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.
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