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）信号传导对于几种血细胞类型的发育和外周维持至关重要。IL-7或其受体的任一组分的缺陷导致主要造血缺陷，在小鼠中引起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-7 Ra）基因中的突变引起T-、B+、NK+ SCID，并且最近IL-7 Ra基因中的单核苷酸多态性已与自身免疫相关。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的多种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，IL-7信号可以调节T细胞发育和外周功能，即通过调节N-糖基化。在这些领域的洞察力可能会导致新的治疗途径的自身免疫和先天性，后天性和诱导淋巴细胞减少症。 IL-7的作用与T细胞受体（TCR）信号传导错综复杂地相关，使得IL-7的许多作用与TCR信号强度的改变相关。N-糖基化是影响几乎所有细胞表面和分泌蛋白的共翻译修饰。细胞表面糖蛋白与半乳糖凝集素的相互作用与连接的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聚集和信号传导以抑制T细胞生长和自身免疫。我们的初步数据表明，IL-7信号转导导致T细胞中N-聚糖分支的显著变化。IL-7 Ra突变小鼠显示T细胞N-聚糖分支增加2至3倍，这是在高尔基体酶的遗传操作之外观察到的最大变化之一。预计这种高糖基化显著抑制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-聚糖分支来调节T细胞的发育和生长。为了检验这一假设，我们提出通过另外敲除Mgat 1和Mgat 5（两种关键的N-聚糖分支酶）来“拯救”IL-7 Ra缺陷小鼠中的高糖基化。这将使我们能够通过比较IL-7 Ra-/-小鼠与糖基化酶有额外缺陷的小鼠中的这些过程来直接评估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。这些研究将提供关于IL-7和TCR信号传导的相互作用的见解，从而更好地理解淋巴区室的发育和维持。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。 公共卫生相关性：白色血细胞发育和生长缺陷会影响许多血液和免疫系统疾病。这项提案研究了控制白色血细胞发育和生长的信号，这可能会导致更好的治疗疾病，如感染，免疫缺陷和自身免疫。

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.