N ‐Glycosylation affects the adhesive function of E‐Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA‐MB‐435

E‐cadherin mediates calcium‐dependent cell–cell adhesion between epithelial cells. The ectodomain of human E‐cadherin contains four potential N ‐glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N ‐glycosylation in E‐cadherin‐mediated cell–cell adhesion was investigated by site‐directed mutagenesis. In MDA‐MB‐435 cells, all four potential N ‐glycosylation sites of human E‐cadherin were N ‐glycosylated. Removal of N ‐glycan at Asn‐633 dramatically affected E‐cadherin stability. In contrast, mutant E‐cadherin lacking the other three N ‐glycans showed similar protein stability in comparison with wild‐type E‐cadherin. Moreover, N ‐glycans at Asn‐554 and Asn‐566 were found to affect E‐cadherin‐mediated calcium‐dependent cell–cell adhesion, and removal of either of the two N ‐glycans caused a significant decrease in calcium‐dependent cell–cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N ‐glycans on E‐cadherin resulted in elevated tyrosine phosphorylation level of β‐catenin and reduced β‐ and α‐catenins at AJs. These findings demonstrate that N ‐glycosylation may affect the adhesive function of E‐cadherin through modifying the composition of AJs. J. Cell. Biochem. 104: 162–175, 2008. © 2007 Wiley‐Liss, Inc.