A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency

Abigail Cline, Ningguo Gao, Heather Flanagan-Steet, Vandana Sharma, Sabrina Rosa, Roberto Sonon, Parastoo Azadi, Kirsten Sadler Edepli, Hudson H. Freeze, Mark A. Lehrman, Richard Steet

    Research output: Contribution to journalArticle

    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.

    Original languageEnglish (US)
    Pages (from-to)4175-4187
    Number of pages13
    JournalMolecular Biology of the Cell
    Volume23
    Issue number21
    DOIs
    StatePublished - Nov 1 2012

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    Neurogenesis
    Zebrafish
    Glycosylation
    Guanosine Diphosphate Mannose
    Congenital Disorders of Glycosylation
    Morpholinos
    Polysaccharides
    Limit of Detection
    Spinal Cord
    Embryonic Structures
    Animal Models
    Phosphates
    lipid-linked oligosaccharides
    mannose-6-phosphate
    Phenotype
    Mutation
    Enzymes
    mannose 1-phosphate

    ASJC Scopus subject areas

    • Molecular Biology
    • Cell Biology

    Cite this

    A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency. / Cline, Abigail; Gao, Ningguo; Flanagan-Steet, Heather; Sharma, Vandana; Rosa, Sabrina; Sonon, Roberto; Azadi, Parastoo; Sadler Edepli, Kirsten; Freeze, Hudson H.; Lehrman, Mark A.; Steet, Richard.

    In: Molecular Biology of the Cell, Vol. 23, No. 21, 01.11.2012, p. 4175-4187.

    Research output: Contribution to journalArticle

    Cline, A, Gao, N, Flanagan-Steet, H, Sharma, V, Rosa, S, Sonon, R, Azadi, P, Sadler Edepli, K, Freeze, HH, Lehrman, MA & Steet, R 2012, 'A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency', Molecular Biology of the Cell, vol. 23, no. 21, pp. 4175-4187. https://doi.org/10.1091/mbc.E12-05-0411
    Cline, Abigail ; Gao, Ningguo ; Flanagan-Steet, Heather ; Sharma, Vandana ; Rosa, Sabrina ; Sonon, Roberto ; Azadi, Parastoo ; Sadler Edepli, Kirsten ; Freeze, Hudson H. ; Lehrman, Mark A. ; Steet, Richard. / A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency. In: Molecular Biology of the Cell. 2012 ; Vol. 23, No. 21. pp. 4175-4187.
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    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.",
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    AU - Cline, Abigail

    AU - Gao, Ningguo

    AU - Flanagan-Steet, Heather

    AU - Sharma, Vandana

    AU - Rosa, Sabrina

    AU - Sonon, Roberto

    AU - Azadi, Parastoo

    AU - Sadler Edepli, Kirsten

    AU - Freeze, Hudson H.

    AU - Lehrman, Mark A.

    AU - Steet, Richard

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    N2 - 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.

    AB - 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.

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