Pathogenic Mechanisms of Congenital Disorders of Glycosylation

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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.