Abstract
Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, 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. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, 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 M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P 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 an M6P accumulation mechanism for underglycosylation.
Publication types
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Research Support, American Recovery and Reinvestment Act
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cartilage / drug effects
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Cartilage / embryology
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Cartilage / pathology
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Cell Shape / drug effects
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Chondrocytes / drug effects
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Chondrocytes / metabolism
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Chondrocytes / pathology
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Congenital Disorders of Glycosylation / enzymology*
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Congenital Disorders of Glycosylation / genetics
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Congenital Disorders of Glycosylation / pathology*
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Craniofacial Abnormalities / embryology
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Craniofacial Abnormalities / pathology
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Disease Models, Animal
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Embryo, Nonmammalian / abnormalities
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Embryo, Nonmammalian / drug effects
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Embryo, Nonmammalian / enzymology
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Gene Expression Regulation, Developmental / drug effects
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Glycosylation / drug effects
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Lipopolysaccharides / metabolism
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Mannose-6-Phosphate Isomerase / metabolism
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Mannosephosphates / metabolism
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Morpholinos / pharmacology
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Motor Neurons / drug effects
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Motor Neurons / pathology
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Movement / drug effects
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Neurogenesis* / drug effects
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Phosphotransferases (Phosphomutases) / deficiency
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Phosphotransferases (Phosphomutases) / genetics
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Phosphotransferases (Phosphomutases) / metabolism*
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Skull / abnormalities
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Skull / drug effects
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Skull / embryology
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Spinal Cord / drug effects
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Spinal Cord / embryology
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Spinal Cord / pathology
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Substrate Specificity / drug effects
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Zebrafish / embryology
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Zebrafish / genetics
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Zebrafish / metabolism*
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Zebrafish Proteins / deficiency
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Zebrafish Proteins / genetics
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Zebrafish Proteins / metabolism*
Substances
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Lipopolysaccharides
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Mannosephosphates
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Morpholinos
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Zebrafish Proteins
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lipid-linked oligosaccharides
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mannose-6-phosphate
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Mannose-6-Phosphate Isomerase
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Phosphotransferases (Phosphomutases)
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phosphomannomutase