Research Project 2

项目总结 先天性糖基化紊乱(CDGs)是一组罕见的遗传性疾病，由 与蛋白质糖基化有关的基因突变。最常见的CDG，PMM2-CDG，结果来自 磷酸甘露糖变位酶2(PMM2)基因突变，编码一种将甘露糖6- 磷酸(M6P)到甘露糖1-磷酸(M1P)。PMM2的缺陷限制了GDP-甘露糖的生产 合成N-连接的糖基化所需的前体所必需的核苷酸。GDP减少- 甘露糖可引起蛋白质低糖基化和多种临床表型。两者之间的联系 低糖蛋白和表型尚不清楚，这造成了我们对CDG的认识上的重大差距。 疾病发病机制。PMM2-CDG患者表现出不同的外显，表明遗传和/或 环境因素改变了疾病。我们对PMM2-CDG的斑马鱼模型进行了表征，并确定了两个 作为候选的酶类，蛋白质原转换酶(PC)和基质金属蛋白酶(MMPs) 病理学的驱动力。对PMM2突变斑马鱼软骨缺陷的分析显示，早期就出现了阻塞 与细胞黏附分子N-加工缺陷有关的软骨细胞发育 钙粘附素。N-钙粘蛋白被Furin PC和MMPs顺序切割，两者在PMM2中都表现出不同的活性 突变的斑马鱼。拟议的研究解决了这样的假设，即减少糖基化会改变 像Furin这样的PC的活动，启动涉及MMP的级联反应，扰乱关键细胞的处理 黏附分子，包括N-钙粘附素。我们将定义蛋白质加工过程中的低糖基化 酶改变组织发育；确定CDG亚型之间是否有共同的机制； 并确定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和基质金属蛋白酶的野生型和缺乏多糖的标志标记形式的斑马鱼 酵素。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 PMM2-CDG的软骨形成受损。在目标2中，我们将对几条斑马鱼进行RNA测序 PMM2、STT3A和STT3B-CDG模型研究致病机制及分子网络 在CDG中常见或独特改变的基因。将这些分析与新果蝇结合使用 我们还将寻求CDG疾病严重程度的遗传修饰物。分子和遗传途径 被确认为对低糖基化敏感的人将提供开发急需的基础信息 治疗。此外，在该提案中建立的平台将创建最终研究的路线图 其他N-糖基化基因的破坏如何导致疾病。

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.