A Zebrafish Model Of PMM2-CDG Reveals Altered Neurogenesis And A Substrate-Accumulation Mechanism For N-Linked Glycosylation Deficiency

Congenital Disorder of Glycosylation PMM2-CDG results from mutations in PMM2, which encodes the phosphomannomutase that converts mannose-6-P to mannose-1-P. Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Though widely proposed that PMM2 deficiency depletes mannose-1-P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here, we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. While mannose-1-P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of mannose-6-P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the mannose-6-P synthesizing enzyme, mannose phosphate isomerase, within the pmm2 background normalized mannose-6-P levels and certain aspects of the craniofacial phenotype, and abrogated pmm2-dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including a mannose-6-P accumulation mechanism for under-glycosylation.