The Function Of N-linked Oligosaccharides Of E-cadherin And Its Mechanism

E-cadherin (also known as uvomorulin, L-CAM, Cell-CAM 120/80 and Arc-1) is a type-1 single-span transmembrane glycoprotein, it mediates cell-cell adhesion by calcium-dependent homotypic intractions between neighboring cells. The full-length human E-cadherin gene (CDH1) spans a region of approximately 100 kb, it contains 16 exons and it is located on chromosome 16q22.1, and was isolated by Berx G et al. in 1995. The full-length cDNA of human E-cadherin is 2815 bp, and was cloned by Rimm DL in 1994. The predicted molecular mass of the unglycosylated and unprocessed protein is 97kDa, after it is glycosylated the molecular weight is about 120kDa.Six N-X-S/T consensus sequences for N-linked glycosylation are found in the human E-cadherin protein. The one in the cytoplasmic domain is probably not utilized, the other five sites is in the extracellular domain. Four of these five sites are conserved in human, mouse and chicken E-cadherins. The remaining site at codons 566-568 is unique to the human protein.E-cadherin consists of three parts: an extracellular portion that contains five homologous segments called cadherin domains; a transmembrane segment; and a highly conserved cytoplasmic domainIn the first extracellular repeat domain, there is a motif at AA229-231 (H-A-V) which is necessary for homotypic interaction. The catenins (pl20^ctn, b-catenin, plakoglobin and a-catenin) bind to the cytoplasmic tails of E-cadherin. b-catenin and plakoglobin compete for binding to the socalled catenin-binding domain (CBD) and mediate the attachment of cadherin to the actin cytoskeleton via a-catenin. In contrast, p120 associates with the cadherin juxtamembrane domain (JMD) and does not bind to a-catenin.E-cadherin is expressed in most epithelial tissues. Several immunohistochemical studies have reported a strong correlation between E-cadherin loss and the initiation and progression of tumors. This loss appears to be a key event in acquisition of invasive capacity, because re-expression of E-cadherin suppresses the invasion of tumor cells in vitro. For these reasons, E-cadherinhas been termed an 'invasion suppressor'.As a type-I transmembrane glycoprotein, the functions of N-glycan chain in E-cadherin is poorly understood. In order to explore the role of E-cadherin N-sugarchain in the initiation and progression of tumors, we prepared mutant human E-cadherin which was analyzed by site-directed replacement of each of the five glycosylation sites, Asn-372, Asn-554, Asn-566, Asn-619 and Asn-634 with Gin, individually. Furthermore, we prepared mutant human E-cadherin was replaced by all of these five glycosylation sites, in order to eliminate N-sugar chains being attached to the extracellular region. To examine the effect of the loss of the sugar chain on the E-cadherin function and observed cell biological behavior, including cell proliferation, cell cycle, cell spreading, cell aggregation and cell invasion, these mutants were constantly expressed in MDA-MB-435 breast cells, which E-cadherin was unexpressed in mRNAand protein level.When we transfected these six mutant plasmids and wild-type E-cadherin into MDA-MB-435cell line we found that Mu-372, Mu-554, Mu-566, Mu-619 and wild-type cell lines can normally express the E-cadherin protein, and Mu-634 and Mu-all can express E-cadherin only at mRNA levels, the protein expression of E-cadherin is too less to be measured by western-blot. So we can speculate that the suar chain at Asn-634 site is important to the E-cadherin stability. In order to further demonstrated the role of the suar chain at Asn-634 site, we construct the Mu-372/554/566/619 cell lines which only reserved the Asn-634 sugar chain on E-cadherin molecule, we found that Mu-372/554/566/619 cell line also can normally express E-cadherin protein, thus we can confirmed that Asn-634 sugar chain is very important to reserve the E-cadherin molecule stability. Additionally, when we delete the sugar chain at Asn372, Asn554, Asn566, Asn619 sites, respectively. The distribution of E-cadherin was changed. In the wild-type cell line, the E-cadherin equably distributed on the cell surface, and in the Mu-372, Mu-554, Mu-566, Mu-619 cell lines, E-cadherin was aggregated on the cell, but we can not confirm it is located on the cell surface or in the cytoplasm. To explore this, we isolated cell membrane protein and cytoplasma protein, and we measured the level of E-cadherin in these two type protein by western-blot, we found that E-cadherin can only detect at membrane protein, in the cytoplasma, we did not find E-cadherin. So we can confirm that E-cadherin was aggregated on the cell surface, and it also imply that the deletion of sugar chain at Asn-372, Asn-554, Asn-566 and Asn-619 sites did not affect the sorting or trafficking of E-cadherin.In order to explore the condition of N-linked glycosylation at this six sites, we measured the glycosylation of E-cadherin by Hrp-hyrazinium, Hrp-ConA and Massspectrum method. We found the mutation of Asn-372, Asn-554, Asn-566 and Asn-619 glycosylation site significantly reduced the sugar contents by Hrp-hyrazinium method. It suggested that there are sugar chains at these sites. Asn-634 site may also have sugar chain because of obviously dying at Mu-372/554/566/619 cell line by Hrp-hyrazinium and the Hrp-ConA method. From the Hrp-ConA dying, we can speculate that the sugar chain at Asn-566, Asn-619 and Asn-634 sites may be biantenna structure, and the sugar chain at Asn-372 and Asn-554 sites are not biantenna structure.In order to determine the role of various N-sugar chain of E-cadherin in cell proliferation, we mearured the proliferation change of the mutant cell lines and wild-type cell line by MTS system, and found the proliferation ability of Mu-634 cell line is significantly lower than that of wild-type cell line, the proliferation ability of Mu-619 and Mu-all cell lines also decreased. When we measured the cell cycle of these mutant cell lines by the flow cytometry we found that the Gl phase of Mu-634, Mu-619 and Mu-all cell lines were increased compare with the wild type cell line. Up to this, we can speculate the reason of decreased proliferation is the Gl arrest in Mu-634, Mu-619 and Mu-all cell lines. To explore the molecular mechanism of these changes, we measured the protein level of CyclinDl, CyclinE, Cdk2, cyclin dependent kinase inhibitor P21 and P27, E-cadherin associated signal protein P-catenin and P120ctn, and other signal molecules such as PKB and Gsk-3p by western-blot. We discovered the expression of CyclinDl was reduced in Mu-634, Mu-619 and Mu-all cell lines, especially in Mu-634, Mu-619, and cyclinE and Cdk2 have no change in their protein level. The cyclin dependent kinase inhibitor P27 level was relatively high in these three cell lines. The P-catenin level was dramaticly decreased in Mu-634 cell line. The expression of Gsk-3p was increased in Mu-634 and Mu-619 compare with wild type cell lines, and the level of PKB was decreased in these two cell lines. It suggest that the deletion of sugar chain at Asn-634 site promoted the expression of Gsk-3p, and this protein make P-catenin unstable in cytoplasma, and inhibit the trancripation of CyclinDl or the increased Gsk-3p directly affect cyclinDl, and caused it degradation. Additionally, the reduced expression of PKB is also one of the reason that caused cyclinDl decreased.We further explored the role N-sugar chain in cell invasion. We observed the changes of invasiveness ability in vitro by Matrigel-transwell method. We discovered that the invasion ability was dramatically decreased in Mu-372, Mu-619 and Mu-634...