A hypomorphic Mpi mutation unlocks an in vivo tool for studying global N-glycosylation deficiency
Elisa B. Lin, Steve Meregini, Zhao Zhang, Avishek Roy, Tandav Argula, James M. Mitchell, William J. Israelsen, Sara Ludwig, Jamie Russell, Jiexia Quan, Sara Hildebrand, Evan Nair-Gill, Bruce Beutler, Jeffrey A. SoRelle
Elisa B. Lin, Steve Meregini, Zhao Zhang, Avishek Roy, Tandav Argula, James M. Mitchell, William J. Israelsen, Sara Ludwig, Jamie Russell, Jiexia Quan, Sara Hildebrand, Evan Nair-Gill, Bruce Beutler, Jeffrey A. SoRelle
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Research Article Gastroenterology Genetics

A hypomorphic Mpi mutation unlocks an in vivo tool for studying global N-glycosylation deficiency

Abstract

Glycans are one of the 4 major macromolecules essential for life and are the most abundant family of organic molecules. However, in contrast with DNA and RNA, glycan structures have no template; this results in limited tools to study this challenging macromolecule with a diversity of glycan structures. A central bottleneck in studying glycosylation in vivo is that inhibitors and complete KOs are lethal. In a forward genetic screen, we identified a viable, hypomorphic mutation at a conserved site in mannose phosphate isomerase (Mpi) that causes a multisystemic phenotype affecting RBCs, liver, stomach, intestines, skin, size, fat, and fluid balance in mice. The phenotype could be rescued with mannose. Analyses of glycopeptides in mice with this mutation showed a 500% increase in unoccupied N-glycan sites. This is equivalent to a “glycan knockdown,” which would be useful for examining the role of glycans in biology and disease. Therefore, we report an in vivo tool to study global N-glycosylation deficiency with tissue-specific targeting and a rescue mechanism with mannose.

Authors

Elisa B. Lin, Steve Meregini, Zhao Zhang, Avishek Roy, Tandav Argula, James M. Mitchell, William J. Israelsen, Sara Ludwig, Jamie Russell, Jiexia Quan, Sara Hildebrand, Evan Nair-Gill, Bruce Beutler, Jeffrey A. SoRelle

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Figure 1

Benadryl has a viable, hypomorphic allele of Mpi, which causes a small body size and ruffled fur phenotype.

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Benadryl has a viable, hypomorphic allele of Mpi, which causes a small ...
(A) Photograph of benadryl mouse (bottom) compared with WT littermate. (B) Manhattan plot. Linkage of a “visible, abnormal phenotype” to a mutation in Mpi −log10 (P values) versus the chromosomal positions of mutations identified in the founder (generation 1, G1) of the affected pedigree, determined by (C) whole-exome sequencing and validated by (D) ion torrent next-generation sequencing of all G3 progeny. (E) Schematic of Mpi domains and the substitution of histidine to arginine at position 54 of 423 total amino acids. Numbers indicate amino acid positions. PMI enzyme, phosphomannose isomerase enzyme domain, also known as MPI. (F) Table showing conservation of amino acids at and adjacent to p.H54 (highlighted) across multiple organisms back to prokaryotes. (G) Structure of Mpi indicating H54 (yellow), active site binding pocket (highlighted in green; darker color is deeper portion and lighter color faces outward), and divalent cation-binding amino acids (blue). (H and I) Intermolecular N-N interaction of H54 with R56 and aromatic ring interactions of H54 and W50 shown from 2 orientations.