N-glycosylation as a downstream effector of Interleukin-7

说明(申请人提供)：白介素7(IL-7)信号对几种血细胞类型的发育和外周维持是必不可少的。IL-7或其受体的任何一种成分的缺陷都会导致严重的造血缺陷，导致小鼠T细胞阴性(T-)、B细胞阴性(B-)、自然杀伤细胞阴性(NK-)的严重联合免疫缺陷(SCID)。在人类中，已发现IL-7受体α(IL-7ra)基因突变导致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细胞动态平衡、生存和功能的关键介质。然而，IL-7不同功能的分子机制仍然知之甚少。我们的目标是揭示一种新的机制，通过IL-7信号调节T细胞发育和外周功能，即通过调节N-糖基化。对这些领域的深入了解可能会为自身免疫和先天性、获得性和诱导性淋巴细胞减少症开辟新的治疗途径。IL-7的作用复杂地涉及T细胞受体(TCR)信号，因此IL-7的许多作用与TCR信号强度的变化相关。N-糖基化是一种共翻译修饰，几乎影响所有细胞表面和分泌的蛋白质。细胞表面糖蛋白与Galectins相互作用的比例与连接的N-糖链的数量和分支成正比，形成一个分子格子，全局调节表面受体的浓度和信号。在T细胞中，N-葡聚糖分支反对TCR聚集和抑制T细胞生长和自身免疫的信号。我们的初步数据表明，IL-7信号导致T细胞中N-糖链分支的戏剧性变化。IL-7Ra突变小鼠的T细胞N-糖链分支增加了两到三倍，这是在高尔基体酶遗传操作之外观察到的最大变化之一。这种高糖基化被预测为显著抑制TCR信号强度，从而抑制TCR信号介导的发育、存活和增殖。我们假设IL-7通过改变N-糖链的分支来调节T细胞的发育和生长。为了验证这一假说，我们建议通过额外敲击两种关键的N-糖链分支酶--mGAT1和mGAT5来“挽救”IL-7Ra缺陷小鼠的高糖基化。这将使我们能够通过比较IL-7Ra-/-小鼠和糖基化酶额外缺陷的小鼠的这些过程，直接评估N-糖基化在IL-7介导的造血发育、外周功能和体内稳定中的作用。这些研究将提供关于IL-7和TCR信号相互作用的见解，从而更好地理解淋巴系统的发育和维持。此外，识别N-糖基化在IL-7信号转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。 与公共卫生相关：白细胞发育和生长的缺陷会影响许多血液和免疫疾病。这项建议研究了控制白细胞发育和增长的信号，这可能会导致对感染、免疫缺陷和自身免疫等各种疾病的更好治疗。

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.