Dolichol-linked oligosaccharide and glycoprotein biosyntheses in glial cells in primary culture: development and enzymatic correlates.

Abstract

Primary cultures of cerebral glia derived from neonatal rat brain were utilized to determine 1) the developmental changes of dolichol-linked oligosaccharide and N-linked glycoprotein biosyntheses, 2) the enzymatic correlates of these developmental changes, and 3) the temporal relations between the biosyntheses of dolichol-oligosaccharide and N-linked glycoproteins and the biochemical expression of astrocytic and oligodendroglial differentiation. Marked, parallel developmental increases in the rates of incorporation of [3H]glucosamine into both dolichol-oligosaccharide and glycoprotein were observed, with maximal rates achieved after 9-10 days in culture and little change occurring over the next 10 days in culture. Concerning the enzymatic correlates, dolichol kinase exhibited a moderate developmental increase with a maximum at 5 days in culture, whereas the activities of the three critical enzymes that utilize dolichyl phosphate in the synthesis of the dolichol-linked oligosaccharide, i.e., N-acetylglucosaminylphosphotransferase (GlcNAc-1-P transferase), mannosylphosphoryldolichol (Man-P-Dol) synthase, and glucosylphosphoryldolichol (Glc-P-Dol) synthase, reached maxima after 6-9 days in culture. Both the activity of Man-P-Dol synthase in vitro and the rate of formation of its product, Man-P-Dol, in intact cells were shown to correlate closely with the rates of oligosaccharide and glycoprotein biosyntheses. An important regulatory role for Man-P-Dol synthase and its product, Man-P-Dol, was suggested further by the demonstration of a maturation-dependent activation by Man-P-Dol of GlcNAc-1-P transferase, the first committed step in the pathway. Two enzymatic markers of astrocytic (glutamine synthetase) and oligodendroglial (2',3'-cyclic nucleotide 3'-phosphohydrolase) differentiation exhibited marked developmental increases in activity with onset at the time of attainment of peak rates of dolichol-oligosaccharide and glycoprotein biosyntheses. Importance of the latter processes for glial differentiation is suggested.