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.

概括 营养不良的肌肉疾病，一部分先天性肌肉营养不良，影响超过1：100,000个人和 涵盖一系列疾病，患者伴有伴有轻度肌营养不良症的疾病 寿命至严重的肌肉营养不良症，具有重大神经和眼部缺陷，最初几年导致死亡 生活。绝大多数遗传定义的多糖层病变是基因编码的突变的结果 O-甘露糖基化途径的酶，特别是所谓的M3途径。迄今为止，M3途径， 它由大约十二种酶组成，仅发现在一个 苏氨酸残基在周围膜蛋白α-二粘膜上的少量子集与参与A 复合物将细胞外基质桥接到质膜上的肌动蛋白细胞骨架上。在先验中 资金期，我们和其他人阐明了以重复二糖为终止的M3结构 作为矩阵。我们最近证明，单独的Matriglycan缺乏基础的M3聚糖和 alpha-dystrolgycan，既需要且足以结合含LG结构域的蛋白质，又足够 某些体育症病毒（例如LASSA病毒）结合和感染。在这里，我们试图解决剩余的关键 基础科学问题，尤其是那些促进早期临床研究的问题，包括基材 增强疗法，AAV基因转移疗法和新型基因鉴定。因此，我们将测试特异性 在M3酶中，包括测试M3糖是否可以基于其他非曼聚糖结构（A1）。 进一步测试特异性，并解决为一个一个M3途径构建整个M3途径的进化问题 蛋白质底物，我们将测试其他蛋白质是否含有M3聚糖结构，这可能参与 某些多糖肿瘤病的表型（A1）。我们渴望理解结构功能的愿望的驱动 M3酶的关系以及为临床合作者提供信息，我们将研究错过的影响 M3酶在稳定性和活动中使用我们已建立的方案（A2）的变体。此外，给予 L276i FKTN变体的高载体频率（北欧的1：150），我们将调查是否是否 携带该变体的杂合子和纯合细胞系不易依赖于矩阵 假病毒感染（A2）。最后，鉴于所有肢体疾病的〜1/3均具有未知的遗传病因学， 我们将在相关的人类细胞系中进行无偏的CRISPR/CAS9屏幕，以识别新型基因产物 α-Dystroglycan（A3）功能性糖基所必需的。加上这种公正的方法，我们 还将研究M3聚糖合成所必需的CDP-核醇合成的途径，以鉴定 可能涉及的基因产物可能导致一部分未知病例的多糖肿瘤病例（A3）。 拟议工作的完成将提供对M3途径酶的更全面的理解 和蛋白质靶标，变体在疾病和感染中的作用，并为临床合作者的工作提供信息。