Biosynthesis of N-glycoprotein gylcan core using chemically synthesized lipid intermediates

The dolichyl pyrophosphates of β-Manp-(1→4)-β-D-G1cpNAc-(1→4)-α-D-GlcpNAc (I), α-D-Manp-(1→3)-β-D-Manp-(1→4)-β-D-GlcpNAc-(1→4)-α-D-GlcpNAc (II), and α-D-Manp-(1→6)-β-D-Manp-(1→4)-β-D-GlcpNAc-(1→4)-α-D-GlcpNAc (III) were chemically synthesized as follows: I from the monosaccharides, II from a disaccharide isolated from human mannosidosis urine by elongation of the sugar chain at the reducing end, and III from a tetrasaccharide isolated from the urine of swainsonine-treated sheep. I, II, and III were incubated with GDP[14C] mannose and calf pancreas microsomes or detergent-solubilized preparations derived therefrom. The products were extracted by chloroform/methanol or chloroform methanol/water and examined by t.1.c. The oligosaccharides obtained by mild hydrolysis were identified by gel filtration, liquid chromatography, enzyme degradation, acetolysis, and Con-A Sepharose chromatography. The principal additions of α-D-Manp residues were as follows: to I, preponderantly (1→3)-linked; to II a first residue (1→6)-linked, followed by other (1→2)-linked residues; and to III, compounds containing 3,4,5 or 6 residues. THus, the preferred mechanism of core assembly is addition of the α-(1→3)-linked Man residue, followed by the α-(1→6)-linked residue and the elongation of the (1→3) branch