CYTOPLASMIC FUCOSYLATION

蛋白质糖基化的突变对细胞有巨大的影响 增殖率、细胞分选、假原体游走、孢子壳 细胞内的形成、孢子萌发和蛋白质运输速率 粘液霉菌盘基网基菌。该项目的长期目标是 在分子水平上了解糖基化如何支持这些 正常细胞中的细胞活动。对一种突变体的分析 国内生产总值-岩藻糖合成的条件已经证明了最优细胞 增殖率和孢子健康取决于正常的岩藻糖基化。 对可能影响这些过程的岩藻糖化途径的检查 导致了发生的岩藻糖化反应的部分表征 在网柄蕨类细胞的细胞质中。因为他们的 区划、受体岩藻蛋白(Fp21)和 对其进行修饰的岩藻糖基转移酶(FP21-岩藻糖基转移酶)是 特别准备影响这些行为。生物化学研究 分离的细胞导致了FP21在细胞中积累的结论 胞质中，活性的FP21-岩藻糖基转移酶积累在 细胞质，岩藻糖基转移酶对 糖核苷酸供体，GDP-岩藻糖。这导致了一种 FP21合成并在细胞质中蓄积的模型 它是由合成的常驻糖基转移酶糖基化的。 在那里，来自细胞质的糖核苷酸池。具体的 有待检验的假设是，FP21是在细胞质中合成的，即 它在细胞质中被α(1-4)岩藻糖基转移酶 也是在那里合成的，即FP21具有独特的结构特征 这标志着它是FTase的岩藻糖基受体，这个反应是 保存在其他生物体中。这些假设将通过以下方式进行验证 开发抗体用于研究FP21和Fp21的生物发生区段 岩藻糖基转移酶，利用免疫显微镜确认定位 在完整的细胞中，比较预测的氨基酸序列 初级翻译产物与已知的细胞质、非膜的翻译产物 蛋白质，确定FP21寡糖的结构，以及 开展岩藻糖基转移酶反应的竞争研究。一次 FP21及其岩藻糖基转移酶是以这种方式定义的，将有一个 以直接测试它们在增殖和孢子健康中的作用，并 识别在其他生物体中以这种方式修饰的糖蛋白。 了解细胞质糖基化是很重要的，因为它 巨大的监管潜力，从以下几个方面可见一斑 丝氨酸和苏氨酸残基的磷酸化对酶活性和构象的影响 修饰的蛋白质。描述这一途径的第二个原因是 对岩藻共轭化合物的分析日益重视， 活体组织和生物体液中岩藻糖基化活性作为 病理学中的细胞活动。对这些问题的理性解读 分析将受益于对多重势能的理解 这些物质的细胞起源。

Mutations in the glycosylation of proteins have dramatic effects on cell proliferation rate, cell sorting, pseudoplasmodial migration, spore coat formation, spore germination, and protein transport rate, in the cellular slime mold Dictyostelium discoideum. The long term goal of the project is to understand at the molecular level how glycosylation supports these cellular activities in normal cells. Analysis of a mutant that is conditional for GDP-Fucose synthesis has demonstrated that optimal cell proliferation rate and spore health depend on normal fucosylation. Examination of fucosylation pathways that may influence these processes has led to the partial characterization of a fucosylation reaction which occurs in the cytoplasm of Dictyostelium cells. Because of their compartmentalization, the acceptor fucoprotein (FP21), and the fucosyltransferase which modifies it (FP21-fucosyltransferase), are specially poised to influence these behaviors. Biochemical studies on fractionated cells have led to the conclusion that FP21 accumulates in the cytoplasm, that active FP21-fucosyltransferase accumulates in the cytoplasm, and that the fucosyltransferase has a very high affinity for the sugar nucleotide donor, GDP-Fucose. This has led to the development of a model that FP21 is synthesized and accumulates in the cytoplasm, and that it is glycosylated by resident glycosyltransferases, which are synthesized there, from cytoplasmic pools of sugar nucleotides. The specific hypotheses to be tested are that FP21 is synthesized in the cytoplasm, that it is fucosylated in the cytoplasm by an alpha(1-4)fucosyltransferase which is also synthesized there, that FP21 possesses unique structural features that mark it as a fucosyl acceptor for the FTase, and that this reaction is conserved in other organisms. These hypotheses will be tested by developing antibodies to study the compartments of biogenesis of FP21 and the fucosyltransferase, utilizing immunomicroscopy to confirm localization in intact cells, comparing the predicted amino acid sequences of the primary translation products with those of known cytoplasmic, non-membrane proteins, determining the structure of the FP21 oligosaccharide, and carrying out competition studies on the fucosyltransferase reaction. Once FP21 and its fucosyltransferase are defined in these ways, there will be a basis to directly test their role in proliferation and spore health, and to identify glycoproteins that are modified in this way in other organisms. It is important to understand cytoplasmic glycosylation because of its tremendous regulatory potential, as evidenced by the roles of phosphorylation of Ser and Thr residues for activity and conformation of modified proteins. A second reason for characterizing this pathway relates to the increasing attention given to the analysis of fucoconjugates and fucosylation activities in biopsies and biological fluids, as markers for cellular activity in pathology. A rational interpretation of these analyses would benefit from an understanding of the multiple potential cellular origins of these substances.