ISOLATION OF OLIGOSACCHARIDES--SUGAR-BINDING PROTEINS

肿瘤胎儿糖脂抗原，由单抗定义 对人类肿瘤细胞，也被发现作为游离糖在 人奶。单特异性兔抗血清的制备 纯化的人乳低聚糖已被用于检测 并在胎儿组织和人类体内鉴定新的神经节苷脂 结直肠癌细胞株SW1116。这些东西的缺失 来自正常成人肠道的抗原与其存在的 肿瘤胎儿抗原。可能是诊断上最重要的 癌胎儿抗原一直是唾液酸-Lea抗原，它是 由抗SW1116细胞的单抗定义。这 抗原存在于血清粘蛋白中，水平升高 与胃肠道癌症有关。一种限制 19-9抗体的诊断价值在于患者 与Lewis阴性的血型不能合成抗原。 针对合成的寡糖的抗血清 与血型抗原无关，但与唾液酸基有关- LEA抗原可能是更有用的诊断试剂。结构性 肿瘤胎儿糖脂抗原分析为临床诊断提供了依据 了解它们在肿瘤细胞中的生物合成。这些分析， 然而，它们仅限于由单克隆检测到的结构 抗体。本提案中概述的工作成果将 是相对关系的结构定义和确定 SW1116细胞的糖脂含量。此信息将 为识别任何有用的肿瘤标志物提供生物信息学 不被抗体检测到，并有助于理解 肿瘤细胞中糖脂的生物合成。这项任务将是 在SW1116中通过代谢标记糖脂实现的 使用(~3H)糖前体的细胞。糖脂的结构研究- 用系列凝集素或抗体纯化衍生的(~3H)-寡糖 亲和层析，将使用已建立的， 结合外切糖苷酶的微量甲基化分析 摘要。虽然这种方法已经成功地应用于 糖蛋白低聚糖的结构测定 从培养的细胞以及低密度脂蛋白和表皮生长因子受体，它从来没有 已被应用于糖脂。利用这一敏感的和 代谢产物分析的明确结构方法 标记的糖脂将是这项提案的主要焦点。这个 这项研究的长期好处将是建立一个 糖脂结构分析的重要方法 在研究中将遇到的细胞数量 胚胎发育过程中的糖共轭代谢。

Oncofetal glycolipid antigens, defined by monoclonal antibodies against human tumors cells, are also found as free sugars in human milk. Monospecific rabbit antisera prepared against purified human milk oligosaccharides have been used to detect and identify new gangliosides in fetal tissue and in a human colorectal carcinoma cell line SW1116. The absence of these antigens from normal adult intestine is consistent with their being oncofetal antigens. Perhaps the most diagnostically important oncofetal antigen has been the Sialyl-Lea antigen which was defined by a monoclonal antibody against SW1116 cells. This antigen occurs in serum mucins, and elevated levels are associated with gastrointestinal cancer. A limitation of the diagnostic usefulness of the 19-9 antibody is the fact that patients with the Lewis negative blood type cannot synthesize the antigen. Antisera directed against oligosaccharides that are synthesized independent of the blood group antigens but related to the sialyl- Lea antigen may be more useful diagnostic reagents. Structural analyses of oncofetal glycolipid antigens provided the basis for understanding their biosynthesis in tumor cells. These analyses, however, are limited to structures detected by monoclonal antibodies. The results of the work outlined in this proposal will be the structural difinition and determination of the relative amounts of glycolipids from SW1116 cells. This information will provide a bisis for identifying any useful tumor markers that were undetected by antibodies and contribute to the understanding of glycolipid biosynthesis in tumor cells. This task will be accomplished by metabolically radiolabeling glycolipids in SW1116 cells using (3H)sugar precursors. Structual studies of glycolipid- derived (3H)-oligosaccharides purified by serial lectin or antibody affinity chromatography, will be performed using established, micro-methylation analyses in combination with exoglycosidase digestions. Although this method has been successfully applied to the structural determination of glycoprotein oligosaccharides from cultured cells and the LDL and EGF receptors, it has never been applied to glycolipids. The exploitation of this sensitive and unequivocal structural method to the analysis of metabolically labeled glycolipids will be a major focus of this proposal. The long term benefits of this research will be to establish an important method for glycolipid structural analyses in the limited numbers of cells that will be encountered in studying glycoconjugate metabolism during embryogenesis.