ISOLATION OF OLIGOSACCHARIDES--SUGAR-BINDING PROTEINS

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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.