Glucosylphosphoryldolichol: Role as a glucosyl donor in the biosynthesis of an oligosaccharide lipid intermediate by calf brain membranes

When membrane preparations from calf brain are incubated with exogenous [ 14 C]-glucosylphosphoryldolichol, [ 14 C]glucose is enzymatically transferred into an endogenous oligosaccharide lipid. Efficient transfer was achieved by dispersing the lipophilic glucosyl donor in Triton X-100. The transfer of [ 14 C]glucose from exogenous [ 14 C]glucosylphosphoryldolichol into [Glc-[ 14 C]oligosaccharide lipid did not require the addition of a divalent cation, and occurred in the presence of EDTA. The inclusion of Ca 2+ in the reaction mixture resulted in a lower level of [Glc- 14 C]oligosaccharide lipid recovery and the incorporation of label into glycoprotein. If Ca 2+ is added following a 10-min preincubation of membranes with exogenous [ 14 C]glucosylphosphoryldolichol, label is lost from the oligosaccharide lipid fraction and there is a corresponding appearance of label in glycoprotein. The [Glc- 14 C]-oligosaccharide lipid glycosylated via [ 14 C]glucosylphosphoryldolichol in brain membranes is recovered by extraction with CHCl 3 -CH 3 OH-H 2 O (10:10:3). The chromatographic behavior of the labeled oligosaccharide lipid on DEAE-cellulose is similar to that of the oligosacharide lipids in which the oligosaccharide units are bound to dolichol by a pyrophosphate linkage. Consistent with this proposed structure, the [Glc- 14 C]oligosaccharide is released from the carrier lipid by treatment with 0.1 n HCl in 80% tetrahydrofuran at 50°C for 20 min. The free [Glc- 14 C]oligosaccharide unit contains approximately 10 glycose residues based on gel filtration analysis. The presence of two N -acetylated sugars is indicated by the formation of two positively charged products by treatment with strong base and the subsequent electrophoretic neutralization under conditions used for N -acetylation. The labeled product recovered in the delipidated membrane residue following incubation of calf brain membranes with exogenous [ 14 C]glucosylphosphoryldolichol in the presence of Ca 2+ can be solubilized with 1% sodium dodecyl sulfate-0.1% mercaptoethanol (100°C, 2 min). The solubilized product is resolved into two large molecular weight fractions by chromatography on a Bio-Gel A-1.5m column eluted with a buffer containing 0.2% sodium dodecyl sulfate-0.1% mercaptoethanol. Both high molecular weight fractions are converted to low molecular weight products by digestion with Pronase or trypsin. The labeled glycopeptide fraction produced by Pronase digestion is eluted as a single peak on Bio-Gel P-6 and appears to be slightly larger than the glucosylated oligosaccharide released from the carrier lipid by mild acid treatment. Strong acid hydrolysis (3 n HCl, 100°C, 6 h) of the [ 14 C]-glycopeptides released a single radioactive product that cochromatographed with authentic glucose. The electrophoretically neutral [Glc- 14 ]glycopeptide fractions by treatment with mild alkali, indicating that the [Glc- 14 C]oligosaccharide units are probably attached to the polypeptide chains by an N -glycosidic linkage. These results show that in calf brain membranes at least one glucose residue in a glucosylated oligosaccharide lipid is acquired via glucosylphosphoryldolichol, and strongly suggest that in the presence of Ca 2+ the glucosylated oligosaccharide unit can be enzymatically transferred from its carrier lipid into glycoprotein