【作者】 苏剑敏；

【导师】 查锡良； 韩泽广；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 E-钙粘蛋白(E-cadherin,又称uvomorulin,cell-CAM 120/80和Arc-1)是一类钙离子依赖的介导相邻细胞间粘附的Ⅰ型跨膜糖蛋白。E-钙粘蛋白包含一个具有5个钙粘蛋白重复序列的胞外结构域,主要控制钙粘蛋白间的相互作用;一个跨膜结构域和一个位于胞浆内的高度保守的C-末端序列。 E-钙粘蛋白分子中有6个潜在的N—糖基化位点(N-X-S/T),其中一个存在于胞内肽段的位点糖基化可能性不大,其余5个均存在于胞外肽段范围内,分别为AA372-374(N-P-T),AA554-556(N-S-T),AA566-568(N-G-S),AA619-621(N-T-S)和AA634-636(N-W-T)。进一步比较研究发现,5个潜在糖基化位点中,只有AA566-568为人独有的,而其它4个潜在糖基化位点较为保守,在小鼠和鸡E-钙粘蛋白中同样存在。 E-钙粘蛋白在许多上皮组织细胞中有表达。许多研究表明肿瘤细胞表面E-钙粘蛋白的异常表达与肿瘤细胞的增殖,去分化,侵袭和转移均有关系。而E-钙粘蛋白缺失可能是肿瘤细胞恶性增殖和侵袭转移的一个重要原因,因为当肿瘤细胞中重新导入E-钙粘蛋白后,会导致细胞增殖和侵袭能力的下降,所以E-钙粘蛋白被认为是一种抑癌基因,同时又是一种侵袭转移抑制基因。 E-钙粘蛋白作为一种糖蛋白,其分子中的N-糖链在肿瘤细胞的增殖、侵袭和转移中的作用还不清楚。为了阐明E-钙粘蛋白N-糖链在肿瘤细胞的增殖、侵袭和转移中的作用,我们首先将E-钙粘蛋白分子中潜在的N-糖基化位点,Asn372,Asn554,Asn566,Asn619和Asn634分别突变成谷氨酰胺Gln,构建突变不同糖基化位点的E-钙粘蛋白基因真核表达质粒,分别去除E-钙粘蛋白不同位点上潜在的N—糖链,我们还构建了一个同时缺失上述5个糖基化位点的E-钙粘蛋白基因真核表达质粒,分别将这些质粒及野生型质粒转入不表达E-钙粘蛋白的MDA-MB-435高侵袭性的乳腺癌细胞株,得到了相应的稳定转染细胞株。研究E-钙粘蛋白糖链缺失前后对细胞间聚集、细胞周期、增殖、迁移和伸展等生物学行为的影响;E-钙粘蛋白N-糖链对E-钙粘蛋白分拣、转运和分泌等的影响;N-糖链对E-钙粘蛋白介导的信号通路的影响,以阐明E-钙粘蛋白分子中N-糖链的生理功能。 为了阐明E-钙粘蛋白各糖基化位点上N-糖链的存在情况,我们用肼解法、凝集素法、糖苷酶酶解法和质谱方法检测了E-钙粘蛋白N-糖链的分布情况,结果发现当Asn-372,Asn-554,Asn-566和Asn-619 N-糖基化位点突变后,从肼解法的结果可以看出E-钙粘蛋白的糖链含量明显下降,提示在这四个位点上均有N-糖链存在。观察Mu-372/554/566/619的情况我们可以看到当Asn-372,Asn-554,更多还原

【Abstract】 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更多还原