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:
- 金额:$ 41.83万
- 依托单位:
- 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:
- 关键词:ActinsAddressAffectAnimalsAntibodiesArenavirusBasic ScienceBindingBiological AssayCRISPR screenCardiacCell LineCell membraneCellsCessation of lifeClinicalComplexCoupledCytoskeletonDataDefectDimerizationDisaccharidesDiseaseDisseminated Malignant NeoplasmDuchenne muscular dystrophyDystrophinEnsureEnzyme TestsEnzymesExtracellular MatrixExtracellular Matrix ProteinsFrequenciesFunctional disorderFundingGene MutationGene TransferGene therapy trialGenesGenetic Predisposition to DiseaseGlycopeptidesGlycoproteinsGoalsHeterozygoteHumanHuman Cell LineImpairmentIn VitroIndividualInfectionInterventionKineticsKnowledgeLamininLassa virusLifeLigand BindingLimb-Girdle Muscular DystrophiesLinkLongevityMammalian CellMannoseMass Spectrum AnalysisMembrane ProteinsMethodsMuscle eye brain diseaseMuscular DystrophiesMutationNeurologicNorthern EuropePathway interactionsPatientsPeripheralPhenotypePolymersPolysaccharidesPopulationPredispositionProteinsProtocols documentationRecombinant ProteinsResearchRoleSeriesSeverity of illnessSpecificityStructureStructure-Activity RelationshipSystemTestingTherapeuticThreonineVariantVesicular stomatitis Indiana virusWalker-Warburg syndromeWorkalpha Dystroglycanautosomecausal variantclinical investigationcognitive functioncongenital muscular dystrophydystroglycanopathyenzyme pathwayfukutingene productglycosylationimprovedneuralnoveloverexpressionprotein complexreceptortool
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.
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Lance Wells其他文献
Lance Wells的其他文献
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{{ truncateString('Lance Wells', 18)}}的其他基金
The Role of the O-GlcNAc Modification in X-linked Intellectual Disability
O-GlcNAc 修饰在 X 连锁智力障碍中的作用
- 批准号:
10607367 - 财政年份:2023
- 资助金额:
$ 41.83万 - 项目类别:
O-GlcNAc dynamics and the OGT interactome in variants causal for X-linked intellectual disability
导致 X 连锁智力障碍的变异中的 O-GlcNAc 动力学和 OGT 相互作用组
- 批准号:
10011894 - 财政年份:2019
- 资助金额:
$ 41.83万 - 项目类别:
SITE-SPECIFIC GLYCOSYLATION OF ALPHA-DYSTROGLYCAN FROM RAT BRAIN
大鼠脑中 α-抗肌聚糖的位点特异性糖基化
- 批准号:
8363022 - 财政年份:2011
- 资助金额:
$ 41.83万 - 项目类别:
O-MANNOSYLATION ON DROSOPHILA ALPHA-DYSTROGLYCAN
果蝇α-抗肌聚糖上的O-甘露糖基化
- 批准号:
8363045 - 财政年份:2011
- 资助金额:
$ 41.83万 - 项目类别:
VALIDATION OF IDAWG IN MESC AND HESC
IDAWG 在 MESC 和 HESC 中的验证
- 批准号:
8363032 - 财政年份:2011
- 资助金额:
$ 41.83万 - 项目类别:
QUANTIFICATION OF GLYCOSYLTRANSFERASE PROTEIN LEVELS IN HESC & DERIVED CELLS
HESC 中糖基转移酶蛋白水平的定量
- 批准号:
8363120 - 财政年份:2011
- 资助金额:
$ 41.83万 - 项目类别:
LAMININ-BINDING O-GLYCANS ON ALPHA-DYSTROGLYCAN
α-抗肌聚糖上的层粘连蛋白结合 O-聚糖
- 批准号:
8363043 - 财政年份:2011
- 资助金额:
$ 41.83万 - 项目类别:
MAPPING SITES OF N-LINKED GLYCOSYLATION ON PGIP
PGIP 上 N-连接糖基化位点的图谱
- 批准号:
8363046 - 财政年份:2011
- 资助金额:
$ 41.83万 - 项目类别:
O-MANNOSYLATION ON DROSOPHILA ALPHA-DYSTROGLYCAN
果蝇α-抗肌聚糖上的O-甘露糖基化
- 批准号:
8170808 - 财政年份:2010
- 资助金额:
$ 41.83万 - 项目类别:
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