Pathogenic Mechanisms of Congenital Disorders of Glycosylation

先天性糖基化紊乱(CDG)是一组日益增多的罕见遗传性疾病，由 涉及蛋白质和脂肪糖基化的基因突变。我们对驱动机制的理解 CDG的发病机制仍然有限，极大地阻碍了新疗法的发展。为了克服这一障碍， 我们小组为最常见的CDG开发并表征了斑马鱼模型，PMM2-CDG。PMM2- CDG是由磷酸甘露糖变异酶2(PMM2)的突变引起的，PMM2编码一种将 甘露糖-6-磷酸(M6P)到甘露糖-1-磷酸(M1P)。PMM2中的缺陷限制了脂质连接的生产 N-糖基化前体，损害蛋白质糖基化，引起多种临床症状。这个 然而，个体糖基化蛋白与疾病表型之间的联系很差。 明白了。使用PMM2-CDG斑马鱼模型(pmm 2m/m)，我们鉴定了两类酶， 蛋白原转化酶和基质金属蛋白酶(MMPs)，作为病理的候选驱动因素。分析了 PMM2突变斑马鱼的软骨缺陷揭示了早期软骨细胞发育的障碍，即 与细胞黏附分子N-钙粘附素的加工缺陷有关，并改变两者的活性 处理N-钙粘附素的基质金属蛋白酶和前转化酶。我们将检验改变糖基化的假说 在功能上损害这些酶中的一个或多个，启动一连串的异常加工，从而阻止N- 钙粘附素裂解并破坏软骨形成。同时进行的工作确定了多种代谢物被改变 在pmm 2m/m胚胎中，包括升高的多元醇山梨醇水平。山梨醇在PMM2-CDG中增加 患者及其水平与疾病的严重程度相关。依帕司他是一种正在评估中的药物，用于治疗 PMM2-CDG，降低山梨醇水平，部分恢复了pmm 2m/m胚胎的软骨发育。 同样，抑制原转换酶活性可以恢复一些软骨表型，但不能缓解 Pmm 2m/m软骨细胞空泡化明显。这些发现表明，多种致病因素 机制--一种与酶功能改变和N-钙粘附素加工有关，另一种与山梨醇驱动 细胞应激--参与PMM2-CDG病的发病机制。这笔赠款将利用一套强大的 斑马鱼在分子水平上解开PMM2-CDG发病机制的新工具，长期目标是 广泛地定义CDG基因缺陷是如何导致疾病的，并利用这些信息来确定治疗方法。这个 目标1的研究将探索原转换酶和MMPs活性变化与 异常的N-钙粘附素处理，解决了蛋白质特定的糖基化错误如何驱动这些表型。在……里面 目标2，将使用多种方法来调节与糖代谢和多元醇有关的酶 以确定它们在PMM2-CDG软骨发病机制中的作用。目标3利用新的斑马鱼 寡糖转移酶(OST)复合体中的突变以研究这些机制在CDG中的相关性 这会扰乱N-糖基化途径中的其他步骤。

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.