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.
Highlights
IntroductionHuman endoglin is a type I integral membrane protein with a large (561 amino acids) glycosylated extracellular region, a single hydrophobic transmembrane domain, and a short cytosolic domain [1]
InInorder to to obtain a fusion protein between endoglin and theand enhanced green fluoorder obtain a fusion protein between endoglin the enhanced green flurescent protein (EGFP), we cloned the extracellular domain of endoglin in expression orescent protein (EGFP), we cloned the extracellular domain of endoglin invecexpression tor pEGFP–N1 to generate the pEGFP–N1/Eng.EC
Soluble endoglin encompasses most of the ectodomain of membrane-bound endoglin, and is involved in several cardiovascular and metabolic pathologies [2,5,52]
Summary
Human endoglin is a type I integral membrane protein with a large (561 amino acids) glycosylated extracellular region, a single hydrophobic transmembrane domain, and a short cytosolic domain [1]. In addition to the membrane-bound form, a soluble form of endoglin (sEng) can be generated by the action of the metalloproteases (MMP) MMP-14 or MMP-12 on the ectodomain of the membrane protein [7,8,9,10]. Abnormal levels of sEng were found in several endothelium-related pathological conditions, including preeclampsia [11,12], hypercholesterolemia and atherosclerosis [5,13], diabetes mellitus [14], hypertension [15], diabetic retinopathy [16], coronary artery disease [17], hereditary hemorrhagic telangiectasia [18,19], acute myocardial infarction [20]
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