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.
