Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, Spain.
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28040 Madrid, Spain.
Int J Mol Sci. 2021 Oct 19;22(20):11282. doi: 10.3390/ijms222011282.
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.
内皮糖蛋白（Eng，CD105）是一种 I 型膜糖蛋白，在血管内皮细胞中作为转化生长因子 β（TGF-β）/骨形态发生蛋白（BMP）家族成员的辅助受体，以及整合素配体发挥作用，调节血管病理生理学。除了膜结合的内皮糖蛋白（Eng）外，还有一种可溶性形式的内皮糖蛋白（sEng），它可以由基质金属蛋白酶（MMP）-14 或 -12 在其细胞外结构域的近膜区域作用产生。已有报道称，子痫前期、高胆固醇血症、动脉粥样硬化和癌症患者体内 sEng 水平升高。此外，sEng 是高血压和糖尿病患者心血管损伤的标志物，在子痫前期中发挥致病作用，并抑制癌症中的血管生成和肿瘤增殖、迁移和侵袭。然而，sEng 的作用机制尚未阐明，需要新的工具和实验方法来推动该领域的发展。为此，我们旨在获得 sEng 的荧光形式，作为生物成像的新工具。因此，我们将内皮糖蛋白的细胞外结构域克隆到 pEGFP-N1 质粒中，生成与绿色荧光蛋白（GFP）融合的融合蛋白，从而产生 pEGFP-N1/Eng.EC。通过转染 CHO-K1 细胞，使用荧光显微镜、SDS-PAGE、免疫检测和 ELISA 技术对重组融合蛋白进行了鉴定。在转染 pEGFP-N1/Eng.EC 后，细胞中很容易检测到荧光，表明重组蛋白中包含的 GFP 已正确折叠到细胞质中。此外，如 Western blot 分析所示，分泌的融合蛋白产生了预期的分子量，并显示出特异性的荧光信号。融合蛋白还能够在体外结合 BMP9 和 BMP10。因此，本文所述的构建体可用作内皮糖蛋白细胞外结构域体外功能研究的工具。
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