A dolichol-linked trisaccharide from central nervous tissue: Structure and biosynthesis

Abstract

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[ 14C]glucosamine from UDP- N-acetyl[ 14C]glucosamine into N-acetyl[ 14C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[ 14C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [ 14C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys. 181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[ 14C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [ 14C]trisaccharide lipid. The intact [ 14C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [ 14C]trisaccharide is converted to N,N′-diacetyl[ 14C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl- N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [ 14C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP- N-acetylglucosamine to exogenous N-acetyl[ 14C] glucosaminylpyrophosphoryldolichol to form [ 14C]disaccharide lipid. The biosynthesis of [ 14C]disaccharide lipid is stimulated by unlabeled UDP- N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [ 14C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [ 14C] trisaccharide lipid. The formation of the [ 14C]trisaccharide lipid from exogenous [ 14C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg 2+, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.