T lymphocyte-specific gene targeting of Notch receptor glycosyltransferase fringe

Notch 受体糖基转移酶边缘的 T 淋巴细胞特异性基因靶向

• 批准号: 16590407
• 负责人: KISHIHARA Kenji
• 金额: $ 2.24万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590407/
• 关键词: Notch; lunatic fringe; glycosylation; T lymphocyte development; gene targeting; cell fate decision; thymic organ culture; cell sutonomous regulation; ジーン・ターゲティング; 糖鎖転移酵素; 細胞系譜決定

Notch1, one of Notch receptors, is essential for T cell development from lymphoid precursor. For instance, Notch1-deficient mice completely lack T cell development. Interactions between Notch receptors and their ligands are generally affected by glycosylation of Notch receptors, and enzymes to glycosylate them are fringes including three members in mammals : lunatic, manic and radical fringes. However, the role of glycosylation of Notch1 during T cell development has not been fully understood. By searching genes that dynamically change their expression during T cell development, we found that lunatic fringe (Lfg) showed different expression levels during the maturation process of thymocytes. To clarify an involvement of Lfg in T cell differentiation, we attempted to generate T lymphocyte-specifically Lfg-deficient mice using conditional gene targeting technology. Unfortunately the other group in Canada has produced the Lfg knockout mice in the way of our project. Finally, we changed our strategy to address our aim and we got the following results. The culture of CD4^-CD8^- thymocytes in which Lfg was ectopically expressed by using fetal thymic organ culture enhanced differentiation of immature CD8^+ T cells while inhibiting CD4^+CD8^- mature T-cell development. we found that Lfg that generally strengthens the interaction between Notch and Notch ligands (Delta family) regulates T cell development probably affecting the glycosylation of Notch1. Interestingly, the high expression of Lfg in lymphoid precursor regulates T cell development by strengthening Notch signaling of their own (cell autonomous) and inhibits the development toward T cells from neighboring precursors by competing limited amounts of Notch ligands in the thymus (non-autonomous). These results indicate that Lfg has novel roles to set the threshold of Notch signaling to promote T cell development in a cell autonomous and non-autonomous manner.

Notch1 是 Notch 受体之一，对于淋巴前体 T 细胞的发育至关重要。例如，Notch1 缺陷的小鼠完全缺乏 T 细胞发育。 Notch受体与其配体之间的相互作用通常受到Notch受体糖基化的影响，而糖基化它们的酶是边缘酶，在哺乳动物中包括三种成员：疯狂边缘、躁狂边缘和激进边缘。然而，Notch1 糖基化在 T 细胞发育过程中的作用尚未完全了解。通过搜索在T细胞发育过程中动态改变表达的基因，我们发现lunatic fringe（Lfg）在胸腺细胞的成熟过程中表现出不同的表达水平。为了阐明 Lfg 在 T 细胞分化中的作用，我们尝试使用条件基因靶向技术产生 T 淋巴细胞特异性 Lfg 缺陷小鼠。不幸的是，加拿大的另一个小组已经按照我们项目的方式生产了 Lfg 基因敲除小鼠。最后，我们改变了策略来实现我们的目标，并得到了以下结果。使用胎儿胸腺器官培养异位表达Lfg的CD4^-CD8^-胸腺细胞的培养物增强了未成熟CD8^+T细胞的分化，同时抑制了CD4^+CD8^-成熟T细胞的发育。我们发现 Lfg 通常会增强 Notch 和 Notch 配体（Delta 家族）之间的相互作用，调节 T 细胞发育，可能影响 Notch1 的糖基化。有趣的是，Lfg在淋巴前体细胞中的高表达通过加强自身的Notch信号传导（细胞自主性）来调节T细胞的发育，并通过竞争胸腺中有限数量的Notch配体（非自主性）来抑制邻近前体细胞向T细胞的发育。这些结果表明，Lfg 具有设定 Notch 信号阈值的新作用，从而以细胞自主和非自主方式促进 T 细胞发育。