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E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with ß1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell-cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression. ; IPATIMUP integrates the I3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors—COMPETE— and National Funds through the FCT—Foundation for Science and Technology, under the projects: PTDC/CVT/111358/2009; EXPL/BIM-MEC/0149/2012; and PTDC/BBB-EBI/0786/2012. SC (SFRH/BD/77386/2011), AMD (SFRH/BI/52380/2013) BiotechHealth Doctoral Programme, and SSP (SFRH/BPD/63094/2009) thank FCT and the Luso-American Foundation (FLAD) for funding. JMS acknowledges FCT (UID/EEA/50009/2013). DK acknowledges support by the Max Planck Society and European Union (Seventh Framework Programme 'Glycoproteomics', grant number PCIG09-GA-2011-293847. CAR and DK acknowledge GastricGlycoExplorer project, grant number 316929). We thank Ola Soderberg and Gaëlle Cane (Department of Genetics and Pathology, University of Uppsala, Uppsala, Sweden) for providing the Streptavidin PLA probe and to Márcia Pereira and Sara Campos for the support in PLA technique and statistical analyses.