Structure and Function in alpha-Dystroglycan Glycosylation

摘要 先天性肌营养不良症的一种，影响超过1：100,000人和 包括患者后遗症的一系列疾病，从轻度肌营养不良症到接近正常的 严重肌营养不良症的寿命，最初几年有严重的神经和眼睛缺陷导致死亡 生活的一部分。绝大多数基因定义的糖代谢不良症是编码基因突变的结果。 O-甘露糖化途径的酶，特别是所谓的M3途径。迄今为止，M3通路， 它由大约12种酶组成，只被发现在一种 外周膜蛋白α-营养不良聚糖上的一小部分苏氨酸残基参与了一种 跨质膜将细胞外基质与肌动蛋白细胞骨架连接起来的复合体。在前一次 在资助期内，我们和其他人阐明了M3结构，该结构终止于参考的重复二糖 为我们的母亲干杯。我们最近已经证明，只有Matriglycan，没有潜在的M3糖蛋白和 α-dystrogycan既是与含有lg结构域的蛋白质结合的必要条件也是充分条件，以及促进 某些病毒的结合和感染，如拉萨病毒。在这里，我们寻求解决剩余的关键问题 基础科学问题，特别是那些有助于早期临床研究的问题，包括底物 增强治疗、AAV基因转移治疗和新的基因识别。因此，我们将测试其特异性 对M3酶的研究，包括测试M3多聚糖是否可以构建在其他非O-Man多聚糖结构上(A1)。 进一步测试特异性，并解决为一个人构建完整的M3通路的进化困境 对于蛋白质底物，我们将测试其他蛋白质是否含有M3糖链结构，这可能参与 某些糖代谢不良症的表型(A1)。受我们理解结构-功能的愿望所驱使 M3酶的关系以及告知临床合作者，我们将调查错义的影响 M3酶的变种在稳定性和活性方面使用我们建立的方案(A2)。此外，给出了 L276I FKTN变种的高载波频率(北欧为1：150)，我们将调查 携带该变异体的杂合子和纯合子细胞系不太容易受到依赖于Matriglycan的影响 假病毒感染(A2)。最后，鉴于约三分之一的血糖营养不良症是由未知的遗传病因引起的， 我们将在相关的人类细胞系中进行无偏见的CRISPR/Cas9筛选，以确定新的基因产物 这是功能性糖基化α-营养不良糖链(A3)所必需的。再加上这种不偏不倚的做法，我们 我还将研究合成CDP-核糖醇的途径，这是合成M3多糖所必需的，以确定 所涉及的基因产品可能导致糖代谢不良症(A3)的一部分未知病例。 拟议工作的完成将提供对M3途径酶的更全面的了解 和蛋白质靶点，变异在疾病和感染中的作用，以及为临床合作者的工作提供信息。

SUMMARY Dystroglycanopathies, a subset of congenital muscular dystrophies, impact more than 1:100,000 individuals and encompass a range of disorders with patient sequalae ranging from mild muscular dystrophy with near normal lifespan to severe muscular dystrophy with major neural and ocular defects leading to death in the first few years of life. The vast majority of genetically-defined dystroglycanopathies are a result of mutations in genes encoding enzymes of the O-mannosylation pathway, specifically the so-called M3 pathway. To date, the M3 pathway, which consists of approximately a dozen enzymes, has only been found to elaborate O-mannose glycans on a small subset of threonine residues on the peripheral membrane protein alpha-dystroglycan that is involved in a complex that bridges the extracellular matrix to the actin cytoskeleton across the plasma membrane. In the prior funding period, we and others elucidated the M3 structure that terminates in a repeating disaccharide referred to as matriglycan. We have recently demonstrated that matriglycan alone, absent the underlying M3 glycan and alpha-dystrolgycan, is both necessary and sufficient to bind LG domain-containing proteins as well as facilitate binding and infection by certain arenaviruses, such as Lassa virus. Here, we seek to address key remaining basic science issues especially those that would facilitate early stage clinical investigations, including substrate enhancement therapy, AAV gene transfer therapy, and novel gene identification. Thus, we will test the specificity of M3 enzymes including testing whether M3 glycans can be built on other non-O-Man glycan structures (A1). Further testing specificity and to address the evolutionary quandary of building an entire M3 pathway for one protein substrate, we will test whether other proteins contain M3 glycan structures, which could be involved in the phenotypes of certain dystroglycanopathies (A1). Driven by our desire to understand the structure-function relationship of M3 enzymes as well as to inform clinical collaborators, we will investigate the impact of missense variants on M3 enzymes with regards to stability and activity using our established protocols (A2). Further, given the high carrier frequency (1:150 in Northern Europe) of the L276I FKTN variant, we will investigate whether heterozygous and homozygous cell lines harboring this variant are less susceptible to matriglycan-dependent pseudovirus infection (A2). Finally, given that ~1/3 of all dystroglycanopathies are of unknown genetic etiology, we will conduct an unbiased CRISPR/Cas9 screen in relevant human cell lines to identify novel gene products that are required for functional glycosylation of alpha-dystroglycan (A3). Coupled to this unbiased approach, we will also investigate the pathway for CDP-ribitol synthesis, that is necessary for M3 glycan synthesis, to identify gene products involved that may be responsible for a subset of the unknown cases of dystroglycanopathy (A3). Completion of the proposed work will provide a more comprehensive understanding of the M3 pathway enzymes and protein targets, the role of variants in disease and infection, as well as inform the work of clinical collaborators.