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

Abstract

Endoglin (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.

Frozen bean curd-inspired xenogeneic acellular dermal matrix with triple pretreatment approach of freeze–thaw, laser drilling and ADSCs pre-culture for promoting early vascularization and integration

“…Angiopoietin-Tie2 signaling plays a critical role in morphogenesis and homeostasis of blood vessels and in vascular remodeling [ 27 ]. CD105 is predominantly expressed on proliferating HUVECs [ 28 ]. Compared with group O, the gene expressions of Tie-2 and CD105 were up-regulated in group F ( Fig.…”

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbβ3 integrin

“…Recombinant human endoglin/CD105 (sEng; R&D Systems, #1097-EN/CF; Supplementary Figure S1) was used for functional in vitro assays with platelets. For measuring protein-protein interactions by surface plasmon resonance, a streptavidin-tagged construct (sEng-Strep-tag II) of soluble endoglin [44] was used.…”

Prognostic value of soluble endoglin in patients with septic shock and severe COVID-19

“…However, infectious complications are the most common cause of death in these patients (35%) ( 17 ). Under certain conditions (ischemia, oxidative stress), the extracellular part of Eng is cleaved by metalloproteinase 14 to form circulating soluble endoglin (sEng) ( 18 , 19 ). sEng is speculated to have rather antiangiogenic effects ( 20 , 21 ) (unlike Eng).…”