Roles of Cell Surface N-Linked Oligosaccharides in Intercellular Cohesion in Dictyostelium Discoideum

盘藻发育的不同阶段，糖蛋白N-连接低聚糖(OLS)的加工过程有很大差异。最近对野生型和突变型菌株的研究为两个细胞表面衍生的OLS在后期聚集过程中的细胞间识别和凝聚中的作用提供了强有力的证据。这些OL在发育的早期和后期都不能被检测到。这项拟议中的研究将通过与麻省理工学院的凯瑟琳·科斯特洛博士合作，结合快速原子轰击/质谱仪，并使用特定的糖苷酶治疗，来确定这些多糖的化学结构。这些结果将通过特定的降解处理扩展到结构/功能关系的分析，以确定OLS对凝聚力抑制的影响，并通过对OLS组装和凝聚力的条件缺陷的特定突变菌株的研究。与已知的与凝聚力有关的糖蛋白相比，这些OL对凝聚力的确切贡献将通过努力抑制与多糖和包括抗体在内的其他干扰剂的组合的聚集来测试。携带OLS的糖肽在与载体蛋白(如BSA)共价连接后，将通过免疫产生单抗。这些抗体还将用于鉴定体内携带OLS的糖蛋白，并用于分离缺乏OLS的突变体和恢复OLS的返回体。高等生物中的很大一部分蛋白质携带复杂的糖分子(低聚糖)作为其结构的一部分。这些低聚糖对蛋白质的功能特性的贡献尚未得到很好的表征，但已知在细胞与细胞之间的黏附中具有重要作用。亨德森博士对低聚糖进行了结构/功能分析，已知的低聚糖参与了细胞发育过程中的聚集。她正在使用一种简单的有机体--圆盘芽孢杆菌作为模型系统。

There are dramatic differences in processing of glycoprotein N- linked oligosaccharides (OLS) at different stages during development of D. discoideum. Recent studies with wild type and mutant strains have provided strong evidence for a role of two cell surface-derived OLS in intercellular recognition and cohesion during late aggregation. These OLS are not detectable at earlier and later developmental stages. The proposed research will establish the chemical structure of these glycans, using a combination of fast atom bombardment/mass spectrometry in collaboration with Dr. Catherine Costello at M.I.T. and using specific glycosidase treatments. These results will be extended to analysis of structure/function relations by specific degradative treatments to ascertain effects on cohesion inhibition by the OLS and by study of specific mutant strains conditionally defective in both OLS assembly and cohesion. The exact contribution of these OLS to cohesion, in comparison with known glycoproteins implicated in cohesion, will be tested by efforts to inhibit aggregation with combinations of glycans and other perturbing agents, including antibodies. Monoclonal antibodies will be generated by immunization with glycopeptides carrying the OLS, after covalent conjugation to a carrier protein such as BSA. These antibodies will also be used for identification of the in vivo glycoproteins which carry the OLS and for isolation of mutants lacking them and of revertants which have recovered them. %%% A large proportion of the proteins in higher organisms carry complex sugar molecules (oligosaccharides) as part of their structure. These oligosaccharides contribute to the functional properties of the proteins in ways that are not well characterized but are known to be important in cell-cell adhesion. Dr. Henderson has undertaken a structure/function analysis of oligosaccharides known to be involved in the aggregation of cells during development. She is using the simple organism, D. discoideum, as a model system