The Effect Of E-cadherin Individual N-glycosylation On Calcium-dependent Cell-cell Adhesion And Cell Cycle Progression And Its Molecular Mechanisms

E-cadherin is well-characterized typeⅠtransmembrane glycoprotein expressed on the surface of epithelial cells,which plays a major role in the establishment of cell-cell adherens junctions(AJs)and the maintenance of cell polarity and tissue morphology.More importantly,it serves as a signaling hub that relays information from the extracellular environment and mediates intercellular communication. E-cadherin inhibits tumor proliferation and metastasis effectively and it is frequently down-regulated or function-deficient in multiple carcinomas,therefore E-cadherin is viewed as an indispensable tumor suppressor.E-cadherin is composed of an extracellular domain,a transmembrane domain and a cytoplasmic domain.Base on amino acid sequence analysis of human E-cadherin (Gene Bank Accession No.L08599),the extracellulr domain of E-cadherin contains four potential N-glycosylation sites at Asn 554,Asn 566,Asn 618,and Asn 633 (N554,N566,N618 and N633).Although E-cadherin plays a crucial role on calcium-dependent cell-cell adhesion and tumor suppression,however,up to date the structure and function of its N-glycosylation remains largely unknown.To elucidate these questions,we previously generated N-glycosylation-deficient mutants of E-cadherin by substituting glutamine for asparagine in each N-glycosylation consensus sequence(NXS/T)of human E-cadherin,either individually or in combinations,by site-directed mutagenesis.Wild type E-cadherin or N-glycosylation-deficient E-cadherin was stably expressed in human highly invasive MDA-MB-435 breast carcinoma cell line which lack completely E-cadherin expression.Previous study found that N633 N-glycosylation was required for E-cadherin stability.Once N633 N-glycosylation was abrogated,E-cadherin failed to express at the protein level.As mentioned before,there are four potential N-glycosylation sites at the extracellular domain of E-cadherin.Not every potential N-glycosylation site of glycoprotein is necessarily N-glycosylated.In the first part of this paper,we therefore identified whether the four potential N-glycosylation sites of E-cadherin were occupied and charactered E-cadherin N-glycosylation structure using sensitivity to PNGaseF,EndoH and MAIDI-TOF MS.The results showed that E-cadherin four potential N-glycosylation sites were all N-glycosylated and the four N-glycans were likely to be complex N-glycans structure.It has been reported that aberrant N-glycosylation affects the adhesive function of cadherin.Overexpression of N-acetylglucosaminyltransferase V(GnT-V),a Golgi resident glycosyltransferase,caused increased modification of N-cadherin withβ1, 6-branched oligosaccharides,which resulted in a decrease in N-cadherin-mediated cell-cell adhesion.Overexpression of N-acetylglucosaminyltransferase(GnT-Ⅲ), another Golgi resident glycosyltransferase,caused increased modifications of E-cadherin with biasecting-branched oligosaccharides,which led to an increase in E-cadherin-mediated cell-cell adhesion.All these results indicate that N-glycosylation probably affects cadherin-associated calcium-dependent cell-cell adhesion,however, how N-glycosylation affect E-cadherin-mediated calcium-dependent cell-cell adhesion and underlying molecular mechanisms remain largely unknown.In the second part of our paper,we analyzed these questions systematically.Using cell aggregation assay,we found that N554 and N566 N-glycosylation affect E-cadherin-mediated cell-cell adhesion;removal of these two N-glycans caused disruption of cell-cell adhesion and elevated cell migration.Nevertheless,N618 N-glycosylation had negligible effect on E-cadherin-mediated cell-cell adhesion and cell migration.In the third part of our paper,we further investigated the molecular mechanisms through which N-glycosylation affected E-cadherin-mediated cell-cell adhesion.We first evaluated the protein stability using cycloheximide(CHX)treatment analysis. Unlike crucial role of N633 N-glycosylation on E-cadherin stability,N554,N566 and N618 N-glycosylation contributed slightly to E-cadherin stability. Immunofluorescence staining analysis and Triton X-100-insoluble fraction analysis demonstrated that N554,N566 and N618 N-glycosylation did not affect E-cadherin expression on the cell surface or at AJs.These results proved that the decrease in cell-cell adhesion did not result from the effect of N-glycosylation on E-cadherin stability or its distribution on the cell surface and at AJs.Then,how N-glycosylation affects E-cadherin-associated cell-cell adhesion? We subsequent found that removal of E-cadherin N554,N566 and N618 N-glycosylation resulted in increased tyrosine phosphorylation levels of p-catenin,which were usually viewed as concomitant with the disassociation of cadherin-catenin complex.Next,Triton X-100-insoluble fraction analysis demonstrated that removal of N554,N566 and N618 N-glycosylation resulted in a decrease inβ- and a-catenin at AJs.From these results we draw a conclusion that E-cadherin N554,N566 and N618 N-glycosylation appears to modulate the tyrosine phosphorylation levels ofβ-catenin,regulate interaction between E-cadherin andβ-catenin,and ultimately affect the strength of cell-cell adhesion.As an important tumor suppressor,E-cadherin suppresses tumor proliferation and matastasis effectively and restrains malignant behaviors of tumor cells.Whether N-glycosylation affects E-cadherin to function as a tumor suppressor? To answer this question,in the fourth part of this paper we investigated the effect of E-cadherin N-glycosylation on cell cycle progression.The results showed that re-expression of wild type E-cadherin restrained the expression of cyclin D1 and G1/S transition, resulted in G1-phase arrest in MDA-MB-435 cells.Moreover,we found that N554, N566,and N618 N-glycosylation was indispensable for the inhibitory effect of E-cadherin on G1/S transition.Once N554,N566 and N618 N-glycosylation was deleted,mutant E-cadherin failed to inhibit G1/S transition.The further study showed that E-cadherin N554,N566 and N618 N-glycosylation affect cell cycle progression through extracellular signal-regulated kinase(ERK)signaling pathway.Notably, E-cadherin N554 and N566 N-glycosylation,especially the former,is crucial for the inhibitory effect of E-cadherin on G1/S transition.Herein we report that E-cadherin individual N-glycosylation present distinctive function charactered as follows:N633 N-glycosylation is required for protein stability whereas N554 N566 and N618 N-glycosylation seems to be dispensable for protein stability.N554,566 N-glycosylation appears to modulate the level ofβ-catenin tyrosine phosphorylation and ultimately affect the strength of cell-cell adhesion.In addition,N554,566 N-glycosylation affects cell cycle progression through ERK signaling pathway.Nevertheless,N618 N-glycosylation plays negligible role on either E-cadherin-mediated cell-cell adhesion or cell cycle progression in MDA-MB-435 cells.