N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

Abstract

Epithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N-glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E-cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N-glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N-glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin-catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with ␥-catenin and vinculin compared with the endogenous E-cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization.

Roles of N-Acetylglucosaminyltransferase III in Epithelial-to-Mesenchymal Transition Induced by Transforming Growth Factor β1 (TGF-β1) in Epithelial Cell Lines

“…However, the modification of E-cadherin with complex N-glycans has been associated with the formation of dynamic, but weak, adherens junctions, whereas E-cadherin modified by high mannose or less N-glycans has been reported to promote the establishment of stable adherens junctions (33). In light of the report showing the importance of ectodomains of E-cadherin in homophilic adhesion, it is tempting to speculate that these N-glycans affect the interactions between the ectodomains through stearic hindrance.…”

“…Several studies have shown that alterations in N-glycosylation could modulate cadherin-dependent cell-cell adhesion (19,(31)(32)(33). Taniguchi and co-workers (19) reported that overexpression of GnT-III enhanced cell-cell adhesion in B16 melanoma cells, because of a delay in the turnover of E-cadherin on the cell surface.…”

Potential roles of N-glycosylation in cell adhesion

“…Cytoplasmic O-glycosylation of the E-cadherin cytosolic tail has been shown to occur in response to endoplasmic reticulum stress and inactivate Ecadherin-mediated intercellular adhesion by preventing its transport to the cell membrane [50]. In addition, E-cadherin can be N-glycosylated and the N-glycosylation at Asn-633 is essential for E-cadherin expression, folding and trafficking [51,52]. In an earlier study, Yoshimura, et al reported that E-cadherin-mediated cell adhesion was regulated by GnT-III.…”

“…However, the modification of E-cadherin with complex N-glycans has been associated with the formation of dynamic, but weak, adherens-junctions, whereas E-cadherin modified by high mannose or less N-glycans has been reported to promote the establishment of stable adherensjunctions [51]. In addition, Pihno, et al reported that Ecadherin underwent extensive modification of its N-glycans with β1-6 branched and sialylated structures during acquisition of the malignant phenotype in a canine mammary tumor cell line model [64].…”

N-Glycosylation Induces the CTHRC1 Protein and Drives Oral Cancer Cell Migration

“…In turn, canonical Wnt activates DPAGT1 expression via ␤-catenin at a transcriptional level. Furthermore, N-glycosylation inhibits E-cadherin adhesion and interferes with E-cadherin antagonism of canonical Wnt signaling (17,18). Aberrant activation of canonical Wnt and N-glycosylation pathways has been associated with tumorigenesis (9, 19 -21).…”