The Effect Of N-glycosylation At Asn-633 Site On E-cadherin Expression, Folding, And Trafficking And Its Molecular Mechanism

The human E-cadherin is well-characterized typeâ… transmembrane glycoprotein expressed on the surface of epithelial cells,which belongs to a classic cadherin family.It is involved in Ca²⁺-dependent cell-cell adhesion, morphogenetic processes,differentiation and establishing and maintaining cell polarity.More importantly,it works as a "bridge" which transduces signaling from the extracellular environment into the nucleolus via the mediation ofβ-catenin.It has been reported that it is frequently down-regulated or function-deficient in multiple carcinomas.When transfected cancer cells with E-cadherin,the proliferation and metastasis of tumor are inhibited by E-cadherin.Therefore,E-cadherin is viewed as a candidate of tumor suppressor.E-cadherin is composed of an extracellular domain,a transmembrane domain and a cytoplasmic domain.Its extracellular domain is composed of five tandemly repeated subdomains of about 110 amino acids each,which together coordinate several Ca²⁺ ions to maintain the rod-like conformation of the entire extracellular region and allow interaction with other cadherins,resulting in their adhesive activity.Human E-cadherin has four potential N-glycosylation sites,two in the extracellular subdomain 4(EC4) at Asn-554,566 residues,two in the extracellular subdomain 5 (EC5) at Asn-618,633 residues.It has been reported that,the changes of N-glycosylation of E-cadherin could affect the molecular organization of adherence junction complexes(AJs),alter the cell-cell adhesive activity,and regulate the intracellular signal pathway.Because of the importance of N-glycosylation in E-cadherin biological function, we previously generated N-glycosylation-deficient mutants of E-cadherin by substituting glutamine for asparagines in each N-glycosylation consensus sequence (N-X-S/T) of human E-cadherin,either individually or in combination,by site-directed mutagenesis.Human MDA-MB-435 breast carcinoma cell line which lacking endogenic E-cadherin were transfected with E-cadherin variant plasmids.And cells stably expressed E-cadherin variants were established by G418 selection.In previously study,N-glycans at Asn-554,566 played an important role in cell-cell adhesion which mediated by E-cadherin.Furthermore,N-glycan at Asn-633 affected the expression of E-cadherin.The elimination of the N-glaycan resulted in the degradation of E-cadherin.It has been reported that N-glycosylation plays a role in glycoprotein folding and maintaining the stability of glycoprotein structure.When eliminated given N-glycans, some glycoproteins would fold incorrectly and sequentially degraded by ERAD pathway(ER-associated degradation).To elucidate the degradation of E-cadherin lacking N-glycan at Asn-633 site in details,in the first part of this paper,we first treated cells stably expressed E-cadherin lacking N-glycan at Asn-633 site with inhibitor of proteasomes,MG132.Results showed that MG132 could efficiently block the degradation of the mutant.Furthermore,E-cadherin associated with ubiquitins in cells stably expressed E-cadherin lacking N-glycan at Asn-633 site,and formed a series of ubiquitinated E-cadherin ladder bands.Next,we treated cells stably expressed E-cadherin lacking N-glycan at Asn-633 site with inhibitor of ERAD pathway,DMM.Results showed that DMM also could efficiently block the degradation of the mutant.Furthermore,association of E-cadherin with p97 was also detected in cells stably expressed E-cadherin lacking N-glycan at Asn-633 site.These results showed that unglycosylation at Asn-633 site resulted in the degradation of E-cadherin via ERAD pathway.In the second part of this paper,we further investigated the effect of unglycosylation at Ash-633 site on E-cadherin folding.First,we examined the association of E-cadherin lacking N-glycan at Asn-633 site withα-catenin,β-catenin, and p120,respectively.Results showed that this mutant could associate withβ-catenin and p120,suggesting that unglycosylation at Asn-633 has few of effect on the E-cadherin cytoplasmic domain,it was still functional.Furthermore,we examined the association of E-cadherin variants with calnexin.Results showed that calnexin participated in E-cadherin folding,all variants associated with calnexin.Compared with other variants,the ration of E-cadherin with calnexin significantly increased in cells stably expressed E-cadherin lacking N-glycan at Asn-633 site,suggesting its N-terminal was misfolded.A prediction three dimensional structure of E-cadherin extracellular domain was constructed using homology molding method.From the structure,Asn-633 site localized at core region of EC5,suggesting N-glaycan at this site likely participated in maintaining the stability of core region structure.It has been reported that,not all ERAD substrates are arrested in ER.The degradation of some misfolded protein are dependent on the ER-Golgi trafficking.So in the third part of this paper,we focused our research on the subcellular distribution of E-cadherin lacking N-glycan at Asn-633 site after its degradation was blocked. Results from Immunofluorescence and TritonX-100 soluble/insoluble extraction showed that,after suppressed the ERAD pathway,E-cadherin lacking N-glycan at Asn-633 site could be re-expressed,however,its subcellular distribution had been changed.This mutant could not be transported to cell surface and insteadly mainly distributed in cytoplasm.In contrast,when eliminated other three N-glycans as well as a single N-glycan at Asn-633 was conserved,E-cadherin could still be transported to cell surface correctly.Analysis of N-glycans type of E-cadherin lacking N-glycan at Asn-633 site was performed using the method of Lectin stain and glycosidase treatment.Results showed that,N-glycans of this mutant were modified with immature high mannose N-glycan type,suggesting that this mutant could not be transported to Golgi apparatus.After E-cadherin lacking N-glycan at Asn-633 site degraded,how would theβ-catenin be? In the 4^th part of this paper,we investigated the effect of E-cadherin mutant degradation onβ-catenin.We found that the degradation of E-cadherin mutant could result in the degradation ofβ-catenin.And theseβ-catenins could not be phosphorylated at serine/threonine residues before its degradation.Further researching showed thatβ-catenin in cells stably expressed E-cadherin lacking N-glycan at Asn-633 site associated with p97,and was packed into retro-translocation complexes.In conclusion,N-glycosylation at Asn-633 site of E-cadherin affects its expression,folding,and trafficking.The elimination of this N-glycan would make E-cadherin fold incorrectly at N-terminal.Then misfolded E-cadherin would be ubiquitinated and sequentially was retro-translocated by p97 to proteasomes.During the process,β-catenin which binds to E-cadherin was also co-degraded together.And the degradation ofβ-catenin was independent on the phosphorylation at serine/threonine residues.