Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein
Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi mathvariant=&q...
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| Format: | Article |
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MDPI AG
2021-10-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/20/11282 |
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| author | Lidia Ruiz-Llorente M. Cristina Vega Francisco J. Fernández Carmen Langa Nicholas W. Morrell Paul D. Upton Carmelo Bernabeu |
| author_facet | Lidia Ruiz-Llorente M. Cristina Vega Francisco J. Fernández Carmen Langa Nicholas W. Morrell Paul D. Upton Carmelo Bernabeu |
| author_sort | Lidia Ruiz-Llorente |
| collection | DOAJ |
| description | Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi mathvariant="sans-serif">β</mi></semantics></math></inline-formula> (TGF-<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi mathvariant="sans-serif">β</mi></semantics></math></inline-formula>)/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. |
| first_indexed | 2024-03-10T06:29:45Z |
| format | Article |
| id | doaj.art-600c446356be499ba3670854f6ecc105 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T06:29:45Z |
| publishDate | 2021-10-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-600c446356be499ba3670854f6ecc1052023-11-22T18:37:00ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-10-0122201128210.3390/ijms222011282Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent ProteinLidia Ruiz-Llorente0M. Cristina Vega1Francisco J. Fernández2Carmen Langa3Nicholas W. Morrell4Paul D. Upton5Carmelo Bernabeu6Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, SpainCentro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, SpainCentro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, SpainCentro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, SpainDepartment of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UKDepartment of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UKCentro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, SpainEndoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi mathvariant="sans-serif">β</mi></semantics></math></inline-formula> (TGF-<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi mathvariant="sans-serif">β</mi></semantics></math></inline-formula>)/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.https://www.mdpi.com/1422-0067/22/20/11282endoglinGFPsoluble endoglinfusion proteinrecombinant proteinfluorescence |
| spellingShingle | Lidia Ruiz-Llorente M. Cristina Vega Francisco J. Fernández Carmen Langa Nicholas W. Morrell Paul D. Upton Carmelo Bernabeu Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein International Journal of Molecular Sciences endoglin GFP soluble endoglin fusion protein recombinant protein fluorescence |
| title | Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein |
| title_full | Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein |
| title_fullStr | Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein |
| title_full_unstemmed | Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein |
| title_short | Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein |
| title_sort | generation of a soluble form of human endoglin fused to green fluorescent protein |
| topic | endoglin GFP soluble endoglin fusion protein recombinant protein fluorescence |
| url | https://www.mdpi.com/1422-0067/22/20/11282 |
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