Dolichol is not a necessary moiety for lipid-linked oligosaccharide substrates of the mannosyltransferases involved in in vitro N-linked-oligosaccharide assembly

Abstract

Dolichol is utilized in vivo as an unusually large anchor on which the precursor for N-linked oligosaccharides is assembled by a series of glycosyltransferases. The role of dolichol in enzyme substrate recognition is investigated. Thus the biosynthetic intermediate NN′-diacetylchitobiose was chemically linked to either dolichol or the much shorter fully saturated tetraisoprenoid phytanol. Both lipids were used as substrates by a recombinant, soluble beta-1,4-mannosyltransferase. beta-[3H]Mannosylated lipids from this reaction were then used as substrates for the subsequent mannosyltransferases from yeast or rat liver microsomes. It was found that both the dolichyl- and phytanyl-linked substrates were easily mannosylated to form Man5GlcNAc2, with some further mannosylation to Man7GlcNAc2 and Man9GlcNAc2 at low concentrations of lipid-linked substrate. It is concluded that dolichol is not necessary in vitro as part of the substrate for the mannosyltransferases in the biosynthetic pathway for N-glycosylation.