To investigate the changes in glycolipid biosynthesis during early embryogenesis mouse F9 teratocarcinoma cells were induced to differentiate in vitro in the presence of retinoic acid. Control embryonal carcinoma cells and their differentiated derivatives, RNF9 cells, were metabolically-radiolabeled with [6-3H]galactose or [6-3H]glucosamine, and their glycolipids were compared. The neutral and acidic glycolipid fractions from both cell lines were subjected to ozonolysis and alkali fragmentation or endoglycoceramidase digestion to release the glycolipid-derived oligosaccharides. The neutral oligosaccharides were separated according to size by gel filtration and high performance liquid chromatography. These analyses indicated that differentiated F9 cells synthesized less high molecular weight oligosaccharides (containing more than 5 sugar residues) relative to controls. Serial lectin affinity chromatography on columns of immobilized Helix pomatia. Wisteria f1oribunda. Griffonia simplicifolia-I and Ricinus communis-' agglutinins followed by reduction and permethylation revealed that globoside (GaINAcβ1, 3Galα1, 4Galβ1, 4Glc) and lactose (Galβ1,4Glc) are the principal glycolipid-derived oligosaccharides synthesized by F9 and RNF9 cells. An increased biosynthesis of these components was observed in RNF9 cells relative to controls. These changes paralleled the reduced biosynthesis of Forssman pentasaccharide (GaiNAcα 1 ,3GaINAcβ1 ,3Galα1 ,4Galβ1 ,4Glc) reported previously. Normalization of the incorporation of 3H-monosaccharides in glycolipid-derived oJigosaccharides to the number of cells indicated a 2-6 fold increase in the incorporation of radioactive precursors in RAlF9 cells relative to F9 controls, suggesting that an enhancement in glycosphingolipid biosynthesis accompanies the differentiation of F9 cells. The monosialylganglioside-derived oligosaccharides obtained from F9 and RAlF9 cells were separated by anion exchange chromatography. A reduced biosynthesis of high molecular weight components was observed in RAlF9 cells when compared with undifferentiated F9. Lectin affinity chromatography on immobilized Maackia amurensis agglutinin followed by reduction and permethylation indicated a dramatic increase in the synthesis of GM1 (Galβ1,3Ga1NAcβ1,4[NeuAcα2,3] Galβ1,4Glc) and GM3 (NeuAcα2,3Galβ1,4Glc) in RAlF9 cells relative to controls. These changes were accompanied by a decrease in the synthesis of sialyltetrasaccharide a (NeuAcα2,3Galβ1 ,3GlcNAcβ1 ,3Galβ1,4Glc) and sialylparagloboside (NeuAcα2,3Galβ1 ,4GlcNAβ1 ,3Galβ1 ,4Glc) in the differentiated cells. These observations are in agreement with previous reports in leukemic and human embryonal carcinoma cell lines and may be related to the growth arrest and antigenic changes associated with F9 differentiation. In the work reported herein, serial lectin affinity chromatography in concert with permethylation analysis prove to be powerful methods for the isolation and characterization of glycolipid-derived oligosaccharides. The application of these methods has allowed the unequivocal identification of main glycosphingolipid components as well as of some representing less than 1 % of the total glycolipids synthesized by two cell lines. This information should provide the basis for further studies involving glycosyltransferas. / Master of Science
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/44226 |
| Date | 14 August 2009 |
| Creators | Gorbea, Carlos M. |
| Contributors | Biochemistry and Nutrition, Smith, David F., Potts, Malcolm, Bevan, David R., Gregory, Eugene M., Schurig, Gerhardt G. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | xvi, 162 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 23739320, LD5655.V855_1991.G672.pdf |
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