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Published on: July 24, 2013
Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic
Charles DeRossi1, Lars Bode, Erik A Eklund
1Glycobiology and Carbohydrate Chemistry Program, Burnham Institute for Medical Research, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract:
MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.
Insights
Mice lacking phosphomannose isomerase (MPI) die in utero, and mannose supplementation, while rescuing glycosylation, proves toxic. Mannose toxicity in Mpi(-/-) embryos stems from mannose-6-phosphate accumulation, inhibiting glucose metabolism and depleting ATP.
Area of Science:
- Biochemistry
- Developmental Biology
- Genetics
Background:
- Phosphomannose isomerase (MPI) is crucial for glycoconjugate biosynthesis, interconverting fructose 6-phosphate and mannose 6-phosphate (Man-6-P).
- Mutations in MPI cause congenital disorder of glycosylation type Ib (CDG-Ib) in humans, treatable with oral mannose.
- A mouse model is needed to study CDG-Ib pathogenesis and mannose response.
Purpose of the Study:
- To create a mouse model for CDG-Ib by ablating the Mpi gene.
- To investigate the effects of mannose supplementation on Mpi-deficient embryos.
- To elucidate the mechanisms underlying embryonic lethality in Mpi(-/-) mice.
Main Methods:
- Gene ablation of Mpi in mice.
- Administration of mannose to pregnant dams.
- Analysis of embryonic development, glycosylation, and placental formation.
- In vitro studies using primary embryonic fibroblasts to assess Man-6-P accumulation, ATP levels, and enzyme inhibition.
Main Results:
- Mpi(-/-) embryos exhibited embryonic lethality around E11.5, despite normal glycosylation.
- Mannose supplementation exacerbated embryonic lethality, indicating mannose toxicity.
- Mpi(-/-) embryos showed growth retardation, placental hyperplasia, and defects in yolk sac vasculature and chorioallantoic fusion.
- In Mpi(-/-) fibroblasts, mannose led to Man-6-P accumulation, significant ATP depletion, and inhibition of key glycolytic enzymes (hexokinase, phosphoglucose isomerase, glucose-6-phosphate dehydrogenase).
Conclusions:
- Complete Mpi ablation results in embryonic lethality, independent of glycosylation status.
- Mannose is toxic to Mpi-deficient embryos due to Man-6-P accumulation, which disrupts glucose metabolism and depletes ATP.
- A hypomorphic Mpi allele is required to establish a viable murine CDG-Ib model.
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