subjectTGF-β
dc.subjectendothelium
dc.titleGeneration of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein.
dc.typeArticle
dcterms.dateAccepted2021-10-15
prism.issueIdentifier20
prism.publicationNameInt J Mol Sci
prism.volume22
pubs.funder-project-idMinisterio de Ciencia, Innovación y Universidades (SAF2013-43421-R)
pubs.funder-project-idConsejo Superior de Investigaciones Científicas (201920E022)
pubs.funder-project-idCentro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (ISCIII-CB06/07/0038)
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0/
rioxxterms.versionVoR
rioxxterms.versionofrecord10.3390/ijms222011282

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