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

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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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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

PY - 2012/11/1

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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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