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J. Biol. Chem., Vol. 281, Issue 9, 5916-5927, March 3, 2006

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Ablation of Mouse Phosphomannose Isomerase (Mpi) Causes Mannose 6-Phosphate Accumulation, Toxicity, and Embryonic Lethality^*

Charles DeRossi¹, Lars Bode¹, Erik A. Eklund², Fangrong Zhang, Joseph A. Davis³, Vibeke Westphal⁴, Ling Wang, Alexander D. Borowsky, and Hudson H. Freeze⁵

From the Burnham Institute for Medical Research, La Jolla, California 92037 and the University of California, Davis, California 95616

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-[³H]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.

Received for publication, November 7, 2005 , and in revised form, December 8, 2005.

^* This work was supported by the National Institutes of Health Grants RO1 DK065091 and RO1 GM55695, a postdoctoral fellowship from STINT/VR (Sweden) (to E. A. E.), and Deutsche Forschungsgemeinschaft Postdoctoral Research Fellowship BO2488/1-1 (to L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The online version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ These two authors contributed equally to this work.

² Present address: Dept. of Cell and Molecular Biology, Lund University, BMC C13, 221 84 Lund, Sweden.

³ Present address: Ranbaxy Laboratories Limited 20, Sector 18, Gurgaon 122001 Haryana, India.

⁴ Present address: Novo Nordisk A/S, DK-2760, Måløv, Denmark.

⁵ To whom correspondence should be addressed: Glycobiology and Carbohydrate Chemistry Program, The Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3142; Fax: 858-713-6281; E-mail: hudson{at}burnham.org.

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