Abstract

Protein N-glycosylation and the Wnt/β-catenin signaling pathways play critical roles in development and cancer. Although N-glycosylation has been shown to influence Wnt signaling through its effects on Wnt ligands, it is unclear whether the Wnt/β-catenin pathway impacts protein N-glycosylation. In this study, we show that promoters of the first N-glycosylation gene, DPAGT1, from Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human epidermoid carcinoma (A253) cells contain the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus sequence. Treatment of cells with a Wnt activator, lithium chloride, up-regulated DPAGT1 transcript levels that correlated with an increase in the β-catenin abundance. Furthermore, exposure of cells to a Wnt receptor ligand, Wnt3a, resulted in an increase in the DPAGT1 transcript levels that was abrogated by the Wnt inhibitor, Dickkopf-1. DNA mobility shift assays revealed specific protein complexes at the DPAGT1 TCF/LEF binding region that were competed off with antibodies to either Tcf3/4 or β-catenin. Chromatin immunoprecipitation analysis confirmed the presence of β-catenin at the DPAGT1 promoter in vivo. In addition, the DPAGT1 TCF/LEF sequence drove the expression of the luciferase reporter gene. Furthermore, up-regulation of DPAGT1 transcripts by Wnt3a led to altered N-glycosylation of E-cadherin. Interestingly, the DPAGT1 TCF/LEF sequence also interacted with γ-catenin, a close homologue of β-catenin, although not in a lithium chloride-dependent manner. Our results provide the first evidence that the Wnt/β-catenin signaling pathway regulates the metabolic pathway of protein N-glycosylation by targeting DPAGT1 expression. Moreover, they suggest the existence of another regulatory mechanism involving the interaction of Tcf with γ-catenin at the DPAGT1 promoter.

Highlights

  • DPAGT1, the gene that initiates the synthesis of the lipid-linked oligosaccharide precursor for protein N-glycosylation in the endoplasmic reticulum and is a key determinant of the extent of protein N-glycosylation [12, 13], is down-regulated with cellular differentiation, suggesting that attenuation of N-glycosylation is required for the establishment of a differentiated phenotype [14]

  • Our studies reveal that the first N-glycosylation gene, DPAGT1, is a target of the Wnt/␤-catenin signaling pathway

  • Under conditions of Wnt activation, DPAGT1 transcription is up-regulated though the binding of ␤-catenin to Tcf at the T-cell factor/lymphoid enhancerbinding factor (TCF/LEF)-binding site in the DPAGT1 promoter

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Summary

The abbreviations used are

T-cell factor; AJ, adherens junctions; ChIP, chromatin immunoprecipitation; CHO, Chinese hamster ovary; CM, conditioned medium; LEF, lymphoid enhancer-binding factor; MDCK, MadinDarby canine kidney; N-glycans, asparagine-linked oligosaccharides; EndoH, endoglycosidase H; PNGase, peptide:N-glycosidase. Inappropriate phosphorylation of ␤-catenin at the tyrosine residues has been shown to lead to its dissociation from, and subsequent disassembly of, AJs [29, 30] Such diminished cadherin-mediated adhesion has been linked to cancer development and progression [10, 31, 32]. Our studies provide the first evidence that protein N-glycosylation is regulated by the canonical Wnt signaling pathway through the DPAGT1 gene

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