Pathogenic Mechanisms of Congenital Disorders of Glycosylation

先天性糖基化障碍的发病机制

基本信息

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
项目类别：
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

项目摘要

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) 是一组不断增长的罕见遗传性疾病，由以下原因引起：我们对参与蛋白质和脂质糖基化的基因突变的理解。 CDG 发病机制仍然有限，极大地阻碍了新疗法的开发。我们的小组开发并表征了最常见的 CDG PMM2-CDG- 斑马鱼模型。 CDG 是磷酸甘露糖变位酶 2 (PMM2) 突变的结果，该酶编码一种酶，可将 PMM2 中的缺陷限制了脂质连接的产生。6-磷酸甘露糖 (M6P) 转变为 1-磷酸甘露糖 (M1P)。 N-糖基化前体，损害蛋白质糖基化并引起多种临床表现。然而，个体错误糖基化蛋白与疾病表型之间的联系却很薄弱。使用 PMM2-CDG 斑马鱼模型 (pmm2m/m)，我们确定了两类酶：蛋白质原转化酶和基质金属蛋白酶（MMP）作为病理学分析的候选驱动因素。 pmm2突变斑马鱼的软骨缺陷揭示了早期软骨细胞发育的阻碍与细胞粘附分子 N-钙粘蛋白的加工缺陷以及两者的活性改变有关处理 N-钙粘蛋白的 MMP 和原转化酶我们将检验改变糖基化的假设。功能上损害这些酶中的一种或多种，启动一系列异常加工，防止 N- 并行的努力确定了多种代谢物，这些代谢物会裂解钙粘蛋白并破坏软骨形成。在 pmm2m/m 胚胎中，PMM2-CDG 中多元醇山梨醇的水平升高。患者及其水平与疾病严重程度相关。依帕司他是一种正在评估的药物。 PMM2-CDG，降低了 pmm2m/m 胚胎中的山梨醇水平并部分恢复了软骨发育。同样，抑制原转化酶活性可以恢复一些软骨表型，但未能缓解 pmm2m/m 软骨中有明显的细胞空泡形成，这些发现表明存在多种致病性。机制 – 一种与改变蛋白酶功能和 N-钙粘蛋白加工有关，另一种与山梨醇驱动有关细胞应激——促成 PMM2-CDG 疾病发病机制这笔赠款将利用一套强大的工具。新型斑马鱼工具在分子水平上揭示 PMM2-CDG 发病机制，长期目标是广泛定义 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。目标 1 中的研究将调查原转化酶和 Mmps 活性改变与异常的 N-钙粘蛋白加工，解决蛋白质特异性糖基化如何驱动这些表型。目标2，将采用多种方法来调节参与糖代谢和多元醇的酶目的 3 利用新的斑马鱼来确定其在 PMM2-CDG 软骨发病机制中的作用。寡糖转移酶 (OST) 复合物中的突变体，以研究这些机制在 CDG 中的相关性破坏 N-糖基化途径中的其他步骤。