SULFATED OLIGOSACCHARIDES OF NORMAL AND MALIGNANT CELLS

以前的研究表明存在一类硫酸化的 真核生物糖蛋白中的N-连接寡糖。 然而， 目前已知它们的详细结构，生物合成，调节， 或生物学意义。 在本提案中，我们描述了 一种定量、不降解酶法测定 完整硫酸化N-连接寡糖直接从全 蛋白质，并将其与所有其他硫酸化 大分子 此方法将用于查找 硫酸化N-连接寡糖在总细胞匀浆中的表达 在随后的连续组织培养中， 系统：（1）CHO和小鼠淋巴瘤细胞，具有特异性缺陷， N-连接的糖基化和/或用加工抑制剂处理 （2）NIH 3T3细胞经特异性β-葡萄糖苷酶转化前后， 癌基因的种类;（3）转移性和非转移性肿瘤细胞;和 (4)\处于诱导分化的各个阶段的白血病细胞。 上 根据筛选结果，将对其中一个或两个系统进行 选择进一步研究。 单个低聚糖将是 分馏和硫酸化单糖的类型鉴定。 这 随后将进行详细的结构分析的硫酸化 低聚糖，关于位置，连接和类型， 成分糖，使用传统的和新开发的方法。 这 将定义在所选系统中发现的任何结构差异 为了学习 从结构分析中获得的信息将 用于预测生物合成途径的产生 硫酸寡糖在这些细胞，并预测可能的水平， 调制，可以解释发现的任何差异。最后，我们将 设计分析，将确定酶和底物参与 硫酸化反应。 这项工作的具体目标是 看看相关酶表达的差异是否可以解释 这些系统中N-连接寡糖硫酸化的差异。 在 从长远来看，我们希望扩展这些研究， N-连接寡糖的硫酸化在 如肿瘤发生和转移形成的过程。 （B）

Previous studies have demonstrated the existence of a class of sulfated N-linked oligosaccharides in eukaryotic glycoproteins. However, little is currently known about their detailed structure, biosynthesis, regulation, or biological significance. In this proposal, we describe the establishment of an enzymatic method for quantitative and nondegradative release of intact sulfated N-linked oligosaccharides directly from whole proteins, and their complete separation from all other sulfated macromolecules. This method will be used to look for differences in the expression of sulfated N-linked oligosaccharides in total cell homogenates and on specific glycoproteins in the following continuous tissue culture systems: (1)\CHO and mouse lymphoma cells with specific defects in N-linked glycosylation and/or treated with inhibitors of processing glycosidases; (2)\NIH 3T3 cells before and after transformation by specific classes of oncogenes; (3)\metastatic and nonmetastatic tumors cells; and (4)\leukemia cells in various stages of induced differentiation. On the basis of the results of this screening, one or two of these systems will be chosen for further study. The individual oligosaccharides will be fractionated and the types of sulfated monosaccharides identified. This will be followed by a detailed structural analysis of the sulfated oligosaccharides, with regard to the location, linkages, and types of component sugars, using conventional and newly developed methods. This will define any differences in structure found within the systems chosen for study. The information obtained from the structural analysis will be used to predict the biosynthetic pathways for the generation of the sulfated oligosaccharides in these cells, and to predict possible levels of modulation that could explain any differences found. Finally, we will design assays that will identify the enzyme and substrates involved in the sulfation reactions in question. The specific aim of this work will be to see if differences in the expression of the enzymes involved could explain differences in sulfation of N-linked oligosaccharides in these systems. In the long run, we hope to extend these studies to attempt to understand potential biological roles of the sulfation of N-linked oligosaccharides in processes such as tumorigenesis and metastasis formation. (B)