Structure and Function in alpha-Dystroglycan Glycosylation

α-肌营养不良聚糖糖基化的结构和功能

基本信息

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
项目类别：
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

项目摘要

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.

概括肌营养不良症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，并且涵盖一系列患者后遗症的疾病，从轻度肌营养不良症到接近正常的肌营养不良症寿命到严重的肌营养不良症，并伴有严重的神经和眼部缺陷，导致在最初几年内死亡的生活。绝大多数基因定义的肌营养不良症是编码基因突变的结果 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，它由大约十几种酶组成，仅被发现可以在外周膜蛋白 α-dystroglycan 上的一小部分苏氨酸残基，参与跨质膜将细胞外基质与肌动蛋白细胞骨架桥接的复合物。在之前的在资助期间，我们和其他人阐明了以重复二糖结尾的 M3 结构作为基质聚糖。我们最近证明，单独的基质聚糖，缺乏潜在的 M3 聚糖和 α-dystrolgycan，对于结合包含 LG 结构域的蛋白质以及促进某些沙粒病毒（例如拉沙病毒）的结合和感染。在这里，我们寻求解决剩下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 M3 酶的研究，包括测试 M3 聚糖是否可以构建在其他非 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完整 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，这可能参与某些肌营养不良症的表型 (A1)。受到我们了解结构功能的渴望的驱动 M3 酶的关系以及告知临床合作者，我们将研究错义的影响使用我们既定的方案 (A2) 对 M3 酶的稳定性和活性进行变体。进一步，给定 L276I FKTN 变体的高载波频率（北欧为 1:150），我们将调查是否携带这种变体的杂合和纯合细胞系对基质聚糖依赖性不太敏感假病毒感染（A2）。最后，考虑到约 1/3 的肌营养不良症的遗传病因未知，我们将在相关人类细胞系中进行公正的 CRISPR/Cas9 筛选，以鉴定新的基因产物 α-肌营养不良聚糖 (A3) 功能性糖基化所需的。结合这种公正的方法，我们还将研究 M3 聚糖合成所必需的 CDP-核糖醇合成途径，以确定所涉及的基因产物可能与部分未知的肌聚糖病病例有关 (A3)。完成拟议的工作将使人们对 M3 途径酶有更全面的了解和蛋白质靶标、变异在疾病和感染中的作用，并为临床合作者的工作提供信息。