ROLE OF N-GLYCOSYLATION IN E-CADHERIN MEDIATED CELL-CELL ADHESION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 E-钙粘蛋白是一种在上皮细胞中表达的120 kDa的膜糖蛋白，在细胞间的黏附连接中起主要作用。贴壁连接具有细胞内和细胞外的锚定点，以调节细胞间的相互作用。[1-2]贴壁连接的组装或拆卸的变化与细胞状态的重大变化有关，包括分化和增殖[3]，以及癌症进展。E-钙粘附素功能丧失是许多类型癌症中的常见事件，通常由E-钙粘附素表达减弱或异常引起。[4]我们自己和其他实验室的结果表明，E-钙粘附素的N-糖基化模式在黏附连接的分子组织中起着作用。具体地说，复杂N-糖链的存在与不稳定的黏附连接有关。[5]研究E-钙粘蛋白的结构特征，特别是其N-糖基化模式和蛋白质结合伙伴，有助于深入了解E-钙粘蛋白介导的黏附和细胞信号功能。 分离这种大的膜蛋白需要开发特定的方法。我们现在已经分离出具有不同结合伙伴和不同N-糖基化状态的E-钙粘素池，这取决于细胞状态。恶性细胞中E-钙粘附素的形式比非恶性细胞中的E-钙粘蛋白形式糖基化程度更高；通过Western印迹和切除的SDS-PAGE条带的多肽质量指纹图谱检测，它们以更高的表观重量迁移。蛋白质组学方法已经被用来确定E-钙粘素复合体在几种不同条件下的组成。[6]我们发现，与非恶性细胞中存在的E-钙粘附素相比，恶性细胞中的E-钙粘附素与不稳定蛋白的关联程度更大。 E-钙粘蛋白N-糖基化也被研究过，特别是位点占有率和N-糖链组成。MALDI-TOF MS和串联ESI-MS/MS分析表明，E-钙粘蛋白主要含有复杂的N-葡聚糖。 这项工作强调了细胞环境、特定蛋白质对黏附复合体的招募以及对E-钙粘附素的特定结构修饰可能定义了E-钙粘附素介导的黏附的整体稳定性。 一份描述结果的手稿正在准备中。 参考资料： 1.Gottardi CJ，Wong E，Gumbiner BM。《细胞生物学》2001；153(5)：1049-60 2.Perez-Moreno M，Jamora C，Fuchs E.Cell 2003；112(4)：-48 3.Gumbiner BM。NAT Rev Mol Cell Biol 2005；6：622-634 4.惠洛克MJ，约翰逊KR。Annu Rev Cell Dev Biol 2003；19：207-35 5.Liwosz A、Lei T和Kukuruzinska MA。生物化学杂志2006年；281：23138-49。 6.Blackmun-Ross K，Kukuruszinska M，Costello CE，2008，在ASMS会议上介绍。和糖类生物学学会会议 7.博士论文，K.Blackmun-Ross，波士顿大学，2008年8月。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. E-cadherin is a 120-kDa membrane glycoprotein expressed in epithelial cells; it is the main player in establishing adherens junctions between cells. The adherens junctions have both intra- and extracellular anchorage points in order to regulate cell-cell interactions.[1-2] Alterations in the assembly or disassembly of adherens junctions occur in association with major changes in the state of cell, including differentiation and proliferation [3], as well as in cancer progression. Loss of E-cadherin function is a frequent event in many types of cancer, commonly caused by diminished or aberrant E-cadherin expression. [4] Results from our own and other laboratories indicate that the N-glycosylation pattern of E-cadherin has a role in the molecular organization of adherens junctions. Specifically, the presence of complex N-glycans is associated with destabilized adherens junctions.[5] Investigation into the structural characteristics of E-cadherin, specifically its N-glycosylation pattern and protein binding partners, provide insight into E-cadherin mediated adhesion and cell signaling functions. Isolation of this large membrane protein has required the development of specific methods. We have now isolated pools of E-cadherin with different binding partners and different N-glycosylation states, depending on cell status. The forms of E-cadherin in malignant cells are more highly glycosylated than are those in non-malignant cells; they migrate at a higher apparent weight, as detected by Western blot and peptide mass fingerprinting of excised SDS-PAGE bands. Proteomic methods have been used to define the composition of the E-cadherin complex under several different conditions. [6] We have found that E-cadherin is associated with destabilizing proteins in malignant cells to a greater extent than when it is present in non-malignant cells. E-cadherin N-glycosylation has also been investigated, specifically site occupancy and N-glycan composition. MALDI-TOF MS and tandem ESI-MS/MS analyses of in-gel enzymatically released N-glycans indicate that E-cadherin contains predominantly complex N-glycans. This work highlights that cell context, the recruitment of specific proteins to the adhesion complex, along with specific structural modifications to E-cadherin may define the overall stability of E-cadherin mediated adhesion. A manuscript describing the results is in preparation. References: 1. Gottardi CJ, Wong E, Gumbiner BM. J Cell Biol 2001; 153(5): 1049-60 2. Perez-Moreno M, Jamora C, Fuchs E. Cell 2003; 112(4): 535-48 3. Gumbiner BM. Nat Rev Mol Cell Biol 2005; 6:622-634 4. Wheelock MJ, Johnson KR. Annu Rev Cell Dev Biol 2003; 19:207-35 5. Liwosz A, Lei T, and Kukuruzinska MA. J Biol Chem 2006; 281:23138-49. 6. Blackmun-Ross K, Kukuruszinska M, Costello CE, 2008, presented at ASMS Conf. and Society for Glycobiology Meeting 7. PhD thesis, K. Blackmun-Ross, Boston University, August 2008.