Research Project 2

研究项目2

• 批准号: 10569663
• 负责人: Heather R Flanagan Steet
• 金额: $ 21.02万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-10 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569663
• 关键词: Address; Affect; Biochemical; Bioinformatics; Biosensor; Cartilage; Cell Adhesion Molecules; Centers of Research Excellence; Chondrocytes; Chondrogenesis; Congenital disorders of glycosylation; Defect; Development; Disease; Drosophila genus; Embryo; Environmental Risk Factor; Enzymes; Exhibits; Fishes; Foundations; Future; Gene Expression; Genes; Genetic; Goals; Guanosine Diphosphate Mannose; Heat-Shock Response; Hereditary Disease; Human Genetics; Impairment; Individual; Knowledge; Laboratories; Link; Maps; Matrix Metalloproteinases; Mendelian disorder; Methods; Modeling; Molecular; Molecular Genetics; Mutation; N-Cadherin; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Penetrance; Peptide Hydrolases; Phenotype; Phosphomannomutase; Polysaccharides; Process; Production; Protein Glycosylation; Proteins; Research Project Grants; Services; Severity of illness; Signal Transduction; Single-Gene Defect; System; Technology; Testing; Tissues; Transgenic Organisms; Viral; Work; Zebrafish; cartilage development; clinical phenotype; confocal imaging; disease-causing mutation; fly; genetic manipulation; glycosylation; in vivo; mannose 1-phosphate; mannose 6 phosphate; mutant; novel; null mutation; pharmacologic; promoter; stem; sugar nucleotide; tool; transcriptome sequencing

PROJECT SUMMARY Congenital disorders of glycosylation (CDGs) are a heterogeneous group of rare inherited diseases caused by mutations in genes involved in protein glycosylation. The most common CDG, PMM2-CDG, results from mutations in the gene phosphomannomutase 2 (PMM2), encoding an enzyme that converts mannose 6- phosphate (M6P) to mannose 1-phosphate (M1P). Defects in PMM2 limit the production of GDP-mannose, a nucleotide sugar essential to synthesize precursors needed for N-linked glycosylation. Reduced GDP- mannose causes protein hypoglycosylation and numerous clinical phenotypes. The connection between hypoglycosylated proteins and phenotypes is unclear, creating a major gap in our knowledge of CDG disease pathogenesis. PMM2-CDG patients exhibit variable penetrance indicating that genetic and/or environmental factors modify disease. We characterized a zebrafish model of PMM2-CDG and identified two classes of enzymes, the protein proconvertases (PCs) and matrix metalloproteinases (MMPs), as candidate drivers of pathology. Analyses of cartilage defects in pmm2 mutant zebrafish revealed a block in early chondrocyte development that is associated with defective processing of the cell adhesion molecule N- cadherin. N-cadherin is sequentially cleaved by furin PCs and MMPs, and both exhibit altered activity in pmm2 mutant zebrafish. The proposed studies address the hypothesis that reduced glycosylation alters the activity of PCs like furin, initiating a cascade involving MMPs that disrupts processing of key cell adhesion molecules, including N-cadherin. We will define how hypoglycosylation of protein processing enzymes alters tissue development; determine whether there is a common mechanism among CDG subtypes; and identify genetic modifiers of CDG disease severity. Toward these goals in Aim 1 we will develop novel zebrafish lines that express wild type and glycan-deficient FLAG-tagged forms of several PCs and MMP enzymes. We will use these tools to define how hypoglycosylation of individual enzymes contributes to impaired chondrogenesis in PMM2-CDG. In Aim 2 we will perform RNA sequencing on several zebrafish models of PMM2, STT3A and STT3B-CDG to identify the pathogenic mechanisms and molecular networks that are commonly or uniquely altered in CDG. Using these analyses in combination with novel Drosophila models we will also pursue genetic modifiers of CDG disease severity. The molecular and genetic pathways identified as sensitive to hypoglycosylation will provide foundational information to develop much needed therapies. Further, the platform established within this proposal will create the road map to ultimately study how disruption of other N-glycosylation genes causes disease.

项目摘要 先天性糖基化障碍（CDG）是一组罕见的遗传性疾病， 参与蛋白质糖基化的基因突变。最常见的CDG，PMM 2-CDG，来自 磷酸甘露变位酶2（PMM 2）基因突变，编码一种将甘露糖6- 磷酸（M6 P）转化为甘露糖1-磷酸（M1 P）。PMM 2的缺陷限制了GDP-甘露糖的生产， 合成N-连接糖基化所需前体所必需的核苷酸糖。GDP下降- 甘露糖引起蛋白质低糖基化和许多临床表型。之间的连接 低糖基化蛋白和表型尚不清楚，这在我们对CDG的认识上造成了一个重大空白 发病机理PMM 2-CDG患者表现出可变的遗传率，表明遗传和/或 环境因素改变疾病。我们表征了PMM 2-CDG的斑马鱼模型，并确定了两个 蛋白质前转化酶（PC）和基质金属蛋白酶（MMPs）作为候选酶 病理学的驱动因素。对pmm 2突变斑马鱼软骨缺损的分析显示， 软骨细胞发育与细胞粘附分子N- 钙粘蛋白N-钙粘蛋白依次被弗林蛋白酶PC和MMPs切割，两者在pmm 2中表现出改变的活性。 突变斑马鱼拟议的研究解决了减少糖基化改变糖基化的假设。 如弗林蛋白酶的PC活性，启动涉及MMPs的级联反应，破坏关键细胞的加工， 粘附分子，包括N-钙粘蛋白。我们将定义如何低糖基化的蛋白质加工 酶改变组织发育;确定CDG亚型之间是否存在共同机制; 并鉴定CDG疾病严重程度的遗传修饰物。为了实现目标1中的这些目标，我们将开发新的 表达几种PC和MMP的野生型和聚糖缺陷FLAG标记形式的斑马鱼品系 内切酶我们将使用这些工具来定义单个酶的低糖基化如何有助于 PMM 2-CDG中软骨形成受损。在目标2中，我们将对几种斑马鱼进行RNA测序 PMM 2、STT 3A和STT 3B-CDG模型，以确定致病机制和分子网络 在CDG中常见或独特的改变。将这些分析与新的果蝇 模型，我们还将寻求遗传修饰剂的CDG疾病的严重程度。分子和遗传途径 鉴定为对低糖基化敏感的蛋白质将为开发急需的 治疗此外，在本提案中建立的平台将创建路线图， 其他N-糖基化基因的破坏如何导致疾病。