Structure and Function in alpha-Dystroglycan Glycosylation

α-肌营养不良聚糖糖基化的结构和功能

• 批准号: 10678139
• 负责人: Lance Wells
• 金额: $ 41.83万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678139
• 关键词: Actins; Address; Affect; Animals; Antibodies; Arenavirus; Basic Science; Binding; Biological Assay; CRISPR screen; Cardiac; Cell Line; Cell membrane; Cells; Cessation of life; Clinical; Complex; Coupled; Cytoskeleton; Data; Defect; Dimerization; Disaccharides; Disease; Disseminated Malignant Neoplasm; Duchenne muscular dystrophy; Dystrophin; Ensure; Enzyme Tests; Enzymes; Extracellular Matrix; Extracellular Matrix Proteins; Frequencies; Functional disorder; Funding; Gene Mutation; Gene Transfer; Gene therapy trial; Genes; Genetic Predisposition to Disease; Glycopeptides; Glycoproteins; Goals; Heterozygote; Human; Human Cell Line; Impairment; In Vitro; Individual; Infection; Intervention; Kinetics; Knowledge; Laminin; Lassa virus; Life; Ligand Binding; Limb-Girdle Muscular Dystrophies; Link; Longevity; Mammalian Cell; Mannose; Mass Spectrum Analysis; Membrane Proteins; Methods; Muscle eye brain disease; Muscular Dystrophies; Mutation; Neurologic; Northern Europe; Pathway interactions; Patients; Peripheral; Phenotype; Polymers; Polysaccharides; Population; Predisposition; Proteins; Protocols documentation; Recombinant Proteins; Research; Role; Series; Severity of illness; Specificity; Structure; Structure-Activity Relationship; System; Testing; Therapeutic; Threonine; Variant; Vesicular stomatitis Indiana virus; Walker-Warburg syndrome; Work; alpha Dystroglycan; autosome; causal variant; clinical investigation; cognitive function; congenital muscular dystrophy; dystroglycanopathy; enzyme pathway; fukutin; gene product; glycosylation; improved; neural; novel; overexpression; protein complex; receptor; tool

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.

总结 肌营养不良聚糖病是先天性肌营养不良症的一个子集，影响超过1：100，000的个体， 包括一系列具有患者后遗症的疾病， 严重肌营养不良伴严重神经和眼部缺陷，导致最初几年死亡 生命绝大多数遗传学定义的肌营养不良蛋白聚糖病是基因突变的结果， 酶的O-甘露糖基化途径，特别是所谓的M3途径。迄今为止，M3途径， 它由大约十几种酶组成，只发现在一种酶上加工O-甘露糖聚糖。 外周膜蛋白α-肌营养不良蛋白聚糖上的一小部分苏氨酸残基参与了 跨质膜将细胞外基质连接到肌动蛋白细胞骨架的复合物。现有 资金期间，我们和其他人阐明了M3结构，终止于重复二糖提到 作为matriglycan。我们最近已经证明，单独的基质聚糖，缺乏潜在的M3聚糖， α-肌营养不良蛋白聚糖是结合含LG结构域的蛋白质以及促进细胞内蛋白质的合成所必需的和足够的。 结合并被某些沙粒病毒如拉沙病毒感染。在这里，我们寻求解决关键的剩余 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 增强疗法、AAV基因转移疗法和新基因鉴定。因此，我们将测试特异性 包括测试M3聚糖是否可以建立在其他非O-Man聚糖结构上（A1）。 进一步测试特异性，并解决为一个人构建整个M3通路的进化困境。 蛋白质底物，我们将测试其他蛋白质是否含有M3聚糖结构，这可能涉及 某些肌营养不良聚糖病的表型（A1）。在我们想要理解结构-功能的欲望的驱使下 M3酶的关系以及通知临床合作者，我们将调查错义的影响 使用我们建立的方案（A2）对M3酶的变体进行关于稳定性和活性的研究。此外，鉴于 L276 I FKTN变体的高载波频率（在北方为1：150），我们将调查是否 含有该变体的杂合和纯合细胞系对基质聚糖依赖性 假病毒感染（A2）。最后，鉴于所有肌营养不良聚糖病的约1/3是未知的遗传病因， 我们将在相关的人类细胞系中进行无偏见的CRISPR/Cas9筛选，以识别新的基因产物。 α-肌营养不良蛋白聚糖（A3）的功能性糖基化所需的蛋白质。结合这种无偏见的方法，我们 还将研究CDP-核糖醇合成的途径，这是M3聚糖合成所必需的，以确定 参与的基因产物可能与一部分未知的肌营养不良症病例有关（A3）。 完成拟议的工作将提供对M3途径酶更全面的了解 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。