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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK030331-07
Application #
3229391
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1982-01-01
Project End
1990-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Virginia Polytechnic Institute and State University
Department
Type
Earth Sciences/Resources
DUNS #
003137015
City
Blacksburg
State
VA
Country
United States
Zip Code
24060
Zatta, P F; Nyame, K; Cormier, M J et al. (1991) A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin. Anal Biochem 194:185-91
Clark, G F; Gorbea, C M; Cummings, R D et al. (1991) Decreased biosynthesis of Forssman glycolipid after retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells. Lectin-affinity chromatography of the glycolipid-derived oligosaccharide. Carbohydr Res 213:155-68
Do, K Y; Smith, D F; Cummings, R D (1990) LAMP-1 in CHO cells is a primary carrier of poly-N-acetyllactosamine chains and is bound preferentially by a mammalian S-type lectin. Biochem Biophys Res Commun 173:1123-8
Nyame, K; Smith, D F; Damian, R T et al. (1989) Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine. J Biol Chem 264:3235-43
Wang, W C; Clark, G F; Smith, D F et al. (1988) Separation of oligosaccharides containing terminal alpha-linked galactose residues by affinity chromatography on Griffonia simplicifolia I bound to concanavalin A-sepharose. Anal Biochem 175:390-6
Tarrago, M T; Tucker, K H; Van Halbeek, H et al. (1988) A novel sialylhexasaccharide from human milk: purification by affinity chromatography on immobilized wheat germ agglutinin. Arch Biochem Biophys 267:353-62
Smith, D F; Prieto, P A; McCrumb, D K et al. (1987) A novel sialylfucopentaose in human milk. Presence of this oligosaccharide is not dependent on expression of the secretor or Lewis fucosyltransferases. J Biol Chem 262:12040-7
Law, K L; Smith, D F (1987) III6NeuAcLc4Cer in human SW1116 colorectal carcinoma cells: a possible oncofetal antigen that is not dependent on Lewis gene expression. Arch Biochem Biophys 258:315-23