This project will emphasize the relation of qualitative and quantitative changes in the oligosaccharides of membrane glycoproteins to glial cell differentiation during the development of the brain. The objectives of this research are to: 1) analyze the changes in the membrane oligosaccharide patterns associated with glial differentiation in primary cultured rat brain cells and in C-6 glial cells, 2) investigate the relation of changes in the membrane glycopeptides to glial differentiation and, 3) provide an insight into the mechanisms operative in such changes and to identify the possible regulatory steps in the biosynthetic pathway of oligosaccharides. The changes accompanying glial differentiation will be studied in cultured C-6 glial cells and in primary cultures of purified astrocytes and oligodendrocytes prepared from newborn rat brain. The glial cells will be induced to undergo differentiation under various experimental conditions and will be labeled with suger precursors of glycoproteins, 3H-glucosamine and 3H-mannose as well as with 32p-phosphate (for phosphoglycoproteins) and with 35S-sulfate (for sulfoglycoproteins). The glycopeptides prepared by proteolytic digestion of the trypsinates and membranes of the glial cells will be fractionated by 1) gel filtration, 2) lectin affinity chromatography, 3) ion exchange chromatography and 4) HPLC. The functional significance of membrane oligosaccharide expression during glial differentiation will be assessed by testing the effect of a) inhibitors of glycosylation: tunicamycin, compactin and swainsonine, b) isolated membrane glycopeptides on biochemical expression of glial properties (2',3', cyclic nucleotide phosphohydrolase, glutamine synthetase and sulfolipid synthesis). The regulation of the pathway of N-linked oligosaccharide biosynthesis attending glial differentiation will be studied, 1) by analyzing the intermediates of the pathway and 2) by assaying the specific glycosidases and glycosyltransferases involved in the processing and modification of the oligosaccharides.
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