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

N-糖基化在 E-钙粘蛋白介导的细胞粘附中的作用

• 批准号: 8170891
• 负责人: MARIA A. KUKURUZINSKA
• 金额: $ 6.06万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170891
• 关键词: Adherens Junction; Adhesions; Binding; Boston; Cadherins; Cell Communication; Cell-Cell Adhesion; Cells; Characteristics; Complex; Computer Retrieval of Information on Scientific Projects Database; Development; Doctor of Philosophy; E-Cadherin; Epithelial Cells; Event; Fingerprint; Funding; Gel; Glycobiology; Grant; Institution; Investigation; Laboratories; Manuscripts; Mediating; Membrane Glycoproteins; Membrane Proteins; Methods; Modification; Molecular; Non-Malignant; Pattern; Peptides; Polysaccharides; Preparation; Protein Binding; Proteins; Proteomics; Research; Research Personnel; Resources; Role; Signal Transduction; Site; Societies; Source; United States National Institutes of Health; Universities; Weight; Western Blotting; Work; cancer cell; cancer type; extracellular; glycosylation; insight; meetings; tumor progression

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 E-钙粘蛋白是一种在上皮细胞中表达的 120 kDa 膜糖蛋白；它是在细胞之间建立粘附连接的主要参与者。粘附连接具有细胞内和细胞外锚定点，以调节细胞间相互作用。[1-2] 粘附连接组装或分解的改变与细胞状态的主要变化有关，包括分化和增殖 [3] 以及癌症进展。 E-钙粘蛋白功能丧失是许多类型癌症中的常见事件，通常是由 E-钙粘蛋白表达减少或异常引起的。 [4] 我们自己和其他实验室的结果表明，E-钙粘蛋白的 N-糖基化模式在粘附连接的分子组织中发挥作用。具体来说，复杂 N-聚糖的存在与不稳定的粘附连接有关。 [5]对 E-钙粘蛋白结构特征的研究，特别是其 N-糖基化模式和蛋白质结合伴侣，可深入了解 E-钙粘蛋白介导的粘附和细胞信号传导功能。 分离这种大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伴侣和不同 N-糖基化状态的 E-钙粘蛋白池，具体取决于细胞状态。 恶性细胞中的 E-钙粘蛋白形式比非恶性细胞中的 E-钙粘蛋白的糖基化程度更高。通过蛋白质印迹和切除的 SDS-PAGE 条带的肽质量指纹分析检测到，它们以更高的表观重量迁移。 蛋白质组学方法已用于定义几种不同条件下 E-钙粘蛋白复合物的组成。 [6] 我们发现，与非恶性细胞中的 E-钙粘蛋白相比，E-钙粘蛋白在恶性细胞中与不稳定蛋白的关联程度更大。 还研究了 E-钙粘蛋白 N-糖基化，特别是位点占用和 N-聚糖组成。对凝胶内酶促释放的 N-聚糖的 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-聚糖。 这项工作强调了细胞环境、特定蛋白质向粘附复合物的募集以及 E-钙粘蛋白的特定结构修饰可能定义 E-钙粘蛋白介导的粘附的整体稳定性。 描述结果的手稿正在准备中。 参考： 1. Gottardi CJ，Wong E，Gumbiner BM。细胞生物学杂志2001； 153（5）：1049-60 2. Perez-Moreno M、Jamora C、Fuchs E. Cell 2003； 112（4）：535-48 3. 甘比纳 BM。自然评论分子细胞生物学2005； 6:622-634 4.惠洛克·MJ，约翰逊·KR。细胞开发生物学年鉴 2003； 19:207-35 5.Liwosz A、Lei T 和 Kukuruzinska MA。生物化学杂志，2006； 281：23138-49。 6. Blackmun-Ross K、Kukuruszinska M、Costello CE，2008 年，在 ASMS Conf 上发表。和糖生物学会会议 7. 博士论文，K. Blackmun-Ross，波士顿大学，2008 年 8 月。