ISOLATION OF OLIGOSACCHARIDES--SUGAR-BINDING PROTEINS

通过单克隆抗体确定的癌胚糖脂抗原 对人类肿瘤细胞，也被发现作为游离糖， 人乳 制备的单特异性兔抗血清 纯化的人乳低聚糖已被用于检测 并在胎儿组织和人体中发现新的神经节苷脂 大肠癌细胞系SW 1116。 没有这些 来自正常成人肠道的抗原与它们的 癌胚抗原 也许最重要的诊断 癌胚抗原是唾液酸-Lea抗原， 由针对SW 1116细胞的单克隆抗体定义。 这 抗原存在于血清粘蛋白中， 与胃肠癌有关。 的限制 19-9抗体的诊断有用性在于， 刘易斯阴性血型的人不能合成抗原。 针对合成的寡糖的抗血清 与血型抗原无关，但与唾液酸相关， 莱亚抗原可能是更有用的诊断试剂。 结构 对癌胚糖脂抗原的分析为 了解它们在肿瘤细胞中的生物合成。 这些分析， 然而，仅限于通过单克隆抗体检测的结构， 抗体的 本提案中概述的工作成果将 是相对的结构定义和确定 量的糖脂从SW 1116细胞。 这些信息将 提供了一个用于鉴定任何有用的肿瘤标志物的BIS， 不被抗体检测到，有助于理解 肿瘤细胞中糖脂的生物合成。 这一任务 通过代谢放射性标记SW 1116中的糖脂完成 使用（3 H）糖前体的细胞。 糖脂的结构研究- 通过系列凝集素或抗体纯化的衍生（3 H）-低聚糖 亲和层析，将使用已建立的， 结合外切糖苷酶的微甲基化分析 数字化。 虽然这种方法已经成功地应用于 糖蛋白寡糖结构测定 从培养的细胞和LDL和EGF受体，它从来没有 应用于糖脂。 利用这一敏感和 代谢分析的明确结构方法 标记的糖脂将是该提议的主要焦点。 的 这项研究的长期利益将是建立一个 糖脂结构分析的重要方法 研究中会遇到的细胞数量 胚胎发育过程中的糖代谢。

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.