Verona Erik V, Tang Yuping, Millstead Thomas K, Hinck Andrew P, Agyin Joseph K, Sun Lu-Zhe
Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, TX 78229, USA.
Protein Eng Des Sel. 2008 Jul;21(7):463-73. doi: 10.1093/protein/gzn023. Epub 2008 May 21.
Transforming growth factor beta (TGFbeta) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFbeta isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFbeta type II (RII) and betaglycan (BG) have been utilized as TGFbeta inhibitors, their antagonistic potency against different TGFbeta isoforms varies considerably because RII does not appreciably bind to TGFbeta2 whereas BG binds weakly to TGFbeta1 and TGFbeta3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BG(E)) and the extracellular domain of RII. The fusion protein (named BG(E)RII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFbeta1 and TGFbeta3 than a commercially available soluble RII and to TGFbeta2 than a commercially available soluble BG. More significantly, whereas BG(E) or RII alone showed no antagonistic activity towards TGFbeta2, BG(E)RII inhibited the signaling of both TGFbeta1 and TGFbeta2 in cell-based assays including TGFbeta-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFbeta-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BG(E)RII was found to have greater activity than other potent TGFbeta inhibitors in blocking the signaling of TGFbeta1 and TGFbeta3. Thus, BG(E)RII is a potent pan-TGFbeta inhibitor in vitro and has potential for blocking TGFbeta-induced pathogenesis in vivo.
已知转化生长因子β（TGFβ）亚型在某些疾病进展过程中会上调。它们在致癌过程中可刺激侵袭和转移，过度刺激时会促进许多病理性纤维化疾病。TGFβ亚型参与晚期癌症和病理性纤维化，使其成为治疗干预的主要靶点。尽管TGFβ II型（RII）和β聚糖（BG）的可溶性胞外域已被用作TGFβ抑制剂，但它们对不同TGFβ亚型的拮抗效力差异很大，因为RII与TGFβ2结合不明显，而BG与TGFβ1和TGFβ3结合较弱。在本研究中，我们成功构建并表达了一种包含BG的内皮糖蛋白结构域（BG(E)）和RII胞外域的重组融合蛋白。该融合蛋白（命名为BG(E)RII）通过固定化金属离子色谱从细菌包涵体中纯化出来，进行重折叠并表征。它与TGFβ1和TGFβ3的结合亲和力高于市售可溶性RII，与TGFβ2的结合亲和力高于市售可溶性BG。更显著的是，尽管单独的BG(E)或RII对TGFβ2没有拮抗活性，但在基于细胞的试验中，包括TGFβ诱导的Smad2和Smad3磷酸化以及TGFβ反应性启动子的转录，BG(E)RII比等摩尔浓度的RII或BG更有效地抑制TGFβ1和TGFβ2的信号传导。通过凝胶过滤色谱进一步纯化后，发现BG(E)RII在阻断TGFβ1和TGFβ3信号传导方面比其他强效TGFβ抑制剂具有更高的活性。因此，BG(E)RII在体外是一种强效的泛TGFβ抑制剂，具有在体内阻断TGFβ诱导的发病机制的潜力。
