This dissertation describes the analysis of the carbohydrate portion of glycosphingolipids from colorectal carcinoma cells, SW1116, by metabolically labelling the cells with radioactive monosaccharide precursors. SW1116 cells (1 x 10⁶) metabolically labelled with 222 μCi/ml of either 6-[³H]-D-galactose (25 Ci/mMol) or 6-[³H]-D-glucosamine (38 Ci/mMol) for 30 hours, incorporated 1%-3% of the radioactivity into their glycoconjugates. Approximately 63% of the radioactivity recovered in the glycoconjugates corresponded to glycolipids when cells were labelled with 6-[³H]-D-galactose, and about 12% when cells were radiolabelled with 6- [³H]-D-glucosamine. Metabolically radiolabelled glycolipids were separated into neutral (88-91% of the radioactivity recovered in glycolipids) and acidic (9-12% of the radioactivity in glycolipids) fractions by ion exchange chromatography. Glycolipids in these fractions were subjected to ozonolysis and alkali fragmentation to release the oligosaccharide chains from the ceramide portion. Oligosaccharides obtained from the neutral glycolipids were separated into single components by a combination of high performance liquid chromatography (HPLC) and Ricinus communis agglutinin I (RCA-I)-agarose affinity chromatography. Oligosaccharides were identified based on the monosaccharide composition, methylation analysis, and exoglycosidase digestions. Major glycolipid components present in the neutral fraction were, glucosylceramide (Glc-Cer), galactosylceramide(Gal-Cer), galabiosylceramide (Galαl-4Gal-Cer), lacto-N-tetraosylceramide (Galβ1-3GIcNAcβ1-3Galβ1-4Glc-Cer), Le<sup>a</sup>- pentaglycosylceramide (Galβ1-3[Fucal-4]GlcNAcβ1-3Galβ1-4Glc-Cer), HIpentaglycosylceramide (Fucal-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), a difucosylated lacto-N-tetraosylceramide, and a fucosylated lacto-Nnorhexaglycosylceramide. Minor components detected in this fraction corresponded to lactosylceramide (Galfp1-4Glc-Cer), lacto-Nneotetraosylceramide (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer), and fucosylated and difucosylated lacto-N-neotetraosylceramides. The acidic fraction was separated into monosialylgangliosides and _ disialylgangliosides by ion exchange chromatography. Monosialyloligosaccharides were further purified on HPLC, and biochemically characterized by methylation analysis, exoglycosidase digestions, and monosaccharide composition. The major component of this fraction corresponded to the sialyl-Le<sup>a</sup> glycolipid (NeuAcα2-3Galβ1-3[Fucαl-4]GlcNAcβ1-3Galβ1-4Glc-Cer) as previously reported by Magnani et al. [183]. GM3 (NeuAcα2-3Galβ1-4Glc-Cer) (0.42% of radioactivity recovered in glycolipids), sialyltetraosylceramide a (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer) (0.46% of radioactivity in glycolipids), sialyltetraosylceramide b (Galβ1-3[NeuAcα2-6]GIcNAcβ1-3Galβ1- 4Glc-Cer) (0.21% of radioactivity in glycolipids), and sialyllated fucosylhexaglycosylceramide, were present in minor quantities.
Results from this study demonstrate that metabolic radiolabelling provides a method for the structural analysis of glycolipids, as sensitive as the immunostaining procedures, as unmistakable as physical techniques (Mass Spectrometry, and Nuclear Magnetic Resonance), and that permits the identification of the majority of glycolipids expressed by a cell line, using relatively small number of cells in culture (6 x 10⁶). Application of this method could be extended to the study of changes in glycolipid accompanying oncogenic transformation and differentiation, glycolipid biosynthesis, intracellular sorting of glycolipids, recycling and turnover. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/39852 |
| Date | 14 October 2005 |
| Creators | Tarrago-Trani, Maria T. |
| Contributors | Biochemistry and Nutrition, Smith, David F., Bevan, David R., Hess, John L., Keenan, Thomas W., Sitz, Thomas O. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | xiv, 198 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 23728777, LD5655.V856_1991.T377.pdf |
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