The Fifth People's Hospital of Shanghai, The Shanghai Key Laboratory of Medical Epigenetics, The International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.
Institute of Pediatrics of Children's Hospital of Fudan University, Shanghai, 201102, China.
Biochem Biophys Res Commun. 2022 Aug 20;617(Pt 1):42-47. doi: 10.1016/j.bbrc.2022.05.067. Epub 2022 May 31.
Hematopoietic stem cell (HSC) transplantation represents an important curative therapy for numerous hematological and immune diseases. Many efforts have been applied to achieve attainable ex vivo HSC expansion. We previously showed that angiopoietin-like proteins 2 (Angptl2) binds and activates the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support the expansion of HSC. However, soluble Angptl2 is unstable and the downstream signaling would be attenuated by ligand-binding triggered receptor endocytosis, compromising the potential of Angptl2 to expand HSCs. We proposed that membrane anchored Angptl2 will overcome these limitations. In this study, we constructed the C-terminal and N-terminal anchored membrane Angptl2 (Cm-Angptl2 and Nm-Angptl2) by adding a transmembrane domain at the C-terminal or an anchor sequence at the N-terminal respectively. Both forms of Angptl2 showed efficient expression on the surface of feeder cells. Nm-Angptl2, but not Cm-Angptl2, induces a potent activation of LILRB2 reporter, indicating the fibronectin (FBN) domain at the C-terminus of Angptl2 is essential to stimulate LILRB2 signaling. Compared to soluble Angptl2, Nm-Angptl2 displays higher activities to activate LILRB2 reporter, and to promote the expansion of mouse HSCs as determined by transplantation and limiting dilution assay. Our study revealed the importance of FBN domain for Angptl2 to activate LILRB2 and demonstrated that Nm-Angptl2 have enhanced activities than the soluble protein in LILRB2 activation and HSC expansion, providing a strategy to explore the mode of ligand induced receptor signaling, and an optimized approach to expand HSCs ex vivo.
造血干细胞（HSC）移植是治疗许多血液系统和免疫系统疾病的重要治疗方法。人们已经做出了许多努力来实现可实现的体外 HSC 扩增。我们之前曾表明，血管生成素样蛋白 2（Angptl2）与免疫抑制受体人类白细胞免疫球蛋白（Ig）样受体 B2（LILRB2）结合并激活，以支持 HSC 的扩增。然而，可溶性 Angptl2 不稳定，配体结合触发的受体内吞会减弱下游信号，从而削弱 Angptl2 扩增 HSC 的潜力。我们提出膜锚定的 Angptl2 将克服这些限制。在这项研究中，我们通过在 C 端添加跨膜结构域或在 N 端添加锚序列，构建了 C 端和 N 端锚定的膜 Angptl2（Cm-Angptl2 和 Nm-Angptl2）。两种形式的 Angptl2 都能有效地在饲养细胞表面表达。Nm-Angptl2 而非 Cm-Angptl2 诱导 LILRB2 报告基因的有效激活，表明 Angptl2 的 C 末端纤维连接蛋白（FBN）结构域对于刺激 LILRB2 信号很重要。与可溶性 Angptl2 相比，Nm-Angptl2 激活 LILRB2 报告基因的活性更高，并且通过移植和有限稀释测定确定，它更能促进小鼠 HSC 的扩增。我们的研究揭示了 FBN 结构域对于 Angptl2 激活 LILRB2 的重要性，并证明 Nm-Angptl2 在激活 LILRB2 和扩增 HSC 方面比可溶性蛋白具有更高的活性，为探索配体诱导受体信号的模式以及优化体外扩增 HSC 的方法提供了策略。
