N-glycosylation as a downstream effector of Interleukin-7

描述（由申请人提供）：白细胞介素-7 （IL-7）信号传导对几种血细胞类型的发育和外周维持至关重要。IL-7或其受体任一组分的缺乏会导致主要的造血缺陷，导致小鼠出现T细胞阴性（T-）、B细胞阴性（B-）、自然杀伤细胞阴性（NK-）严重联合免疫缺陷（SCID）。在人类中，已发现IL-7受体α (IL-7Ra)基因突变可引起T-、B+、NK+ SCID，最近发现IL-7Ra基因的单核苷酸多态性与自身免疫有关。此外，IL-7已被证明是外周T细胞稳态、存活和功能的关键介质。然而，IL-7多种功能背后的分子机制仍然知之甚少。我们的目标是揭示IL-7信号通路调节t细胞发育和外周功能的新机制，即通过调节n -糖基化。在这些领域的洞察力可能导致新的治疗途径，无论是自身免疫和先天性，获得性和诱导淋巴细胞减少症。IL-7的作用与t细胞受体（TCR）信号传导错综复杂，因此IL-7的许多作用与TCR信号强度的改变有关。n -糖基化是一种影响几乎所有细胞表面和分泌蛋白的共翻译修饰。细胞表面糖蛋白与凝集素的相互作用与附着的n -聚糖的数量和分支成比例，形成一个分子晶格，全局调节表面受体的浓度和信号传导。在t细胞中，n -聚糖分支对抗TCR聚集和信号传导，抑制t细胞生长和自身免疫。我们的初步数据表明，IL-7信号传导导致t细胞中n -聚糖分支的剧烈变化。IL-7Ra突变小鼠的t细胞n -聚糖分支增加了两到三倍，这是除高尔基酶基因操作外观察到的最大变化之一。预计这种高糖基化会显著抑制TCR信号强度，从而抑制TCR信号介导的发育、生存和增殖。我们假设IL-7通过改变n -聚糖分支来调节t细胞的发育和生长。为了验证这一假设，我们提出通过额外敲除Mgat1和Mgat5这两个关键的n -聚糖分支酶来“拯救”IL-7Ra缺陷小鼠的超糖基化。这将使我们能够通过比较IL-7Ra-/-小鼠与糖基化酶额外缺陷小鼠的这些过程，直接评估n -糖基化在IL-7介导的造血发育、外周功能和体内平衡中的作用。这些研究将提供有关IL-7和TCR信号相互作用的见解，从而更好地了解淋巴细胞室的发育和维持。此外，识别n -糖基化在IL-7信号传导中的作用可能为免疫缺陷和自身免疫性疾病提供新的治疗途径。

DESCRIPTION (provided by applicant): Interleukin-7 (IL-7) signaling is essential for the development and peripheral maintenance of several blood cell types. Deficiencies in IL-7 or either component of its receptor result in major hematopoietic defects, causing T-cell-negative (T-), B-cell-negative (B-), natural killer cell-negative (NK-) severe combined immunodeficiency (SCID) in mice. In humans, mutations in the IL-7 receptor alpha (IL-7Ra) gene have been found to cause T-, B+, NK+ SCID, and more recently a single nucleotide polymorphism in the IL-7Ra gene has been associated with autoimmunity. In addition, IL-7 has been shown to be a critical mediator of peripheral T- cell homeostasis, survival, and function. Nevertheless, the molecular mechanism underlying the diverse functions of IL-7 remain poorly understood. We aim to uncover a new mechanism by which IL-7 signaling may regulate both T-cell development and peripheral function, namely through regulation of N-glycosylation. Insight in these areas may lead to new therapeutic avenues for both autoimmunity and congenital, acquired and induced lymphopenias. The roles of IL-7 are intricately involved with T-cell receptor (TCR) signaling, such that many of the effects of IL-7 correlate with alterations in TCR signal strength. N-glycosylation is a co-translational modification that affects nearly all cell-surface and secreted proteins. Cell surface glycoproteins interact with galectins in proportion to the number and branching of attached N-glycans, forming a molecular lattice that globally regulates the concentration and signaling of surface receptors. In T-cells, N-glycan branching opposes TCR clustering and signaling to suppress T-cell growth and autoimmunity. Our preliminary data demonstrate that IL-7 signaling leads to dramatic changes in N-glycan branching in T-cells. IL-7Ra mutant mice display a two to three fold increase in T-cell N-glycan branching, among the largest changes observed outside of genetic manipulation of Golgi enzymes. This hyper-glycosylation is predicted to markedly inhibit TCR signal strength and thus TCR signal mediated development, survival, and proliferation. We hypothesize that IL-7 regulates T-cell development and growth by altering N-glycan branching. To examine this hypothesis we propose to "rescue" hyper-glycosylation in IL-7Ra deficient mice by additionally knocking Mgat1 and Mgat5, two key N-glycan branching enzymes. This will allow us to directly asses the role of N-glycosylation in IL-7 mediated hematopoietic development, peripheral function, and homeostasis by comparing these processes in IL-7Ra-/- mice to mice with additional defect in glycosylation enzymes. These studies will provide insights about the interaction of IL-7 and TCR signaling, leading to a greater understanding of the development and maintenance of the lymphoid compartment. Furthermore, discerning the role of N-glycosylation in IL-7 signaling may provide new therapeutic avenues for both immunodeficiencies and autoimmune disease. PUBLIC HEALTH RELEVANCE: Defects in the development and growth of white blood cells impact many blood and immunological disorders. This proposal examines the signals that control the development and growth of white blood cells, which may lead to better therapies for disorders as diverse as infection, immunodeficiency and autoimmunity.