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Mouse WIF1 Is Only Modified with O-Fucose in Its EGF-like Domain III Despite Two Evolutionarily Conserved Consensus
Florian Pennarubia1,2, Emilie Pinault1,3, Bilal Al Jaam1
1Glycosylation and cell differentiation, PEIRENE, EA 7500, Faculty of Sciences and Technology, University of Limoges, F-87060 Limoges, France.
Biomolecules
|September 3, 2020
Summary
Wnt Inhibitory Factor 1 (WIF1) protein undergoes O-fucosylation at EGF-like domain III, not domain V. This modification impacts WIF1 secretion, suggesting a role in Wnt signaling regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Evolutionary Biology
Background:
- Wnt Inhibitory Factor 1 (WIF1) protein inhibits Wnt signaling pathways and contains WIF and EGF-like domains (EGF-LDs).
- Potential O-fucosylation sites exist in WIF1's EGF-LDs III and V, but their occupancy has not been confirmed.
- O-fucosylation is a post-translational modification crucial for protein function and interactions.
Purpose of the Study:
- To investigate the phylogenetic distribution and conservation of Wif1 proteins.
- To determine the O-fucosylation site occupancy of recombinant mouse WIF1.
- To elucidate the functional impact of O-fucosylation on WIF1 secretion.
Main Methods:
- Phylogenetic analysis of Wif1 protein sequences across gnathostomes.
- Biochemical assays including click chemistry and mass spectrometry for O-fucosylation detection.
- Site-directed mutagenesis of the O-fucosylation site in mouse WIF1 EGF-LD III.
- Analysis of WIF1 secretion from CHO cells using wild-type and mutant WIF1.
Main Results:
- The consensus sequence for O-fucose modification by Pofut1 is conserved in WIF1 EGF-LD III but divergent in EGF-LD V across gnathostomes.
- Mouse WIF1 is modified with non-extended O-fucose exclusively on EGF-LD III.
- Mutation of the O-fucosylation site in EGF-LD III reduced WIF1 secretion into the cell secretome.
- EGF-LD V likely cannot interact with POFUT1, explaining the absence of O-fucosylation.
Conclusions:
- WIF1 undergoes specific O-fucosylation on EGF-LD III, a modification conserved in gnathostomes.
- This O-fucosylation influences WIF1 secretion, highlighting its role in regulating Wnt signaling.
- The structural divergence of EGF-LD V likely prevents its O-fucosylation by POFUT1.
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