Human Autoimmunity and Genetic Defects in N-Glycosylation

描述(申请人提供)：遗传信息从DNA流向大分子结构；-生命分子组织中的主导力量。然而，几乎所有后生动物的细胞表面和分泌的蛋白质都是通过在内质网/高尔基体分泌途径中添加复杂的碳水化合物来修饰的。我们发现，代谢产物对高尔基体N-糖基化途径的可获得性对细胞表面大分子复合体的组装施加自主控制，并在这种能力中，作用于信号和基因表达的上游，影响细胞的生长、分化和疾病状态。每个蛋白质分子的支链和N-糖链的数量共同调节与Galectins的结合，从而以可预测的方式调节表面糖蛋白的分布、聚集和内吞。N-糖基化的遗传干扰通过增强T细胞受体聚集/信号和减少生长抑制物CTLA-4的表面滞留而促进小鼠T细胞高活性和自身免疫性疾病。在人类中，将糖蛋白中N-糖链分支减少~20%的MGAT1单倍型与减少N-糖链数量~50%的CTLA-4等位基因协同作用，使多发性硬化症(MS)和类风湿性关节炎(RA)的风险增加~2倍。这两个变异体有望独立地降低CTLA-4与Galectins的亲和力，实际上，它们协同作用限制表面CTLA-4的表面水平。只有携带这两种变异的受试者才能观察到疾病的增加，而且似乎是通过有缺陷的CTLA-4等位基因的拷贝数来滴定的。对高尔基体的代谢补充抑制了小鼠的T细胞功能和自身免疫，并挽救了与MS和RA相关的T细胞生长中的N-糖基化缺陷和CTLA-4表面滞留。我们的数据表明，在人类自身免疫的病因学中，条件性抑制高尔基体GlcNAc分支的多态性和CTLA-4中N-X-S/T位点的使用之间存在协同作用，并为环境和遗传相互作用提供了治疗策略和分子机制。为了将这些结果推广到人类身上，我们提出了以下目标。具体目标1将确定改变N-糖链分支的遗传变异。特定目标2将研究MS相关等位基因对N-糖链分支的调节以及它们与目标1中确定的变异体的相互作用。特定目标3将研究改变N-糖链分支的变异体与CTLA-4 Ala17及相关等位基因之间的合作相互作用。特定目标4将检查多发性硬化症和1型糖尿病患者中特定目标1-3的变异之间的遗传相互作用。 公共卫生相关性：多发性硬化症和1型糖尿病是自身免疫性疾病，由个体的遗传背景和他/她的环境之间复杂的相互作用引起。我们的综合数据表明，控制蛋白质中特定糖添加的途径(即蛋白质糖基化)的遗传缺陷会导致免疫亢进，并促进小鼠和人类的自身免疫。用单糖代谢补充该途径可抑制免疫亢进和自身免疫的发展，这表明人类疾病可以通过代谢治疗来治疗/预防。

DESCRIPTION (provided by applicant): Genetic information flows from DNA to macromolecular structures;- the dominant force in the molecular organization of life. However, virtually all cell surface and secreted proteins in metazoans are modified by the addition of complex carbohydrates in the ER/Golgi secretory pathway. We find that metabolite availability to the Golgi N- glycosylation pathway exerts autonomous control over the assembly of macromolecular complexes on the cell surface, and in this capacity, acts upstream of signaling and gene expression to influence cell growth, differentiation and disease states. The branching and number of N-glycans per protein molecule cooperate to regulate binding to galectins and thereby the distribution, clustering and endocytosis of surface glycoproteins in a predictable manner. Genetic disruption of N-glycosylation promotes T cell hyper-activity and autoimmune disease in mice by enhancing T cell receptor clustering/signaling and reducing surface retention of the growth inhibitor CTLA-4. In humans, a haplotype of MGAT1 that reduces N-glycan branching in glycoproteins by ~20% synergistically interacts with an allele of CTLA-4 that reduces N-glycan number by ~50%, increasing the risk of Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA) by ~2 fold. The two variants are expected to independently reduce CTLA-4 affinity for galectins and indeed, they act cooperatively to limit surface CTLA-4 surface levels. Disease promotion is observed only in subjects who harbor both variants, and appears to be titrated by the number of copies of the defective CTLA-4 allele. Metabolic supplementation to the Golgi inhibits T cell function and autoimmunity in mice and rescues the N-glycosylation defects in T cell growth and CTLA-4 surface retention associated with MS and RA. Our data suggests synergism in the etiology of human autoimmunity between polymorphisms that conditionally suppress Golgi GlcNAc branching and N-X-S/T site usage in CTLA-4, and provides a therapeutic strategy and molecular mechanism for environmental and genetic interactions. To extend these results in humans we propose the following aims. Specific Aim 1 will identify genetic variants that alter N-glycan branching. Specific Aim 2 will investigate the regulation of N-glycan branching by MS associated alleles and their interaction with variants identified in Aim 1. Specific Aim 3 will investigate for co- operative interactions between variants that alter N-glycan branching and CTLA-4 Ala17 and related alleles. Specific Aim 4 will examine for genetic interaction of variants from Specific Aim's 1-3 in Multiple Sclerosis and Type 1 Diabetes co-horts. PUBLIC HEALTH RELEVANCE: Multiple Sclerosis and Type 1 Diabetes are autoimmune diseases resulting from complex interactions between genetic background of the individual and his/her environment. Our combined data suggests that genetic deficiency in a pathway that controls the addition of specific sugars to proteins (i.e. protein glycosylation) leads to immune hyperactivity and promotes autoimmunity in mice and humans. Metabolically supplementing the pathway with a simple sugar suppresses immune hyperactivity and development of autoimmunity, suggesting human disease may be treated/prevented with metabolic therapy.