Pathogenic Mechanisms of Congenital Disorders of Glycosylation

先天性糖基化障碍的发病机制

• 批准号: 10633548
• 负责人: Heather R Flanagan Steet
• 金额: $ 23.51万
• 依托单位: GREENWOOD GENETIC CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10633548
• 关键词: Address; Affect; Aldehyde Reductase; Automobile Driving; Biosensor; Cartilage; Cell Adhesion Molecules; Cellular Stress; Chondrocytes; Chondrogenesis; Clinical; Complex; Congenital disorders of glycosylation; Defect; Development; Disease; Embryo; Enzymes; Evaluation; FRAP1 gene; Genes; Genetic; Goals; Grant; Guanosine Diphosphate Mannose; Hereditary Disease; Image; Impairment; In Situ; In Vitro; Individual; Light; Link; Lipids; Matrix Metalloproteinases; Metabolic dysfunction; Metabolism; Modeling; Molecular; Mutation; N-Cadherin; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Phosphomannomutase; Polysaccharides; Process; Production; Proprotein Convertases; Protein Glycosylation; Proteins; Rare Diseases; Role; Severity of illness; Sorbitol; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stress; Substrate Specificity; Symptoms; System; Testing; Toxic effect; Transgenic Organisms; Work; Zebrafish; cartilage development; disease phenotype; disease-causing mutation; functional disability; glycosylation; inhibitor; insight; mannose 1-phosphate; mannose 6 phosphate; metabolomics; mutant; novel; novel therapeutics; pharmacologic; polyol; prevent; sugar; sugar nucleotide; therapy development; tool

The Congenital Disorders of Glycosylation (CDG) are a growing group of rare inherited diseases caused by mutations in genes involved in protein and lipid glycosylation. Our understanding of the mechanisms driving CDG pathogenesis remains limited, greatly impeding development of new therapies. To overcome this barrier, our group developed and characterized a zebrafish model for the most common CDG, PMM2-CDG. PMM2- CDG results from mutations in phosphomannomutase 2 (PMM2), which encodes an enzyme that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Defects in PMM2 limit production of lipid-linked N-glycosylation precursors, impairing protein glycosylation and causing numerous clinical manifestations. The connection between individual misglycosylated proteins and disease phenotypes, however, is poorly understood. Using the PMM2-CDG zebrafish model (pmm2m/m), we identified two classes of enzymes, the protein proconvertases 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, and altered activity of both MMPs and proconvertases that process N-cadherin. We will test the hypothesis that altered glycosylation functionally impairs one or more of these enzymes, initiating a cascade of aberrant processing that prevents N- cadherin cleavage and disrupts chondrogenesis. Parallel efforts identified multiple metabolites that are altered in pmm2m/m embryos, including elevated levels of the polyol sorbitol. Sorbitol is increased in PMM2-CDG patients and its level correlates with disease severity. Treatment with epalrestat, a drug under evaluation for PMM2-CDG, reduced sorbitol levels and partially restored cartilage development in pmm2m/m embryos. Likewise, inhibiting proconvertase activity restored some of the cartilage phenotypes, but failed to alleviate the pronounced cellular vacuolation in pmm2m/m cartilage. These findings indicate that multiple pathogenic mechanisms – one related to altered protease function and N-cadherin processing, another to sorbitol-driven cellular stress – contribute to PMM2-CDG disease pathogenesis. This grant will leverage a powerful suite of novel zebrafish tools to unravel PMM2-CDG pathogenesis at the molecular level, with the long-term goal of broadly defining how defects in CDG genes cause disease and using this information to identify therapies. The studies in Aim 1 will investigate the mechanisms linking altered activity of proconvertases and Mmps to aberrant N-cadherin processing, addressing how protein-specific misglycosylation drives these phenotypes. In Aim 2, multiple approaches will be used to modulate enzymes involved in sugar metabolism and polyol production to define their role in PMM2-CDG cartilage pathogenesis. Aim 3 takes advantage of new zebrafish mutants in the oligosaccharyltransferase (OST) complex to study the relevance of these mechanisms in CDG that disrupt other steps within the N-glycosylation pathway.

先天性糖基化障碍（CDG）是一组日益增长的罕见遗传性疾病， 参与蛋白质和脂质糖基化的基因突变。我们对驱动人类行为的机制的理解 CDG的发病机制仍然有限，极大地阻碍了新疗法的开发。为了克服这一障碍， 我们的小组开发并表征了用于最常见的CDG PMM 2-CDG的斑马鱼模型。PMM2- CDG由磷酸甘露糖变位酶2（PMM 2）突变引起，PMM 2编码一种酶， 甘露糖-6-磷酸（M6 P）转化为甘露糖-1-磷酸（M1 P）。PMM 2中的缺陷限制了脂质连接的 N-糖基化前体，损害蛋白质糖基化并引起许多临床表现。的 然而，个别糖基化蛋白质和疾病表型之间的联系很差， 明白使用PMM 2-CDG斑马鱼模型（pmm 2 m/m），我们鉴定了两类酶， 蛋白质前转化酶和基质金属蛋白酶（MMP），作为病理学的候选驱动因素。分析 pmm 2突变斑马鱼的软骨缺陷揭示了早期软骨细胞发育的阻滞， 与细胞粘附分子N-钙粘蛋白的加工缺陷有关， 加工N-钙粘蛋白的MMP和前转化酶。我们将检验糖基化改变的假设 在功能上损害这些酶中的一种或多种，启动异常处理的级联反应，阻止N- 钙粘蛋白裂解并破坏软骨形成。平行的努力确定了多种代谢物， 在pmm 2 m/m胚胎中，包括多元醇山梨醇水平升高。PMM 2-CDG中山梨醇增加 其水平与疾病严重程度相关。依帕司他治疗，一种正在评估的药物， PMM 2-CDG，降低山梨醇水平和部分恢复软骨发育的pmm 2 m/m胚胎。 同样地，抑制前转化酶活性恢复了一些软骨表型，但未能减轻软骨细胞的损伤。 pmm 2 m/m软骨中明显的细胞空泡化。这些发现表明， 机制-一个与改变的蛋白酶功能和N-钙粘蛋白加工有关，另一个与胆固醇驱动的 细胞应激-有助于PMM 2-CDG疾病的发病机制。这笔赠款将利用一套强大的 新的斑马鱼工具在分子水平上解开PMM 2-CDG发病机制，长期目标是 广泛地定义了CDG基因缺陷如何导致疾病，并利用这些信息来确定治疗方法。的 目的1中的研究将研究前转化酶和Mmps的活性改变与 异常的N-钙粘蛋白加工，解决了蛋白质特异性错糖基化如何驱动这些表型。在 目的2，多种方法将用于调节参与糖代谢和多元醇的酶 以确定它们在PMM 2-CDG软骨发病机制中的作用。AIM 3利用新的斑马鱼 突变体中的寡糖基转移酶（OST）复合物，研究这些机制在CDG的相关性 破坏N-糖基化途径中的其他步骤。