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Abstract
SUMMARYIndividuals with congenital disorders of glycosylation (CDG) have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO), which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI) enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf), which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy and the broader molecular and developmental abnormalities that contribute to disorders associated with defective protein glycosylation.
Highlights
Chu et al - 3; Introduction Congenital disorders of glycosylation (CDG) are rare, under-diagnosed monogenic disorders with over 1000 patients identified worldwide (Freeze, 2006; Freeze et al, 2012; Haeuptle and Hennet, 2009; Jaeken, 2010)
mannose-6-phosphate via the phosphomannose isomerase (MPI)-Congenital Disorders of Glycosylation (CDG) patients primarily develop gastrointestinal problems including diarrhea from protein losing enteropathy, gastrointestinal bleeding due to coagulopathy resulting from failed hepatocyte secretion or instability of clotting factors, and underlying portal hypertension caused by congenital hepatic fibrosis
20% mortality of mpi morphants occurred by 24 hpf and 50% of embryos injected with 6.7 ng morpholino were dead by 4 dpf, with a range of 14-81% mortality across 14 experiments (Fig. 1B; p
Summary
Chu et al - 3; Introduction Congenital disorders of glycosylation (CDG) are rare, under-diagnosed monogenic disorders with over 1000 patients identified worldwide (Freeze, 2006; Freeze et al, 2012; Haeuptle and Hennet, 2009; Jaeken, 2010). Type I CDG patients typically develop protein hypoglycosylation and multi-systemic pathologies, including cardiac, neurological, musculoskeletal, gastrointestinal and hepatic disease with high morbidity and mortality (Freeze, 2001; Freeze et al, 2012; Jaeken, 1980). MPI-CDG patients primarily develop gastrointestinal problems including diarrhea from protein losing enteropathy, gastrointestinal bleeding due to coagulopathy resulting from failed hepatocyte secretion or instability of clotting factors (which are glycoproteins), and underlying portal hypertension caused by congenital hepatic fibrosis (de Lonlay and Seta, 2009; Freeze, 2001). Zebrafish models of CDG will provide muchneeded tools to study CDG pathology, as development of mice with hypomorphic alleles is costly and time consuming and the mice generated far have failed to adequately replicate the abnormal gene expression, developmental abnormalities, protein hypoglycosylation, and loss of LLOs associated with human disease
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